Human hypofunctional NCF1 variants promote pulmonary fibrosis in the bleomycin-induced mouse model and patients with systemic sclerosis via expansion of SPP1 monocytes-derived macrophages.

Objective: We assessed the role of a systemic lupus erythematosus causal hypofunctional variant, neutrophil cytosolic factor 1 (NCF1)-p.Arg90His (p.R90H) substitution, in systemic sclerosis (SSc).

Methods: Association of NCF1-H90 with SSc was performed in case-control cohorts, bleomycin (BLM)-treated Ncf1-R90 C57BL/6 wildtype and Ncf1-H90 knock-in (KI) littermates. Peripheral blood mononuclear cell (PBMC) subsets were analysed by cytometry by time-of-flight.

Results: The NCF1-H90 allele is associated with risk for diffuse cutaneous SSc (dcSSc) in Chinese and European Americans, and lung fibrosis in Chinese patients with SSc (OR=2.09, p=7.96E-10). Low copy number of NCF1 associated with lung fibrosis in European Americans (OR=4.33, p=2.60E-2). BLM-treated KI mice demonstrated increased pulmonary fibrosis, exhibiting activated type I interferon signature, elevated Spp1, Ccl2, Arg1, Timp1 and Il6 expression, enriched macrophage scores in lung tissues. In a longitudinal observation cohort, homozygous H90 patients with SSc at baseline had increased anti-nuclear antibody titres, anti-topoisomerase antibody seropositivity and anti-centromere antibody seronegativity, increased incidence of lung fibrosis and Gender-Age-lung Physiology index, elevated modified Rodnan Skin Score (mRSS) and elevated plasma osteopontin (OPN, SPP1), CCL2, ARG1, TIMP-1 and IL-6. These H90 patients with SSc sustained elevated mRSS during follow-up years with decreased survival. The 0, 1 and 2 copies of H90 carriage in SSc PBMCs exhibited dose-dependent increases in profibrotic CD14CD68CD11bTim3monocytes. Elevated OPN, CCL2 and ARG1 in CD68CD11bmonocyte-derived macrophages from H90 patients were decreased after co-culturing with anti-CCL2 antibody.

Conclusion: Low NCF1 activity increases the risk for the development of dcSSc and lung fibrosis via expanding profibrotic SPP1MoMs in a CCL2-dependent manner, contributing to the severity of lung fibrosis in both BLM-treated mice and patients with SSc.