Background We previously identified a p.Arg90His (p.R90H) hypomorphic variant of neutrophil cytosolic factor 1 (NCF1, a regulatory subunit of phagocyte NADPH oxidase 2 complex, NOX2) predisposes to multiple autoimmune diseases including systemic lupus erythematosus (SLE). We established a C57BL/6 (B6) mouse model with a knock-in (KI) H90 variant in the Ncf1 locus by CRISPR/Cas9 editing to study how this common NCF1 variant promotes the development of lupus manifestations.
Materials and Methods Wild type (WT) and KI littermates were assessed either for spontaneously-developed or pristane-induced immune profiles and lupus-like features. Efferocytosis was assessed using irradiated WT thymocytes or Jurkat cells as apoptotic cells (AC) to co-culture with murine bone marrow-derived macrophages or human circulating monocyte-derived macrophages, respectively. Disease activity and renal damage of SLE patients were assessed by SLEDAI and renal items of SLICC, respectively.
Results Compared to WT littermates, 5-week-old homozygous KI mice had reduced oxidative burst, splenomegaly, elevated type I interferon (IFN-I) scores, increased ratios of splenic follicular T helper 2 (Tfh2) to either T follicular regulatory (Tfr) or Tfh1 cell numbers, increased ANA+ follicular, germinal center B cells and plasma cells, but no spontaneous kidney disease up to one-year of age. Pristane treatment induced kidney disease development in 36-week-old H90 KI B6 female mice, exhibiting increased Tfh2 coupled with decreased Tfr and Tfh1 proportions, robust germinal center formation and IgG autoantibody production. Decreased efferocytosis of macrophages derived from KI mice and homozygous H90 SLE patients promoted elevated ratios of Tfh2/Tfr and Tfh2/Tfh1 as well as dysregulated humoral responses due to reduced Hv1-dependent acidification of phagosome pH to neutralize the decreased electrogenic effect of the H90 variant, resulting in impaired maturation and proteolysis of phagosome. SLE patients carrying homozygous H90 genotype had elevated circulating Tfh2/Tfr and Tfh2/Tfh1 ratios, positive correlations of circulating Tfh2 percentage with plasmablast frequency and disease activity, deposition of IgG and complement C3 in kidney biopsies, and increased kidney damage in multiple ethnic populations.
Conclusion The same links between the NCF1 H90 hypofunctional genotype to lupus-like phenotype in a mouse model and SLE patients demonstrates it is the causal variant in the NCF1 locus associated with SLE.
Acknowledgements This study was supported by Lupus Research Alliance (grant to Betty Tsao) and NRF-2017M3A9B40500335 (grant to Sang-Cheol Bae).
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