368 Epithelial-mesenchymal crosstalk communication promotes fibroblast activation and fibrosis in RDEB

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating progressive skin fragility disorder caused by a deficiency in type VII collagen (C7) and favored by a pro-fibrotic and pro-tumorigenic dermal stroma. RDEB has served as a paradigmatic model of injury-driven fibrosis. Nevertheless, the contribution of epithelial cells to dermal fibrosis has been scarcely addressed in RDEB progression. To study the effect of keratinocytes on fibroblast activation and fibrosis, we approached epithelial-mesenchymal communication in an in vitro surrogate model. Co-culture of RDEB keratinocytes on fibroblast-derived ECM promoted an activated and contractile phenotype in RDEB fibroblasts. This effect was counteracted by co-cultivating RDEB fibroblast-populated ECM with healthy keratinocytes. Moreover, co-culture of RDEB keratinocytes with healthy fibroblasts induced an activated phenotype, contrary to what was observed when co-cultivating healthy fibroblasts and keratinocytes. These results raise the question of whether the observed differences are due to C7 and, therefore, whether correction of C7 in RDEB keratinocytes would be sufficient to reverse their pro-fibrotic effect. Using genome editing tools, we edited RDEB keratinocytes with CRISPR/Cas9 technology to restore C7 expression. Interestingly, RDEB-edited keratinocytes partially reverted fibroblast activation compared with their non-edited counterparts. However, minimum activation levels were not reached as when dermal fibroblasts were co-cultured with healthy keratinocytes. This suggests that RDEB keratinocytes promote fibroblast activation beyond the presence or absence of C7 and that there are other factors influencing the pro-fibrotic environment in RDEB. This preliminary finding may be useful for the development of future therapeutic strategies, as it suggests the plausible need for adjuvant therapies, in addition to the correction of the primary defect, to achieve an efficient curative approach.