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

Skeletal muscle of mdx/utro(−/−) mice develops higher level of muscle stem cell exhaustion, immune cell infiltration, and cellular senescence

Gastrocnemius muscles from hindlimbs were collected from 8-week old WT and mdx/utro(−/−) mice for histological analyses (Figure 1). Immunofluorescent staining of Pax7 and collagen type IV in muscle tissues revealed that, there is significantly reduced number of Pax7+ muscle stem cells in mdx/utro(−/−) muscles (Figure 1A); while in contrast, immunofluorescent staining of CD68 showed that, the number of CD68+ macrophages is greatly increased in mdx/utro(−/−) muscles (Figure 1B). Meanwhile, SA-β-Gal (senescence-associated β-Galactosidase, a marker for cellular senescence) staining further revealed that, there is obviously higher number of SA-β-Gal+ senescent cells in mdx/utro(−/−) muscles (Figure 1C).

Figure 1. mdx/utro(−/−) muscle exhibits obvious stem cell exhaustion, immune cell infiltration and cellular senescence. (A) Immunofluorescent staining of Pax7 and Collagen type IV in muscle tissues of WT and mdx/utro(−/−) mice. (B) Immunofluorescent staining of CD68 in muscle tissues of WT and mdx/utro(−/−) mice. (C) Staining of SA-β-Gal in muscle tissues of WT and mdx/utro(−/−) mice. n = 8 mice/group for statistics, and data are presented as mean +/− SD.

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 β-galactosidase) 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.

Figure 2. Macrophages in mdx/utro(−/−) muscle exhibits higher level of mTOR activation. (A) Immunofluorescent staining of CD68 and p-4E-BP1 in muscle tissues of WT and mdx/utro(−/−) mice. (B) Immunofluorescent staining of p-4E-BP1 in macrophages isolated from muscle tissues of WT and mdx/utro(−/−) mice. (C) Statistics of results in vivo. n = 8 mice/group. Data are presented as mean +/− SD. (D) Statistics of result in vitro, n = 6 (3 cell lineages x 2 replicates/group). Data are presented as mean +/− SD.

Also, CD68+ macrophages were isolated from WT and mdx/utro(−/−) muscles via flow cytometry based on CD68 expression, to compare their expression of p-4E-BP1 protein, and we observed significantly higher percentage of p-4E-BP1+ cells in CD68+ macrophages from mdx/utro(−/−) muscle (Figure 2B, 2D).

CD68+ macrophages from mdx/Utro(−/−) muscle features elevated expression of SASP factors and increased cellular senescence

The higher activation of mTOR signaling in mdx/utro(−/−) macrophage may suggest a potential development of cellular senescence. Thus, we then compared the expression of SASP factors in CD68+ macrophages isolated from WT and mdx/utro(−/−) muscles. RT-PCR result showed that, CD68+ macrophages from mdx/utro(−/−) muscle displayed up-regulated expression of SASP factors (i.e., TNF-α, IL-1α, IL-1β, IL-6, CXCL1, MCP1, and IFN-γ) and p16, and down-regulated expression of anti-inflammation factor IL10 (Figure 3A). Meanwhile, the fluorescent staining of SA-β-Gal revealed that there was higher percentage of SA-β-Gal+ cells in mdx/utro(−/−) macrophages, compared with WT macrophages (Figure 3B, 3C).

Figure 3. Fisetin treatment specifically remove senescent macrophages from mdx/utro(−/−) mouse. (A) Real time-PCR result of gene expression level of SASP factors, p16 and IL-10 in WT and mdx/utro(−/−) macrophages (n = 3 cell lineages). (B) Staining of SA-β-Gal in 4 groups of macrophages with fluorescent Senescence Assay Kit. Fisetin was applied to treat the WT and mdx/utro(−/−) macrophages for 48 hr at 20 μM of concentration. (C) Statistics of SA-β-Gal+ cells in 4 groups of cells. n = 6 (3 cell lineages x 2 replicates/group). Data are presented as mean +/− SD.

Further, we examined whether the senolytic drug fisetin may specifically remove SA-β-Gal+ senescent cells in mdx/utro(−/−) macrophages. Fisetin was applied to treat the WT and mdx/utro(−/−) macrophages at 20 μM of concentration for 48 hr. Result showed that, the percentage of SA-β-Gal+ senescent cells in mdx/utro(−/−) macrophages was obviously reduced after fisetin treatment (Figure 3B, 3C).

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).

Figure 4. Macrophages from mdx/utro(−/−) muscle impair the function of co-cultured MPCs, which can be rescued with senolytic drug. (A) The progression of myotube formation was tracked in the myogenesis assay of WT MPCs co-cultured with WT or mdx/utro(−/−) macrophages, with or without fisetin treatment (20 μM, 96 h). (B) Schematic demonstration of the cell co-culture system of WT or mdx/utro(−/−) macrophages (upper chamber) and WT MPCs (lower chamber). (C) Statistics of result for myogenesis assay. (D) Statistics of result for proliferation assay. n = 6 (3 cell lineages x 2 replicates/group). Data are presented as mean +/− SD.

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).

Figure 5. In vivo results of fisetin administration in mdx/utro(−/−) mice. (A) mdx/utro(−/−) mice were treated with fisetin via oral gavage (20 mg/kg for 5 consecutive days each week) for 4 weeks and muscle tissues were analyzed. Immunofluorescent staining of CD68 showed that the number of macrophages was decreased with fisetin administration; staining of SA-β-Gal showed that the number of senescent cells was decreased with fisetin administration; immunofluorescent staining of Pax7 showed that the number of MPCs was increased with fisetin administration; trichrome staining of collage deposition (fibrosis formation) showed that the amount of fibrosis formation was decreased with fisetin administration. (B) Statistics of in vivo results. n = 8 mice/group. Data are presented as mean +/− SD.

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 co-cultured in the upper chamber (8000 cells/cm²); cells were co-cultured in the DMEM medium containing 2% HS (horse serum), and allowed for differentiation for 96 h with or without fisetin (Selleckchem, Houston, TX, USA), before being fixed and imaged to compare the number of myotubes formed by WT MPCs in different groups (WT MPCs + WT macrophages, WT MPCs + WT macrophages + fisetin, WT MPCs + mdx/utr(−/−) macrophages, and WT MPCs + mdx/utr(−/−) macrophages + fisetin). The concentration of fisetin used for cells was 20 μM, which was based upon previous studies that reported its senolytic effect [16, 24]. For proliferation assay, WT MPCs were seeded as 2000 cells/cm² in the lower chamber of transwell, with macrophages being co-cultured in the upper chamber (5000 cells/cm²); cells were co-cultured in the DMEM medium containing 20% FBS and 1% CEE (Chicken Embryo Extracts) and allowed for proliferation for 72 h with or without fisetin (20 μM) before being observed to compare the number of WT MPCs in different groups and calculate cell population doubling time (PDT).

Fisetin treatment of cell co-culture system and mice

For in vitro cell treatment, fisetin (Selleckchem) was applied to treat the cell co-culture system of mdx/utr(−/−) macrophages and WT MPCs (n = 3, with 2 replicates for each cell population) at 20 μM of concentration, and the proliferation and myogenesis potential of WT MPCs was then compared to non-treated controls. For in vivo treatment of mdx/utr(−/−) mice [n = 8 for WT or mdx/utr(−/−) mice] , fisetin was dissolved in vehicle (10% ethanol, 30% polyethylene glycol, 60% phosal 50 pg). Mice were weighed and given fisetin (20 mg/kg for 5 consecutive days each week) or vehicle control alone by oral gavage for 4 weeks.

mRNA analysis via reverse transcriptase-PCR

Total RNA was obtained from MPCs or the skeletal muscles of mice (n = 8) using the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA, USA) according to the manufacturer’s instructions. Reverse transcription was performed using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The sequences of primers are given in Table 1 for SASP genes (CXCL2, MCP1, IL-1α, IL-1β, IL-6 and IFN-γ), p16, IL-10, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Real-Time PCR reactions were performed using an iCycler thermal cycler (Bio-Rad Laboratories, Inc.). The cycling parameters used for all primers were as follows: 95°C for 10 minutes; PCR, 40 cycles of 30 seconds at 95°C for denaturation, 1 minute at 54°C for annealing, and 30 seconds at 72°C for extension. All data were normalized to the expression of GAPDH.

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.

Immunofluorescent staining and imaging

Cultured cells (n = 3, with 2 replicates for each cell population) were fixed with 4% paraformaldehyde, and frozen muscle tissue sections were fixed with 10% formalin. The primary antibodies used – CD68 (Abcam, Boston, MA, USA), p-4E-BP1(Cell Signaling), Pax7 (DSHB, Iowa City, IA, USA), Collagen IV (Abcam), and fMHC (fast-type myosin heavy chain, R&D Systems, Minneapolis, MN, USA) – were all applied at a 1:100 to 1:300 dilution. Cell nucleus was stained with DNA binding reagent and 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescent images of myofibers were imaged and photographed with a Nikon high resolution microscope.

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.