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

Andrea Ottria, Maili Zimmermann, Laurent M. Paardekooper, Tiago Carvalheiro, Nadia Vazirpanah, Sandra Silva-Cardoso, Alsya J. Affandi, Eleni Chouri, Maarten V D Kroef, Ralph G. Tieland, Cornelis P. J. Bekker, Catharina G. K. Wichers, Marzia Rossato, Enric Mocholi-Gimeno, Janneke Tekstra, Evelien Ton, Jaap M. van Laar, Marta Cossu, Lorenzo Beretta, Samuel Garcia PerezAridaman Pandit, Femke Bonte-Mineur, Kris A. Reedquist, Geert van den Bogaart, Timothy R. D. J. Radstake, Wioleta Marut

Research output: Contribution to journalArticleAcademicpeer-review

6 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)2682-2693
Number of pages12
JournalRheumatology (Oxford, England)
Issue number6
Early online date24 Sept 2021
Publication statusPublished - 1 Jun 2022
Externally publishedYes


  • CXCL4
  • HIF-2α
  • TLRs
  • hypoxia
  • mtROS
  • plasmacytoid dendritic cells
  • systemic sclerosis

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