Lupus-associated causal mutation in neutrophil cytosolic factor 2 (NCF2) brings unique insights to the structure and function of NADPH oxidase

The Lupus Genetic Group, Department of Medicine, University of Southern California, Los Angeles, CA 90089, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2011; 109(2):E59-67. DOI: 10.1073/pnas.1113251108
Source: PubMed


Systemic lupus erythematosus (SLE), the prototypic systemic autoimmune disease, is a debilitating multisystem autoimmune disorder characterized by chronic inflammation and extensive immune dysregulation in multiple organ systems, resulting in significant morbidity and mortality. Here, we present a multidisciplinary approach resulting in the identification of neutrophil cytosolic factor 2 (NCF2) as an important risk factor for SLE and the detailed characterization of its causal variant. We show that NCF2 is strongly associated with increased SLE risk in two independent populations: childhood-onset SLE and adult-onset SLE. The association between NCF2 and SLE can be attributed to a single nonsynonymous coding mutation in exon 12, the effect of which is the substitution of histidine-389 with glutamine (H389Q) in the PB1 domain of the NCF2 protein, with glutamine being the risk allele. Computational modeling suggests that the NCF2 H389Q mutation reduces the binding efficiency of NCF2 with the guanine nucleotide exchange factor Vav1. The model predicts that NCF2/H389 residue interacts with Vav1 residues E509, N510, E556, and G559 in the ZF domain of Vav1. Furthermore, replacing H389 with Q results in 1.5 kcal/mol weaker binding. To examine the effect of the NCF2 H389Q mutation on NADPH oxidase function, site-specific mutations at the 389 position in NCF2 were tested. Results show that an H389Q mutation causes a twofold decrease in reactive oxygen species production induced by the activation of the Vav-dependent Fcγ receptor-elicited NADPH oxidase activity. Our study completes the chain of evidence from genetic association to specific molecular function.

Download full-text


Available from: Andreas Reiff,
  • Source
    • "In humans and mice alike, NOX2 mutation in gp91 phox results in X-linked chronic granulomatous disease, which renders the patients simultaneously prone to serious infections by microbial pathogens, sterile chronic inflammation, and occasionally SLE (Campbell et al., 2012; Sareila et al., 2011). Consistently, a mutation in neutrophil cytosolic factor 2 (NCF2), which results in a reduction of Nox2 activity and ROS production, confers substantially increased SLE (Jacob et al., 2012). Interestingly, rodents bearing Ncf1 mutation are susceptible to autoimmune arthritis (Hultqvist et al., 2004). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Here, we examine the mechanism by which plasmacytoid dendritic cells (pDCs) and type I interferons promote humoral autoimmunity. In an amyloid-induced experimental autoimmune model, neutrophil depletion enhanced anti-nuclear antibody development, which correlated with heightened IFN-γ production by natural killer (NK) cells. IFN-α/β produced by pDCs activated NK cells via IL-15 induction. Neutrophils released reactive oxygen species (ROS), which negatively modulated the levels of IL-15, thereby inhibiting IFN-γ production. Mice deficient in NADPH oxidase 2 produced increased amounts of IFN-γ and developed augmented titers of autoantibodies. Both the pDC-IFN-α/β pathway and IFN-γ were indispensable in stimulating humoral autoimmunity. Male NZB/W F1 mice expressed higher levels of superoxide than their female lupus-prone siblings, and depletion of neutrophils resulted in spontaneous NK cell and autoimmune B cell activation. Our findings suggest a regulatory role for neutrophils in vivo and highlight the importance of an NK-IFN-γ axis downstream of the pDC-IFN-α/β pathway in systemic autoimmunity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 08/2015; 12(7). DOI:10.1016/j.celrep.2015.07.021 · 8.36 Impact Factor
  • Source
    • "In addition to this well-established connection between CGD and autoimmunity, genes encoding oxidase subunits have been associated with autoinflammatory and autoimmune disorders in genome-wide association studies (GWAS). A variation in NCF2, the gene encoding p67phox, has been identified as an important risk factor for SLE (18). NCF4, encoding p40phox, has been associated with rheumatoid arthritis (19) and Crohn’s disease (20, 21). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expressed in phagocytes is a multi-subunit enzyme complex that generates superoxide (O2 (.-)). This radical is an important precursor of hydrogen peroxide (H2O2) and other reactive oxygen species needed for microbicidal activity during innate immune responses. Inherited defects in NADPH oxidase give rise to chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent infections and granulomatous inflammation. Interestingly, CGD, CGD carrier status, and oxidase gene polymorphisms have all been associated with autoinflammatory and autoimmune disorders, suggesting a potential role for NADPH oxidase in regulating adaptive immune responses. Here, NADPH oxidase function in antigen processing and presentation is reviewed. NADPH oxidase influences dendritic cell (DC) crosspresentation by major histocompatibility complex class I molecules through regulation of the phagosomal microenvironment, while in B lymphocytes, NADPH oxidase alters epitope selection by major histocompatibility complex class II molecules.
    Frontiers in Immunology 09/2013; 4:295. DOI:10.3389/fimmu.2013.00295
  • Source
    • "We also found that B6.Sle2c2 myeloid cells and granulocytes produced more ROS at steady-state than B6 cells. A recent study found a strong association between a coding mutation in the human NCF2 gene with SLE susceptibility, and determined that the disease associated allele was associated with a lower ROS production [24]. This is consistent with decreased levels of Ncf1, a gene encoding for another unit of the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase complex, being associated with collagen-induced arthritis [25] and experimental allergic encephalomyelitis [26]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Introduction An NZB-derived genetic locus (Sle2c2) that suppresses autoantibody production in a mouse model of induced systemic lupus erythematosus contains a polymorphism in the gene encoding the G-CSF receptor. This study was designed to test the hypothesis that the Sle2c2 suppression is associated with an impaired G-CSF receptor function that can be overcome by exogenous G-CSF. Methods Leukocytes from B6.Sle2c2 and B6 congenic mice, which carry a different allele of the G-CSF receptor, were compared for their responses to G-CSF. Autoantibody production was induced with the chronic graft-versus-host-disease (cGVHD) model by adoptive transfer of B6.bm12 splenocytes. Different treatment regimens varying the amount and frequency of G-CSF (Neulasta®) or carrier control were tested on cGVHD outcomes. Autoantibody production, immune cell activation, and reactive oxygen species (ROS) production were compared between the two strains with the various treatments. In addition, the effect of G-CSF treatment was examined on the production autoantibodies in the B6.Sle1.Sle2.Sle3 (B6.TC) spontaneous model of lupus. Results B6.Sle2c2 and B6 leukocytes responded differently to G-CSF. G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment. G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils. In contrast, the expression of G-CSF responsive genes indicated a higher G-CSF receptor signaling in B6.Sle2c2 cells. G-CSF treatment restored the ability of B6.Sle2c2 mice to produce autoantibodies in a dose-dependent manner upon cGVHD induction, which correlated with restored CD4+ T cells activation, as well as dendritic cell and granulocyte expansion. Steady-state ROS production was higher in B6.Sle2c2 than in B6 mice. cGVHD induction resulted in a larger increase in ROS production in B6 than in B6.Sle2c2 mice, and this difference was eliminated with G-CSF treatment. Finally, a low dose G-CSF treatment accelerated the production of anti-dsDNA IgG in young B6.TC mice. Conclusion The different in vivo and in vitro responses of B6.Sle2c2 leukocytes are consistent with the mutation in the G-CSFR having functional consequences. The elimination of Sle2c2 suppression of autoantibody production by exogenous G-CSF indicates that Sle2c2 corresponds to a loss of function of G-CSF receptor. This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus. Overall, these results suggest that the G-CSF pathway regulates the production of autoantibodies in murine models of lupus.
    Arthritis research & therapy 04/2013; 15(2):R49. DOI:10.1186/ar4208 · 3.75 Impact Factor
Show more