Hypoxia and TLR9 activation drive CXCL4 production in systemic sclerosis plasmacytoid dendritic cells via mtROS and HIF-2α

Rheumatology (Oxford). 2022 May 30;61(6):2682-2693. doi: 10.1093/rheumatology/keab532.


Objective: SSc is a complex disease characterized by vascular abnormalities and inflammation culminating in hypoxia and excessive fibrosis. Previously, we identified chemokine (C-X-C motif) ligand 4 (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 toll-like receptor (TLR) agonists. Further, pro-inflammatory cytokine production, CXCL4, hypoxia-inducible factor (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 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.

Keywords: CXCL4; HIF-2α; TLRs; hypoxia; mtROS; plasmacytoid dendritic cells; systemic sclerosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Basic Helix-Loop-Helix Transcription Factors / metabolism
  • Dendritic Cells / metabolism
  • Humans
  • Hypoxia / metabolism
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Platelet Factor 4 / metabolism*
  • Reactive Oxygen Species / metabolism*
  • Scleroderma, Systemic*
  • Toll-Like Receptor 9*


  • Basic Helix-Loop-Helix Transcription Factors
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • PF4 protein, human
  • Reactive Oxygen Species
  • TLR9 protein, human
  • Toll-Like Receptor 9
  • Platelet Factor 4