The NCF1 variant p.R90H aggravates autoimmunity by facilitating the activation of plasmacytoid dendritic cells
Yao Meng, Jianyang Ma, Chao Yao, Zhizhong Ye, Huihua Ding, Can Liu, Jun Li, Guanhua Li, Yuke He, Jia Li, Zhihua Yin, Li Wu, Haibo Zhou, Nan Shen
Yao Meng, Jianyang Ma, Chao Yao, Zhizhong Ye, Huihua Ding, Can Liu, Jun Li, Guanhua Li, Yuke He, Jia Li, Zhihua Yin, Li Wu, Haibo Zhou, Nan Shen
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Research Article Autoimmunity

The NCF1 variant p.R90H aggravates autoimmunity by facilitating the activation of plasmacytoid dendritic cells

Abstract

Plasmacytoid dendritic cells (pDCs) are a professional type I IFN producer that play critical roles in the pathogenesis of autoimmune diseases. However, both genetic regulation of the function of pDCs and their relationships with autoimmunity are largely undetermined. Here, we investigated the causality of the neutrophil cytosolic factor 1 (NCF1) missense variant, which is one of the most significant associated risk variants for lupus, and found that the substitution of arginine (R) for histidine (H) at position 90 in the NCF1 protein (NCF1 p.R90H) led to excessive activation of pDCs. A mechanism study demonstrated that p.R90H reduced the affinity of NCF1 for phospholipids, thereby impairing endosomal localization of NCF1. As NCF1 is a subunit of the NADPH oxidase 2 (NOX2) complex, this impairment led to an acidified endosomal pH and facilitated downstream TLR signaling. Consistently, the homozygous knockin mice manifested aggravated lupus progression in a pDC-dependent lupus model. More important, pharmaceutical intervention revealed that hydroxychloroquine (HCQ) could antagonize the detrimental function of NCF1 p.R90H in the lupus model and systemic lupus erythematosus samples, supporting the idea that NCF1 p.R90H could be identified as a genetic biomarker for HCQ application. Therefore, our study provides insights into the genetic control of pDC function and a paradigm for applying genetic variants to improve targeted therapy for autoimmune diseases.

Authors

Yao Meng, Jianyang Ma, Chao Yao, Zhizhong Ye, Huihua Ding, Can Liu, Jun Li, Guanhua Li, Yuke He, Jia Li, Zhihua Yin, Li Wu, Haibo Zhou, Nan Shen

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Figure 2

NCF1 p.R90H impairs endosomal localization of NCF1 and acidifies the endosomal pH.

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NCF1 p.R90H impairs endosomal localization of NCF1 and acidifies the end...
pDCs were stimulated with CpG for 2 hours. (A) Colocalization of NCF1 (green) and EEA1 (red) was detected by confocal microscopy and analyzed by ImageJ. Scale bar: 5 μm. One data point in the plot indicates the mean of Pearson’s R values from 5 cells, and a total of 50 cells per group were calculated . (B) The pH of late endosomes/lysosomes in CpG-activated pDCs was indicated by a lysosome sensor, detected by confocal microscopy, and measured by ImageJ. Scale bar: 10 μm. One data point in the plot indicates the average MFI of 5 cells, and a total of 50 cells per group were calculated. (C) Immunoblot analysis of cleavage of TLR7 and TLR9. G/G and A/A pDCs were stimulated with R848 or 5 μM CpG for 1 hour, respectively. The levels of intact TLR7 and the cleaved TLR7 fragment (TLR7-F) in R848-treated groups and the levels of intact TLR9 and the cleaved TLR9 fragment (TLR9-F) in the CpG-treated groups were analyzed. The ratios of cleaved TLR to intact TLR were calculated. (D) Structure of the PX domain of NCF1 90R and 90H. R90 and R43 are amino acids responsible for PtdIns(3,4)p2 binding. (E) Immunoblot analysis of GST-NCF1. S, protein in supernatant, P, protein in precipitation. The P/S ratios were calculated. Experiments were repeated 3 times. Error bars represent the SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed, unpaired t test (A, B, and E) or 1-way ANOVA with Tukey’s multiple-comparison test (C).