Background/Purpose: Systemic Sclerosis (SSc) is an immune driven condition leading to skin fibrosis, as well as internal organs. Dermal fibroblasts are the key cellular elements of skin fibrosis, which cause the accumulation of extracellular matrix (ECM) proteins, altering tissue architecture and function. The triggers responsible for re-establishing tissue homeostasis and functional skin following inflammation vs. scarring and loss of function are not known.Recently we have shown that dermal fibroblast can induce a Type I IFN response in keratinocytes, partially through their secreted exosomes.
RNAseqanalysis revealed that lncRNA H19 is the most significantly increased gene in SSc fibroblast exosomes compared to Healthy fibroblast derived-exosomes. Here we aimed to understand the role of lncRNA H19 mediating the fibrotic signature and skin homeostasis in SSc skin.
 

Methods: Skin biopsies were obtained from the forearms of adult patients with diffuse SSc or healthy controls. Fibroblasts were isolated and immortalized using human telomerase reverse transcriptase (hTERT). In situ Hybridization was performed to detect H19 levels in skin from SSc and co-immunohistochemistry for COL1A1. H19 was overexpressed by retroviral transduction and knockdown using siRNA. RT-qPCR assay was used to measure transcript levels of H19 and profibrotic markers. Gel contraction assays used to analyse myofibroblast activity in vitro. Co-culture of fibroblasts and Hacats was performed by Transwell assays. Phosphorylation and total expression of STAT3 and STAT1 protein and profibrotic markers was assessed by immunoblotting.  

Results: RNAscope assay demonstrated higher H19 levels in SSc skin compared to Healthy, with detectable signal spread across dermal fibroblast, keratinocytes, endothelial cells and fat tissue in the skin. H19 lncRNA expression was highly variable across SSc explanted fibroblasts, and strongly correlated with mRNA levels of Col1A1 (p=0.007), SMA (p=0.049) and CTGF/CNN2 (p=0.022). 
Knockdown of H19 in SSc fibroblasts had a 0.75 fold decrease in CTGF/CNN2 (p= 0.023) and 0.6 in COL1A1 (p= 0.019) gene expression relative to SCR SSc fibroblast. Accordingly, H19 knockdown significantly reduced the ability of SSc fibroblasts to contract collagen matrix (p=0.038). On the contrary, overexpression of H19 in healthy fibroblasts significantly increased pSMAD3, pSTAT3 and CTGF/CNN2 protein levels.  Importantly, in keratinocytes/fibroblast co-culture models, knockdown of H19 in SSc fibroblast prevented keratinocytes activation as assessed by total and phosphorylation protein levels of STAT3 and STAT1. 
 

Conclusion: All together we show that H19 lncRNA is upregulated in skin from SSc patients and  SSc primary dermal fibroblast comparing to Healthy. Our studies indicate that H19 contributes to the profibrotic activation of dermal fibroblasts and the proinflammatory activation of keratinocytes through STAT3 dependent signalling. 
 

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/role-of-long-non-coding-rna-h19-in-mediating-skin-fibrosis-in-systemic-sclerosis/