Senolytic elimination of senescent macrophages restores muscle stem cell function in severely dystrophic muscle

The aging of the immune system, or immunosenescence, was recently verified to have a causal role in driving the aging of solid organs, while the senolytic elimination of senescent immune cells was found to effectively delay systemic aging. Our recent study also showed that immune cells in severely dystrophic muscles develop senescence-like phenotypes, including the increased expression of senescence-associated secretory phenotype (SASP) factors and senescence markers. Here we further investigated whether the specific clearance of senescent immune cells in dystrophic muscle may effectively improve the function of muscle stem cells and the phenotypes of dystrophic muscle. We observed increased percentage of senescent cells in macrophages from mdx/utro(−/−) mice (a murine model for muscular dystrophy disease, dystrophin−/−; utrophin−/−), while the treatment of mdx/utro(−/−) macrophages with senolytic drug fisetin resulted in reduced number of senescent cells. We administrated fisetin to mdx/utro(−/−) mice for 4 weeks, and observed obviously reduced number of senescent immune cells, restored number of muscle cells, and improve muscle phenotypes. In conclusion, our results reveal that senescent immune cells, such as macrophages, are greatly involved in the development of muscle dystrophy by impacting the function of muscle stem cells, and the senolytic ablation of these senescent cells with fisetin can be an effective therapeutic strategy for improving function of muscle stem cells and phenotypes of dystrophic muscles.


INTRODUCTION
Duchenne Muscular Dystrophy (DMD) is caused by mutations of the dystrophin gene, which leads to progressive muscle fiber damage and degeneration, resulting in cardiac or respiratory failure and ultimately premature death [1,2]. DMD patients develop severe chronic inflammation in their muscles, which is characterized by the massive infiltration of immune cells into muscles and elevated activation of pro-inflammatory signaling in muscle cells [1,2]. Our previous studies of mdx/Utro(−/−) mice (a severe murine model for DMD; dystrophin−/−; utrophin−/−) revealed the greatly increased number of SA-β-gal (Senescence-associated β-Galactosidase) + senescent cells in the dystrophic muscles, which is closely associated with severe dystrophic phenotypes [3,4]. Our recent study further revealed that, macrophages in dystrophic muscles have aberrant activation of RhoA signaling, which is associated with upregulated expression of SASP (Senescence-associated secretary phenotypes) factors and senescence-like features of macrophages [3]. Therefore, it would be interesting to find out whether the repression or elimination of these senescence-like macrophages can be effective in recuing dystrophic muscle phenotypes.
One recent important progression in the studying of ageing mechanism has been the observation that the ageing immune system, or immunosenescence, has a causal role in driving systemic ageing and therefore represents a key therapeutic target to delay ageing [5]. It was verified that the senolytic ablation of senescent immune cells in the aged organism can effectively delay the systemic aging [5]. In normal conditions, senescent cells accumulated in body are specifically identified and removed by immune system [6,7]; however, when the immune cell become senescent too, their ability to clear senescent cells would be greatly compromised, leading to excessive accumulation of senescent cells and agingrelated disorders [5][6][7]. Therefore, potential clearance or repression of senescent immune cells would be greatly beneficial for body health.
Therefore, in the current study, we investigated whether the treatment of utrophin−/−mdx mice with senolytic drug (fisetin) can be effective in eliminating the senescent immune cells and improve muscle phenotypes. We also performed in vitro treatment of cell co-culture system of immune cells and muscle stem cells to verify our observation in vivo. Our results will help verify the potential causal role of senescent immune cells in leading to severe muscle dystrophy and present a novel strategy for therapeutic treatment of DMD disease with senolytic drugs.

CD68+ macrophages in mdx/Utro(−/−) muscle features higher level of mTORC1 activation
Our previous study of mdx/utro(−/−) mice revealed that, CD68+ macrophages in dystrophic muscle develop senescence-like phenotypes, including higher level of positive C12FDG (a fluorogenic substrate for βgalactosidase) signaling [3]. mTORC1 activation has been known as the central regulator of cellular senescence, which occurs in senescent human cells independent of growth factor signaling [22,23]. In order to further verify whether CD68+ macrophages in mdx/utro(−/−) muscle may develop higher level of cellular senescence, co-immunostaining of CD68 and p-4E-BP1 (a substrate for mTORC1 signaling) in muscle tissues was performed. Results showed that, CD68+ macrophages in mdx/utro(−/−) muscle express higher amount of p-4E-BP1 protein (Figure 2A, 2C), indicating a high level of mTOR activation and senescence.

CD68+ macrophages isolated from mdx/utro(−/−) muscle negatively impact the function of muscle stem cell in a cell co-culture system
A cell coculture system was setup, to verify the potential direct impact of CD68+ macrophages in mdx/utro(−/−) muscle may exert on muscle progenitor/stem cells (MPCs). FAPs and MPCs were isolated from muscles of WT and mdx/utro(−/−) mice, and CD68+ macrophages from WT or mdx/utro(−/−) muscles and WT MPCs were co-cultured in a transwell system, with CD68+ macrophages from WT or mdx/utro(−/−) being at the upper chamber and WT MPCs being at lower chamber. Cell myogenesis and proliferation assays of WT MPCs showed that, the WT MPCs co-cultured with CD68+ macrophages from mdx/utro(−/−) developed impaired myogenesis and proliferation potentials ( Figure 4).

Treatment of co-culture system of mdx/utro(−/−) macrophages and WT MPCs with fisetin rescues the impaired function of WT MPCs
We then examined whether the removal of senescent cells in mdx/utro(−/−) macrophages with senolytic drug may rescue the function of muscle stem cells in the cell co-culture system. Fisetin was applied to treat the cell co-culture system of mdx/utro(−/−) macrophages and WT MPCs for 48 hr at 20 µM of concentration, and we observed improved myogenesis and proliferation potential of WT MPCs, compared to non-treated controls ( Figure 4).

Treatment of mdx/utro(−/−) mice with fisetin results in increased number of muscle stem cells and improved muscle phenotypes
Finally, in order to investigate the efficacy of senolytics on muscle health in vivo, mdx/utro(−/−) mice were treated with fisetin via oral gavage (20 mg/kg for 5 consecutive days each week)) for 4 weeks. Immunostaining of muscle cryosections revealed that, compared to non-treated mice, the number of CD68+ macrophages and SA-β-Gal+ senescent cells was  decreased and the number of Pax7+ MPCs was elevated in fisetin treated mice, while muscle phenotypes were also improved by observation of reduced fibrosis formation (collagen deposition) ( Figure 5).

DISCUSSION
Cellular senescence is a defined hallmark of aging, contributing to the aging process and age-related diseases and disorders. Senotherapeutics, including senolytics and senomorphics, has emerged as a promising anti-aging strategy, and was found to be able to prolong healthspan and lifespan of animals [12]. In recent years, senolytics have been widely studied in various types of tissues and disease models and have been proven for their beneficial effect in delaying aging or treating age-related diseases [11][12][13][14]. Interestingly, our recent study of mdx/utr(−/−) mice revealed that, macrophages are highly prevalent in severely dystrophic muscle of mdx/utr(−/−) mice and display senescencelike phenotypes, including the expression of SASP factors and positive staining for C12FDG [3]. However, the potential effect and mechanism of senolytics in regulating the function of MPCs in severely dystrophic muscles remains largely unclear. It was recently discovered that the ageing immune system, or immunosenescence, has a causal role in driving systemic ageing and therefore represents a key therapeutic target to delay ageing [5]. Therefore, here we investigated whether senolytic ablation of senescent immune cells in dystrophic muscle of mdx/utr(−/−) mice may promote the restoration of exhausted muscle stem cell pool and improve muscle phenotypes. Our current results show that, a substantial fraction of mdx/utr(−/−) macrophages develop senescence in the severely dystrophic muscles, which dominantly repress the proper function of MPCs by releasing SASPs; while the elimination of senescent macrophages with fisetin is able to rescue the function of MPCs.
Senescent cells are known as a source of age-related chronic inflammation via secreting multiple proinflammatory factors of SASPs [9,10]. As senescent cells accumulate in the aged tissues, they can exert profound effects on the growth and function of normal cells by releasing SASP factors including exosomes  [18,20]. Senescent cells play a critical role in inducing or mediating various types of aging-related diseases [16][17][18][19][20]. A reduction in the senescent cell burden can be achieved with several different classes of senolytics including the combination of dasatinib and quercetin (D+Q), navitoclax (ABT263) and related Bcl-2 family member inhibitors, HSP90 inhibitors such as 17-DMAG and the flavonoid fisetin [16,[22][23][24][25][26][27]. Fisetin is a naturally occurring anti-oxidant that scavenges free radicals as well as upregulates synthesis of glutathione, and it also has been reported to inhibit apoptosis-related signaling BCL-XL [26], which may also be important for its senolytic activity. The positive effect of fisetin in removing senescent cell in aged tissues has been welldemonstrated, our current results have further demonstrated that fisetin is effective in removing senescent immune cells in dystrophic muscles. Senescent macrophages are in fact also found to express senescence-related markers p16(Ink4a) and β-galactosidase (β-gal), and promote inflammation in diseased tissues [25,26]. Our previous work has indicated increased cellular senescence in dystrophic muscles of mdx/utr(−/−) mice [3], however, whether or not macrophages in particular develop cellular senescence and promote senescence associated phenotypes was still unknown. To this end, here we further examined mdx/utr(−/−) mice and solved these puzzles.
Immune cells in the skeletal muscle are activated during muscle injury and promote the process of muscle regeneration by coordinating with muscle stem cells. However, studies with severely diseased muscles further demonstrate that immune cells can become dominantly activated and is inductive of increased fatty infiltration and fibrosis formation, while at the same time potently repress the proliferation and function of muscle stem cells [27]. Our current results in severely dystrophic muscle reveal a similar situation of interaction between macrophages and MPCs, showing that the function of MPCs is repressed by the senescent macrophages. As senescent cells accumulate in the aged or diseased tissues, it can exert profound effects on the growth and function of normal cells by releasing SASPs [9,10]. Fisetin is a naturally-occurring flavone with low toxicity, a selective BCL-XL inhibitor [15], and our current results demonstrate the effective rescuing of muscle stem cell function and dystrophic muscle phenotype with fisetin administration. Because senescent cell usually takes weeks to reaccumulate, intermit administration of senolytics is efficient to achieve ideal results. Here in our study, it shows the weekly administration of fisetin in mice is effective in eliminating many of senescent cells and rescue the growth and function of MPCs.
Still, there is some limitation in our current study, such as the lack of results for other types of senolytic drugs than fisetin in potentially having better senolytic effect and improving severely dystrophic muscle, and results for other types of immune cells than senescent macrophage in potentially impacting the function of muscle stem cells, which can be further studied in our future researches. Also, here we have studied cells from a murine model of DMD, and the potential effect of fisetin in dystrophic muscle cells from human patients is to be further verified. Therefore, our current results reveal that the senescence of macrophages play an important role in negatively regulating MPC function in severely dystrophic muscles of murine model of DMD, and the elimination of senescent macrophages with senolytics is effective in rescuing MPC function. Our results demonstrate that senescent macrophages may potentially serve as a new therapeutic target for treatment of muscular dystrophy or delaying the aging of skeletal muscle tissues.

Animal models
WT (C57BL/10J, male) mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Mdx/utr(−/−) mice (dystrophin/utrophin double knock out, dKO, or dystrophin −/− :utrophin −/− ; male mice) were derived from our in-house colony. Mice were housed in groups of 4 on a 12:12-hour light-dark cycle at 20-23°C. At least 8 mice were used in each experimental sample group. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh (IACUC-1109718) and the University of Texas Health Science Center at Houston (AWC-18-0068). All mice were housed and maintained in the Center for Laboratory Animal Medicine and Care (CLAMC) at UTHealth (University of Texas Health Science Center at Houston) in accordance with established guidelines and protocols approved by the UTHealth Animal Welfare Committee.

Isolation of macrophages from skeletal muscles
Gastrocnemius (GM) muscle tissues were harvested from WT and mdx/utr(−/−) mice (8-week old, n = 3 for each group), and digested by serial 1-hr incubations at 37°C in 0.2% type XI collagenase (Sigma-Aldrich, Burlington, MA, USA), dispase (grade II, 240 units; Sigma-Aldrich), and 0.1% trypsin (Thermo Fisher Scientific, Waltham, MA, USA), as previously described [6]. Primary cells from muscle tissues were harvested after digestion, and macrophages were then isolated from these primary cells by Fluorescence activated cell sorting (FACS) of CD68+ cells with a cell sorter (BD FACSAria, San Jose, CA, USA). Macrophages were cultured and immunostaining with CD68 antibody was performed to validate the identity of the isolated cells to be macrophages; the primary muscle cells were also immunostained with CD68 antibody to serve as negative control.

SA-β-gal staining for senescent cells
The presence of senescent cells in skeletal muscle tissue [n = 8 for WT or mdx/utr(−/−) mice] and cells [n = 3 for WT or mdx/utr(−/−) cells, with 2 replicates for each cell population] cultured in vitro was individually measured using the regular senescence-associated β-Galactosidase (SA-β-gal) Staining Kit (Cell Signaling Technology, Danvers, MA, USA) and CellEvent Senescence Green Detection Kit (fluorescent) (Thermo Fisher Scientific), following the manufacturers' protocol. The number of cells positive for β-gal activity at pH 6, a known characteristic of senescent cells, was determined.

Cell co-culture system of FAPs and MPCs
MPCs from WT muscle (n = 3, with 2 replicates for each cell population) were seeded in the lower chamber of a transwell (Thermo Fisher Scientific), with macrophage from WT or mdx/utr(−/−) mice (n = 3, with 2 replicates for each cell population) being seeded in the upper chamber. The pore size of the transwell membrane was 0.4 µm, and coated with a layer of Matrigel (20% of Matrigel in water, 0.2cm of thickness).

Myogenesis and proliferation assays
For myogenic differentiation assay, WT MPCs (n = 3, with 2 replicates for each cell population) were seeded as ~90% confluence in the lower chamber of transwell, with macrophages from WT and mdx/utr(−/−) mice (n = 3, with 2 replicates for each cell population) being

Trichrome staining
Fibrosis formation in muscle tissues of WT or mdx/utr(−/−) mice (n = 8) was visualized by Masson trichrome staining with the Trichrome Stain (Masson) Kit (Sigma-Aldrich). Sections were incubated in Weigert's iron hematoxylin working solution for 10 min and rinsed under running water for 10 min. Slides were transferred to Biebrich scarlet-acid fuchsin solution for 15 min before incubation in aniline blue solution for another 5 min. Slides were then rinsed, dehydrated, and mounted as earlier. The ratio of the area of fibrotic collagen (blue) to the area of normal muscle (red) was quantified to measure fibrosis formation.

Measurements of results and statistical analysis
Image analysis was performed using ZEN 2.3 imaging software (ZEISS Microscopy, Germany) and Image J software (version 1.32j; National Institutes of Health, Bethesda, MD, USA). Data from at least six samples from each subject were pooled for statistical analysis. Results are given as the mean ± SEMx. The statistical significance of any difference was calculated using Student's t-test or two-way ANOVA test. P values < 0.05 were considered statistically significant.

AUTHOR CONTRIBUTIONS
PDR, JH, ZW and XM performed literature search, study design, and paper writing; LL, XY, ZS, WSH, QF, and YC performed experiments and data collection; LL, WSH, YC and XM performed data analysis.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest related to this study.

ETHICAL STATEMENT
Procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Health Science Center at Houston (AWC-18-0068). Mice were housed and maintained in the Center for Laboratory Animal Medicine and Care (CLAMC) in accordance with established guidelines and protocols approved by the UTHealth Animal Welfare Committee.