Background GWAS have linked loss-of-function mutations in phagocytic NADPH oxidase complex (NOX2) genes, including NCF1 and NCF2, to lupus pathogenesis. The prevailing model holds that reduced NOX2 promotes SLE via defective efferocytosis, the immunologically silent clearance of apoptotic cells. We previously showed that defects in endolysosomal flux increase B cell TLR signals, resulting in humoral autoimmunity.1–3 Since NCF1/NCF2 are known regulators of myeloid endosomal trafficking, we hypothesized that a parallel B cell-intrinsic mechanism contributes to lupus risk.
Methods We tested the impact of NOX2 family gene deletion on B cell TLR signaling using in vivo animal models, primary murine B cells, NCF1-null human B cell lymphoma lines, and CRISPR-edited primary human B cells.
Results NOX2-deficient mice exhibited increased humoral responses to nucleic acid-containing antigens, findings which correlate with enhanced B cell signals downstream of endosomal TLRs. In keeping with important roles for B cell TLRs in lupus pathogenesis, B cell-intrinsic NADPH oxidase deletion promoted humoral autoimmunity in mice. To understand the underlying mechanisms, we quantified TLR-induced intracellular trafficking in B cells using live cell microscopy. Following CpG stimulation, NADPH oxidase activation promoted trafficking of TLR-containing endosomes to lysosomes, resulting in TLR signal termination. Whereas initial uptake and aggregation of fluorescent CpG in early endosomes was preserved in NCF1-null B cell lymphomas, loss of NADPH oxidase activity limited signal degradation resulting in enhanced downstream NFkB activation. Finally, CRISPR-mediated disruption of NCF1 in primary human B cells confirmed a direct role for NOX2 in regulating endosomal TLR signaling in B cells. Following TLR9 activation in vitro, NCF1-deficient human B cells exhibited increased differentiation into IgM- and IgG-producing plasma cells, supporting a mechanistic link between B cell NADPH oxidase activity and human lupus pathogenesis.
Conclusions Loss of function NCF1/NCF2 variants exert a B cell-intrinsic contribution to lupus pathogenesis.
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