Table1_Lupus-associated NCF2 variant p.R395W in the NADPH oxidase 2 complex results in a reduced production of reactive oxygen species by myeloid cells.docx

The leukocyte NADPH oxidase 2 (NOX2) generates superoxide, and derivative reactive oxygen species play important roles in both host defense and immunoregulation. The rs13306575 genetic variant, resulting in an Arginine395→Tryptophan (R395W) substitution in the NOX2 NCF2 subunit, is associated with an increased risk of lupus in patients of Hispanic-American or of Korean ancestry. Arginine395 resides within the NCF2 PB1 domain and participates in a constitutive high-affinity interaction with the NOX2 NCF4 subunit to stabilize their expression. However, whether this variant impacts NCF2 function and NOX2 activity is unknown. To answer this question, mice expressing NCF2-R395W were generated using CRISPR/Cas9. NCF2 and NCF4 expression were reduced by twofold in neutrophils, macrophages, and dendritic cells homozygous for NCF2-R395W. Moreover, following stimulation with soluble or particulate stimuli, reactive oxygen species production at the plasma membrane and within cells was reduced in all three myeloid lineages expressing NCF2-R395W. Additional studies on Ncf2+/− mice, which have a reduced expression of wild-type NCF2 but not of NCF4, suggest that the reduced expression of both NCF2 and NCF4 contributes to the diminished NOX2 activity in NCF2-R395 mice. These results establish that the lupus-associated rs13306575 p.R395W allele is a functional hypomorph. The findings add to growing evidence implicating deficient NOX2 activity in the pathogenesis of lupus.

白细胞NADPH氧化酶2（NOX2）产生超氧化物，其衍生的活性氧物种在宿主防御和免疫调节中发挥着重要作用。遗传变异rs13306575，导致NOX2 NCF2亚基中Arginine395→Tryptophan（R395W）的替换，与西班牙裔美国人或韩国血统的患者患系统性红斑狼疮的风险增加相关。Arginine395位于NCF2 PB1结构域内，并参与与NOX2 NCF4亚基的构成性高亲和力相互作用，以稳定其表达。然而，此变异是否影响NCF2功能及NOX2活性尚不明确。为解答此问题，利用CRISPR/Cas9技术构建了表达NCF2-R395W的小鼠模型。在NCF2-R395W同型纯合的嗜中性粒细胞、巨噬细胞和树突状细胞中，NCF2和NCF4的表达量减少了一倍。此外，在受到可溶性或颗粒性刺激后，表达NCF2-R395W的三个髓系谱系细胞在细胞膜和细胞内产生的活性氧物种减少。针对Ncf2+/−小鼠的进一步研究，该小鼠野生型NCF2表达量降低，但NCF4未受影响，表明NCF2和NCF4表达量的降低共同导致了NCF2-R395小鼠中NOX2活性的下降。这些研究结果确立了与狼疮相关的rs13306575 p.R395W等位基因是一个功能低等位基因。这些发现进一步证实了NOX2活性不足在狼疮发病机制中的作用。