OBJECTIVE: Systemic sclerosis (SSc) is a complex disease characterized by vascular abnormalities and inflammation culminating in hypoxia and excessive fibrosis. Previously, we identified CXCL4 as a novel predictive biomarker in SSc. Although CXCL4 is well-studied, the mechanisms driving its production are unclear. The aim of this study was to elucidate the mechanisms leading to CXCL4 production.
METHODS: Plasmacytoid dendritic cells (pDCs) from 97 healthy controls and 70 SSc patients were cultured in the presence of hypoxia or atmospheric oxygen level and/or stimulated with several TLR-agonists. Further, pro-inflammatory cytokine production, CXCL4, HIF-1α and HIF-2α gene and protein expression were assessed using ELISA, Luminex, qPCR, FACS and western blot assays.
RESULTS: CXCL4 release was potentiated only when pDCs were simultaneously exposed to hypoxia and TLR9 agonist (p < 0.0001). Here, we demonstrated that CXCL4 production is dependent on the overproduction of mitochondrial reactive oxygen species (mtROS) (p = 0.0079) leading to stabilization of HIF-2α (p = 0.029). In addition, we show that hypoxia is fundamental for CXCL4 production by umbilical cord (uc)CD34 derived pDCs.
CONCLUSION: TLR-mediated activation of immune cells in the presence of hypoxia underpins the pathogenic production of CXCL4 in SSc. Blocking either mtROS or HIF-2α pathways may therapeutically attenuate the contribution of CXCL4 to SSc and other inflammatory diseases driven by CXCL4.