Muscle Injury and Aging Differentially Shape Immune Responses to Influenza Vaccination

Muscle Injury and Aging Differentially Shape Immune Responses to Influenza Vaccination | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Muscle Injury and Aging Differentially Shape Immune Responses to Influenza Vaccination Moataz Noureddine, Eleanor Burgess, Johanna Vandekerckhove, Farah El-Ayache, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8544011/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The muscle environment during intramuscular vaccination has the potential to shape downstream immune responses, yet this relationship remains poorly defined. We hypothesize that muscle condition at the time of vaccination matters for the outcome of vaccination. In this study, we examined how glycerol-induced muscle injury at the time of quadrivalent inactivated influenza vaccine (QIV) administration can skew host immune responses to vaccination and modulate systemic and mucosal immunity following subsequent H1N1 challenge three weeks later. Given that seasonal influenza disproportionately affects older adults, and that muscle regenerative capacity is both sex- and age-dependent, we addressed sex and age as biological variables. We found that muscle injury during QIV vaccination promoted regulatory T cell and M2 macrophage levels in the lungs upon subsequent H1N1 infection three weeks later, with responses more pronounced in young females and diminished with age in both sexes. Muscle injury also shifted antibody isotype distribution away from IgG2c toward IgG1 in young male and aged mice, without altering total hemagglutinin-specific antibody titers. In young influenza virus-infected mice, prior QIV vaccination resulted in a type 2–skewed mucosal immune response, marked by IL-4, IL-5, IL-13, and eosinophilia, whereas this response was attenuated in aged cohorts. Finally, because IL-33 is a pro-regenerative alarmin reduced in aging muscle, we tested the impact of IL-33 supplementation. IL-33 enhanced vaccine-specific antibody production across age groups and restored type 2 mucosal immunity in aged mice, thereby partially rescuing the age-associated impairment of the host response to infection, although it did not enhance glycerol damage-dependent regulatory T-cell influx into the lungs after infection as observed in young mice. Our data show that muscle health at the time of vaccination can impact host vaccine responses that shape the lung immune environment during subsequent respiratory infection and highlight IL-33 as a target for vaccine adjuvants. Biological sciences/Immunology/Vaccines Biological sciences/Immunology/Adaptive immunity/Cellular immunity Biological sciences/Microbiology/Virology/Influenza virus Vaccination Muscle regeneration Aging Influenza Macrophages IL-33 Tregs Type II immunity Sex Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Skeletal muscle is an immunocompetent and highly regenerative organ 1 – 4 . Under homeostatic conditions, the muscle contains few immune cells; however, upon injury like trauma, immune cells can rapidly infiltrate 5 . The muscle’s response to injury follows a well-coordinated sequence beginning with an early pro-inflammatory phase dominated by CX3CR1⁺Ly6C hi macrophages, which clear debris and dying cells and activate myoblasts 6 , 7 . This is followed by a pro-repair phase characterized by CX3CR1⁺Ly6Cˡᵒ macrophages and regulatory T-cells (Tregs) that orchestrate myoblast differentiation and myotube formation 8 , 9 . These stages are accompanied by eosinophils 10 , neutrophils 11 and dendritic cells 12 , 13 , which provide key cues such as IL-4 for macrophage repolarization and contribute to antigen surveillance. The muscle is also the primary site for administration of most parenteral vaccines, yet its intrinsic immunological properties remain understudied in the context of vaccine and adjuvant usage. The regenerative muscle environment is rich in cytokines such as IFNγ, TNFα, IL-4, and IL-33, all of which influence the type and magnitude of T- and B-cell responses to vaccination 14 – 17 . Despite this, how the local immune milieu of the muscle contributes to vaccine efficacy and how it differs across sex and age remain poorly understood. Here, we hypothesized that inducing muscle injury at the time of vaccination modulates vaccine responses in a sex- and age-dependent manner via regenerative immune pathways. To test this, we used an established glycerol injection-based injury model combined with vaccination with the seasonal quadrivalent inactivated influenza vaccine (QIV). We examined sex-dependent effects, given that estrogen alters regenerative responses in females 18 , 19 , and age-dependent effects, since muscle regeneration declines with age which has been linked to reduced IL-33 levels 20 – 23 . To address this, we investigated whether enhancing regeneration through supplementation with 0.3 µg of IL-33, a previously defined reparative dose, could restore vaccine responses in aged mice 20 – 23 . In this study, we show that female mice mount a stronger regenerative immune response to muscle injury than young and aged males. Muscle injury during vaccination modulates humoral and cellular immune responses in both a sex- and age-dependent manner, and has longlasting effects as it enhances Treg and M2 macrophage accumulation in the lungs after subsequent influenza infection, particularly in female mice. Muscle injury also increases the IgG1:IgG2c ratio in young male and aged mice. Furthermore, IL-33 supplementation rescues the reparative response in aged mice, reduces sex-dependent variability in local immune responses, enhances anti-vaccine total IgG antibody titers, and restores type II vaccine-specific immune responses in aged animals. Results Glycerol induces cell death in C2C12 myotubes, and induces a sex-dependent pro-inflammatory regenerative response in quadriceps muscles. Glycerol has been used to induce muscle injury in mouse models to study subsequent muscle regeneration 24 – 28 . To assess its effects on muscle cells in vitro , we cultured C2C12 myoblasts in 24-well plates and differentiated them into myotubes. Once differentiated, cells were treated for 24 hours in serum-free media with one of the following: 25% PBS, 25% glycerol, 0.6 µg QIV, or 25% glycerol plus 0.6 µg QIV. To quantify cell viability, we performed flow cytometry staining 29 , 30 .The number of viable myotubes was significantly reduced and approached zero, in wells treated with glycerol or glycerol + QIV, compared to an average of 8,365 ± 1,944 live cells with QIV and 13,811 ± 7,389 live cells with PBS (Fig. 1 A). Additionally, in the glycerol + QIV group 4 out of 5 wells had enhanced levels of IL-6, which was a significant increase compared to glycerol alone (Fig. 1 B). Further, we observed increased IFNγ and IL-18 compared to PBS (Fig. 1 C,D), QIV, and glycerol; increased TNFα compared to QIV and glycerol (Fig. 1 E); and increased IL-2 compared to QIV and PBS (Fig. 1 F). To evaluate the effects of muscle injury on vaccine response in vivo , we induced controlled muscle damage using a previously established model that uses a concentration of 50% glycerol in mice during vaccination 31 . We also investigated the combination of 50% glycerol with IL-33, an alarmin extensively reported to initiate pro-regenerative pathways in muscle injury and shown to decline with age 32 . To do so, we vaccinated young and aged, male and female mice in the quadriceps with 3 µg of the seasonal QIV vaccine (designated group Q) alone, 50% glycerol alone (designated group G), QIV + 50% glycerol (designated group Q + G), or Q + G supplemented with 0.3 µg of IL-33 (designated group Q + G + IL-33). We collected injected quadriceps on day 2 to capture the early pro-inflammatory phase following injury 33 . We utilized two spectral flow cytometry panels that allowed the characterization of the myeloid and lymphocyte compartments in the muscle (Gating schemes are provided in Supplementary Figs. 1 and 2). First, we investigated sex-dependent muscle immune composition differences in the control P group. In the P groups on day 2, young female and male mice had a comparable myeloid cell distribution, with no significant differences in neutrophil, eosinophil, macrophage, and cDC2 counts per gram of muscle between both sexes (Fig. 1 G, Supplementary Table 2). On the other hand, lymphocytes were significantly more enriched in male P-group quadriceps on day 2, including CD4⁺ T helper cells, CD8⁺ T cells, and B cells compared to female P quadriceps (Fig. 1 H, Supplementary Table 2). Next, we characterized the immune composition on day 2 after a 50% glycerol injection. In the G group, glycerol induced a significant increase in immune cell counts per gram in male (19-fold) and female (13-fold) mice compared to their respective P groups (Fig. 1 G, Supplementary Table 1). After glycerol injection, female quadriceps showed 764,052 ± 321,691 immune cells per gram of muscle, compared to 197,708 ± 25,156 in male quadriceps (Fig. 1 G, Supplementary Fig. 3A and B, Supplementary Table 1). The significant increase in immune cell counts in the G group was driven by increases in eosinophils, neutrophils, macrophages, and cDC2s (Fig. 1 G; see Supplementary Fig. 3 for individually plotted populations). There was no significant difference in all quantified immune populations between male and female G groups (Supplementary Table 2). In the G groups, eosinophils comprised 20% ± 13% and 18% ± 12% of total immune cells per gram in females and males, respectively (Fig. 1 G; Supplementary Table 1). Neutrophils were significantly increased in the male G group compared with the male P group, whereas no such increase was observed in females. Accordingly, neutrophils constituted a significantly greater fraction of total immune cells in male G mice (24% ± 3%) than in female G mice (11% ± 2%) (Fig. 1 G; Supplementary Fig. 4A; Supplementary Table 1). Macrophages were the most abundant immune cell population, accounting for 39% ± 16% and 45% ± 16% of total immune cells per gram in female and male G groups, respectively (Supplementary Table 1). Conventional dendritic cells 2 (cDC2s) represented approximately 5% ± 7% of total immune cells in both sexes (Fig. 1 H; Supplementary Table 1). No significant increase in T-cell counts per gram was observed at this time point in either sex (Fig. 1 K). Compared to the G group, the Q group showed significantly lower immune cell counts per gram in males and a downward trend in females (Fig. 1 G). However, immune cell counts were still approximately twofold higher in Q than in P in both sexes (Supplementary Table 1). This increasing trend of immune cell count in Q group was driven by increasing trends in cDC2s, eosinophils, CX3CR1⁺Ly6C hi and CX3CR1⁺Ly6Cˡᵒ macrophages and neutrophils (Supplementary Table 1, see Supplementary Fig. 3 for individually plotted populations). Q groups differed between sexes, where male Q mice had significantly fewer neutrophils, CX3CR1⁺Ly6C hi and CX3CR1⁺Ly6Cˡᵒ macrophages, and cDC2s than female Q mice (Supplementary Table 2). The Q + G group showed no significant difference in immune cell counts per gram compared to G in either sex with significant increases in eosinophils, cDC2s, and both CX3CR1⁺Ly6C hi and CX3CR1⁺Ly6Cˡᵒ macrophages compared to P (Fig. 1 G, see Supplementary Fig. 3 for individually plotted populations). Direct comparison between sexes revealed that Q + G immune composition exhibited variability unlike G alone. In Q + G, males had significantly fewer eosinophils, CX3CR1⁺Ly6C hi macrophages, and cDC2s, and a greater enrichment of CD8⁺ T cells and CD4⁺ T helper cells relative to female Q + G (Fig. 1 H, Supplementary Table 2). The Q + G + IL-33 group showed no significant differences compared to G or Q + G immune composition (see Supplementary Fig. 3 for individually plotted populations). Notably, IL-33 reduced sex-associated variability observed in Q + G, with only cDC2s and CX3CR1⁺Ly6C hi macrophages remaining significantly lower in males than in females (Fig. 1 H, Supplementary Table 2). Next, we investigated the macrophage phenotype in all conditions. In both sexes, macrophages increased significantly in the G, Q + G, and Q + G + IL-33 groups compared to P (Supplementary Fig. 3). The infiltrating macrophages consisted of pro-inflammatory CX3CR1⁺Ly6C hi and pro-repair CX3CR1⁺Ly6Cˡᵒ subsets (Fig. 1 G,I and J). The increase in CX3CR1 + Ly6C hi macrophage counts was significantly more robust in female Q, Q + G and Q + G + IL-33 groups compared to male respective groups (Fig. 1 H). To assess the inflammatory state of the macrophage compartment, we computed the proportions of CX3CR1⁺Ly6C hi and CX3CR1⁺Ly6Cˡᵒ macrophages among total macrophages (Fig. 1 I). We found a significant enrichment of pro-inflammatory CX3CR1⁺Ly6C hi macrophages In female Q, G, Q + G and Q + G + IL-33 groups compared to P group (Fig. 1 I, Supplementary Fig. 4B). Meanwhile, In males, the proportion was comparable between all groups (Supplementary Fig. 4B). Because dendritic cells are well established as key inducers of adaptive immune responses to intramuscular vaccination 34 , 35 , we investigated their relationship with CX3CR1⁺Ly6C hi macrophages, which are critical for muscle regeneration following injury 36 . Pearson’s correlation analysis revealed a strong positive linear relationship between CX3CR1⁺Ly6C hi macrophages and cDC2 counts per gram of muscle in females (R² = 0.878, y = 5.687 + 8682) and males (R² = 0.699, y = 9.206–9292) (Fig. 1 L). Furthermore, there were significant positive and linear correlations between cDC2s and eosinophils (female: R² = 0.94; male: R² = 0.9)(Supplementary Fig. 4C) and between CX3CR1⁺Ly6C hi macrophages and eosinophils (female: R² = 0.79; male: R² = 0.77) (Supplementary Fig. 4D), regardless of condition or sex. CX3CR1 + Ly6C hi macrophages and lymphocytes are increased in draining inguinal lymph nodes in female mice after Q + G vaccination, and IL-33 increases general immune infiltration to the draining lymph nodes irrespective of sex. The adaptive immune response to intramuscularly delivered vaccine antigens is mounted in the draining lymph nodes, one of which is the inguinal draining lymph node 37 .To investigate whether muscle injury modulates immune response in the draining lymph node after vaccination, we harvested inguinal draining lymph nodes on day 2 after P, Q, G, Q + G, or Q + G + IL-33 injections. We characterized their immune composition using two spectral flow cytometry panels enabling the quantification of multiple myeloid and lymphocyte populations (Gating schemes are provided in Supplementary Figs. 5 and 6). In female mice on day 2 after vaccination, a significant threefold increase in total immune cell counts was observed in Q + G + IL-33 compared to P. In Q + G, we observed a twofold increasing trend in immune cell counts compared to P in female lymph nodes (Supplementary Table 3). The increase in immune cell number in Q + G + IL-33 was driven by enrichments in neutrophil, macrophage, lymphocyte, and cDC2 counts (Fig. 2 A and B, Supplementary Table 1, see individually plotted populations per condition in Supplementary Fig. 7). Meanwhile, in Q + G, the enrichment was mainly confined to the macrophage compartment—particularly the Ly6C hi CX3CR1⁺ macrophage subset—and lymphocytes (Fig. 2 C-G). Within the lymphocyte compartment of female draining lymph nodes, Tregs increased significantly by twofold after Q + G and three-fold after Q + G + IL-33 compared to P, whereas Q alone did not result in significant differences over P (Fig. 2 C, Supplementary Table 3). B cells increased by three-fold after Q + G and four-fold after Q + G + IL-33 relative to P, and B cell numbers were also significantly higher in the Q + G + IL-33 group compared to Q only (Fig. 2 D, Supplementary Table 3). CD8⁺ T cells increased by two-fold after both Q + G and Q + G + IL-33 compared to P (Fig. 2 E, Supplementary Table 3). CD4⁺ T helper cells also increased by two-fold after Q + G and Q + G + IL-33 compared to P (Fig. 2 F, Supplementary Table 3). When the myeloid compartment is considered in female mice, CX3CR1⁺Ly6C hi macrophages increased by three-fold following Q + G, and this effect was further amplified to a sixteenfold increase with Q + G + IL-33 (Fig. 2 G, Supplementary Table 3). Excluding the Q + G + IL-33 condition, Q + G alone induced a significant increase in total immune cells and in CX3CR1⁺Ly6C hi macrophages compared to P and G. Additionally, CX3CR1⁺Ly6C hi macrophage counts correlated positively with B-cell counts in lymph nodes across P, G, Q, and Q + G conditions (linear regression: Y = 97.19 × X + 47,185; R² = 0.527) (Fig. 2 H). Downstream of Q + G + IL-33, the regression slope did not significantly differ from zero, indicating the loss of correlation between these two populations (Fig. 2 H). cDC2s and CX3CR1⁺Ly6Cˡᵒ macrophages increased only downstream of Q + G + IL-33 by 11-fold and 4-fold, respectively, and did not change significantly in Q, G, or Q + G compared to P (Fig. 2 I). In male mice, immune kinetics differed markedly from females on day 2. We found a twofold higher total immune cell count in male P lymph nodes compared to female P lymph nodes (Fig. 2 A and 2 B, Supplementary Table 3). Immune cell counts were significantly lower downstream of Q (61% of P, SD = 16%), G (62% of P, SD = 15%), and Q + G (43% of P, SD = 16%) compared to P (Fig. 2 A and 2 B, Supplementary Table 3, Supplementary Fig. 8A). In contrast, Q + G + IL-33 restored immune cell counts to baseline, with values comparable to P (128% of P, SD = 19%) (Fig. 2 A and 2 B, Supplementary Table 3, Supplementary Fig. 8A). We did not observe significant increases in myeloid or lymphocyte populations in male draining lymph nodes on day 2 compared to P (Supplementary Fig. 8A-I). However, if only vaccinated groups were considered in comparisons, Q + G + IL-33 led to a significant enrichment of neutrophils and eosinophils compared to Q. Moreover, Q + G + IL-33 significantly increased CX3CR1⁺Ly6Cˡᵒ macrophages and B cells compared to Q and Q + G. Tregs, CD8⁺ T cells, and CD4⁺ T helper cells were also significantly enriched downstream of Q + G + IL-33 compared to Q, G, and Q + G. Comparing male and female draining lymph nodes, male P lymph nodes were significantly more enriched in macrophages, cDC2s, CD4⁺ T helper cells, CD8⁺ T cells, and Tregs compared to female P lymph nodes (Supplementary Table 4). However, eosinophils were significantly lower in male P lymph nodes relative to females on day 2 (Supplementary Table 4). Glycerol triggers a muscle pro-regenerative response by day 4 in young female and male mice, amplified by IL-33 supplementation in young mice and independent of IFNAR signaling. Since muscle regenerative response consists of a pro-inflammatory response followed by a later pro-repair immune response, we characterized the muscle response on day 4 in young female, young male, and aged male mice. By day 4, the regenerative phase has been described to shift toward a pro-repair or anti-inflammatory milieu 38 ,characterized by an influx of regulatory T cells (Tregs) and an increase in CX3CR1⁺Ly6Cˡᵒ macrophages 39 , 40 . By day 4, the immune infiltrate consisted mainly of macrophages, eosinophils, T cells, and cDC2s in the G, Q + G, and Q + G + IL-33 groups for both sexes (Fig. 3 A and 3 B, see Supplementary Figs. 9 and 10 for individually plotted myeloid and lymphocyte populations per condition, respectively). Neutrophil counts declined by more than 83% across all conditions compared to day 2 after vaccination suggesting the transition to the anti-inflammatory stage (Supplementary Table 7). On day 4, the muscle immune composition appeared to be more comparable in P, Q, Q + G, and Q + G + IL-33 between males and females than on day 2 (Fig. 3 A and 3 B). In G groups, the immune influx in the males averaged at 322,892 immune cells per gram (SD ± 158,467) which is a 98% (± 40%, P = 0.02) increase compared to the female G group (162,918 ± 103,277 SD) (Supplementary Table 6). This pattern differed from day 2, when female G exhibited sixfold higher immune cell counts than male G (Supplementary Table 2). Compared to day 2, female mice showed a significant decrease on day 4 in total immune cell count per gram in P, Q, G and Q + G groups while Q + G + IL-33 showed no change (Supplementary Table 7). Meanwhile, male mice showed a significant increase in total immune cell count per gram in G, Q + G and Q + G + IL-33 and a significant decrease in P and Q groups compared to day 2 (Supplementary Table 7). This led to male G group having significantly higher counts in eosinophils, cDC2s and CX3CR1 + Ly6C lo macrophages compared to female G on day 4 (Supplementary Table 6). Otherwise, no significant sex-dependent differences were observed in P, Q, Q + G and Q + G + IL-33 immune populations other than a significantly higher neutrophil count in Q + G in males compared to females (Supplementary Table 6). With these changes compared to day 2, G, Q + G, and Q + G + IL-33 groups still maintained significantly elevated macrophage counts compared to P in both sexes (Supplementary Fig. 9). Macrophages represented 22–48% and 37–42% of total immune cells in female and male mice, respectively (Supplementary Table 5), and only decreased in density in P and Q groups of both sexes (Supplementary Table 7). Additionally, CX3CR1⁺Ly6Cˡᵒ-to-CX3CR1⁺Ly6C hi macrophage ratio was significantly altered in both males and females compared to day 2 (Fig. 3 C, Supplementary Table 7). In both sexes, CX3CR1⁺Ly6Cˡᵒ macrophages increased significantly in G, Q + G, and Q + G + IL-33 compared to P (Fig. 3 D). Unlike day 2, CX3CR1⁺Ly6Cˡᵒ macrophages became the dominant subset at 4 days after vaccination, representing 73% ±11% of macrophages in female and 72% ±8% in male G muscles, and 74% ±10% (female) and 66% ±9% (male) in Q + G (Fig. 3 D). In Q + G + IL-33, CX3CR1⁺Ly6Cˡᵒ macrophages comprised 73% ±10% of macrophages in males but were balanced with CX3CR1⁺Ly6C hi macrophages (49% ±12%) in females (Fig. 3 D). On day 4, eosinophil and cDC2 counts were significantly reduced compared to day 2 in P and Q groups for both sexes (Supplementary Table 7). For the G, Q + G, and Q + G + IL-33 groups, Female eosinophil counts remained unchanged while in males they increased compared to day 2 (Supplementary Table 7). cDC2s also remained elevated and unchanged in G, Q + G and Q + G + IL-33 regardless of sex but in the female Q + G group where they decreased by 53% (± 19%) compared to day 2 (Supplementary Table 7). Unlike day 2 after vaccination, CD3⁺ T-cell densities increased significantly in male G and Q + G muscles compared to P and Q, and in male Q + G + IL-33 compared to P (Supplementary Fig. 10A and 10B). In females, G and Q + G + IL-33 groups also showed significant CD3⁺ T-cell enrichment compared to P and Q, while Q + G displayed a 40-fold increasing trend (Supplementary Table 5). This increase reflected elevated Tregs, CD8⁺ T cells, and CD4⁺ T helper cells in both sexes (Fig. 3 B, see Supplementary Fig. 10 for individually plotted lymphocyte populations per condition). Tregs increased robustly in male G, Q + G, and Q + G + IL-33 compared to P and Q (Supplementary Fig. 10C), and in female G and Q + G + IL-33 compared to P (Supplementary Fig. 10D). Between day 2 and 4, Q + G + IL-33 induced the largest Treg expansion: +3072% (± 825%) in females and + 2557% (± 529%) in males (Supplementary Table 7). In Q + G, Tregs increased + 56.8% (± 77%) in females and + 520% (± 151%) in males compared to day 2 (Supplementary Table 7). CD8⁺ T-cell counts per gram increased significantly in male G and Q + G + IL-33 versus P and Q (Supplementary Fig. 10G), and in male Q + G versus P. In females, CD8⁺ T cells rose significantly in G (vs. P) and Q + G + IL-33 (vs. P and Q) (Supplementary Fig. 10H). From day 2 to day 4, Q + G + IL-33 showed the largest CD8⁺ T-cell increase—+512% (± 130%) in males and + 1697% (± 626%) in females (Supplementary Table 7). Q + G also caused a 277% (± 121%) increase in females and 117% (± 49%) in males compared to day 2 (Supplementary Table 7). We next computed the CD8⁺ T-cell to Treg ratio in muscle to evaluate whether IL-33 altered the inflammatory-to-regulatory lymphocyte balance (Supplementary Fig. 10I). No significant differences were detected between G, Q + G, and Q + G + IL-33 in either sex. Finally, we were interested in investigating type I interferon role in muscle regeneration. To do so, we injected IFNAR knockout (IFNAR⁻/⁻) female mice with P or Q + G and analyzed muscle tissue on day 4. No significant changes in immune composition were observed between IFNAR⁻/⁻ and wild-type females under either condition ( Fig. 3 A and 3 B, Supplementary Table 6). Muscle pro-regenerative response is replaced with an increased CD8 + T-cell response in aged mice which is reversed with the supplementation of IL-33. With age, muscle health deteriorates and muscles become sarcopenic 41 , which can be assessed in mice by calculating the muscle-to-body weight ratio 42 , 43 . To evaluate muscle health in young, aged, and IFNAR⁻/⁻ mice, we computed their muscle-to-body ratio. We found a significant decrease in this ratio in aged male mice, compared to young male, young female, and IFNAR⁻/⁻ female mice (Fig. 3 E). Young female, male, and IFNAR⁻/⁻ female mice exhibited comparable muscle-to-body ratios (Fig. 3 E). In aged male mice on day 4, we observed a significant enrichment of CD3⁺ lymphocyte counts in the aged P group (597 ± 302) compared to young male P mice (7 ± 7 cells per gram of muscle; Supplementary Tables 5 and 8). The age-associated increase in lymphocyte density in P mice was driven by elevated CD8⁺ T cells, CD4⁺ T-helper cells, and Tregs (Supplementary Table 8). Additionally, the aged P group showed a significant enrichment in CX3CR1⁺Ly6Cˡᵒ macrophage counts (344 ± 154) compared to the young P group (101 ± 50; Supplementary Tables 5 and 8). In the aged male Q + G group, total immune cell counts were significantly higher than in Q and P groups (Fig. 3 A and 3 B, Supplementary Fig. 11A). In aged Q + G mice, this increase relative to P reflected significant enrichment of macrophages (Supplementary Fig. 11B) including CX3CR1 + Ly6C lo macrophages (Fig. 3 F), CX3CR1 + Ly6C hi macrophages (Fig. 3 G), neutrophils (Supplementary Fig. 11C), eosinophils (Fig. 3 H), CD8⁺ T cells (Supplementary Fig. 11D), Tregs (Fig. 3 I), and cDC2s (Fig. 3 J). However, when compared to young male Q + G mice, total immune cell density was significantly lower by 75% (± 39%)(Supplementary Table 8). Compared to young male Q + G mice on day 4, macrophage density in aged Q + G muscle was significantly lower by 75% (± 37%). This reduction was driven primarily by CX3CR1⁺Ly6Cˡᵒ macrophages, which was 81% lower (± 38%), whereas CX3CR1⁺Ly6C hi macrophages showed a nonsignificant decreasing trend of 65% (± 37%)(Supplementary Table 8). Relative to young Q + G males, eosinophil counts were significantly lower by 94% (± 43%) and cDC2 by 90% (± 39%) in aged males at the same time point (Supplementary Table 8). In the lymphocyte compartment, no increase in B-cell counts was detected in aged Q + G mice compared to P (Supplementary Fig. 11E). Moreover, B-cell numbers were significantly lower in aged Q + G males relative to young Q + G males by 97% (± 30%) (Supplementary Table 8). In contrast, CD3⁺ T cells increased significantly in aged Q + G mice compared to P and Q (Supplementary Fig. 11F). Specifically, CD8⁺ T cells increased significantly in aged Q + G mice compared to P and Q, mirroring trends in young Q + G mice (Supplementary Fig. 11D). CD4⁺ T-helper cells showed a nonsignificant enrichment compared to P and Q (Supplementary Fig. 11G). When compared to young Q + G males on day 4, aged Q + G mice displayed significant enrichment of CD3⁺ T cells (+ 505% ± 183%), CD8⁺ T cells (+ 1376% ± 458%), and CD4⁺ T-helper cells (+ 856% ± 303%) ( Fig. 3 L, Supplementary Table 8). Treg counts increased significantly in aged Q + G mice compared to Q (Fig. 3 I), and were comparable between aged and young Q + G males on day 4 (Supplementary Table 8). However, the Treg:CD8⁺ T-cell ratio was significantly lower in aged Q + G than in young Q + G males (Supplementary Fig. 10L). Q + G + IL-33 did not elicit a significant increase in total immune cells in aged mice compared to P or Q (Supplementary Fig. 11A). In aged male Q + G + IL-33 mice, CD3⁺ T cells—including CD8⁺ and CD4⁺ subsets—were not significantly different from young Q + G or Q + G + IL-33 mice on day 4 (Supplementary Table 8). B cells did not increase in Q + G + IL-33 relative to P or Q, unlike in young male Q + G + IL-33 (Supplementary Fig. 11E). Similar to aged male Q + G, Q + G + IL-33 showed a significant reduction in total immune cell counts, neutrophils, eosinophils, CX3CR1⁺Ly6C hi macrophages, CX3CR1⁺Ly6Cˡᵒ macrophages, and cDC2s compared to young male Q + G and Q + G + IL-33 groups (Supplementary Table 8). However, aged male Q + G + IL-33 mice exhibited an increased Treg:CD8⁺ T-cell ratio compared to aged Q + G males, restoring the ratio to comparable levels to young male G, Q + G, or Q + G + IL-33 groups (Supplementary Fig. 10L). Increased macrophage levels in the draining lymph nodes downstream of Q + G subside by day 4 in female but not in male mice and are lower in aged mice but rescuable with IL-33 supplementation. Next, we collected inguinal draining lymph nodes on day 4 from young female, young male, aged male, and IFNAR⁻/⁻ female mice and characterized their immune myeloid and lymphocyte composition (Fig. 4 A and 4 B). On day 4, we found neutrophils, CX3CR1⁺Ly6Cʰⁱ macrophages, and B cells to be significantly enriched in Q + G + IL-33 compared to P in young female draining lymph nodes (Fig. 4 C, Supplementary Fig. 12A and 12B). Young female Q draining lymph nodes were also enriched in CD3⁺ T cells and CD8⁺ T cells compared to P and Q + G (Supplementary Fig. 13A and 13B). Additionally, compared to young female Q + G, Q was enriched in Tregs on day 4 (Supplementary Fig. 13C). In young male mice, no statistically significant changes were detected in myeloid or lymphocyte populations across groups in the draining lymph nodes on day 4 (Supplementary Figs. 12C-I and 13D-H). Compared to day 2, the composition of draining lymph nodes changed significantly by day 4 in both sexes (Supplementary Table 10). In female mice, total immune cell counts per lymph node were significantly lower in Q + G and Q + G + IL-33 on day 4 compared to day 2 (Supplementary Table 10). Within the myeloid compartment, all young female groups showed a significant reduction in macrophages and CX3CR1⁺Ly6Cˡᵒ macrophages on day 4 compared to day 2 (Supplementary Table 10). The magnitude of decrease varied, with the largest change observed in the Q + G + IL-33 group (-11,787 ± 2,899 cells per lymph node), followed by Q + G (-2,198 ± 522), and smaller but comparable reductions in Q (-1,083 ± 305), P (-830 ± 173), and G (-701 ± 286). Furthermore, cDC2 counts per lymph node were significantly lower in all female groups except Q, where they remained stable (Supplementary Table 10). Similar to macrophages, cDC2 counts changed most in the Q + G + IL-33 group (-14,663 ± 2,814) compared to smaller decreases in Q + G (-1,276 ± 371), G (-842 ± 450), and P (-796 ± 303). In the lymphocyte compartment, CD3⁺ T-cell counts remained constant in Q + G and Q + G + IL-33 on day 4 compared to day 2, while Tregs increased in both Q + G and Q + G + IL-33 groups (Supplementary Table 10). In young male mice, macrophage, CX3CR1⁺Ly6Cˡᵒ macrophage, and cDC2 counts per lymph node were significantly reduced only in P, G, and Q + G + IL-33 groups, while they remained stable in Q and Q + G (Supplementary Table 10). Treg and CD3⁺ T-cell counts increased in Q + G on day 4 compared to day 2 (Supplementary Table 10). When comparing male and female draining lymph nodes on day 4, we found no significant differences in any quantified immune populations in P and Q groups between sexes (Supplementary Table 11). In G groups, male draining lymph nodes were enriched for neutrophils, CX3CR1⁺Ly6C hi macrophages, cDC2s, CD8⁺ T cells, and B cells compared to female lymph nodes (Supplementary Table 11). In Q + G, eosinophils, macrophages—particularly CX3CR1⁺Ly6Cˡᵒ macrophages—and Tregs were enriched in females on day 4 compared to males (Supplementary Table 11). Male Q + G draining lymph nodes had macrophage compositions on day 4 comparable to female Q + G draining lymph nodes on day 2 (Supplementary Table 11). Tregs were more enriched in male Q + G draining lymph nodes on day 4 than in female Q + G on day 2 (+ 180% ± 95%) (Supplementary Table 11). In Q + G + IL-33 groups, neutrophils, macrophages, and CX3CR1⁺Ly6C hi macrophages were significantly more enriched in male draining lymph nodes than in female Q + G + IL-33 nodes (Supplementary Table 11). When comparing aged and young male mice, we found a significant decrease in total immune cell counts per lymph node on day 4 in P and Q + G groups (Supplementary Table 12). In P, total immune cell numbers were 74% (± 34%) lower and included reductions in neutrophils (-93% ± 36%), CD3⁺ T cells (-66% ± 21%), CD8⁺ T cells (-54% ± 25%), CD4⁺ T-helper cells (-80% ± 16%), B cells (-84% ± 32%), and Tregs (-60% ± 22%) in aged males compared to young males on day 4 (Supplementary Table 12). In Q groups, we observed significant reductions in neutrophils (-89% ± 42%) and eosinophils (-93% ± 49%) in aged males compared to young males (Supplementary Table 12). In Q + G groups, there were significant decreases in neutrophils (-91% ± 44%), eosinophils (-93% ± 49%), CX3CR1⁺Ly6Cˡᵒ macrophages (-84% ± 41%), CD3⁺ T cells (-68% ± 30%), CD4⁺ T-helper cells (-82% ± 28%), and B cells (-71% ± 34%) in aged male mice compared to young male mice on day 4 (Supplementary Table 12). In Q + G + IL-33, only CD4⁺ T-helper cells were significantly reduced by 69% (± 17%, P = 0.0007) in aged males compared to young males on day 4 (Supplementary Table 12). In contrast, macrophages, including CX3CR1⁺Ly6C hi and CX3CR1⁺Ly6Cˡᵒ subsets, were significantly enriched in aged male lymph nodes by + 382% (± 126%) and + 112% (± 65%), respectively, compared to young male Q + G + IL-33 lymph nodes (Supplementary Table 12). In fact, macrophage composition in aged Q + G + IL-33 lymph nodes was not significantly different from that of young male Q + G lymph nodes (Supplementary Table 12). In aged male mice, we observed an increase in total immune cell counts, macrophages (including CX3CR1⁺Ly6C hi and CX3CR1⁺Ly6Cˡᵒ macrophages), and B cells in Q + G + IL-33 draining lymph nodes compared to P (Fig. 4 D-G, Supplementary Fig. 14A). No significant changes were detected in Q + G or Q compared to P in aged male draining lymph nodes on day 4 (Fig. 4 D-G, Supplementary Fig. 14A-H). In young female IFNAR⁻/⁻ mice, we observed no changes in immune cell populations between P and Q + G, consistent with wild-type female draining lymph nodes on day 4 (Supplementary Fig. 12J, Supplementary Fig. 13I). Furthermore, no differences were detected between IFNAR⁻/⁻ and wild-type females when comparing P and Q + G groups (Supplementary Table 12). Muscle Injury skews antibody subtypes to IgG1 in young male and aged mice of both sexes, and IL-33 increases total IgG response. Next, we quantified the vaccine-specific anti-QIV total IgG titers in female and male, young and aged mice, 14 days post-vaccination. We computed the endpoint anti-QIV titers as percentages relative to the Q group to normalize three independent experiments from log₁₀-transformed anti-QIV antibody endpoint titers (Supplementary Table 14, Supplementary Fig. 15). We found aged female mice mounted a significantly reduced antibody response following QIV compared to young females, showing a 30% decrease (± 6.3%) (Fig. 5 A, Supplementary Table 14). In aged male mice, total anti-QIV IgG titers downstream of QIV showed a decreasing trend of 14% (± 8.2%) compared to young males (Fig. 5 B, Supplementary Table 14). Overall, most vaccination regimens in males yielded comparable total IgG titers across age groups, with the exception of Q + IL-33 in young males and Q + G + IL-33 in aged males, both of which significantly increased antibody titers compared to the aged Q group (Fig. 5 B, Supplementary Table 14). In young female mice, the addition of damage-inducing glycerol to QIV (Q + G) did not significantly alter total anti-QIV IgG titers compared to QIV alone (Fig. 5 A). The Q + IL-33 group exhibited a significant 17.5% (± 8.2%) increase compared to Q + G and a trend toward a 16% increase compared to Q alone (Fig. 5 A, Supplementary Table 14). Q + G + IL-33 showed an increasing trend of 8% (± 2.3%) compared to Q (Fig. 5 A, Supplementary Table 14). When comparing young and aged female mice, Q + G + IL-33 and Q + IL-33 in young females induced significantly higher antibody titers than aged female Q and Q + G groups (Fig. 5 A). Aged females receiving Q + G + IL-33 or Q + IL-33 achieved total anti-QIV IgG levels comparable to young females vaccinated with QIV alone (Fig. 5 A). Next, we characterized the antibody response in aged IFNAR⁻/⁻ female mice receiving Q + G or Q + G + IL-33. Total IgG titers were comparable between aged wild-type and IFNAR⁻/⁻ females in both vaccination groups (Fig. 5 A), suggesting IFN signaling does not drive the observations made in WT mice. We then analyzed the anti-QIV-specific IgG subclasses IgG1 and IgG2c across all experimental groups. In young female mice, Q and Q + G groups showed comparable anti-QIV IgG1 titers 14 days post-vaccination (Fig. 5 C). Q + G + IL-33 and Q + IL-33 groups displayed increasing trends of + 13.2% (± 2.1%) and + 23.4% (± 2.3%), respectively, compared to young female Q (Fig. 5 C, Supplementary Table 14). In aged females, QIV alone induced a significant 43.9% (± 6.7%) reduction in anti-QIV IgG1 titers relative to young Q (Fig. 5 C, Supplementary Table 14). Aged Q + G and Q + G + IL-33 groups showed non-significant decreases of -31.2% (± 3.2%) and − 15.2% (± 6.8%) compared to young Q, while aged Q + IL-33 mice maintained IgG1 titers equivalent to young female Q (100.9% ± 4.8%) (Fig. 5 C, Supplementary Table 14). Aged IFNAR⁻/⁻ females displayed no differences in IgG1 titers in Q + G or Q + G + IL-33 compared to their aged wild-type counterparts(Fig. 5 C). In male mice Q, anti-QIV IgG1 titers were 35% (± 8.6%) lower in aged males compared to young male Q (Fig. 5 D, Supplementary Table 14). In young males, Q + G + IL-33 and Q + IL-33 both showed increasing trends in IgG1 titers—27.5% (± 4.2%) and 24.9% (± 5.9%) higher than young Q, respectively—and were significantly elevated compared to aged male Q (Fig. 5 , Supplementary Table 14). Young male Q + G did not significantly alter IgG1 titers compared to young Q, but resulted in higher titers than aged male Q (Fig. 5 D). We next characterized anti-QIV IgG2c titers across all groups. In females, anti-QIV IgG2c titers dropped significantly only in aged Q + G groups in both IFNAR⁻/⁻ and wild-type mice, showing decreases of 69.4% (± 14.8%) and 64.4% (± 7.4%), respectively, compared to young female Q (Fig. 5 E, Supplementary Table 14). Similarly, in young male mice, Q, Q + G, Q + G + IL-33, and Q + IL-33 produced comparable IgG2c titers 14 days post-vaccination (Fig. 5 F). In aged males, the Q + G group showed a significant reduction in IgG2c titers compared to young male Q, whereas aged Q, Q + G + IL-33, and Q + IL-33 groups did not differ significantly from young Q (Fig. 5 F). Finally, we computed the IgG1:IgG2c ratio to evaluate whether the vaccine formulations differentially influenced antibody subtype responses. Q + G significantly increased the IgG1:IgG2c ratio in young males, aged females, and aged males compared to Q, but not in young females (Fig. 5 G-J). Q + G + IL-33 provides enhanced protection against subsequent lethal H1N1 infection compared to P, in young mice only. To evaluate the protective efficacy of the different vaccine formulations, we infected all groups 21 days post-vaccination with a 3LD₅₀ dose of mouse-adapted H1N1 influenza virus (A/Michigan/45/2015) via intranasal instillation. Following infection, body weights were recorded daily to monitor weight loss until day 6 post-infection, at which point lungs were collected to quantify replicating viral titers using plaque assays. By day 6, unvaccinated P groups exhibited average weight losses of 14.7% (± 2.4%), 17% (± 0.8%), 16.3% (± 3.6%), and 10.8% (± 2.1%) in young female, young male, aged female, and aged male mice, respectively (Fig. 6 A-D). Compared to P, weight loss was significantly reduced in vaccinated young male mice, with losses of 7.8% (± 1.8%) in Q, 6.1% (± 1.48%) in Q + G, and 3.9% (± 1.9%) in Q + G + IL-33 (Fig. 6 C). In young females, the Q + G + IL-33 group also showed significantly reduced weight loss (6% ± 2.5%) compared to the female P group (Fig. 6 A). In aged mice, no significant differences in weight loss were observed among vaccination groups in either sex (Fig. 6 B and 6 D). Next, we quantified titers of replicating virus (plaque-forming units, PFUs) from lung homogenates collected on day 6 post-infection (Fig. 6 E-H). There is a relatively large spread in lung virus titers, which most likely is due to the late time point of sample collection (6DPI), thereby allowing some mice to start controlling the replicating virus titers. Young male Q + G + IL-33 mice exhibited a significant reduction in PFUs compared to P (Fig. 6 G). Notably, 2 of 5 young male mice in the Q + G + IL-33 group had undetectable viral titers, whereas all other groups showed detectable PFUs (Fig. 6 G). In young female mice, the Q + G + IL-33 group displayed the highest number of mice with undetectable lung PFUs (3 of 5 mice) compared to none in the P group (Fig. 6 E). In aged females, Q, Q + G, and Q + G + IL-33 groups exhibited a trend toward reduced PFUs compared to P, with 2 of 5, 3 of 5, and 2 of 5 lungs, respectively, showing no detectable viral titers, whereas all P-group lungs contained detectable PFUs (Fig. 6 F and 6 H). Q, Q + G and Q + G + IL-33 show type II immune response skewing in the lungs after subsequent H1N1 viral infection in young mice, which is reduced in aged mice. We then characterized the lung inflammatory milieu using a Th1/Th2 multiplex cytokine assay to determine whether P, Q, G, Q + G, and Q + G + IL-33 treatments skewed the immune response to QIV toward a type 1 or type 2 profile. To do so, we segregated lungs with detectable viral titers (PFUs) from those without detectable PFUs and compiled all lungs with non-detectable PFUs into a single group (ND PFUs) (Fig. 7 ). This approach allowed us to examine the cytokine environment associated with ongoing viral infection separately from that of mice that effectively cleared virus from their lungs. Because no significant differences in cytokine concentrations were found between sexes within each age group, data from male and female mice were color-coded by sex and combined and presented as young and aged cohorts (Fig. 7 ). In young mice, we observed a significant increase in IL-4 concentrations in Q (61.1 ± 9.8 pg/mL), Q + G (53.2 ± 14.7 pg/mL), and Q + G + IL-33 (67.2 ± 21.2 pg/mL) compared to ND PFUs (2.1 ± 0.4 pg/mL) (Fig. 7 A). Similarly, IL-5 concentrations increased significantly in Q (864 ± 75 pg/mL), Q + G (807.2 ± 157.3 pg/mL), and Q + G + IL-33 (889.6 ± 225.7 pg/mL) compared to ND PFUs (20.2 ± 8.6 pg/mL) (Fig. 7 B). Additionally, IL-4 levels were significantly higher in Q compared to P (8.4 ± 0.8 pg/mL) and G (7.7 ± 1.0 pg/mL), and IL-5 was significantly elevated in Q compared to G (195.7 ± 40.1 pg/mL) (Fig. 7 A and 7 B). We also found an increasing trend in IL-13 concentrations (all following concentrations are in pg/mL) in Q (149.4 ± 16.5), Q + G (118.8 ± 29.3), and Q + G + IL-33 (157.7 ± 52.7) compared to P (19.3 ± 1.6 pg/mL), G (16.9 ± 2.5), and ND PFUs (6.4 ± 1.2) (Fig. 7 C). IFNγ, IL-18, and IL-1β concentrations increased significantly in all infected groups compared to ND PFUs (Fig. 7 D-F). TNFα levels were significantly elevated in P (85.8 ± 5.1) and G (69.4 ± 7.8) relative to ND PFUs (12.3 ± 1.7) (Fig. 7 G). GM-CSF concentrations also increased significantly in P (28.6 ± 1.5), Q (25.3 ± 1.5 pg/mL), and G (23.6 ± 1.9) compared to ND PFUs (6.5 ± 0.3) (Fig. 7 H). IL-12p70 concentrations were elevated in Q (12.9 ± 0.7), Q + G (12.7 ± 1.0), and Q + G + IL-33 (12.0 ± 2.0 pg/mL) compared to ND PFUs (3.8 ± 0.2) (Fig. 7 I). IL-6 concentrations were significantly higher in P (3,865 ± 417) and Q (3,516 ± 415) than in ND PFUs (40.7 ± 8.6) (Fig. 7 J). In aged mice, unlike in young mice, no significant increase in IL-4 was observed downstream of Q + G compared to ND PFUs (Fig. 7 K). Q and Q + G + IL-33 groups exhibited significant increases in both IL-4 and IL-13 concentrations compared to ND PFUs, with Q + G + IL-33 displaying the highest levels of both cytokines, which were significantly higher than P (Fig. 7 K and 7 L). When comparing aged and young groups, IL-4 levels were reduced in aged mice by 37.6% (± 12.5%, P = 0.017) in P, by 46.3% (± 21.7%, P = 0.09) in Q, and by 49.8% (± 30.5%, P = 0.13) in Q + G, while aged Q + G + IL-33 mice maintained IL-4 concentrations comparable to young Q + G + IL-33 (Supplementary Table 15). IL-13 levels were comparable between aged and young mice in P, Q, and Q + G, but trended higher (+ 86.9% ± 46.7%, P = 0.09)in aged Q + G + IL-33 compared to young Q + G + IL-33 (Supplementary Table 15). In aged mice, IL-5 concentrations increased significantly in Q, Q + G, and Q + G + IL-33 compared to ND PFUs, similar to trends in young mice (Fig. 7 M). When considering age as a variable, IL-5 levels were significantly altered in aged mice, showing reductions in P (-49% ± 34%, P = 0.04) and increases in Q (+ 52.4% ± 28%) and Q + G + IL-33 (+ 115% ± 45%) compared to their respective young groups (Supplementary Table 15). IFNγ concentrations increased significantly in aged Q, Q + G, and Q + G + IL-33 compared to ND PFUs, but not in aged P (Fig. 7 N). When comparing across ages, IFNγ was markedly reduced in aged P (-52.2% ± 20.3%) and increased in aged Q + G (+ 166.6% ± 102.2%) relative to their young counterparts (Supplementary Table 15). Unlike young mice, TNFα concentrations increased in all aged groups (P, Q, Q + G, and Q + G + IL-33) compared to ND PFUs (Fig. 7 O). Moreover, TNFα was significantly higher in aged Q (+ 36.5% ± 11.6%) and aged Q + G (+ 57.2% ± 29.9%) compared to young Q and Q + G, respectively (Supplementary Table 15). IL-6 concentrations increased significantly in aged P, Q, and Q + G + IL-33 compared to ND PFUs (Fig. 7 P). Between ages, IL-6 was significantly elevated in aged P (+ 75.1% ± 36.1%) and aged Q + G + IL-33 (+ 231% ± 70%) relative to young groups, while Q and Q + G showed increasing trends (+ 77.5% ± 33.6%, P = 0.056; +128.5% ± 55.8%, P = 0.055) (Supplementary Table 15). IL-2, IL-12p70, GM-CSF, and IL-1β concentrations did not differ between young and aged mice (Supplementary Table 15). IL-18 concentrations were reduced in aged P by 24.3% (± 10.2%, P = 0.019) compared to young P (Supplementary Table 15, Supplementary Fig. 16A-F). 6 days post H1N1 infection, Tregs and M2 prevalence increases in young and aged Q + G lungs, and IL-33 rescues eosinophil recruitment to the lungs in aged mice. Using two spectral flow cytometry panels, we characterized the myeloid and lymphocyte composition of H1N1–infected lungs harvested 6 days post-infection from young and aged, male and female mice across the different treatment groups (Supplementary Figs. 17 and 18). On day 6 post-infection, in young female mice, we observed a significant increase in total immune cell counts in the Q + G group (8,081,404 ± 257,145 cells per lung) compared to P (3,557,550 ± 985,043) and ND PFUs (3,557,550 ± 985,043) (Fig. 8 A). Q and Q + G groups induced significant increases in eosinophil counts, including both Siglec-F hi and Siglec-Fˡᵒ subpopulations, compared to ND PFUs, with comparable levels between the two vaccinated groups (Fig. 8 C-D). Additionally, in young female Q + G lungs, there was a significant increase in non-alveolar macrophages,that are Ly6Cˡᵒ, Arginase I⁺, and iNOS − compared to P and ND PFUs (Fig. 8 G-H). In addition, Q and Q + G young female mice exhibited a reduced M1/M2 macrophage ratio relative to P and G groups (Fig. 8 I). Furthermore, Treg counts per lung also increased significantly in young female Q + G compared to both ND PFUs and P (Fig. 8 J) In young male mice, Q + G and Q produced comparable increases in total immune cell counts relative to P and G (Fig. 8 K). Treg counts were significantly elevated in male Q + G compared to ND PFUs, but not in Q (Fig. 8 L). Eosinophil counts showed increasing trends in both Q and Q + G compared to P, G, and ND PFUs (Fig. 8 M). Furthermore, Arginase I + M2 non-alveolar macrophages increased significantly in male Q + G compared to ND PFUs (Fig. 8 N). M1/M2 ratio was therefore significantly reduced in male Q + G compared to P (Fig. 8 O). However, male Q + G lungs exhibited significantly lower immune cell numbers than young female Q + G lungs, including total immune cells (-36.1% ± 12.2%), Tregs (-59.9% ± 17.7%), and B cells (-54.5% ± 27.4%) (Supplementary Table 16). Additionally, male Q + G lungs displayed decreasing trends in Ly6Cˡᵒ macrophages (-42.1% ± 22.2%, P = 0.06), Ly6C hi macrophages (-35.9% ± 15.1%, P = 0.06), and Arginase I⁺ macrophages (-46.5% ± 22.5%, P = 0.06) relative to young female Q + G (Supplementary Table 16). No statistically significant differences in lung immune composition were observed among P, Q, G, or ND PFUs groups 6 days post-infection (Supplementary Table 16). Next, we characterized the immune composition of aged P, Q, G, and Q + G + IL-33 lungs at day 6 post-infection. No significant differences were observed between aged male and female mice across any group or immune population, so data were combined and presented as aged mice. In aged mice, non-alveolar macrophages increased in P, Q, and Q + G compared to ND PFUs (Fig. 8 P). The macrophage inflammatory phenotype varied by group: iNOS⁺ macrophages were enriched exclusively in the P group compared to Q + G + IL-33 and ND PFUs (Fig. 8 Q). Additionally, Q, Q + G, and Q + G + IL-33 groups displayed significantly increased eosinophil counts, particularly Siglec-Fʰⁱ eosinophils, compared to ND PFUs (Fig. 8 R and 8 S). Q + G + IL-33 exhibited the highest eosinophil numbers per lung, including Siglec-Fʰⁱ subsets, compared to Q and Q + G (Fig. 8 R and 8 S). Treg counts also increased significantly in aged Q, Q + G, and Q + G + IL-33 compared to ND PFUs (Fig. 8 T). However, Treg levels were comparable across Q, Q + G, and Q + G + IL-33 groups in aged mice, unlike young female and male mice, where Q + G induced the highest Treg numbers (Fig. 8 T). We then compared the lung immune composition of aged and young male mice within each group, 6 days post-infection. In the P groups, aged mice exhibited significant increases in CD3⁺ T cells (+ 266.2% ± 90%), including CD8⁺ T cells (+ 341.3% ± 108.4%) and CD4⁺ T-helper cells (+ 348.6% ± 151.9%) compared to young male P mice (Supplementary Table 17). Tregs showed a non-significant decreasing trend (-41.3% ± 30.6%, P = 0.14) compared to young P (Supplementary Table 17). Eosinophils were significantly lower in aged P (-59.2% ± 27.3%), including Siglec-F hi eosinophils (-68.1% ± 27.4%) relative to young male P (Supplementary Table 17). In the aged Q group, we observed significant increases in CD3⁺ T cells (+ 279.7% ± 94.9%), including CD4⁺ T-helper cells (+ 175.8% ± 71.8%) and CD8⁺ T cells (+ 546.2% ± 162.7%) compared to young male Q (Supplementary Table 17). Eosinophil counts did not differ significantly between aged and young Q groups (Supplementary Table 17). Aged Q + G lungs showed no significant differences in immune composition compared to young Q + G (Supplementary Table 17). In contrast, aged ND PFUs lungs displayed significant decreases in CD4⁺ T-helper cells (-54.7% ± 22.2%, P = 0.04), B cells (-79.9% ± 25.5%), and Tregs (-79.1% ± 38.3%) compared to young ND PFUs males. Conversely, iNOS⁺ macrophages were markedly enriched in aged ND PFUs (+ 496.4% ± 212.2%, P = 0.011)(Supplementary Table 17). We next compared aged and young female mice 6 days post-infection. Tregs were significantly reduced in aged P mice (-74.8% ± 20.3%) compared to young female P (Supplementary Table 18). Similarly, aged Q and Q + G groups showed significant decreases in Tregs of -80.4% (± 24%) and − 67.4% (± 19.2%) compared to young female Q and Q + G, respectively (Supplementary Table 18). No significant differences were observed between aged and young female ND PFUs groups (Supplementary Table 18). We then investigated whether IL-33 supplementation without glycerol could induce eosinophil increase in the lungs post-infection. To test this, we vaccinated young and aged mice with Q or Q + IL-33, followed by intranasal infection with 3LD₅₀ H1N1 IVR-180. Lungs were collected 6 days post-infection to evaluate inflammatory cytokine profiles and immune cell composition via multiplex assay and flow cytometry. IL-33 supplementation significantly increased eosinophil counts in young mice from 52,557 ± 13,190 (Q) to 94,158 ± 10,277 (Q + IL-33) (Fig. 8 U). In aged mice, eosinophils also trended higher—from 36,827 ± 13,190 (Q) to 112,667 ± 51,031 (Q + IL-33; P = 0.065)(Fig. 8 V). IL-33 supplementation did not alter Treg counts in either young or aged lungs 6 days post-infection compared to Q alone (Fig. 8 W-Z). Because Tregs depend on the IL-33–ST2 signaling axis for recruitment to injured muscle 32 , we examined whether IL-33 was locally upregulated in the lung. IL-33 mean fluorescence intensity (MFI) was significantly increased in young female Q + G lungs compared to P, consistent with the higher Treg numbers observed in Q + G lungs (Supplementary Fig. 19L, Fig. 8 AA). In aged mice, however, IL-33 expression did not increase in Q + G compared to P, but increased in Q + G + IL-33 compared to young female P, and G (Fig. 8 AA). Discussion In this study, we investigated how muscle injury influences immune responses to seasonal influenza vaccination using a glycerol-induced injury model and the seasonal quadrivalent inactivated influenza vaccine (QIV), examining the effects in a sex- and age-dependent manner. We further assessed whether IL-33 supplementation, known to mitigate age-related impairments in muscle regeneration 20 , 21 , modulates these responses in young and aged mice. We found that muscle injury alters immune kinetics at the vaccination site and draining lymph nodes, thereby remodeling both humoral and cellular vaccine responses. Furthermore, IL-33 supplementation modified several aspects of vaccine-induced immunity, reduced sex-associated variability at the injection site, enhanced humoral responses, and partially restored age-associated impairments in type II immunity. We first confirmed that glycerol induces cell death in C2C12 myotubes in vitro . When glycerol was combined with QIV, we observed an increase in pro-inflammatory and T-cell activating cytokines —including IFNγ, TNFα, IL-18, and IL-2—that were not elevated with glycerol or QIV treatment alone. The reason why the combination of QIV and glycerol gives higher cytokine responses compared to QIV and glycerol conditions is unclear. It is possible that glycerol allows cytoplasmic delivery of QIV proteins 44 , thereby triggering innate immune pathways. To our knowledge, myotubes have not been described before as a source of IL-2. From a mechanistic point of view, IL-2 is important in muscle repair pathways as it maintains Treg presence and activity 45 . In vivo , glycerol-induced muscle injury triggered an early and coordinated infiltration of myeloid cells dominated by inflammatory CX3CR1⁺Ly6C hi macrophages and neutrophils, accompanied by eosinophils and cDC2s. This early phase was followed by a day 4 transition toward a reparative profile, characterized by reduced neutrophil counts, expansion of CX3CR1⁺Ly6Cˡᵒ macrophages, and Treg enrichment, consistent with the regenerative trajectory described during muscle healing. Correlations between macrophage, cDC2, and eosinophil densities suggest coordinated crosstalk between regenerative and antigen-presenting cell populations that may link tissue repair to adaptive priming. These results align with prior evidence that macrophage–dendritic cell interactions bridge local repair and adaptive activation, but extend this concept to a vaccination context 46 , 47 . We found that muscle repair-associated immune events differed between sexes, with females mounting a more robust early inflammatory response by day 2 and repolarizing toward a reparative profile by day 4. This supports earlier reports indicating that female mice regenerate muscle more robustly than male mice 48 . Male mice exhibited a reduced early inflammatory response but a comparable delayed reparative phase, marked by similar CX3CR1⁺Ly6Cˡᵒ macrophage and Treg densities, yet with persistent neutrophil presence. Sex-based differences in intramuscular immune kinetics were mirrored in the draining lymph nodes, where glycerol-supplemented QIV induced macrophage and lymphocyte expansion on day 2 in females but not in males. Muscle injury during vaccination did not alter anti-QIV total IgG antibody titers but increased the IgG1:IgG2c ratio in young male, aged male, and female mice, suggesting a shift toward a type II–skewed humoral response 49 . Following intranasal infection with a 3LD₅₀ dose of H1N1, muscle injury during vaccination promoted regulatory T-cell accumulation in the lungs, accompanied by Ly6Cˡᵒ and Arginase I⁺ macrophages previously described as non-canonical or anti-inflammatory in infected lungs 50 – 52 . This response was more pronounced in females, highlighting a novel sex-dependent relationship between muscle injury during vaccination and a subsequent anti-inflammatory lung response. Treg density increases were accompanied by an increase in IL-33 levels in the infected lungs of young female mice, IL-33 to be a potential recruiter of Tregs into the lungs after infection similarly to the muscle post-injury 53 . IL-33 has been shown to play a key role in repolarizing pro-inflammatory toward reparative responses through Treg recruitment following muscle injury, and its decline with age contributes to chronic inflammation and sarcopenia. We found that IL-33 supplementation broadly amplified immune populations on day 2 in draining lymph nodes of both sexes when co-administered with QIV and glycerol, thereby reducing sex-dependent variability. By day 4, IL-33 increased Treg and CX3CR1⁺Ly6Cˡᵒ macrophage accumulation in injured muscles of young male and female mice. At the humoral level, IL-33 supplementation elevated anti-QIV antibody titers, maintaining an IgG1:IgG2c balance both with and without muscle injury. The potential adjuvant effect derived from co-delivered IL-33 has already been described for mucosal vaccination strategies 54 , 55 . IL-33–treated mice also exhibited enhanced protection following infection. At the mucosal level, IL-33 supplementation with QIV alone increased eosinophil numbers, supporting a link between local IL-33 expression and type II skewing of the vaccine response. In aged mice, muscles were more sarcopenic and exhibited diminished reparative capacity compared to young mice. The reduced regenerative response was evident in both magnitude and quality, with CD8⁺ T cells—rather than Tregs—dominating day 4 immune profiles in injured muscle. IL-33 supplementation did not fully restore reparative responses in aged mice but rescued the Treg:CD8⁺ T-cell ratio to levels comparable to young animals. In draining lymph nodes, IL-33 enhanced macrophage and B-cell responses compared to vaccine-alone or vaccine-plus-glycerol groups. At the humoral level, aged mice exhibited reduced anti-QIV total IgG titers compared to young mice, and muscle injury during vaccination further decreased IgG2c titers. Importantly, IL-33 supplementation rescued total IgG titers in aged mice to levels comparable to those of young controls, indicating that IL-33 may improve antibody responses in geriatric vaccination. We also found that intramuscular vaccination alone elicits a type II immune response in the lungs after infection, and that increases in Tregs and M2-like macrophages did not diminish this type II signature, which included elevated IL-4, IL-5, IL-13, and eosinophil levels in vaccinated young mice. This is in line with previous reports from our laboratory 56 , 57 and reflects potentially vaccine-induced Th2 responses that are recalled in the lung upon virus infection. At the mucosal level, aged mice displayed a diminished type II immune response to QIV, marked by lower IL-4 levels post-infection, a defect reversed by IL-33 supplementation, which restored IL-4 concentrations and increased lung eosinophil densities. However, IL-33 was insufficient to restore reduced Treg counts in aged infected lungs. In conclusion, muscle injury during vaccination promotes an anti-inflammatory response to subsequent vaccine-matched infections, correlating with the magnitude of the muscle’s regenerative response. Female mice, which mount the strongest regenerative and inflammatory responses to muscle injury, developed the highest Treg accumulation in the lungs after respiratory infection with influenza virus, followed by males, whereas aged mice exhibited diminished regeneration and reduced Treg responses. IL-33, currently explored as a therapy for sarcopenia, showed beneficial effects on intramuscular vaccine responses by increasing antibody titers in aged mice and restoring type II immunity observed in young animals, thereby highlighting a link between vaccine response and muscle health. The findings from this work have implications for both vaccination strategies in different age groups, and provide an avenue for further investigation into the underlying mechanisms of action of vaccines and their adjuvants. This work provides a foundation for exploring the link between tissue repair mechanisms and vaccine efficacy. However, several questions remain. Our findings were generated primarily in C57BL/6J mice, and future studies should assess strain-dependent variability, such as comparisons with BALB/c mice, which display distinct immune polarization biases 58 . Additionally, while our data identify immune kinetics and key cell populations associated with IL-33–enhanced vaccination, mechanistic studies are required to define the causal pathways connecting muscle injury to downstream anti-inflammatory responses, ideally using conditional knockout models. Nevertheless, these findings highlight the muscle and muscle repair pathways as a novel potential target for modulating peripheral immunity. For example, IL-33 supplementation in aged mice previously shown to have a positive reparative effect in aged muscle repair, can also rescue humoral responses and boost the type II immune response seen in younger mice. This emphasizes the importance of targeting the aging muscle environment as a strategy to enhance geriatric vaccine formulations. Additionally, and outside of the context of aging, we found that inducing muscle injury during vaccination enhances a long lived regulatory T-cell response that can be recalled at the mucosa. This novel observation can be harnessed for the induction of Tregs to counter lung fibrosis previously proven to be due to failure of Treg accumulation in the injured lungs 59 , 60 . It can also be expanded beyond infectious diseases and investigated as a venue to enrich Tregs in the context of autoimmunity where immunogenic self antigens have been previously like the case of keratin in the context of psoriasis 61 , 62 . We believe that this study is a promising introduction to ways muscle pathways can be incorporated into the field of vaccinology to develop personalized vaccines. Methods Mice C57BL6/J and BALB/cJ 4-8-week-old female and male mice were purchased from Jackson Laboratory. The aging mice were aged in house to 18-24 months old. All mice were housed in our animal facilities at Icahn School of Medicine at Mount Sinai. All experiments were performed under protocols approved by Icahn School of Medicine at Mount Sinai’s institutional Animal Care and Use Committee. Mice were vaccinated with 50 μL of the Fluzone High-Dose ® (2023-2024) quadrivalent inactivated flu vaccine diluted 1:1 sterile PBS. With this concentration, each injection delivered 3 μg of each of the four HAs included in the formulation. Mice that received glycerol were injected with 50 μL of 50% Glycerol diluted in PBS, or with QIV. 0.3μg of recombinant mouse IL-33 (purchased from Biolegend, Cat. #580502) was supplemented into vaccine formulations. During injections, mice were anesthetized using 5% isoflurane induction through a precision vaporizer. For infections, mice were anesthetized with a ketamine/xylazine mixture (90-120/2-5 mg/kg), and then infected with 25μL of 3xLD50 containing 500 plaque forming units or PFUs of IVR-180 (A/Michigan/25/2015, H1N1) intranasally. At the endpoint, mice were euthanized using pentobarbital at a concentration of >150 mg/Kg delivered into the peritoneal cavity. Serum collection For antibody titers, blood was collected via the submandibular vein and allowed to coagulate overnight at 4 degrees celsius. After coagulation, the blood was spun down at 450g for 5 minutes, and serum was transferred to a new tube. The process was repeated to insure no cell contamination. CD45 + intravenous labeling Mice were euthanized using pentobarbital at a concentration of >150 mg/Kg delivered into the peritoneal cavity. Once unresponsive, but before the heart arrests, we injected the retro orbital sinus with 100 uL of 0.02 mg/mL of anti-CD45 PE (30F-11). After a 3-minute incubation, we harvested the tissue of interest. The tissue was then stained with CD45.2 Alexa Fluor 532™ (104). A different clone of CD45 was used for tissue staining to limit epitope-specific antibody binding competition. Tissue processing Lungs and muscles were processed using the same protocol. Lungs and muscles were harvested and placed in 2.5 mL of pre-chilled digestion buffer containing 2 mg/mL Gibco™ Type II Collagenase, and 25 μg of DNase I (STEMCELL technologies, Cat: 07469) in gentleMACS™ C Tubes (cat:130-093-237). Lungs and Muscles were sheared manually and then loaded onto the gentleMACS Octo Dissociator with heaters. To digest them, 37_m_LDK_1 program was used. The digested mixture was then blocked with pre-chilled 7.5 mL of PBS supplemented with 2 mM EDTA to halt enzyme activity, and strained through a 70μm ASI™ Cell Strainer (Cat:TS70). Cells were then spun down at 450g for 5 minutes and resuspended in 200μL of staining buffer, and the flow cytometry protocol was then followed. Draining lymph nodes were digested by shearing through a 70μm filter and into a 6-well tissue culture plate containing 2.5 mL of DMEM. Cell mixture was then moved to a 15 mL falcon tube, and spun down at 450g for 5 minutes, and the staining protocol followed. Flow cytometry For processed mouse tissue including lungs, muscles, and draining inguinal lymph nodes, two panels were used to characterize the myeloid and lymphoid compartments separately. A two-laser (B/V) northern lights spectral cytometer from Cytek was used to derive the counts. For the myeloid and lymphoid panels, surface staining was performed using a staining buffer consisting of 10% Fetal bovine serum, and 0.1% Sodium Aside in PBS. Cellular fixation and permeabilization was performed using BD Cytofix/Cytoperm™ Fixation/Permeabilization Kit as per manufacturer’s instructions for the myeloid panel. For the lymphoid panel, FoxP3 intranuclear staining was performed using eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set as per manufacturer’s instructions. Surface staining and intracellular staining were performed for 20 minutes at room temperature in the dark and were followed by two washes. Surface staining washes were performed with the staining buffer. Intracellular washes were performed using kit-specific washes as per manufacturer instructions. The following antibodies were used for flow cytometric staining with anti-I-A/I-E (M5.114.15.2), anti-CD172a or anti-SIRPα (P84), anti-F4/80 (BM8), anti-CD11b (M1/70), anti-Ly6C (HK1.4), anti-CD19 (6D5), anti-CD8a (53-6.7), anti-CD4 (RM4-5) all from Biolegend; anti-Ly6G (1A8-Ly6g), anti-Arginase I (A1exF5), anti-CD3 (17A2), anti-CD25 (PC61.5), anti-CD45.2 (104), anti-CD11c (N418), anti-iNOS (CXNFT), anti-FoxP3 (FJK-16s) all from eBioscience TM ; anti-Siglec-F (E50-2440), anti-CX3CR1 (Z8-50) all from BD; anti-CD45 (30F-11) from Cytek. All antibodies were tittered and diluted to working concentration in the staining buffer. Viability was measured using fixable viability dye 520 from eBioscience diluted according to manufacturer instructions. Sample blocking was performed prior to surface and intracellular staining for 10 minutes using Purified Rat Anti-Mouse CD16/CD32 from BD Pharmingen TM . Gating strategy examples per panel and organ are provided in supplementary figures 1, 2, 5, 6, 17 and 18. Reported immune cells counts were normalized in the muscle per gram of muscle tissue to account for harvesting variability. For C2C12 cells, viability was measured using fixable viability dye 780 from eBioscience diluted according to manufacturer instructions. Mean counts are provided with standard error of mean. Culturing C2C12 myotubes C2C12 mouse myoblasts were obtained from the Yizhou Lab at Icahn School of Medicine at Mount Sinai. C2C12 cells were cultured in T75 flasks in 14 mL of Corning™ DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate supplemented with 10% HyClone™ Standard Fetal Bovine Serum (FBS) and 1% Cytiva HyClone™ Penicillin Streptomycin 100X Solution (growth media). Once 70% confluent, cells were detached using 4 mL Corning™ 0.05% Trypsin/0.53mM EDTA in HBSS w/o Calcium, Magnesium or Sodium Bicarbonate blocked with 10 mL of growth media in a Falcon™ 15 mL Conical Centrifuge Tube. Cells were then plated at 300,000 cells per T75 flask in 14 mL of growth medium. Once all plates reached 70% confluence, cells were detached and plated in 24-well cell culture plates with 300,000 cells per well, and allowed to grow to 100% confluence in the growth medium. Once confluent, the medium was swapped with 2 mL per well of serum-free Corning™ DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate media (differentiation media). Differentiation media was refreshed every day for 7 days. At day 7, media was replaced with stimulation conditions which were all diluted in differentiation medium and incubated for 24 hours. Cells were then detached with trypsin and stained for flow cytometry. Vaccine specific antibody titers The coating buffer consisted of 32 mM Na2CO3 and 64 mM NaHCO3. Fluzone High-Dose ® (2023-2024) was diluted 1:250 in the coating buffer and 100μL of coating mixture was pipetted into each well of a MaxiSorp flat-bottom 96-well plate from Thermofisher Scientific TM (Cat: 456537). Plates were incubated overnight at 4 degrees celsius. The next day, PBS-T solution was made from PBS-TWEEN ® Tablets from Millipore Sigma as per manufacturer instructions, and plates were washed twice with 200μL of PBS-T per well. Plates were then blocked with 200 μL of PBS-T supplemented with 5% MP Biomedicals™ Skim Milk Powder (Cat: MP290288705) for 1 hour at room temperature. During the blocking, sera was serially-diluted in blocking solution to create an 8-step 4-fold dilution started with a 1:100 initial dilution. After blocking, the plates were washed twice with 200μL of PBS-T per well. 100μL of sera was then added per well and incubated for 1 hour at room temperature. After incubation, the plates were washed twice with 200μL of PBS-T per well. Based on the antibody subtype, different detection buffers were made. For total IgG detection, goat F(ab) anti-mouse IgG H&L (HRP) (ab6823) was used from Abcam. For IgG1 detection, we used goat anti-mouse IgG2c-HRP (1078-05) from SouthernBiotech as per manufacturer instructions in blocking solution. 100μL of detection solution was added per well and incubated for 1 hour. Plates were then washed twice with 200μL of PBS-T per well. Plates were then developed using 1-Step™ Turbo TMB-ELISA Substrate Solution, and ELISA Stop Solution from Thermofisher as per provider instructions. Plates were then read using a Biotek plate reader using 450 nm (signal) and 650 nm (subtracted background) wavelengths absorbance. Multiplex cytokine assay Left lungs were harvested for cytokine quantification. They were moved into homogenizing tubes containing 500μL of sterile PBS solution containing 3 mm triple-pure high impact zirconium beads from Benchmark Scientific. They were flash frozen by placing them on dry ice and stored at -80°C until the assay time. On assay day, they were thawed on ice, and then loaded onto a homogenizer and run at 6 MP/S, MP 24X2 for 30 seconds. Tubes were then spun down at 450g for 10 minutes and supernatant was transferred to new 1.5 mL eppendorf tubes. The multiplex cytokines assay chosen was a ProcartaPlex™ Mouse Th1/Th2 Cytokine Panel, 11plex as per manufacturer instructions. Data were acquired on a Luminex 100/200 analyzer (Millipore) with xPONENT software (version 4.3). Data visualization and analysis were conducted using GraphPad Prism (version 9.4.1). Lung viral titers quantification Lung viral titers were specified using the same left lung lobes, and experiments were performed on the same day as that of the multiplex cytokine assay. Plaque assays were performed to quantify viral titers. To do so, a confluent monolayer of MDCK cells were incubated with 250μL of 10-fold dilutions of sample in PBS at 37°C. After a 1 hour incubation, the inoculum was aspirated and replaced by an overlay with 2% oxoid agar (Oxoid, Basingstoke, UL) mixed with an equal volume of NaHCO3-buffered 2xMEM supplemented with DEAE/Dextran and TPCK-treated trypsine at a concentration of 1μg/mL. Cells were incubated in this overlay for 48 hours in a CO2 cell incubator at 37°C. To quantify plaques, cell surfaces were fixed using a 4% formaldehyde solution for 5 minutes at room temperature. Cells were then stained with a 0.001x diluted post-challenge mouse serum matching viral infection (IVR-180, H1N1) followed by 0.001x diluted anti-mouse IgG sheep serum conjugated to horseradish peroxidase (GE Healthcare) and addition of TrueBlue substrate (KPL- Seracare, Milford, MA, USA) Statistical analysis All statistical analysis was done on GraphPad Prism software. The data is presented as mean ± SEM provided in the text unless stated otherwise. Statistical significance was computed through one-way ANOVA or non-parametric Mann-Whitney tests. To compare experimental groups, Kruskall-Wallis test was performed followed by Dunn's multiple comparison test to derive significance. P < 0.05 was considered significant. Declarations Data availability FCS files, ELISA absorbance measurements, multiplex cytokine assay tables, and mice weight tables are available for immediate release upon request from the corresponding author. Acknowledgements Research in the M.S. laboratory is funded by NIH/NIAID grant R01AI160706, and partly funded by CRIPT (Center for Research on Influenza Pathogenesis and Transmission), a NIH NIAID-funded Center of Excellence for Influenza Research and Response (CEIRR, contract number 75N93021C00014). J.V. is supported by a fellowship from the Belgian American Educational Foundation (BAEF). Author contributions M.N. conceived and designed the study, performed the experiments, analyzed the data, and wrote the manuscript. E.B., J.V., F.A., L.A.C., G.L., E.M.T., S.P., V.Y., L.G., G.J., performed experiments. P.W. secured funding, and M.S. secured funding, provided overall project direction, and edited the manuscript with input from all authors. Competing interests The M.S. laboratory has received unrelated funding support in sponsored research agreements from Phio Pharmaceuticals, 7Hills Pharma, ArgenX NV, Ziphius and Moderna. The other authors declare they have no conflicts of interest. 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Cells, 8 (8), 807. https://doi.org/10.3390/cells8080807 Pietraforte, I., & Frasca, L. (2023). Autoreactive T-cells in psoriasis: Are they spoiled Tregs and can therapies restore their functions? International Journal of Molecular Sciences, 24 (5), 4348. https://doi.org/10.3390/ijms24054348 Additional Declarations Yes there is potential Competing Interest. The M.S. laboratory has received unrelated funding support in sponsored research agreements from Phio Pharmaceuticals, 7Hills Pharma, ArgenX NV, Ziphius and Moderna. The other authors declare they have no conflicts of interest. Supplementary Files SupplementaryFigures.docx Supplementary Figures SupplementaryTables.docx Supplementary Tables Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8544011","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":586649732,"identity":"0d361228-b9dc-4e8f-9a85-a85c623c6522","order_by":0,"name":"Moataz Noureddine","email":"","orcid":"","institution":"Icahn School of Medicine at Mount Sinai","correspondingAuthor":false,"prefix":"","firstName":"Moataz","middleName":"","lastName":"Noureddine","suffix":""},{"id":586649733,"identity":"4337261b-3320-4d33-b446-85ffec0aba31","order_by":1,"name":"Eleanor Burgess","email":"","orcid":"","institution":"Icahn School of Medicine at Mount 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17:10:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8544011/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8544011/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103406884,"identity":"9bc9d2a2-a21d-419f-9287-b069cc849e77","added_by":"auto","created_at":"2026-02-25 10:13:10","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":473207,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlycerol induces C2C12 cell death in vitro, and initiates the pro-inflammatory response of muscle regeneration in vivo. (A) \u003c/strong\u003eFlow quantification of C2C12 myotube viability after a 24 hour incubation with\u0026nbsp; 25% PBS, 25% glycerol, 0.6 µg QIV, or 25% glycerol plus 0.6 µg QIV.\u003cstrong\u003e (B-F)\u003c/strong\u003e Multiplex cytokine assay results showing the concentrations of IL-4, IFNγ, IL-18, TNFα, and IL-2 respectively in the supernatant of C2C12 myotubes after a 24 hour incubation with 5% PBS, 25% glycerol, 0.6 µg QIV, or 25% glycerol plus 0.6 µg QIV. \u003cstrong\u003e(G) \u003c/strong\u003eFlow cytometry data showing the immune myeloid and granulocyte composition of quadriceps 2 days after P, Q, Q+G, and Q+G+IL-33 injections in male and female mice. \u003cstrong\u003e(H) \u003c/strong\u003eQuantification of CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e macrophage counts per gram in the quadriceps 2 days after P, Q, G, Q+G, and Q+G+IL-33 injections in male and female mice. \u003cstrong\u003e(I) \u003c/strong\u003eRatio of CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e to CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003elo\u003c/sup\u003e macrophages in the quadriceps on day 2 after P, Q, Q+G, and Q+G+IL-33 injections in male and female mice. \u003cstrong\u003e(J) \u003c/strong\u003eStatistically significant percentage changes in male mice muscle immune cell means compared to female mean per population on day 2 P, Q, Q+G, and Q+G+IL-33 groups. \u003cstrong\u003e(K) \u003c/strong\u003eQuantification of CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e macrophage counts per gram per condition comparing male and female densities. \u003cstrong\u003e(L) \u003c/strong\u003eQuantification of CD3\u003csup\u003e+\u003c/sup\u003e T-cell counts per gram in the quadriceps 2 days after P, Q, G, Q+G, and Q+G+IL-33 injections in male and female mice. \u003cstrong\u003e(M)\u003c/strong\u003e Correlation Pearson’s test shows a linear regression correlation between intramuscular cDC2 count per gram of muscle and CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e macrophage count per gram of muscle 2 days post-injection with P, Q, Q+G, and Q+G+IL-33 (color-matched with other panels). * P ≤ 0.05 ** P ≤ 0.01 *** P ≤ 0.001 **** P ≤ 0.0001 (Kruskall-Wallis followed by Dunn’s multiple comparisons test).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8544011/v1/54b51c3a3d9979f908953275.jpeg"},{"id":103406899,"identity":"f5ca63ce-bfb1-4ea8-81b5-5dc6e8bddfd7","added_by":"auto","created_at":"2026-02-25 10:13:13","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":483923,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlycerol in female mice and IL-33 in female and male mice modulate draining inguinal lymph nodes on day 2. (A) \u003c/strong\u003eFlow cytometry data showing the immune myeloid and granulocyte composition of inguinal draining lymph nodes 2 days after P, Q, Q+G, and Q+G+IL-33 injections in male and female mice.\u0026nbsp; \u003cstrong\u003e(B) \u003c/strong\u003eFlow cytometry data showing the immune lymphocyte composition of inguinal draining lymph nodes 2 days after P, Q, Q+G, and Q+G+IL-33 injections in male and female mice. \u003cstrong\u003e(C-F) \u003c/strong\u003eQuantification of Treg, B-cell, CD8\u003csup\u003e+\u003c/sup\u003e T-cell, and CD4\u003csup\u003e+\u003c/sup\u003e T-helper cell counts in the draining inguinal lymph nodes, respectively, 2 days after P, Q, G, Q+G, and Q+G+IL-33 injections in male and female mice. \u003cstrong\u003e(J)\u003c/strong\u003e Quantification of CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e macrophage counts in the draining inguinal lymph nodes 2 days after P, Q, G, Q+G, and Q+G+IL-33 injections in male and female mice. \u003cstrong\u003e(I) \u003c/strong\u003eQuantification of cDC2 counts in the draining inguinal lymph nodes 2 days after P, Q, G, Q+G, and Q+G+IL-33 injections in male and female mice. \u003cstrong\u003e(H)\u003c/strong\u003e Correlation Pearson’s test shows a linear regression correlation between CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e macrophage counts and B-cell counts in the draining inguinal lymph nodes 2 days post-injection with P, Q, Q+G, and Q+G+IL-33 in females. * P ≤ 0.05 ** P ≤ 0.01 *** P ≤ 0.001 **** P ≤ 0.0001 (Kruskall-Wallis followed by Dunn’s multiple comparisons test).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8544011/v1/95aa8ae1222e6831cb8f1473.jpeg"},{"id":103406903,"identity":"c34248e7-bfcc-4a9f-a475-078baeffd2c3","added_by":"auto","created_at":"2026-02-25 10:13:14","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":511695,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDay 4 shows a pro-repair immune response in G, Q+G, and Q+G+IL-33; impaired with age but improved with IL-33 supplementation. (A) \u003c/strong\u003eFlow cytometry data showing the immune myeloid and granulocyte composition of quadriceps 4 days after P, Q, Q+G, and Q+G+IL-33 injections in young male, young female, aged male, and IFNAR⁻/⁻ female\u0026nbsp; mice.\u0026nbsp; \u003cstrong\u003e(B)\u0026nbsp; \u003c/strong\u003eFlow cytometry data showing the lymphocyte composition of quadriceps 4 days after P, Q, Q+G, and Q+G+IL-33 injections in young male, young female, aged male, and IFNAR⁻/⁻ female\u0026nbsp; mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eRatio of CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e to CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003elo\u003c/sup\u003e macrophages in the quadriceps on day 4 after P, Q, Q+G, and Q+G+IL-33 injections in male and female mice. \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003elo\u003c/sup\u003e macrophage counts in quadriceps 4 days after P, Q, G, Q+G, and Q+G+IL-33 injections in male and female mice.\u003cstrong\u003e (E) \u003c/strong\u003eQuantification of muscle to body ratios in young male, young female, IFNAR⁻/⁻ female, and aged male mice. \u003cstrong\u003e(F-J) \u003c/strong\u003eQuantification of CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003elo\u003c/sup\u003e macrophages, CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e macrophages, eosinophil, Treg, and cDC2 cell counts in the quadriceps, respectively, 4 days after P, Q,\u0026nbsp; Q+G, and Q+G+IL-33 injections in aged male mice. \u003cstrong\u003e(K) \u003c/strong\u003eQuantification of Tregs:CD8\u003csup\u003e+\u003c/sup\u003e T-cells counts per gram ratio in Q+G, and Q+G+IL-33 conditions on day 4 in aged male mice muscles with detectable CD8\u003csup\u003e+\u003c/sup\u003e T-cells and Tregs. \u003cstrong\u003e(L) \u003c/strong\u003eQuantification of CD3\u003csup\u003e+\u003c/sup\u003e T-cell, CD8\u003csup\u003e+\u003c/sup\u003e T-cell and CD4\u003csup\u003e+\u003c/sup\u003e T-helper cell counts in the quadriceps, respectively, 4 days after Q+G injection in young and aged male mice. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;* P ≤ 0.05 ** P ≤ 0.01 *** P ≤ 0.001 **** P ≤ 0.0001 (Kruskall-Wallis followed by Dunn’s multiple comparisons test).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8544011/v1/f371f25bad50e0e047653b60.jpeg"},{"id":103406902,"identity":"01baa05f-3992-46db-92a6-9d2cb2d1fbf2","added_by":"auto","created_at":"2026-02-25 10:13:14","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":407426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDay 4 shows a pro-repair immune response in G, Q+G, and Q+G+IL-33; impaired with age but improved with IL-33 supplementation. (A) \u003c/strong\u003eFlow cytometry data showing the immune myeloid and granulocyte composition of draining inguinal lymph nodes 4 days after P, Q, Q+G, and Q+G+IL-33 injections in young male, young female, aged male, and IFNAR⁻/⁻ female\u0026nbsp; mice. \u003cstrong\u003e(B)\u0026nbsp; \u003c/strong\u003eFlow cytometry data showing the lymphocyte composition of draining inguinal lymph nodes 4 days after P, Q, G, Q+G, and Q+G+IL-33 injections in young male, young female, aged male, and IFNAR⁻/⁻ female\u0026nbsp; mice.\u003cstrong\u003e (C) \u003c/strong\u003eQuantification of B-cell counts in the inguinal draining lymph nodes on day 4 after P, Q, Q+G, and Q+G+IL-33 injections in female mice. \u003cstrong\u003e(D-G) \u003c/strong\u003eQuantification of Immune cell, B-cell, CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e macrophage, CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003elo\u003c/sup\u003e macrophage counts in inguinal draining lymph nodes 4 days after P, Q, Q+G, and Q+G+IL-33 injections in aged male mice. * P ≤ 0.05 ** P ≤ 0.01 *** P ≤ 0.001 **** P ≤ 0.0001 (Kruskall-Wallis followed by Dunn’s multiple comparisons test).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8544011/v1/a82eacbd9868720ad234e963.jpeg"},{"id":103508690,"identity":"3ec26df5-bb1c-42e2-be81-98f5e829e25e","added_by":"auto","created_at":"2026-02-26 13:52:58","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":852094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e14 days after-vaccination, glycerol skews anti-QIV antibody IgG isotype response to IgG1, and IL-33 reverses geriatric decreases in anti-QIV IgG endpoint titers. (A) \u003c/strong\u003eQuantification of anti-QIV total IgG antibody endpoint titers on day 14 after vaccination in young, aged Wildtype, and aged IFNAR⁻/⁻ female mice; \u003cstrong\u003e(B)\u003c/strong\u003e in young and aged male mice. \u003cstrong\u003e(C) \u003c/strong\u003eQuantification of anti-QIV total IgG1 antibody endpoint titers on day 14 after vaccination in young, aged Wildtype, and aged IFNAR⁻/⁻ female mice; \u003cstrong\u003e(D)\u003c/strong\u003e in young and aged male mice. (\u003cstrong\u003eE) \u003c/strong\u003eQuantification of anti-QIV total IgG2c antibody endpoint titers on day 14 after vaccination in young, aged Wildtype, and aged IFNAR⁻/⁻ female mice; \u003cstrong\u003e(F)\u003c/strong\u003e in young and aged male mice. \u003cstrong\u003e(G-J) \u003c/strong\u003eQuantification of anti-QIV IgG1:IgG2c ratio in young female, young male, aged female, and aged male mice respectively on day 14 post-vaccination. * P ≤ 0.05 ** P ≤ 0.01 *** P ≤ 0.001 **** P ≤ 0.0001 (Kruskall-Wallis followed by Dunn’s multiple comparisons test).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8544011/v1/ccff45e6ab6c49506b446d34.jpeg"},{"id":103508692,"identity":"9d10a7b7-f515-4efa-9f39-d38b52d3aa30","added_by":"auto","created_at":"2026-02-26 13:53:07","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":335705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtection against 3LD\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e50 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH1N1 infection 21 days after infection enhance in Q+G+IL-33 compared to Q, and Q+G groups in young mice. (A-D) \u003c/strong\u003eQuantification weight as a percentage of weight on day of intranasal infection with a 3LD\u003csub\u003e50 \u003c/sub\u003e(300 PFUs/mouse) dose IVR-180 (A/Michigan/25/2015, H1N1) in young female, aged female, young male, and aged male mice respectively up to 6 days post-infection. \u003cstrong\u003e(E-H) \u003c/strong\u003eQuantification of Log\u003csub\u003e10\u003c/sub\u003e plaque forming units of IVR-180 in young female, aged female, young male, and aged male mice lung homogenates respectively, 6 days post-infection. * P ≤ 0.05 ** P ≤ 0.01 *** P ≤ 0.001 **** P ≤ 0.0001 (Kruskall-Wallis followed by Dunn’s multiple comparisons test).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8544011/v1/46fdd4a1069fd1ad950fc062.jpeg"},{"id":103406897,"identity":"ff03375f-8f11-4ab6-902c-6346175062f9","added_by":"auto","created_at":"2026-02-25 10:13:12","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":951591,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultiplex cytokine assay shows type II immune response skewing downstream of Q, Q+G and Q+G+IL-33 in young mice, reduced in aged Q and Q+G, but not in Q+G+IL-33. (A-J)\u003c/strong\u003e Quantification of IL-4, IL-5, IL-13, IFNγ,\u0026nbsp; IL-1β, IL-18, TNFα, GM-CSF, IL12-p70, and IL-6 concentrations (pg/mL), respectively in young female (open circles) and male mice (filled circles) lung homogenates 6 days post-intranasal infection with 3LD\u003csub\u003e50 \u003c/sub\u003e(300 PFUs/mouse) dose IVR-180 (A/Michigan/25/2015, H1N1) 21 days after vaccination with P, G, Q+G, or Q+G+IL-33. \u003cstrong\u003e(K-P)\u003c/strong\u003e Quantification of IL-4, IL-13, IL-5, IFNγ, TNFα, and IL-6 concentrations (pg/mL), respectively in aged female (open circles) and male mice (filled circles) lung homogenates 6 days post-intranasal infection with 3LD\u003csub\u003e50 \u003c/sub\u003e(300 PFUs/mouse) dose IVR-180 (A/Michigan/25/2015, H1N1) 21 days after vaccination with P, Q+G, or Q+G+IL-33. * P ≤ 0.05 ** P ≤ 0.01 *** P ≤ 0.001 **** P ≤ 0.0001 (Kruskall-Wallis followed by Dunn’s multiple comparisons test).\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8544011/v1/6fd100e791f54ec642ff22cd.jpeg"},{"id":103406865,"identity":"5343811e-df93-4157-a7a8-e377562e3f82","added_by":"auto","created_at":"2026-02-25 10:13:09","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1237478,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune characterization of lungs post-infection shows Treg and M2 increases in Q+G groups, and a type II immune eosinophilia downstream of Q, Q+G and Q+G+IL-33, reduced with age, but can be increased with IL-33 supplementation. (A-H)\u003c/strong\u003e Quantification of Immune cell, eosinophil, Siglec-F\u003csup\u003ehi\u003c/sup\u003e eosinophil, Siglec-F\u003csup\u003elo\u003c/sup\u003e eosinophil, non-alveolar macrophage, Ly6C\u003csup\u003elo\u003c/sup\u003e non-alveolar macrophage, ArgI\u003csup\u003e+\u003c/sup\u003e non-alveolar macrophage, and iNOS\u003csup\u003e+\u003c/sup\u003e non-alveolar macrophage counts per sample, respectively 6 days post-infection with 3LD\u003csub\u003e50 \u003c/sub\u003e(300 PFUs/mouse) dose IVR-180 (A/Michigan/25/2015, H1N1) in lungs of P, Q, Q+G, Q+G+IL-33, G young female mice.\u003cstrong\u003e (I)\u003c/strong\u003e Ratio of ArgI\u003csup\u003e+\u003c/sup\u003e and iNOS\u003csup\u003e+\u003c/sup\u003e non-alveolar macrophages in young female lungs of P, Q, Q+G, Q+G+IL-33 and G groups,\u0026nbsp; 6 days post-infection with 3LD\u003csub\u003e50 \u003c/sub\u003e(300 PFUs/mouse) dose IVR-180 (A/Michigan/25/2015, H1N1). \u003cstrong\u003e(J)\u003c/strong\u003e Quantification of Treg counts per sample 6 days post-infection with 3LD\u003csub\u003e50 \u003c/sub\u003e(300 PFUs/mouse) dose IVR-180 (A/Michigan/25/2015, H1N1) in lungs of P, Q, Q+G, Q+G+IL-33, G young female mice.\u003cstrong\u003e (K-N)\u003c/strong\u003e Quantification of Immune cell,Treg,\u0026nbsp; eosinophil, and ArgI\u003csup\u003e+\u003c/sup\u003e non-alveolar macrophage counts per sample, respectively 6 days post-infection with 3LD\u003csub\u003e50 \u003c/sub\u003e(300 PFUs/mouse) dose IVR-180 (A/Michigan/25/2015, H1N1) in lungs of P, Q, Q+G, Q+G+IL-33, G young male mice.\u003cstrong\u003e (O) \u003c/strong\u003eRatio of ArgI\u003csup\u003e+\u003c/sup\u003e and iNOS\u003csup\u003e+\u003c/sup\u003e non-alveolar macrophages in young male lungs of P, Q, Q+G, Q+G+IL-33 and G groups,\u0026nbsp; 6 days post-infection with 3LD\u003csub\u003e50 \u003c/sub\u003e(300 PFUs/mouse) dose IVR-180 (A/Michigan/25/2015, H1N1). \u003cstrong\u003e(P-T) \u003c/strong\u003eQuantification of non-alveolar macrophage, iNOS\u003csup\u003e+\u003c/sup\u003e non-alveolar macrophages, eosinophil, Siglec-F\u003csup\u003ehi\u003c/sup\u003e eosinophil and Treg counts per sample, respectively 6 days post-infection with 3LD\u003csub\u003e50 \u003c/sub\u003e(300 PFUs/mouse) dose IVR-180 (A/Michigan/25/2015, H1N1) in lungs of P, Q, Q+G, and Q+G+IL-33 aged male mice.\u003cstrong\u003e (U,V) \u003c/strong\u003eQuantification of eosinophil counts per sample, respectively 6 days post-infection with 3LD\u003csub\u003e50 \u003c/sub\u003e(300 PFUs/mouse) dose IVR-180 (A/Michigan/25/2015, H1N1) in lungs of Q, Q+IL-33 in aged and young mice, respectively.\u003cstrong\u003e (W-Z)\u0026nbsp; \u003c/strong\u003eQuantification of Treg counts per sample, respectively 6 days post-infection with 3LD\u003csub\u003e50 \u003c/sub\u003e(300 PFUs/mouse) dose IVR-180 (A/Michigan/25/2015, H1N1) in lungs of Q, Q+IL-33 in young female, aged female, young male and aged male mice, respectively.\u003cstrong\u003e (AA) \u0026nbsp;\u003c/strong\u003eQuantification of IL-33 MFIs 6 days post-infection with 3LD\u003csub\u003e50 \u003c/sub\u003e(300 PFUs/mouse) dose IVR-180 (A/Michigan/25/2015, H1N1) in lungs of P, Q, Q+G, Q+G+IL-33, and G in young female mice and aged mice.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8544011/v1/21291c8e4fb9e6f94e7c17cb.jpeg"},{"id":107706216,"identity":"d3378bba-6624-4d98-9038-a20b1ca04358","added_by":"auto","created_at":"2026-04-24 09:17:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5808814,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8544011/v1/3a7c9688-97c3-4dc7-9684-868ce477b346.pdf"},{"id":103406864,"identity":"fd73e3fc-acd8-47a0-b775-356ed02b2c72","added_by":"auto","created_at":"2026-02-25 10:13:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6362011,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8544011/v1/4f86f6dc2b69ebcdb2aaea6a.docx"},{"id":103406874,"identity":"d712081e-404b-4375-b582-093091609dbe","added_by":"auto","created_at":"2026-02-25 10:13:09","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10864679,"visible":true,"origin":"","legend":"Supplementary Tables","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8544011/v1/c2ebd22106d8856162c5f30a.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nThe M.S. laboratory has received unrelated funding support in sponsored research agreements from Phio Pharmaceuticals, 7Hills Pharma, ArgenX NV, Ziphius and Moderna. The other authors declare they have no conflicts of interest.","formattedTitle":"Muscle Injury and Aging Differentially Shape Immune Responses to Influenza Vaccination","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSkeletal muscle is an immunocompetent and highly regenerative organ\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Under homeostatic conditions, the muscle contains few immune cells; however, upon injury like trauma, immune cells can rapidly infiltrate\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The muscle\u0026rsquo;s response to injury follows a well-coordinated sequence beginning with an early pro-inflammatory phase dominated by CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages, which clear debris and dying cells and activate myoblasts\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This is followed by a pro-repair phase characterized by CX3CR1⁺Ly6Cˡᵒ macrophages and regulatory T-cells (Tregs) that orchestrate myoblast differentiation and myotube formation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. These stages are accompanied by eosinophils\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, neutrophils\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and dendritic cells\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, which provide key cues such as IL-4 for macrophage repolarization and contribute to antigen surveillance.\u003c/p\u003e \u003cp\u003eThe muscle is also the primary site for administration of most parenteral vaccines, yet its intrinsic immunological properties remain understudied in the context of vaccine and adjuvant usage. The regenerative muscle environment is rich in cytokines such as IFNγ, TNFα, IL-4, and IL-33, all of which influence the type and magnitude of T- and B-cell responses to vaccination\u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Despite this, how the local immune milieu of the muscle contributes to vaccine efficacy and how it differs across sex and age remain poorly understood.\u003c/p\u003e \u003cp\u003eHere, we hypothesized that inducing muscle injury at the time of vaccination modulates vaccine responses in a sex- and age-dependent manner via regenerative immune pathways. To test this, we used an established glycerol injection-based injury model combined with vaccination with the seasonal quadrivalent inactivated influenza vaccine (QIV). We examined sex-dependent effects, given that estrogen alters regenerative responses in females\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, and age-dependent effects, since muscle regeneration declines with age which has been linked to reduced IL-33 levels\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. To address this, we investigated whether enhancing regeneration through supplementation with 0.3 \u0026micro;g of IL-33, a previously defined reparative dose, could restore vaccine responses in aged mice\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we show that female mice mount a stronger regenerative immune response to muscle injury than young and aged males. Muscle injury during vaccination modulates humoral and cellular immune responses in both a sex- and age-dependent manner, and has longlasting effects as it enhances Treg and M2 macrophage accumulation in the lungs after subsequent influenza infection, particularly in female mice. Muscle injury also increases the IgG1:IgG2c ratio in young male and aged mice. Furthermore, IL-33 supplementation rescues the reparative response in aged mice, reduces sex-dependent variability in local immune responses, enhances anti-vaccine total IgG antibody titers, and restores type II vaccine-specific immune responses in aged animals.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGlycerol induces cell death in C2C12 myotubes, and induces a sex-dependent pro-inflammatory regenerative response in quadriceps muscles.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGlycerol has been used to induce muscle injury in mouse models to study subsequent muscle regeneration\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. To assess its effects on muscle cells \u003cem\u003ein vitro\u003c/em\u003e, we cultured C2C12 myoblasts in 24-well plates and differentiated them into myotubes. Once differentiated, cells were treated for 24 hours in serum-free media with one of the following: 25% PBS, 25% glycerol, 0.6 \u0026micro;g QIV, or 25% glycerol plus 0.6 \u0026micro;g QIV. To quantify cell viability, we performed flow cytometry staining\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.The number of viable myotubes was significantly reduced and approached zero, in wells treated with glycerol or glycerol\u0026thinsp;+\u0026thinsp;QIV, compared to an average of 8,365\u0026thinsp;\u0026plusmn;\u0026thinsp;1,944 live cells with QIV and 13,811\u0026thinsp;\u0026plusmn;\u0026thinsp;7,389 live cells with PBS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Additionally, in the glycerol\u0026thinsp;+\u0026thinsp;QIV group 4 out of 5 wells had enhanced levels of IL-6, which was a significant increase compared to glycerol alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Further, we observed increased IFNγ and IL-18 compared to PBS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC,D), QIV, and glycerol; increased TNFα compared to QIV and glycerol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE); and increased IL-2 compared to QIV and PBS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the effects of muscle injury on vaccine response \u003cem\u003ein vivo\u003c/em\u003e, we induced controlled muscle damage using a previously established model that uses a concentration of 50% glycerol in mice during vaccination\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. We also investigated the combination of 50% glycerol with IL-33, an alarmin extensively reported to initiate pro-regenerative pathways in muscle injury and shown to decline with age\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To do so, we vaccinated young and aged, male and female mice in the quadriceps with 3 \u0026micro;g of the seasonal QIV vaccine (designated group Q) alone, 50% glycerol alone (designated group G), QIV\u0026thinsp;+\u0026thinsp;50% glycerol (designated group Q\u0026thinsp;+\u0026thinsp;G), or Q\u0026thinsp;+\u0026thinsp;G supplemented with 0.3 \u0026micro;g of IL-33 (designated group Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33). We collected injected quadriceps on day 2 to capture the early pro-inflammatory phase following injury\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. We utilized two spectral flow cytometry panels that allowed the characterization of the myeloid and lymphocyte compartments in the muscle (Gating schemes are provided in Supplementary Figs.\u0026nbsp;1 and 2).\u003c/p\u003e \u003cp\u003eFirst, we investigated sex-dependent muscle immune composition differences in the control P group. In the P groups on day 2, young female and male mice had a comparable myeloid cell distribution, with no significant differences in neutrophil, eosinophil, macrophage, and cDC2 counts per gram of muscle between both sexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, Supplementary Table\u0026nbsp;2). On the other hand, lymphocytes were significantly more enriched in male P-group quadriceps on day 2, including CD4⁺ T helper cells, CD8⁺ T cells, and B cells compared to female P quadriceps (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eNext, we characterized the immune composition on day 2 after a 50% glycerol injection. In the G group, glycerol induced a significant increase in immune cell counts per gram in male (19-fold) and female (13-fold) mice compared to their respective P groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, Supplementary Table\u0026nbsp;1). After glycerol injection, female quadriceps showed 764,052\u0026thinsp;\u0026plusmn;\u0026thinsp;321,691 immune cells per gram of muscle, compared to 197,708\u0026thinsp;\u0026plusmn;\u0026thinsp;25,156 in male quadriceps (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, Supplementary Fig.\u0026nbsp;3A and B, Supplementary Table\u0026nbsp;1). The significant increase in immune cell counts in the G group was driven by increases in eosinophils, neutrophils, macrophages, and cDC2s (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG; see Supplementary Fig.\u0026nbsp;3 for individually plotted populations). There was no significant difference in all quantified immune populations between male and female G groups (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eIn the G groups, eosinophils comprised 20% \u0026plusmn; 13% and 18% \u0026plusmn; 12% of total immune cells per gram in females and males, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG; Supplementary Table\u0026nbsp;1). Neutrophils were significantly increased in the male G group compared with the male P group, whereas no such increase was observed in females. Accordingly, neutrophils constituted a significantly greater fraction of total immune cells in male G mice (24% \u0026plusmn; 3%) than in female G mice (11% \u0026plusmn; 2%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG; Supplementary Fig.\u0026nbsp;4A; Supplementary Table\u0026nbsp;1). Macrophages were the most abundant immune cell population, accounting for 39% \u0026plusmn; 16% and 45% \u0026plusmn; 16% of total immune cells per gram in female and male G groups, respectively (Supplementary Table\u0026nbsp;1). Conventional dendritic cells 2 (cDC2s) represented approximately 5% \u0026plusmn; 7% of total immune cells in both sexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH; Supplementary Table\u0026nbsp;1). No significant increase in T-cell counts per gram was observed at this time point in either sex (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003eCompared to the G group, the Q group showed significantly lower immune cell counts per gram in males and a downward trend in females (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). However, immune cell counts were still approximately twofold higher in Q than in P in both sexes (Supplementary Table\u0026nbsp;1). This increasing trend of immune cell count in Q group was driven by increasing trends in cDC2s, eosinophils, CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e and CX3CR1⁺Ly6Cˡᵒ macrophages and neutrophils (Supplementary Table\u0026nbsp;1, see Supplementary Fig.\u0026nbsp;3 for individually plotted populations). Q groups differed between sexes, where male Q mice had significantly fewer neutrophils, CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e and CX3CR1⁺Ly6Cˡᵒ macrophages, and cDC2s than female Q mice (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eThe Q\u0026thinsp;+\u0026thinsp;G group showed no significant difference in immune cell counts per gram compared to G in either sex with significant increases in eosinophils, cDC2s, and both CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e and CX3CR1⁺Ly6Cˡᵒ macrophages compared to P (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, see Supplementary Fig.\u0026nbsp;3 for individually plotted populations). Direct comparison between sexes revealed that Q\u0026thinsp;+\u0026thinsp;G immune composition exhibited variability unlike G alone. In Q\u0026thinsp;+\u0026thinsp;G, males had significantly fewer eosinophils, CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages, and cDC2s, and a greater enrichment of CD8⁺ T cells and CD4⁺ T helper cells relative to female Q\u0026thinsp;+\u0026thinsp;G (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eThe Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 group showed no significant differences compared to G or Q\u0026thinsp;+\u0026thinsp;G immune composition (see Supplementary Fig.\u0026nbsp;3 for individually plotted populations). Notably, IL-33 reduced sex-associated variability observed in Q\u0026thinsp;+\u0026thinsp;G, with only cDC2s and CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages remaining significantly lower in males than in females (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eNext, we investigated the macrophage phenotype in all conditions. In both sexes, macrophages increased significantly in the G, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups compared to P (Supplementary Fig.\u0026nbsp;3). The infiltrating macrophages consisted of pro-inflammatory CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e and pro-repair CX3CR1⁺Ly6Cˡᵒ subsets (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG,I and J). The increase in CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e macrophage counts was significantly more robust in female Q, Q\u0026thinsp;+\u0026thinsp;G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups compared to male respective groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). To assess the inflammatory state of the macrophage compartment, we computed the proportions of CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e and CX3CR1⁺Ly6Cˡᵒ macrophages among total macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). We found a significant enrichment of pro-inflammatory CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages In female Q, G, Q\u0026thinsp;+\u0026thinsp;G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups compared to P group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, Supplementary Fig.\u0026nbsp;4B). Meanwhile, In males, the proportion was comparable between all groups (Supplementary Fig.\u0026nbsp;4B).\u003c/p\u003e \u003cp\u003eBecause dendritic cells are well established as key inducers of adaptive immune responses to intramuscular vaccination\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, we investigated their relationship with CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages, which are critical for muscle regeneration following injury\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Pearson\u0026rsquo;s correlation analysis revealed a strong positive linear relationship between CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages and cDC2 counts per gram of muscle in females (R\u0026sup2; = 0.878, y\u0026thinsp;=\u0026thinsp;5.687\u0026thinsp;+\u0026thinsp;8682) and males (R\u0026sup2; = 0.699, y\u0026thinsp;=\u0026thinsp;9.206\u0026ndash;9292) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL). Furthermore, there were significant positive and linear correlations between cDC2s and eosinophils (female: R\u0026sup2; = 0.94; male: R\u0026sup2; = 0.9)(Supplementary Fig.\u0026nbsp;4C) and between CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages and eosinophils (female: R\u0026sup2; = 0.79; male: R\u0026sup2; = 0.77) (Supplementary Fig.\u0026nbsp;4D), regardless of condition or sex.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCX3CR1\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eLy6C\u003c/b\u003e \u003csup\u003e \u003cb\u003ehi\u003c/b\u003e \u003c/sup\u003e \u003cb\u003emacrophages and lymphocytes are increased in draining inguinal lymph nodes in female mice after Q\u0026thinsp;+\u0026thinsp;G vaccination, and IL-33 increases general immune infiltration to the draining lymph nodes irrespective of sex.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe adaptive immune response to intramuscularly delivered vaccine antigens is mounted in the draining lymph nodes, one of which is the inguinal draining lymph node\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.To investigate whether muscle injury modulates immune response in the draining lymph node after vaccination, we harvested inguinal draining lymph nodes on day 2 after P, Q, G, Q\u0026thinsp;+\u0026thinsp;G, or Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 injections. We characterized their immune composition using two spectral flow cytometry panels enabling the quantification of multiple myeloid and lymphocyte populations (Gating schemes are provided in Supplementary Figs.\u0026nbsp;5 and 6).\u003c/p\u003e \u003cp\u003eIn female mice on day 2 after vaccination, a significant threefold increase in total immune cell counts was observed in Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to P. In Q\u0026thinsp;+\u0026thinsp;G, we observed a twofold increasing trend in immune cell counts compared to P in female lymph nodes (Supplementary Table\u0026nbsp;3). The increase in immune cell number in Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 was driven by enrichments in neutrophil, macrophage, lymphocyte, and cDC2 counts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B, Supplementary Table\u0026nbsp;1, see individually plotted populations per condition in Supplementary Fig.\u0026nbsp;7). Meanwhile, in Q\u0026thinsp;+\u0026thinsp;G, the enrichment was mainly confined to the macrophage compartment\u0026mdash;particularly the Ly6C\u003csup\u003ehi\u003c/sup\u003eCX3CR1⁺ macrophage subset\u0026mdash;and lymphocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWithin the lymphocyte compartment of female draining lymph nodes, Tregs increased significantly by twofold after Q\u0026thinsp;+\u0026thinsp;G and three-fold after Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to P, whereas Q alone did not result in significant differences over P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Supplementary Table\u0026nbsp;3). B cells increased by three-fold after Q\u0026thinsp;+\u0026thinsp;G and four-fold after Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 relative to P, and B cell numbers were also significantly higher in the Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 group compared to Q only (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, Supplementary Table\u0026nbsp;3). CD8⁺ T cells increased by two-fold after both Q\u0026thinsp;+\u0026thinsp;G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, Supplementary Table\u0026nbsp;3). CD4⁺ T helper cells also increased by two-fold after Q\u0026thinsp;+\u0026thinsp;G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, Supplementary Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eWhen the myeloid compartment is considered in female mice, CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages increased by three-fold following Q\u0026thinsp;+\u0026thinsp;G, and this effect was further amplified to a sixteenfold increase with Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, Supplementary Table\u0026nbsp;3). Excluding the Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 condition, Q\u0026thinsp;+\u0026thinsp;G alone induced a significant increase in total immune cells and in CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages compared to P and G. Additionally, CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophage counts correlated positively with B-cell counts in lymph nodes across P, G, Q, and Q\u0026thinsp;+\u0026thinsp;G conditions (linear regression: Y\u0026thinsp;=\u0026thinsp;97.19 \u0026times; X\u0026thinsp;+\u0026thinsp;47,185; R\u0026sup2; = 0.527) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Downstream of Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33, the regression slope did not significantly differ from zero, indicating the loss of correlation between these two populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). cDC2s and CX3CR1⁺Ly6Cˡᵒ macrophages increased only downstream of Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 by 11-fold and 4-fold, respectively, and did not change significantly in Q, G, or Q\u0026thinsp;+\u0026thinsp;G compared to P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eIn male mice, immune kinetics differed markedly from females on day 2. We found a twofold higher total immune cell count in male P lymph nodes compared to female P lymph nodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Supplementary Table\u0026nbsp;3). Immune cell counts were significantly lower downstream of Q (61% of P, SD\u0026thinsp;=\u0026thinsp;16%), G (62% of P, SD\u0026thinsp;=\u0026thinsp;15%), and Q\u0026thinsp;+\u0026thinsp;G (43% of P, SD\u0026thinsp;=\u0026thinsp;16%) compared to P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Supplementary Table\u0026nbsp;3, Supplementary Fig.\u0026nbsp;8A). In contrast, Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 restored immune cell counts to baseline, with values comparable to P (128% of P, SD\u0026thinsp;=\u0026thinsp;19%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Supplementary Table\u0026nbsp;3, Supplementary Fig.\u0026nbsp;8A).\u003c/p\u003e \u003cp\u003eWe did not observe significant increases in myeloid or lymphocyte populations in male draining lymph nodes on day 2 compared to P (Supplementary Fig.\u0026nbsp;8A-I). However, if only vaccinated groups were considered in comparisons, Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 led to a significant enrichment of neutrophils and eosinophils compared to Q. Moreover, Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 significantly increased CX3CR1⁺Ly6Cˡᵒ macrophages and B cells compared to Q and Q\u0026thinsp;+\u0026thinsp;G. Tregs, CD8⁺ T cells, and CD4⁺ T helper cells were also significantly enriched downstream of Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to Q, G, and Q\u0026thinsp;+\u0026thinsp;G.\u003c/p\u003e \u003cp\u003eComparing male and female draining lymph nodes, male P lymph nodes were significantly more enriched in macrophages, cDC2s, CD4⁺ T helper cells, CD8⁺ T cells, and Tregs compared to female P lymph nodes (Supplementary Table\u0026nbsp;4). However, eosinophils were significantly lower in male P lymph nodes relative to females on day 2 (Supplementary Table\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGlycerol triggers a muscle pro-regenerative response by day 4 in young female and male mice, amplified by IL-33 supplementation in young mice and independent of IFNAR signaling.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince muscle regenerative response consists of a pro-inflammatory response followed by a later pro-repair immune response, we characterized the muscle response on day 4 in young female, young male, and aged male mice. By day 4, the regenerative phase has been described to shift toward a pro-repair or anti-inflammatory milieu\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e,characterized by an influx of regulatory T cells (Tregs) and an increase in CX3CR1⁺Ly6Cˡᵒ macrophages\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBy day 4, the immune infiltrate consisted mainly of macrophages, eosinophils, T cells, and cDC2s in the G, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups for both sexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, see Supplementary Figs.\u0026nbsp;9 and 10 for individually plotted myeloid and lymphocyte populations per condition, respectively). Neutrophil counts declined by more than 83% across all conditions compared to day 2 after vaccination suggesting the transition to the anti-inflammatory stage (Supplementary Table\u0026nbsp;7).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn day 4, the muscle immune composition appeared to be more comparable in P, Q, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 between males and females than on day 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In G groups, the immune influx in the males averaged at 322,892 immune cells per gram (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;158,467) which is a 98% (\u0026plusmn;\u0026thinsp;40%, P\u0026thinsp;=\u0026thinsp;0.02) increase compared to the female G group (162,918\u0026thinsp;\u0026plusmn;\u0026thinsp;103,277 SD) (Supplementary Table\u0026nbsp;6). This pattern differed from day 2, when female G exhibited sixfold higher immune cell counts than male G (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eCompared to day 2, female mice showed a significant decrease on day 4 in total immune cell count per gram in P, Q, G and Q\u0026thinsp;+\u0026thinsp;G groups while Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 showed no change (Supplementary Table\u0026nbsp;7). Meanwhile, male mice showed a significant increase in total immune cell count per gram in G, Q\u0026thinsp;+\u0026thinsp;G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 and a significant decrease in P and Q groups compared to day 2 (Supplementary Table\u0026nbsp;7). This led to male G group having significantly higher counts in eosinophils, cDC2s and CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003elo\u003c/sup\u003e macrophages compared to female G on day 4 (Supplementary Table\u0026nbsp;6). Otherwise, no significant sex-dependent differences were observed in P, Q, Q\u0026thinsp;+\u0026thinsp;G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 immune populations other than a significantly higher neutrophil count in Q\u0026thinsp;+\u0026thinsp;G in males compared to females (Supplementary Table\u0026nbsp;6).\u003c/p\u003e \u003cp\u003eWith these changes compared to day 2, G, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups still maintained significantly elevated macrophage counts compared to P in both sexes (Supplementary Fig.\u0026nbsp;9). Macrophages represented 22\u0026ndash;48% and 37\u0026ndash;42% of total immune cells in female and male mice, respectively (Supplementary Table\u0026nbsp;5), and only decreased in density in P and Q groups of both sexes (Supplementary Table\u0026nbsp;7). Additionally, CX3CR1⁺Ly6Cˡᵒ-to-CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophage ratio was significantly altered in both males and females compared to day 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Supplementary Table\u0026nbsp;7). In both sexes, CX3CR1⁺Ly6Cˡᵒ macrophages increased significantly in G, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to P (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Unlike day 2, CX3CR1⁺Ly6Cˡᵒ macrophages became the dominant subset at 4 days after vaccination, representing 73% \u0026plusmn;11% of macrophages in female and 72% \u0026plusmn;8% in male G muscles, and 74% \u0026plusmn;10% (female) and 66% \u0026plusmn;9% (male) in Q\u0026thinsp;+\u0026thinsp;G (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33, CX3CR1⁺Ly6Cˡᵒ macrophages comprised 73% \u0026plusmn;10% of macrophages in males but were balanced with CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages (49% \u0026plusmn;12%) in females (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eOn day 4, eosinophil and cDC2 counts were significantly reduced compared to day 2 in P and Q groups for both sexes (Supplementary Table\u0026nbsp;7). For the G, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups, Female eosinophil counts remained unchanged while in males they increased compared to day 2 (Supplementary Table\u0026nbsp;7). cDC2s also remained elevated and unchanged in G, Q\u0026thinsp;+\u0026thinsp;G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 regardless of sex but in the female Q\u0026thinsp;+\u0026thinsp;G group where they decreased by 53% (\u0026plusmn;\u0026thinsp;19%) compared to day 2 (Supplementary Table\u0026nbsp;7).\u003c/p\u003e \u003cp\u003eUnlike day 2 after vaccination, CD3⁺ T-cell densities increased significantly in male G and Q\u0026thinsp;+\u0026thinsp;G muscles compared to P and Q, and in male Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to P (Supplementary Fig.\u0026nbsp;10A and 10B). In females, G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups also showed significant CD3⁺ T-cell enrichment compared to P and Q, while Q\u0026thinsp;+\u0026thinsp;G displayed a 40-fold increasing trend (Supplementary Table\u0026nbsp;5). This increase reflected elevated Tregs, CD8⁺ T cells, and CD4⁺ T helper cells in both sexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, see Supplementary Fig.\u0026nbsp;10 for individually plotted lymphocyte populations per condition).\u003c/p\u003e \u003cp\u003eTregs increased robustly in male G, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to P and Q (Supplementary Fig.\u0026nbsp;10C), and in female G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to P (Supplementary Fig.\u0026nbsp;10D). Between day 2 and 4, Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 induced the largest Treg expansion: +3072% (\u0026plusmn;\u0026thinsp;825%) in females and +\u0026thinsp;2557% (\u0026plusmn;\u0026thinsp;529%) in males (Supplementary Table\u0026nbsp;7). In Q\u0026thinsp;+\u0026thinsp;G, Tregs increased\u0026thinsp;+\u0026thinsp;56.8% (\u0026plusmn;\u0026thinsp;77%) in females and +\u0026thinsp;520% (\u0026plusmn;\u0026thinsp;151%) in males compared to day 2 (Supplementary Table\u0026nbsp;7).\u003c/p\u003e \u003cp\u003eCD8⁺ T-cell counts per gram increased significantly in male G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 versus P and Q (Supplementary Fig.\u0026nbsp;10G), and in male Q\u0026thinsp;+\u0026thinsp;G versus P. In females, CD8⁺ T cells rose significantly in G (vs. P) and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 (vs. P and Q) (Supplementary Fig.\u0026nbsp;10H). From day 2 to day 4, Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 showed the largest CD8⁺ T-cell increase\u0026mdash;+512% (\u0026plusmn;\u0026thinsp;130%) in males and +\u0026thinsp;1697% (\u0026plusmn;\u0026thinsp;626%) in females (Supplementary Table\u0026nbsp;7). Q\u0026thinsp;+\u0026thinsp;G also caused a 277% (\u0026plusmn;\u0026thinsp;121%) increase in females and 117% (\u0026plusmn;\u0026thinsp;49%) in males compared to day 2 (Supplementary Table\u0026nbsp;7).\u003c/p\u003e \u003cp\u003eWe next computed the CD8⁺ T-cell to Treg ratio in muscle to evaluate whether IL-33 altered the inflammatory-to-regulatory lymphocyte balance (Supplementary Fig.\u0026nbsp;10I). No significant differences were detected between G, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 in either sex.\u003c/p\u003e \u003cp\u003eFinally, we were interested in investigating type I interferon role in muscle regeneration. To do so, we injected IFNAR knockout (IFNAR⁻/⁻) female mice with P or Q\u0026thinsp;+\u0026thinsp;G and analyzed muscle tissue on day 4. No significant changes in immune composition were observed between IFNAR⁻/⁻ and wild-type females under either condition ( Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplementary Table\u0026nbsp;6).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMuscle pro-regenerative response is replaced with an increased CD8\u003c/b\u003e \u003csup\u003e \u003cb\u003e+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eT-cell response in aged mice which is reversed with the supplementation of IL-33.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWith age, muscle health deteriorates and muscles become sarcopenic\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, which can be assessed in mice by calculating the muscle-to-body weight ratio\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. To evaluate muscle health in young, aged, and IFNAR⁻/⁻ mice, we computed their muscle-to-body ratio. We found a significant decrease in this ratio in aged male mice, compared to young male, young female, and IFNAR⁻/⁻ female mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Young female, male, and IFNAR⁻/⁻ female mice exhibited comparable muscle-to-body ratios (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). In aged male mice on day 4, we observed a significant enrichment of CD3⁺ lymphocyte counts in the aged P group (597\u0026thinsp;\u0026plusmn;\u0026thinsp;302) compared to young male P mice (7\u0026thinsp;\u0026plusmn;\u0026thinsp;7 cells per gram of muscle; Supplementary Tables\u0026nbsp;5 and 8). The age-associated increase in lymphocyte density in P mice was driven by elevated CD8⁺ T cells, CD4⁺ T-helper cells, and Tregs (Supplementary Table\u0026nbsp;8). Additionally, the aged P group showed a significant enrichment in CX3CR1⁺Ly6Cˡᵒ macrophage counts (344\u0026thinsp;\u0026plusmn;\u0026thinsp;154) compared to the young P group (101\u0026thinsp;\u0026plusmn;\u0026thinsp;50; Supplementary Tables\u0026nbsp;5 and 8).\u003c/p\u003e \u003cp\u003eIn the aged male Q\u0026thinsp;+\u0026thinsp;G group, total immune cell counts were significantly higher than in Q and P groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplementary Fig.\u0026nbsp;11A). In aged Q\u0026thinsp;+\u0026thinsp;G mice, this increase relative to P reflected significant enrichment of macrophages (Supplementary Fig.\u0026nbsp;11B) including CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003elo\u003c/sup\u003e macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), CX3CR1\u003csup\u003e+\u003c/sup\u003eLy6C\u003csup\u003ehi\u003c/sup\u003e macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), neutrophils (Supplementary Fig.\u0026nbsp;11C), eosinophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), CD8⁺ T cells (Supplementary Fig.\u0026nbsp;11D), Tregs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), and cDC2s (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). However, when compared to young male Q\u0026thinsp;+\u0026thinsp;G mice, total immune cell density was significantly lower by 75% (\u0026plusmn;\u0026thinsp;39%)(Supplementary Table\u0026nbsp;8). Compared to young male Q\u0026thinsp;+\u0026thinsp;G mice on day 4, macrophage density in aged Q\u0026thinsp;+\u0026thinsp;G muscle was significantly lower by 75% (\u0026plusmn;\u0026thinsp;37%). This reduction was driven primarily by CX3CR1⁺Ly6Cˡᵒ macrophages, which was 81% lower (\u0026plusmn;\u0026thinsp;38%), whereas CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages showed a nonsignificant decreasing trend of 65% (\u0026plusmn;\u0026thinsp;37%)(Supplementary Table\u0026nbsp;8). Relative to young Q\u0026thinsp;+\u0026thinsp;G males, eosinophil counts were significantly lower by 94% (\u0026plusmn;\u0026thinsp;43%) and cDC2 by 90% (\u0026plusmn;\u0026thinsp;39%) in aged males at the same time point (Supplementary Table\u0026nbsp;8).\u003c/p\u003e \u003cp\u003eIn the lymphocyte compartment, no increase in B-cell counts was detected in aged Q\u0026thinsp;+\u0026thinsp;G mice compared to P (Supplementary Fig.\u0026nbsp;11E). Moreover, B-cell numbers were significantly lower in aged Q\u0026thinsp;+\u0026thinsp;G males relative to young Q\u0026thinsp;+\u0026thinsp;G males by 97% (\u0026plusmn;\u0026thinsp;30%) (Supplementary Table\u0026nbsp;8). In contrast, CD3⁺ T cells increased significantly in aged Q\u0026thinsp;+\u0026thinsp;G mice compared to P and Q (Supplementary Fig.\u0026nbsp;11F). Specifically, CD8⁺ T cells increased significantly in aged Q\u0026thinsp;+\u0026thinsp;G mice compared to P and Q, mirroring trends in young Q\u0026thinsp;+\u0026thinsp;G mice (Supplementary Fig.\u0026nbsp;11D). CD4⁺ T-helper cells showed a nonsignificant enrichment compared to P and Q (Supplementary Fig.\u0026nbsp;11G).\u003c/p\u003e \u003cp\u003eWhen compared to young Q\u0026thinsp;+\u0026thinsp;G males on day 4, aged Q\u0026thinsp;+\u0026thinsp;G mice displayed significant enrichment of CD3⁺ T cells (+\u0026thinsp;505% \u0026plusmn; 183%), CD8⁺ T cells (+\u0026thinsp;1376% \u0026plusmn; 458%), and CD4⁺ T-helper cells (+\u0026thinsp;856% \u0026plusmn; 303%) ( Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL, Supplementary Table\u0026nbsp;8). Treg counts increased significantly in aged Q\u0026thinsp;+\u0026thinsp;G mice compared to Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), and were comparable between aged and young Q\u0026thinsp;+\u0026thinsp;G males on day 4 (Supplementary Table\u0026nbsp;8). However, the Treg:CD8⁺ T-cell ratio was significantly lower in aged Q\u0026thinsp;+\u0026thinsp;G than in young Q\u0026thinsp;+\u0026thinsp;G males (Supplementary Fig.\u0026nbsp;10L).\u003c/p\u003e \u003cp\u003eQ\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 did not elicit a significant increase in total immune cells in aged mice compared to P or Q (Supplementary Fig.\u0026nbsp;11A). In aged male Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 mice, CD3⁺ T cells\u0026mdash;including CD8⁺ and CD4⁺ subsets\u0026mdash;were not significantly different from young Q\u0026thinsp;+\u0026thinsp;G or Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 mice on day 4 (Supplementary Table\u0026nbsp;8). B cells did not increase in Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 relative to P or Q, unlike in young male Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 (Supplementary Fig.\u0026nbsp;11E). Similar to aged male Q\u0026thinsp;+\u0026thinsp;G, Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 showed a significant reduction in total immune cell counts, neutrophils, eosinophils, CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages, CX3CR1⁺Ly6Cˡᵒ macrophages, and cDC2s compared to young male Q\u0026thinsp;+\u0026thinsp;G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups (Supplementary Table\u0026nbsp;8). However, aged male Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 mice exhibited an increased Treg:CD8⁺ T-cell ratio compared to aged Q\u0026thinsp;+\u0026thinsp;G males, restoring the ratio to comparable levels to young male G, Q\u0026thinsp;+\u0026thinsp;G, or Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups (Supplementary Fig.\u0026nbsp;10L).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIncreased macrophage levels in the draining lymph nodes downstream of Q\u0026thinsp;+\u0026thinsp;G subside by day 4 in female but not in male mice and are lower in aged mice but rescuable with IL-33 supplementation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we collected inguinal draining lymph nodes on day 4 from young female, young male, aged male, and IFNAR⁻/⁻ female mice and characterized their immune myeloid and lymphocyte composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). On day 4, we found neutrophils, CX3CR1⁺Ly6Cʰⁱ macrophages, and B cells to be significantly enriched in Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to P in young female draining lymph nodes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;12A and 12B). Young female Q draining lymph nodes were also enriched in CD3⁺ T cells and CD8⁺ T cells compared to P and Q\u0026thinsp;+\u0026thinsp;G (Supplementary Fig.\u0026nbsp;13A and 13B). Additionally, compared to young female Q\u0026thinsp;+\u0026thinsp;G, Q was enriched in Tregs on day 4 (Supplementary Fig.\u0026nbsp;13C). In young male mice, no statistically significant changes were detected in myeloid or lymphocyte populations across groups in the draining lymph nodes on day 4 (Supplementary Figs.\u0026nbsp;12C-I and 13D-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCompared to day 2, the composition of draining lymph nodes changed significantly by day 4 in both sexes (Supplementary Table\u0026nbsp;10). In female mice, total immune cell counts per lymph node were significantly lower in Q\u0026thinsp;+\u0026thinsp;G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 on day 4 compared to day 2 (Supplementary Table\u0026nbsp;10). Within the myeloid compartment, all young female groups showed a significant reduction in macrophages and CX3CR1⁺Ly6Cˡᵒ macrophages on day 4 compared to day 2 (Supplementary Table\u0026nbsp;10). The magnitude of decrease varied, with the largest change observed in the Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 group (-11,787\u0026thinsp;\u0026plusmn;\u0026thinsp;2,899 cells per lymph node), followed by Q\u0026thinsp;+\u0026thinsp;G (-2,198\u0026thinsp;\u0026plusmn;\u0026thinsp;522), and smaller but comparable reductions in Q (-1,083\u0026thinsp;\u0026plusmn;\u0026thinsp;305), P (-830\u0026thinsp;\u0026plusmn;\u0026thinsp;173), and G (-701\u0026thinsp;\u0026plusmn;\u0026thinsp;286).\u003c/p\u003e \u003cp\u003eFurthermore, cDC2 counts per lymph node were significantly lower in all female groups except Q, where they remained stable (Supplementary Table\u0026nbsp;10). Similar to macrophages, cDC2 counts changed most in the Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 group (-14,663\u0026thinsp;\u0026plusmn;\u0026thinsp;2,814) compared to smaller decreases in Q\u0026thinsp;+\u0026thinsp;G (-1,276\u0026thinsp;\u0026plusmn;\u0026thinsp;371), G (-842\u0026thinsp;\u0026plusmn;\u0026thinsp;450), and P (-796\u0026thinsp;\u0026plusmn;\u0026thinsp;303). In the lymphocyte compartment, CD3⁺ T-cell counts remained constant in Q\u0026thinsp;+\u0026thinsp;G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 on day 4 compared to day 2, while Tregs increased in both Q\u0026thinsp;+\u0026thinsp;G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups (Supplementary Table\u0026nbsp;10).\u003c/p\u003e \u003cp\u003eIn young male mice, macrophage, CX3CR1⁺Ly6Cˡᵒ macrophage, and cDC2 counts per lymph node were significantly reduced only in P, G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups, while they remained stable in Q and Q\u0026thinsp;+\u0026thinsp;G (Supplementary Table\u0026nbsp;10). Treg and CD3⁺ T-cell counts increased in Q\u0026thinsp;+\u0026thinsp;G on day 4 compared to day 2 (Supplementary Table\u0026nbsp;10).\u003c/p\u003e \u003cp\u003eWhen comparing male and female draining lymph nodes on day 4, we found no significant differences in any quantified immune populations in P and Q groups between sexes (Supplementary Table\u0026nbsp;11). In G groups, male draining lymph nodes were enriched for neutrophils, CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages, cDC2s, CD8⁺ T cells, and B cells compared to female lymph nodes (Supplementary Table\u0026nbsp;11). In Q\u0026thinsp;+\u0026thinsp;G, eosinophils, macrophages\u0026mdash;particularly CX3CR1⁺Ly6Cˡᵒ macrophages\u0026mdash;and Tregs were enriched in females on day 4 compared to males (Supplementary Table\u0026nbsp;11). Male Q\u0026thinsp;+\u0026thinsp;G draining lymph nodes had macrophage compositions on day 4 comparable to female Q\u0026thinsp;+\u0026thinsp;G draining lymph nodes on day 2 (Supplementary Table\u0026nbsp;11). Tregs were more enriched in male Q\u0026thinsp;+\u0026thinsp;G draining lymph nodes on day 4 than in female Q\u0026thinsp;+\u0026thinsp;G on day 2 (+\u0026thinsp;180% \u0026plusmn; 95%) (Supplementary Table\u0026nbsp;11). In Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups, neutrophils, macrophages, and CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages were significantly more enriched in male draining lymph nodes than in female Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 nodes (Supplementary Table\u0026nbsp;11).\u003c/p\u003e \u003cp\u003eWhen comparing aged and young male mice, we found a significant decrease in total immune cell counts per lymph node on day 4 in P and Q\u0026thinsp;+\u0026thinsp;G groups (Supplementary Table\u0026nbsp;12). In P, total immune cell numbers were 74% (\u0026plusmn;\u0026thinsp;34%) lower and included reductions in neutrophils (-93% \u0026plusmn; 36%), CD3⁺ T cells (-66% \u0026plusmn; 21%), CD8⁺ T cells (-54% \u0026plusmn; 25%), CD4⁺ T-helper cells (-80% \u0026plusmn; 16%), B cells (-84% \u0026plusmn; 32%), and Tregs (-60% \u0026plusmn; 22%) in aged males compared to young males on day 4 (Supplementary Table\u0026nbsp;12).\u003c/p\u003e \u003cp\u003eIn Q groups, we observed significant reductions in neutrophils (-89% \u0026plusmn; 42%) and eosinophils (-93% \u0026plusmn; 49%) in aged males compared to young males (Supplementary Table\u0026nbsp;12). In Q\u0026thinsp;+\u0026thinsp;G groups, there were significant decreases in neutrophils (-91% \u0026plusmn; 44%), eosinophils (-93% \u0026plusmn; 49%), CX3CR1⁺Ly6Cˡᵒ macrophages (-84% \u0026plusmn; 41%), CD3⁺ T cells (-68% \u0026plusmn; 30%), CD4⁺ T-helper cells (-82% \u0026plusmn; 28%), and B cells (-71% \u0026plusmn; 34%) in aged male mice compared to young male mice on day 4 (Supplementary Table\u0026nbsp;12).\u003c/p\u003e \u003cp\u003eIn Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33, only CD4⁺ T-helper cells were significantly reduced by 69% (\u0026plusmn;\u0026thinsp;17%, P\u0026thinsp;=\u0026thinsp;0.0007) in aged males compared to young males on day 4 (Supplementary Table\u0026nbsp;12). In contrast, macrophages, including CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e and CX3CR1⁺Ly6Cˡᵒ subsets, were significantly enriched in aged male lymph nodes by +\u0026thinsp;382% (\u0026plusmn;\u0026thinsp;126%) and +\u0026thinsp;112% (\u0026plusmn;\u0026thinsp;65%), respectively, compared to young male Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 lymph nodes (Supplementary Table\u0026nbsp;12). In fact, macrophage composition in aged Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 lymph nodes was not significantly different from that of young male Q\u0026thinsp;+\u0026thinsp;G lymph nodes (Supplementary Table\u0026nbsp;12).\u003c/p\u003e \u003cp\u003eIn aged male mice, we observed an increase in total immune cell counts, macrophages (including CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e and CX3CR1⁺Ly6Cˡᵒ macrophages), and B cells in Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 draining lymph nodes compared to P (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-G, Supplementary Fig.\u0026nbsp;14A). No significant changes were detected in Q\u0026thinsp;+\u0026thinsp;G or Q compared to P in aged male draining lymph nodes on day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-G, Supplementary Fig.\u0026nbsp;14A-H).\u003c/p\u003e \u003cp\u003eIn young female IFNAR⁻/⁻ mice, we observed no changes in immune cell populations between P and Q\u0026thinsp;+\u0026thinsp;G, consistent with wild-type female draining lymph nodes on day 4 (Supplementary Fig.\u0026nbsp;12J, Supplementary Fig.\u0026nbsp;13I). Furthermore, no differences were detected between IFNAR⁻/⁻ and wild-type females when comparing P and Q\u0026thinsp;+\u0026thinsp;G groups (Supplementary Table\u0026nbsp;12).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMuscle Injury skews antibody subtypes to IgG1 in young male and aged mice of both sexes, and IL-33 increases total IgG response.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we quantified the vaccine-specific anti-QIV total IgG titers in female and male, young and aged mice, 14 days post-vaccination. We computed the endpoint anti-QIV titers as percentages relative to the Q group to normalize three independent experiments from log₁₀-transformed anti-QIV antibody endpoint titers (Supplementary Table\u0026nbsp;14, Supplementary Fig.\u0026nbsp;15). We found aged female mice mounted a significantly reduced antibody response following QIV compared to young females, showing a 30% decrease (\u0026plusmn;\u0026thinsp;6.3%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Table\u0026nbsp;14). In aged male mice, total anti-QIV IgG titers downstream of QIV showed a decreasing trend of 14% (\u0026plusmn;\u0026thinsp;8.2%) compared to young males (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, Supplementary Table\u0026nbsp;14). Overall, most vaccination regimens in males yielded comparable total IgG titers across age groups, with the exception of Q\u0026thinsp;+\u0026thinsp;IL-33 in young males and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 in aged males, both of which significantly increased antibody titers compared to the aged Q group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, Supplementary Table\u0026nbsp;14).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn young female mice, the addition of damage-inducing glycerol to QIV (Q\u0026thinsp;+\u0026thinsp;G) did not significantly alter total anti-QIV IgG titers compared to QIV alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The Q\u0026thinsp;+\u0026thinsp;IL-33 group exhibited a significant 17.5% (\u0026plusmn;\u0026thinsp;8.2%) increase compared to Q\u0026thinsp;+\u0026thinsp;G and a trend toward a 16% increase compared to Q alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Table\u0026nbsp;14). Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 showed an increasing trend of 8% (\u0026plusmn;\u0026thinsp;2.3%) compared to Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Table\u0026nbsp;14).\u003c/p\u003e \u003cp\u003eWhen comparing young and aged female mice, Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 and Q\u0026thinsp;+\u0026thinsp;IL-33 in young females induced significantly higher antibody titers than aged female Q and Q\u0026thinsp;+\u0026thinsp;G groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Aged females receiving Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 or Q\u0026thinsp;+\u0026thinsp;IL-33 achieved total anti-QIV IgG levels comparable to young females vaccinated with QIV alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eNext, we characterized the antibody response in aged IFNAR⁻/⁻ female mice receiving Q\u0026thinsp;+\u0026thinsp;G or Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33. Total IgG titers were comparable between aged wild-type and IFNAR⁻/⁻ females in both vaccination groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), suggesting IFN signaling does not drive the observations made in WT mice.\u003c/p\u003e \u003cp\u003eWe then analyzed the anti-QIV-specific IgG subclasses IgG1 and IgG2c across all experimental groups. In young female mice, Q and Q\u0026thinsp;+\u0026thinsp;G groups showed comparable anti-QIV IgG1 titers 14 days post-vaccination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 and Q\u0026thinsp;+\u0026thinsp;IL-33 groups displayed increasing trends of +\u0026thinsp;13.2% (\u0026plusmn;\u0026thinsp;2.1%) and +\u0026thinsp;23.4% (\u0026plusmn;\u0026thinsp;2.3%), respectively, compared to young female Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, Supplementary Table\u0026nbsp;14). In aged females, QIV alone induced a significant 43.9% (\u0026plusmn;\u0026thinsp;6.7%) reduction in anti-QIV IgG1 titers relative to young Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, Supplementary Table\u0026nbsp;14). Aged Q\u0026thinsp;+\u0026thinsp;G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups showed non-significant decreases of -31.2% (\u0026plusmn;\u0026thinsp;3.2%) and \u0026minus;\u0026thinsp;15.2% (\u0026plusmn;\u0026thinsp;6.8%) compared to young Q, while aged Q\u0026thinsp;+\u0026thinsp;IL-33 mice maintained IgG1 titers equivalent to young female Q (100.9% \u0026plusmn; 4.8%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, Supplementary Table\u0026nbsp;14). Aged IFNAR⁻/⁻ females displayed no differences in IgG1 titers in Q\u0026thinsp;+\u0026thinsp;G or Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to their aged wild-type counterparts(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eIn male mice Q, anti-QIV IgG1 titers were 35% (\u0026plusmn;\u0026thinsp;8.6%) lower in aged males compared to young male Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, Supplementary Table\u0026nbsp;14). In young males, Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 and Q\u0026thinsp;+\u0026thinsp;IL-33 both showed increasing trends in IgG1 titers\u0026mdash;27.5% (\u0026plusmn;\u0026thinsp;4.2%) and 24.9% (\u0026plusmn;\u0026thinsp;5.9%) higher than young Q, respectively\u0026mdash;and were significantly elevated compared to aged male Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Supplementary Table\u0026nbsp;14). Young male Q\u0026thinsp;+\u0026thinsp;G did not significantly alter IgG1 titers compared to young Q, but resulted in higher titers than aged male Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eWe next characterized anti-QIV IgG2c titers across all groups. In females, anti-QIV IgG2c titers dropped significantly only in aged Q\u0026thinsp;+\u0026thinsp;G groups in both IFNAR⁻/⁻ and wild-type mice, showing decreases of 69.4% (\u0026plusmn;\u0026thinsp;14.8%) and 64.4% (\u0026plusmn;\u0026thinsp;7.4%), respectively, compared to young female Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, Supplementary Table\u0026nbsp;14).\u003c/p\u003e \u003cp\u003eSimilarly, in young male mice, Q, Q\u0026thinsp;+\u0026thinsp;G, Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33, and Q\u0026thinsp;+\u0026thinsp;IL-33 produced comparable IgG2c titers 14 days post-vaccination (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). In aged males, the Q\u0026thinsp;+\u0026thinsp;G group showed a significant reduction in IgG2c titers compared to young male Q, whereas aged Q, Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33, and Q\u0026thinsp;+\u0026thinsp;IL-33 groups did not differ significantly from young Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eFinally, we computed the IgG1:IgG2c ratio to evaluate whether the vaccine formulations differentially influenced antibody subtype responses. Q\u0026thinsp;+\u0026thinsp;G significantly increased the IgG1:IgG2c ratio in young males, aged females, and aged males compared to Q, but not in young females (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-J).\u003c/p\u003e \u003cp\u003e \u003cb\u003eQ\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 provides enhanced protection against subsequent lethal H1N1 infection compared to P, in young mice only.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the protective efficacy of the different vaccine formulations, we infected all groups 21 days post-vaccination with a 3LD₅₀ dose of mouse-adapted H1N1 influenza virus (A/Michigan/45/2015) via intranasal instillation. Following infection, body weights were recorded daily to monitor weight loss until day 6 post-infection, at which point lungs were collected to quantify replicating viral titers using plaque assays.\u003c/p\u003e \u003cp\u003eBy day 6, unvaccinated P groups exhibited average weight losses of 14.7% (\u0026plusmn;\u0026thinsp;2.4%), 17% (\u0026plusmn;\u0026thinsp;0.8%), 16.3% (\u0026plusmn;\u0026thinsp;3.6%), and 10.8% (\u0026plusmn;\u0026thinsp;2.1%) in young female, young male, aged female, and aged male mice, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D). Compared to P, weight loss was significantly reduced in vaccinated young male mice, with losses of 7.8% (\u0026plusmn;\u0026thinsp;1.8%) in Q, 6.1% (\u0026plusmn;\u0026thinsp;1.48%) in Q\u0026thinsp;+\u0026thinsp;G, and 3.9% (\u0026plusmn;\u0026thinsp;1.9%) in Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). In young females, the Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 group also showed significantly reduced weight loss (6% \u0026plusmn; 2.5%) compared to the female P group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In aged mice, no significant differences in weight loss were observed among vaccination groups in either sex (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we quantified titers of replicating virus (plaque-forming units, PFUs) from lung homogenates collected on day 6 post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-H). There is a relatively large spread in lung virus titers, which most likely is due to the late time point of sample collection (6DPI), thereby allowing some mice to start controlling the replicating virus titers. Young male Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 mice exhibited a significant reduction in PFUs compared to P (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Notably, 2 of 5 young male mice in the Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 group had undetectable viral titers, whereas all other groups showed detectable PFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eIn young female mice, the Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 group displayed the highest number of mice with undetectable lung PFUs (3 of 5 mice) compared to none in the P group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). In aged females, Q, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups exhibited a trend toward reduced PFUs compared to P, with 2 of 5, 3 of 5, and 2 of 5 lungs, respectively, showing no detectable viral titers, whereas all P-group lungs contained detectable PFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003cb\u003eQ, Q\u0026thinsp;+\u0026thinsp;G and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 show type II immune response skewing in the lungs after subsequent H1N1 viral infection in young mice, which is reduced in aged mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe then characterized the lung inflammatory milieu using a Th1/Th2 multiplex cytokine assay to determine whether P, Q, G, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 treatments skewed the immune response to QIV toward a type 1 or type 2 profile. To do so, we segregated lungs with detectable viral titers (PFUs) from those without detectable PFUs and compiled all lungs with non-detectable PFUs into a single group (ND PFUs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This approach allowed us to examine the cytokine environment associated with ongoing viral infection separately from that of mice that effectively cleared virus from their lungs. Because no significant differences in cytokine concentrations were found between sexes within each age group, data from male and female mice were color-coded by sex and combined and presented as young and aged cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn young mice, we observed a significant increase in IL-4 concentrations in Q (61.1\u0026thinsp;\u0026plusmn;\u0026thinsp;9.8 pg/mL), Q\u0026thinsp;+\u0026thinsp;G (53.2\u0026thinsp;\u0026plusmn;\u0026thinsp;14.7 pg/mL), and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 (67.2\u0026thinsp;\u0026plusmn;\u0026thinsp;21.2 pg/mL) compared to ND PFUs (2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 pg/mL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Similarly, IL-5 concentrations increased significantly in Q (864\u0026thinsp;\u0026plusmn;\u0026thinsp;75 pg/mL), Q\u0026thinsp;+\u0026thinsp;G (807.2\u0026thinsp;\u0026plusmn;\u0026thinsp;157.3 pg/mL), and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 (889.6\u0026thinsp;\u0026plusmn;\u0026thinsp;225.7 pg/mL) compared to ND PFUs (20.2\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6 pg/mL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Additionally, IL-4 levels were significantly higher in Q compared to P (8.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 pg/mL) and G (7.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 pg/mL), and IL-5 was significantly elevated in Q compared to G (195.7\u0026thinsp;\u0026plusmn;\u0026thinsp;40.1 pg/mL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eWe also found an increasing trend in IL-13 concentrations (all following concentrations are in pg/mL) in Q (149.4\u0026thinsp;\u0026plusmn;\u0026thinsp;16.5), Q\u0026thinsp;+\u0026thinsp;G (118.8\u0026thinsp;\u0026plusmn;\u0026thinsp;29.3), and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 (157.7\u0026thinsp;\u0026plusmn;\u0026thinsp;52.7) compared to P (19.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 pg/mL), G (16.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5), and ND PFUs (6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eIFNγ, IL-18, and IL-1β concentrations increased significantly in all infected groups compared to ND PFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-F). TNFα levels were significantly elevated in P (85.8\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1) and G (69.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7.8) relative to ND PFUs (12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). GM-CSF concentrations also increased significantly in P (28.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5), Q (25.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 pg/mL), and G (23.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9) compared to ND PFUs (6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). IL-12p70 concentrations were elevated in Q (12.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7), Q\u0026thinsp;+\u0026thinsp;G (12.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0), and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 (12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 pg/mL) compared to ND PFUs (3.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI). IL-6 concentrations were significantly higher in P (3,865\u0026thinsp;\u0026plusmn;\u0026thinsp;417) and Q (3,516\u0026thinsp;\u0026plusmn;\u0026thinsp;415) than in ND PFUs (40.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003eIn aged mice, unlike in young mice, no significant increase in IL-4 was observed downstream of Q\u0026thinsp;+\u0026thinsp;G compared to ND PFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK). Q and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups exhibited significant increases in both IL-4 and IL-13 concentrations compared to ND PFUs, with Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 displaying the highest levels of both cytokines, which were significantly higher than P (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003eWhen comparing aged and young groups, IL-4 levels were reduced in aged mice by 37.6% (\u0026plusmn;\u0026thinsp;12.5%, P\u0026thinsp;=\u0026thinsp;0.017) in P, by 46.3% (\u0026plusmn;\u0026thinsp;21.7%, P\u0026thinsp;=\u0026thinsp;0.09) in Q, and by 49.8% (\u0026plusmn;\u0026thinsp;30.5%, P\u0026thinsp;=\u0026thinsp;0.13) in Q\u0026thinsp;+\u0026thinsp;G, while aged Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 mice maintained IL-4 concentrations comparable to young Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 (Supplementary Table\u0026nbsp;15). IL-13 levels were comparable between aged and young mice in P, Q, and Q\u0026thinsp;+\u0026thinsp;G, but trended higher (+\u0026thinsp;86.9% \u0026plusmn; 46.7%, P\u0026thinsp;=\u0026thinsp;0.09)in aged Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to young Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 (Supplementary Table\u0026nbsp;15).\u003c/p\u003e \u003cp\u003eIn aged mice, IL-5 concentrations increased significantly in Q, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to ND PFUs, similar to trends in young mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eM). When considering age as a variable, IL-5 levels were significantly altered in aged mice, showing reductions in P (-49% \u0026plusmn; 34%, P\u0026thinsp;=\u0026thinsp;0.04) and increases in Q (+\u0026thinsp;52.4% \u0026plusmn; 28%) and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 (+\u0026thinsp;115% \u0026plusmn; 45%) compared to their respective young groups (Supplementary Table\u0026nbsp;15).\u003c/p\u003e \u003cp\u003eIFNγ concentrations increased significantly in aged Q, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to ND PFUs, but not in aged P (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eN). When comparing across ages, IFNγ was markedly reduced in aged P (-52.2% \u0026plusmn; 20.3%) and increased in aged Q\u0026thinsp;+\u0026thinsp;G (+\u0026thinsp;166.6% \u0026plusmn; 102.2%) relative to their young counterparts (Supplementary Table\u0026nbsp;15).\u003c/p\u003e \u003cp\u003eUnlike young mice, TNFα concentrations increased in all aged groups (P, Q, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33) compared to ND PFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eO). Moreover, TNFα was significantly higher in aged Q (+\u0026thinsp;36.5% \u0026plusmn; 11.6%) and aged Q\u0026thinsp;+\u0026thinsp;G (+\u0026thinsp;57.2% \u0026plusmn; 29.9%) compared to young Q and Q\u0026thinsp;+\u0026thinsp;G, respectively (Supplementary Table\u0026nbsp;15).\u003c/p\u003e \u003cp\u003eIL-6 concentrations increased significantly in aged P, Q, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to ND PFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eP). Between ages, IL-6 was significantly elevated in aged P (+\u0026thinsp;75.1% \u0026plusmn; 36.1%) and aged Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 (+\u0026thinsp;231% \u0026plusmn; 70%) relative to young groups, while Q and Q\u0026thinsp;+\u0026thinsp;G showed increasing trends (+\u0026thinsp;77.5% \u0026plusmn; 33.6%, P\u0026thinsp;=\u0026thinsp;0.056; +128.5% \u0026plusmn; 55.8%, P\u0026thinsp;=\u0026thinsp;0.055) (Supplementary Table\u0026nbsp;15).\u003c/p\u003e \u003cp\u003eIL-2, IL-12p70, GM-CSF, and IL-1β concentrations did not differ between young and aged mice (Supplementary Table\u0026nbsp;15). IL-18 concentrations were reduced in aged P by 24.3% (\u0026plusmn;\u0026thinsp;10.2%, P\u0026thinsp;=\u0026thinsp;0.019) compared to young P (Supplementary Table\u0026nbsp;15, Supplementary Fig.\u0026nbsp;16A-F).\u003c/p\u003e \u003cp\u003e \u003cb\u003e6 days post H1N1 infection, Tregs and M2 prevalence increases in young and aged Q\u0026thinsp;+\u0026thinsp;G lungs, and IL-33 rescues eosinophil recruitment to the lungs in aged mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUsing two spectral flow cytometry panels, we characterized the myeloid and lymphocyte composition of H1N1\u0026ndash;infected lungs harvested 6 days post-infection from young and aged, male and female mice across the different treatment groups (Supplementary Figs.\u0026nbsp;17 and 18).\u003c/p\u003e \u003cp\u003eOn day 6 post-infection, in young female mice, we observed a significant increase in total immune cell counts in the Q\u0026thinsp;+\u0026thinsp;G group (8,081,404\u0026thinsp;\u0026plusmn;\u0026thinsp;257,145 cells per lung) compared to P (3,557,550\u0026thinsp;\u0026plusmn;\u0026thinsp;985,043) and ND PFUs (3,557,550\u0026thinsp;\u0026plusmn;\u0026thinsp;985,043) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Q and Q\u0026thinsp;+\u0026thinsp;G groups induced significant increases in eosinophil counts, including both Siglec-F\u003csup\u003ehi\u003c/sup\u003e and Siglec-Fˡᵒ subpopulations, compared to ND PFUs, with comparable levels between the two vaccinated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-D). Additionally, in young female Q\u0026thinsp;+\u0026thinsp;G lungs, there was a significant increase in non-alveolar macrophages,that are Ly6Cˡᵒ, Arginase I⁺, and iNOS\u003csup\u003e\u0026minus;\u003c/sup\u003e compared to P and ND PFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG-H). In addition, Q and Q\u0026thinsp;+\u0026thinsp;G young female mice exhibited a reduced M1/M2 macrophage ratio relative to P and G groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI). Furthermore, Treg counts per lung also increased significantly in young female Q\u0026thinsp;+\u0026thinsp;G compared to both ND PFUs and P (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn young male mice, Q\u0026thinsp;+\u0026thinsp;G and Q produced comparable increases in total immune cell counts relative to P and G (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK). Treg counts were significantly elevated in male Q\u0026thinsp;+\u0026thinsp;G compared to ND PFUs, but not in Q (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eL). Eosinophil counts showed increasing trends in both Q and Q\u0026thinsp;+\u0026thinsp;G compared to P, G, and ND PFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eM). Furthermore, Arginase I\u003csup\u003e+\u003c/sup\u003e M2 non-alveolar macrophages increased significantly in male Q\u0026thinsp;+\u0026thinsp;G compared to ND PFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eN). M1/M2 ratio was therefore significantly reduced in male Q\u0026thinsp;+\u0026thinsp;G compared to P (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eO).\u003c/p\u003e \u003cp\u003eHowever, male Q\u0026thinsp;+\u0026thinsp;G lungs exhibited significantly lower immune cell numbers than young female Q\u0026thinsp;+\u0026thinsp;G lungs, including total immune cells (-36.1% \u0026plusmn; 12.2%), Tregs (-59.9% \u0026plusmn; 17.7%), and B cells (-54.5% \u0026plusmn; 27.4%) (Supplementary Table\u0026nbsp;16).\u003c/p\u003e \u003cp\u003eAdditionally, male Q\u0026thinsp;+\u0026thinsp;G lungs displayed decreasing trends in Ly6Cˡᵒ macrophages (-42.1% \u0026plusmn; 22.2%, P\u0026thinsp;=\u0026thinsp;0.06), Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages (-35.9% \u0026plusmn; 15.1%, P\u0026thinsp;=\u0026thinsp;0.06), and Arginase I⁺ macrophages (-46.5% \u0026plusmn; 22.5%, P\u0026thinsp;=\u0026thinsp;0.06) relative to young female Q\u0026thinsp;+\u0026thinsp;G (Supplementary Table\u0026nbsp;16). No statistically significant differences in lung immune composition were observed among P, Q, G, or ND PFUs groups 6 days post-infection (Supplementary Table\u0026nbsp;16).\u003c/p\u003e \u003cp\u003eNext, we characterized the immune composition of aged P, Q, G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 lungs at day 6 post-infection. No significant differences were observed between aged male and female mice across any group or immune population, so data were combined and presented as aged mice. In aged mice, non-alveolar macrophages increased in P, Q, and Q\u0026thinsp;+\u0026thinsp;G compared to ND PFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eP). The macrophage inflammatory phenotype varied by group: iNOS⁺ macrophages were enriched exclusively in the P group compared to Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 and ND PFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eQ). Additionally, Q, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups displayed significantly increased eosinophil counts, particularly Siglec-Fʰⁱ eosinophils, compared to ND PFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eR and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eS). Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 exhibited the highest eosinophil numbers per lung, including Siglec-Fʰⁱ subsets, compared to Q and Q\u0026thinsp;+\u0026thinsp;G (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eR and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eS).\u003c/p\u003e \u003cp\u003eTreg counts also increased significantly in aged Q, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to ND PFUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eT). However, Treg levels were comparable across Q, Q\u0026thinsp;+\u0026thinsp;G, and Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 groups in aged mice, unlike young female and male mice, where Q\u0026thinsp;+\u0026thinsp;G induced the highest Treg numbers (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eT).\u003c/p\u003e \u003cp\u003eWe then compared the lung immune composition of aged and young male mice within each group, 6 days post-infection. In the P groups, aged mice exhibited significant increases in CD3⁺ T cells (+\u0026thinsp;266.2% \u0026plusmn; 90%), including CD8⁺ T cells (+\u0026thinsp;341.3% \u0026plusmn; 108.4%) and CD4⁺ T-helper cells (+\u0026thinsp;348.6% \u0026plusmn; 151.9%) compared to young male P mice (Supplementary Table\u0026nbsp;17). Tregs showed a non-significant decreasing trend (-41.3% \u0026plusmn; 30.6%, P\u0026thinsp;=\u0026thinsp;0.14) compared to young P (Supplementary Table\u0026nbsp;17). Eosinophils were significantly lower in aged P (-59.2% \u0026plusmn; 27.3%), including Siglec-F\u003csup\u003ehi\u003c/sup\u003e eosinophils (-68.1% \u0026plusmn; 27.4%) relative to young male P (Supplementary Table\u0026nbsp;17).\u003c/p\u003e \u003cp\u003eIn the aged Q group, we observed significant increases in CD3⁺ T cells (+\u0026thinsp;279.7% \u0026plusmn; 94.9%), including CD4⁺ T-helper cells (+\u0026thinsp;175.8% \u0026plusmn; 71.8%) and CD8⁺ T cells (+\u0026thinsp;546.2% \u0026plusmn; 162.7%) compared to young male Q (Supplementary Table\u0026nbsp;17). Eosinophil counts did not differ significantly between aged and young Q groups (Supplementary Table\u0026nbsp;17). Aged Q\u0026thinsp;+\u0026thinsp;G lungs showed no significant differences in immune composition compared to young Q\u0026thinsp;+\u0026thinsp;G (Supplementary Table\u0026nbsp;17).\u003c/p\u003e \u003cp\u003eIn contrast, aged ND PFUs lungs displayed significant decreases in CD4⁺ T-helper cells (-54.7% \u0026plusmn; 22.2%, P\u0026thinsp;=\u0026thinsp;0.04), B cells (-79.9% \u0026plusmn; 25.5%), and Tregs (-79.1% \u0026plusmn; 38.3%) compared to young ND PFUs males. Conversely, iNOS⁺ macrophages were markedly enriched in aged ND PFUs (+\u0026thinsp;496.4% \u0026plusmn; 212.2%, P\u0026thinsp;=\u0026thinsp;0.011)(Supplementary Table\u0026nbsp;17).\u003c/p\u003e \u003cp\u003eWe next compared aged and young female mice 6 days post-infection. Tregs were significantly reduced in aged P mice (-74.8% \u0026plusmn; 20.3%) compared to young female P (Supplementary Table\u0026nbsp;18). Similarly, aged Q and Q\u0026thinsp;+\u0026thinsp;G groups showed significant decreases in Tregs of -80.4% (\u0026plusmn;\u0026thinsp;24%) and \u0026minus;\u0026thinsp;67.4% (\u0026plusmn;\u0026thinsp;19.2%) compared to young female Q and Q\u0026thinsp;+\u0026thinsp;G, respectively (Supplementary Table\u0026nbsp;18). No significant differences were observed between aged and young female ND PFUs groups (Supplementary Table\u0026nbsp;18).\u003c/p\u003e \u003cp\u003eWe then investigated whether IL-33 supplementation without glycerol could induce eosinophil increase in the lungs post-infection. To test this, we vaccinated young and aged mice with Q or Q\u0026thinsp;+\u0026thinsp;IL-33, followed by intranasal infection with 3LD₅₀ H1N1 IVR-180. Lungs were collected 6 days post-infection to evaluate inflammatory cytokine profiles and immune cell composition via multiplex assay and flow cytometry. IL-33 supplementation significantly increased eosinophil counts in young mice from 52,557\u0026thinsp;\u0026plusmn;\u0026thinsp;13,190 (Q) to 94,158\u0026thinsp;\u0026plusmn;\u0026thinsp;10,277 (Q\u0026thinsp;+\u0026thinsp;IL-33) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eU). In aged mice, eosinophils also trended higher\u0026mdash;from 36,827\u0026thinsp;\u0026plusmn;\u0026thinsp;13,190 (Q) to 112,667\u0026thinsp;\u0026plusmn;\u0026thinsp;51,031 (Q\u0026thinsp;+\u0026thinsp;IL-33; P\u0026thinsp;=\u0026thinsp;0.065)(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eV). IL-33 supplementation did not alter Treg counts in either young or aged lungs 6 days post-infection compared to Q alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eW-Z).\u003c/p\u003e \u003cp\u003eBecause Tregs depend on the IL-33\u0026ndash;ST2 signaling axis for recruitment to injured muscle\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, we examined whether IL-33 was locally upregulated in the lung. IL-33 mean fluorescence intensity (MFI) was significantly increased in young female Q\u0026thinsp;+\u0026thinsp;G lungs compared to P, consistent with the higher Treg numbers observed in Q\u0026thinsp;+\u0026thinsp;G lungs (Supplementary Fig.\u0026nbsp;19L, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e AA). In aged mice, however, IL-33 expression did not increase in Q\u0026thinsp;+\u0026thinsp;G compared to P, but increased in Q\u0026thinsp;+\u0026thinsp;G\u0026thinsp;+\u0026thinsp;IL-33 compared to young female P, and G (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eAA).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated how muscle injury influences immune responses to seasonal influenza vaccination using a glycerol-induced injury model and the seasonal quadrivalent inactivated influenza vaccine (QIV), examining the effects in a sex- and age-dependent manner. We further assessed whether IL-33 supplementation, known to mitigate age-related impairments in muscle regeneration\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, modulates these responses in young and aged mice. We found that muscle injury alters immune kinetics at the vaccination site and draining lymph nodes, thereby remodeling both humoral and cellular vaccine responses. Furthermore, IL-33 supplementation modified several aspects of vaccine-induced immunity, reduced sex-associated variability at the injection site, enhanced humoral responses, and partially restored age-associated impairments in type II immunity.\u003c/p\u003e \u003cp\u003eWe first confirmed that glycerol induces cell death in C2C12 myotubes \u003cem\u003ein vitro\u003c/em\u003e. When glycerol was combined with QIV, we observed an increase in pro-inflammatory and T-cell activating cytokines \u0026mdash;including IFNγ, TNFα, IL-18, and IL-2\u0026mdash;that were not elevated with glycerol or QIV treatment alone. The reason why the combination of QIV and glycerol gives higher cytokine responses compared to QIV and glycerol conditions is unclear. It is possible that glycerol allows cytoplasmic delivery of QIV proteins\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, thereby triggering innate immune pathways. To our knowledge, myotubes have not been described before as a source of IL-2. From a mechanistic point of view, IL-2 is important in muscle repair pathways as it maintains Treg presence and activity\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vivo\u003c/em\u003e, glycerol-induced muscle injury triggered an early and coordinated infiltration of myeloid cells dominated by inflammatory CX3CR1⁺Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages and neutrophils, accompanied by eosinophils and cDC2s. This early phase was followed by a day 4 transition toward a reparative profile, characterized by reduced neutrophil counts, expansion of CX3CR1⁺Ly6Cˡᵒ macrophages, and Treg enrichment, consistent with the regenerative trajectory described during muscle healing. Correlations between macrophage, cDC2, and eosinophil densities suggest coordinated crosstalk between regenerative and antigen-presenting cell populations that may link tissue repair to adaptive priming. These results align with prior evidence that macrophage\u0026ndash;dendritic cell interactions bridge local repair and adaptive activation, but extend this concept to a vaccination context\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe found that muscle repair-associated immune events differed between sexes, with females mounting a more robust early inflammatory response by day 2 and repolarizing toward a reparative profile by day 4. This supports earlier reports indicating that female mice regenerate muscle more robustly than male mice\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Male mice exhibited a reduced early inflammatory response but a comparable delayed reparative phase, marked by similar CX3CR1⁺Ly6Cˡᵒ macrophage and Treg densities, yet with persistent neutrophil presence. Sex-based differences in intramuscular immune kinetics were mirrored in the draining lymph nodes, where glycerol-supplemented QIV induced macrophage and lymphocyte expansion on day 2 in females but not in males.\u003c/p\u003e \u003cp\u003eMuscle injury during vaccination did not alter anti-QIV total IgG antibody titers but increased the IgG1:IgG2c ratio in young male, aged male, and female mice, suggesting a shift toward a type II\u0026ndash;skewed humoral response\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Following intranasal infection with a 3LD₅₀ dose of H1N1, muscle injury during vaccination promoted regulatory T-cell accumulation in the lungs, accompanied by Ly6Cˡᵒ and Arginase I⁺ macrophages previously described as non-canonical or anti-inflammatory in infected lungs\u003csup\u003e\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. This response was more pronounced in females, highlighting a novel sex-dependent relationship between muscle injury during vaccination and a subsequent anti-inflammatory lung response. Treg density increases were accompanied by an increase in IL-33 levels in the infected lungs of young female mice, IL-33 to be a potential recruiter of Tregs into the lungs after infection similarly to the muscle post-injury\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. IL-33 has been shown to play a key role in repolarizing pro-inflammatory toward reparative responses through Treg recruitment following muscle injury, and its decline with age contributes to chronic inflammation and sarcopenia. We found that IL-33 supplementation broadly amplified immune populations on day 2 in draining lymph nodes of both sexes when co-administered with QIV and glycerol, thereby reducing sex-dependent variability. By day 4, IL-33 increased Treg and CX3CR1⁺Ly6Cˡᵒ macrophage accumulation in injured muscles of young male and female mice. At the humoral level, IL-33 supplementation elevated anti-QIV antibody titers, maintaining an IgG1:IgG2c balance both with and without muscle injury. The potential adjuvant effect derived from co-delivered IL-33 has already been described for mucosal vaccination strategies\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. IL-33\u0026ndash;treated mice also exhibited enhanced protection following infection. At the mucosal level, IL-33 supplementation with QIV alone increased eosinophil numbers, supporting a link between local IL-33 expression and type II skewing of the vaccine response.\u003c/p\u003e \u003cp\u003eIn aged mice, muscles were more sarcopenic and exhibited diminished reparative capacity compared to young mice. The reduced regenerative response was evident in both magnitude and quality, with CD8⁺ T cells\u0026mdash;rather than Tregs\u0026mdash;dominating day 4 immune profiles in injured muscle. IL-33 supplementation did not fully restore reparative responses in aged mice but rescued the Treg:CD8⁺ T-cell ratio to levels comparable to young animals. In draining lymph nodes, IL-33 enhanced macrophage and B-cell responses compared to vaccine-alone or vaccine-plus-glycerol groups. At the humoral level, aged mice exhibited reduced anti-QIV total IgG titers compared to young mice, and muscle injury during vaccination further decreased IgG2c titers. Importantly, IL-33 supplementation rescued total IgG titers in aged mice to levels comparable to those of young controls, indicating that IL-33 may improve antibody responses in geriatric vaccination.\u003c/p\u003e \u003cp\u003eWe also found that intramuscular vaccination alone elicits a type II immune response in the lungs after infection, and that increases in Tregs and M2-like macrophages did not diminish this type II signature, which included elevated IL-4, IL-5, IL-13, and eosinophil levels in vaccinated young mice. This is in line with previous reports from our laboratory\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e and reflects potentially vaccine-induced Th2 responses that are recalled in the lung upon virus infection. At the mucosal level, aged mice displayed a diminished type II immune response to QIV, marked by lower IL-4 levels post-infection, a defect reversed by IL-33 supplementation, which restored IL-4 concentrations and increased lung eosinophil densities. However, IL-33 was insufficient to restore reduced Treg counts in aged infected lungs.\u003c/p\u003e \u003cp\u003eIn conclusion, muscle injury during vaccination promotes an anti-inflammatory response to subsequent vaccine-matched infections, correlating with the magnitude of the muscle\u0026rsquo;s regenerative response. Female mice, which mount the strongest regenerative and inflammatory responses to muscle injury, developed the highest Treg accumulation in the lungs after respiratory infection with influenza virus, followed by males, whereas aged mice exhibited diminished regeneration and reduced Treg responses. IL-33, currently explored as a therapy for sarcopenia, showed beneficial effects on intramuscular vaccine responses by increasing antibody titers in aged mice and restoring type II immunity observed in young animals, thereby highlighting a link between vaccine response and muscle health. The findings from this work have implications for both vaccination strategies in different age groups, and provide an avenue for further investigation into the underlying mechanisms of action of vaccines and their adjuvants.\u003c/p\u003e \u003cp\u003eThis work provides a foundation for exploring the link between tissue repair mechanisms and vaccine efficacy. However, several questions remain. Our findings were generated primarily in C57BL/6J mice, and future studies should assess strain-dependent variability, such as comparisons with BALB/c mice, which display distinct immune polarization biases\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Additionally, while our data identify immune kinetics and key cell populations associated with IL-33\u0026ndash;enhanced vaccination, mechanistic studies are required to define the causal pathways connecting muscle injury to downstream anti-inflammatory responses, ideally using conditional knockout models.\u003c/p\u003e \u003cp\u003eNevertheless, these findings highlight the muscle and muscle repair pathways as a novel potential target for modulating peripheral immunity. For example, IL-33 supplementation in aged mice previously shown to have a positive reparative effect in aged muscle repair, can also rescue humoral responses and boost the type II immune response seen in younger mice. This emphasizes the importance of targeting the aging muscle environment as a strategy to enhance geriatric vaccine formulations.\u003c/p\u003e \u003cp\u003eAdditionally, and outside of the context of aging, we found that inducing muscle injury during vaccination enhances a long lived regulatory T-cell response that can be recalled at the mucosa. This novel observation can be harnessed for the induction of Tregs to counter lung fibrosis previously proven to be due to failure of Treg accumulation in the injured lungs\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. It can also be expanded beyond infectious diseases and investigated as a venue to enrich Tregs in the context of autoimmunity where immunogenic self antigens have been previously like the case of keratin in the context of psoriasis\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. We believe that this study is a promising introduction to ways muscle pathways can be incorporated into the field of vaccinology to develop personalized vaccines.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMice\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57BL6/J and BALB/cJ 4-8-week-old female and male mice were purchased from Jackson Laboratory. The aging mice were aged in house to 18-24 months old. All mice were housed in our animal facilities at Icahn School of Medicine at Mount Sinai. All experiments were performed under protocols approved by Icahn School of Medicine at Mount Sinai\u0026rsquo;s institutional Animal Care and Use Committee. Mice were vaccinated with 50 \u0026mu;L of the Fluzone High-Dose \u0026reg; (2023-2024) quadrivalent inactivated flu vaccine diluted 1:1 sterile PBS. With this concentration, each injection delivered 3 \u0026mu;g of each of the four HAs included in the formulation. Mice that received glycerol were injected with 50 \u0026mu;L of 50% Glycerol diluted in PBS, or with QIV. 0.3\u0026mu;g of recombinant mouse IL-33 (purchased from Biolegend, Cat. #580502) was supplemented into vaccine formulations. During injections, mice were anesthetized using 5% isoflurane induction through a precision vaporizer. For infections, mice were anesthetized with a ketamine/xylazine mixture (90-120/2-5 mg/kg), and then infected with 25\u0026mu;L of 3xLD50 containing 500 plaque forming units or PFUs of IVR-180 (A/Michigan/25/2015, H1N1) intranasally. At the endpoint, mice were euthanized using pentobarbital at a concentration of \u0026nbsp; \u0026gt;150 mg/Kg delivered into the peritoneal cavity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerum collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor antibody titers, blood was collected via the submandibular vein and allowed to coagulate overnight at 4 degrees celsius. After coagulation, the blood was spun down at 450g for 5 minutes, and serum was transferred to a new tube. The process was repeated to insure no cell contamination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD45\u003csup\u003e+\u003c/sup\u003e intravenous labeling\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were euthanized using pentobarbital at a concentration of \u0026nbsp;\u0026gt;150 mg/Kg delivered into the peritoneal cavity. Once unresponsive, but before the heart arrests, we injected the retro orbital sinus with 100 uL of 0.02 mg/mL of anti-CD45 PE (30F-11). After a 3-minute incubation, we harvested the tissue of interest. The tissue was then stained with CD45.2 Alexa Fluor 532\u0026trade; (104). A different clone of CD45 was used for tissue staining to limit epitope-specific antibody binding competition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLungs and muscles were processed using the same protocol. Lungs and muscles were harvested and placed in 2.5 mL of pre-chilled digestion buffer containing 2 mg/mL Gibco\u0026trade; Type II Collagenase, and 25 \u0026mu;g of DNase I (STEMCELL technologies, Cat: 07469) in gentleMACS\u0026trade; C Tubes (cat:130-093-237). Lungs and Muscles were sheared manually and then loaded onto the gentleMACS Octo Dissociator with heaters. To digest them, 37_m_LDK_1 program was used. The digested mixture was then blocked with pre-chilled 7.5 mL of PBS supplemented with 2 mM EDTA to halt enzyme activity, and strained through a 70\u0026mu;m ASI\u0026trade; Cell Strainer (Cat:TS70). Cells were then spun down at 450g for 5 minutes and resuspended in 200\u0026mu;L of staining buffer, and the flow cytometry protocol was then followed. Draining lymph nodes were digested by shearing through a 70\u0026mu;m filter and into a 6-well tissue culture plate containing 2.5 mL of DMEM. Cell mixture was then moved to a 15 mL falcon tube, and spun down at 450g for 5 minutes, and the staining protocol followed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor processed mouse tissue including lungs, muscles, and draining inguinal lymph nodes, two panels were used to characterize the myeloid and lymphoid compartments separately. A two-laser (B/V) northern lights spectral cytometer from Cytek was used to derive the counts. For the myeloid and lymphoid panels, surface staining was performed using a staining buffer consisting of 10% Fetal bovine serum, and 0.1% Sodium Aside in PBS. Cellular fixation and permeabilization was performed using BD Cytofix/Cytoperm\u0026trade; Fixation/Permeabilization Kit as per manufacturer\u0026rsquo;s instructions for the myeloid panel. For the lymphoid panel, FoxP3 intranuclear staining was performed using eBioscience\u0026trade; Foxp3 / Transcription Factor Staining Buffer Set as per manufacturer\u0026rsquo;s instructions. Surface staining and intracellular staining were performed for 20 minutes at room temperature in the dark and were followed by two washes. Surface staining washes were performed with the staining buffer. Intracellular washes were performed using kit-specific washes as per manufacturer instructions. The following antibodies were used for flow cytometric staining with anti-I-A/I-E (M5.114.15.2), anti-CD172a or anti-SIRP\u0026alpha; (P84), anti-F4/80 (BM8), anti-CD11b (M1/70), anti-Ly6C (HK1.4), anti-CD19 (6D5), anti-CD8a (53-6.7), anti-CD4 (RM4-5) all from Biolegend; \u0026nbsp;anti-Ly6G (1A8-Ly6g), anti-Arginase I (A1exF5), anti-CD3 (17A2), anti-CD25 (PC61.5), anti-CD45.2 (104), anti-CD11c (N418), anti-iNOS (CXNFT), anti-FoxP3 (FJK-16s) all from eBioscience\u003csup\u003eTM\u003c/sup\u003e; anti-Siglec-F (E50-2440), anti-CX3CR1 (Z8-50) all from BD; anti-CD45 (30F-11) from Cytek. All antibodies were tittered and diluted to working concentration in the staining buffer. Viability was measured using fixable viability dye 520 from eBioscience diluted according to manufacturer instructions. Sample blocking was performed prior to surface and intracellular staining for 10 minutes using Purified Rat Anti-Mouse CD16/CD32 from BD Pharmingen\u003csup\u003eTM\u003c/sup\u003e. Gating strategy examples per panel and organ are provided in supplementary figures 1, 2, 5, 6, 17 and 18. Reported immune cells counts were normalized in the muscle per gram of muscle tissue to account for harvesting variability. For C2C12 cells, viability was measured using fixable viability dye 780 from eBioscience diluted according to manufacturer instructions. Mean counts are provided with standard error of mean.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulturing C2C12 myotubes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC2C12 mouse myoblasts were obtained from the Yizhou Lab at Icahn School of Medicine at Mount Sinai.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eC2C12 cells were cultured in T75 flasks in 14 mL of Corning\u0026trade; DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate supplemented with 10% HyClone\u0026trade; Standard Fetal Bovine Serum (FBS) and 1% Cytiva HyClone\u0026trade; Penicillin Streptomycin 100X Solution (growth media). Once 70% confluent, cells were detached using 4 mL Corning\u0026trade; 0.05% Trypsin/0.53mM EDTA in HBSS w/o Calcium, Magnesium or Sodium Bicarbonate blocked with 10 mL of growth media in a Falcon\u0026trade; 15 mL Conical Centrifuge Tube. Cells were then plated at 300,000 cells per T75 flask in 14 mL of growth medium. Once all plates reached 70% confluence, cells were detached and plated in 24-well cell culture plates with 300,000 cells per well, and allowed to grow to 100% confluence in the growth medium. Once confluent, the medium was swapped with 2 mL per well of serum-free Corning\u0026trade; DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate media (differentiation media). Differentiation media was refreshed every day for 7 days. At day 7, media was replaced with stimulation conditions which were all diluted in differentiation medium and incubated for 24 hours. Cells were then detached with trypsin and stained for flow cytometry.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVaccine specific antibody titers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe coating buffer consisted of \u0026nbsp;32 mM Na2CO3 and 64 mM NaHCO3. Fluzone High-Dose \u0026reg; (2023-2024) was diluted 1:250 in the coating buffer and 100\u0026mu;L of coating mixture was pipetted into each well of a MaxiSorp flat-bottom 96-well plate from Thermofisher Scientific\u003csup\u003eTM\u003c/sup\u003e (Cat: 456537). Plates were incubated overnight at 4 degrees celsius. The next day, PBS-T solution was made from PBS-TWEEN\u003csup\u003e\u0026reg;\u003c/sup\u003e Tablets from Millipore Sigma as per manufacturer instructions, and plates were washed twice with 200\u0026mu;L of PBS-T per well. Plates were then blocked with 200 \u0026mu;L of PBS-T supplemented with 5% MP Biomedicals\u0026trade; Skim Milk Powder (Cat: MP290288705) for 1 hour at room temperature. During the blocking, sera was serially-diluted in blocking solution to create an 8-step 4-fold dilution started with a 1:100 initial dilution. After blocking, the plates were washed twice with 200\u0026mu;L of PBS-T per well. 100\u0026mu;L of sera was then added per well and incubated for 1 hour at room temperature. After incubation, the plates were washed twice with \u0026nbsp;200\u0026mu;L of PBS-T per well. Based on the antibody subtype, different detection buffers were made. For total IgG detection, goat F(ab) anti-mouse IgG H\u0026amp;L (HRP) (ab6823) was used from Abcam. For IgG1 detection, we used goat anti-mouse IgG2c-HRP (1078-05) from SouthernBiotech as per manufacturer instructions in blocking solution. 100\u0026mu;L of detection solution was added per well and incubated for 1 hour. Plates were then washed twice with 200\u0026mu;L of PBS-T per well. Plates were then developed using 1-Step\u0026trade; Turbo TMB-ELISA Substrate Solution, and ELISA Stop Solution from Thermofisher as per provider instructions. Plates were then read using a Biotek plate reader using 450 nm (signal) and 650 nm (subtracted background) wavelengths absorbance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMultiplex cytokine assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLeft lungs were harvested for cytokine quantification. They were moved into homogenizing tubes containing 500\u0026mu;L of sterile PBS solution containing \u0026nbsp;3 mm triple-pure high impact zirconium beads from Benchmark Scientific. They were flash frozen by placing them on dry ice and stored at -80\u0026deg;C until the assay time. On assay day, they were thawed on ice, and then loaded onto a homogenizer and run at 6 MP/S, MP 24X2 for 30 seconds. Tubes were then spun down at 450g for 10 minutes and supernatant was transferred to new 1.5 mL eppendorf tubes. The multiplex cytokines assay chosen was a ProcartaPlex\u0026trade; Mouse Th1/Th2 Cytokine Panel, 11plex as per manufacturer instructions. Data were acquired on a Luminex 100/200 analyzer (Millipore) with xPONENT software (version 4.3). Data visualization and analysis were conducted using GraphPad Prism (version 9.4.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLung viral titers quantification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLung viral titers were specified using the same left lung lobes, and experiments were performed on the same day as that of the multiplex cytokine assay. Plaque assays were performed to quantify viral titers. To do so, a confluent monolayer of MDCK cells were incubated with 250\u0026mu;L of 10-fold dilutions of sample in PBS at 37\u0026deg;C. After a 1 hour incubation, the inoculum was aspirated and replaced by an overlay with 2% oxoid agar (Oxoid, Basingstoke, UL) mixed with an equal volume of NaHCO3-buffered 2xMEM supplemented with DEAE/Dextran and TPCK-treated trypsine at a concentration of 1\u0026mu;g/mL. Cells were incubated in this overlay for 48 hours in a CO2 cell incubator at 37\u0026deg;C. To quantify plaques, cell surfaces were fixed using a 4% formaldehyde solution for 5 minutes at room temperature. Cells were then stained with a 0.001x diluted post-challenge mouse serum matching viral infection (IVR-180, H1N1) followed by 0.001x diluted anti-mouse IgG sheep serum conjugated to horseradish peroxidase (GE Healthcare) and addition of TrueBlue substrate (KPL- Seracare, Milford, MA, USA)\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analysis was done on GraphPad Prism software. The data is presented as mean \u0026plusmn; SEM provided in the text unless stated otherwise. Statistical significance was computed through one-way ANOVA or non-parametric Mann-Whitney tests. To compare experimental groups, Kruskall-Wallis test was performed followed by Dunn\u0026apos;s multiple comparison test to derive significance. P \u0026lt; 0.05 was considered significant.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFCS files, ELISA absorbance measurements, multiplex cytokine assay tables, and mice weight tables are available for immediate release upon request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch in the M.S. laboratory is funded by NIH/NIAID grant R01AI160706, and partly funded by CRIPT (Center for Research on Influenza Pathogenesis and Transmission), a NIH NIAID-funded Center of Excellence for Influenza Research and Response (CEIRR, contract number 75N93021C00014). J.V. is supported by a fellowship from the Belgian American Educational Foundation (BAEF).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.N. conceived and designed the study, performed the experiments, analyzed the data, and wrote the manuscript. E.B., J.V., F.A., L.A.C., G.L., E.M.T., S.P., V.Y., L.G., G.J., performed experiments. P.W. secured funding, and M.S. secured funding, provided overall project direction, and edited the manuscript with input from all authors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe M.S. laboratory has received unrelated funding support in sponsored research agreements from Phio Pharmaceuticals, 7Hills Pharma, ArgenX NV, Ziphius and Moderna. The other authors declare they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col start=\"1\" type=\"1\"\u003e\n\u003cli\u003eGayraud-Morel B, Chr\u0026eacute;tien F, Tajbakhsh S. Skeletal muscle as a paradigm for regenerative biology and medicine. \u003cem\u003eRegenerative Medicine.\u003c/em\u003e 2009;4(2):293-319. doi:10.2217/17460751.4.2.293\u003c/li\u003e\n\u003cli\u003eTurner NJ, Badylak SF. Regeneration of skeletal muscle. \u003cem\u003eCell Tissue Res.\u003c/em\u003e 2011;347(3):759-774. doi:10.1007/s00441-011-1185-7\u003c/li\u003e\n\u003cli\u003eYusuf F, Brand-Saberi B. Myogenesis and muscle regeneration. \u003cem\u003eHistochem Cell Biol.\u003c/em\u003e 2012;138(2):187-199. doi:10.1007/s00418-012-0972-x\u003c/li\u003e\n\u003cli\u003eTidball JG. 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(2023). \u003cem\u003eAutoreactive T-cells in psoriasis: Are they spoiled Tregs and can therapies restore their functions?\u003c/em\u003e \u003cem\u003eInternational Journal of Molecular Sciences, 24\u003c/em\u003e(5), 4348. https://doi.org/10.3390/ijms24054348\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Vaccination, Muscle regeneration, Aging, Influenza, Macrophages, IL-33, Tregs, Type II immunity, Sex","lastPublishedDoi":"10.21203/rs.3.rs-8544011/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8544011/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe muscle environment during intramuscular vaccination has the potential to shape downstream immune responses, yet this relationship remains poorly defined. We hypothesize that muscle condition at the time of vaccination matters for the outcome of vaccination. In this study, we examined how glycerol-induced muscle injury at the time of quadrivalent inactivated influenza vaccine (QIV) administration can skew host immune responses to vaccination and modulate systemic and mucosal immunity following subsequent H1N1 challenge three weeks later. Given that seasonal influenza disproportionately affects older adults, and that muscle regenerative capacity is both sex- and age-dependent, we addressed sex and age as biological variables. We found that muscle injury during QIV vaccination promoted regulatory T cell and M2 macrophage levels in the lungs upon subsequent H1N1 infection three weeks later, with responses more pronounced in young females and diminished with age in both sexes. Muscle injury also shifted antibody isotype distribution away from IgG2c toward IgG1 in young male and aged mice, without altering total hemagglutinin-specific antibody titers. In young influenza virus-infected mice, prior QIV vaccination resulted in a type 2\u0026ndash;skewed mucosal immune response, marked by IL-4, IL-5, IL-13, and eosinophilia, whereas this response was attenuated in aged cohorts. Finally, because IL-33 is a pro-regenerative alarmin reduced in aging muscle, we tested the impact of IL-33 supplementation. IL-33 enhanced vaccine-specific antibody production across age groups and restored type 2 mucosal immunity in aged mice, thereby partially rescuing the age-associated impairment of the host response to infection, although it did not enhance glycerol damage-dependent regulatory T-cell influx into the lungs after infection as observed in young mice. Our data show that muscle health at the time of vaccination can impact host vaccine responses that shape the lung immune environment during subsequent respiratory infection and highlight IL-33 as a target for vaccine adjuvants.\u003c/p\u003e","manuscriptTitle":"Muscle Injury and Aging Differentially Shape Immune Responses to Influenza Vaccination","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-25 10:13:01","doi":"10.21203/rs.3.rs-8544011/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6098146c-b7db-4473-ada9-21fb6023f715","owner":[],"postedDate":"February 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":62416722,"name":"Biological sciences/Immunology/Vaccines"},{"id":62416723,"name":"Biological sciences/Immunology/Adaptive immunity/Cellular immunity"},{"id":62416724,"name":"Biological sciences/Microbiology/Virology/Influenza virus"}],"tags":[],"updatedAt":"2026-04-23T09:01:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-25 10:13:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8544011","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8544011","identity":"rs-8544011","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}