Lung environment in healthy old age shapes the phenotype and CCR2-mediated recruitment of a subset of apoptotic, high-turnover alveolar macrophages

Lung environment in healthy old age shapes the phenotype and CCR2-mediated recruitment of a subset of apoptotic, high-turnover alveolar macrophages | 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 Lung environment in healthy old age shapes the phenotype and CCR2-mediated recruitment of a subset of apoptotic, high-turnover alveolar macrophages Larry Schlesinger, Susanta Pahari, Miranda Lumbreras, Arkajyoti Paul, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7123735/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Immune system changes with age lead to chronic systemic inflammation termed "inflammaging", contributing to age-related pathologies. Alveolar macrophages (AMs) maintain lung homeostasis and health. The impact of inflammaging on AM populations requires further definition. Herein, we examined the effect of age on the phenotype and ontogeny of AMs from mice, non-human primates and humans. We identify three AM subpopulations in old age, two of which increase more than 10-fold, leading to significant functional consequences associated with heightened inflammation and immune dysregulation. RNA-seq analysis identifies unique transcriptional AM subpopulation profiles. Adoptive transfer experiments reveal the importance of the alveolar environment in AM recruitment and phenotypic change in old age. Monocyte-derived AM recruitment in old age requires CCR2 and leads to relatively short-lived AMs with high turnover due to Fas-mediated apoptosis. These studies provide new insight on the impact of the alveolar environment in healthy old age on AM phenotype and function. Biological sciences/Immunology/Inflammation/Chronic inflammation Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages/Alveolar macrophages Biological sciences/Cell biology/Cell death/Apoptosis alveolar macrophages inflammaging CCR2 Fas apoptosis adoptive transfer mice human non-human primates Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights The alveolar microenvironment’s unique inflammatory profile impacts alveolar macrophage recruitment and phenotype There is a significant increase in two recruited alveolar macrophage subpopulations in old age observed across mammalian species Recruitment of monocyte-derived CD11c + CD11b + alveolar macrophages (MoAMs) in old age is CCR2-dependent Recruited MoAMs in old age are relatively short-lived and undergo Fas-mediated apoptosis Introduction By 2030, one in six people globally will be 60 or older, totaling 1.4 billion. By 2050, this group will double to 2.1 billion, and the number of individuals aged 80 and older will triple 1 . Old individuals are more susceptible to severe illnesses and increased mortality due to various diseases 2 – 5 . Aging is associated with a decline in immune function, a phenomenon known as immunosenescence, which has been studied primarily within the adaptive immune system 6 , 7 . In contrast, "inflammaging," or chronic inflammation linked to innate immune system dysfunction with age, is less well understood, especially in the lung alveoli 2 , 8 . Aging lungs are influenced by various biological pathways due to environmental exposures, leading to oxidative stress, inflammation, telomere shortening, DNA damage, mitochondrial dysfunction, epigenetic instability, immune dysregulation, and impaired proteostasis 9 . Oxidative stress and mitochondrial dysfunction in old age, even in the absence of disease, collectively contribute to an inflammatory alveolar environment significantly affecting the phenotype and recruitment of alveolar macrophages (AMs). AMs, the first immune cells to interact with airborne pathogens and inhaled particulates, are essential for lung health. Despite this knowledge, little is known about the characteristics of AMs recruited to the lung in old age. AM development and function is influenced by signals from the lung microenvironment. Shortly after birth, fetal monocytes quickly transform into a stable, self-renewing population called tissue-resident AMs (TRAMs). In mice, TRAMs persist for extended periods without the need for input from bone marrow-derived monocytes 10 – 12 . TRAMs are located within epithelial surface lining fluid, where they attach to the lung epithelium via integrins and play a crucial role in sampling, responding to, and eliminating pathogens and particulates in the alveoli 13 , 14 . With TRAM depletion or inflammatory conditions, monocytes are recruited to the alveoli, where they undergo differentiation into monocyte-derived alveolar macrophages (MoAMs) through a progressive reshaping of their epigenome and transcriptome 15 – 17 . We recently identified distinct AM subpopulations in old mice that are significantly increased relative to young mice 2 . AMs in young mice consist mainly of CD11c + CD11b − TRAMs 2 , 18 whereas in old mice, there are marked increases in subset populations that are CD11c + CD11b + and CD11c − CD11b + . The importance of these increased subpopulations relates to their unique inflammatory profile and inability to control Mycobacterium tuberculosis growth, suggesting a potential sentinel population for tuberculosis susceptibility in old age 2 . The appearance of these AM subpopulations in old age result from 3 potential mechanisms: i) cell-autonomous changes, ii) alterations in the lung microenvironment, and/or iii) the replacement of TRAMs with MoAMs due to repeated insults over the lifespan. Herein we investigate these mechanisms in AMs from old mice (mAMs), humans (hAMs) and non-human primates (NHPs; baboons, bAMs; Rhesus macaque, rAMs) using several approaches. Flow cytometry and fluorescence microscopy indicate similar AM phenotype changes with old age across species, i.e. , increased CD11c + CD11b + and CD11c − CD11b + AM subpopulations. RNA-seq and qRT-PCR analyses reveal unique transcriptional profiles of these subpopulations, providing insight into their unique functions. Our experiments in mice demonstrate the importance of the altered alveolar environment in old age in AM recruitment and phenotypic change, including adoptive transfer experiments in mice to confirm monocyte origin. Additionally, we found that recruitment of MoAMs to the alveolar space requires the chemokine CCR2. The recruited MoAMs are relatively short-lived, undergoing increased apoptosis through Fas activation. This work enhances our understanding of the cellular and molecular processes associated with old age, paving the way for novel approaches to interventions that are more effective in this unique tissue environment for elderly patients with lung diseases. Results Characterization of old/elderly AMs from mice (mAMs), humans (hAMs) and non-human primates (NHPs; baboons: bAMs and rhesus macaque: rAMs ): increased CD11c + CD11b + subpopulations To assess AM subpopulations across the species in mice, humans and NHPs, we performed bronchoalveolar lavage (BAL) to obtain their AMs ( Figure 1 ). We focused on samples from young and old mice (3 months and 18 months old, respectively), healthy adults versus healthy elderly humans (ages 20-50 and ≥ 65 years old), and young and old baboons/rhesus (3-5 years and over 15 years old). We identified the presence of distinct cell populations of AMs, lymphocytes, neutrophils, and eosinophils in the BAL of old/elderly subjects compared to young/adults on cytospun samples. AMs were the most prominent population in both groups ( Figure S1 ). mAM, hAM and bAM subgroups were distinguished by their respective cell surface receptor expression using flow cytometry: mAMs were CD45 + SiglecF + CD11c + , hAMs were CD206 + CD64 + CD11c + , and bAMs were CD45 + CD64 + CD206 + CD163 + CD11c + 19-22 . To identify CD11c + CD11b + AM subpopulations, cells were pre-gated on CD45 + SiglecF + (mAMs) and CD45 + CD64 + (hAMs, bAMs) populations ( Figure 1 & Figure S2, 3, 4 ). TRAMs were the predominant population of cells that express CD11c cell surface receptors. We previously identified an increased subpopulation of CD11c + CD11b + monocyte-derived AMs (MoAM) and a very small subpopulation of CD11c - CD11b + cells in old mice 2 . CD11c + CD11b + MoAMs exhibit a unique inflammatory signature based on defined markers and enhanced growth of Mycobacterium tuberculosis 2 . Our current results confirm these increased subpopulations in young and old mice ( Figure 1a, b ). Our findings in hAMs/bAMs revealed three distinct markedly increased AM subpopulations in the old/elderly groups ( Figure 1c-f ). The most prominent subpopulation was designated as CD11c + CD11b - TRAMs. The 2 smaller subpopulations were CD11c + CD11b + and CD11c - CD11b + . Notably, the presence of these two latter subpopulations increased more than ten-fold in old baboons and elderly humans when compared to their younger counterparts ( Figure 1c-f ). Further analysis indicated that the expression levels of CD11b, CD11c, CCR2, Fas, HLA-DR, CD14, and CD16 were elevated in the CD11c + CD11b + elderly hAM subpopulation ( Figure 1g ). There was no significant change in CX3CR1 expression in the CD11c + CD11b + subpopulation of elderly hAMs ( Figure 1g ). CD14 + CD16 + cells are recognized as inflammatory monocytes 23 . We hypothesize that these inflammatory monocytes are recruited to the lungs and in situ differentiated into the CD11c + CD11b + MoAM subpopulation in elderly individuals. Flow cytometry analysis of bAMs revealed that CD11b, CD86, HLA-DR, CD206, CD163, CD64, and CD36 were upregulated in old age when compared to bAMs from young baboons ( Figure S4 ). Like hAMs, the chemokine receptor CCR2, but not CX3CR1, was upregulated in old age bAMs when compared to young bAMs ( Figure S4; Figure S5 ). Further analysis indicated that the expression levels of CD64, CD11c, CD11b, HLA-DR, CD163, and CCR2 were elevated in the CD11c + CD11b + elderly bAM subpopulation ( Figure S5 ). We also examined the expression levels of AM surface proteins in rhesus macaques. Confocal microscopy showed increased expression of CD163, CD206, CD11b and CD11c in old age rAMs ( Figure S6 ). Our previous research found that CD11c + CD11b + mAMs from old mice exhibit elevated levels of cell surface receptors Ly6C and CD115, consistent with a monocytic origin 2 . We extended this analysis in mice to examine at what age changes in cell surface receptor expression occur. As age progresses, we observed a gradual increase in CD11c + CD11b + recruited AMs with CD95 (Fas) expression ( Figure 1h ). Overall, these findings demonstrate significant changes in AM surface receptor expression in healthy old age and the finding of increased subpopulations is consistent across mice, humans and NHPs. Additionally, the findings suggest that the newly recruited AM subpopulations in old age differentiate from blood monocytes and that the chemokine CCR2 plays an important role in this recruitment. Unique transcriptional profiles of the 3 mAM subpopulations in old mice To further characterize the AM subpopulations in mice, we conducted a transcriptomic analysis using bulk-RNA sequencing (RNA-seq) on four flow cytometry-sorted populations: TRAMs in young mice (CD11c + CD11b - ), TRAMs in old mice (CD11c + CD11b - ), the increased subpopulation of CD11c - CD11b + mAMs in old mice, and the increased subpopulation of CD11c + CD11b + mAMs in old mice ( Figure 2a ; Figure S7a ). CD11c - CD11b + mAMs in old mice were present in very low numbers and exhibited a transcriptional profile like that of the CD11c + CD11b + populations. Thus, we did not include them in our further analyses. Our primary focus was on comparing three subpopulations of mAMs (CD11c + CD11b - mAMs in young and old mice (TRAMs) and CD11c + CD11b + mAMs in old mice). Principal component analysis (PCA) indicated a high degree of similarity among biological replicates within each group ( Figure 2b ). Volcano plots illustrated the false discovery rate (FDR) in relation to the magnitude of change in gene expression across a total of 14,509 genes. 577 out of 5,300 genes (FDR 1, in the CD11c + CD11b + mAMs ( Table S1a ). Moreover, 10 genes were downregulated (logFC < -1) when compared to TRAMs (CD11c + CD11b - ) in old mice ( Figure 2c and Table S1b ). We also compared TRAM subpopulations in young and old mice. 311 out of 4,796 genes (FDR 1) in old TRAMs ( Table S2a ). Moreover, 36 genes were downregulated (logFC < -1, FDR < 0.05) in old mice when compared to TRAMs in young mice ( Figure S7b). Furthermore, we compared the three subpopulations of mAMs in both young and old mice. TNF signaling-related genes, including inflammatory genes, e.g., Ccl3 , Ccl8 , Csf1 , Cd40 , Tnf , Ccl22 , Ccl2 , Ccl5 , Tlr1 , Tlr9 , S100a8 , and S100a9 were highly upregulated in CD11c + CD11b + mAMs in old mice ( Figure 2d, e ). Other immune response-related genes, including those involved in positive regulation of the ERK1/2 pathway, cell migration and chemotaxis were also upregulated in the old mice ( Figure S8a-c ). Notably, Hif1a was upregulated in CD11c + CD11b + mAMs which suggests induction of glycolysis in this AM subpopulation. Inflammatory genes were observed in CD11c + CD11b + mAMs ( Figure 2f ), highlighting a unique inflammatory profile consistent with a previous gene expression study 2 . Quantitative PCR analysis in hAMs confirmed significantly higher levels of inflammatory, immunoregulatory, and cell death markers in elderly compared to adult hAMs ( Figure S9 ). Metabolic and oxidative changes of AMs in old age mice RNAseq results suggested major changes in metabolism and oxidative stress in AMs in old age. HIF-1α regulates glucose metabolism to promote anaerobic glycolysis and conserve ATP 24 . Healthy adult AMs are classically driven primarily through oxidative phosphorylation (OXPHOS) 25 , but there is evidence for a metabolic shift in AMs from OXPHOS to glycolysis in old age 26 . We posited that increased HIF-1α expression in old AMs will lead to increased glycolysis and decreased OXPHOS, and that the complex inflammatory and oxidative alveolar environment 27 in old age changes AM phenotype. We measured glycolysis and OXPHOS by quantifying the ECAR and OCR in old versus young mAMs. Our findings revealed a significant increase in both the basal and maximal respiratory capacity and ATP production, and spare respiratory capacity, along with a moderate increase in proton leak in mAMs from old mice compared to those from young mice ( Figure S10a-f ). Additionally, glycolysis (ECAR) in the mAMs from old mice was significantly higher than that in young mice ( Figure S10 g-j) . The basal mRNA levels of key glycolytic genes ( Glut1, Hk2, Gls1, Pfkfb3 ) were also elevated in mAMs from old versus young mice ( Figure S10j-m ). Previous data indicated a significant increase in TLR2 expression (both RNA and protein levels) in AMs from old mice, with higher levels observed specifically in the CD11c + CD11b + AM subset 2 . Similarly, we found that TLR2 expression was elevated in hAMs from elderly individuals ( Figure S9d ). To investigate the TLR2 response in mAMs from old mice, we stimulated these cells in vitro with the TLR2 agonist Pam 3 CSK 4 . Following stimulation, we observed elevated RNA expression levels of aerobic metabolism-related genes, including Hif1α, Cox2, CD74, Mif, Glut1, Hk2, Pfkb3, Gls1, Slc25a, and Acly ( Figure S11 ). In old mAMs, there was an increase in both OXPHOS (OCR) and glycolysis (ECAR), attributed to heightened glucose utilization, mitochondrial respiration, and ATP production. However, this pattern was not observed in hAMs, where the glycolysis-related gene (LDHA) was elevated, and the OXPHOS-related gene (NDUFAF6) was decreased in old age ( Figure S12 ). One possible explanation for these differences is that the metabolic pathways (OXPHOS versus glycolysis) in mice operate independently. Another possibility is that old mAMs have high glucose availability to drive glycolysis, while simultaneously having low ATP levels, which activates the citric acid cycle to produce significantly more ATP through OXPHOS 25 . Finally, we observed a significant increase in mitochondrial ROS in old mAMs as compared to young mAMs ( Figure S13a-b ). The inflammatory alveolar environment in old age shapes the AM phenotype AMs within the alveolar environment are bathed in an aqueous alveolar hypophase, recycle surfactant, and contact Type I and type II epithelial cells 28 . Delicate alveolar air sacs enable gas exchange. Such an environment requires a highly regulated inflammatory response to enable normal lung function. Recent studies in old age have shown significant changes to protein production and function in the alveolar hypophase, e.g., pro-inflammatory cytokines, altered surfactant proteins and lipids, and modifications to complement components. These changes contribute to an environment with heightened oxidative stress, leading to an enhanced lung inflammatory state 3,29 . We next explored the role of this altered alveolar environment in old age in the phenotype and recruitment of the AM subpopulations. As a first approach, we depleted TRAMs from both young and old mice by administering two doses of clodronate liposomes (clodrolip) intratracheally (i.t.) on Day 0 and Day 3 to induce apoptotic cell death ( Figure 3a-b ) 30,31 . We achieved ˃ 85-90% AM depletion by Day 5 to 8 by Cytospin ( Figure 3b; Figure S14 ) and flow cytometry ( Figure 3c-d ). On Day 5, we performed adoptive transfers of CD11c + CD11b - BAL isolated TRAMs from young mice by introducing them i.t. (1x10 6 cells) into both young and old mice ( Figure 3a ) to assess the effect of the lung environment on the phenotypic changes of recruited AMs in old mice. The adoptive transferred TRAMs from young mice more effectively converted to a CD11c + CD11b + mAMs in old mice after 48h ( Figure 3e ). Thus, we conclude that the phenotype changes of newly transferred TRAMs are due to the inflammatory alveolar milieu in old age. In concert with this, we observed elevated levels of soluble TNF, FasL (CD178), and S100A9 proteins in the isolated BAL fluid from old mice ( Figure 3f ). We next employed adoptive transfer using CD45.1 congenic mice ( Figure 4a ). CD45.1 bone marrow monocytes were isolated from young mice and transferred to young and old CD45.2 mice via tail vein injection. After 5 days, BAL was performed, and the recovered CD45.1 mAMs were labeled for flow cytometry analysis which revealed that they effectively migrated into the alveolar spaces of old mice, but much less so in young mice ( Figure 4b ). Finally, we employed the Ms4a3 Cre -Rosa TdT fate mapper mouse model to assess the origin of recruited MoAMs ( Figure 4a ). The Ms4a3 gene is specifically expressed in monocyte-committed progenitors 32 , and in this model, the bone marrow cells are labeled with an irreversible tdTomato red fluorescent marker, which identifies macrophage progenitor cells while excluding dendritic cell progenitors 32 . We adoptively transferred BM monocytes from the Ms4a3 Cre -Rosa TdT mice into both young and old mice via tail vein injection ( Figure 4a ). In young mice, we observed the presence of Ms4a3 Cre -Rosa TdT monocytes in the peripheral circulation, but these cells did not reach the lungs. In contrast, old mice exhibited fewer monocytes in circulation, suggesting that these cells likely migrated to the lungs and other organs ( Figure S15 ). Flow cytometry, confocal microscopy, and histopathology analyses revealed a higher recruitment of Ms4a3-derived MoAMs in old mice into the alveolar space ( Figure 4c-e; Figure S15 ). Furthermore, the Siglec F+Ms4A3 double-positive AM population corresponded to the CD11b + CD11c + mAM subpopulation ( Figure 4f ). Additional flow cytometry data corroborated that after the adoptive transfer of Ms4a3 Cre -Rosa TdT monocytes, old mice showed increased recruitment of both the Ms4a3 Cre -Rosa TdT and CD11c + CD11b + AM subpopulations ( Figure 4g-h ). RNA sequencing analysis revealed that several Ms4a-related gene sets (Ms4a4c, Ms4a4a, Ms4a6b, Ms4a7, and Ms4a14) 33 are upregulated in CD11c + CD11b + MoAMs from old mice compared to the TRAMs in both young and old mice ( Figure 2d, 4i ). These data correlate with the evidence that the MoAMs in old mice originate from bone marrow monocytes (CD11b + , Ly6c + , CD11c - ), which subsequently differentiate into CD11c + CD11b + MoAMs within the lung environment. Overall, the findings demonstrate the importance of the altered alveolar environment in old age in recruiting new AM subpopulations derived from peripheral monocytes. Recruitment of MoAMs in old age is dependent on a CCR2-mediated pathway To investigate the mechanism(s) involved in the recruitment of MoAMs to the lungs in old age, we focused on C-C chemokine receptor type 2 (CCR2), which in adult mice is essential for activating and recruiting monocytes and macrophages to kidney, liver, myocardium and skin injury tissue sites, contributing to tissue inflammation 34,35 . TRAMs are known to lack CCR2 expression 36 . In contrast, our results show that the CD11c + CD11b + MoAMs express CCR2 on their surface in the alveolus, which increases with age ( Figure 5a ), a finding also shown on hAMs ( Figure 1g ) and bAMs ( Figure S4b; S5b ). There was no significant change in surface expression of CX3CR1 with age in CD11c + CD11b + MoAMs, although gene expression increased in hAM ( Figure 1g; Figure 5b; Figure S4b; Figure S9k ). RNA-seq data also demonstrated significant upregulation of both Ccr2 and Ccl2 genes in CD11c + CD11b + MoAMs ( Figure 2f; 5 c, d ) and increased CCR2 expression in old hAMs ( Figure S9l ). We conducted adoptive transfers of BM monocytes from CCR2 knockout-GFP knockin mice via tail vein injections into both young and old recipient mice ( Figure 5e ). The majority of these CCR2-KO-GFP bone marrow monocytes remained in the bloodstream ( Figure 5f, g ) and did not reach the alveolar space in young or old mice, indicating an essential role of CCR2 in facilitating this process in old age ( Figure 5h ). Recruited MoAMs are relatively short-lived when compared to resident AMs To investigate the turnover of recruited MoAMs, we adoptively transferred CD45.1 young monocytes into both young and old mice via tail vein injection. At specified time points (Days 1, 3, 5, 7, or 10), we isolated and analyzed the mAM subpopulations in the alveolar space, identified by the markers SiglecF + CD45.2 for TRAMs and CD45.1 for recruited cells ( Figure 6a ). As shown earlier ( Figure 4b ), CD45.1 monocytes actively migrated into the alveoli and differentiated into MoAMs in old age. Peak migration occurs on Day 5, and following this peak, we noted a gradual reduction in the number of recruited MoAMs over time ( Figure 6 b, c ). In contrast, the population of TRAMs remained stable throughout the observed period ( Figure 6d, e ). Based on these observations and induction of Fas expression on MoAMs ( Figures 1g, h; S9h ), we hypothesized that recruited MoAMs have a transient existence characterized by rapid turnover, primarily driven by Fas-mediated apoptosis and continuous turnover of CD11c + CD11b + MoAMs. Moreover, RNA-seq analysis of flow cytometry sorted CD11c + CD11b + MoAMs revealed a significant increase in Mmp2 (matrix metallopeptidase-2) and Mmp14 ( Figure 6f, g ), suggesting their involvement in cell migration and turnover within the alveoli. During cell migration, MMP-2 breaks down extracellular matrix components to enable movement, while MMP-14 activates MMP-2 at the cell surface, initiating the process 37 . RNA-seq data also showed upregulation of Mmp9 gene expression in CD11c + CD11b + MoAMs ( Figure 2d ). Mmp9 cleaves the extracellular region of FasL (CD178), leading to the release of a soluble form of FasL (sFasL) 38 . Overall, our findings support the notion of a dynamic balance between resident and recruited AM populations in old age, where newly recruited MoAMs exhibit high turnover due to the inflammatory environment they encounter. Role of Fas in monocyte recruitment to the lungs and controlling the inflammatory environment of the alveolus in old age Our results indicate a significant increase in Fas receptor (CD95) expression in old mice ( Figure 1h ; Figure 7 a, b ) associated with turnover of recruited MoAMs. Fas signaling can induce the production of pro-inflammatory cytokines 39 , including those from macrophages, and higher Fas expression in the elderly correlates with an increase in inflammatory cytokines 39 . Chronic inflammation driven by Fas in older adults, particularly observed for alveolar epithelial cells, is associated with age-related diseases 40-42 . The role of resident and newly immigrated AMs in Fas activation has not been explored. We first hypothesized that enhanced Fas-mediated signaling of resident AMs in old age creates a more favorable inflammatory environment for the recruitment of monocytes to the lungs, facilitating their differentiation into MoAMs. To test this, we combined Fas Ligand (FasL, CD178) neutralizing (NA/LE) Ab administered i.t. with the adoptive transfer of CD45.1 monocytes by i.v. on Day 0 in young and old mice ( Figure 7c ). We observed that FasL neutralization effectively reduced Fas expression and the recruitment of CD45.1 MoAMs in old mice ( Figure 7d-e ). TNF levels were increased significantly in the isolated day 5 BAL fluid of old mice and administration of FasL Ab administered on day 0 effectively reduced TNF levels in these old mice ( Figure 7f ). These studies suggested that Fas neutralization can reverse the inflammatory alveolar environment of old mice to more closely resemble the environment of young mice leading to reduced MoAM recruitment. We next hypothesized that the turnover of recruited MoAMs in old mice can be slowed by the addition of FasL neutralizing Ab. To test this, we administered FasL Ab 5 days after the adoptive transfer of CD45.1 monocytes via i.v. [day in which recruited MoAMs is maximal ( Figure 6b-c ; Figure 7g )]. With Ab treatment, the recruited MoAMs are maintained for a longer period relative to those in the isotype control group ( Figure 7h ). To further understand how Fas regulates the lung microenvironment, we first depleted TRAMs by Clodrolip treatment and then added FasL neutralizing Ab along with adoptively transferring TRAMs from young mice to young and old mice via i.t., then assessed the phenotype of donor AMs ( Figure 7i ). Ab treatment had no effect on the newly added TRAMs in young recipient mice. In contrast, Ab treatment nearly abolished the increased CD11c + CD11b + AM population in the control group in old mice ( Figure 7j ). The role of FasL NA/LE Ab treatment in controlling AM apoptosis in old age Our results on Fas/FasL expression and AM turnover indicated that one mechanism for the observed turnover is FasL-mediated induction of apoptosis 43,44 . The use of FasL NA/LE Ab may improve macrophage turnover and functionality by preventing premature apoptosis 12 . We extended the experimental approach noted above where we combined FasL NA/LE Ab administration by i.t. with the adoptive transfer of CD45.1 monocytes via i.v. on Day 0 in young and old mice ( Figure 7c, S16a ) to assess the degree of apoptosis using the Annexin-V assay. The results showed that the addition of FasL NA/LE Ab reduced the level of apoptosis in MoAMs ( Figure S16 a-c ), with the apoptotic pathway mediated through caspase 3/7 ( Figure S16d ). Fas-mediated apoptosis occurs through the extrinsic pathway rather than the intrinsic pathway 43 . Caspase 3/7 are executor caspases involved in both pathways. Our results provide evidence that recruited MoAMs primarily undergo apoptosis through the extrinsic pathway, specifically those mediated by FasL and Caspase-8, followed by the downstream activation of Caspases-3/7 ( Figure 7k-l ). In concert with this, RNA-seq analysis of flow cytometry sorted CD11c + CD11b + MoAMs revealed a significant increase in Caspase-3 ( Figure 7m ). Overall, our data provide evidence that enhanced Fas-mediated signaling in old age leads to extrinsic apoptosis and consequently, high turnover of recruited MoAMs ( Figure 7n ). Discussion The impact of healthy aging on the nature of cellular function in tissue environments is significant, particularly in the lung, which is constantly exposed to inhaled particulates and microbes. This study offers important new insights into how aging, in the absence of disease, affects the phenotype, recruitment, and turnover of AMs across mammalian species and lays the groundwork for developing interventions to mitigate respiratory diseases in older adults. A significant finding is the increased presence of CD11c + CD11b + MoAM subpopulations in healthy elderly subjects, which exhibit elevated inflammatory, immunoregulatory, and cell death markers compared to the resident CD11c + CD11b - TRAMs. The inflammatory alveolar environment in old age can more effectively 1) convert TRAMs from young to a CD11c + CD11b + phenotype in old age, and 2) recruit MoAMs from monocytes in the periphery that undergo continuous replenishment due to a relatively short half-life resulting from increased apoptosis in the inflammatory milieu ( Figure 8 ). This shift towards a more inflammatory AM phenotype underscores the role of aging as a driving force behind heightened inflammatory responses and likely contributes to the increased susceptibility to respiratory diseases in older individuals. RNA sequencing analysis demonstrates significant transcriptional shifts in mAM subpopulations in healthy old age, particularly regarding CD11c + CD11b + mAMs. These cells exhibit significant upregulation of inflammatory genes, e.g., Ccl2, Ccl3, Ccl8, Cd40, Hif1α, Il6, Tnf, S100a8, S100a9, Csf1, Mmps and Tlr1, 2, 9 . The identified inflammatory profile, characterized by increased expression of TNF signaling-related genes and pro-inflammatory cytokines, aligns with the chronic low-grade inflammation commonly observed in age-associated diseases such as COPD and asthma 45 . Another notable finding is the metabolic shift towards glycolysis in old age mAMs, consistent with the Warburg effect, supporting the energetic demands of their inflammatory role and may contribute to tissue remodeling in the lungs in old age. Quantitative PCR analysis of hAMs corroborates our mouse RNAseq data, revealing higher levels of inflammatory and cell death markers in elderly individuals, indicating common mechanisms of age-related inflammation across mammalian species. As we age, baseline oxidative stress and inflammation significantly contribute to the development of an inflammatory microenvironment. These two factors are interconnected and can accelerate the aging process, increasing the risk of chronic diseases 46,47 . Increased baseline inflammation is linked to mitochondrial dysfunction and increased mitochondrial ROS production 48 . The metabolic reprogramming of mAMs in old mice, characterized by a shift from OXPHOS to glycolysis, has important implications for the heightened inflammatory responses within the lungs. Our findings align with studies that have demonstrated shifts in cellular metabolism with age in other cells and tissues. Previous research has shown that M1 macrophages or aged BMDMs display altered metabolic profiles, supporting our notion of a glycolytic shift contributing to inflammation 49,50 . Unexpectedly, we found that OXPHOS and glycolysis are both elevated in old mAMs, indicating that these pathways operate independently in mice. The interplay between increased ROS production and mitochondrial dysfunction is consistent with previous work regarding the relationship between oxidative stress and inflammation 51 . Incorporating comparative studies across mammalian species provides a deeper understanding of the cellular dynamics at play and their common underlying mechanisms. Adoptive transfer experiments provide evidence that with old age, the alveolar environment becomes less favorable for maintaining TRAM homeostasis, leading to the recruitment and activation of MoAMs. The Ms4a3 Cre -Rosa TdT fate mapper mouse model has proven instrumental in studying the origin of tissue macrophages 32 . The observed upregulation of Ms4a-related gene sets in MoAMs suggests that old age is associated with a shift in the immune landscape within the lungs, potentially driven by chronic inflammatory microenvironment. Our studies corroborate those indicating that the aging process or exposure to other environmental insults alters the immune cell composition within the lungs 5,8,15,29,52 . In all, the findings further support the premise that an inflammatory microenvironment drives differentiation of circulating monocytes into unique tissue macrophage populations 53,54 . Resident AMs lack CCR2 expression 36 , whereas CCR2 expression is high in monocytes in peripheral circulation 55 . We find that CD11c + CD11b + MoAMs express CCR2. Adoptively transferred CCR2-KO-GFP bone marrow monocytes remained in circulation and did not reach the alveoli. This suggests that CCR2 is essential for the migration of MoAMs into the lungs (and likely to other organs) in healthy old age. Increased levels of circulating monocytes in elderly populations correlate with hyperactivation immune cells 56 . Our data provides evidence for the unique role of CCR2 in monocyte migration in healthy old age. In contrast, we did not observe consistent differences in CX3CR1 protein levels among old/elderly individuals across species. Our experiments reveal that old age leads to the recruitment of short-lived MoAMs, which are prone to rapid apoptosis via Fas activation through the extrinsic Caspase-8, 3/7 pathway. Thus, while recruitment of these immune cells is increased, their lifespan is constrained, with ongoing turnover, potentially as a strategy (albeit unsuccessful) to mitigate excessive inflammation. Our findings regarding Fas (CD95), a member of the TNF receptor superfamily, contribute to the increasingly recognized role of Fas signaling in regulating apoptosis and inflammatory responses 57 in the context of aging 58,59 . This contrasts with apoptosis in adults, which is considered anti-inflammatory and promotes antigen presentation and microbial control 60,61 . Previous research has demonstrated that Fas ligand (FasL)-mediated activation of Fas on immune cells, e.g., human monocytes and monocyte-derived macrophages or epithelial cell types during acute lung injury, leads to increased apoptosis and the production of pro-inflammatory cytokines 8,39,62 . In older individuals, Fas activation is associated with the dysregulation of immune homeostasis, which contributes to the chronic low-grade inflammation frequently observed in aging. Our evidence suggests that Fas signaling is crucial in maintaining the inflammatory environment in the lungs associated with old age, reinforcing the concept that Fas-induced apoptosis in immune cells is a significant contributor to age-associated inflammatory responses. Our findings emphasize the detrimental effects of Fas in the aging context, where its activation appears to skew the immune response toward chronic inflammation and accelerate immune dysfunction. Indeed, Yu et al. (2011) demonstrated that Fas-deficient mice exhibit decreased inflammatory cell infiltration and reduced production of pro-inflammatory cytokines in an acute spinal cord injury model 57,63 . The implications of this research are significant, opening new potential therapeutic strategies aimed at improving immune function and tissue homeostasis in the healthy elderly population. By reducing the alveolar inflammatory environment and the presence of short-lived, apoptotic AMs, we may be able to reverse some age-associated changes within the lung, ultimately leading to healthier aging and lessening the incidence of age-related respiratory conditions. This study focuses exclusively on comparing young and old age groups. It does not evaluate the effects of Fas in regulating Fas-mediated inflammatory conditions across the ageing spectrum. Also, although we provide evidence for the role of the Fas-mediated extrinsic pathway contributing to macrophage apoptosis associated with old age, we have not ruled out the involvement of the intrinsic apoptosis pathway. This study focuses on the lung alveolus and AMs; it did not examine the tissue immune response in other organs. Finally, future studies will need to directly link our findings to their impact on host susceptibility to airborne infections, which is our next goal. Declarations Resource availability Lead contact If you need additional information or resources, please contact the lead person in charge for assistance. Lead contact: Larry S. Schlesinger, Email: [email protected] Materials availability This study did not generate new unique reagents. Data availability Statement RNA-seq data can be found in the NCBI GEO database: GSE297392 Competing Interest Statement. All authors declare no competing interests. Acknowledgments This work was supported by National Institute on Aging (NIA), NIH [P01 AG-051428] (to LSS, JBT, JT), NIH award [AI136831] (to LSS), Texas Biomed Cowles and Forum Postdoctoral Fellowships, and the Interdisciplinary NexGen TB Research Advancement Center (IN-TRAC) Pilot Grant (to SP). Research reported in this publication was supported by the NIH-NIAID under IN-TRAC Award Number P30 AI-168439. Research was also supported by the Office of The Director, NIH Award [S10 OD-028653] for the BD FACSymphony flow cytometry machine. RNA sequencing data were generated in the Genome Sequencing Facility, supported by UT Health San Antonio, NIH-NCI P30 CA-054174, NIH Shared Instrument grant [1S10 OD-021805-01] (S10 grant), and CPRIT Core Facility Award [RP-160732]. Acquisition of old mice was supported by an NIH grant to the University of Texas Health Science Center, San Antonio, The Barshop Institute for Longevity and Aging Studies, and the Nathan Shock Center of Excellence in the Biology of Aging [5P30-AG-013319-28] to John Randy Strong. Fluorescence/confocal microscopy imaging was conducted with instruments at the Biology Core at Texas Biomed. Author contributions L.S.S and J.T. contributed to the early conceptual development of the project. S.P. and L.S.S. designed the studies and generated protocols. S.P., M.L., A.P., A.A., H. Z., H. C., Z. L., J. M., J. P., W.P.L., Y.W., conducted experiments, acquired the data, analyzed them, and generated figures. S.P. wrote the manuscript with input from LSS. S.P., L.S.S, E.A., Y.W, F.G, J.B.T., and J.T. edited the manuscript and performed a critical review. Methods Human Subjects and Ethics Statement Human subject studies were conducted in strict accordance with the US Code of Federal and Local Regulations, as overseen by The Ohio State University (OSU) institutional review board with the number 2012H0135. The studies were also overseen by the Texas Biomedical Research Institute with IRB number HSC20170673H. Most samples were collected from the Division of Pulmonary and Critical Care Medicine, UT Health Science Center, San Antonio. Bronchoalveolar lavage (BAL) samples were collected from healthy adult (20-50 years) and elderly (≥65 years) individuals of both sexes, without discrimination of race or ethnicity, following written consent. The donors had a preoperative diagnosis of lung nodule, lung nodule with mass, or abnormal parathyroid and thymus, without clinical features. BAL was performed on the healthy lobe of each donor's lung. Individuals with certain comorbidities, such as smokers, drug users, excessive alcohol users, acute illnesses, chronic conditions, and various other health conditions were excluded from the study. Each "n" value represents a different human BAL donor as specified in the figure legends. Mice Specific pathogen-free C57BL/6 female mice were obtained from Charles River Laboratories (Wilmington, MA) at either 3 months of age (young) or 18 months of age (old) through a contract with the National Institute on Aging (NIA). Mice were housed in individually ventilated cages (IVC) and allowed to acclimate to the facility for one week before being used for the study. All procedures were approved by The Texas Biomedical Research Institute (Texas Biomed) Institutional Laboratory Animal Care and Use Committee (Texas Biomed-IACUC number 1608-MU). C57BL/6 young CD45.1 congenic mice (3 months age), B6.SJL-Ptprc a Pepc b /BoyJ (B6.CD45.1, Strain # 002014) and CByJ.SJL-Ptprc a /J (CD45.1, Strain # 006584) BALB/c mice were purchased from The Jackson Laboratory. Ms4a3 Cre -Rosa TdT mice bone marrow was acquired from Florent Ginhoux, Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR). Ccr2 gfp/gfp KI/KO mice (C57BL/6, Strain # 027619) were purchased from The Jackson Laboratory. Collection and isolation of human alveolar macrophages (hAMs) To isolate and culture hAMs from BAL 64 samples collected within 6h were centrifuged (250 g) and washed twice in PBS at 4°C. The cell pellet was then resuspended in RPMI 1640 with Penicillin-G (Pen-G stock diluted to 1:50 to achieve 10,000 U/mL), and cells were allowed to adhere for 2 h in 24 well tissue culture wells (Falcon/ Corning Life Sciences). After adherence, Pen-G was removed by washing. A portion of the cell suspension underwent cytospin, followed by staining and microscopy to determine the percentage of hAMs in BAL (>98%). Following cell counting on a hemocytometer, hAMs (1x10 5 ) were plated for morphometric microscopic analysis. In a separate experiment, hAMs (5x10 5 ) were immediately lysed in TRIzol reagent (Invitrogen) for qRT-PCR analysis. Another set of cells was utilized for flow cytometry for phenotypic analysis. Collection and isolation of baboon and Rhesus alveolar macrophages (bAMs; rAMs) Olive baboons ( Papio anubis ) and Rhesus macaque ( Macaca mulatta ), both young (ages 3-5 years) and old (ages 15+ years), were obtained from our colony at the Southwest National Primate Research Center (SNPRC) at Texas Biomed. These animals were acquired through a biomaterials request, involving opportunistic BAL collection conducted without any disease conditions. This occurred during routine necropsies due to age or through live animal BAL collections, following Texas Biomed-IACUC Protocol number 1516 PC and 1516 MM. BAL was performed by experienced pathologists in 0.9% saline at the SNPRC-Pathology lab. AMs were collected through centrifugation at 300 x g for 10 min and washed. Depending on the experimental requirements, BAL cells from individual baboons were analyzed using confocal microscopy or flow cytometry. Isolation of mouse alveolar macrophages (mAM) and Alveolar Lining Fluid (ALF) Both young and old mice were euthanized using CO 2 following an approved protocol (Texas Biomed-IACUC 1608-MU). AMs and BAL fluid (BALF) were obtained by washing the lungs of mice with sterile, endotoxin-free saline containing 100 µM EDTA (0.5M), and either 0.2% BSA (for BAL fluid needed for Luminex assay) or 5% FBS. After washing the lungs 6-10 times with 0.5 ml of the saline solution, AMs were collected through centrifugation at 300 x g for 10 min. Depending on the experimental requirements, individual BAL cells were analyzed using confocal microscopy or flow cytometry. For qRT-PCR, BALs from 6 to10 mice were pooled, and the RNA was isolated using TRIzol reagent (Invitrogen) for measuring basal RNA levels. The supernatant fraction containing ALF from each individual mouse was promptly frozen and stored at −80°C until used for the Luminex assay. Cytospin analysis Healthy adult and elderly hAMs were collected for cytospin analysis. Shandon non-coated cytoslides (Thermo Scientific) were placed in a Shandon EZ single cytofunnel with white filter cards. A single cell suspension was created, and 200 μL of cells (5x10 4 ) were placed into the cytofunnel and centrifuged at 150 x g for 5 min using Program 1. The cells on cytoslides were dried and then stained with HEMA 3 differential staining (Fisher Health Care). Afterward, the slides were washed with water and dried. The slides were placed in Hema 3 fixative solution for 30 s. They were then dipped 30 times for 1 s each in Hema 3 Solution I (eosin Y) and subsequently dipped 30 times for 1 s each in Hema 3 Solution II (methylene blue). It was important to allow excess stains to drain during each step. After staining, the slides were washed with water and left to dry. Hema 3 Solution I (eosin Y) was used to stain cytosolic proteins, while Hema 3 Solution II (methylene blue) stained the nuclear membrane. Finally, cells were examined using a Motic AE2000 microscope. Isolation of mouse monocytes and adoptive transfer Bone marrow cells (BMCs) from the femurs and tibias of C57BL/6 wild type, CD45.1, Ms4a3 Cre -Rosa TdT , and Ccr2 gfp/gfp KI/KO young mice were flushed aseptically. The Ly6C-positive monocytes were isolated by negative selection using the EasySep™ Mouse Monocyte Isolation Kit (Cat#19861RF, Stem Cell Technologies, Cologne, Germany), according to the manufacturer’s instructions. The isolated monocytes were then processed for the adoptive transfer experiments. To study macrophage turnover, monocytes from B6.CD45.1 young mouse (donor cells) were injected intravenously (tail vein injections, i.v.) [1.5x10 6 donor monocytes/mouse] in recipient young or old C57BL/6 mice (CD45.2). After 0, 3, 5, 7, and 10 days, depending on the experimental strategy shown in the figures (4-6), 5-8 recipient mice in each group were sacrificed, and AMs obtained by BAL to perform flow cytometry to verify cell turnover. The immigrated MoAMs were distinguished through CD45.1 cell labeling, while resident AMs were identified from CD45.2 labeled cell populations. To identify the origin of recruited cells in the alveolar region and parenchyma ( Figures 4 & S15 ), the fate mapper model (Ms4a3 Cre -Rosa TdT ) was used for adoptive transfer to track monocytes and their bone marrow progenies 32 . The Ms4a3 Cre -Rosa TdT cells utilize a Cre-loxP recombination system, where the Ms4a3 Cre promoter drives Cre recombinase expression, leading to activation of the TdTomato (TdT) reporter in specific cell lineages. The purified young mice Ms4a3 Cre -Rosa TdT bone marrow monocytes (donor cells) were adoptively transferred (1.5x10 6 donor monocytes/mice) via i.v. in recipient young or old C57BL/6 mice. On day 5, BAL cells were isolated to analyze recruited cells in the alveolar space by flow cytometry, confocal microscopy and histopathological analysis. For histopathological analysis, entire lung lobes were collected from the recipient mice. The lung lobes were preserved in 10% formalin and sent to the Texas Biomed path lab for histopathological analysis by H & E staining. The recruited dense, accumulated cells (blue hematoxylin-stained cells) and Rosa TdTomato-labeled cells were identified through HALO analysis. To assess the role of CCR2 in cell recruitment in the alveolar region and parenchyma ( Figure 5 ), purified Ccr2 gfp/gfp knockout mice bone marrow monocytes were adoptive transferred (1.5x10 6 donor monocytes/mice) through the i.v. route into young or old C57BL/6 recipient mice. On day 5, during the necropsy, blood was collected from the heart using heparin (1,000 U/mL) to prevent coagulation. PBMCs were isolated from the blood of recipient mice using Ficoll-Hypaque density cushion centrifugation, while AMs were isolated by BAL. The presence of GFP+ cells was confirmed in both BAL cells and blood-derived cells. Cells (6 x 10 4 / 200 μL) were then placed in a cytofunnel and centrifuged in the Shandon Cytospin 4 at 150 x g for 5 min using Program 1. Subsequently, the cells were fixed with 2% paraformaldehyde for 3 min. The slides were washed three times with 1X DPBS (Gibco) and mounted on coverslips with prolong gold along with DAPI (Thermo Fisher Scientific). The stained slides were examined using a Zeiss LSM 800 confocal microscope (20X, 63X magnification). Mean fluorescence intensity (MFI) and percentage of positive cells were quantified using ZEN 2.3 blue edition (Zeiss) and ImageJ software (NIH, Bethesda, MA). Depletion of AMs in mice by treatment with clodronate liposomes Young and old mice were treated with Clodronate liposomes or control liposomes (Liposoma research, Cat#CP-005-005) at a dose of 100 µL/mouse (500 µg/mouse) via the i.t. route on day 0 and 3 (two doses). On day 3, 5, 6, and 7, the cells were isolated by BAL. The cells were counted, and cytospin analysis was performed to demonstrate the percentage of AMs that are depleted from the alveolar space. For experimental purposes, AMs from a separate set of young mice (donor mice) were isolated by BAL. 1x10 6 AMs were i.t. installed on Day 5 in clodronate-treated young and old recipient mice. On day 7, BAL was performed on young and old recipient mice to assess the impact of the lung environment on changing the phenotype and function of the AMs (donor young mice) in old mice in the alveolar space. FasL NA/LE treatment in mice For CD178 FasL NA/LE treatment study, donor bone marrow monocytes (1.5x10 6 donor monocytes/mice) were isolated from B6.CD45.1 mice and adoptive transferred via the i.v. route in recipient young or old C57BL/6 mice (CD45.2). After day 0 or day 5, depending on the experimental strategy shown in the Figures 7 and S16 , FasL NA/LE ab was i.t. installed to block the Fas-mediated inflammatory response 57,65 . After 0, 3, 5, 7, and 10 days, depending on the experimental strategy shown in Figure 7 and S16 , 5 to 8 recipient mice in each group were sacrificed, and AMs were obtained by BAL to perform a flow cytometry assay. Fas-mediated apoptosis via Caspase 3, 7 & 8, as well as cell turnover were verified. Confocal Microscopy For confocal microscopy analysis, Ms4a3 Cre -Rosa TdT (Red) and Ccr2 gfp/gfp KI/KO (Green) positive cells (BAL and blood derived) (6x10 4 / 200 μL) were placed in a cytofunnel and centrifuged in the Shandon Cytospin 4 at 150 x g for 5 min using Program 1. Subsequently, the cells were fixed with 2% paraformaldehyde for 3 min. Slides were washed three times with 1X DPBS (Gibco) and mounted on coverslips with prolong gold along with DAPI (Thermo Fisher Scientific). The stained slides were examined using a Zeiss LSM 800 confocal microscope (20X, 63X magnification). Mean fluorescence intensity (MFI) and percentage of GFP-positive cells were quantified using ZEN 2.3 blue edition (Zeiss) and ImageJ software (NIH, Bethesda, MA). Annexin V and EthD-2 cell viability assay Monocytes were taken from B6.CD45.1 mice and 1.5x10 6 donor monocytes were i.v. injected (via tail vein injections) into recipient young or old C57BL/6 mice (CD45.2). On day 0, FasL NA/LE antibodies were administered directly into the lungs to block Fas-mediated apoptosis. On days 3 and 4, recipient mice in each group were sacrificed, and AMs were obtained by BAL to perform a flow cytometry viability assay. The cells were then suspended in Annexin binding buffer [0.01 M HEPES (pH 7.4), 0.14 M NaCl, and 2.5 mM CaCl 2 ]. APC-labeled Annexin V (4 μL per tube) was added to all samples and incubated for 15 min in the dark at room temperature. After washing, Ethidium Homodimer-2 (EthD-2, 4 µM) was added and incubated for another 10 min at room temperature. The cells were fixed with 2% paraformaldehyde (PFA). After washing with 1% binding buffer (400 μL), cells were analyzed by the BD FACS Symphony instrument immediately, and the data were analyzed using FlowJo software. Flow Cytometry AMs (1-2 × 10 5 ) were placed into FACS tubes (Falcon round-bottom polypropylene tubes, Cat# 352063) and centrifuged at 250 x g for 10 min. The cell pellets were then resuspended in 50 µL of cell staining buffer from Biolegend (Cat# 420201) along with TruStain FcX™ PLUS (anti-mouse CD16/32) Antibody (Ab) from Biolegend (Cat# 156604) for FC receptor blocking. After a 30 min incubation on ice, the cells were stained with fluorochrome-tagged antibodies and corresponding isotype-matched control antibodies in BD Horizon Brilliant Stain Buffer Plus (BD, Cat#566385) for 40 min in the dark at 4°C. Following this, the cells were washed once, fixed in 2% paraformaldehyde (Thermo Scientific, Cat# J19943-K2) in cell staining buffer for 10 min, centrifuged (250 x g for 10 min), and then resuspended in 300 µL cells staining buffer. Cells were then filtered through a round-bottom polystyrene tube with a cell strainer snap cap (Falcon, Cat#352235). Samples were then acquired using a BD FACSymphony multi-color flow cytometer, and compensation, analysis, and data visualization were carried out using FlowJo 10.8.1 software (BD Biosciences). Isotype and "Fluorescence minus one" controls were utilized as needed to establish gates. The gating strategy employed for macrophage analysis is depicted in Figures S2-5 . The fluorochrome-tagged antibodies were purchased from BD Biosciences, San Jose, CA, and Biolegend, San Diego, CA. The list of mouse Abs were: BV421 anti-mouse/human CD11b (clone M1/70, Cat# 101236), APC-Cy7 anti-mouse CD11c (clone N418, Cat#117324), BV786 anti-mouse CD64 (clone X54-5/7.1, Cat#741024), BUV395 anti- mouse CD95 (Fas, Clone Jo2, Cat#740254), BUV496 anti-Mouse CD192 (CCR2, Clone 475301, Cat#750043), Alexa Fluor® 488 anti-mouse CD45.1 (Clone A20, Cat#110718), PerCP/Cyanine5.5 anti-mouse CD45.1 (Clone A20, Cat# 110728), CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit (Cat# C10427). The antibody and respective isotypes are indicated in the “Key resources table”. Luminex multiplex analysis The release of cytokines, chemokines, and other secretory factors in the culture supernatant was measured using Luminex assays. The following analytes were measured using the Luminex mouse Discovery Assay (10-Plex, code mxmbhKZn) LXSAMSM-10kit (R&D Systems, Inc.): GM-CSF, TNF-alpha, S100A9, S100A8, CCL2/JE/MCP-1, Fas Ligand/TNFSF6, IL-1 beta/IL-1F2, MMP-2, CCL3/MIP-1a, and IL-10. BAL was performed, and the BALF and AMs were separated by centrifugation at 250 x g for 10 min. The 4 to 5 mL BALF was concentrated by passing it through a 10kDa filter (Amicon, EMD Millipore, MWCO 10 kDa, Ref. UFC9010) and then centrifuged at 2,800 x g , at 4°C, for 30 min with a brake at 2. The upper concentrated ALF was collected, and a protease inhibitor cocktail (A+L) was added to prevent protein degradation. Selected analytes were measured from ALF (1:2 dilutions in 50 µL) in the Luminex® 100/200™ System (Luminex Corporation) following the manufacturer’s protocol. Data were analyzed using Belysa™ Immunoassay Curve Fitting Software (Millipore Sigma). Analytes/mL concentrations were calculated and plotted. Cell sorting using flow cytometry for bulk-RNA-seq analysis AMs were isolated from both 10 young and 10 old mice and then sorted into CD11c + CD11b - populations from young and old mice, and CD11c - CD11b + and CD11c + CD11b + populations from old mice using the BD FACSAria II flow cytometer. CD11c - CD11b + populations were found in very low numbers and displayed a transcriptional signature like the CD11c + CD11b + populations. Consequently, we did not include them in further analyses. Samples were collected in three different batches as three replicates for bulk-RNAseq. This was achieved by flow cytometry staining and sorting, using BV421 anti-CD11b (clone M1/70) and PE anti-CD11c (clone N418) antibodies. The post-sorted cells were then verified based on the gating strategy, and the purity of the populations was confirmed through gating. Bulk RNA-Seq from flow-sorted populations and analyses Flow-sorted CD11c + CD11b - AMs from young mice and CD11c + CD11b - and CD11c + CD11b + AM populations from old mice were collected and washed at 250 x g for 10 min with 1% PBS. The RNA was isolated using TRIzol reagent (Invitrogen) and a Direct-zol RNA Microprep kit (Zymo Research) according to the manufacturer's instructions. The isolated RNA was quantified using the Qubit 4 Fluorimeter (Invitrogen), and its quality was assessed with the 4200 TapeStation System (Agilent). Samples with an RNA integrity number (RIN) higher than 7 were used for RNA-seq. RNA-seq libraries were prepared from 300 ng of total RNA using the NEB Next RNA Ultra Kit (Qiagen, Redwood City, CA) with poly(A) enrichment, and 50-bp single-read sequencing with approximately 25-30M reads per sample. The RNA sequencing was performed at the Genome Sequencing Facility (GSF) at UT Health San Antonio using the HiSeq 3000 platform (Illumina). The raw sequence reads were performed using Trim Galore! to remove adapters and low-quality sequences 66 . Then, the trimmed reads were mapped to the mouse mm10 reference genome using HISAT2 67 . After that, read counts for each sample were obtained using featureCounts 68 . The next step involved conducting differential gene expression analysis using DESeq2 69 with control of the false discovery rate (FDR) using the Benjamini-Hochberg procedure. Shrunken log2 fold changes (LFCs) were calculated using the adaptive shrinkage (ash) estimator with an Empirical Bayes approach. Genes with FDR-adjusted p-value <0.05 and LFC of more than 1 or less than -1 were considered differentially expressed. Finally, heat maps of specific genes were generated using the pheatmap package in R. RNA isolation, quantification and qRT-PCR hAMs from adult and elderly donors or mAMs from young and old mice were collected by BAL. In select experiments, mAMs were cultured with Pam 3 CSK 4 (100 ng/mL) for 24h. Cells were washed at 250 x g for 10 min with 1% PBS (Gibco) and then treated with TRIzol reagent (Invitrogen). RNA was extracted using a Direct-zol RNA Microprep kit (Zymo Research) according to the manufacturer's instructions and quantified using the Qubit 4 Fluorimeter (Invitrogen). The precipitated RNA was reconstituted with DNase/RNase-free water and reverse transcribed using random primers and SuperScript III Reverse Transcriptase (Invitrogen). cDNA synthesis using reverse transcriptase was carried out at 65°C for 5 min, followed by 25°C for 5 min, 50°C for 60 min, 70°C for 15 min, and then cooled to 4°C. mRNA expression was analyzed using quantitative real-time RT-PCR (qRT-PCR) with TaqMan Universal PCR Master Mix (Applied Biosystems). The amplification conditions were selected at 50°C for 10 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s in the Applied Biosystems 7500 Real-Time PCR System. Thermo Fisher Scientific, the TaqMan human primers with the best coverage and most citations were used. Relative expression was calculated using the ΔΔCT method, with β-actin (ACTB) as the housekeeping gene. The selected genes for humans are MRC1, CD11B, CD11C, TLR2, NOS2, HIF1A, PIEZO1, FAS, CX3CR1, CCR2 . Mouse Hif1α, Cox2, Cd74, Mif, Glut1, Hk2, Pfkb3, Gls1, Slc25a, Acly, and Actb were analyzed for qRT-PCR expression using IQ SYBR Green Supermix (BioRad). β-actin ( Actb ) was used as the housekeeping gene. Validated mouse primers listed on PrimerBank 70 were used and primer sequences listed in Key resources table. Extracellular Flux Analysis (Agilent Seahorse) The real-time cell metabolism of mouse AMs was measured using a Seahorse XF Extracellular Flux Analyzer XFe96 (Agilent Technologies). This involved determining the oxygen consumption rate (OCR, pmol/min) and extracellular acidification rate (ECAR, mPh/min) according to the manufacturer’s instructions. To investigate the metabolic changes in old mAMs, we assessed mitochondrial respiration, ATP production, OCR, and ECAR using the Seahorse XFe96 analyzer. For the mitochondrial stress assays, mAMs were placed in a supplemented XF assay medium, followed by sequential injections of oligomycin (O), FCCP, and rotenone/antimycin (R/A). Under the same setup, injections of O and R/A alone were used to determine the ATP production rate specifically from mitochondria. First, 3-month-old and 18-month-old mice (5x10 4 /well) were adhered in 96-well Seahorse plates for 2 h. The cells were then washed and replenished with XF DMEM Seahorse media supplemented with 25 mM D-Glucose, 1 mM Sodium pyruvate, and 2 mM L-glutamine. After incubating in a non-CO 2 incubator at 37˚C for 1h, the basal levels of OCR and ECAR were measured. A Mito stress assay was performed with sequential addition of 5 μM oligomycin (an ATP synthesis inhibitor), 4 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, an uncoupling agent), and 2 μM rotenone and antimycin A (inhibitors of complex I and III of the respiratory chain). For glycolysis stress analysis, cells were injected with 2 μM rotenone and 2 μM antimycin A followed by 100 mM 2-deoxyglucose (2-DG) to determine glycolytic rate. Each of these components has specific roles. Oligomycin decreases electron flow through the ETC, verifying the basal OCR measurement impacts on mitochondrial respiration or OCR. FCCP interrupts the mitochondrial membrane potential, resulting in continuous electron flow through the ETC and maximal oxygen consumption by complex IV. The spare respiratory capacity (SRC) was calculated by measuring the difference between FCCP-induced maximal respiration and basal respiration. The third injection uses a combination of rotenone and antimycin A to block mitochondrial respiration, enabling the measurement of non-mitochondrial respiration in the cells. In the ECAR glycolytic rate assay, glycolytic pathway inhibitor 2-DG is used to inhibit glycolysis through competitive binding to glucose hexokinase. MitoSox assay for mitochondrial ROS detection To assess the production of mitochondrial reactive oxygen species (ROS) in AMs from both young and old mice, cells were stained with 5μM MitoSOX (Thermo Fisher Scientific, Inc.) for 30 min. After staining, the cells were washed with cell staining buffer and centrifuged at 250 x g for 10 min at room temperature. Following centrifugation, the stained cells (5 x 10 4 / 200 μL) were loaded into a cytofunnel and centrifuged in the Shandon Cytospin 4 at 150 x g for 5 min using Program 1. The cells were then fixed with 2% paraformaldehyde for 3 min. Subsequently, the slides were washed three times with 1X DPBS (Gibco) and mounted on coverslips with prolong gold containing DAPI (Thermo Fisher Scientific). The stained slides were visualized using a Ziess LSM 800 confocal microscope at 20X and 63X magnification. Mean fluorescence intensity (MFI) and percentage of positive cells were quantified using ZEN 2.3 blue edition (Zeiss) and ImageJ software (NIH, Bethesda, MA). Statistical analyses Graphs were created and statistical comparisons were conducted using GraphPad Prism version 10. Unpaired two-tailed Student’s T-test was used for statistical comparisons of two groups. Where applicable (as indicated in figure legends), ordinary one-way and two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test (GraphPad Prism vr. 10) was applied for multiple testing. Spearman’s rank test was used for Correlation analysis. Statistical significance was reported to have a p-value of ≤ 0.05. 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HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12 , 357-360 (2015). https://doi.org:10.1038/nmeth.3317 Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30 , 923-930 (2014). https://doi.org:10.1093/bioinformatics/btt656 Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15 , 550 (2014). https://doi.org:10.1186/s13059-014-0550-8 Wang, X., Spandidos, A., Wang, H. & Seed, B. PrimerBank: a PCR primer database for quantitative gene expression analysis, 2012 update. Nucleic Acids Res 40 , D1144-1149 (2012). https://doi.org:10.1093/nar/gkr1013 Additional Declarations There is NO Competing Interest. Supplementary Files TableS1.xlsx Table S1. Differential expression of RNAs in flow cytometry-sorted CD11c + CD11b + mAMs versus TRAMs from old mice (CD11c + ). (a) Upregulated genes with false discovery rate (FDR) adjusted p-value 1. (b) Downregulated genes with false discovery rate (FDR) adjusted p-value <0.05 and log2 fold change < -1. TableS2.xlsx Table S2. Differential expression of RNAs in TRAMs in old mice versus young mice. (a) Upregulated genes with false discovery rate (FDR) adjusted p-value 1. (b) Downregulated genes with false discovery rate (FDR) adjusted p-value <0.05 and log2 fold change < -1. TableS3.docx Table S3. Key Resources Table. A detailed listing of reagents or resources, their sources, and corresponding identifiers. SupplementaryData.pdf Supplementalinformation.docx Cite Share Download PDF Status: Under Review 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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18:10:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7123735/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7123735/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87545355,"identity":"bf467f12-5d1e-4c31-a7a3-71841ccc7300","added_by":"auto","created_at":"2025-07-25 04:45:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":454154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of AM subpopulations in old mice (mAMs), elderly humans (hAMs), and old non-human primates (baboons, bAMs). \u003c/strong\u003e(a \u0026amp; b) Murine AMs (mAMs) were isolated from young (3-month-old) and old (18 month-old) mice using bronchoalveolar lavage (BAL). The cells were then stained with fluorochrome-labeled antibodies and analyzed by flow cytometry, specifically gating on the CD45\u003csup\u003e+\u003c/sup\u003eSiglecF\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e AM subpopulations. (c \u0026amp; d) Human AMs (hAMs) were isolated by BAL from healthy adult volunteers aged 20-50 years and elderly individuals (≥60 years). The flow-labeled cells were analyzed by gating on the CD45\u003csup\u003e+\u003c/sup\u003eCD64\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e AM subpopulations. (e \u0026amp; f) Baboon AMs (bAMs) were isolated by BAL from healthy adult baboons (3-5 years old) and elderly baboons (≥15 years). The flow-labeled cells were also analyzed by gating on the CD45\u003csup\u003e+\u003c/sup\u003eCD64\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e AM subpopulations. (a, c, e) Overlay dot plots include isotype-matched controls (in blue). (b, d, f) Violin plot illustrates the percentage of CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e AM subpopulations from hAMs (N=7 adult and 12 elderly human donors) and mAMs (n=10 young and 14 old mice, n=4 young and 4 old baboons). Each dot indicates individual donors. In elderly individuals, both hAMs and bAMs exhibit a dominant CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e-\u003c/sup\u003e subpopulation, alongside two smaller subpopulations: CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e and CD11c\u003csup\u003e-\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e. mAMs primarily show a dominant CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e-\u003c/sup\u003e subpopulation with an increase in the smaller CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e subpopulation. (g) Flow cytometry analysis of CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e- \u003c/sup\u003eand CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e hAM subpopulations revealed the expression of several cell surface receptors, including CD11b, CD11c, CCR2, Fas (CD95), HLA-DR, CD14, and CD16. (h) Kinetic analysis of CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e mAM subpopulations was conducted in 3 month, 9 month, and 17-month-old mice, pre-gated on CD45\u003csup\u003e+\u003c/sup\u003eSiglecF\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e cells using flow cytometry. (b, d, f-h) Data are presented as mean ± SEM and were analyzed using an unpaired Student’s T-test for two comparisons and an ordinary one-way ANOVA with Sidak's multiple comparisons test. Each dot indicates an individual donor. Significance levels are indicated as follows: **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. \u003cstrong\u003eSee also Figures S1-4. \u003c/strong\u003eThe cartoon was created in BioRender. Pahari, S. (2025) https://BioRender.com/x07c111.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/c2027f18d39d5b3aa7d52e23.png"},{"id":87545825,"identity":"49ce8050-71af-44b1-a61d-96032559ed47","added_by":"auto","created_at":"2025-07-25 04:53:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":346069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of transcriptional signatures in flow cytometry-sorted young and old mAM subpopulations. \u003c/strong\u003eThe figure presents a transcriptomic analysis using bulk-RNA sequencing (RNA-seq) on four flow cytometry-sorted populations: resident AMs from young mice (CD11c\u003csup\u003e+\u003c/sup\u003e), resident AMs from old mice (CD11c\u003csup\u003e+\u003c/sup\u003e), immigrated AMs from old mice (CD11b\u003csup\u003e+\u003c/sup\u003e) and immigrated AMs from old mice (CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e). (a) The schematic diagram illustrates the procedure for bulk RNA sequencing. The schematic diagram was created in Created in BioRender. Pahari, S. (2025) https://BioRender.com/x726985. (b) Principal component analysis (PCA) of the transcriptomes from the four different subpopulations of mAMs in young and old mice shows distinct transcriptional signatures and a high degree of similarity among biological replicates within each group (N=10 mice per group, with 3 flow-sorted experiments). (c) The volcano plot displays the false discovery rate (FDR) in relation to the magnitude of gene expression changes across a total of 14,509 genes. The analysis indicates that 577 out of 5,300 significant genes (FDR \u0026lt; 0.05) were significantly upregulated (red), with a log2 fold change (logFC) \u0026gt;1, and 10 genes were downregulated (blue) in the CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e AM subpopulations, with a log2 fold change (logFC) \u0026lt; -1. (d, e) The heatmaps show relative expression levels for TNF-signaling related and inflammatory signature genes in AM subpopulations from old mice (resident CD11c\u003csup\u003e+\u003c/sup\u003e and immigrated CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e) and resident CD11C\u003csup\u003e+\u003c/sup\u003e from young mice. The red arrow highlights certain inflammatory genes that are highly upregulated in the CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e mAM subpopulations from old mice, indicating unique signature genes (in red) compared to CD11c\u003csup\u003e+\u003c/sup\u003e AMs from either young or old mice. Blue represents low expression, while red represents higher expression. (f) Inflammatory gene signatures (Hif1α, Tnf, Tnfrsf9, Il6, S1008, S1009) were identified through RNA-seq analysis shown by violin plot. Data represents 3 independent experiments and were analyzed using an ordinary one-way ANOVA with Sidak's multiple comparisons test. Statistical significance is indicated as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. **** p \u0026lt;0.0001. \u003cstrong\u003eSee also Figure S5-7.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/f9fd6ef29fd1f305822b0bfe.png"},{"id":87544788,"identity":"b69d112c-3ca0-4427-955e-6534a7e33cb7","added_by":"auto","created_at":"2025-07-25 04:37:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":319078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDepletion of resident mAMs to assess the alveolar environment in old age.\u003c/strong\u003e To characterize the Mo-AM subpopulation and investigate the role of the alveolar environment in old mice, two doses of clodronate liposomes (clodrolip.) were administered i.t. on Day 0 and Day 3. This treatment effectively depleted resident AMs from the alveolar compartments of both young and old mice. On Day 5, CD11c\u003csup\u003e+\u003c/sup\u003e bronchoalveolar lavage (BAL) isolated resident AMs (donor cells) from young mice were adoptively transferred i.t. (using 1X10\u003csup\u003e6\u003c/sup\u003e cells) into both young and old recipient mice. (a) Schematic representation outlines the experimental approaches used. The schematic diagram was created in BioRender. Pahari, S. (2025) https://BioRender.com/b41u275. (b) Cytospin staining analysis presented in the violin plot shows the percentage of AM depletion on Days 3, 5, 7, and 8. Similar AM populations were replenished by Day 7 and 8, indicating immigrating cells 48 h and 72 h after the adoptive transfer. (c) Flow cytometry dot plot analysis shows the percentage of CD11c\u003csup\u003e+\u003c/sup\u003e Siglec F\u003csup\u003e+\u003c/sup\u003e AMs depleted (82.8% to 9.39%) after clodronate liposome (Clodrolip.) treatment at day 5. (d) The violin plot shows the percentage of AM depleted in the clodrolip-treated group. Data pooled from 4 mice/group. (e) Flow cytometry dot plot analysis reveals the presence of CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e MoAMs (Q2 gate: 8.58%) in old mice, while the percentage of MoAMs in the clodrolip-treated group was 3.2% (Q2 gate). Following the adoptive transfer of CD11c\u003csup\u003e+\u003c/sup\u003e BAL isolated resident AMs from young mice, the phenotype transformed into CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e MoAMs (Q2 gate: 12.1%) in old mice. (f) Elevated levels of TNFa, FasL, and S100A9 proteins were detected in the BAL fluid of old mice. Each dot indicates individual mice/group. Data represent 3-4 independent experiments and are expressed as mean ± SEM, analyzed using an ordinary one-way ANOVA with Sidak's multiple comparisons test. Statistical significance is indicated as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. **** p \u0026lt;0.0001.\u003cstrong\u003e\u0026nbsp; See also Figure S8-12.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/fea3e8eb9c7ecdead845d3aa.png"},{"id":87545361,"identity":"839a0715-8226-473a-8d93-5dbfe825aa3b","added_by":"auto","created_at":"2025-07-25 04:45:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":467180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdoptive transfer of monocytes from young mice into recipient young and old mice. \u003c/strong\u003eCD45.1 or Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosta\u003csup\u003eTdT\u003c/sup\u003e monocytes (donor cells) were isolated by magnetic sorting (Ly6c\u003csup\u003e+ \u003c/sup\u003enegative selection kit) from the bone marrow of young mice and transferred into CD45.2 (recipient) young and old mice via tail-vein injection (i.v.). After 5 days, BAL was performed. Recovered AMs were labeled for flow cytometry analysis. (a) Schematic representation of the experimental approaches is shown. Created in BioRender. Pahari, S. (2024) \u003ca href=\"https://BioRender.com/p76z776\"\u003ehttps://BioRender.com/p76z776\u003c/a\u003e. (b) Flow cytometry dot plot data indicate that CD45.2 positive cells are consistent among young and old mice AMs. CD45.1 cells were successfully recruited into the alveolar space of old mice, as indicated by the red arrow highlighting the recruited CD45.1 positive cells. (c, d) Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosta\u003csup\u003eTdT\u003c/sup\u003e macrophages (in red) were identified using confocal microscopy. The white arrow points to Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosta\u003csup\u003eTdT\u003c/sup\u003e positive cells. Scale bar represents 10µm at 63x magnification. In (d), the quantification of Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosta\u003csup\u003eTdT\u003c/sup\u003e positive cells (as a percentage) was performed from over 100 macrophages (DAPI positive in blue) per microscopic field. (e) The contour plot from flow cytometry illustrates the presence of Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosta\u003csup\u003eTdT\u003c/sup\u003e AMs detected in old mice within the CD45\u003csup\u003e+\u003c/sup\u003eSiglecF\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e populations. Further gating reveals that Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosta\u003csup\u003eTdT\u003c/sup\u003e positive AMs are also part of the CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e AM population. (f) A bar graph summarizes cumulative data of Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosta\u003csup\u003eTdT\u003c/sup\u003e macrophages from 4 mice per group. (g) Another contour plot shows that old mice exhibited an increased recruitment of the CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e AM subpopulations. (h) A bar graph demonstrates cumulative data for the CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e AM subpopulations from 4 mice per group. (i) RNA sequencing analysis indicates that several Ms4a-related gene sets (Ms4a4c, Ms4a4a, Ms4a6b, Ms4a7, Ms4a14) were upregulated in CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e monocyte-derived alveolar macrophages (MoAMs) from old mice compared to the resident AMs (CD11c\u003csup\u003e+\u003c/sup\u003e) in both young and old mice. Data are representative of 3 independent experiments and are expressed as the mean ± SEM, with an Unpaired Student’s t-test for two comparisons and an ordinary one-way ANOVA with Sidak's multiple comparisons test. *p \u0026lt;0.05, ** p \u0026lt;0.01, *** p \u0026lt;0.001, **** p \u0026lt;0.0001. \u003cstrong\u003eSee also Figure S13.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/f7072646b919bcc096eb55ff.png"},{"id":87545358,"identity":"10879091-3f8a-47bb-a2c9-16101b501bb8","added_by":"auto","created_at":"2025-07-25 04:45:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1226205,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRecruitment of monocyte-derived macrophages (MoAMs) through a CCR2-dependent pathway. \u003c/strong\u003eMo-AM were isolated from mice aged 3 months (3M), 9 months (9M), and 17 months (17M). (a, b) Kinetic analysis of CCR2 and CX3CR1 expression in 3M, 9M, and 17M old mice was conducted using flow cytometry, pre-gated on CD45\u003csup\u003e+\u003c/sup\u003eSiglecF\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e cells. (c, d) RNA sequencing (RNA-seq) data showed increased expression of Ccr2 and Ccl2 genes in CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e MoAMs from old mice compared to the resident AMs (CD11c\u003csup\u003e+\u003c/sup\u003e) in both young and old mice. (e) Schematic representation of the experimental approaches used. Adoptive transfers of bone marrow monocytes from CCR2 knockout-GFP mice were performed via tail vein injections (i.v.) into both young and old recipient mice. On day 5, BAL was collected from the recipients. Created in BioRender. Pahari, S. (2025) https://BioRender.com/p14c931. (f) Confocal microscopy revealed GFP-positive cells (GFP: green, Nucleus: DAPI-blue) primarily observed in the bloodstream, rather than the alveolar compartment. (g) A bar graph summarizes the cumulative data from 6 young (3M) and 5 old (18M) mice, presented as mean ± SEM; **** p \u0026lt; 0.0001. Each dot represents an individual mouse, with 5-7 microscopic fields (20X) containing more than 100 (BAL cells) to over 500 (blood cells). (h) Pictorial diagram illustrates a significant migration defect in CCR2-KO-GFP bone marrow monocytes, identifying the critical role of CCR2 in this recruitment process. Created in BioRender. Pahari, S. (2025) \u003ca href=\"https://BioRender.com/z00e129\"\u003ehttps://BioRender.com/z00e129\u003c/a\u003e. The data are representative of 2-3 independent experiments and expressed as mean ± SEM, with ordinary one-way ANOVA and Sidak's multiple comparisons test applied. Statistical significance is indicated as ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/3ceeff18f63b6bf1f5764908.png"},{"id":87545362,"identity":"51f624ed-c3f3-449c-a458-eb67de5d6d6b","added_by":"auto","created_at":"2025-07-25 04:45:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":436912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKinetic adoptive transfer study determines mAM turnover.\u003c/strong\u003e CD45.1 monocytes (donor cells) were isolated from the bone marrow of young mice and transferred (at 1.5x10\u003csup\u003e6\u003c/sup\u003e cells per mouse) via tail-vein injections (i.v.) into young and old CD45.2 recipient mice. After 1, 3, 5, 7, and 10 days, BAL was performed. Recovered CD45.1 AMs were labeled for flow cytometry analysis. (a) Panel provides a schematic representation of the experimental approaches used. Created in BioRender. Pahari, S. (2025) \u003ca href=\"https://BioRender.com/p76z776\"\u003ehttps://BioRender.com/p76z776\u003c/a\u003e. (b) Flow cytometry dot plot data indicate that CD45.1 monocytes actively migrated into the alveolar region and differentiated into AMs, with peak differentiation occurring by Day 5. The red arrow highlights the maximum recruitment of CD45.1 positive cells at Day 5, followed by a gradual reduction in the percentage of recruited AMs over time in old mice. (c) The bar graph presents cumulative data showing the percentage of CD45.1 cells pre-gated on SiglecF\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e AMs from five mice per group. (d) Flow cytometry dot plot illustrates CD45.2 resident AMs pre-gated on SiglecF\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e populations. (e) Bar graph demonstrates cumulative data on CD45.2 positive resident AMs, which remain consistent in both young and old mice. (f) RNA-seq analysis reveals that the genes Mmp2 and Mmp14 are upregulated in CD11c+CD11b+ MoAMs from old mice compared to the resident AMs (CD11c\u003csup\u003e+\u003c/sup\u003e) in both age groups. Data are representative of 3 independent experiments and are expressed as mean ± SEM, analyzed using ordinary one-way ANOVA with Sidak's multiple comparisons test. Statistical significance is indicated as follows: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/1ec16faff1560c6f79864fbb.png"},{"id":87545359,"identity":"ac074799-82fb-4d71-a43f-a1e392075be0","added_by":"auto","created_at":"2025-07-25 04:45:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":306008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdoptive transfer of CD45.1 monocytes and treatment with FasL NA/LE antibody extends the lifespan of mAMs. \u003c/strong\u003e(a\u0026amp;b) Murine AMs (mAMs) were isolated through BAL from young (3 months) and old (18 months) mice, then stained with fluorochrome-labeled Fas (CD95) antibodies. mAM subpopulations were analyzed by flow cytometry, using CD45\u003csup\u003e+\u003c/sup\u003eSiglecF\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e gating. Bar graph presents cumulative data from 4 mice per group, expressed as mean ± SEM. Statistical analysis was performed using an unpaired Student’s ‘t’ test where *p ≤ 0.05 is considered significant. (c) Schematic representation of the experimental approach is shown. Young and old CD45.2 mice were treated i.t. with FasL neutralizing antibody (CD178, FasL NA/LE ab) (100 µL, 0.5 mg/mL), or an isotype control antibody. Concurrently, CD45.1 young mice bone marrow monocytes (isolated by magnetic sorting for Ly6c\u003csup\u003e+\u003c/sup\u003e, negative selection) were transferred i.v. AMs were isolated from BAL of each group on day 3. Created in BioRender. Pahari, S. (2025) \u003ca href=\"https://BioRender.com/q98c015\"\u003ehttps://BioRender.com/q98c015\u003c/a\u003e. (d) Flow cytometry analysis histogram plots demonstrate a reduction in Fas (CD95) receptor expression following treatment with FasL ab. (e) Adoptive transfer of CD45.1 monocytes, combined with the blockade of FasL by FasL ab, effectively reduced the recruitment of CD45.1 MoAMs in old mice compared to the isotype-treated group. Data are expressed as mean ± SEM, analyzed by ordinary one-way ANOVA with Sidak's multiple comparisons test. Statistical significance is indicated as *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001. (f) TNF levels were measured in young and old mice treated with FasL ab in the isolated day 5 BAL fluid. (g) CD45.1 young monocytes were transferred to both young and old CD45.2 mice by i.v., with i.t. treatment using FasL ab on day 5 to assess the survival of already immigrated CD45.1 cells in the alveolus. BAL conducted 2- and 5-days post i.t. Ab administration. The experimental approach is depicted. (h) Flow cytometry analysis shows CD45.1 cells were maintained with FasL ab treatment. In contrast, MoAM numbers were reduced in the isotype control ab-treated group. (i) Another experimental approach aimed at depleting resident AMs by Clodrolip. and subsequent i.t. installation of FasL ab together with CD11c\u003csup\u003e+\u003c/sup\u003e AMs from young mice. BAL was conducted 2 days after AM and Ab administration and donor AMs were analyzed by flow cytometry. \u0026nbsp;(j) Flow cytometry analyzed bar graph shows the conversion of CD11c\u003csup\u003e+\u003c/sup\u003e donor AMs to a CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e phenotype in old but not young mice. Clodrolip- treated old mice treated with FasL ab show less conversion into CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e mAMs. (k, l) Flow cytometry-generated bar graphs illustrate the induction of Caspase-8 and Caspase-3/7 mediated apoptosis in the isotype-treated groups, which was reduced following treatment with FasL ab. (m) RNA-seq analysis revealed increased Caspase-3 gene expression in CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e MoAMs from old mice compared to resident AMs (CD11c+) in both young and old mice. (n) A schematic representation shows the extrinsic pathway of apoptosis, specifically those mediated by FasL, Caspase-8 and Caspase-3/7 in CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e MoAMs. Created in BioRender. Pahari, S. (2025) \u003ca href=\"https://BioRender.com/f26m685\"\u003ehttps://BioRender.com/f26m685\u003c/a\u003e. Data are representative of 3 independent experiments and are expressed as mean ± SEM, analyzed by ordinary one-way ANOVA with Sidak's multiple comparisons test. Statistical significance is marked as *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001. Each dot indicates an individual mouse/group. \u003cstrong\u003eSee also Figure S16.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/3ec9f084462ddba4d47c40a4.png"},{"id":87545360,"identity":"51b67f35-f6f3-4da6-af81-3e9d2182d517","added_by":"auto","created_at":"2025-07-25 04:45:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":521968,"visible":true,"origin":"","legend":"\u003cp\u003eThe pictorial model compares the lung alveolar space and alveolar macrophages (AMs) in young/adult versus old/elderly individuals. In young/adult lungs, the environment is less inflammatory, resulting in lower recruitment of CD11b\u003csup\u003e+\u003c/sup\u003e Ly6C\u003csup\u003e+\u003c/sup\u003e CD11c\u003csup\u003e-\u003c/sup\u003e monocytes via the CCR2 receptor. Most resident AMs (TRAM) are CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e-\u003c/sup\u003e phenotypically. In elderly lungs, inflammation increases due to molecules like Fas, CCL2, and CCL3, enhancing monocyte recruitment and leading to more monocyte-derived AMs (MoAMs) with a CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e Ly6C\u003csup\u003e+\u003c/sup\u003e phenotype. This shift occurs in mouse AMs (mAMs), human AMs (hAMs), and baboon/rhesus AMs (bAMs/rAMs). AMs show a metabolic shift from OXPHOS to glycolysis in old age. CCR2-deficient monocytes (in green) remain in circulation and cannot reach the alveolar region. Neutralizing Fas reduces inflammation and caspase-mediated apoptosis in mAMs and recruits fewer inflammatory cells. This model was created using Created in Created in BioRender. Pahari, S. (2025) https://BioRender.com/r77q644.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/5f3abf30e07c568f81c266d5.png"},{"id":87546087,"identity":"6fd5cc27-f2e7-4a2f-a52f-bf658f4417f6","added_by":"auto","created_at":"2025-07-25 05:01:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6524364,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/869f7491-5867-40e8-8c69-e70a69c222c0.pdf"},{"id":87544785,"identity":"b5051e23-3be2-4c68-9455-93e5d6ef9788","added_by":"auto","created_at":"2025-07-25 04:37:41","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":84136,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1. Differential expression of RNAs in flow cytometry-sorted CD11c\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eCD11b\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mAMs versus TRAMs from old mice (CD11c\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e). \u003c/strong\u003e(a) Upregulated genes with false discovery rate (FDR) adjusted p-value \u0026lt;0.05 and log2 fold change \u0026gt;1. (b) Downregulated genes with false discovery rate (FDR) adjusted p-value \u0026lt;0.05 and log2 fold change \u0026lt; -1.\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/09c15e553b8c26e0b519139d.xlsx"},{"id":87545357,"identity":"9939bab2-5272-46e3-84c0-8bf5c58990a4","added_by":"auto","created_at":"2025-07-25 04:45:41","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":54549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2. Differential expression of RNAs in TRAMs in old mice versus young mice. \u003c/strong\u003e(a) Upregulated genes with false discovery rate (FDR) adjusted p-value \u0026lt;0.05 and log2 fold change \u0026gt;1. (b) Downregulated genes with false discovery rate (FDR) adjusted p-value \u0026lt;0.05 and log2 fold change \u0026lt; -1.\u003c/p\u003e","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/4e0130d6baf46d6953837c36.xlsx"},{"id":87544791,"identity":"e6142971-2390-4ab6-8ade-091008af8879","added_by":"auto","created_at":"2025-07-25 04:37:41","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":76878,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3. Key Resources Table. \u003c/strong\u003eA detailed listing of reagents or resources, their sources, and corresponding identifiers.\u003c/p\u003e","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/8fcdbc2d290c1c4d3467f271.docx"},{"id":87544799,"identity":"66acbec8-adbb-4185-ae9e-b6bcde0d8d6d","added_by":"auto","created_at":"2025-07-25 04:37:41","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3260389,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/68c03a7dd9062591102bb919.pdf"},{"id":87544797,"identity":"ba372b44-a622-4bb2-905a-b2d60d18a4a7","added_by":"auto","created_at":"2025-07-25 04:37:41","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":20859,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7123735/v1/10f7d873020668ad2da44c9d.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Lung environment in healthy old age shapes the phenotype and CCR2-mediated recruitment of a subset of apoptotic, high-turnover alveolar macrophages","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eThe alveolar microenvironment\u0026rsquo;s unique inflammatory profile impacts alveolar macrophage recruitment and phenotype\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eThere is a significant increase in two recruited alveolar macrophage subpopulations in old age observed across mammalian species\u003c/li\u003e\n \u003cli\u003eRecruitment of monocyte-derived CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e alveolar macrophages (MoAMs) in old age is CCR2-dependent\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRecruited MoAMs in old age are relatively short-lived and undergo Fas-mediated apoptosis\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eBy 2030, one in six people globally will be 60 or older, totaling 1.4\u0026nbsp;billion. By 2050, this group will double to 2.1\u0026nbsp;billion, and the number of individuals aged 80 and older will triple \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Old individuals are more susceptible to severe illnesses and increased mortality due to various diseases\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Aging is associated with a decline in immune function, a phenomenon known as immunosenescence, which has been studied primarily within the adaptive immune system\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In contrast, \"inflammaging,\" or chronic inflammation linked to innate immune system dysfunction with age, is less well understood, especially in the lung alveoli\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Aging lungs are influenced by various biological pathways due to environmental exposures, leading to oxidative stress, inflammation, telomere shortening, DNA damage, mitochondrial dysfunction, epigenetic instability, immune dysregulation, and impaired proteostasis\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Oxidative stress and mitochondrial dysfunction in old age, even in the absence of disease, collectively contribute to an inflammatory alveolar environment significantly affecting the phenotype and recruitment of alveolar macrophages (AMs). AMs, the first immune cells to interact with airborne pathogens and inhaled particulates, are essential for lung health. Despite this knowledge, little is known about the characteristics of AMs recruited to the lung in old age.\u003c/p\u003e\u003cp\u003eAM development and function is influenced by signals from the lung microenvironment. Shortly after birth, fetal monocytes quickly transform into a stable, self-renewing population called tissue-resident AMs (TRAMs). In mice, TRAMs persist for extended periods without the need for input from bone marrow-derived monocytes \u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. TRAMs are located within epithelial surface lining fluid, where they attach to the lung epithelium via integrins and play a crucial role in sampling, responding to, and eliminating pathogens and particulates in the alveoli \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. With TRAM depletion or inflammatory conditions, monocytes are recruited to the alveoli, where they undergo differentiation into monocyte-derived alveolar macrophages (MoAMs) through a progressive reshaping of their epigenome and transcriptome \u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. We recently identified distinct AM subpopulations in old mice that are significantly increased relative to young mice \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. AMs in young mice consist mainly of CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e\u0026minus;\u003c/sup\u003e TRAMs \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e whereas in old mice, there are marked increases in subset populations that are CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e and CD11c\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e. The importance of these increased subpopulations relates to their unique inflammatory profile and inability to control \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e growth, suggesting a potential sentinel population for tuberculosis susceptibility in old age \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The appearance of these AM subpopulations in old age result from 3 potential mechanisms: i) cell-autonomous changes, ii) alterations in the lung microenvironment, and/or iii) the replacement of TRAMs with MoAMs due to repeated insults over the lifespan.\u003c/p\u003e\u003cp\u003eHerein we investigate these mechanisms in AMs from old mice (mAMs), humans (hAMs) and non-human primates (NHPs; baboons, bAMs; Rhesus macaque, rAMs) using several approaches. Flow cytometry and fluorescence microscopy indicate similar AM phenotype changes with old age across species, \u003cem\u003ei.e.\u003c/em\u003e, increased CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e and CD11c\u003csup\u003e\u0026minus;\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e AM subpopulations. RNA-seq and qRT-PCR analyses reveal unique transcriptional profiles of these subpopulations, providing insight into their unique functions. Our experiments in mice demonstrate the importance of the altered alveolar environment in old age in AM recruitment and phenotypic change, including adoptive transfer experiments in mice to confirm monocyte origin. Additionally, we found that recruitment of MoAMs to the alveolar space requires the chemokine CCR2. The recruited MoAMs are relatively short-lived, undergoing increased apoptosis through Fas activation.\u003c/p\u003e\u003cp\u003eThis work enhances our understanding of the cellular and molecular processes associated with old age, paving the way for novel approaches to interventions that are more effective in this unique tissue environment for elderly patients with lung diseases.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCharacterization of old/elderly AMs from mice (mAMs), humans (hAMs) and non-human primates (NHPs; baboons: bAMs and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003erhesus\u003c/strong\u003e \u003cstrong\u003emacaque: rAMs\u003c/strong\u003e\u003cstrong\u003e): increased CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e subpopulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess AM subpopulations across the species in mice, humans and NHPs, we performed bronchoalveolar lavage (BAL) to obtain their AMs (\u003cstrong\u003eFigure 1\u003c/strong\u003e). We focused on samples from young and old mice (3 months and 18 months old, respectively), healthy adults \u003cem\u003eversus\u0026nbsp;\u003c/em\u003ehealthy elderly humans (ages 20-50 and \u0026ge; 65 years old), and young and old baboons/rhesus (3-5 years and over 15 years old). We identified the presence of distinct cell populations of AMs, lymphocytes, neutrophils, and eosinophils in the BAL of old/elderly subjects compared to young/adults on cytospun samples. AMs were the most prominent population in both groups (\u003cstrong\u003eFigure S1\u003c/strong\u003e). mAM, hAM and bAM subgroups were distinguished by their respective cell surface receptor expression using flow cytometry: mAMs were CD45\u003csup\u003e+\u003c/sup\u003eSiglecF\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e, hAMs were CD206\u003csup\u003e+\u003c/sup\u003eCD64\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e, and bAMs were CD45\u003csup\u003e+\u003c/sup\u003eCD64\u003csup\u003e+\u003c/sup\u003eCD206\u003csup\u003e+\u003c/sup\u003eCD163\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e \u003csup\u003e19-22\u003c/sup\u003e. To identify CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e AM subpopulations, cells were pre-gated on CD45\u003csup\u003e+\u003c/sup\u003eSiglecF\u003csup\u003e+\u003c/sup\u003e (mAMs) and CD45\u003csup\u003e+\u003c/sup\u003eCD64\u003csup\u003e+\u003c/sup\u003e (hAMs, bAMs) populations (\u003cstrong\u003eFigure 1 \u0026amp; Figure S2, 3, 4\u003c/strong\u003e). TRAMs were the predominant population of cells that express CD11c cell surface receptors. We previously identified an increased subpopulation of CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e monocyte-derived AMs (MoAM) and a very small subpopulation of CD11c\u003csup\u003e-\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e cells in old mice \u003csup\u003e2\u003c/sup\u003e. CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e MoAMs exhibit a unique inflammatory signature based on defined markers and enhanced growth of \u003cem\u003eMycobacterium tuberculosis\u0026nbsp;\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e. Our current results confirm these increased subpopulations in young and old mice (\u003cstrong\u003eFigure 1a, b\u003c/strong\u003e). Our findings in hAMs/bAMs revealed three distinct markedly increased AM subpopulations in the old/elderly groups (\u003cstrong\u003eFigure 1c-f\u003c/strong\u003e). The most prominent subpopulation was designated as CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e-\u003c/sup\u003e TRAMs. The 2 smaller subpopulations were CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e and CD11c\u003csup\u003e-\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e. Notably, the presence of these two latter subpopulations increased more than ten-fold in old baboons and elderly humans when compared to their younger counterparts (\u003cstrong\u003eFigure 1c-f\u003c/strong\u003e). Further analysis indicated that the expression levels of CD11b, CD11c, CCR2, Fas, HLA-DR, CD14, and CD16 were elevated in the CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e elderly hAM subpopulation (\u003cstrong\u003eFigure 1g\u003c/strong\u003e). There was no significant change in CX3CR1 expression in the CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e subpopulation of elderly hAMs (\u003cstrong\u003eFigure 1g\u003c/strong\u003e). CD14\u003csup\u003e+\u003c/sup\u003e CD16\u003csup\u003e+\u003c/sup\u003e cells are recognized as inflammatory monocytes\u003csup\u003e23\u003c/sup\u003e. We hypothesize that these inflammatory monocytes are recruited to the lungs and \u003cem\u003ein situ\u003c/em\u003e differentiated into the CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e MoAM subpopulation in elderly individuals. Flow cytometry analysis of bAMs revealed that CD11b, CD86, HLA-DR, CD206, CD163, CD64, and CD36 were upregulated in old age when compared to bAMs from young baboons (\u003cstrong\u003eFigure S4\u003c/strong\u003e). Like hAMs, the chemokine receptor CCR2, but not CX3CR1, was upregulated in old age bAMs when compared to young bAMs (\u003cstrong\u003eFigure S4; Figure S5\u003c/strong\u003e). Further analysis indicated that the expression levels of CD64, CD11c, CD11b, HLA-DR, CD163, and CCR2 were elevated in the CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e elderly bAM subpopulation (\u003cstrong\u003eFigure S5\u003c/strong\u003e). We also examined the expression levels of AM surface proteins in rhesus macaques. Confocal microscopy showed increased expression of CD163, CD206, CD11b and CD11c in old age rAMs (\u003cstrong\u003eFigure S6\u003c/strong\u003e). Our previous research found that CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e mAMs from old mice exhibit elevated levels of cell surface receptors Ly6C and CD115, consistent with a monocytic origin\u0026nbsp;\u003csup\u003e2\u003c/sup\u003e. We extended this analysis in mice to examine at what age changes in cell surface receptor expression occur. As age progresses, we observed a gradual increase in CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e recruited AMs with CD95 (Fas) expression (\u003cstrong\u003eFigure 1h\u003c/strong\u003e). Overall, these findings demonstrate significant changes in AM surface receptor expression in healthy old age and the finding of increased subpopulations is consistent across mice, humans and NHPs. Additionally, the findings suggest that the newly recruited AM subpopulations in old age differentiate from blood monocytes and that the chemokine CCR2 plays an important role in this recruitment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUnique transcriptional profiles of the 3 mAM subpopulations in old mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further characterize the AM subpopulations in mice, we conducted a transcriptomic analysis using bulk-RNA sequencing (RNA-seq) on four flow cytometry-sorted populations: TRAMs in young mice (CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e-\u003c/sup\u003e), TRAMs in old mice (CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e-\u003c/sup\u003e), the increased subpopulation of CD11c\u003csup\u003e-\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e mAMs in old mice, and the increased subpopulation of CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e mAMs in old mice (\u003cstrong\u003eFigure 2a\u003c/strong\u003e; \u003cstrong\u003eFigure S7a\u003c/strong\u003e). CD11c\u003csup\u003e-\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e mAMs in old mice were present in very low numbers and exhibited a transcriptional profile like that of the CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e populations. Thus, we did not include them in our further analyses. Our primary focus was on comparing three subpopulations of mAMs (CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e-\u003c/sup\u003e mAMs in young and old mice (TRAMs) and CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e mAMs in old mice).\u003c/p\u003e\n\u003cp\u003ePrincipal component analysis (PCA) indicated a high degree of similarity among biological replicates within each group (\u003cstrong\u003eFigure 2b\u003c/strong\u003e). Volcano plots illustrated the false discovery rate (FDR) in relation to the magnitude of change in gene expression across a total of 14,509 genes. 577 out of 5,300 genes (FDR \u0026lt; 0.05) were significantly upregulated, with a log2 fold change (logFC) \u0026gt;1, in the CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e mAMs (\u003cstrong\u003eTable S1a\u003c/strong\u003e). Moreover, 10 genes were downregulated (logFC \u0026lt; -1) when compared to TRAMs (CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e-\u003c/sup\u003e) in old mice (\u003cstrong\u003eFigure 2c\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eTable S1b\u003c/strong\u003e). We also compared TRAM subpopulations in young and old mice. 311 out of 4,796 genes (FDR \u0026lt; 0.05) were significantly upregulated (logFC \u0026gt; 1) in old TRAMs (\u003cstrong\u003eTable S2a\u003c/strong\u003e). Moreover, 36 genes were downregulated (logFC \u0026lt; -1, FDR \u0026lt; 0.05) in old mice when compared to TRAMs in young mice (\u003cstrong\u003eFigure S7b).\u003c/strong\u003e Furthermore, we compared the three subpopulations of mAMs in both young and old mice. \u0026nbsp;TNF signaling-related genes, including inflammatory genes, e.g., \u003cem\u003eCcl3\u003c/em\u003e, \u003cem\u003eCcl8\u003c/em\u003e, \u003cem\u003eCsf1\u003c/em\u003e, \u003cem\u003eCd40\u003c/em\u003e, \u003cem\u003eTnf\u003c/em\u003e, \u003cem\u003eCcl22\u003c/em\u003e, \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eCcl5\u003c/em\u003e, \u003cem\u003eTlr1\u003c/em\u003e, \u003cem\u003eTlr9\u003c/em\u003e, \u003cem\u003eS100a8\u003c/em\u003e, and \u003cem\u003eS100a9\u003c/em\u003e were highly upregulated in CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e mAMs in old mice (\u003cstrong\u003eFigure 2d, e\u003c/strong\u003e). Other immune response-related genes, including those involved in positive regulation of the ERK1/2 pathway, cell migration and chemotaxis were also upregulated in the old mice (\u003cstrong\u003eFigure S8a-c\u003c/strong\u003e). Notably, \u003cem\u003eHif1a\u003c/em\u003e was upregulated in CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e mAMs which suggests induction of glycolysis in this AM subpopulation. Inflammatory genes were observed in CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e mAMs (\u003cstrong\u003eFigure 2f\u003c/strong\u003e), highlighting a unique inflammatory profile consistent with a previous gene expression study \u003csup\u003e2\u003c/sup\u003e. \u0026nbsp;Quantitative PCR analysis in hAMs confirmed significantly higher levels of inflammatory, immunoregulatory, and cell death markers in elderly compared to adult hAMs (\u003cstrong\u003eFigure S9\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolic and oxidative changes of AMs in old age mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNAseq results suggested major changes in metabolism and oxidative stress in AMs in old age. HIF-1\u0026alpha; regulates glucose metabolism to promote anaerobic glycolysis and conserve ATP \u003csup\u003e24\u003c/sup\u003e. Healthy adult AMs are classically \u0026nbsp;driven primarily through oxidative phosphorylation (OXPHOS)\u003csup\u003e25\u003c/sup\u003e, but\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ethere is evidence for a metabolic shift in AMs from OXPHOS to glycolysis in old age\u003csup\u003e26\u003c/sup\u003e.\u0026nbsp;We posited that increased HIF-1\u0026alpha; expression in old AMs will lead to increased glycolysis and decreased OXPHOS, and that the complex inflammatory and oxidative alveolar environment\u0026nbsp;\u003csup\u003e27\u003c/sup\u003e in old age changes AM phenotype.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe measured glycolysis and OXPHOS by quantifying the ECAR and OCR in old \u003cem\u003eversus\u003c/em\u003e young mAMs. \u0026nbsp;Our findings revealed a significant increase in both the basal and maximal respiratory capacity and ATP production, and spare respiratory capacity, along with a moderate increase in proton leak in mAMs from old mice compared to those from young mice (\u003cstrong\u003eFigure S10a-f\u003c/strong\u003e). Additionally, glycolysis (ECAR) in the mAMs from old mice was significantly higher than that in young mice (\u003cstrong\u003eFigure S10 g-j)\u003c/strong\u003e. The basal mRNA levels of key glycolytic genes (\u003cem\u003eGlut1, Hk2, Gls1, Pfkfb3\u003c/em\u003e) were also elevated in mAMs from old \u003cem\u003eversus\u0026nbsp;\u003c/em\u003eyoung mice (\u003cstrong\u003eFigure S10j-m\u003c/strong\u003e). Previous data indicated a significant increase in TLR2 expression (both RNA and protein levels) in AMs from old mice, with higher levels observed specifically in the CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e AM subset \u003csup\u003e2\u003c/sup\u003e. Similarly, we found that TLR2 expression was elevated in hAMs from elderly individuals (\u003cstrong\u003eFigure S9d\u003c/strong\u003e). To investigate the TLR2 response in mAMs from old mice, we stimulated these cells \u003cem\u003ein vitro\u003c/em\u003e with the TLR2 agonist Pam\u003csub\u003e3\u003c/sub\u003eCSK\u003csub\u003e4\u003c/sub\u003e. Following stimulation, we observed elevated RNA expression levels of aerobic metabolism-related genes, including \u003cem\u003eHif1\u0026alpha;, Cox2, CD74, Mif, Glut1, Hk2, Pfkb3, Gls1, Slc25a,\u003c/em\u003e and \u003cem\u003eAcly\u003c/em\u003e (\u003cstrong\u003eFigure S11\u003c/strong\u003e). In old mAMs, there was an increase in both OXPHOS (OCR) and glycolysis (ECAR), attributed to heightened glucose utilization, mitochondrial respiration, and ATP production. However, this pattern was not observed in hAMs, where the glycolysis-related gene (LDHA) was elevated, and the OXPHOS-related gene (NDUFAF6) was decreased in old age (\u003cstrong\u003eFigure S12\u003c/strong\u003e). One possible explanation for these differences is that the metabolic pathways (OXPHOS \u003cem\u003eversus\u0026nbsp;\u003c/em\u003eglycolysis) in mice operate independently. Another possibility is that old mAMs have high glucose availability to drive glycolysis, while simultaneously having low ATP levels, which activates the citric acid cycle to produce significantly more ATP through OXPHOS \u003csup\u003e25\u003c/sup\u003e. Finally, we observed a significant increase in mitochondrial ROS in old mAMs as compared to young mAMs (\u003cstrong\u003eFigure S13a-b\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe inflammatory alveolar environment in old age shapes the AM phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAMs within the alveolar environment are bathed in an aqueous alveolar hypophase, recycle surfactant, and contact Type I and type II epithelial cells \u003csup\u003e28\u003c/sup\u003e. Delicate alveolar air sacs enable gas exchange. Such an environment requires a highly regulated inflammatory response to enable normal lung function. Recent studies in old age have shown significant changes to protein production and function in the alveolar hypophase, e.g., pro-inflammatory cytokines, altered surfactant proteins and lipids, and modifications to complement components. These changes contribute to an environment with heightened oxidative stress, leading to an enhanced lung inflammatory state\u003csup\u003e3,29\u003c/sup\u003e. We next explored the role of this altered alveolar environment in old age in the phenotype and recruitment of the AM subpopulations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs a first approach, we depleted TRAMs from both young and old mice by administering two doses of clodronate liposomes (clodrolip) intratracheally (i.t.) on Day 0 and Day 3 to induce apoptotic cell death (\u003cstrong\u003eFigure 3a-b\u003c/strong\u003e) \u003csup\u003e30,31\u003c/sup\u003e. We achieved ˃ 85-90% AM depletion by Day 5 to 8 by Cytospin (\u003cstrong\u003eFigure 3b; Figure S14\u003c/strong\u003e) and flow cytometry (\u003cstrong\u003eFigure 3c-d\u003c/strong\u003e). On Day 5, we performed adoptive transfers of CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e-\u003c/sup\u003e BAL isolated TRAMs from young mice by introducing them i.t. (1x10\u003csup\u003e6\u003c/sup\u003e cells) into both young and old mice (\u003cstrong\u003eFigure 3a\u003c/strong\u003e) to assess the effect of the lung environment on the phenotypic changes of recruited AMs in old mice. The adoptive transferred TRAMs from young mice more effectively converted to a CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e mAMs in old mice after 48h (\u003cstrong\u003eFigure 3e\u003c/strong\u003e). Thus, we conclude that the phenotype changes of newly transferred TRAMs are due to the inflammatory alveolar milieu in old age. In concert with this, we observed elevated levels of soluble TNF, FasL (CD178), and S100A9 proteins in the isolated BAL fluid from old mice (\u003cstrong\u003eFigure 3f\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next employed adoptive transfer using CD45.1 congenic mice (\u003cstrong\u003eFigure 4a\u003c/strong\u003e). CD45.1 bone marrow monocytes were isolated from young mice and transferred to young and old CD45.2 mice via tail vein injection. After 5 days, BAL was performed, and the recovered CD45.1 mAMs were labeled for flow cytometry analysis which revealed that they effectively migrated into the alveolar spaces of old mice, but much less so in young mice (\u003cstrong\u003eFigure 4b\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, we employed the Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosa\u003csup\u003eTdT\u0026nbsp;\u003c/sup\u003efate mapper mouse model to assess the origin of recruited MoAMs (\u003cstrong\u003eFigure 4a\u003c/strong\u003e).\u0026nbsp;The Ms4a3 gene is specifically expressed in monocyte-committed progenitors \u003csup\u003e32\u003c/sup\u003e, and in this model, the bone marrow cells are labeled with an irreversible tdTomato red fluorescent marker, which identifies macrophage progenitor cells while excluding dendritic cell progenitors \u003csup\u003e32\u003c/sup\u003e. We adoptively transferred BM monocytes from the Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosa\u003csup\u003eTdT\u0026nbsp;\u003c/sup\u003emice into both young and old mice via tail vein injection (\u003cstrong\u003eFigure 4a\u003c/strong\u003e). In young mice, we observed the presence of Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosa\u003csup\u003eTdT\u003c/sup\u003e monocytes in the peripheral circulation, but these cells did not reach the lungs. In contrast, old mice exhibited fewer monocytes in circulation, suggesting that these cells likely migrated to the lungs and other organs (\u003cstrong\u003eFigure S15\u003c/strong\u003e). Flow cytometry, confocal microscopy, and histopathology analyses revealed a higher recruitment of Ms4a3-derived MoAMs in old mice into the alveolar space (\u003cstrong\u003eFigure 4c-e; Figure S15\u003c/strong\u003e). Furthermore, the Siglec F+Ms4A3 double-positive AM population corresponded to the CD11b\u003csup\u003e+\u003c/sup\u003eCD11c\u003csup\u003e+\u003c/sup\u003e mAM subpopulation (\u003cstrong\u003eFigure 4f\u003c/strong\u003e). Additional flow cytometry data corroborated that after the adoptive transfer of Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosa\u003csup\u003eTdT\u003c/sup\u003e monocytes, old mice showed increased recruitment of both the Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosa\u003csup\u003eTdT\u003c/sup\u003e and CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e AM subpopulations (\u003cstrong\u003eFigure 4g-h\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eRNA sequencing analysis revealed that several Ms4a-related gene sets (Ms4a4c, Ms4a4a, Ms4a6b, Ms4a7, and Ms4a14) \u003csup\u003e33\u003c/sup\u003e are upregulated in CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e MoAMs from old mice compared to the TRAMs in both young and old mice (\u003cstrong\u003eFigure 2d, 4i\u003c/strong\u003e). These data correlate with the evidence that the MoAMs in old mice originate from bone marrow monocytes (CD11b\u003csup\u003e+\u003c/sup\u003e, Ly6c\u003csup\u003e+\u003c/sup\u003e, CD11c\u003csup\u003e-\u003c/sup\u003e), which subsequently differentiate into CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e MoAMs within the lung environment. Overall, the findings demonstrate the importance of the altered alveolar environment in old age in recruiting new AM subpopulations derived from peripheral monocytes. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecruitment of MoAMs in old age is dependent on a CCR2-mediated pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the mechanism(s) involved in the recruitment of MoAMs to the lungs in old age, we focused on C-C chemokine receptor type 2 (CCR2), which in adult mice is essential for activating and recruiting monocytes and macrophages to\u0026nbsp;kidney, liver, myocardium and skin injury tissue sites, contributing to tissue inflammation \u003csup\u003e34,35\u003c/sup\u003e. TRAMs are known to lack CCR2 expression \u003csup\u003e36\u003c/sup\u003e. In contrast, our results show that the CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e MoAMs express CCR2 on their surface in the alveolus, which increases with age (\u003cstrong\u003eFigure 5a\u003c/strong\u003e), a finding also shown on hAMs (\u003cstrong\u003eFigure 1g\u003c/strong\u003e) and bAMs (\u003cstrong\u003eFigure S4b; S5b\u003c/strong\u003e). There was no significant change in surface expression of CX3CR1 with age in CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e MoAMs, although gene expression increased in hAM (\u003cstrong\u003eFigure 1g;\u003c/strong\u003e \u003cstrong\u003eFigure 5b;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFigure S4b; Figure S9k\u003c/strong\u003e). RNA-seq data also demonstrated significant upregulation of both Ccr2 and Ccl2 genes in CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e MoAMs (\u003cstrong\u003eFigure 2f; 5 c, d\u003c/strong\u003e) and increased CCR2 expression in old hAMs (\u003cstrong\u003eFigure S9l\u003c/strong\u003e). We conducted adoptive transfers of BM monocytes from CCR2 knockout-GFP knockin mice via tail vein injections into both young and old recipient mice (\u003cstrong\u003eFigure 5e\u003c/strong\u003e). The majority of these CCR2-KO-GFP bone marrow monocytes remained in the bloodstream (\u003cstrong\u003eFigure 5f, g\u003c/strong\u003e) and did not reach the alveolar space in young or old mice, indicating an essential role of CCR2 in facilitating this process in old age (\u003cstrong\u003eFigure 5h\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecruited MoAMs are relatively short-lived when compared to resident AMs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the turnover of recruited MoAMs, we adoptively transferred CD45.1 young monocytes into both young and old mice via tail vein injection. At specified time points (Days 1, 3, 5, 7, or 10), we isolated and analyzed the mAM subpopulations in the alveolar space, identified by the markers SiglecF\u003csup\u003e+\u003c/sup\u003eCD45.2 for TRAMs and CD45.1 for recruited cells (\u003cstrong\u003eFigure 6a\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs shown earlier (\u003cstrong\u003eFigure 4b\u003c/strong\u003e), CD45.1 monocytes actively migrated into the alveoli and differentiated into MoAMs in old age. Peak migration occurs on Day 5, and following this peak, we noted a gradual reduction in the number of recruited MoAMs over time (\u003cstrong\u003eFigure 6 b, c\u003c/strong\u003e). In contrast, the population of TRAMs remained stable throughout the observed period (\u003cstrong\u003eFigure 6d, e\u003c/strong\u003e). Based on these observations and induction of Fas expression on MoAMs (\u003cstrong\u003eFigures 1g, h; S9h\u003c/strong\u003e), we hypothesized that recruited MoAMs have a transient existence characterized by rapid turnover, primarily driven by Fas-mediated apoptosis and continuous turnover of CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e MoAMs. Moreover, RNA-seq analysis of flow cytometry sorted CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e MoAMs revealed a significant increase in \u003cem\u003eMmp2\u003c/em\u003e (matrix metallopeptidase-2) and \u003cem\u003eMmp14\u003c/em\u003e (\u003cstrong\u003eFigure 6f, g\u003c/strong\u003e), suggesting their involvement in cell migration and turnover within the alveoli.\u0026nbsp;During cell migration, MMP-2 breaks down extracellular matrix components to enable movement, while MMP-14 activates MMP-2 at the cell surface, initiating the process \u003csup\u003e37\u003c/sup\u003e.\u0026nbsp;\u0026nbsp;RNA-seq data also showed upregulation of Mmp9 gene expression in CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e MoAMs (\u003cstrong\u003eFigure 2d\u003c/strong\u003e). Mmp9 cleaves the extracellular region of FasL (CD178), leading to the release of a soluble form of FasL (sFasL)\u0026nbsp;\u003csup\u003e38\u003c/sup\u003e. Overall, our findings support the notion of a dynamic balance between resident and recruited AM populations in old age, where newly recruited MoAMs exhibit high turnover due to the inflammatory environment they encounter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRole of Fas in monocyte recruitment to the lungs and controlling the inflammatory environment of the alveolus in old age\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results indicate a significant increase in Fas receptor (CD95) expression in old mice (\u003cstrong\u003eFigure 1h\u003c/strong\u003e; \u003cstrong\u003eFigure 7 a, b\u003c/strong\u003e) associated with turnover of recruited MoAMs. Fas signaling can induce the production of pro-inflammatory cytokines \u003csup\u003e39\u003c/sup\u003e, including those from macrophages, and higher Fas expression in the elderly correlates with an increase in inflammatory cytokines \u003csup\u003e39\u003c/sup\u003e. Chronic inflammation driven by Fas in older adults, particularly observed for alveolar epithelial cells, is associated with age-related diseases \u003csup\u003e40-42\u003c/sup\u003e. The role of resident and newly immigrated AMs in Fas activation has not been explored.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe first hypothesized that enhanced Fas-mediated signaling of resident AMs in old age creates a more favorable inflammatory environment for the recruitment of monocytes to the lungs, facilitating their differentiation into MoAMs. To test this, we combined Fas Ligand (FasL, CD178) neutralizing (NA/LE) Ab administered i.t. with the adoptive transfer of CD45.1 monocytes by i.v. on Day 0 in young and old mice (\u003cstrong\u003eFigure 7c\u003c/strong\u003e). We observed that FasL neutralization effectively reduced Fas expression and the recruitment of CD45.1 MoAMs in old mice (\u003cstrong\u003eFigure 7d-e\u003c/strong\u003e). TNF levels were increased significantly in the isolated day 5 BAL fluid of old mice and administration of FasL Ab administered on day 0 effectively reduced TNF levels in these old mice (\u003cstrong\u003eFigure 7f\u003c/strong\u003e). These studies suggested that Fas neutralization can reverse the inflammatory alveolar environment of old mice to more closely resemble the environment of young mice leading to reduced MoAM recruitment.\u003c/p\u003e\n\u003cp\u003eWe next hypothesized that the turnover of recruited MoAMs in old mice can be slowed by the addition of FasL neutralizing Ab. To test this, we administered FasL Ab 5 days after the adoptive transfer of CD45.1 monocytes via i.v. [day in which recruited MoAMs is maximal (\u003cstrong\u003eFigure 6b-c\u003c/strong\u003e; \u003cstrong\u003eFigure 7g\u003c/strong\u003e)]. With Ab treatment, the recruited MoAMs are maintained for a longer period relative to those in the isotype control group (\u003cstrong\u003eFigure 7h\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further understand how Fas regulates the lung microenvironment, we first depleted TRAMs by Clodrolip treatment and then added FasL neutralizing Ab along with adoptively transferring TRAMs from young mice to young and old mice \u003cem\u003evia\u003c/em\u003e i.t., then assessed the phenotype of donor AMs (\u003cstrong\u003eFigure 7i\u003c/strong\u003e). Ab treatment had no effect on the newly added TRAMs in young recipient mice. In contrast, Ab treatment nearly abolished the increased CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e AM population in the control group in old mice (\u003cstrong\u003eFigure 7j\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe role of FasL NA/LE Ab treatment in controlling AM apoptosis in old age \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results on Fas/FasL expression and AM turnover indicated that one mechanism for the observed turnover is FasL-mediated induction of apoptosis \u003csup\u003e43,44\u003c/sup\u003e. The use of FasL NA/LE Ab may improve macrophage turnover and functionality by preventing premature apoptosis \u003csup\u003e12\u003c/sup\u003e. We extended the experimental approach noted above where we combined FasL NA/LE Ab administration by i.t. with the adoptive transfer of CD45.1 monocytes \u003cem\u003evia\u003c/em\u003e i.v. on Day 0 in young and old mice (\u003cstrong\u003eFigure 7c, S16a\u003c/strong\u003e) to assess the degree of apoptosis using the Annexin-V assay. The results showed that the addition of FasL NA/LE Ab reduced the level of apoptosis in MoAMs (\u003cstrong\u003eFigure S16 a-c\u003c/strong\u003e), with the apoptotic pathway mediated through caspase 3/7 (\u003cstrong\u003eFigure S16d\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eFas-mediated apoptosis occurs through the extrinsic pathway rather than the intrinsic pathway \u003csup\u003e43\u003c/sup\u003e. Caspase 3/7 are executor caspases involved in both pathways. Our results provide evidence that recruited MoAMs primarily undergo apoptosis through the extrinsic pathway, specifically those mediated by FasL and Caspase-8, followed by the downstream activation of Caspases-3/7 (\u003cstrong\u003eFigure 7k-l\u003c/strong\u003e). In concert with this, RNA-seq analysis of flow cytometry sorted CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e MoAMs revealed a significant increase in Caspase-3 (\u003cstrong\u003eFigure 7m\u003c/strong\u003e). Overall, our data provide evidence that enhanced Fas-mediated signaling in old age leads to extrinsic apoptosis and consequently, high turnover of recruited MoAMs (\u003cstrong\u003eFigure 7n\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe impact of healthy aging on the nature of cellular function in tissue environments is significant, particularly in the lung, which is constantly exposed to inhaled particulates and microbes. This study offers important new insights into how aging, in the absence of disease, affects the phenotype, recruitment, and turnover of AMs across mammalian species and lays the groundwork for developing interventions to mitigate respiratory diseases in older adults.\u0026nbsp;A significant finding is the increased presence of CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e MoAM subpopulations in healthy elderly subjects, which exhibit elevated inflammatory, immunoregulatory, and cell death markers compared to the resident CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e-\u003c/sup\u003e TRAMs. The inflammatory alveolar environment in old age can more effectively 1) convert TRAMs from young to a CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e phenotype in old age, and 2) recruit MoAMs from monocytes in the periphery that undergo continuous replenishment due to a relatively short half-life resulting from increased apoptosis in the inflammatory milieu (\u003cstrong\u003eFigure 8\u003c/strong\u003e). This shift towards a more inflammatory AM phenotype underscores the role of aging as a driving force behind heightened inflammatory responses and likely contributes to the increased susceptibility to respiratory diseases in older individuals.\u003c/p\u003e\n\u003cp\u003eRNA sequencing analysis demonstrates significant transcriptional shifts in mAM subpopulations in healthy old age, particularly regarding CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e mAMs. These cells exhibit significant upregulation of inflammatory genes, e.g., \u003cem\u003eCcl2, Ccl3, Ccl8, Cd40, Hif1\u0026alpha;, Il6, Tnf, S100a8, S100a9, Csf1, Mmps\u003c/em\u003e and \u003cem\u003eTlr1, 2, 9\u003c/em\u003e. The identified inflammatory profile, characterized by increased expression of TNF signaling-related genes and pro-inflammatory cytokines, aligns with the chronic low-grade inflammation commonly observed in age-associated diseases such as COPD and asthma \u003csup\u003e45\u003c/sup\u003e. Another notable finding is the metabolic shift towards glycolysis in old age mAMs, consistent with the Warburg effect, supporting the energetic demands of their inflammatory role and may contribute to tissue remodeling in the lungs in old age. Quantitative PCR analysis of hAMs corroborates our mouse RNAseq data, revealing higher levels of inflammatory and cell death markers in elderly individuals, indicating common mechanisms of age-related inflammation across mammalian species.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs we age, baseline oxidative stress and inflammation significantly contribute to the development of an inflammatory microenvironment. These two factors are interconnected and can accelerate the aging process, increasing the risk of chronic diseases\u003csup\u003e46,47\u003c/sup\u003e. Increased baseline inflammation is linked to mitochondrial dysfunction and increased mitochondrial ROS production\u003csup\u003e48\u003c/sup\u003e. The metabolic reprogramming of mAMs in old mice, characterized by a shift from OXPHOS to glycolysis, has important implications for the heightened inflammatory responses within the lungs. Our findings align with studies that have demonstrated shifts in cellular metabolism with age in other cells and tissues. \u0026nbsp;Previous research has shown that M1 macrophages or aged BMDMs display altered metabolic profiles, supporting our notion of a glycolytic shift contributing to inflammation\u003csup\u003e49,50\u003c/sup\u003e. Unexpectedly, we found that OXPHOS and glycolysis are both elevated in old mAMs, indicating that these pathways operate independently in mice. The interplay between increased ROS production and mitochondrial dysfunction is consistent with previous work regarding the relationship between oxidative stress and inflammation\u003csup\u003e51\u003c/sup\u003e. Incorporating comparative studies across mammalian species provides a deeper understanding of the cellular dynamics at play and their common underlying mechanisms.\u003c/p\u003e\n\u003cp\u003eAdoptive transfer experiments provide evidence that with old age, the alveolar environment becomes less favorable for maintaining TRAM homeostasis, leading to the recruitment and activation of MoAMs. The Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosa\u003csup\u003eTdT\u003c/sup\u003e fate mapper mouse model has proven instrumental in studying the origin of tissue macrophages \u003csup\u003e32\u003c/sup\u003e. The observed upregulation of Ms4a-related gene sets in MoAMs suggests that old age is associated with a shift in the immune landscape within the lungs, potentially driven by chronic inflammatory microenvironment. Our studies corroborate those indicating that the aging process or exposure to other environmental insults alters the immune cell composition within the lungs\u003csup\u003e5,8,15,29,52\u003c/sup\u003e. \u0026nbsp;In all, the findings further support the premise that an inflammatory microenvironment drives differentiation of circulating monocytes into unique tissue macrophage populations\u003csup\u003e53,54\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResident AMs lack CCR2 expression \u003csup\u003e36\u003c/sup\u003e, whereas CCR2 expression is high in monocytes in peripheral circulation\u003csup\u003e55\u003c/sup\u003e. We find that CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e MoAMs express CCR2. Adoptively transferred CCR2-KO-GFP bone marrow monocytes remained in circulation and did not reach the alveoli. This suggests that CCR2 is essential for the migration of MoAMs into the lungs (and likely to other organs) in healthy old age. Increased levels of circulating monocytes in elderly populations correlate with hyperactivation immune cells\u003csup\u003e56\u003c/sup\u003e. Our data provides evidence for the unique role of CCR2 in monocyte migration in healthy old age. In contrast, we did not observe consistent differences in CX3CR1 protein levels among old/elderly individuals across species.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur experiments reveal that old age leads to the recruitment of short-lived MoAMs, which are prone to rapid apoptosis \u003cem\u003evia\u003c/em\u003e Fas activation through the extrinsic Caspase-8, 3/7 pathway. Thus, while recruitment of these immune cells is increased, their lifespan is constrained, with ongoing turnover, potentially as a strategy (albeit unsuccessful) to mitigate excessive inflammation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur findings regarding Fas (CD95), a member of the TNF receptor superfamily, contribute to the increasingly recognized role of Fas signaling in regulating apoptosis and inflammatory responses \u003csup\u003e57\u003c/sup\u003e in the context of aging \u003csup\u003e58,59\u003c/sup\u003e. This contrasts with apoptosis in adults, which is considered anti-inflammatory and promotes antigen presentation and microbial control\u003csup\u003e60,61\u003c/sup\u003e. Previous research has demonstrated that Fas ligand (FasL)-mediated activation of Fas on immune cells, e.g., human monocytes and monocyte-derived macrophages or epithelial cell types during acute lung injury, leads to increased apoptosis and the production of pro-inflammatory cytokines \u003csup\u003e8,39,62\u003c/sup\u003e. In older individuals, Fas activation is associated with the dysregulation of immune homeostasis, which contributes to the chronic low-grade inflammation frequently observed in aging. Our evidence suggests that Fas signaling is crucial in maintaining the inflammatory environment in the lungs associated with old age, reinforcing the concept that Fas-induced apoptosis in immune cells is a significant contributor to age-associated inflammatory responses. Our findings emphasize the detrimental effects of Fas in the aging context, where its activation appears to skew the immune response toward chronic inflammation and accelerate immune dysfunction. Indeed, Yu et al. (2011) demonstrated that Fas-deficient mice exhibit decreased inflammatory cell infiltration and reduced production of pro-inflammatory cytokines in an acute spinal cord injury model \u003csup\u003e57,63\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe implications of this research are significant, opening new potential therapeutic strategies aimed at improving immune function and tissue homeostasis in the healthy elderly population. By reducing the alveolar inflammatory environment and the presence of short-lived, apoptotic AMs, we may be able to reverse some age-associated changes within the lung, ultimately leading to healthier aging and lessening the incidence of age-related respiratory conditions.\u003c/p\u003e\n\u003cp\u003eThis study focuses exclusively on comparing young and old age groups. It does not evaluate the effects of Fas in regulating Fas-mediated inflammatory conditions across the ageing spectrum. Also, although we provide evidence for the role of the Fas-mediated extrinsic pathway contributing to macrophage apoptosis associated with old age, we have not ruled out the involvement of the intrinsic apoptosis pathway. This study focuses on the lung alveolus and AMs; it did not examine the tissue immune response in other organs. Finally, future studies will need to directly link our findings to their impact on host susceptibility to airborne infections, which is our next goal.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eResource availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLead contact\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIf you need additional information or resources, please contact the lead person in charge for assistance. Lead contact: Larry S. Schlesinger, Email: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not generate new unique reagents.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-seq data can be found in the NCBI GEO database: GSE297392\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest Statement.\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Institute on Aging (NIA), NIH [P01 AG-051428] (to LSS, JBT, JT), NIH award [AI136831] (to LSS), Texas Biomed Cowles and Forum Postdoctoral Fellowships, and the Interdisciplinary NexGen TB Research Advancement Center (IN-TRAC) Pilot Grant (to SP). Research reported in this publication was supported by the NIH-NIAID under IN-TRAC Award Number P30 AI-168439. Research was also supported by the Office of The Director, NIH Award [S10 OD-028653] for the BD FACSymphony flow cytometry machine. RNA sequencing data were generated in the Genome Sequencing Facility, supported by UT Health San Antonio, NIH-NCI P30 CA-054174, NIH Shared Instrument grant [1S10 OD-021805-01] (S10 grant), and CPRIT Core Facility Award [RP-160732]. Acquisition of old mice was supported by an NIH grant to the University of Texas Health Science Center, San Antonio, The Barshop Institute for Longevity and Aging Studies, and the Nathan Shock Center of Excellence in the Biology of Aging [5P30-AG-013319-28] to John Randy Strong. Fluorescence/confocal microscopy imaging was conducted with instruments at the Biology Core at Texas Biomed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.S.S and J.T. contributed to the early conceptual development of the project. S.P. and L.S.S. designed the studies and generated protocols. S.P., M.L., A.P., A.A., H. Z., H. C., Z. L., J. M., J. P., W.P.L., Y.W., conducted experiments, acquired the data, analyzed them, and generated figures. S.P. wrote the manuscript with input from LSS. S.P., L.S.S, E.A., Y.W, F.G, J.B.T., and J.T. edited the manuscript and performed a critical review.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eHuman Subjects and Ethics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman subject studies were conducted in strict accordance with the US Code of Federal and Local Regulations, as overseen by The Ohio State University (OSU) institutional review board with the number 2012H0135. The studies were also overseen by the Texas Biomedical Research Institute with IRB number HSC20170673H. Most samples were collected from the Division of Pulmonary and Critical Care Medicine, UT Health Science Center, San Antonio. Bronchoalveolar lavage (BAL) samples were collected from healthy adult (20-50 years) and elderly (≥65 years) individuals of both sexes, without discrimination of race or ethnicity, following written consent. The donors had a preoperative diagnosis of lung nodule, lung nodule with mass, or abnormal parathyroid and thymus, without clinical features. BAL was performed on the healthy lobe of each donor's lung. Individuals with certain comorbidities, such as smokers, drug users, excessive alcohol users, acute illnesses, chronic conditions, and various other health conditions were excluded from the study. Each \"n\" value represents a different human BAL donor as specified in the figure legends.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecific pathogen-free C57BL/6 female mice were obtained from Charles River Laboratories (Wilmington, MA) at either 3 months of age (young) or 18 months of age (old) through a contract with the National Institute on Aging (NIA). Mice were housed in individually ventilated cages (IVC) and allowed to acclimate to the facility for one week before being used for the study. All procedures were approved by The Texas Biomedical Research Institute (Texas Biomed) Institutional Laboratory Animal Care and Use Committee (Texas Biomed-IACUC number 1608-MU). C57BL/6 young CD45.1 congenic mice (3 months age), B6.SJL-Ptprc\u003csup\u003ea\u003c/sup\u003ePepc\u003csup\u003eb\u003c/sup\u003e/BoyJ (B6.CD45.1, Strain # 002014) and CByJ.SJL-Ptprc\u003csup\u003ea\u003c/sup\u003e/J (CD45.1, Strain # 006584) BALB/c mice were purchased from The Jackson Laboratory. Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosa\u003csup\u003eTdT\u003c/sup\u003e mice bone marrow was acquired from Florent Ginhoux, Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR). Ccr2\u003csup\u003egfp/gfp\u0026nbsp;\u003c/sup\u003eKI/KO mice (C57BL/6, Strain # 027619) were purchased from The Jackson Laboratory.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection and isolation of human alveolar macrophages (hAMs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo isolate and culture hAMs from BAL \u003csup\u003e64\u003c/sup\u003e samples collected within 6h were centrifuged (250 g) and washed twice in PBS at 4°C. The cell pellet was then resuspended in RPMI 1640 with Penicillin-G (Pen-G stock diluted to 1:50 to achieve 10,000 U/mL), and cells were allowed to adhere for 2 h in 24 well tissue culture wells (Falcon/\u0026nbsp;Corning Life Sciences). After adherence, Pen-G was removed by washing. A portion of the cell suspension underwent cytospin, followed by staining and microscopy to determine the percentage of hAMs in BAL (\u0026gt;98%). Following cell counting on a hemocytometer, hAMs (1x10\u003csup\u003e5\u003c/sup\u003e) were plated for morphometric microscopic analysis. In a separate experiment, hAMs (5x10\u003csup\u003e5\u003c/sup\u003e) were immediately lysed in TRIzol reagent (Invitrogen) for qRT-PCR analysis. Another set of cells was utilized for flow cytometry for phenotypic analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection and isolation of baboon and Rhesus alveolar macrophages (bAMs; rAMs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOlive baboons (\u003cem\u003ePapio anubis\u003c/em\u003e) and Rhesus macaque (\u003cem\u003eMacaca mulatta\u003c/em\u003e), both young (ages 3-5 years) and old (ages 15+ years), were obtained from our colony at the Southwest National Primate Research Center (SNPRC) at Texas Biomed. These animals were acquired through a biomaterials request, involving opportunistic BAL collection conducted without any disease conditions. This occurred during routine necropsies due to age or through live animal BAL collections, following Texas Biomed-IACUC Protocol number 1516 PC and 1516 MM. BAL was performed by experienced pathologists in 0.9% saline at the SNPRC-Pathology lab. AMs were collected through centrifugation at 300 x \u003cem\u003eg\u003c/em\u003e for 10 min and washed. Depending on the experimental requirements, BAL cells from individual baboons were analyzed using confocal microscopy or flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of mouse alveolar macrophages (mAM) and Alveolar Lining Fluid (ALF)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth young and old mice were euthanized using CO\u003csub\u003e2\u003c/sub\u003e following an approved protocol (Texas Biomed-IACUC 1608-MU). AMs and BAL fluid (BALF) were obtained by washing the lungs of mice with sterile, endotoxin-free saline containing 100 µM EDTA (0.5M), and either 0.2% BSA (for BAL fluid needed for Luminex assay) or 5% FBS. After washing the lungs 6-10 times with 0.5 ml of the saline solution, AMs were collected through centrifugation at 300 x g for 10 min. Depending on the experimental requirements, individual BAL cells were analyzed using confocal microscopy or flow cytometry. For qRT-PCR, BALs from 6 to10 mice were pooled, and the RNA was isolated using TRIzol reagent (Invitrogen) for measuring basal RNA levels. The supernatant fraction containing ALF from each individual mouse was promptly frozen and stored at −80°C until used for the Luminex assay.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytospin analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHealthy adult and elderly hAMs were collected for cytospin analysis. Shandon non-coated cytoslides (Thermo Scientific) were placed in a Shandon EZ single cytofunnel with white filter cards. A single cell suspension was created, and 200 μL of cells (5x10\u003csup\u003e4\u003c/sup\u003e) were placed into the cytofunnel and centrifuged at 150 x \u003cem\u003eg\u003c/em\u003e for 5 min using Program 1. The cells on cytoslides were dried and then stained with HEMA 3 differential staining (Fisher Health Care). Afterward, the slides were washed with water and dried. The slides were placed in Hema 3 fixative solution for 30 s. They were then dipped 30 times for 1 s each in Hema 3 Solution I (eosin Y) and subsequently dipped 30 times for 1 s each in Hema 3 Solution II (methylene blue). It was important to allow excess stains to drain during each step. After staining, the slides were washed with water and left to dry. Hema 3 Solution I (eosin Y) was used to stain cytosolic proteins, while Hema 3 Solution II (methylene blue) stained the nuclear membrane. Finally, cells were examined using a Motic AE2000 microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of mouse monocytes and adoptive transfer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBone marrow cells (BMCs) from the femurs and tibias of C57BL/6 wild type, CD45.1, Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosa\u003csup\u003eTdT\u003c/sup\u003e, and Ccr2\u003csup\u003egfp/gfp\u0026nbsp;\u003c/sup\u003eKI/KO young mice were flushed aseptically. The Ly6C-positive monocytes were isolated by negative selection using the EasySep™ Mouse Monocyte Isolation Kit (Cat#19861RF, Stem Cell Technologies, Cologne, Germany), according to the manufacturer’s instructions. The isolated monocytes were then processed for the adoptive transfer experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo study macrophage turnover, monocytes from B6.CD45.1 young mouse (donor cells) were injected intravenously (tail vein injections, i.v.) [1.5x10\u003csup\u003e6\u003c/sup\u003e donor monocytes/mouse] in recipient young or old C57BL/6 mice (CD45.2). \u0026nbsp;After 0, 3, 5, 7, and 10 days, depending on the experimental strategy shown in the figures (4-6), 5-8 recipient mice in each group were sacrificed, and AMs obtained by BAL to perform flow cytometry to verify cell turnover. The immigrated MoAMs were distinguished through CD45.1 cell labeling, while resident AMs were identified from CD45.2 labeled cell populations.\u003c/p\u003e\n\u003cp\u003eTo identify the origin of recruited cells in the alveolar region and parenchyma (\u003cstrong\u003eFigures 4 \u0026amp; S15\u003c/strong\u003e), the fate mapper model (Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosa\u003csup\u003eTdT\u003c/sup\u003e) was used for adoptive transfer to track monocytes and their bone marrow progenies \u003csup\u003e32\u003c/sup\u003e. The Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosa\u003csup\u003eTdT\u003c/sup\u003e cells utilize a Cre-loxP recombination system, where the Ms4a3\u003csup\u003eCre\u003c/sup\u003e promoter drives Cre recombinase expression, leading to activation of the TdTomato (TdT) reporter in specific cell lineages. The purified young mice Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosa\u003csup\u003eTdT\u003c/sup\u003e bone marrow monocytes (donor cells) were adoptively transferred (1.5x10\u003csup\u003e6\u003c/sup\u003e donor monocytes/mice) \u003cem\u003evia\u003c/em\u003e i.v. in recipient young or old C57BL/6 mice. On day 5, BAL cells were isolated to analyze recruited cells in the alveolar space by flow cytometry, confocal microscopy and histopathological analysis. For histopathological analysis, entire lung lobes were collected from the recipient mice. The lung lobes were preserved in 10% formalin and sent to the Texas Biomed path lab for histopathological analysis by H \u0026amp; E staining. The recruited dense, accumulated cells (blue hematoxylin-stained cells) and Rosa TdTomato-labeled cells were identified through HALO analysis.\u003c/p\u003e\n\u003cp\u003eTo assess the role of CCR2 in cell recruitment in the alveolar region and parenchyma (\u003cstrong\u003eFigure 5\u003c/strong\u003e), purified Ccr2\u003csup\u003egfp/gfp\u0026nbsp;\u003c/sup\u003eknockout mice bone marrow monocytes were adoptive transferred (1.5x10\u003csup\u003e6\u003c/sup\u003e donor monocytes/mice) through the i.v. route into young or old C57BL/6 recipient mice. On day 5, during the necropsy, blood was collected from the heart using heparin (1,000 U/mL) to prevent coagulation. PBMCs were isolated from the blood of recipient mice using Ficoll-Hypaque density cushion centrifugation, while AMs were isolated by BAL. The presence of GFP+ cells was confirmed in both BAL cells and blood-derived cells. Cells (6 x 10\u003csup\u003e4\u003c/sup\u003e/ 200 μL) were then placed in a cytofunnel and centrifuged in the Shandon Cytospin 4 at 150 x\u003cem\u003e\u0026nbsp;g\u003c/em\u003e for 5 min using Program 1. Subsequently, the cells were fixed with 2% paraformaldehyde for 3 min. The slides were washed three times with 1X DPBS (Gibco) and mounted on coverslips with prolong gold along with DAPI (Thermo Fisher Scientific). The stained slides were examined using a Zeiss LSM 800 confocal microscope (20X, 63X magnification). Mean fluorescence intensity (MFI) and percentage of positive cells were quantified using ZEN 2.3 blue edition (Zeiss) and ImageJ software (NIH, Bethesda, MA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepletion of AMs in mice by treatment with clodronate liposomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYoung and old mice were treated with Clodronate liposomes or control liposomes (Liposoma research, Cat#CP-005-005) at a dose of 100 µL/mouse (500 µg/mouse) via the i.t. route on day 0 and 3 (two doses). On day 3, 5, 6, and 7, the cells were isolated by BAL. The cells were counted, and cytospin analysis was performed to demonstrate the percentage of AMs that are depleted from the alveolar space. For experimental purposes, AMs from a separate set of young mice (donor mice) were isolated by BAL. 1x10\u003csup\u003e6\u003c/sup\u003e AMs were i.t. installed on Day 5 in clodronate-treated young and old recipient mice. On day 7, BAL was performed on young and old recipient mice to assess the impact of the lung environment on changing the phenotype and function of the AMs (donor young mice) in old mice in the alveolar space.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFasL NA/LE treatment in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor CD178 FasL NA/LE treatment study, donor bone marrow monocytes (1.5x10\u003csup\u003e6\u003c/sup\u003e donor monocytes/mice) were isolated from B6.CD45.1 mice and adoptive transferred via the i.v. route in recipient young or old C57BL/6 mice (CD45.2). \u0026nbsp;After day 0 or day 5,\u0026nbsp;depending on the experimental strategy shown in the \u003cstrong\u003eFigures 7\u003c/strong\u003e and\u003cstrong\u003e\u0026nbsp;S16\u003c/strong\u003e, FasL NA/LE ab was i.t. installed to block the Fas-mediated inflammatory response\u0026nbsp;\u003csup\u003e57,65\u003c/sup\u003e. After 0, 3, 5, 7, and 10 days, depending on the experimental strategy shown in \u003cstrong\u003eFigure 7\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eS16\u003c/strong\u003e, 5 to 8 recipient mice in each group were sacrificed, and AMs were obtained by BAL to perform a flow cytometry assay.\u0026nbsp;Fas-mediated apoptosis via Caspase 3, 7 \u0026amp; 8, as well as cell turnover were verified.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfocal Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor confocal microscopy analysis, Ms4a3\u003csup\u003eCre\u003c/sup\u003e-Rosa\u003csup\u003eTdT\u003c/sup\u003e (Red) and Ccr2\u003csup\u003egfp/gfp\u0026nbsp;\u003c/sup\u003eKI/KO (Green) positive cells (BAL and blood derived) (6x10\u003csup\u003e4\u003c/sup\u003e/ 200 μL) were placed in a cytofunnel and centrifuged in the Shandon Cytospin 4 at 150 x g for 5 min using Program 1. Subsequently, the cells were fixed with 2% paraformaldehyde for 3 min. Slides were washed three times with 1X DPBS (Gibco) and mounted on coverslips with prolong gold along with DAPI (Thermo Fisher Scientific). The stained slides were examined using a Zeiss LSM 800 confocal microscope (20X, 63X magnification). Mean fluorescence intensity (MFI) and percentage of GFP-positive cells were quantified using ZEN 2.3 blue edition (Zeiss) and ImageJ software (NIH, Bethesda, MA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnnexin V and EthD-2 cell viability assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMonocytes were taken from B6.CD45.1 mice and 1.5x10\u003csup\u003e6\u003c/sup\u003e donor monocytes were i.v. injected (via tail vein injections) into recipient young or old C57BL/6 mice (CD45.2). On day 0, FasL NA/LE antibodies were administered directly into the lungs to block Fas-mediated apoptosis. On days 3 and 4, recipient mice in each group were sacrificed, and AMs were obtained by BAL to perform a flow cytometry viability assay. The cells were then suspended in Annexin binding buffer [0.01 M HEPES (pH 7.4), 0.14 M NaCl, and 2.5 mM CaCl\u003csub\u003e2\u003c/sub\u003e]. APC-labeled Annexin V (4 μL per tube) was added to all samples and incubated for 15 min in the dark at room temperature. After washing, Ethidium Homodimer-2 (EthD-2, 4 µM) was added and incubated for another 10 min at room temperature. The cells were fixed with 2% paraformaldehyde (PFA). After washing with 1% binding buffer (400 μL), cells were analyzed by the BD FACS Symphony instrument immediately, and the data were analyzed using FlowJo software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow Cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAMs (1-2 × 10\u003csup\u003e5\u003c/sup\u003e) were placed into FACS tubes (Falcon round-bottom polypropylene tubes, Cat#\u0026nbsp;352063) and centrifuged at 250 x g for 10 min. The cell pellets were then resuspended in 50 µL of cell staining buffer from Biolegend (Cat# 420201) along with TruStain FcX™ PLUS (anti-mouse CD16/32) Antibody (Ab) from Biolegend (Cat# 156604) for FC receptor blocking. After a 30 min incubation on ice, the cells were stained with fluorochrome-tagged antibodies and corresponding isotype-matched control antibodies in BD Horizon Brilliant Stain Buffer Plus (BD, Cat#566385) for 40 min in the dark at 4°C. Following this, the cells were washed once, fixed in 2% paraformaldehyde (Thermo Scientific, Cat# J19943-K2) in cell staining buffer for 10 min, centrifuged (250 x g for 10 min), and then resuspended in 300 µL cells staining buffer. Cells were then filtered through a round-bottom polystyrene tube with a cell strainer snap cap (Falcon, Cat#352235). Samples were then acquired using a BD FACSymphony multi-color flow cytometer, and compensation, analysis, and data visualization were carried out using FlowJo 10.8.1 software (BD Biosciences). Isotype and \"Fluorescence minus one\" controls were utilized as needed to establish gates. The gating strategy employed for macrophage analysis is depicted in\u0026nbsp;\u003cstrong\u003eFigures S2-5\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe fluorochrome-tagged antibodies were purchased from BD Biosciences, San Jose, CA, and Biolegend, San Diego, CA. The list of mouse Abs were: BV421 anti-mouse/human CD11b (clone M1/70, Cat# 101236), APC-Cy7 anti-mouse CD11c (clone N418, Cat#117324), BV786 anti-mouse CD64 (clone X54-5/7.1, Cat#741024), BUV395 anti- mouse CD95 (Fas, Clone Jo2, Cat#740254), BUV496 anti-Mouse CD192 (CCR2, Clone 475301, Cat#750043), \u0026nbsp;Alexa Fluor® 488 anti-mouse CD45.1 (Clone A20, Cat#110718), PerCP/Cyanine5.5 anti-mouse CD45.1 (Clone A20, Cat# 110728), CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit (Cat# C10427).\u003c/p\u003e\n\u003cp\u003eThe antibody and respective isotypes are indicated in the “Key resources table”.\u003c/p\u003e\n\u003ch2\u003eLuminex multiplex analysis\u003c/h2\u003e\n\u003cp\u003eThe release of cytokines, chemokines, and other secretory factors in the culture supernatant was measured using Luminex assays. The following analytes were measured using the Luminex mouse Discovery Assay (10-Plex, code mxmbhKZn) LXSAMSM-10kit (R\u0026amp;D Systems, Inc.): GM-CSF, TNF-alpha, S100A9, S100A8, CCL2/JE/MCP-1, Fas Ligand/TNFSF6, IL-1 beta/IL-1F2, MMP-2, CCL3/MIP-1a, and IL-10. BAL was performed, and the BALF and AMs were separated by centrifugation at 250 x \u003cem\u003eg\u003c/em\u003e for 10 min. The 4 to 5 mL BALF was concentrated by passing it through a 10kDa filter (Amicon, EMD Millipore, MWCO 10 kDa, Ref. UFC9010) and then centrifuged at 2,800 x \u003cem\u003eg\u003c/em\u003e, at 4°C, for 30 min with a brake at 2. The upper concentrated ALF was collected, and a protease inhibitor cocktail (A+L) was added to prevent protein degradation. Selected analytes were measured from ALF (1:2 dilutions in 50 µL) in the Luminex® 100/200™ System (Luminex Corporation) following the manufacturer’s protocol. Data were analyzed using Belysa™ Immunoassay Curve Fitting Software (Millipore Sigma). Analytes/mL concentrations were calculated and plotted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell sorting using flow cytometry for bulk-RNA-seq analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAMs were isolated from both 10 young and 10 old mice and then sorted into CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e-\u003c/sup\u003e populations from young and old mice, and CD11c\u003csup\u003e-\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e and CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e populations from old mice using the BD FACSAria II flow cytometer. CD11c\u003csup\u003e-\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e populations were found in very low numbers and displayed a transcriptional signature like the CD11c\u003csup\u003e+\u003c/sup\u003eCD11b\u003csup\u003e+\u003c/sup\u003e populations. Consequently, we did not include them in further analyses. \u0026nbsp;Samples were collected in three different batches as three replicates for bulk-RNAseq. This was achieved by flow cytometry staining and sorting, using BV421 anti-CD11b (clone M1/70) and PE anti-CD11c (clone N418) antibodies. The post-sorted cells were then verified based on the gating strategy, and the purity of the populations was confirmed through gating.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBulk RNA-Seq from flow-sorted populations and analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow-sorted CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e-\u003c/sup\u003e AMs from young mice and CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e-\u003c/sup\u003e and CD11c\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e AM populations from old mice were collected and washed at 250 x g for 10 min with 1% PBS. The RNA was isolated using TRIzol reagent (Invitrogen) and a Direct-zol RNA Microprep kit (Zymo Research) according to the manufacturer's instructions. The isolated RNA was quantified using the Qubit 4 Fluorimeter (Invitrogen), and its quality was assessed with the 4200 TapeStation System (Agilent). Samples with an RNA integrity number (RIN) higher than 7 were used for RNA-seq. RNA-seq libraries were prepared from 300 ng of total RNA using the NEB Next RNA Ultra Kit (Qiagen, Redwood City, CA) with poly(A) enrichment, and 50-bp single-read sequencing with approximately 25-30M reads per sample. The RNA sequencing was performed at the Genome Sequencing Facility (GSF) at UT Health San Antonio using the HiSeq 3000 platform (Illumina).\u003c/p\u003e\n\u003cp\u003eThe raw sequence reads were performed using Trim Galore! to remove adapters and low-quality sequences \u003csup\u003e66\u003c/sup\u003e. Then, the trimmed reads were mapped to the mouse mm10 reference genome using HISAT2 \u003csup\u003e67\u003c/sup\u003e. After that, read counts for each sample were obtained using featureCounts \u003csup\u003e68\u003c/sup\u003e. The next step involved conducting differential gene expression analysis using DESeq2 \u003csup\u003e69\u003c/sup\u003e with control of the false discovery rate (FDR) using the Benjamini-Hochberg procedure. Shrunken log2 fold changes (LFCs) were calculated using the adaptive shrinkage (ash) estimator with an Empirical Bayes approach. Genes with FDR-adjusted p-value \u0026lt;0.05 and LFC of more than 1 or less than -1 were considered differentially expressed. Finally, heat maps of specific genes were generated using the pheatmap package in R.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation, quantification and qRT-PCR\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ehAMs from adult and elderly donors or mAMs from young and old mice were collected by BAL. In select experiments, mAMs were cultured with Pam\u003csub\u003e3\u003c/sub\u003eCSK\u003csub\u003e4\u003c/sub\u003e (100 ng/mL) for 24h. Cells were washed at 250 x g for 10 min with 1% PBS (Gibco) and then treated with TRIzol reagent (Invitrogen). RNA\u0026nbsp;was extracted using a Direct-zol RNA Microprep kit (Zymo Research) according to the manufacturer's instructions and quantified using the Qubit 4 Fluorimeter (Invitrogen). The precipitated RNA was reconstituted with DNase/RNase-free water and\u0026nbsp;reverse transcribed using random primers and SuperScript III Reverse Transcriptase (Invitrogen). cDNA synthesis using reverse transcriptase was carried out at 65°C for 5 min, followed by 25°C for 5 min, 50°C for 60 min, 70°C for 15 min, and then cooled to 4°C. mRNA expression was analyzed using quantitative real-time RT-PCR (qRT-PCR) with TaqMan Universal PCR Master Mix (Applied Biosystems). The amplification conditions were selected at 50°C for 10 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s in the Applied Biosystems 7500 Real-Time PCR System. Thermo Fisher Scientific, the TaqMan human primers with the best coverage and most citations were used. Relative expression was calculated using the ΔΔCT method, with β-actin (ACTB) as the housekeeping gene. The selected genes for humans are \u003cem\u003eMRC1, CD11B, CD11C, TLR2, NOS2, HIF1A, PIEZO1, FAS, CX3CR1, CCR2\u003c/em\u003e. Mouse \u003cem\u003eHif1α, Cox2, Cd74, Mif, Glut1, Hk2, Pfkb3, Gls1, Slc25a, Acly,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Actb\u003c/em\u003e were analyzed for qRT-PCR expression using IQ SYBR Green Supermix (BioRad). β-actin (\u003cem\u003eActb\u003c/em\u003e) was used as the housekeeping gene. Validated mouse primers listed on PrimerBank\u0026nbsp;\u003csup\u003e70\u003c/sup\u003e were used and primer sequences listed in Key resources table.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtracellular Flux Analysis (Agilent Seahorse)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe real-time cell metabolism of mouse AMs was measured using a Seahorse XF Extracellular Flux Analyzer XFe96 (Agilent Technologies). This involved determining the oxygen consumption rate (OCR, pmol/min) and extracellular acidification rate (ECAR, mPh/min) according to the manufacturer’s instructions. To investigate the metabolic changes in old mAMs, we assessed mitochondrial respiration, ATP production, OCR, and ECAR using the Seahorse XFe96 analyzer. For the mitochondrial stress assays, mAMs were placed in a supplemented XF assay medium, followed by sequential injections of oligomycin (O), FCCP, and rotenone/antimycin (R/A). Under the same setup, injections of O and R/A alone were used to determine the ATP production rate specifically from mitochondria. First, 3-month-old and 18-month-old mice (5x10\u003csup\u003e4\u003c/sup\u003e/well) were adhered in 96-well Seahorse plates for 2 h. The cells were then washed and replenished with XF DMEM Seahorse media supplemented with 25 mM D-Glucose, 1 mM Sodium pyruvate, and 2 mM L-glutamine. After incubating in a non-CO\u003csub\u003e2\u003c/sub\u003e incubator at 37˚C for 1h, the basal levels of OCR and ECAR were measured. A Mito stress assay was performed with sequential addition of 5 μM oligomycin (an ATP synthesis inhibitor), 4 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, an uncoupling agent), and 2 μM rotenone and antimycin A (inhibitors of complex I and III of the respiratory chain). For glycolysis stress analysis, cells were injected with 2 μM rotenone and 2 μM antimycin A followed by 100 mM 2-deoxyglucose (2-DG) to determine glycolytic rate.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEach of these components has specific roles. Oligomycin decreases electron flow through the ETC, verifying the basal OCR measurement impacts on mitochondrial respiration or OCR. FCCP interrupts the mitochondrial membrane potential, resulting in continuous electron flow through the ETC and maximal oxygen consumption by complex IV. The spare respiratory capacity (SRC) was calculated by measuring the difference between FCCP-induced maximal respiration and basal respiration. The third injection uses a combination of rotenone and antimycin A to block mitochondrial respiration, enabling the measurement of non-mitochondrial respiration in the cells. In the ECAR glycolytic rate assay, glycolytic pathway inhibitor 2-DG is used to inhibit glycolysis through competitive binding to glucose hexokinase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitoSox assay for mitochondrial ROS detection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the production of mitochondrial reactive oxygen species (ROS) in AMs from both young and old mice, cells were stained with 5μM MitoSOX (Thermo Fisher Scientific, Inc.) for 30 min. After staining, the cells were washed with cell staining buffer and centrifuged at 250 x g for 10 min at room temperature. Following centrifugation, the stained cells (5 x 10\u003csup\u003e4\u003c/sup\u003e/ 200 μL) were loaded into a cytofunnel and centrifuged in the Shandon Cytospin 4 at 150 x g for 5 min using Program 1. The cells were then fixed with 2% paraformaldehyde for 3 min. Subsequently, the slides were washed three times with 1X DPBS (Gibco) and mounted on coverslips with prolong gold containing DAPI (Thermo Fisher Scientific). The stained slides were visualized using a Ziess LSM 800 confocal microscope at 20X and 63X magnification. Mean fluorescence intensity (MFI) and percentage of positive cells were quantified using ZEN 2.3 blue edition (Zeiss) and ImageJ software (NIH, Bethesda, MA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraphs were created and statistical comparisons were conducted using GraphPad Prism version 10. Unpaired two-tailed Student’s T-test was used for statistical comparisons of two groups. Where applicable (as indicated in figure legends), ordinary one-way and two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test (GraphPad Prism vr. 10) was applied for multiple testing. Spearman’s rank test was used for Correlation analysis. Statistical significance was reported to have a p-value of ≤ 0.05. Data are presented as mean ± SEM for individual mice/donors and mean ± SD for representative data plots.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHamelink, C. J. \u0026amp; Keizer, B. in \u003cem\u003eGlobal Health and Human Rights\u003c/em\u003e 149-159 (Routledge, 2025).\u003c/li\u003e\n\u003cli\u003eLafuse, W. 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PrimerBank: a PCR primer database for quantitative gene expression analysis, 2012 update. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e40\u003c/strong\u003e, D1144-1149 (2012). https://doi.org:10.1093/nar/gkr1013\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"alveolar macrophages, inflammaging, CCR2, Fas, apoptosis, adoptive transfer, mice, human, non-human primates","lastPublishedDoi":"10.21203/rs.3.rs-7123735/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7123735/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImmune system changes with age lead to chronic systemic inflammation termed \"inflammaging\", contributing to age-related pathologies. Alveolar macrophages (AMs) maintain lung homeostasis and health. The impact of inflammaging on AM populations requires further definition. Herein, we examined the effect of age on the phenotype and ontogeny of AMs from mice, non-human primates and humans. We identify three AM subpopulations in old age, two of which increase more than 10-fold, leading to significant functional consequences associated with heightened inflammation and immune dysregulation. RNA-seq analysis identifies unique transcriptional AM subpopulation profiles. Adoptive transfer experiments reveal the importance of the alveolar environment in AM recruitment and phenotypic change in old age. Monocyte-derived AM recruitment in old age requires CCR2 and leads to relatively short-lived AMs with high turnover due to Fas-mediated apoptosis. These studies provide new insight on the impact of the alveolar environment in healthy old age on AM phenotype and function.\u003c/p\u003e","manuscriptTitle":"Lung environment in healthy old age shapes the phenotype and CCR2-mediated recruitment of a subset of apoptotic, high-turnover alveolar macrophages","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-25 04:37:36","doi":"10.21203/rs.3.rs-7123735/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"15989a79-ce64-464b-89eb-58a0714368cb","owner":[],"postedDate":"July 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52020253,"name":"Biological sciences/Immunology/Inflammation/Chronic inflammation"},{"id":52020254,"name":"Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages/Alveolar macrophages"},{"id":52020255,"name":"Biological sciences/Cell biology/Cell death/Apoptosis"}],"tags":[],"updatedAt":"2025-12-01T10:15:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-25 04:37:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7123735","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7123735","identity":"rs-7123735","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}