Impaired DNA repair mechanism promote the progression of systemic sclerosis by novel FOXO1-dependent mechanism in women

Introduction: Systemic sclerosis (SSc) is a deadly, incurable disease characterized by immune dysregulation, vasculopathy and fibrosis. Among all rheumatic diseases, it is associated with a 40 % risk of mortality and there is no effective cure. Hence, it is an area of high unmet clinical need. SSc most commonly affects women, however the reason is unknown. The primary cells promoting fibrosis in SSc are human dermal fibroblasts (HDFs) which develop a myofibroblast phenotype associated with increased resistance to apoptosis. We recently showed that HDFs from patients with SSc have increased genomic instability associated with persistent signals γ-H2AX which is associated with double-stranded DNA breaks (DSB). In cancer, dysregulated DNA damage and repair mechanisms (DDR/R) are associated with the activation of the transcription factor forkhead box transcription factor (FOXO). As a result, I hypothesized that HDFs with increased DSBs from women with SSc have increased apoptosis resistance promoted by FOXO1-dependent signals. Methods: Using 4 mm skin biopsies obtained 5 cm from the ulnar styloid, primary human dermal fibroblasts (HDF) was generated from healthy volunteers (HC), pre-fibrotic female patients with early limited SSc patients (elSSc) and female patients with early diffuse severe scleroderma (edSSc). DDR/R activation was determined via immunoblot (IB) detection of γ-H2AX (a DNA damage sensor). Nuclear FOXO1 activation was detected by immunofluorescence microscope (IF) using low passage (< P5) in vitro cultured HDFs from each group. Pro-fibrotic signals (e.g. fibronectin) were determined in edSSc in the presence or absence of a FOXO1 inhibitor. In addition, HDFs from HC were treated with the DNA damage inducing agent etoposide then nuclear FOXO1, and myofibroblast marker were quantified using IB and qRT-PCR. Results: We found that female patients with aggressive edSSc have the highest levels of γ-H2AX compared to HC and elSSc patients. edSSc HDFs also had a substantial nuclear accumulation of FOXO1. This was associated with increased mRNA expression of the known FOXO1 target, pyruvate dehydrogenase kinase 4 (PDK4). FOXO1 inhibition of edSSc HDFs resulted in decreased levels of fibronectin. Intriguingly, etoposide treatment of HDFs from healthy volunteers also resulted in FOXO1 activation with associated increased expression of the myofibroblast marker alpha-smooth muscle actin. Conclusion: Female patients with severe rapidly progressive scleroderma have the highest levels of spontaneous DNA damage. This was associated with activation of FOXO1 and downstream pro-fibrotic signals in a FOXO1-dependent manner. Future studies assessing a role for DNA damage signals and FOXO1 in promoting fibrotic signals and resistance to apoptosis may reveal a novel mechanism that preferentially affects patients with severe SSc. Together, our novel findings may lead to the development of novel therapeutic strategies for patients with SSc which may improve patient outcomes.