P15 Dermal papillary fibroblasts promote junctional epidermolysis bullosa pathogenesis

Abstract Introduction and aims Junctional epidermolysis bullosa (JEB) is a hereditary skin disorder characterized by severe skin and mucus membrane fragility, caused by loss-of-function mutations in skin basement membrane proteins including laminin-332, type XVII collagen or integrin α6β4. Skin blistering leads to a deregulated epidermal keratinocyte–dermal fibroblast crosstalk involving multiple cytokines, transforming growth factors (TGFs) and immune signals. The skin dermis is composed of multiple fibroblast subpopulations with different functions in skin homeostasis and repair; however, their involvement in JEB is largely unknown. Methods Three-dimensional (3D) organotypic (OT) skin cultures combining short hairpin (sh)RNA knockdown of LAMA3 (JEB) keratinocytes or shC controls with distinct fibroblast subpopulations were developed and analysed by immunohistochemistry, cytokine profiling, and RNA sequencing. Identified disease promoting epidermal–dermal signalling pathways were targeted in our two-dimensional (2D) and 3D cell culture models, before testing selected candidates in our inducible JEB mouse model for preclinical validation. Results JEB OTs with papillary fibroblasts from different anatomical sites show increased epidermal growth and invagination and deregulated differentiation compared with reticular fibroblasts. JEB OTs with papillary fibroblasts showed increased TGF-β signalling by cytokine array, enzyme-linked immunosorbent assay and TGF-β pathway activation by pSMAD2 staining. Treatment of 2D and 3D models with the TGF-β receptor inhibitor RepSox efficiently normalized aberrant cell proliferation, differentiation and cytokine signalling in a concentration dependent manner. TGF-β signalling was also strongly increased in the inducible JEB disease model around fibrotic blistered skin lesions. Two weeks of topical RepSox treatment significantly improved skin fibrosis, reduced proliferation and inflammation in Lama3-depleted mice. Conclusions Our 2D and 3D in vitro and in vivo JEB disease model revealed that papillary fibroblasts are key drivers of JEB pathogenesis, by promoting aberrant TGF-β signalling leading to increased skin epidermal proliferation, inflammation and fibrosis. Disrupting the pathological signalling interactions by inhibiting TGF-β signalling significantly improved skin health and regeneration in JEB.