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.

Chronic inflammation, promoted by an upregulated NF-kappa B (NF-kB) pathway, has a key role in Duchenne muscular dystrophy (DMD) patients' pathogenesis. Blocking the NF-kB pathway has been shown to be a viable approach to diminish chronic inflammation and necrosis in the dystrophin-defective mdx mouse, a murine DMD model. In this study, we used the recombinant adeno-associated virus serotype 9 (AAV9) carrying an short hairpin RNA (shRNA) specifically targeting the messenger RNA of NF-kB/p65 (p65-shRNA), the major subunit of NF-kB associated with chronic inflammation in mdx mice. We examined whether i.m. AAV9-mediated delivery of p65-shRNA could decrease NF-kB activation, allowing for amelioration of muscle pathologies in 1-and 4-month-old mdx mice. At 1 month after treatment, NF-kB/p65 levels were significantly decreased by AAV gene transfer of p65-shRNA in the two ages of treatment groups, with necrosis significantly decreased compared with controls. Quantitative analysis revealed that central nucleation (CN) of the myofibers of p65-shRNA-treated 1-month-old mdx muscles was reduced from 67 to 34%, but the level of CN was not significantly decreased in treated 4-month-old mdx mice. Moreover, delivery of the p65-shRNA enhanced the capacity of myofiber regeneration in old mdx mice treated at 4 months of age when the dystrophic myofibers were most exhausted; however, such p65 silencing diminished the myofiber regeneration in young mdx mice treated at 1 month of age. Taken together, these findings demonstrate that the AAV-mediated delivery of p65-shRNA has the capacity to ameliorate muscle pathologies in mdx mice by selectively reducing NF-kB/p65 activity.

Recently, we have reported that intramuscular (i.m.) injection of muscle‐derived stem cells (MDSCs) heterozygous for the NF‐kB subunit p65 (p65+/−) reduces host inflammation and fiber necrosis following muscle injury more so than wild type (WT) MDSC injection. In vitro inflammation assays demonstrated that MDSCs secrete factors that modulate cytokine expression in LPS‐activated macrophages, and genetic reduction of p65 enhances this effect. We have also found that the anti‐inflammatory protein hepatocyte growth factor is more highly expressed in p65+/−cells. In order to investigate the impact of these observations in vivo, we performed i.m. injections of WT or p65+/−MDSCs into WT mouse muscle 24 hrs post injury. We followed cell fate at 1, 3, and 7 days post‐injection. At 24 hrs we observed a higher number of engrafting WT cells, but a higher percentage of proliferating (Ki67+) p65+/− MDSCs. By 3, and 7 days, the number of p65+/−MDSCs exceeded that of WT cells. These preliminary results suggest a novel role for NF‐kB in stem cells, one in which the anti‐inflammatory properties of MDSCs are regulated by NF‐kB via growth factor expression and cell proliferation kinetics.Grant Funding Source: Department of Defense