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

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

Andrea Ottria  1   2 , Maili Zimmermann  1   2 , Laurent M Paardekooper  3 , Tiago Carvalheiro  1   2 , Nadia Vazirpanah  1   2 , Sandra Silva-Cardoso  1   2 , Alsya J Affandi  1   2 , Eleni Chouri  1   2 , Maarten V D Kroef  1   2 , Ralph G Tieland  1   2 , Cornelis P J Bekker  1   2 , Catharina G K Wichers  1   2 , Marzia Rossato  1   2 , Enric Mocholi-Gimeno  1   2 , Janneke Tekstra  2 , Evelien Ton  2 , Jaap M van Laar  2 , Marta Cossu  1   2 , Lorenzo Beretta  4 , Samuel Garcia Perez  1   2 , Aridaman Pandit  1   2 , Femke Bonte-Mineur  5 , Kris A Reedquist  1   2 , Geert van den Bogaart  3   6 , Timothy R D J Radstake  1   2 , Wioleta Marut  1   2
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Hypoxia and TLR9 activation drive CXCL4 production in systemic sclerosis plasmacytoid dendritic cells via mtROS and HIF-2α

Andrea Ottria et al. Rheumatology (Oxford). .

Abstract

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.

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