Sema4A neutralization reduces fibrosis and inflammation in a mouse model of systemic sclerosis and a 3D model of immunity‐driven fibrosis

OBJECTIVE: We previously showed that Semaphorin4A (Sema4A) is elevated in patients with systemic sclerosis (SSc) and promotes inflammation and fibrosis in vitro. Here, we examined the effect of Sema4A neutralization in vivo and in 3D fibrosis models. METHODS: Sema4A plasma levels were measured by ELISA in healthy controls (n=31) and patients with SSc (n=30). In the bleomycin SSc-model, mice were treated with an anti-Sema4A neutralizing antibody or its respective isotype control (n=6-7). The 3D fibrosis models, consisting of collagen hydrogel, dermal fibroblasts, and peripheral blood mononuclear cells, were stimulated with recombinant Sema4A, rapamycin, or antibodies neutralizing Sema4A, PlexinD1, PlexinB2, Neuropilin-1 or their respective isotype controls (n=6-10). Collagen content was determined by trichrome staining and hydroxyproline assay. Gene expression was determined by RT-qPCR and obtained from GEO-NCBI expression arrays, while protein expression by immunohistochemistry, immunoblotting and flow cytometry. RESULTS: Circulating Sema4A was elevated in patients with SSc (p<0.0001), while Sema4A receptors were elevated in SSc skin and lung (p ranged from <0.05 to 0.0001). Anti-Sema4A administration significantly reduced skin and lung collagen content in bleomycin-treated mice (p<0.01), as well as the expression of extracellular matrix components, fibrotic and inflammatory mediators, and immune cell infiltration. In the 3D models, SSc serum and SSc PBMCs induced cell contraction, which was drastically abrogated by blocking Sema4A (p<0.01). This effect was associated with reduced expression of fibrotic mediators and attenuated mTORC1 pathway activation. CONCLUSIONS: We confirmed the pro-fibrotic and pro-inflammatory role of Sema4A, postulating Sema4A neutralization as a novel approach for the treatment of SSc.