CTLA4-Ig reduces proliferation and inflammatory gene expression in muscle fibroblasts, corresponding to less fibrosis and inflammation in mdx muscular dystrophy

Muscle pathology in Duchenne muscular dystrophy (DMD) is greatly amplified by the immune response to dystrophic muscle, which provides the rationale for targeting the immune system in DMD therapies. Much of immune-driven pathology in the mdx mouse model of DMD is caused by T-lymphocytes and macrophages; thus, preventing T-cell activation by blocking pathways that cause their activation has the potential to reduce muscle damage and fibrosis in muscular dystrophy. CTLA4-Ig is a recombinant protein that blocks co-stimulatory signaling between T-cells and antigen presenting cells, such as macrophages. In this investigation, we tested whether treatment of mdx mice until they reach advanced, fibrotic stages of the disease reduces pathology. Our findings show that CTLA4-Ig treatments reduced muscle damage and inflammation and also reduced numbers of fibrogenic cells and fibrosis of muscles in aging, mdx mice. However, the treatments did not reduce numbers of CD8+ T-cells or activated CD25+ cells, suggesting that the reduced pathology was not mediated by affecting T-cell activation. Complete blood counts and clinical histopathological scoring also showed that the treatments had little effect on hematopoietic tissues. In vitro, CTLA4-Ig acted directly on muscle fibroblasts, reducing their expression of pro-inflammatory genes without affecting the expression of genes encoding connective tissue proteins, assayed by qPCR. However, CTLA4-Ig treated fibroblasts were less proliferative in vitro. Collectively, these findings show that CTLA4-Ig acts directly on fibroblasts to produce changes in proliferation and gene expression that are consistent with the reductions in muscle pathology that occurs in aging, mdx mice treated with CTLA4-Ig.