In vivo GDF3 administration abrogates aging related muscle regeneration delay following acute sterile injury

Abstract Tissue regeneration is a highly coordinated process with sequential events including immune cell infiltration, clearance of damaged tissues, and immune‐supported regrowth of the tissue. Aging has a well‐documented negative impact on this process globally; however, whether changes in immune cells per se are contributing to the decline in the body’s ability to regenerate tissues with aging is not clearly understood. Here, we set out to characterize the dynamics of macrophage infiltration and their functional contribution to muscle regeneration by comparing young and aged animals upon acute sterile injury. Injured muscle of old mice showed markedly elevated number of macrophages, with a predominance for Ly6Chigh pro‐inflammatory macrophages and a lower ratio of the Ly6Clow repair macrophages. Of interest, a recently identified repair macrophage‐derived cytokine, growth differentiation factor 3 (GDF3), was markedly downregulated in injured muscle of old relative to young mice. Supplementation of recombinant GDF3 in aged mice ameliorated the inefficient regenerative response. Together, these results uncover a deficiency in the quantity and quality of infiltrating macrophages during aging and suggest that in vivo administration of GDF3 could be an effective therapeutic approach.

. In addition to satellite cells, there is clear evidence supporting the essential role of immune cells both in the clearance of damaged tissue and enhancing tissue regeneration upon injury (Tidball, 2017). However, age-related changes in the immune cell functions and its therapeutic potential remain elusive. Here, we demonstrate that innate immune cells are an important component of age-related delay in muscle regeneration. As a proof of concept, we show that the number of reparative macrophages and the level of growth differentiation factor 3 (GDF3) produced by these cells are severely decreased with aging in regenerating muscles, leading to delayed repair. Supplementation of the cytokine alone can restore the normal recovery time following acute injury, and thus, it provides a new therapeutic approach to treat muscle injury in elderly people.
In line with previous studies (Bernet et al., 2014;Brack et al., 2007;Chakkalakal, Jones, Basson, & Brack, 2012;Conboy, Conboy, Smythe, & Rando, 2003;Cosgrove et al., 2014;Lee et al.,  To test, whether innate immune responses, in addition to previously identified age-related changes in satellite cell function, could also contribute to impairment in muscle regeneration, we set out to characterize the cellular dynamics of the myeloid cell infiltration in uninjured tissues and during muscle regeneration. We could detect increased expression level of macrophage activation markers in aged uninjured and regenerating muscles compared to young controls ( Figure 1f). Next, we isolated myeloid cells from CTX-injured TA muscles at Days 0, 1, 2, 4, and 6 after the injury. In an interesting manner, we found a statistically significant increase in the number of invading myeloid cells (CD45 + ) in the aged versus young muscles at Day 4 (repair phase; Figure 1e). These findings suggested the existence of age-related changes in the cellular composition and differentiation profile of the infiltrating myeloid cells. Indeed, the ratio of Ly6C high F4/80 low (inflammatory) macrophages to Ly6C low F4/80 high (repair) macrophages in injured muscle between young versus aged animals showed remarkable differences (Figure 1g-h), suggesting a delay in the phenotypic transition of infiltrating myeloid cells to repair macrophages in the aged muscles.
Several members of the TGFβ family (Egerman et al., 2015) are known regulators of muscle regeneration, whose members are secreted by repair macrophages acting in a paracrine manner (Massague, Cheifetz, Endo, & Nadal-Ginard, 1986;McPherron, Lawler, & Lee, 1997), including GDF3 (Varga et al., 2016). We selected GDF3 for a proof-of-concept experiment to evaluate whether the observed impaired phenotypic transition in macrophage phenotype from inflammatory to repair type (Patsalos et al., 2017) can contribute to age-related delay in muscle regeneration. In line with previous findings (Varga et al., 2016) Figure 2f). These findings suggest that the F I G U R E 1 Impaired skeletal muscle regeneration and delayed phenotypic transition of infiltrating myeloid cells in aged animals following CTX injury. (a) Representative images of H&E-stained skeletal muscle from young adult (2-month-old) and aged (23-month-old) male mice at Days 0, 8, 12, and 16 post-CTX-induced injury. Scale bars in the upper left represent 100 μm. (b) Mean myofiber cross-sectional area (CSA) of regenerating muscles in young adult (2-month-old) and aged (23-month-old) mice (number of fibers counted > 20,000) at Days 0, 8, 12, and 16 post-CTX-induced injury (n = 6 per group). (c) The ratio of necrotic fibers relative to regeneration area (in mm 2 ) at Day 8 of regeneration in young adult (2-month-old) and aged (23-month-old) muscle sections is shown. (d) Normalized tibialis anterior (TA) muscle mass-to-body weight ratio from young adult (2-month-old) and aged (23-month-old) mice at indicated time points following CTX injury (n = 6 per group). (e) Number of infiltrating myeloid (CD45 + ) cells in regenerating muscle from young (2-month-old) or aged (28-month-old) muscles at indicated time points prior and post-CTX injury (n = 8 muscles per group). (f) Heatmap representations of atrophy and macrophage-related genes (measured by qPCR) from young and old uninjured (left panel) and regenerating (Day 8 post-CTX; right panel) TA muscles. Relative mRNA expression (calculated using the 2 -ΔΔCT method) is shown as log 10 (fold change) (n = 6 muscles per group). (g and h) Percentage of inflammatory (Ly6C high F4/80 low ) and repair (Ly6C low F4/80 high ) MFs from young (2-month-old) or aged (28-month-old) muscles at indicated time points following CTX injury (n = 8 mice per group). In all bar and line graphs, bars and data points represent mean ± SEM PATSALOS ET AL.  In conclusion, our findings suggest that aging has a negative effect on the ability of macrophages to perform their phenotypic transition, leading to reduced production of growth factors (including GDF3), and this impacts the muscle regeneration potential. This work provides strong evidence that the immune axis should be considered for future therapeutic interventions.

ACKNOWLEDGMENTS
The authors acknowledge the discussions and comments on the manuscript by members of the Nagy laboratory. The aged mouse lines were kindly provided by GlaxoSmithKline as a gift.

CONF LICT OF I NTEREST
None declared.