Characterization of the PKCθ-dependent Immune Response in Muscular Dystrophy

Our previous observations showed that lack of PKCθ in mdx, greatly improved muscle maintenance, regeneration and strength. The observed phenotype was primarily due to lack of PKCθ in BM derived cells. PKCθ is currently proposed as a target to selectively manipulate T cell functions. Scid/mdx mice, which lack functional T and B-cells in the mdx background, present a milder dystrophic phenotype than the mdx, supporting the pathogenic role of T cells in mdx muscle. The aims we addressed were: (1) characterize the PKCθ-dependent immune response in the pathogenesis of muscular dystrophy in mdx, using mdx/θ-/- mice; (2) investigate the T-cell dependent contribution to the pathogenesis of muscular dystrophy, using Scid/mdx mice as recipient of PKCθ expressing or not expressing T cells; (3) investigate the T/macrophage cells interactions by using an in vitro system. We found that PKCθ depletion is sufficient to counteract dystrophic features progression, already from the early stages, decreasing muscle inflammation and preventing fibrotic tissue deposition. PKCθ depletion in mdx avoided an excessive accumulation of M1 or M2 macrophages in muscle, modifying macrophage balance. Wild type T cells reintroduced into Scid/mdx mice specifically invaded dystrophic muscle, co-localizing with infiltrate and surrounding necrotic fibers, thus worsening dystrophic phenotype. Exogenous wild type T cells increased M1 macrophages in muscle, cell infiltrate and fibrosis. PKCθ inhibition decreases T cells activation in vitro, increasing Treg differentiation. By contrast, PKCθ-/- T cells reintroduction into Scid/mdx mice did not worsen dystrophic phenotype, neither increasing cell infiltrate nor affecting M1/M2 balance. By co-culturing T cells and Raw 264.7 macrophage cell line we found that lack of PKCθ in T cells affects macrophage responsiveness to LPS or IL-4. By PKCθ inhibition in macrophages we found that PKCθ is necessary for MMP-9 activity, but not for iNOS expression, in response to LPS, while its inhibition increased CD206 and CD163 expression in response to IL-4. According to our studies T cells and macrophages are key players inmuscular dystrophy. Taken together these results suggest that lack of PKCθ in mdx may improve Treg activity in the inflammatory infiltrate, which, in turn, reduces M1 pro-inflammatory activity while enhancing M2 pro-healing activity, but avoiding accumulation of M2 pro-fibrotic macrophages in late stages, resulting in attenuation of dystrophic features. For its role in T cells, particularly in Treg, in macrophages and in T/macrophage cross-talk, PKCθ could be a good pharmacological target in immune disorders.