Immunoresolvents Support Skeletal Myofiber Regeneration via Actions on Myeloid and Muscle Stem Cells

Specialized pro-resolving mediators (SPMs) actively limit inflammation and expedite its resolution. Here we profiled intramuscular lipid mediators following injury and investigated the role of SPMs in skeletal muscle inflammation and repair. Both eicosanoids and SPMs increased following myofiber damage induced by intramuscular injection of barium chloride or functional overload. Daily systemic administration of resolvin D1 (RvD1) limited the degree and duration of inflammation, enhanced regenerating myofiber growth, and improved recovery of muscle strength. RvD1 suppressed inflammatory cytokines, enhanced polymorphonuclear cell clearance, modulated muscle stem cells, and polarized macrophages to a more pro-regenerative subset. RvD1 had minimal direct impact on in-vitro myogenesis but directly suppressed myokine production and stimulated macrophage phagocytosis, showing that SPMs influence modulate both infiltrating myeloid and resident muscle cells. These data reveal the efficacy of immunoresolvents as a novel alternative to classical anti-inflammatory interventions in the management of muscle injuries to modulate inflammation while stimulating tissue repair.


Abstract (150-word limit):
Specialized pro-resolving mediators (SPMs) actively limit inflammation and expedite its resolution. Here we profiled intramuscular lipid mediators following injury and investigated the role of SPMs in skeletal muscle inflammation and repair. Both eicosanoids and SPMs increased following myofiber damage induced by intramuscular injection of barium chloride or functional overload. Daily systemic administration of resolvin D1 (RvD1) limited the degree and duration of inflammation, enhanced regenerating myofiber growth, and improved recovery of muscle strength. RvD1 suppressed inflammatory cytokines, enhanced polymorphonuclear cell clearance, modulated muscle stem cells, and polarized macrophages to a more pro-regenerative subset. RvD1 had minimal direct impact on in-vitro myogenesis but directly suppressed myokine production and stimulated macrophage phagocytosis, showing that SPMs influence modulate both infiltrating myeloid and resident muscle cells. These data reveal the efficacy of immunoresolvents as a novel alternative to classical anti-inflammatory interventions in the management of muscle injuries to modulate inflammation while stimulating tissue repair. differ fundamentally from this classical anti-inflammatory approach (26). Administration of resolution agonists, coined immunoresolvents, such as native SPMs or drug mimetics, can limit inflammation and expedite its resolution, while simultaneously relieving pain in many clinically relevant models of acute and chronic inflammation (27). Unlike NSAIDs, SPMs have been recently found to have pro-regenerative tissue actions following dermal wound healing (28-30), spinal cord injury (31), bone fracture (32, 33), and myocardial infarction (34-37).
SPMs are locally produced and appear systemically following skeletal muscle injury in both mice and humans, suggesting that they may play an important role in control of the inflammatory and adaptive responses to myofiber damage (18,(38)(39)(40)(41)(42). Recently, intramuscular injection to MuSC deficient mice of one native SPM, resolvin D2 (RvD2), was reported to accelerate MΦ transitions following degenerative muscle injury, resulting in improved recovery of muscle mass and strength (38). However, the endogenous role of SPMs and the therapeutic applicability of immunoresolvents to modulate muscle inflammation and repair under physiological circumstances where MuSCs play an indispensable role in successful myofiber regeneration remains unknown (43). Moreover, this prior study focused on immunological endpoints, thus there remains a complete lack of data concerning the impact of immunoresolvents on cellular and molecular indices of myofiber growth/regeneration or the MuSC response (38). Recently, Annexin A1, a key pro-resolving peptide which shares its cell surface receptor (FPR2/ALX) with the SPM resolvin D1 (RvD1), was also shown to control muscle regeneration in healthy young mice by muscle cell direct and indirect actions (44).
In the current study, we investigated the potential role of SPMs in modulating inflammatory and regenerative responses in physiological models of skeletal muscle growth and repair including both adaptive myofiber hypertrophy and myofiber degeneration/regeneration. Moreover, we tested whether daily systemic administration of RvD1, as an exogenous immunoresolvent, regulates cellular and molecular indices of myofiber regeneration and myogenic MuSC responses to muscle injury in healthy young mice in vivo and whether RvD1 directly modulates myogenesis and myokine production in vitro.

Dynamic Changes in Muscle Lipid Mediators Are Conserved Across Different Species and Models of Injury
We also performed LC-MS based lipidomic profiling of the plantaris muscle following synergist ablationinduced functional overload in rats (Figure 2A). This is a milder, but potentially more physiologically relevant model of myofiber damage when compared to BaCl2 induced injury (45) (Figure 2A). Synergist ablation resulted in a compensatory increase in the mass of the overloaded plantaris at 28 days' post-surgery ( Figure 2B-C). This was attributable to an increase in muscle fiber size ( Figure 2D), and was most evident for slow-twitch Type I and IIa fiber types (46) ( Figure 2E). Control plantaris muscles contained many resident ED2 (CD163 + ) MΦ, few scattered ED1 (CD68 + ) MΦ, and very few PMNs (HIS-48 + cells). Therefore, unlike in mice, the resident MΦ in rat muscle were predominantly CD163 (ED2 + ), but CD68 (ED1 -) (47) (Supplemental Figure 1 Figure 2F). Scattered HIS48 + cells could still be seen at day 7, but were absent by day 28. In contrast, apparent effect of RvD1 to specifically reduce muscle PMNs, TA muscles were collected at day 3 post-BaCl2 injury and the intramuscular single-cell population was isolated for analysis by flow cytometry ( Figure 3D).
Approximately 80% of the live single-cells within injured muscle stained positive for the pan leukocyte marker CD45 and RvD1 treatment did not impact total intramuscular leukocytes. Nevertheless, the relative proportion of gated live CD45 + cells that co-stained positive for the PMN marker Ly6G was lower in RvD1 treated mice ( Figure   3D). This was accompanied by a parallel increase in the relative proportion of intramuscular CD45 + Ly6Gcells, the vast majority of which are most likely MΦ at this time-point.

Resolvin D1 Directly Stimulates MΦ Phagocytosis
MΦ play an important role in the active resolution of inflammation via their phagocytic uptake and removal of pathogens, cellular debris, and apoptopic PMNs. Therefore, we assessed whether RvD1 could directly influence MΦ phagocytic activity in-vitro ( Figure 4A). Treatment with RvD1 at doses between 10-100 nM stimulated phagocytosis of E. Coli Bio Particles by GM-CSF derived mouse bone marrow MΦ (BMMs) ( Figure   4B). RvD1 (1-10 nM) also stimulated E. coli phagocytosis by human peripheral blood monocyte (PBMC) derived M1 polarized MΦ, although higher doses of RvD1 (100 nM) were apparently refractory in this cell type ( Figure   4C).

Expedited Resolution of Muscle Inflammation Supports Regenerating Myofiber Growth
To test whether stimulating resolution influenced myofiber regeneration, mice were treated daily with systemic (IP) administration of RvD1 and TA muscles were collected at day 5 of recovery from BaCl2 injury.
Hematoxylin and eosin (H&E) staining of TA muscle cross-sections did not reveal any gross histological differences in overall muscle morphology between vehicle and RvD1 treated mice ( Figure 5A). Thus, RvD1 did not appear to obviously perturb normal muscle regeneration as has been reported previously with systemic NSAID treatment (20-25). To more precisely measure the extent of myofiber regeneration, tissue sections were stained with an antibody against embryonic myosin heavy chain (eMHC) ( Figure 5A, Supplemental Figure 3). RvD1 did not influence the absolute or relative number of eMHC + fibers within the regenerating TA, but did increase the average cross-sectional area (CSA) of the regenerating (eMHC + ) myofiber population ( Figure 5B).

Immunoresolvent Treatment Modulates Muscle Stem Cell Responses to Injury
Skeletal muscle regeneration is predominantly, if not entirely, dependent on the function of resident MuSCs (43). Therefore, we also stained muscle cross-sections with an antibody against the MuSC marker Pax7 ( Figure 5A). The number of Pax7 + cells increased ~20 fold at day 5 following BaCl2 injury and RvD1 treatment reduced MuSC number ( Figure 5C). Since myogenic regulatory factors control the activation, proliferation and differentiation of MuSCs, we questioned whether RvD1 influenced myogenic gene expression. Whole TA muscle mRNA expression of myogenin increased markedly at day 5 post-injury, and increased even further in mice that received RvD1 ( Figure 5C). Taken together with the effect of RvD1 to increase regenerating myofiber size, a   lower muscle Pax7 + cell density at day 5 post-injury may thus be interpreted as RvD1 stimulating myogenic commitment, differentiation, and/or fusion of MuSCs with regenerating myofibers.

Resolvin D1 Shifts Intramuscular MΦ Activation State
Many MΦ persisted in muscle at day 5 post-BaCl2 injury and RvD1 did not influence the total number of muscle MΦ ( Figure 5E), or muscle mRNA expression of MΦ markers including CD11b, CD68, F4/80 or CD206 (data not shown). Only a small proportion of intramuscular MΦ at day 5 post-injury co-expressed the CD163 antigen (≤5%) ( Figure 5D). Nevertheless, RvD1 treated mice had greater numbers of CD163 + MΦ in regenerating muscle ( Figure 5E). RvD1 also increased muscle mRNA expression of the M2 MΦ marker arginase-1 (Arg-1) and platelet-type 12-lipoxygenase (12-LOX), a key SPM biosynthetic enzyme that is highly expressed by mature MΦ ( Figure 5F). Expression of the MΦ related pro-inflammatory cytokine IL-1β was also ~3 fold higher in regenerating muscle of RvD1 treated mice ( Figure 5F), although other M1-related cytokines such as IL-6, MCP-1 and TNFα were not affected (data not shown).

Improved Recovery of Muscle Function in Resolvin D1 Treated Mice
BaCl2 induced muscle injury resulted in a ~40% decrease in nerve-stimulated in-situ maximal isometric contractile force (Po) at day 14 post-injury and much of force deficit persisted when normalized to muscle size (specific force, sPo) ( Figure 6A). Treatment with RvD1 improved recovery of Po by ~15%, but did not significantly influence recovery of sPo. Histological analysis of regenerating muscles showed that RvD1 did not influence muscle fiber type composition, but the size of fast-twitch type IIb fibers was larger in RvD1 treated mice ( Figure 6C). There was also an improved recovery of overall TA muscle size (CSA) in mice receiving RvD1 treatment ( Figure 6D). ~60% of the muscle fibers contained centrally located myonuclei at this time-point. RvD1 did not influence the absolute or relative number of these regenerating myofibers but regenerating myofiber CSA was larger in mice treated with RvD1 ( Figure 6D). Regenerating muscles still contained ~3-fold more MΦ than uninjured TAs ( Figure 6E). Only a minority (~20%) of these CD68 + cells expressed CD163, however, suggesting that even by 14 days post-BaCl2 injury, the bulk of muscle MΦ are not M2-like in the traditional sense. RvD1 did not impact the number of intramuscular CD163 + MΦ, but did reduce total MΦ numbers back towards the numbers typically seen in uninured muscle ( Figure 6G).

Related to Muscle-Immune Interactions:
In order to gain mechanistic insight into the underlying mechanisms by which RvD1 enhanced muscle regeneration, we performed transcriptome wide profiling of gene expression of MuSCs isolated from day 3 post-BaCl2 injured TA muscles via fluorescence activated cell sorting (FACS) (CD45 -, CD11b -, CD31 -, Sca-1 -, Ter-119 -, CXCR4 + , Integrin beta-1 + cells) followed by RNA-sequencing. The absolute MuSC yield from injured TA muscles was lower in RvD1 treated mice ( Figure 7A), while TA muscle mass was not affected ( Figure 7B), indicating relative muscle MuSC number was reduced in RvD1 treated mice at this time-point ( Figure 7C).
Overall the global transcriptomic profile of isolated MuSCs from vehicle and RvD1 treated mice at day 3 postinjury was very similar ( Figure 7D), and differed markedly from uninjured MuSCs (Supplemental Figure 4A).
Nevertheless, assessment of differentially expressed genes (>2-fold change, irrespective of p-value) between RvD1 and vehicle treated mice did reveal an enrichment of genes associated with response to wounding (76 genes), the inflammatory response (54 genes), response to external stimulus (98 genes), and cytokine secretion (17 genes), amongst others ( Figure 7E). A heat map of the 100 most different annotated genes in isolated MuSCs between vehicle and RvD1 treated mice is shown in Supplemental Figure 4B.

Physiological Doses of Resolvin D1 Neither Promote nor Perturb In-Vitro Myogenesis:
We also tested whether RvD1 could directly modulate muscle cell growth in-vitro. Treatment with a single dose of 100 nM RvD1 [based on established bioactivity in murine MΦ (Figure 4)] at the time of myogenic differentiation did not influence cellular density, percentage of myoblast differentiation, or fused myotube size ( Figure 8A). Replenishing the culture media with fresh RvD1 every 24 hours, in an attempt to account for any potential instability of RvD1 also did not influence in-vitro myogenesis (data not shown). Since NSAIDs have direct suppressive effects on in-vitro myogenesis (61), we questioned whether higher doses of RvD1 would also interfere with myogenesis. The non-specific COX pathway inhibitors ibuprofen and indomethacin, as well as the COX-2 selective inhibitor NS-398, impaired myotube development in a dose-dependent manner (Supplemental  Figure 8B). In contrast, muscle cells treated with RvD1 at doses of 0.1-1 µM, showed no such deleterious effects and a modest yet statistically significant increase in fused myotube size (+5%) was observed with 1 µM RvD1 ( Figure 8B).

Resolvin D1 Directly Limits Myokine Production and Protects Muscle Cells Under Conditions of Chronic Inflammatory Stress:
RvD1 is well established to suppress cytokine production by a variety of cell types, most notably MΦ (62-67). Therefore, we questioned whether RvD1 could directly modulate inflammatory cytokines in muscle cells (myokines). Exposure of C2C12 myotubes to lipopolysaccharide (LPS) markedly increased production of IL-6, MCP-1 and TNFα at the gene and protein level ( Figure 8C-D). Pre-treatment with RvD1 blunted LPS stimulated (3 h) mRNA expression of IL-6 and TNFα, but did not influence MCP-1 ( Figure 8C). Following more prolonged treatment (24 h), RvD1 also reduced LPS stimulated secretion of MCP-1 ( Figure 8D). Despite this, prolonged RvD1 treatment did not statistically reduce IL-6 secretion and TNFα was not present at detectable concentrations from LPS stimulated C2C12 cells with or without RvD1 treatment. In order to determine whether RvD1 may directly influence myogenesis under conditions of chronic non-resolving inflammation, C2C12 myoblasts were also induced to differentiate in the presence of the pro-inflammatory cytokine TNFα (20 ng/mL) ( Figure 8E).
Long-term TNFα exposure reduced the number of myoblasts successfully undergoing myogenic differentiation and lead to formation of thin elongated myotubes. Co-treatment with RvD1 (100 nM) protected against the deleterious effect of TNFα on developing myotube size, but did not rescue TNFα induced effects on myoblast cellular density or myogenic differentiation ( Figure 8E).

Discussion:
Here we investigated the role of specialized pro-resolving mediators (SPMs) in inflammatory and adaptive responses to muscle damage and tested the efficacy of systemic immunoresolvent treatment as a novel therapeutic strategy to the treatment of muscular injuries. SPMs were locally produced in muscle in response to both chemical injury and functional overload, coinciding with peak MΦ infiltration, clearance of tissue PMNs, and the onset of adaptive myofiber remodeling. Daily systemic treatment with the native SPM RvD1 as an immunoresolvent suppressed muscle inflammatory cytokines, expedited tissue PMN clearance, and shifted intramuscular MΦ to a more M2-like activation state. These immunomodulatory effects were associated with enhanced regenerating myofiber growth and improved recovery of muscle strength. While RvD1 had little direct impact on myogenesis in-vitro, it directly suppressed myokine production and enhanced MΦ phagocytic activity, indicative of both muscle direct and indirect actions. These findings show that SPMs play an important supportive role in myofiber growth and regeneration and highlight their therapeutic potential in the context of muscle injuries as a novel alternative to classic anti-inflammatory interventions (e.g. NSAIDs), which are known to inhibit endogenous cellular regenerative mechanisms.
Intramuscular injection of BaCl2 results in wide-spread myofiber necrosis and a robust inflammatory response (68-70). In this model, restoration of muscle function is dependent on de-novo muscle fiber formation via the proliferation, differentiation, and fusion of MuSCs (myofiber regeneration) (43). In contrast, functional muscle overload induced by synergist ablation results in mild myofiber damage, and ensuing adaptive tissue remodeling occurs mainly as a result of compensatory growth of pre-existing muscle cells (myofiber hypertrophy) (71). We show here that both interventions resulted in rapid infiltration of muscle by PMNs, their subsequent disappearance, and a persistent intramuscular MΦ presence. In both models, there was an initial increase in expression of inducible COX-2, leading to increased intramuscular concentrations of classical pro-inflammatory eicosanoids. COX pathway products including PGE2 (72, 73), PGF2α (74-76) and PGI2 (77, 78) have all been previously implicated in stimulating various stages of skeletal muscle regeneration. PGE2 also plays a key role in signaling the onset of the resolution phase by inducing transcription of 15-LOX, a phenomenon termed lipid mediator class switching (79). Consistent with this concept, expression of 5-, 12-and 15-LOX, as well as intramuscular concentrations of many LOX derived monohydroxylated fatty acid intermediates in SPM biosynthesis pathways increased locally during muscle regeneration. COX-2 exhibited a bimodal response, which is consistent with the proposed dual role of prostaglandins in both the induction and resolution of acute inflammation (80). Heightened local 5-and 15-LOX expression and increased tissue abundance of many SPM pathway markers were also observed following functional overload of the rat plantaris muscle for the first time.
Bioactive SPMs themselves were generally not present at detectable concentrations in uninjured muscle, but intramuscular protectin D1 and maresin 1 were detected in response to both BaCl2 injury and functional overload, while resolvin D6 and lipoxin A4 were also detected in the latter model. SPMs and/or their monohydroxylated pathway markers have also recently been found to increase in mouse muscle following experimental ischemia (81), intramuscular cardiotoxin injection (38), and eccentric exercise (38), as well as in human muscle biopsies following an acute bout of damaging muscular contractions (39). Furthermore, acute exercise transiently increases SPMs in human blood (41, 42), and repeated exercise training was recently found to prime murine MΦ to release greater amounts of RvD1 in response to a subsequent inflammatory challenge (82). PMNs (Ly6G + ), inflammatory (Ly6C hi F4/80 lo ) monocytes, and reparative (Ly6C lo F4/80 hi ) MΦ populations isolated from cardiotoxin injured mouse muscle were also recently found to display distinct time-dependent modulation of bioactive lipid mediators (38). Overall, these findings show that dynamic regulation of the SPM bioactive metabolome is an endogenous mechanism that is conserved across multiple different species and models of muscle damage.
We did not detect endogenous RvD1 within muscle before or after injury, but we did observe increased expression of the enzymatic machinery required in RvD1 biosynthesis (15-and 5-LOX) (48, 49), as well as intramuscular concentrations of 17-HDoHE, the primary 15-LOX intermediate product of n-3 DHA produced during D-series resolvin biosynthesis (48, 49). We (41) and others (42) previously found that circulating RvD1 does increase following strenuous exercise. Notably, primary monohydroxylated 5-, 12, and 15-LOX metabolites were far more abundant in muscle homogenates in the current study than bioactive SPMs themselves. This finding is consistent with our prior study of metabolipidomic profiling of human muscle biopsies (39), as well as recent reports by others in mouse muscle (38,81). This suggests that the lipid metabolome of muscle may predominantly reflect intracellular metabolites whereas bioactive SPMs may be relatively enriched in the extracellular environment where they bind to their cell surface receptors. Another possibility is that bioactive SPMs in tissues are rapidly converted to downstream enzymatic inactivation products. Consistent with this concept, we observed 8-oxo-RvD1 [a bioactive 15-hydroxy prostaglandin dehydrogenase metabolite of RvD1 (48)] in rat muscle 3 days post-synergist ablation at levels ~2-fold higher than baseline.
Prior studies have shown that PMNs can directly damage muscle cells in-vitro (83), and that blockade of PMN influx following muscle damage, either by knockout of CD18 (7) or depletion of circulating PMNs (84), protects muscle from leukocyte-induced secondary injury. Therefore, limiting PMN recruitment and hastening their removal has been proposed to be organ protective in the context of sterile skeletal muscle injury. A class defining action of SPMs is the selective inhibition of further PMN recruitment to the site of inflammation (12, 13). Indeed, systemic injection of resolvins displays similar potency in reducing PMN infiltration in the murine peritonitis model as the NSAID indomethacin (13, 48, 49). On this basis, we hypothesized that treatment with RvD1 at the time of BaCl2 injury would limit the initial appearance of PMNs within injured muscle. RvD1 treatment did not influence peak intramuscular PMN, but did reduce intramuscular PMNs at day 3 of recovery.
In prior studies, RvD1 also did not reduce peak PMN infiltration of cardiac muscle following myocardial infarction, and rather specifically accelerated PMN egress from the infarcted left ventricle (34, 35). Overall, these data are consistent with the notion that in addition to their anti-inflammatory actions, SPMs actively promote PMN clearance, thus accelerating resolution back to a non-inflamed state (85). One potential mechanism, is the ability of SPMs to stimulate MΦ mediated phagocytic uptake and removal of apoptopic PMNs (86). Consistent with prior studies (87), RvD1 proved to be a potent stimulator of MΦ phagocytic activity in our hands. Indeed, administration of RvD2 at the peak of inflammation (24 h) following ligation of the femoral artery reduced PMN numbers in the hamstring musculature of mice when assessed 48 h later (day 3 post-ischemia) (81). Notably, traditional anti-inflammatory drugs such as the NSAIDs, which lack the pro-resolving bioactivity that we and others have observed for SPMs, can inadvertently delay timely resolution of inflammation by limiting monocyte recruitment and interfering with MΦ mediated clearance of tissue PMNs (80).
One key molecular mechanism by which resolvins function is by counteracting production of proinflammatory cytokines (13, 49). Indeed, we found that systemic treatment with RvD1 at the time of muscle injury suppressed local cytokine expression. RvD1 also directly reduced LPS stimulated mRNA expression of IL-6 and TNFα, as well as secretion of MCP-1 protein by C2C12 myotubes cultured in-vitro. Consistent with these observations, n-3 EPA derived resolvin E1 (RvE1) directly inhibits cytokine production by muscle cells (88).
While the overall impact of systemic RvD1 treatment on the global MuSC transcriptome of following injury was minimal in the current study, clusters of genes with strong relevance to muscle-immune interactions such as those involved in the response to wounding, the inflammatory response, and cytokine secretion were amongst those most influenced by RvD1 based on magnitude of change. Collectively, these data suggest that resolvins may have some direct modulatory effects on the production and/or release of inflammatory mediators by cells resident to the musculature such as post-mitotic myofibers and resident MuSCs.
The chemokine MCP-1 is indispensable for recruitment of blood monocytes to the injured musculature (89). Given the suppressive effect of RvD1 on muscle MCP-1 expression, the initial reduction observed in the current study in intramuscular MΦ following RvD1 treatment is not surprising. Despite this, the recovery of muscle MΦ numbers to normal levels during the resolution phase suggests that RvD1 treatment may have either stimulated later monocyte recruitment or promoted expansion of tissue MΦ. Mice receiving RvD1 also displayed heightened muscle expression of the alternative MΦ activation marker arginase-1, as well as a greater number of intramuscular MΦ expressing the M2-like marker CD163. Overall, these data are consistent with prior studies showing that RvD1 can induce a M2-like polarization state of MΦ in-vitro (52, 67, 90). In turn, M2-polarized MΦ produce more SPMs than M1 activated MΦ (91-94), which may explain why muscle expression of platelet-type 12-LOX, a key SPM biosynthetic enzyme expressed by MΦ, was increased in muscle in response to RvD1 treatment in the current study. Whilst most M1 signature cytokines did not differ between vehicle and RvD1 treated mice during the regenerative phase, IL-1β, a classical pro-inflammatory cytokine, was ~3 fold higher in muscle of RvD1 treated mice at day 5 post-injury. This may potentially be explained by recent studies showing that resolution phase MΦ are neither classically nor alternatively activated, but possess certain aspects of both phenotypes (52, 95).
NSAIDs have been extensively shown to have deleterious effects on muscle repair (20-25). Given that SPMs also possess some anti-inflammatory actions, it was important to determine whether repeated daily immunoresolvent treatment would impact the efficiency of muscle regeneration. Overall, we found no evidence Recently, a single intramuscular injection of resolvin D2 (RvD2) was reported to improve recovery of muscle mass and strength following muscle injury in mice (38). However, that study focused primarily on immunological outcomes and the underlying cellular and molecular basis for these apparent effects on muscle regeneration were not investigated. Moreover, the mice were depleted of resident MuSCs by irradiation, which is known to markedly deregulate the normal inflammatory and regenerative responses to muscle injury (97), as further evidenced by the marked weakness at day 14 of recovery from injury compared with that measured for the healthy young mice in the present study (900 vs. 200 mN Po) (38). Under the physiological relevant circumstances in which MuSCs play a well-established and indispensable role in de-novo myofiber formation (43), we observed that the beneficial effects of RvD1 on recovery of muscle size and strength were mechanistically attributable to hypertrophy of regenerating myofibers. This was accompanied by enhanced myogenic gene expression and an accelerated fusion of proliferated MuSCs, indicative of modulatory effects on this key resident myogenic stem cell population. Furthermore, transcriptome-wide profiling of MuSCs isolated from injured muscle showed that RvD1 modulated expression of clusters of genes relevant to muscle-immune interactions in-vivo and directly modulated myokine production in-vitro. In addition to recently reported effects of RvD2 on MΦ (38), we further show an impact of immunoresolvent treatment on intramuscular PMN clearance from injured muscle, which provides one more mechanism that may explain the therapeutic benefit of immunoresolvents in the context of muscle regeneration (7). In support of our findings, the pro-resolving peptide Annexin A1, a distinct agonist of the RvD1 receptor (FPR2/ALX), was also recently shown to be required for effective myofiber regeneration in healthy young mice (44).
The demonstrated benefit of systemic immunoresolvent treatment on muscle inflammation and regeneration in the current study has important implications for future therapeutic translation to human studies, especially given that RvD1 is orally bioavailable in mice (98). Moreover, the ability of daily dosing with RvD1 throughout the entire time-course of recovery from injury to ultimately improve muscle regeneration rather than compromise it, as is the case with daily systemic NSAID treatment (20-25), is in itself novel. This clear advantage of RvD1 treatment compared with NSAIDs is of great clinical importance given that such frequent repeated dosing would likely be required to effectively exploit the analgesic potential of SPMs for pain management (99, 100), which is a key therapeutic goal in the clinical treatment of soft tissue injuries. An additional novel finding of the current study is the demonstration for the first time that SPM biosynthetic circuits are also locally induced in response to overload of skeletal muscle, in close association with intramuscular immune cell transitions and the onset of the adaptive hypertrophy of pre-existing myofibers. In addition to their key supportive role in muscle regeneration, MΦ also regulate overload-induced myofiber hypertrophy (101) and myofiber re-growth during recovery from disuse (102). Thus, our observation of SPM biosynthesis in muscle with increased load suggests that immunoresolvents may also have potential therapeutic applicability to modulate adaptive responses to functional unloading and loading of muscle in physiologically and clinically relevant settings. Therefore, further studies should address the effects of immunoresolvents on muscle in models of adaptive remodeling of preexisting post-mitotic myofibers such as limb immobilization or bed rest, and subsequent return to ambulation and rehabilitation. Immunoresolvents may also be an effective novel therapeutic in physiological and clinically relevant settings characterized by sustained non-resolving acute inflammatory responses and limited regenerative efficiency such as volumetric muscle loss (103). Finally, states of chronic unresolved inflammation which negatively impact upon muscle mass and myofiber regenerative capacity including aging (104), muscular dystrophies (19), and metabolic disease (e.g. obesity/diabetes) (105, 106) may also benefit from this novel therapeutic strategy and should be investigated.
In conclusion, SPMs are locally produced in response to myofiber damage and play an important role in the control of muscle inflammation, its active resolution, and cellular transitions of MΦ, MuSCs and myofibers to enable effective muscle tissue repair. These data implicate SPMs in controlling adaptive muscle remodeling and highlight the potential efficacy of systemic immunoresolving therapies as a novel treatment of muscular injuries which can enhance tissue regenerative capacity by stimulating endogenous resolution mechanisms.

Materials and Methods:
Animal Handling: C57BL/6 mice and Sprague-Dawley rats were obtained from Charles River Laboratories and housed under specific pathogen free conditions with ad-libitum access to food and water. 4-6 month-old female mice were used for chemical muscle injury and 6-month-old male rats were used for functional muscle overload experiments.
Chemical Induced Muscle Injury: Mice were anesthetized with 2% isoflurane and received bilateral intramuscular injection of the tibialis anterior (TA) muscle with 50 µL per limb of 1.2% BaCl2 in sterile saline (68-70). In some experiments mice randomized to receive sham injury via intramuscular injection of sterile saline alone instead of BaCl2. Mice were returned to their home cage to recover and monitored until ambulatory.

Functional Muscle Overload:
Synergist ablation was used to assess the inflammatory response to functional overload of the plantaris muscle (71). To minimize the impact of surgical manipulation on tissue inflammation, we used a mild protocol in which only the soleus/gastrocnemius tendon is surgically ablated. Rats were anesthetized with 2% isoflurane and preemptive analgesia provided by subcutaneous injection of buprenorphine (0.03 mg/kg) and carprofen (5 mg/kg). The skin overlying the posterior hind-limb was shaved and scrubbed with chlorhexidine and ethyl alcohol. A midline incision was made to visualize the gastrocnemius/soleus tendon and a full thickness tenectomy performed while leaving the plantaris tendon intact. The incision was closed using 4-0 Vicryl sutures. The procedure was then repeated on the contralateral limb to induce bilateral functional overload of both plantaris muscles. Following surgery, rats were returned to their cage to recover and monitored until ambulatory with free access to food and water. Postoperative analgesia was provided via subcutaneous injection of buprenorphine (0.03 mg/kg) at 12 h post-surgery and animals were monitored daily for any signs of pain or distress for 7 days. Age and gender matched rats served as non-surgical controls. with the first dose administered ~5 min prior to muscle injury. Mice were allowed to recover for up to two-weeks post-injury with daily IP injection of 100 ng of RvD1 or vehicle.

Muscle Tissue Collection:
Animals were euthanized via induction of bilateral pneumothorax while under deep isoflurane anesthesia. Muscles were rapidly dissected, blotted dry, weighed, and snap frozen in liquid nitrogen. Muscles for histological analysis were cut transversely at the mid-belly with a scalpel blade, oriented longitudinally on a plastic support, covered with a thin layer of optimal cutting temperature (OCT) compound, and rapidly frozen by plunging into isopentane cooled on liquid nitrogen. Samples were stored at -80°C until further analysis. ReadyProbes, R37112) were used to counterstain cell nuclei, extracellular matrix, and muscle fibers respectively in particular experiments. Image Analysis: Muscle tissue morphology was analyzed on stitched panoramic images of the entire muscle cross-section by high-throughput fully automated image analysis with the MuscleJ plugin for FIJI (108), with few exceptions. At day 5 post-BaCl2 injury, regenerating (eMHC + ) fiber number and size was specifically quantified on the stitched panoramic images of the entire TA muscle cross-section by in-house semi-automated image analysis using ImageJ/FIJI (Supplemental Figure 3). In all cases the cross-sectional area (CSA) measurement of each individual myofiber (technical replicates) were averaged to obtain a single biological replicate for each muscle sample. Immune cells were manually counted throughout the entire mouse TA crosssection and normalized to tissue area as determined by MuscleJ or from five non-overlapping 20 x fields of view captured from within the inflammatory lesion of the rat plantaris (Supplemental Figure 1). In the latter case, the results of the five images were averaged to obtain a single biological replicate for each muscle sample. In all cases, the experimenter was blinded to the experimental group.

Muscle Force Testing:
Mice were anesthetized with 2% isoflurane and placed on a heated platform. The distal half of the TA muscle was isolated by dissecting the overlying skin and fascia. The knee joint was immobilized and a 4-0 silk suture tied around the distal TA tendon which was severed from its boney insertion and tied to the lever arm of a servomotor (6650LR, Cambridge Technology). A saline drip warmed to 37°C was continuously applied to the exposed muscle. The peroneal nerve was stimulated with 0.2 ms pulses using platinum electrodes with the stimulation voltage and muscle length adjusted to obtain optimal muscle length (Lo) maximum isometric twitch force (Pt). The TA was then stimulated at increasing frequencies while held at Lo until maximum isometric tetanic force (Po) was achieved. One-minute rest was allowed between each tetanic contraction. Muscle length then measured with calipers and optimum fiber length (Lf) determined by multiplying Lo by the TA muscle Lf/Lo ratio of 0.6 (109). The cross-sectional area (CSA) of the muscle was calculated by dividing muscle mass by the product of Lf and 1.06 mg/mm 3 (110). Specific Po (sPo) was calculated by dividing Po by muscle CSA.

LC-MS/MS Based Metabolipidomic Profiling of Muscle Tissue:
TA muscle samples were mechanically homogenized in 1 mL of 50 mM phosphate, pH 7.4 with 0.9% saline (PBS) using a bead mill with reinforced tubes and zirconium beads (Precellys). The tissue homogenates were centrifuged at 3,000 x g for 5 min and the supernatant was collected. Supernatants (0.85 ml) were spiked with 5 ng each of 15(S)-HETE-d8, 14(15)-EpETrE-d8, Resolvin D2-d5, Leukotriene B4-d4, and Prostaglandin E1-d4 as internal standards (in 150 µl methanol) for recovery and quantitation and mixed thoroughly. The samples were then extracted for polyunsaturated fatty acid metabolites using C18 extraction columns as described earlier (39, 41, [111][112][113]. Briefly, the internal standard spiked samples were applied to conditioned C18 cartridges, washed with 15% methanol in water followed by hexane and then dried under vacuum. The cartridges were eluted with 2 x 0.5 ml methanol with 0.1% formic acid. The eluate was dried under a gentle stream of nitrogen. The residue was redissolved in 50 µl methanol-25 mM aqueous ammonium acetate (1:1) and subjected to LC-MS analysis.
HPLC was performed on a Prominence XR system (Shimadzu) using Luna C18 (3µ, 2.1x150 mm) column. The mobile phase consisted of a gradient between A: methanol-water-acetonitrile (10:85:5 v/v) and B: methanol-water-acetonitrile (90:5:5 v/v), both containing 0.1% ammonium acetate. The gradient program with respect to the composition of B was as follows: 0-1 min, 50%; 1-8 min, 50-80%; 8-15 min, 80-95%; and 15-17 min, 95%. The flow rate was 0.2 ml/min. The HPLC eluate was directly introduced to ESI source of QTRAP5500  (Table 1). Duplicate technical replicates were averaged to obtain single Ct value from individual muscle sample as a biological replicate. Relative mRNA expression was determined using the 2 -ΔΔCT method with Actb and Gapdh serving as endogenous controls from mouse (BaCl2 induced injury) and rat (synergist ablation) experiments respectively. Primer sequences used are listed in Table 1.

RNA-Seq Data Processing and Analysis:
Single-stranded RNAseq data was aligned to the mm10 reference genome with the STAR algorithm (STAR_2.5.0a) and RSEM quantification applied to the aligned reads. Differentially expressed genes were identified in R using Limma-Voom analysis. Expected counts were first Voom transformed to counts per million (CPM) and then surrogate variable analysis was performed with the SVA package. Surrogate variables were quantified and removed from the data matrix. All pairwise contrasts were examined to identify differentially expressed genes between vehicle and RvD1 treated mice. Thresholds of padjusted < 0.05 and log2-fold-change >=1 were used to call differential genes. Enrichment analysis of gene lists Statistics: Data is presented as the mean ± SEM with raw data from each individual biological replicate also shown. Statistical analysis was performed in GraphPad Prism 7. Between group differences were tested by two-tailed unpaired students t-tests (2 groups) or by a one-way analysis of variance (ANOVA) followed by pairwise Holm-Sidak post-hoc tests (≥3 groups). For time-course experiments, multiple comparison testing was made compared to a single baseline control group. p≤0.05 was used to determine statistical significance.                Data is presented at the percentage change in fluorescence intensity relative to matching vehicle treated macrophages from the same host. Bars show the mean ± SEM of cells originating from 3-4 different donors with dots with representing data from each host mouse or human subject (biological replicates). P-values were determined by one-way ANOVA followed by Holm-Sidak post-hoc tests with vehicle treated cells serving as a control group. . Cell nuclei and the basal lamina were counterstained with DAPI and a laminin antibody respectively. Scale bars are 200 µm. B: Quantitative analysis of total regenerating (eMHC + ) myofiber number, relative eMHC + fiber number (as % of total fibers), mean eMHC + fiber crosssectional area (CSA), and the percentage frequency distribution of the CSA of the eMHC + fiber population. C: Quantification of MuSC number (Pax7 + /DAPI + nuclei) and whole muscle mRNA expression of the myogenic regulatory factor myogenin at day 5 post-injury. D: Cross-sections of TA muscles at day 5 post-injury were stained for total macrophages (MΦ, CD68) and M2-like MΦ (CD163). E: Quantification of total intramuscular MΦ (CD68) and M2-like MΦ (CD163 + cells). F: Whole muscle mRNA expression of MΦ-related genes including arginase-1 (Arg1), platelet-type 12-lipoxygenase (12-LOX), and interleukin 1 beta (IL-1β) as determined by RT-PCR. Gene of interest expression was normalized to beta-actin (Actb). Bars show the mean ± SEM of 5-8 mice per group with dots representing data for each individual mouse (biological replicates). P-values were determined by one-way ANOVA followed by pair-wise Holm-Sidak posthoc tests (panels C and E) or by two-tailed unpaired t-tests (panels B and F).

Figure 6 -Resolvin D1 improves recovery of isometric muscle strength:
A: Female C57BL/6 mice (4-6 mo) received bilateral intramuscular injection of the tibialis anterior (TA) with 50 µL of 1.2% barium chloride (BaCl2) to induce myofiber injury. Mice were then treated daily with intraperitoneal (IP) injection of resolvin D1 (RvD1, 100 ng) or vehicle (0.1% ethanol) for 14 days with the first dose administered ~5 min prior to injury. TA muscle function was then tested for maximal isometric nerve-stimulated in-situ contractile force (Po) with age and gender matched mice serving as uninjured controls. Absolute maximal isometric force (mN) generated by the TA muscle was measured and used to calculate maximal specific isometric contractile force (sPo, mN/mm 2 ). Representative force traces obtained from uninjured and injured TA muscles are shown. B: TA cross-sections were stained with conjugated phalloidin to label the total muscle fiber population (actin filaments) or for muscle fiber type with antibodies against type myosin heavy chain I, IIa, and IIb. Type IIx fibers remain unstained (black) and are identified by lack of fluorescent staining. Cell nuclei and the basal lamina were counterstained with DAPI and a laminin antibody respectively. Scale bars are 200 µm. C: Quantitative analysis of percent muscle fiber type composition and mean fiber cross-sectional area (CSA) split by muscle fiber type as determined by MuscleJ software. D: Quantification of overall TA muscle CSA, the percentage of centrally nucleated (regenerating) myofibers, mean regenerating myofiber CSA, and percent frequency distribution of regenerating fiber CSA as determined by MuscleJ software. E: TA cross-sections were stained for hematoxylin & eosin (H&E), total macrophages (MΦ, CD68 + cells), and M2-like MΦ (CD163 + cells). Scale bars are 200 µm. F: Quantification of the histological presence of total muscle MΦ and M2-like MΦ. Cell counts were performed manually throughout the entire muscle cross-section and then normalized to tissue surface area as determined by MuscleJ software. Bars show the mean ± SEM of 5-10 mice per group with dots representing data from each individual mouse (biological replicates). P-values were determined by one-way ANOVA followed by pairwise Holm-Sidak post-hoc tests (panels A and F) or two-tailed unpaired t-tests (panels C and D).

Figure 8 -Resolvin D1 neither enhances nor perturbs basal in-vitro myogenesis, but directly modulates myokine production and protects muscle cells against the deleterious effects chronic inflammation:
A: Murine C2C12 myoblasts were treated with resolvin D1 (RvD1, 100 nM) at onset of myogenic differentiation. At 3-days post-differentiation, myotubes were fixed in 4% paraformaldehyde and stained for sarcomeric myosin. Cell nuclei were counterstained with DAPI. Quantitative analysis was performed on 6 non-consecutive fields of view per well to determine mean myotube diameter, percentage of myogenic differentiation, and overall cell density. B: Confluent C2C12 myoblasts were induced to differentiate in the presence of RvD1 (0.1-1 µM), or non-steroidal antiinflammatory drugs (NSAIDs) including NS-398 (50 µM), ibuprofen (500 µM), and indomethacin (200 µM). C: C2C12 myotubes at day 3 post-differentiation were pre-treated with RvD1 (100 nM) for 30 min before stimulation with lipopolysaccharide (LPS, 100 ng/mL) for 3 h in the continued presence of RvD1. Cellular mRNA expression of cytokines including interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), and tumor necrosis factor alpha (TNFα) was determined by RT-PCR. D: C2C12 myotubes at day 3 post-differentiation were pre-treated with RvD1 (100 nM) for 30 min and then stimulated with LPS (100 ng/mL) for 24 h in the continued presence of RvD1. Conditioned culture media was collected from the cells and analyzed for concentrations of the cytokines IL-6, MCP-1 and TNFα by ELISA. E: Confluent C2C12 myoblasts were induced to differentiate in the presence of exogenous TNFα (20 ng/mL), with or without RvD1 cotreatment (100 nM). At day 3 post-differentiation, myotubes were fixed, stained, and quantified as described in panel A. Scale bars are 400 µm. Bars show the mean ± SEM of 3-8 replicates per group with each dot representing data from a single independent culture well considered as a biological replicate. P-values were determined by two-tailed unpaired t-tests (panel A) or one-way ANOVA followed by pair-wise Holm-Sidak post-hoc tests (panels B-E). Values are mean ± SEM of 5 mice/group. ND = Below limits of detection of the assay.  Values are mean ± SEM of 8-12 muscles from 4-6 rats per group. ND = Below limits of detection of the assay.