Dysfunctional Keratinocytes Increase Dermal Inflammation in Systemic Sclerosis: Results From Studies Using Tissue‐Engineered Scleroderma Epidermis

Abstract

Evidence suggests that keratinocyte-fibroblast interactions are abnormal in systemic sclerosis (SSc). The present study was undertaken to investigate potential epidermal dysfunction in SSc and its effects on dermal homeostasis. Epidermal equivalents (EEs) were generated using keratinocytes from 6 healthy donors and 4 individuals with SSc. Skin and EE expression of markers of proliferation, differentiation, and activation was evaluated by immunohistochemistry. The transcriptomic profile of SSc EEs and healthy donor EEs was identified by RNA sequencing. EE conditioned medium (CM) was used to stimulate fibroblasts, and their production of interleukin-6 (IL-6), IL-8, matrix metalloproteinase 1 (MMP-1), type I collagen, and fibronectin was assessed by enzyme-linked immunosorbent assay. Compared to healthy donor EEs, SSc EEs exhibited aberrant differentiation, enhanced expression of activation markers, and a lower rate of basal keratinocyte mitosis, reproducing most of the abnormalities observed in SSc epidermis. RNA sequencing analysis revealed that, compared to healthy donor EEs, SSc EEs were characterized by lower expression of homeobox gene family members and by enhanced metabolic and oxidative stress molecular pathways. EE CM enhanced fibroblast production of IL-6, IL-8, MMP-1, type I collagen, and fibronectin (P < 0.05). Except for type I collagen and fibronectin, this effect was 2-fold higher in the presence of CM generated form SSc EEs. IL-1 was responsible, at least in part, for keratinocyte-dependent fibroblast activation. SSc EEs recapitulate the in vivo characteristics of SSc epidermis, demonstrating that SSc keratinocytes have an intrinsically altered differentiation program, possibly due to the dysregulation of genes from the homeobox family. The increased metabolic and oxidative stress associated with SSc epidermis may contribute to chronic inflammation and fibrosis of the dermis.