Regulatory T Cell Attracting Therapy Accelerates Skeletal Muscle Functional Recovery Following Injury

Regulatory T Cell Attracting Therapy Accelerates Skeletal Muscle Functional Recovery Following Injury | 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 Regulatory T Cell Attracting Therapy Accelerates Skeletal Muscle Functional Recovery Following Injury Matthew A. Borrelli, Jordan JP. Warunek, Steven R. Little, Heth R. Turnquist This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7237053/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Skeletal muscle injuries are a common consequence of physical activity, repetitive movements, and trauma. Regulatory T cells (Treg) have recently been identified as critical mediators of immune repair response after injury, and treatments effectively targeting Treg may accelerate injury resolution. CCL22 is a chemokine that recruits CCR4-expressing cells, particularly Treg, to sites of inflammation or immune regulation, such as tumor microenvironments. When a sustained release formulation of polymeric microparticles (MP) delivering CCL22 (CCL22MP), was administered after cardiotoxin (CTx)-mediated muscle injury, significantly improved limb function was observed on days 3 and 5 post injury. Histologic evaluation of the injured limbs showed reduced area of injury in CCL22MP treated limbs. Analysis of the local immune populations revealed augmented Treg concentrations, as well as increased myeloid derived suppressor cell and neutrophil frequency. These findings reveal that amplifying local Treg to damaged areas improves outcomes, thus offering a translationally promising approach after muscle injury. Health sciences/Diseases Biological sciences/Immunology Health sciences/Medical research Skeletal Muscle Treg Injury Microparticles Tissue Repair Immunomodulation Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Skeletal muscle injuries are a common and often unavoidable consequence of prolonged physical activity, intense exercise, and trauma. Muscle damage encompasses over 40% of all sports and exercise-induced injuries, and nearly 20% of injuries sustained during basic training of armed forces 1 . Among the general population, more than 21 million Americans over the age of 18 seek medical care for muscle injuries each year 2 , amounting to hundreds of billions in lost yearly productivity 3 . Muscle injuries of differing severity occur when myofibers undergo structural damage leading to rupture or tear, resulting in swelling, pain and lost limb function 4 . Muscle injuries are often treated through a combination of rest, ice, compression elevation (RICE) and broad spectrum anti-inflammatory medications, such as non-steroidal anti-inflammatory drugs (NSAIDs) or corticosteroids 4 . While NSAIDs and corticosteroids can reduce pain and swelling associated with inflammation, they also impair critical functions of the infiltrating immune cells that remove cellular debris and promote regeneration 5 . For this reason, non-specific anti-inflammatory therapeutics often have poor efficacy and, in some cases, impair muscle regeneration 6 , 7 . This has led to growing concerns about the overuse of NSAIDs for post-injury management in physically active populations 8 . Single-cell and spatial transcriptomics, precise lineage tracing and cell-specific reporter constructs have enabled a detailed understanding of the mechanisms underlying the immune responses to skeletal muscle injury and how individual cell types contribute to the repair, remodeling and resolution of the injured tissue 4 , 5 , 9 – 16 . Damage to the myofiber bundles can lead to necrosis, or inflammatory cellular death, of myocytes, which contributes to the release of damage associated molecular patterns (DAMPs), pro-inflammatory cytokines 17 , and chemoattractant molecules (chemokines), such as MCP-1 (CCL2), MIP-1α (CCL3), and MIP-1ß (CCL4) 18 . These chemokines signal through CCR2 and CCR5 receptors to attract neutrophils and monocytes to the injured tissue 18 . There is some evidence that they also act directly on myocytes to initiate proliferation 18 , 19 . Attracted neutrophils provide necessary clearance of cellular debris 16 while monocytes differentiate into inflammatory Ly6C hi macrophages that are critical for early satellite cell activation, myogenesis, and preventing maladaptive fibrosis 20 . While engulfing cellular debris from the injured tissue, macrophages gradually receive signaling to differentiate into Ly6C lo macrophages between day 3 and 6 post-injury 21 , 22 , which exhibit a reparative phenotype 20 , and begin orchestrating tissue remodeling 10 , 11 , 23 . Specifically, they coordinate myogenesis and tissue remodeling through extracellular matrix deposition and stimulating tissue vascularization 24 . Importantly, effective tissue repair requires a balance of pro-inflammatory and reparative pathways, as chronic influx or overexuberant activity of these early inflammatory cells also have the potential to delay or impair the tissue repair. Situations may occur where monocytes differentiate into osteoclasts and reduce bone density 9 or collateral damage by neutrophils and their release of NETs can damage healthy cells 16 . However, countering chemoattraction of neutrophils and monocytes via antiserum blockade of CCL2 or genetic ablation of CCR2 and CCR5 receptors results in severely diminished repair 18 . Additionally, early addition of the anti-inflammatory cytokine, IL-10, can also impair skeletal muscle regeneration by inhibiting macrophage proliferation and necessary cytokine expression 22 . Precise regulation is required to facilitate appropriate expansion of the macrophage population while avoiding the negative consequences of a chronic or overexuberant response. Regulatory T cells (Treg) are recognized endogenous controllers of inflammatory immune cells that prevent autoimmunity and maintain tissue homeostasis 13 , 14 , 25 . Treg utilize several immunomodulatory mechanisms to suppress inflammatory responses, including the secretion of immunomodulatory proteins (IL-10 and TGFβ) 26 , scavenging IL-2, which is needed to perpetuate effector T cells 27 , and controlling the co-stimulation available to T cells 28 . Treg can also exert control over neutrophils 25 and macrophages 14 to reduce their production of inflammatory cytokines. It has also emerged, however, that Treg are not only immunosuppressive, but can be programmed by local injury signals to fulfill important reparative functions 14 . After injury to skeletal muscle, as well as other tissues 29 – 31 and organs 32 – 35 , Treg detection of interleukin-33 (IL-33) leads to their proliferation and release of reparative effector molecules, such as IL-13, a cytokine that polarizes local monocytes and macrophages towards a pro-repair subsets 33 , 36 . IL-33 also stimulates Treg to produce potent growth factors, such as the epidermal growth factor amphiregulin (Areg) or vascular endothelial growth factor A (VEGF), among others 14 , 37 , 38 . Treg production of Areg is a particularly important mechanism for muscle repair, as Areg directly stimulates satellite cell proliferation after injury 14 . IL-33-mediated Tregs production of Areg has been shown to be of importance in other models of tissue injury including transplantation 39 , acute lung injury 33 , and myocardial infarction 34 . Treg repair functions are critical after injury, as global depletion of Treg following injury results in failed repair marked by lingering inflammation and disrupted reparative macrophage differentiation 14 . Unfortunately, Treg in injured muscles are of limited frequency, especially in aged individuals 14 , 38 , and slow to infiltrate or expand following injury 12 . Likewise, severe traumatic injuries cause the excessive release of inflammatory mediators that can cause inordinate inflammation and injury that overburden local reparative cells 40 . Novel therapies able to limit inflammation, while promoting repair are thought to be especially promising. Given their function in regulating immune cell inflammatory activity and ability to stimulate direct and indirect tissue repair, Tregs have been gaining increasing interest as a potential therapeutic target in skeletal muscle injury 41 . We have developed a Treg-attracting formulation of microparticles capable of directing Treg chemotaxis to the site of administration via the controlled release of encapsulated CCL22 42 . CCL22 is a chemokine primarily involved in recruiting CCR4-expressing cells, particularly Tregs 43 , 44 , as well as Th2 cells, and some myeloid cell subsets, to sites of inflammation or immune regulation 45 . CCL22 plays a critical role in maintaining immune homeostasis, promoting anti-inflammatory responses, and has been implicated in tumor immune evasion 43 , 46 – 48 and transplant tolerance by facilitating Treg accumulation 49 , 50 . CCL22 releasing microparticles (CCL22MP) have been employed successfully to control inflammation causing or perpetuating disorders including periodontitis 51 , dry-eye disease 52 , and transplantation rejection 49 , but CCL22MP have not yet been evaluated for their ability to improve regeneration after muscle injury by attracting Treg. As such, we assessed if the regulatory and direct regenerative functions of Treg could be harnessed for accelerated repair following acute skeletal muscle injury using CCL22MP treatment. Using a murine model of skeletal muscle injury, we clearly show that CCL22MP treatment is a highly promising and novel therapeutic that enhances functional strength recovery by rapidly amplifying the presence of Treg in the injured limbs to stimulate regeneration and reduce the area of injury. RESULTS Development and characterization of CCL22 releasing microparticles Microparticles (MP) formulated with a blend of PEG terminated PLGA and RG502H, in which the PEG-PLGA fraction was gradually increased, were loaded with CCL22 (“CCL22MP”) or water (“BlankMP”) and 15 mOsm of NaCl ( Supplementary Fig. 1 ). 55% PEG-PLGA (Balance RG502H) was selected for functional characterization and use in vivo due to its favorable release kinetics. More specifically, the release profile for this formulation was compatible with our desire to promote rapid and accelerated Treg infiltration by simulating an initial boost in chemotactic signal followed by sustained signaling to establish a stable chemotactic gradient. Scanning electron microscopy (SEM) imaging shows that both CCL22 and BlankMP had expected spherical morphology and that the surface is populated with pores to facilitate early release as reported previously 42 (Fig. 1 A). Both formulations show similar size distributions, in which the mean diameter ranges from 15–25 µm (Fig. 1 B). The cumulative release profile of CCL22 from CCL22MP was quantified for 15 days (Fig. 1 C). Following an initial rapid burst release of 0.2 ng CCL22/mg of MP, this formulation produces a sustained and controlled rate of release amounting to 20 (pg of CCL22)/(mg of MP)/(day) for 15 days. CCL22 releasing microparticles improve limb function following acute muscle injury The impact of CCL22MP treatments were evaluated in a variant of the well characterized cardiotoxin (CTx)-induced muscle injury model 53 , 54 . Specifically, mice received a single injection of CTx in PBS (“CTx”) or PBS alone (“PBS”) into the gastrocnemius of the right and left hindlimb and the extensor carpi of the forelimbs to generate injury to all extremities. 24 hours post injury, a subset of mice received intramuscular injections of CCL22MP suspended in PBS (“CCL22MP”) to the injured muscles, the CTx subsets received a control treatment of PBS alone, and others were treated with empty or “BlankMP” suspended in PBS. Limb function was then quantified on days 3, 5, 7, 10, and 14 following injury induction (Fig. 2 A). Here, limb function and motor coordination were tested using the inverted wire hang method (reference image in Fig. 2 B). Hang duration was normalized to baseline measurements taken prior to CTx injury and plotted in Fig. 2 C. As expected, mice in the uninjured PBS treated control group did not display a functional defect. Mice in the CTx group that were administered CTx and then treated with PBS exhibited significantly reduced functions compared to PBS only controls at days 3, 5, and 7 (Fig. 2 C). Mice administered CTx and then treated with CCL22MP had significantly improved hang duration at day 3 and 5 timepoints, relative to the CTx injury group. By the day 7 timepoint, hang duration for CCL22MP treated mice remained elevated, but other control groups began to show increasingly improved performance. By day 14 post-injury, all groups exhibited a return of function to baseline measurements. Thus, CCL22MP accelerates functional strength and recovery during the acute phase following muscle injury. CCL22MP treated limbs exhibit reduced injury in CTx treated skeletal muscles To define how CCL22MP-mediated improvements in function relates to skeletal muscle injury repair, we completed histologic assessment of cross-sectioned gastrocnemius muscles at day 14 post-CTx administration. Figure 3 A shows representative images of the regenerating muscle for each treatment group. PBS alone tissues exhibit normal muscle architecture consisting of well-organized and tightly packed fibers with peripheral nuclei as expected. CTx treated muscles, however, displayed disrupted and irregularly shaped fibers with centrally located nuclei (Fig. 3 A). In samples from CCL22MP-treated mice, the diameter of the myofibers appear increased relative to CTx control. Myofiber cross-sections from BlankMP-treated limbs exhibited an increased frequency of peripheral nuclei, which corresponds to the final phase of regeneration. Quantification of the total number of centrally nucleated, regenerating myocytes and their area percentage relative to the entire gastrocnemius is presented in Fig. 3 B-C. CCL22MP treatment results in a significant reduction in the number and area of regenerating cells, relative to the CTx injury control. Taken together, CCL22MP treatment improves muscle regeneration and reduces injury more effectively than BlankMP, supporting its reparative potential after muscle injury. CCL22MP enrich for immunoregulatory cell populations at the site of injury We next defined how CCL22MP treatment shapes immune responses following CTx injury using multispectral flow cytometry on immune cells isolated from the skeletal muscles and nearest draining lymph nodes (see Supp. Figure 2 for a representative gating strategy). In these studies, mice were injected with CTx or PBS and then administered CCL22MP in PBS, BlankMP in PBS, or PBS alone (Fig. 4 A). Assessment of CD45 + leukocyte populations isolated from the injured gastrocnemius muscle 5 days after injury and treatment reveals an increased frequency of CD3 + CD4 + CD25 + cells expressing the Treg transcription factor Foxp3 for CCL22MP treated limbs (Fig. 4 B and Supplementary Fig. 4A ). The total number of Tregs normalized to the mass of hindlimb tissue was calculated and also revealed a significant increase relative to the BlankMP or CTx alone control groups (Fig. 4 C). The level of expression of the IL-33 receptor, serum stimulation‑2 (ST2), was increased on local Foxp3 + CD3 + CD4 + CD25 + by muscle injury, however, there was not a difference noted between treatment groups (Fig. 4 D). Further assessment of the myeloid compartment found that there was an elevated frequency of rare CD45 + Ly6G + CD11b Lo cells that are phenotypically consistent with those described as monocyte-derived suppressor cell (MDSC) 55 (Fig. 4 D-E and Supp. Figure 3 A). There was also an elevated frequency of neutrophils in the CCL22MP treated limbs compared to CTx alone (Fig. 4 D and Supp. Figure 3 A). Quantification of the total number of MDSC and neutrophils showed a significant increase in response to CCL22MP treatment relative to BlankMP-treated limbs (Fig. 4 E). We did not detect significant differences in the frequency of intramuscular B cells, dendritic cells, as well as macrophages ( Supp. Figure 3 ), however, intramuscular B cells and macrophages exhibited a trend towards increased frequency ( Supp. Figure 3 ) and number ( Supp. Figure 4 ) with CCL22MP treatment. To better understand changes in the local immune populations supporting tissue healing versus any treatment-induced distant immunomodulatory impacts, we assessed CD45 + leukocytes from the draining popliteal lymph nodes. Similar flow cytometric analysis found minimal elevations in the frequency of immune cells assessed ( Supp. Figure 4 ). CCL22MP treatment was associated with increased numbers of macrophages in the draining LN ( Supp. Figure 4 F). There was not an increase in the frequency of Treg, nor did we observe changes in ST2 in the draining lymph nodes ( Supp. Figure 4 A). In total, these findings demonstrate that CCL22MP treatment enhances local accumulation of Tregs, presumed MDSC, and neutrophils at the site of muscle injury, which was associated with an accelerated restoration of muscular function. DISCUSSION The present study reports how enhancing the local population of Tregs through the administration of a Treg-attracting microparticle formulation affects the recovery of injured skeletal muscle. Prior studies have delineated that Treg play critical roles in skeletal muscle injury 13 , 14 , 38 by not only regulating infiltrating leukocytes 25 , but also secreting growth factors like Areg that stimulates muscle satellite cell proliferation 14 . Despite increasing interest in leveraging Tregs as a therapeutic target in skeletal muscle injury 41 , to our knowledge, there have not been prior studies that have investigated the amplification of Tregs through local drug delivery in the injury site as a potential treatment. In this first-of-its-kind study amplifying the local population of Tregs in the injured muscle, we found that treatment with microparticles releasing CCL22 (CCL22MP), a Treg chemoattractant, significantly improved the limb function of mice at early timepoints. Subsequent investigations revealed a reduced area of injury in the limb, as well as significantly elevated amounts of Treg, presumed MDSC, and neutrophils, locally, but not in the adjacent lymphoid tissues. Together, these findings establish a novel and effective strategy for modulating the immune microenvironment at the site of injury through local Treg recruitment, highlighting the therapeutic potential of CCL22MPs in accelerating muscle repair and functional recovery. The importance of Treg chemoattraction to local immunomodulation was originally defined in studies developing an understanding how tumors evade immune clearance 43 , 46 – 48 , 56 . These studies revealed that the chemokine, CCL22, is secreted by tumor associated macrophages and dendritic cells 46 – 48 . This directs Treg that are enriched for the CCL22 receptor, CCR4, to infiltrate the tumor site 43 , 56 . Taking inspiration from this, we have developed a PLGA microparticle-based platform that has demonstrated the ability to attract adoptively transferred Tregs to the CCL22MP injection site 42 . Subsequent studies demonstrated CCL22MP treatment to be efficacious in models of inflammatory disease including periodontitis 51 , dry-eye disease 52 , and transplantation rejection 49 . In each of these studies, RG502H – an acid terminated PLGA polymer containing equal ratio of glycolic and lactic acid repeating units – was utilized as the encapsulating polymer. CCL22 release from this polymer formulation has produced linear release profiles that delivered 80–4500 pg CCL22/mg of MP by day 14, which is compatible with Treg functional immunobiology. PLGA polymer is one of the most widely explored degradable polymers for delivery of protein therapeutics because of its biocompatible degradation products, glycolic and lactic acid, and its prior use in FDA-approved formulations 57 . Recently, PLGA polymers with various terminal groups have become more accessible 58 and, in some cases, facilitate a gain of function such as improved cellular targeting of nanoparticles 59 or reduced phagocytic clearance 59 , 60 . For the current study, we included poly(ethylene glycol) (PEG) terminated PLGA in the encapsulating polymer due to its ability to confer several benefits. Specifically, PEG has been reported to slow the clearance rate of polymeric particles 60 , 61 , and, following injury, it has been demonstrated to provide membrane sealing 62 and to reduce apoptosis 63 . The resultant formulation of 55 wt.% PEG-PLGA and 45 wt.% RG502H produced linear release behavior amounting to 500 pg of CCL22/mg of MP delivered by day 14, which is consistent with our previously reported release profiles that produced rapid augmentation of Treg at the site of injury. That we did not witness systemic changes in Treg suggests precisely controlled local delivery. The importance of a well-coordinated immune response in skeletal muscle regeneration has been routinely characterized using murine models, including CTx injections. The regenerative program after CTx administration depends on a temporally regulated sequence of events, beginning with an acute inflammatory phase dominated by pro-inflammatory macrophages, followed by a reparative phase characterized by restorative macrophages 53 . However, Tregs also play critical roles in shaping skeletal muscle repair. Recent work investigating Tregs functions in muscle repair documented a subset of skeletal muscle-resident Treg that highly express the helios transcription factor and the neuropilin transmembrane receptor 14 , 38 . Their helios and neuropilin expression indicate that these Tregs are most likely thymic derived and not peripherally induced. These Treg were also reported to highly express ST2 receptor and respond to IL-33 with production of Areg 14 , 38 . Transgenic mice where Treg lack Areg were exploited to determine that Areg contribute a significant role in shaping the Treg response to skeletal injury by stimulating satellite cell proliferation 14 , 38 . While Areg has also be implicated in suppressive functions 64 , several studies using Treg lacking Areg have found that systemic immune responses to virus 65 and alloantigen 39 are not modified in the absence of Treg-expressed Areg. Tregs typically begin to amass in the injury site 4 days following initial injury via CCL3 chemoattraction 12 . Thus, CCL22MP treatment one day following injury has the potential to augment and accelerate the normal attraction of Treg to injured skeletal muscles. Indeed, our experiment administering CCL22MP after CTx-mediated injury resulted in a significant improvement to limb function on post-injury days 3 and 5 – timepoints where Treg are typically only just arriving. Consistent with improved function, subsequent histologic analysis on post-injury day 14 showed that CCL22MP treated limbs have reduced injury area, suggesting that CCL22MP treatment facilitated a reduction in damage spreading or accelerated repair relative to the control group. Whether this is the result of Areg-mediated repair or local immunomodulation by attracted Treg, however, will require further investigations using precise transgenic mice allowing the targeting of Areg or molecules implicated in Treg immunomodulation. BlankMP show a slight reduction in the injury area, though it is not statistically significant, indicating the PEG may be providing an anti-apoptotic effect, which would be consistent with prior reports 62 , 63 . Additionally, lactic acid has beneficial effects following muscle injury 66 . Thus, lactic acid degradation products from locally injected microparticles may also be promoting the observed differences in the BlankMP-treated limbs. These effects would be conserved for CCL22MP treatment and may be providing a synergistic benefit that complements the effects of attracted Treg. Our flow cytometry assessments define how CCL22MP influenced the immune populations in the injury site and the draining lymph nodes, Treg in the injury site, but not the nearest draining lymph node, were significantly expanded for mice receiving CCL22MP. This result indicates that CCL22MP exerts a targeted effect, enhancing Treg recruitment or retention specifically within the tissue microenvironment of the injury, without inducing systemic or regional immune changes. This localized immunomodulation is predicted to be therapeutically advantageous because it will enhance repair and suppression at the site of injury, while minimizing any risk of systemic immunosuppression. Notably, there was an increased expression of ST2 on Treg at the site of injury, but no difference in the ST2 expression among the treatment groups. This suggests that ST2 expression is a response to the injury microenvironment itself, independent of CCL22MP treatment. This aligns with our recent studies showing that ST2 + Treg emerge upon infiltrating in inflamed or damaged tissues, where IL-33 is often released by stressed or necrotic cells 39 . Thus, the injury environment, not CCL22, appears to be sufficient to induce this ST2, and presumably allow IL-33 to support the reparative Treg phenotype. The changes to B cell and macrophage populations identified in the hindlimb and lymph node are largely not statistically significant, but do suggest these populations are attracted or expanded in response to BlankMP and CCL22MP treatments. It is possible that the microparticles caused a foreign body response in the hindlimbs. It is well known that large microparticles (> 20–30 µm 67 , 68 ) are not readily phagocytosed resulting in a state of frustrated phagocytosis 69 and activating macrophages 70 . Notably, we did observe CCL22MP treatment caused an expanded Ly6G + CD11b Lo myeloid cell and neutrophil populations. The Ly6G + CD11b Lo phenotype is associated with the monocyte derived suppressor cell phenotype, which possess potent immunosuppressive and reparative capabilities in injury 10 , 11 , 23 . The increase in neutrophil population 5 days following injury was unexpected. These cells typically undergo apoptosis following clearance of cellular debris 2–3 days after injury 15 . However, in vitro studies have shown Tregs can act to reduce neutrophil IL-6 expression and skew them towards a suppressive and pro-repair phenotype that secretes IL-10 and TGF-ß, heme oxygenase-1, and indoleamine 2,3-dioxygenase 25 . Thus, it is possible that the attracted Treg are sustaining the neutrophil population to perform anti-inflammatory and pro-repair roles. Another possible explanation is that these neutrophils and MDSCs are co-migrating into the injured limb in response to CCL22. Neutrophils have been reported to express an array of chemokine receptors including CCR1, CCR2, CCR3, CCR5, CXCR1, CXCR2, CXCR3, and CXCR4, and that the expression of these receptors differs among injury infiltrating neutrophils and peripheral blood neutrophils 71 . Of interest is CCR2 expression, which is the target receptor or CCL2. Interestingly, CCL2 can also bind to CCR4 72 ; because CCR4 is the target receptor for CCL22, it is possible CCL22 could bind to the CCR2 receptor, reciprocally. Further, it has been reported that chemokines can interact with one another to produce synergistic migratory effects 73 . For instance, in the context of allergic dermatitis, CCL22 was shown to interact with CXCL10 resulting in amplified migration of CCR4 + T cells into the tissue 74 . A similar phenomenon has been observed for neutrophil migration, in which CCL2 and CCL7 synergize with CXCL8 to enhance their migration during acute respiratory distress syndrome 75 . The present study provides insights into how localized regulatory T cell amplification following muscle injury can affect limb functional recovery. One important limitation of this work is that it lacked quantification of secreted reparative, inflammatory, and anti-inflammatory factors from the local immune populations. Logically, increased numbers of Treg that are phenotypically identical should result in a proportional increase in secreted factors. However, it is possible that the activity of the expanded Treg population is also increased. Likewise, the studies completed did not quantify how the expanded Treg population affects the local balance of inflammatory (e.g. IFNγ, TNFα, IL-6, etc.) and anti-inflammatory (e.g. TGFß, IL-10, etc.) signals. Thus, future experiments that investigate how CCL22MP treatment may alter these secreted factors following muscle injury are merited. Further, this study lacks a full investigation into the expanded neutrophil population observed in CCL22MP treated limbs. Future studies could investigate the neutrophil populations to inform what role they are playing in the injured limb. Another limitation of this work, however, is a lack of precise understanding of the mechanism these Treg improved functional repair. Without blockade of the reparative effectors, IL-13 and Areg, secreted from Treg or the suppressive mechanisms of these cells, such as IL-10 or CTLA-4, it is unclear what their individual contributions are to the observed functional recovery and reduction to injury. Most likely benefits require both suppressive and reparative Treg functions. Therefore, future studies employing the Cre-loxP system to alter genetic expression of the repair and suppressive genes in Foxp3 + Treg in this injury model with CCL22MP treatment may provide greater insight. Additionally, this study focused on a CTx model of muscle injury which does not interfere with basal repair functions. While the improvement that CCL22MP treatment conferred was considerable in this model, repeating this work in injury models that have limited regenerative capabilities, such as the volumetric muscle loss model, or in mice that express poor or dysregulated muscle regeneration, such as mdx mice that exhibit muscular dystrophy, may provide a greater understanding of the potential benefits of this treatment. Despite these limitations, the work reported in this manuscript has broad implications for the field of regenerative medicine. Because the immune response is critical to tissue repair and remodeling, this Treg attracting treatment has potential to be beneficial in other injury and regenerative models. In total our study establishes that CCL22-enriched Tregs have a significant ability to shape the injury microenvironment. Intervening within the first 24 hours of an acute skeletal muscle injury with a Treg-attracting microparticle formulation resulted in early improvements to limb function and a reduction in total injury. This study is the first of its kind to manipulate Tregs in the injury site, directly, and may provide similar efficacy in other models of muscle injury and trauma. Future work exploring how early intervention with Treg-targeting therapies affects the cellular microenvironment following injury in muscles and other tissue types will be insightful and may lead to the discovery of new therapeutic targets or establish if local Treg could be replaced by controlled release of their delivered reparative or immunoregulatory effectors. METHODS Materials : Polymers : RG502H Poly(D,L-lactic-co-glycolic acid) (PLGA) polymer, with 50:50 lactide:glycolide composition was obtained from Evonik (Essen, Germany). Poly(ethylene glycol) (PEG) terminated PLGA (PEG-PLGA) and Poly(vinyl alcohol) (PVA, 98 mol% hydrolyzed, M w = 25000 g mol − 1 ) were purchased from PolySciences (Warrington, PA). Protein : Carrier-free recombinant murine CCL22 (mCCL22) was obtained from R&D systems (Minneapolis, MN). Naja pallida cardiotoxin (CTx) was obtained from Sigma-Aldrich (St. Louis, MO) and diluted to a working concentration of 0.03 mg/mL prior to injection. Bovine Serum Albumin (BSA) was obtained from Fisher Chemical (Waltham, MA). Reagents : Dichloromethane (DCM), Acetonitrile (ACN), Sodium Dodecyl Sulfate (SDS), was obtained from Fisher Chemical (Waltham, MA) Cell Reagents and Buffers : Phosphate buffered saline (PBS), fetal bovine serum (FBS), Hanks’ balanced salt solution (HBSS), Roswell Park Memorial Institute 1640 medium (RPMI), Cytiva percoll centrifugation media, and eBioscience Foxp3 / Transcription Factor Fixation/Permeabilization buffer from Fisher Scientific (Waltham, MA). FACS and perm buffer were produced in house by supplementing PBS with 5% Fetal Bovine Serum (FBS) and 0.1% sodium azide; 1% FBS, 0.1% sodium azide, and 0.1% saponin; and 5% FBS, respectively. Microparticle (MP) formulation : Microparticles (MPs) encapsulating CCL22 or blanks were formulated via a double emulsion-evaporation method, as described in our previous work 42 , 76 . Briefly, MP formulation involves the formation of an emulsion between an inner, aqueous phase (200 µL vol) that contains CCL22 protein (5 µg) (CCL22MP) or water (BlankMP) and the outer, organic phase that contains PLGA polymer (200 mg, 55 wt.% PEG-PLGA & balance RG502H) solvated in dichloromethane (DCM) (4 mL). The aqueous phase for CCL22MP and BlankMP was formulated with 0.375 mg BSA to stabilize the encapsulated protein, and an osmolarity of 15 mOsm to produce surface pores. The resulting mixture of protein and polymer was sonicated (Active Motif EpiShear probe sonicator, 110V) at 55% amplitude for 10s to form the first emulsion (water-in-oil, w/o), and then transferred to a 2% PVA solution (60 mL) and homogenized (L4RT-A; Silverson, East Longmeadow, MA) at 3000 rpm for 1 minute, forming the second emulsion (w/o/w). Following homogenization, the solution was transferred to a 1% (w/v) PVA solution (80 mL) and stirred at 600 RPM for 3 hours on ice to allow the DCM to evaporate. Freshly formed MPs were centrifuged (Eppendorf 5810R 15 amp version, 200 xg for 5 min at 4°C) and washed four times (4x) with 4°C MilliQ water (Milli-Q IQ 7000, Millipore Sigma) (35 mL). The MPs were then re-suspended in MilliQ water (5 mL), flash-frozen with liquid nitrogen (5 minutes), and lyophilized (Benchtop Pro, Virtis SP Scientific) (< 100 mTorr, 48–72 hours). Microparticle characterization and release assays : Scanning electron micrographs of MPs were obtained using a scanning electron microscope (ZEISS Sigma500 VP), located in the University of Pittsburgh Nanofabrication and Characterization Core Facility. Size distributions of MPs were determined using volume impedance measurements on a Beckman Coulter Counter (Multisizer-3; Beckman Coulter; Brea, CA). In vitro release behavior was characterized by incubating 10 mg of MPs in 1 mL of release media (1% (w/v) bovine serum albumin (BSA) in PBS) and incubated on a roto-shaker (Thermo Scientific™ Tube Revolver Rotator, 1.5–2 mL Eppendorf tube paddles, speed 10) at 37°C. At specified time intervals, MP suspensions were centrifuged (580 g for 5 mins at room temperature) (Eppendorf 5417R), 800 µL of the supernatant was removed and replaced with 800 µL of fresh release media, and the MPs were resuspended and placed back on the roto-shaker. Supernatant concentrations of CCL22 were determined via enzyme-linked immunosorbent assay (ELISA; R&D Systems), and subsequently the ELISA results were interpreted through quantification of optical density (SpectraMax M5; Molecular Devices; Sunnyvale, CA). Release profiles generated from measured concentrations of CCL22 had any background signal from BlankMP subtracted and then normalized to the total amount loaded within MPs (quantified by solvating MPs and extracting the protein liberated) and MP mass. CTx Muscle Injury Model & MP Treatment : Limb injury was conducted following a variant of the well characterized cardiotoxin skeletal muscle injury model 53 , 54 . Mice (B6 Wildtype, Male, 8–10 wks old) were randomly assigned to one of 4 groups: Healthy control (PBS), injury and treatment control (CTx), Vehicle control (BlankMP), and treatment group (CCL22MP). Mice were anesthetized with 4% Isoflurane and injected with 50 µL of CTx (0.03 mg/mL) (CTx, BlankMP, and CCL22MP groups) or 50 µL of saline (PBS group) into each forelimb (Extensor Carpi) and hindlimb (Gastrocnemius). Injured limbs were administered 50 µL of CCL22MP (10 mg/mL) (CCL22MP group), BlankMP (10 mg/mL) (BlankMP group), or saline (CTx group) was injected into each forelimb and hindlimb. For functional studies, treatment occurred 24 hours after injury and for flow cytometry studies treatment occurred simultaneously with injury induction. At the conclusion of a study, mice (age: 8–16 weeks, weight: 22–30g) were euthanized via CO 2 (99% purity) and subsequent cervical dislocation. The Gastrocnemius was then collected for further analysis. Study approval and reporting : All procedures completed at the University of Pittsburgh were approved by the IACUC of the University of Pittsburgh (protocol 22051178) and complied with the NIH’s Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011). This study is reported in accordance with ARRIVE guidelines ( https://arriveguidelines.org ). Limb function and histologic injury assessment : At specified timepoints following CTx injury (POD 3, 5, 7, 10, & 14) limb function was assessed via the commonly used inverted wire hang method 77 – 79 . Briefly, mice were placed on a wire cage, the cage was inverted, and the time it took for the mice to fall off the cage was recorded. For each mouse, this assessment was repeated in triplicate with at least 60 seconds rest between each trial. The maximum value of the three trials was calculated and normalized to the maximum value measured during a baseline wire hang test performed prior to injury. The data was tabulated and evaluated for statistical differences to the CTx injury control. Histology : After the functional measures were completed on Day 14, the gastrocnemius muscles were collected for histologic analysis. The muscle tissue was fixed in 4% paraformaldehyde for 2 hours followed by a 2-step dehydration utilizing an overnight incubation in 30% sucrose and final storage in 70% ethanol (balance water). Samples were sectioned (10µm thick) at the midpoint of the gastrocnemius perpendicular to the origin and insertion, and hematoxylin and eosin (H&E) staining was then performed. Regenerating myofibers have centrally located nuclei and at later stages of regeneration and later mature into normal sized myofibers with peripheral nuclei 80 . Thus, quantification of the count and area of centrally nucleated, regenerating muscle cells was performed using QuPath open-source software 81 . Tissue Collection : Muscle : C57BL/6 wildtype mice were euthanized and gastrocnemius skeletal muscle were carefully excised and then minced by scissors in RPMI 1640 digestion buffer (2% FBS, 12.5mg collagenase IV (Thermo Fisher; 17104019) and DNAse I (Sigma Aldrich; DN25-1G)). The tissue was further homogenized using Miltenyi gentleMACS Dissociator and incubated at 250 rpm and 37°C for 45 minutes. After digestion, homogenate was passed through a 70µm nylon mesh strainer and processed for mononuclear cells by using a 37.5% Percoll gradient (GE Healthcare). Pellets were resuspended in 2% RPMI, counted by hemacytometer and prepared for flow cytometry staining. Lymph nodes : The nearest draining lymph node (popliteal) to the gastrocnemius was collected simultaneously. Each replicate represents the aggregate collection of both popliteal lymph nodes collected from each limb. The collected lymph nodes were minced by scissors in RPMI 1640 and passed through a 70µm nylon mesh strainer. The cells were pelleted by centrifugation and resuspended in RPMI 1640 for counting and prepared for flow cytometry staining. Flow Cytometry : Harvested cells were incubated in 5% normal goat serum, stained with surface antibodies for 25 minutes on ice, washed and placed in Foxp3/Transcription Factor Staining Fixation/Permeabilization Buffer Set (eBioscience) overnight. For intracellular staining, cells were permeabilized (eBioscience) and stained with antibodies for 45 minutes on ice. Data was acquired using a Cytek Aurora spectral flow cytometer (Cytek Biosciences) and analyzed using FlowJo v10.9.0 (BD Life Sciences). The final stained samples were then washed 2x with FACS buffer and run on Cytek’s Aurora Cytometer (Fremont, CA). Flow Cytometry Antibodies : BUV496 rat anti-mouse CD45 (1:300 BD Biosciences Cat: 569673; Clone: 30-F11), Pacific Blue anti-mouse F4/80 (1:150 BioLegend Cat: 123124; Clone: BM8), Brilliant Violet 785 rat anti-mouse CD4 (1:200 BioLegend Cat: 100552; Clone: GK1.5), Brilliant Violet 650 rat anti-mouse CD86 (1:200 BD Biosciences Cat: 564200; Clone: GL1), Brilliant Violet 605 rat anti-mouse CD25 (1:200 BD Biosciences Cat: 563061; Clone: PC61), PE rat anti-mouse Ly6C (1:200 BD Pharmigen Cat: 560592; Clone: AL-21), PEcy5 rat anti-mouse B220 (1:200 BD Pharmingen Cat: 553091; Clone: RA3-6B2), Alexa Fluor 700 rat anti-mouse CD3 Molecular Complex (1:200 BD Pharmigen Cat: 561388; Clone: 17A2), APC/Fire 750 anti-mouse/human CD11b (1:200 BioLegend Cat: 101262; Clone: M1/70), BUV395 anti-mouse I-A/I-E (1:200 BD OptiBuild Cat: 743876; Clone: 2G9), BUV805 rat anti-mouse Ly6G (1:200 BD OptiBuild Cat: 741994; Clone: 1A8), Brilliant Violet 711 Hamster anti-mouse CD11c (1:200 BD Biosciences Cat: 563048; Clone: HL3), BUV737 rat anti-mouse IL33R (ST2) (1:100 BD OptiBuild Cat: 749323; Clone: U29-93), Brilliant Violet 421 rat anti-mouse CD8a (1:200 BioLegend Cat: 100737; Clone: 53 − 6.7), APC Armenian Hamster anti-mouse CD194 (CCR4) (1:100 BioLegend Cat: 131212; Clone: 2G12), FITC rat anti-mouse/rat FoxP3 (1:100 Thermo Fisher Cat: 11-5773-82; Clone: FJK-16s), PE-Cyanine7 iNOS rat anti-mouse antibody (1:400 Thermo Fisher Cat: 25-5920-82; Clone: CXNFT), Alexa Fluor 647 anti-mouse CD206 (1:200 BioLegend Cat: 141712; Clone: 068C2), PE-Dazzle 594 anti-mouse CD301b (1:300 BioLegend Cat: 146816; Clone: URA-1), PerCP/Cyanine5.5 rat anti-mouse T-bet (1:200 BioLegend Cat: 5760; Clone: 4B10), rat anti-mouse CD16/32 (TruStain FcX) (1:200 BioLegend Cat: 101320; Clone: 93), eBioscience Fixable Viability Dye eFluor 506 (1:500 Thermo Fisher Cat: 65-0866-14). Statistics and reproducibility : CCL22MP release experiments were performed in triplicate, and data represent means with standard deviation error bars. For multiple comparisons, One-Way Anova was performed followed by Šídák's multiple comparisons test. Differences in means were considered to be significant if p ≤ 0.05. Declarations DATA AVAILABILITY Data associated with this manuscript is maintained on an internal data repository at the University of Pittsburgh. Data will be made available upon reasonable request to the corresponding author. AUTHOR CONTRIBUTIONS Matthew A. Borrelli – Conceptualization; Data Curation; Formal analysis, Investigation, Methodology, Visualization, Writing – Original Drat, Writing – Review & Editing Jordan JP. Warunek – Conceptualization; Data Curation; Formal analysis, Investigation, Methodology, Visualization, Writing – Original Draft, Writing – Review & Editing Steven R. Little – Funding acquisition, Resources, Supervision, Writing – Review & Editing Hēth R. Turnquist – Funding acquisition, Resources, Supervision, Writing – Review & Editing ACKNOWLEDGEMENTS Work performed in the University of Pittsburgh Nanofabrication and Characterization Core Facility (RRID:SCR_05124) and services and instruments used in this project were graciously supported, in part, by the University of Pittsburgh. SOURCES OF FUNDING This work was supported by funding from National Institutes of Health (R01HL122489, F31HL170514, and T32HL076124), the Department of Defense (RT200012, RT230018, and PR230360), and the American Heart Association (23PRE1026713). These funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. COMPETING INTERESTS All authors declare no financial or non-financial competing interests. References Edouard, P. et al. Traumatic muscle injury. Nat. Rev. Dis. 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We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7237053","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":503848512,"identity":"c07d2c78-6665-4fc6-a4fb-cf0110749b88","order_by":0,"name":"Matthew A. Borrelli","email":"","orcid":"","institution":"University of Pittsburgh","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"A.","lastName":"Borrelli","suffix":""},{"id":503848513,"identity":"6f81157f-141a-48ec-8398-f10eb4de4e0a","order_by":1,"name":"Jordan JP. Warunek","email":"","orcid":"","institution":"University of Pittsburgh School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jordan","middleName":"JP.","lastName":"Warunek","suffix":""},{"id":503848514,"identity":"ae97b052-1bad-464e-850f-ecf4024af15b","order_by":2,"name":"Steven R. Little","email":"","orcid":"","institution":"University of Pittsburgh","correspondingAuthor":false,"prefix":"","firstName":"Steven","middleName":"R.","lastName":"Little","suffix":""},{"id":503848515,"identity":"ace4e265-142e-49e5-86ca-a32c6f21f4a9","order_by":3,"name":"Heth R. Turnquist","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqUlEQVRIiWNgGAWjYBACAwYG9g8fGCTAHAkitTCzMc5gkJDgIUkLM1A5CVrM2fuPPbbdYVFnz8B88DYPMVosew6zG+eeATmMLdmaKC0GN5IZpHPbQFp4zKSJ03L/MYO0JVgL/zcitdxgZpNmhNjCRqSWM8nGhr1nJCR7DrMZW84hSsvxgw8f/NxRx8/e3vzwxhtitIABYwOQYCZaOVzLKBgFo2AUjAJcAAB1Uid6pVU/UQAAAABJRU5ErkJggg==","orcid":"","institution":"University of Pittsburgh School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Heth","middleName":"R.","lastName":"Turnquist","suffix":""}],"badges":[],"createdAt":"2025-07-28 20:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7237053/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7237053/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89648117,"identity":"3397de69-f129-4783-aee6-9d4df2d69958","added_by":"auto","created_at":"2025-08-22 09:19:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":208323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCL22 encapsulated in PLGA MP releases for 15 days\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProposed Treg attracting microparticles designed to release CCL22 protein were characterized. \u003cstrong\u003ea)\u003c/strong\u003e SEM images of 55 wt.% polyethylene glycol (PEG) terminated poly(lactic-co-glycolic) acid (balance RG502H PLGA) microparticles (MP) show spherical morphology with a rough, porous surface. Left – 800x, scale bar = 10 µm; Right – 3500x, scale bar = 3 µm. \u003cstrong\u003eb)\u003c/strong\u003e Particle size distributions of unloaded MP (BlankMP) and MP loaded with 5 µg of CCL22 (CCL22MP) show particles are between 15-25 µm in diameter. \u003cstrong\u003ec)\u003c/strong\u003eQuantification of CCL22MP release shows sustained and controlled release of CCL22 for a period of at least 15 days. Data shown represents the mean ± SD for N = 3 independent experiments.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7237053/v1/aa2da8ba1df2f4be9cdc0de8.png"},{"id":89649490,"identity":"e08bc29d-1c7f-4b32-b042-6e41af2858fc","added_by":"auto","created_at":"2025-08-22 09:27:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":369409,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCL22MP treatment improves limb function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea)\u003c/strong\u003e Experimental scheme – MP treatments were administered 24 hours after injury with limb function assessments evaluated on days 3, 5, 7, 10, and 14 via inverted cage hanging. \u003cstrong\u003eb)\u003c/strong\u003e Example image of a mouse hanging on an inverted cage. Additional images and video are contained in the supplemental data. \u003cstrong\u003ec)\u003c/strong\u003e The normalized hang duration (minutes) was significantly elevated in CCL22MP treated mice for days 3 and 5. Data shown represents mean ± SD of N = 14 to 22 mice. One-way anova was performed with Dunnet’s multiple comparisons correction to determine differences between each group and the CTX only control. * p \u0026lt; 0.05, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7237053/v1/6b1a06736c7164487719caf5.png"},{"id":89649493,"identity":"adba1725-ea58-433e-b550-18a35b5c8456","added_by":"auto","created_at":"2025-08-22 09:27:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":601882,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCL22MP reduces area of injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistologic evaluation of hindlimb muscles harvested on day 14 post-CTx, following functional measurements. \u003cstrong\u003ea)\u003c/strong\u003e Representative H\u0026amp;E staining – Scale bar (red) is 20 µm. \u003cstrong\u003eb)\u003c/strong\u003e Cell count of regenerating (centrally nucleated) myofibers within tissue sections. \u003cstrong\u003ec)\u003c/strong\u003e Area % of regenerating muscle. Data shown represents mean ± SD for N = 14 mice and each data point is the average of both hindlimbs. One-way Anova was performed with Dunnet’s multiple comparisons correction to determine differences between each group. * p \u0026lt; 0.05, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7237053/v1/6d5440262bb3ee191e2e258f.png"},{"id":89648124,"identity":"51d86ff8-4543-407e-9b9f-3a4c562d224d","added_by":"auto","created_at":"2025-08-22 09:19:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":318122,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCL22MP treatment augments Treg and myeloid cells within the injured muscle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea)\u003c/strong\u003e Experimental scheme – Mice were co-administered microparticle (MP) therapeutics and cardiotoxin (CTx); after 5 days the limb muscles and popliteal draining lymph node were harvested, digested, and leukocytes were isolated. \u003cstrong\u003eb)\u003c/strong\u003e Representative quantitation of Foxp3\u003csup\u003e+\u003c/sup\u003e CD3\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e Treg population in the CD45\u003csup\u003e+ \u003c/sup\u003eB220\u003csup\u003e- \u003c/sup\u003eCD11b\u003csup\u003e- \u003c/sup\u003eCD11c\u003csup\u003e-\u0026nbsp;\u0026nbsp; \u003c/sup\u003egate is shown \u003cstrong\u003ec)\u003c/strong\u003e Quantification of the number of Tregs present in the treated limbs. \u003cstrong\u003ed)\u003c/strong\u003e Assessment of ST2 expression on Treg. \u003cstrong\u003ee)\u003c/strong\u003e Representative assessment of myeloid populations from muscle digests. CD45\u003csup\u003e+ \u003c/sup\u003eB220\u003csup\u003e-\u003c/sup\u003e CD3\u003csup\u003e-\u003c/sup\u003e CD11b\u003csup\u003e+\u003c/sup\u003e gated cells is shown. \u003cstrong\u003ef)\u003c/strong\u003e Quantification of the number of\u0026nbsp; CD11b\u003csup\u003e+\u003c/sup\u003e Ly6G\u003csup\u003ehi \u003c/sup\u003eneutrophils and CD11b\u003csup\u003e+ \u003c/sup\u003eLy6G\u003csup\u003elo \u003c/sup\u003eMDSCs in each group. Data shown is mean ± SD and represents the aggregate digest of both hindlimbs for N = 9 to 12 mice per group. One-way Anova was performed with Dunnet’s multiple comparisons correction to determine differences between each group. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, **** p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7237053/v1/3e6be8794aab5a9486a8610a.png"},{"id":89651631,"identity":"8086aabb-f9f0-4a63-af2c-394bff24df01","added_by":"auto","created_at":"2025-08-22 09:51:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2401720,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7237053/v1/3ba1607c-8441-4506-9b97-3b339d71434d.pdf"},{"id":89649872,"identity":"1325d504-865b-4398-af0a-97746bb7bdff","added_by":"auto","created_at":"2025-08-22 09:35:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":817468,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7237053/v1/959675d3907cf52155ba729c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regulatory T Cell Attracting Therapy Accelerates Skeletal Muscle Functional Recovery Following Injury","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSkeletal muscle injuries are a common and often unavoidable consequence of prolonged physical activity, intense exercise, and trauma. Muscle damage encompasses over 40% of all sports and exercise-induced injuries, and nearly 20% of injuries sustained during basic training of armed forces\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Among the general population, more than 21\u0026nbsp;million Americans over the age of 18 seek medical care for muscle injuries each year\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, amounting to hundreds of billions in lost yearly productivity\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Muscle injuries of differing severity occur when myofibers undergo structural damage leading to rupture or tear, resulting in swelling, pain and lost limb function\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Muscle injuries are often treated through a combination of rest, ice, compression elevation (RICE) and broad spectrum anti-inflammatory medications, such as non-steroidal anti-inflammatory drugs (NSAIDs) or corticosteroids\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. While NSAIDs and corticosteroids can reduce pain and swelling associated with inflammation, they also impair critical functions of the infiltrating immune cells that remove cellular debris and promote regeneration\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. For this reason, non-specific anti-inflammatory therapeutics often have poor efficacy and, in some cases, impair muscle regeneration\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. This has led to growing concerns about the overuse of NSAIDs for post-injury management in physically active populations\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSingle-cell and spatial transcriptomics, precise lineage tracing and cell-specific reporter constructs have enabled a detailed understanding of the mechanisms underlying the immune responses to skeletal muscle injury and how individual cell types contribute to the repair, remodeling and resolution of the injured tissue\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14 CR15\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Damage to the myofiber bundles can lead to necrosis, or inflammatory cellular death, of myocytes, which contributes to the release of damage associated molecular patterns (DAMPs), pro-inflammatory cytokines\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and chemoattractant molecules (chemokines), such as MCP-1 (CCL2), MIP-1α (CCL3), and MIP-1\u0026szlig; (CCL4)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. These chemokines signal through CCR2 and CCR5 receptors to attract neutrophils and monocytes to the injured tissue\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. There is some evidence that they also act directly on myocytes to initiate proliferation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Attracted neutrophils provide necessary clearance of cellular debris\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e while monocytes differentiate into inflammatory Ly6C\u003csup\u003ehi\u003c/sup\u003e macrophages that are critical for early satellite cell activation, myogenesis, and preventing maladaptive fibrosis\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. While engulfing cellular debris from the injured tissue, macrophages gradually receive signaling to differentiate into Ly6C\u003csup\u003elo\u003c/sup\u003e macrophages between day 3 and 6 post-injury\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, which exhibit a reparative phenotype\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and begin orchestrating tissue remodeling\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Specifically, they coordinate myogenesis and tissue remodeling through extracellular matrix deposition and stimulating tissue vascularization\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Importantly, effective tissue repair requires a balance of pro-inflammatory and reparative pathways, as chronic influx or overexuberant activity of these early inflammatory cells also have the potential to delay or impair the tissue repair. Situations may occur where monocytes differentiate into osteoclasts and reduce bone density\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e or collateral damage by neutrophils and their release of NETs can damage healthy cells\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. However, countering chemoattraction of neutrophils and monocytes via antiserum blockade of CCL2 or genetic ablation of CCR2 and CCR5 receptors results in severely diminished repair\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Additionally, early addition of the anti-inflammatory cytokine, IL-10, can also impair skeletal muscle regeneration by inhibiting macrophage proliferation and necessary cytokine expression\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Precise regulation is required to facilitate appropriate expansion of the macrophage population while avoiding the negative consequences of a chronic or overexuberant response.\u003c/p\u003e\u003cp\u003eRegulatory T cells (Treg) are recognized endogenous controllers of inflammatory immune cells that prevent autoimmunity and maintain tissue homeostasis\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Treg utilize several immunomodulatory mechanisms to suppress inflammatory responses, including the secretion of immunomodulatory proteins (IL-10 and TGFβ)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, scavenging IL-2, which is needed to perpetuate effector T cells\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and controlling the co-stimulation available to T cells\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Treg can also exert control over neutrophils\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e and macrophages\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e to reduce their production of inflammatory cytokines. It has also emerged, however, that Treg are not only immunosuppressive, but can be programmed by local injury signals to fulfill important reparative functions\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. After injury to skeletal muscle, as well as other tissues\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and organs\u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, Treg detection of interleukin-33 (IL-33) leads to their proliferation and release of reparative effector molecules, such as IL-13, a cytokine that polarizes local monocytes and macrophages towards a pro-repair subsets\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. IL-33 also stimulates Treg to produce potent growth factors, such as the epidermal growth factor amphiregulin (Areg) or vascular endothelial growth factor A (VEGF), among others\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Treg production of Areg is a particularly important mechanism for muscle repair, as Areg directly stimulates satellite cell proliferation after injury\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. IL-33-mediated Tregs production of Areg has been shown to be of importance in other models of tissue injury including transplantation\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, acute lung injury\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and myocardial infarction\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Treg repair functions are critical after injury, as global depletion of Treg following injury results in failed repair marked by lingering inflammation and disrupted reparative macrophage differentiation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Unfortunately, Treg in injured muscles are of limited frequency, especially in aged individuals\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, and slow to infiltrate or expand following injury\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Likewise, severe traumatic injuries cause the excessive release of inflammatory mediators that can cause inordinate inflammation and injury that overburden local reparative cells\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNovel therapies able to limit inflammation, while promoting repair are thought to be especially promising. Given their function in regulating immune cell inflammatory activity and ability to stimulate direct and indirect tissue repair, Tregs have been gaining increasing interest as a potential therapeutic target in skeletal muscle injury\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. We have developed a Treg-attracting formulation of microparticles capable of directing Treg chemotaxis to the site of administration via the controlled release of encapsulated CCL22\u003csup\u003e42\u003c/sup\u003e. CCL22 is a chemokine primarily involved in recruiting CCR4-expressing cells, particularly Tregs\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, as well as Th2 cells, and some myeloid cell subsets, to sites of inflammation or immune regulation\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. CCL22 plays a critical role in maintaining immune homeostasis, promoting anti-inflammatory responses, and has been implicated in tumor immune evasion\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e and transplant tolerance by facilitating Treg accumulation\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. CCL22 releasing microparticles (CCL22MP) have been employed successfully to control inflammation causing or perpetuating disorders including periodontitis\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, dry-eye disease\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, and transplantation rejection\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, but CCL22MP have not yet been evaluated for their ability to improve regeneration after muscle injury by attracting Treg. As such, we assessed if the regulatory and direct regenerative functions of Treg could be harnessed for accelerated repair following acute skeletal muscle injury using CCL22MP treatment. Using a murine model of skeletal muscle injury, we clearly show that CCL22MP treatment is a highly promising and novel therapeutic that enhances functional strength recovery by rapidly amplifying the presence of Treg in the injured limbs to stimulate regeneration and reduce the area of injury.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cem\u003eDevelopment and characterization of CCL22 releasing microparticles\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMicroparticles (MP) formulated with a blend of PEG terminated PLGA and RG502H, in which the PEG-PLGA fraction was gradually increased, were loaded with CCL22 (\u0026ldquo;CCL22MP\u0026rdquo;) or water (\u0026ldquo;BlankMP\u0026rdquo;) and 15 mOsm of NaCl (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). 55% PEG-PLGA (Balance RG502H) was selected for functional characterization and use \u003cem\u003ein vivo\u003c/em\u003e due to its favorable release kinetics. More specifically, the release profile for this formulation was compatible with our desire to promote rapid and accelerated Treg infiltration by simulating an initial boost in chemotactic signal followed by sustained signaling to establish a stable chemotactic gradient. Scanning electron microscopy (SEM) imaging shows that both CCL22 and BlankMP had expected spherical morphology and that the surface is populated with pores to facilitate early release as reported previously\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Both formulations show similar size distributions, in which the mean diameter ranges from 15\u0026ndash;25 \u0026micro;m (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The cumulative release profile of CCL22 from CCL22MP was quantified for 15 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Following an initial rapid burst release of 0.2 ng CCL22/mg of MP, this formulation produces a sustained and controlled rate of release amounting to 20 (pg of CCL22)/(mg of MP)/(day) for 15 days.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCCL22 releasing microparticles improve limb function following acute muscle injury\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe impact of CCL22MP treatments were evaluated in a variant of the well characterized cardiotoxin (CTx)-induced muscle injury model\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Specifically, mice received a single injection of CTx in PBS (\u0026ldquo;CTx\u0026rdquo;) or PBS alone (\u0026ldquo;PBS\u0026rdquo;) into the gastrocnemius of the right and left hindlimb and the extensor carpi of the forelimbs to generate injury to all extremities. 24 hours post injury, a subset of mice received intramuscular injections of CCL22MP suspended in PBS (\u0026ldquo;CCL22MP\u0026rdquo;) to the injured muscles, the CTx subsets received a control treatment of PBS alone, and others were treated with empty or \u0026ldquo;BlankMP\u0026rdquo; suspended in PBS. Limb function was then quantified on days 3, 5, 7, 10, and 14 following injury induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Here, limb function and motor coordination were tested using the inverted wire hang method (reference image in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Hang duration was normalized to baseline measurements taken prior to CTx injury and plotted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. As expected, mice in the uninjured PBS treated control group did not display a functional defect. Mice in the CTx group that were administered CTx and then treated with PBS exhibited significantly reduced functions compared to PBS only controls at days 3, 5, and 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Mice administered CTx and then treated with CCL22MP had significantly improved hang duration at day 3 and 5 timepoints, relative to the CTx injury group. By the day 7 timepoint, hang duration for CCL22MP treated mice remained elevated, but other control groups began to show increasingly improved performance. By day 14 post-injury, all groups exhibited a return of function to baseline measurements. Thus, CCL22MP accelerates functional strength and recovery during the acute phase following muscle injury.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCCL22MP treated limbs exhibit reduced injury in CTx treated skeletal muscles\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTo define how CCL22MP-mediated improvements in function relates to skeletal muscle injury repair, we completed histologic assessment of cross-sectioned gastrocnemius muscles at day 14 post-CTx administration. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA shows representative images of the regenerating muscle for each treatment group. PBS alone tissues exhibit normal muscle architecture consisting of well-organized and tightly packed fibers with peripheral nuclei as expected. CTx treated muscles, however, displayed disrupted and irregularly shaped fibers with centrally located nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In samples from CCL22MP-treated mice, the diameter of the myofibers appear increased relative to CTx control. Myofiber cross-sections from BlankMP-treated limbs exhibited an increased frequency of peripheral nuclei, which corresponds to the final phase of regeneration. Quantification of the total number of centrally nucleated, regenerating myocytes and their area percentage relative to the entire gastrocnemius is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C. CCL22MP treatment results in a significant reduction in the number and area of regenerating cells, relative to the CTx injury control. Taken together, CCL22MP treatment improves muscle regeneration and reduces injury more effectively than BlankMP, supporting its reparative potential after muscle injury.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eCCL22MP enrich for immunoregulatory cell populations at the site of injury\u003c/em\u003e\u003c/p\u003e\u003cp\u003eWe next defined how CCL22MP treatment shapes immune responses following CTx injury using multispectral flow cytometry on immune cells isolated from the skeletal muscles and nearest draining lymph nodes (see \u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e for a representative gating strategy). In these studies, mice were injected with CTx or PBS and then administered CCL22MP in PBS, BlankMP in PBS, or PBS alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Assessment of CD45\u003csup\u003e+\u003c/sup\u003e leukocyte populations isolated from the injured gastrocnemius muscle 5 days after injury and treatment reveals an increased frequency of CD3\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e cells expressing the Treg transcription factor Foxp3 for CCL22MP treated limbs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u003cb\u003eand Supplementary Fig.\u0026nbsp;4A\u003c/b\u003e). The total number of Tregs normalized to the mass of hindlimb tissue was calculated and also revealed a significant increase relative to the BlankMP or CTx alone control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The level of expression of the IL-33 receptor, serum stimulation‑2 (ST2), was increased on local Foxp3\u003csup\u003e+\u003c/sup\u003e CD3\u003csup\u003e+\u003c/sup\u003e CD4\u003csup\u003e+\u003c/sup\u003e CD25\u003csup\u003e+\u003c/sup\u003e by muscle injury, however, there was not a difference noted between treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Further assessment of the myeloid compartment found that there was an elevated frequency of rare CD45\u003csup\u003e+\u003c/sup\u003e Ly6G\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003eLo\u003c/sup\u003e cells that are phenotypically consistent with those described as monocyte-derived suppressor cell (MDSC)\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E and \u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). There was also an elevated frequency of neutrophils in the CCL22MP treated limbs compared to CTx alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Quantification of the total number of MDSC and neutrophils showed a significant increase in response to CCL22MP treatment relative to BlankMP-treated limbs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). We did not detect significant differences in the frequency of intramuscular B cells, dendritic cells, as well as macrophages (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), however, intramuscular B cells and macrophages exhibited a trend towards increased frequency (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and number (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) with CCL22MP treatment. To better understand changes in the local immune populations supporting tissue healing versus any treatment-induced distant immunomodulatory impacts, we assessed CD45\u003csup\u003e+\u003c/sup\u003e leukocytes from the draining popliteal lymph nodes. Similar flow cytometric analysis found minimal elevations in the frequency of immune cells assessed (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). CCL22MP treatment was associated with increased numbers of macrophages in the draining LN (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). There was not an increase in the frequency of Treg, nor did we observe changes in ST2 in the draining lymph nodes (\u003cb\u003eSupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In total, these findings demonstrate that CCL22MP treatment enhances local accumulation of Tregs, presumed MDSC, and neutrophils at the site of muscle injury, which was associated with an accelerated restoration of muscular function.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study reports how enhancing the local population of Tregs through the administration of a Treg-attracting microparticle formulation affects the recovery of injured skeletal muscle. Prior studies have delineated that Treg play critical roles in skeletal muscle injury\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e by not only regulating infiltrating leukocytes\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, but also secreting growth factors like Areg that stimulates muscle satellite cell proliferation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Despite increasing interest in leveraging Tregs as a therapeutic target in skeletal muscle injury\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, to our knowledge, there have not been prior studies that have investigated the amplification of Tregs through local drug delivery in the injury site as a potential treatment. In this first-of-its-kind study amplifying the local population of Tregs in the injured muscle, we found that treatment with microparticles releasing CCL22 (CCL22MP), a Treg chemoattractant, significantly improved the limb function of mice at early timepoints. Subsequent investigations revealed a reduced area of injury in the limb, as well as significantly elevated amounts of Treg, presumed MDSC, and neutrophils, locally, but not in the adjacent lymphoid tissues. Together, these findings establish a novel and effective strategy for modulating the immune microenvironment at the site of injury through local Treg recruitment, highlighting the therapeutic potential of CCL22MPs in accelerating muscle repair and functional recovery.\u003c/p\u003e\u003cp\u003eThe importance of Treg chemoattraction to local immunomodulation was originally defined in studies developing an understanding how tumors evade immune clearance\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. These studies revealed that the chemokine, CCL22, is secreted by tumor associated macrophages and dendritic cells\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. This directs Treg that are enriched for the CCL22 receptor, CCR4, to infiltrate the tumor site\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Taking inspiration from this, we have developed a PLGA microparticle-based platform that has demonstrated the ability to attract adoptively transferred Tregs to the CCL22MP injection site\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Subsequent studies demonstrated CCL22MP treatment to be efficacious in models of inflammatory disease including periodontitis\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, dry-eye disease\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e, and transplantation rejection\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In each of these studies, RG502H \u0026ndash; an acid terminated PLGA polymer containing equal ratio of glycolic and lactic acid repeating units \u0026ndash; was utilized as the encapsulating polymer. CCL22 release from this polymer formulation has produced linear release profiles that delivered 80\u0026ndash;4500 pg CCL22/mg of MP by day 14, which is compatible with Treg functional immunobiology. PLGA polymer is one of the most widely explored degradable polymers for delivery of protein therapeutics because of its biocompatible degradation products, glycolic and lactic acid, and its prior use in FDA-approved formulations\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Recently, PLGA polymers with various terminal groups have become more accessible\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e and, in some cases, facilitate a gain of function such as improved cellular targeting of nanoparticles\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e or reduced phagocytic clearance\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. For the current study, we included poly(ethylene glycol) (PEG) terminated PLGA in the encapsulating polymer due to its ability to confer several benefits. Specifically, PEG has been reported to slow the clearance rate of polymeric particles\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, and, following injury, it has been demonstrated to provide membrane sealing\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e and to reduce apoptosis\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. The resultant formulation of 55 wt.% PEG-PLGA and 45 wt.% RG502H produced linear release behavior amounting to 500 pg of CCL22/mg of MP delivered by day 14, which is consistent with our previously reported release profiles that produced rapid augmentation of Treg at the site of injury. That we did not witness systemic changes in Treg suggests precisely controlled local delivery.\u003c/p\u003e\u003cp\u003eThe importance of a well-coordinated immune response in skeletal muscle regeneration has been routinely characterized using murine models, including CTx injections. The regenerative program after CTx administration depends on a temporally regulated sequence of events, beginning with an acute inflammatory phase dominated by pro-inflammatory macrophages, followed by a reparative phase characterized by restorative macrophages\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. However, Tregs also play critical roles in shaping skeletal muscle repair. Recent work investigating Tregs functions in muscle repair documented a subset of skeletal muscle-resident Treg that highly express the helios transcription factor and the neuropilin transmembrane receptor\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Their helios and neuropilin expression indicate that these Tregs are most likely thymic derived and not peripherally induced. These Treg were also reported to highly express ST2 receptor and respond to IL-33 with production of Areg\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Transgenic mice where Treg lack Areg were exploited to determine that Areg contribute a significant role in shaping the Treg response to skeletal injury by stimulating satellite cell proliferation\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. While Areg has also be implicated in suppressive functions\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, several studies using Treg lacking Areg have found that systemic immune responses to virus\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e and alloantigen\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e are not modified in the absence of Treg-expressed Areg. Tregs typically begin to amass in the injury site 4 days following initial injury via CCL3 chemoattraction\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Thus, CCL22MP treatment one day following injury has the potential to augment and accelerate the normal attraction of Treg to injured skeletal muscles. Indeed, our experiment administering CCL22MP after CTx-mediated injury resulted in a significant improvement to limb function on post-injury days 3 and 5 \u0026ndash; timepoints where Treg are typically only just arriving. Consistent with improved function, subsequent histologic analysis on post-injury day 14 showed that CCL22MP treated limbs have reduced injury area, suggesting that CCL22MP treatment facilitated a reduction in damage spreading or accelerated repair relative to the control group. Whether this is the result of Areg-mediated repair or local immunomodulation by attracted Treg, however, will require further investigations using precise transgenic mice allowing the targeting of Areg or molecules implicated in Treg immunomodulation. BlankMP show a slight reduction in the injury area, though it is not statistically significant, indicating the PEG may be providing an anti-apoptotic effect, which would be consistent with prior reports\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Additionally, lactic acid has beneficial effects following muscle injury\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Thus, lactic acid degradation products from locally injected microparticles may also be promoting the observed differences in the BlankMP-treated limbs. These effects would be conserved for CCL22MP treatment and may be providing a synergistic benefit that complements the effects of attracted Treg.\u003c/p\u003e\u003cp\u003eOur flow cytometry assessments define how CCL22MP influenced the immune populations in the injury site and the draining lymph nodes, Treg in the injury site, but not the nearest draining lymph node, were significantly expanded for mice receiving CCL22MP. This result indicates that CCL22MP exerts a targeted effect, enhancing Treg recruitment or retention specifically within the tissue microenvironment of the injury, without inducing systemic or regional immune changes. This localized immunomodulation is predicted to be therapeutically advantageous because it will enhance repair and suppression at the site of injury, while minimizing any risk of systemic immunosuppression. Notably, there was an increased expression of ST2 on Treg at the site of injury, but no difference in the ST2 expression among the treatment groups. This suggests that ST2 expression is a response to the injury microenvironment itself, independent of CCL22MP treatment. This aligns with our recent studies showing that ST2\u003csup\u003e+\u003c/sup\u003e Treg emerge upon infiltrating in inflamed or damaged tissues, where IL-33 is often released by stressed or necrotic cells\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Thus, the injury environment, not CCL22, appears to be sufficient to induce this ST2, and presumably allow IL-33 to support the reparative Treg phenotype.\u003c/p\u003e\u003cp\u003eThe changes to B cell and macrophage populations identified in the hindlimb and lymph node are largely not statistically significant, but do suggest these populations are attracted or expanded in response to BlankMP and CCL22MP treatments. It is possible that the microparticles caused a foreign body response in the hindlimbs. It is well known that large microparticles (\u0026gt;\u0026thinsp;20\u0026ndash;30 \u0026micro;m\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e) are not readily phagocytosed resulting in a state of frustrated phagocytosis\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e and activating macrophages\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Notably, we did observe CCL22MP treatment caused an expanded Ly6G\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003eLo\u003c/sup\u003e myeloid cell and neutrophil populations. The Ly6G\u003csup\u003e+\u003c/sup\u003e CD11b\u003csup\u003eLo\u003c/sup\u003e phenotype is associated with the monocyte derived suppressor cell phenotype, which possess potent immunosuppressive and reparative capabilities in injury\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The increase in neutrophil population 5 days following injury was unexpected. These cells typically undergo apoptosis following clearance of cellular debris 2\u0026ndash;3 days after injury\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, \u003cem\u003ein vitro\u003c/em\u003e studies have shown Tregs can act to reduce neutrophil IL-6 expression and skew them towards a suppressive and pro-repair phenotype that secretes IL-10 and TGF-\u0026szlig;, heme oxygenase-1, and indoleamine 2,3-dioxygenase\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Thus, it is possible that the attracted Treg are sustaining the neutrophil population to perform anti-inflammatory and pro-repair roles. Another possible explanation is that these neutrophils and MDSCs are co-migrating into the injured limb in response to CCL22. Neutrophils have been reported to express an array of chemokine receptors including CCR1, CCR2, CCR3, CCR5, CXCR1, CXCR2, CXCR3, and CXCR4, and that the expression of these receptors differs among injury infiltrating neutrophils and peripheral blood neutrophils\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Of interest is CCR2 expression, which is the target receptor or CCL2. Interestingly, CCL2 can also bind to CCR4\u003csup\u003e72\u003c/sup\u003e; because CCR4 is the target receptor for CCL22, it is possible CCL22 could bind to the CCR2 receptor, reciprocally. Further, it has been reported that chemokines can interact with one another to produce synergistic migratory effects\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. For instance, in the context of allergic dermatitis, CCL22 was shown to interact with CXCL10 resulting in amplified migration of CCR4\u0026thinsp;+\u0026thinsp;T cells into the tissue\u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. A similar phenomenon has been observed for neutrophil migration, in which CCL2 and CCL7 synergize with CXCL8 to enhance their migration during acute respiratory distress syndrome\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe present study provides insights into how localized regulatory T cell amplification following muscle injury can affect limb functional recovery. One important limitation of this work is that it lacked quantification of secreted reparative, inflammatory, and anti-inflammatory factors from the local immune populations. Logically, increased numbers of Treg that are phenotypically identical should result in a proportional increase in secreted factors. However, it is possible that the activity of the expanded Treg population is also increased. Likewise, the studies completed did not quantify how the expanded Treg population affects the local balance of inflammatory (e.g. IFNγ, TNFα, IL-6, etc.) and anti-inflammatory (e.g. TGF\u0026szlig;, IL-10, etc.) signals. Thus, future experiments that investigate how CCL22MP treatment may alter these secreted factors following muscle injury are merited. Further, this study lacks a full investigation into the expanded neutrophil population observed in CCL22MP treated limbs. Future studies could investigate the neutrophil populations to inform what role they are playing in the injured limb. Another limitation of this work, however, is a lack of precise understanding of the mechanism these Treg improved functional repair. Without blockade of the reparative effectors, IL-13 and Areg, secreted from Treg or the suppressive mechanisms of these cells, such as IL-10 or CTLA-4, it is unclear what their individual contributions are to the observed functional recovery and reduction to injury. Most likely benefits require both suppressive and reparative Treg functions. Therefore, future studies employing the Cre-loxP system to alter genetic expression of the repair and suppressive genes in Foxp3\u003csup\u003e+\u003c/sup\u003e Treg in this injury model with CCL22MP treatment may provide greater insight. Additionally, this study focused on a CTx model of muscle injury which does not interfere with basal repair functions. While the improvement that CCL22MP treatment conferred was considerable in this model, repeating this work in injury models that have limited regenerative capabilities, such as the volumetric muscle loss model, or in mice that express poor or dysregulated muscle regeneration, such as mdx mice that exhibit muscular dystrophy, may provide a greater understanding of the potential benefits of this treatment. Despite these limitations, the work reported in this manuscript has broad implications for the field of regenerative medicine. Because the immune response is critical to tissue repair and remodeling, this Treg attracting treatment has potential to be beneficial in other injury and regenerative models.\u003c/p\u003e\u003cp\u003eIn total our study establishes that CCL22-enriched Tregs have a significant ability to shape the injury microenvironment. Intervening within the first 24 hours of an acute skeletal muscle injury with a Treg-attracting microparticle formulation resulted in early improvements to limb function and a reduction in total injury. This study is the first of its kind to manipulate Tregs in the injury site, directly, and may provide similar efficacy in other models of muscle injury and trauma. Future work exploring how early intervention with Treg-targeting therapies affects the cellular microenvironment following injury in muscles and other tissue types will be insightful and may lead to the discovery of new therapeutic targets or establish if local Treg could be replaced by controlled release of their delivered reparative or immunoregulatory effectors.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMaterials\u003c/span\u003e:\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePolymers\u003c/span\u003e: RG502H Poly(D,L-lactic-co-glycolic acid) (PLGA) polymer, with 50:50 lactide:glycolide composition was obtained from Evonik (Essen, Germany). Poly(ethylene glycol) (PEG) terminated PLGA (PEG-PLGA) and Poly(vinyl alcohol) (PVA, 98 mol% hydrolyzed, \u003cem\u003eM\u003c/em\u003e\u003csub\u003e\u003cem\u003ew\u003c/em\u003e\u003c/sub\u003e = 25000 g mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) were purchased from PolySciences (Warrington, PA).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eProtein\u003c/span\u003e: Carrier-free recombinant murine CCL22 (mCCL22) was obtained from R\u0026amp;D systems (Minneapolis, MN). Naja pallida cardiotoxin (CTx) was obtained from Sigma-Aldrich (St. Louis, MO) and diluted to a working concentration of 0.03 mg/mL prior to injection. Bovine Serum Albumin (BSA) was obtained from Fisher Chemical (Waltham, MA).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eReagents\u003c/span\u003e: Dichloromethane (DCM), Acetonitrile (ACN), Sodium Dodecyl Sulfate (SDS), was obtained from Fisher Chemical (Waltham, MA)\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCell Reagents and Buffers\u003c/span\u003e: Phosphate buffered saline (PBS), fetal bovine serum (FBS), Hanks\u0026rsquo; balanced salt solution (HBSS), Roswell Park Memorial Institute 1640 medium (RPMI), Cytiva percoll centrifugation media, and eBioscience Foxp3 / Transcription Factor Fixation/Permeabilization buffer from Fisher Scientific (Waltham, MA). FACS and perm buffer were produced in house by supplementing PBS with 5% Fetal Bovine Serum (FBS) and 0.1% sodium azide; 1% FBS, 0.1% sodium azide, and 0.1% saponin; and 5% FBS, respectively.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMicroparticle (MP) formulation\u003c/span\u003e: Microparticles (MPs) encapsulating CCL22 or blanks were formulated via a double emulsion-evaporation method, as described in our previous work\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Briefly, MP formulation involves the formation of an emulsion between an inner, aqueous phase (200 \u0026micro;L vol) that contains CCL22 protein (5 \u0026micro;g) (CCL22MP) or water (BlankMP) and the outer, organic phase that contains PLGA polymer (200 mg, 55 wt.% PEG-PLGA \u0026amp; balance RG502H) solvated in dichloromethane (DCM) (4 mL). The aqueous phase for CCL22MP and BlankMP was formulated with 0.375 mg BSA to stabilize the encapsulated protein, and an osmolarity of 15 mOsm to produce surface pores. The resulting mixture of protein and polymer was sonicated (Active Motif EpiShear probe sonicator, 110V) at 55% amplitude for 10s to form the first emulsion (water-in-oil, w/o), and then transferred to a 2% PVA solution (60 mL) and homogenized (L4RT-A; Silverson, East Longmeadow, MA) at 3000 rpm for 1 minute, forming the second emulsion (w/o/w). Following homogenization, the solution was transferred to a 1% (w/v) PVA solution (80 mL) and stirred at 600 RPM for 3 hours on ice to allow the DCM to evaporate. Freshly formed MPs were centrifuged (Eppendorf 5810R 15 amp version, 200 xg for 5 min at 4\u0026deg;C) and washed four times (4x) with 4\u0026deg;C MilliQ water (Milli-Q IQ 7000, Millipore Sigma) (35 mL). The MPs were then re-suspended in MilliQ water (5 mL), flash-frozen with liquid nitrogen (5 minutes), and lyophilized (Benchtop Pro, Virtis SP Scientific) (\u0026lt;\u0026thinsp;100 mTorr, 48\u0026ndash;72 hours).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMicroparticle characterization and release assays\u003c/span\u003e: Scanning electron micrographs of MPs were obtained using a scanning electron microscope (ZEISS Sigma500 VP), located in the University of Pittsburgh Nanofabrication and Characterization Core Facility. Size distributions of MPs were determined using volume impedance measurements on a Beckman Coulter Counter (Multisizer-3; Beckman Coulter; Brea, CA). \u003cem\u003eIn vitro\u003c/em\u003e release behavior was characterized by incubating 10 mg of MPs in 1 mL of release media (1% (w/v) bovine serum albumin (BSA) in PBS) and incubated on a roto-shaker (Thermo Scientific\u0026trade; Tube Revolver Rotator, 1.5\u0026ndash;2 mL Eppendorf tube paddles, speed 10) at 37\u0026deg;C. At specified time intervals, MP suspensions were centrifuged (580 g for 5 mins at room temperature) (Eppendorf 5417R), 800 \u0026micro;L of the supernatant was removed and replaced with 800 \u0026micro;L of fresh release media, and the MPs were resuspended and placed back on the roto-shaker. Supernatant concentrations of CCL22 were determined via enzyme-linked immunosorbent assay (ELISA; R\u0026amp;D Systems), and subsequently the ELISA results were interpreted through quantification of optical density (SpectraMax M5; Molecular Devices; Sunnyvale, CA). Release profiles generated from measured concentrations of CCL22 had any background signal from BlankMP subtracted and then normalized to the total amount loaded within MPs (quantified by solvating MPs and extracting the protein liberated) and MP mass.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCTx Muscle Injury Model \u0026amp; MP Treatment\u003c/span\u003e: Limb injury was conducted following a variant of the well characterized cardiotoxin skeletal muscle injury model\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Mice (B6 Wildtype, Male, 8\u0026ndash;10 wks old) were randomly assigned to one of 4 groups: Healthy control (PBS), injury and treatment control (CTx), Vehicle control (BlankMP), and treatment group (CCL22MP). Mice were anesthetized with 4% Isoflurane and injected with 50 \u0026micro;L of CTx (0.03 mg/mL) (CTx, BlankMP, and CCL22MP groups) or 50 \u0026micro;L of saline (PBS group) into each forelimb (Extensor Carpi) and hindlimb (Gastrocnemius). Injured limbs were administered 50 \u0026micro;L of CCL22MP (10 mg/mL) (CCL22MP group), BlankMP (10 mg/mL) (BlankMP group), or saline (CTx group) was injected into each forelimb and hindlimb. For functional studies, treatment occurred 24 hours after injury and for flow cytometry studies treatment occurred simultaneously with injury induction. At the conclusion of a study, mice (age: 8\u0026ndash;16 weeks, weight: 22\u0026ndash;30g) were euthanized via CO\u003csub\u003e2\u003c/sub\u003e (99% purity) and subsequent cervical dislocation. The Gastrocnemius was then collected for further analysis.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eStudy approval and reporting\u003c/span\u003e: All procedures completed at the University of Pittsburgh were approved by the IACUC of the University of Pittsburgh (protocol 22051178) and complied with the NIH\u0026rsquo;s Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011). This study is reported in accordance with ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eLimb function and histologic injury assessment\u003c/span\u003e: At specified timepoints following CTx injury (POD 3, 5, 7, 10, \u0026amp; 14) limb function was assessed via the commonly used inverted wire hang method\u003csup\u003e\u003cspan additionalcitationids=\"CR78\" citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Briefly, mice were placed on a wire cage, the cage was inverted, and the time it took for the mice to fall off the cage was recorded. For each mouse, this assessment was repeated in triplicate with at least 60 seconds rest between each trial. The maximum value of the three trials was calculated and normalized to the maximum value measured during a baseline wire hang test performed prior to injury. The data was tabulated and evaluated for statistical differences to the CTx injury control. \u003cem\u003eHistology\u003c/em\u003e: After the functional measures were completed on Day 14, the gastrocnemius muscles were collected for histologic analysis. The muscle tissue was fixed in 4% paraformaldehyde for 2 hours followed by a 2-step dehydration utilizing an overnight incubation in 30% sucrose and final storage in 70% ethanol (balance water). Samples were sectioned (10\u0026micro;m thick) at the midpoint of the gastrocnemius perpendicular to the origin and insertion, and hematoxylin and eosin (H\u0026amp;E) staining was then performed. Regenerating myofibers have centrally located nuclei and at later stages of regeneration and later mature into normal sized myofibers with peripheral nuclei\u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. Thus, quantification of the count and area of centrally nucleated, regenerating muscle cells was performed using QuPath open-source software\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTissue Collection\u003c/span\u003e: \u003cem\u003eMuscle\u003c/em\u003e: C57BL/6 wildtype mice were euthanized and gastrocnemius skeletal muscle were carefully excised and then minced by scissors in RPMI 1640 digestion buffer (2% FBS, 12.5mg collagenase IV (Thermo Fisher; 17104019) and DNAse I (Sigma Aldrich; DN25-1G)). The tissue was further homogenized using Miltenyi gentleMACS Dissociator and incubated at 250 rpm and 37\u0026deg;C for 45 minutes. After digestion, homogenate was passed through a 70\u0026micro;m nylon mesh strainer and processed for mononuclear cells by using a 37.5% Percoll gradient (GE Healthcare). Pellets were resuspended in 2% RPMI, counted by hemacytometer and prepared for flow cytometry staining. \u003cem\u003eLymph nodes\u003c/em\u003e: The nearest draining lymph node (popliteal) to the gastrocnemius was collected simultaneously. Each replicate represents the aggregate collection of both popliteal lymph nodes collected from each limb. The collected lymph nodes were minced by scissors in RPMI 1640 and passed through a 70\u0026micro;m nylon mesh strainer. The cells were pelleted by centrifugation and resuspended in RPMI 1640 for counting and prepared for flow cytometry staining.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFlow Cytometry\u003c/span\u003e: Harvested cells were incubated in 5% normal goat serum, stained with surface antibodies for 25 minutes on ice, washed and placed in Foxp3/Transcription Factor Staining Fixation/Permeabilization Buffer Set (eBioscience) overnight. For intracellular staining, cells were permeabilized (eBioscience) and stained with antibodies for 45 minutes on ice. Data was acquired using a Cytek Aurora spectral flow cytometer (Cytek Biosciences) and analyzed using FlowJo v10.9.0 (BD Life Sciences). The final stained samples were then washed 2x with FACS buffer and run on Cytek\u0026rsquo;s Aurora Cytometer (Fremont, CA).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFlow Cytometry Antibodies\u003c/span\u003e: BUV496 rat anti-mouse CD45 (1:300 BD Biosciences Cat: 569673; Clone: 30-F11), Pacific Blue anti-mouse F4/80 (1:150 BioLegend Cat: 123124; Clone: BM8), Brilliant Violet 785 rat anti-mouse CD4 (1:200 BioLegend Cat: 100552; Clone: GK1.5), Brilliant Violet 650 rat anti-mouse CD86 (1:200 BD Biosciences Cat: 564200; Clone: GL1), Brilliant Violet 605 rat anti-mouse CD25 (1:200 BD Biosciences Cat: 563061; Clone: PC61), PE rat anti-mouse Ly6C (1:200 BD Pharmigen Cat: 560592; Clone: AL-21), PEcy5 rat anti-mouse B220 (1:200 BD Pharmingen Cat: 553091; Clone: RA3-6B2), Alexa Fluor 700 rat anti-mouse CD3 Molecular Complex (1:200 BD Pharmigen Cat: 561388; Clone: 17A2), APC/Fire 750 anti-mouse/human CD11b (1:200 BioLegend Cat: 101262; Clone: M1/70), BUV395 anti-mouse I-A/I-E (1:200 BD OptiBuild Cat: 743876; Clone: 2G9), BUV805 rat anti-mouse Ly6G (1:200 BD OptiBuild Cat: 741994; Clone: 1A8), Brilliant Violet 711 Hamster anti-mouse CD11c (1:200 BD Biosciences Cat: 563048; Clone: HL3), BUV737 rat anti-mouse IL33R (ST2) (1:100 BD OptiBuild Cat: 749323; Clone: U29-93), Brilliant Violet 421 rat anti-mouse CD8a (1:200 BioLegend Cat: 100737; Clone: 53\u0026thinsp;\u0026minus;\u0026thinsp;6.7), APC Armenian Hamster anti-mouse CD194 (CCR4) (1:100 BioLegend Cat: 131212; Clone: 2G12), FITC rat anti-mouse/rat FoxP3 (1:100 Thermo Fisher Cat: 11-5773-82; Clone: FJK-16s), PE-Cyanine7 iNOS rat anti-mouse antibody (1:400 Thermo Fisher Cat: 25-5920-82; Clone: CXNFT), Alexa Fluor 647 anti-mouse CD206 (1:200 BioLegend Cat: 141712; Clone: 068C2), PE-Dazzle 594 anti-mouse CD301b (1:300 BioLegend Cat: 146816; Clone: URA-1), PerCP/Cyanine5.5 rat anti-mouse T-bet (1:200 BioLegend Cat: 5760; Clone: 4B10), rat anti-mouse CD16/32 (TruStain FcX) (1:200 BioLegend Cat: 101320; Clone: 93), eBioscience Fixable Viability Dye eFluor 506 (1:500 Thermo Fisher Cat: 65-0866-14).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eStatistics and reproducibility\u003c/span\u003e: CCL22MP release experiments were performed in triplicate, and data represent means with standard deviation error bars. For multiple comparisons, One-Way Anova was performed followed by Š\u0026iacute;d\u0026aacute;k's multiple comparisons test. Differences in means were considered to be significant if p\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData associated with this manuscript is maintained on an internal data repository at the University of Pittsburgh. Data will be made available upon reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMatthew A. Borrelli \u0026ndash; Conceptualization; Data Curation; Formal analysis, Investigation, Methodology, Visualization, Writing \u0026ndash; Original Drat, Writing \u0026ndash; Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003eJordan JP. Warunek \u0026ndash; Conceptualization; Data Curation; Formal analysis, Investigation, Methodology, Visualization, Writing \u0026ndash; Original Draft, Writing \u0026ndash; Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003eSteven R. Little \u0026ndash; Funding acquisition, Resources, Supervision, Writing \u0026ndash; Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003eHēth R. Turnquist \u0026ndash; \u0026nbsp;Funding acquisition, Resources, Supervision, Writing \u0026ndash; Review \u0026amp; Editing\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWork performed in the University of Pittsburgh Nanofabrication and Characterization Core Facility (RRID:SCR_05124) and services and instruments used in this project were graciously supported, in part, by the University of Pittsburgh.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSOURCES OF FUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by funding from\u0026nbsp;National Institutes of Health\u0026nbsp;(R01HL122489, F31HL170514, and\u0026nbsp;T32HL076124), the Department of Defense (RT200012, RT230018, and PR230360), and\u0026nbsp;the\u0026nbsp;American Heart Association (23PRE1026713). These funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no financial or non-financial competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEdouard, P. et al. Traumatic muscle injury. \u003cem\u003eNat. Rev. Dis. Primers\u003c/em\u003e. \u003cb\u003e9\u003c/b\u003e \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41572-023-00469-8\u003c/span\u003e\u003cspan address=\"10.1038/s41572-023-00469-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCenters for Disease, C. \u003cem\u003e\u0026amp; Prevention. 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Rep.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 16878. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-017-17204-5\u003c/span\u003e\u003cspan address=\"10.1038/s41598-017-17204-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Skeletal Muscle, Treg, Injury, Microparticles, Tissue Repair, Immunomodulation","lastPublishedDoi":"10.21203/rs.3.rs-7237053/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7237053/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSkeletal muscle injuries are a common consequence of physical activity, repetitive movements, and trauma. Regulatory T cells (Treg) have recently been identified as critical mediators of immune repair response after injury, and treatments effectively targeting Treg may accelerate injury resolution. CCL22 is a chemokine that recruits CCR4-expressing cells, particularly Treg, to sites of inflammation or immune regulation, such as tumor microenvironments. When a sustained release formulation of polymeric microparticles (MP) delivering CCL22 (CCL22MP), was administered after cardiotoxin (CTx)-mediated muscle injury, significantly improved limb function was observed on days 3 and 5 post injury. Histologic evaluation of the injured limbs showed reduced area of injury in CCL22MP treated limbs. Analysis of the local immune populations revealed augmented Treg concentrations, as well as increased myeloid derived suppressor cell and neutrophil frequency. These findings reveal that amplifying local Treg to damaged areas improves outcomes, thus offering a translationally promising approach after muscle injury.\u003c/p\u003e","manuscriptTitle":"Regulatory T Cell Attracting Therapy Accelerates Skeletal Muscle Functional Recovery Following Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 09:19:48","doi":"10.21203/rs.3.rs-7237053/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-26T04:09:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-25T07:06:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-21T19:53:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305662456392937254044055497070118853654","date":"2025-08-21T09:47:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170026559657794958384146464202982521247","date":"2025-08-18T13:13:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"244869492469276559665728008776168155206","date":"2025-08-16T18:40:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"260362036370209675728924118933937196432","date":"2025-08-16T17:49:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"319071293261703534219083445019038119829","date":"2025-08-15T15:45:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-14T16:18:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-14T16:02:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-13T12:31:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-31T19:14:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-31T19:10:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8b354dc7-bed7-43e8-b278-21f7011cdff6","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53531664,"name":"Health sciences/Diseases"},{"id":53531665,"name":"Biological sciences/Immunology"},{"id":53531666,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-05-13T03:54:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 09:19:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7237053","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7237053","identity":"rs-7237053","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}