NF-κB inhibition reveals a novel role for HGF during skeletal muscle repair

Abstract

The transcription factor nuclear factor κB (NF-κB)/p65 is the master regulator of inflammation in Duchenne muscular dystrophy (DMD). Disease severity is reduced by NF-κB inhibition in the mdx mouse, a murine DMD model; however, therapeutic targeting of NF-κB remains problematic for patients because of its fundamental role in immunity. In this investigation, we found that the therapeutic effect of NF-κB blockade requires hepatocyte growth factor (HGF) production by myogenic cells. We found that deleting one allele of the NF-κB subunit p65 (p65+/-) improved the survival and enhanced the anti-inflammatory capacity of muscle-derived stem cells (MDSCs) following intramuscular transplantation. Factors secreted from p65+/- MDSCs in cell cultures modulated macrophage cytokine expression in an HGF-receptor-dependent manner. Indeed, we found that following genetic or pharmacologic inhibition of basal NF-κB/p65 activity, HGF gene transcription was induced in MDSCs. We investigated the role of HGF in anti-NF-κB therapy in vivo using mdx;p65+/- mice, and found that accelerated regeneration coincided with HGF upregulation in the skeletal muscle. This anti-NF-κB-mediated dystrophic phenotype was reversed by blocking de novo HGF production by myogenic cells following disease onset. HGF silencing resulted in increased inflammation and extensive necrosis of the diaphragm muscle. Proteolytic processing of matrix-associated HGF is known to activate muscle stem cells at the earliest stages of repair, but our results indicate that the production of a second pool of HGF by myogenic cells, negatively regulated by NF-κB/p65, is crucial for inflammation resolution and the completion of repair in dystrophic skeletal muscle. Our findings warrant further investigation into the potential of HGF mimetics for the treatment of DMD.

Figure 1

MDSC-CM reduces cytokine expression by LPS-activated RAW cells. (a) Real-time RT-PCR demonstrated that at 24 h, Il1β, Tnfα and Il6 gene expression was attenuated by WT-CM (left), but the expression of Il6 was even further reduced in p65^+/−-CM-treated RAW cells (right) (*versus No LPS, P⩽0.05; ^#versus +LPS, P⩽0.05; ⁺versus LPS+WT-CM, P⩽0.05). At an earlier timepoint of 3 h, (b) Il6 and (c) Il10 gene expression is induced in RAW cells by exposure to LPS and MDSC-CM, an effect that is enhanced in p65^+/− MDSCs (*versus LPS in SF medium, P⩽0.05; ⁺versus LPS+WT-CM, P⩽0.05). Data are displayed from a representative experiment as mean±S.E.M. Each experiment was performed a minimum of three times

Figure 2

Donor p65^+/− MDSC engraftments promote the repair of recipient muscle. (a) Immunofluorescent staining of tissue for the macrophage marker CD68 (green) indicated that RFP+ donor cell (red) engraftments in injured muscle were infiltrated by macrophages within 24 h postinjection (48 h postinjury), which continued to persist at 7 days (bottom). (b) eMyHC+ fibers (green) could be identified in or around donor cell engraftments at 7 days. (c) Quantification of CD68 positivity within x20 images indicated that p65^+/− MDSC engraftments have significantly less CD68+ cells present at 7 days (*P⩽0.05), and (d) demonstrated a trend towards higher numbers of total eMyHC+ fibers (host+donor) (P=0.12). Data are displayed as mean±S.E.M.; n=3–4 mice per group. Scale bar: 100 μm

Figure 3

Haploinsufficiency of p65 improves MDSC survival in vivo. (a) Immunofluorescent staining of tissue sections for the proliferation marker RFP (donor cells, red) and Ki-67 (green) demonstrated improved survival of p65^+/− MDSCs up to 1 week postinjection. (b) Quantification of RFP+ cells indicated a significant decline in WT cells within the first week, while p65^+/− MDSCs displayed a much slower decline in number (**versus WT, P⩽0.001; *versus day 1, P⩽0.05). (c) Ki-67 positivity indicated that there were no differences in proliferation at days 1 and 3 (*P⩽0.05; ⁺versus day 1, P⩽0.10). (d) When cocultured with RAW cells (1 : 10) in the presence of LPS, the population doubling time of p65^+/− MDSCs significantly increased, reflecting a decreased rate of proliferation. WT MDSCs demonstrated no significant changes (*P<0.05). For (a): Scale bar: 100 μm; n=8–9 mice per group. Data are displayed as mean±S.E.M. Data in (d) are from one experiment representative of three

Figure 4

Reducing basal NF-κB activity upregulates Hgf expression in MDSCs. (a) Real-time RT-PCR analysis revealed that Hgf was significantly upregulated in p65^+/− compared with WT MDSCs (*P<0.05). (b) Hgf transcription could also be induced in WT cells by treatment with an IKKi for 24 h (left). IKKi treatment of p65^+/− MDSCs induced only a modest increase in Hgf at 2 h (*versus vehicle, P<0.05; ⁺versus time=2 h; P<0.05). (c) p-Met in RAW cells can be detected within 5 min of stimulation with HGF (left), or p65^+/− CM (right), but not WT-CM (middle). (d) Inhibiton of Met activation on RAW cells using SU11274 significantly decreased Il6 induction 3 h following stimulation with LPS and p65^+/−-CM, but not LPS and WT-CM. (e) Il10 induction by LPS in both WT-CM or p65^+/− CM was decreased by SU11274, but to a much greater degree in the p65^+/− CM group (*versus vehicle, P<0.05). Values from a representative experiment are displayed as mean±S.E.M. Each experiment was performed at least three times

Figure 5

p65^+/−-CM activates an HGF/Met/GSK3β pathway in RAW cells. (a) Western blot demonstrated that activation of RAW cells in PM induced an increase in pS9-GSK3β within 30 min (left), a response that was amplified by both WT- (middle) and p65^+/−-CM (right). (b) Densitometric analysis revealed that when activated in p65^+/−-CM, the fraction of pS9-GSK3β increased by 3.5-fold in 30 min, an amount significantly higher than WT-CM and PM groups (*P⩽0.05). (c) Inhibition of Met by SU11274 blocked pS9-GSK3β in RAW cells 30 min after exposure to LPS and p65^+/−-CM (d) in a dose-dependent manner (*versus SF+LPS, ⁺versus no LPS, P<0.05) Data are represented as mean±S.E.M. of at least three independent experiments

Figure 6

Hgf upregulation correlates with accelerated muscle regeneration in vivo. (a) Hgf expression was significantly upregulated in p65^+/− muscle at 3 days after CTX injury (*P⩽0.05 versus 3 days WT; ⁺P<0.10 versus day 0 WT). (b) Representative images from hemotoxylin and eosin staining indicated that compared with WT muscle, the GAS muscle of p65^+/− mice regenerated more rapidly (arrows) following CTX injury. (c) Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis of muscle extracts showed a slight increase in total HGF in uninjured p65^+/− muscles. (d) pS9-GSK3β+ macrophages were identified in injured skeletal muscle by immunofluorescent costaining for pS9-GSK3β (green) and CD68 (red). Arrows indicate examples of colocalization and regions outlined with dashes are digitally enlarged and separated by color channel to the right. (e) Quantification of the number of pS9-GSK3β+ macrophages per high power field (HPF, x600) indicated that a significantly higher number of pS9-GSK3β⁺/CD68⁺ macrophages were found in p65^+/− skeletal muscle compared with WT skeletal muscle at 1, 3 and 5 days after injury (P⩽0.05). For (b), scale bar: 50 μm, n=6–8 mice per group. For (d), scale bar: 20 μm, n=3 mice per group. Data are displayed as mean±S.E.M.

Figure 7

Upregulation of Hgf in mdx;p65^+/− skeletal muscle at 4 weeks of age correlates with accelerated regeneration. (a) Real-time RT-PCR demonstrated that Hgf expression was elevated in mdx:p65^+/− muscle at 4 weeks, coinciding with enhanced regeneration, quantified as the percent of centrally nucleated muscle fibers in (b) (*versus WT, P⩽0.05; ⁺versus WT, P⩽0.10; ^#versus mdx, P⩽0.05). (c) ZsGreen+ centrally nucleated fibers indicated that muscle progenitor cells were transduced by the AAV vector (arrows, top). (d) After 4 weeks, Hgf expression was significantly reduced in the DIA and GAS muscles of HGF-shRNA-treated mdx;p65^+/− mice compared with the ct-shRNA-treated group (P⩽0.05, ⁺shHGF versus ct-shRNA; *GAS, ⁺DIA). (e) Silencing of Hgf worsens the histopathology of the DIA muscle from treated mdx;p65^+/− mice (top) and mdx mice. (f) We quantified the necrotic/inflammatory lesions in H&E-stained DIA tissue sections from treated mice by measuring the lesions as percent area; n=4–6 mice per group. Data are displayed as mean±S.E.M.

Figure 8

(a) Immunofluorescent staining revealed a striking increase in CD68+ macrophages (left) and IgG+ necrotic fibers (right) in the DIA of mdx;p65^+/−-treated with HGF-shRNA compared with ct-shRNA mdx;p65^+/−. (b) The DIA of HGF-shRNA-treated mdx mice did not demonstrate a statistically significant increase in inflammation (left), but did demonstrate a significant increase in fiber necrosis (right), quantified in (c) (*P⩽0.05 versus CD68 in ct-shRNA group; ⁺P⩽0.05 versus IgG in ct-shRNA group). (d) We propose a model in which NF-κB inhibition improves dystrophic muscle regeneration not only by directly promoting lineage progression of muscle progenitor cells but also by increasing progenitor cell survival and upregulating the expression of Hgf. In turn, HGF may promote muscle fiber survival and inactivate myeloid cells to alleviate chronic inflammation in dystrophic muscle and promote repair. Scale bar: 100 μm; n=4–6 mice per group. Data are displayed as mean±S.E.M.