NRBF2, a regulatory subunit of the ATG14-BECN1/Beclin 1-PIK3C3/VPS34 complex, positively regulates macroautophagy/autophagy. In this study, we report that NRBF2 is required for the clearance of apoptotic cells and alleviation of inflammation during colitis in mice. NRBF2-deficient mice displayed much more severe colitis symptoms after the administration of ulcerative colitis inducer, dextran sulfate sodium salt (DSS), accompanied by prominent intestinal inflammation and apoptotic cell accumulation. Interestingly, we found that nrbf2^−/- mice and macrophages displayed impaired apoptotic cell clearance capability, while adoptive transfer of nrbf2^+/+ macrophages to nrbf2^−/- mice alleviated DSS-induced colitis lesions. Mechanistically, NRBF2 is required for the generation of the active form of RAB7 to promote the fusion between phagosomes containing engulfed apoptotic cells and lysosomes via interacting with the MON1-CCZ1 complex and regulating the guanine nucleotide exchange factor (GEF) activity of the complex. Evidence from clinical samples further reveals the physiological role of NRBF2 in maintaining intestinal homeostasis. In biopsies of UC patient colon, we observed upregulated NRBF2 in the colon macrophages and the engulfment of apoptotic cells by NRBF2-positive cells, suggesting a potential protective role for NRBF2 in UC. To confirm the relationship between apoptotic cell clearance and IBD development, we compared TUNEL-stained cell counts in the UC with UC severity (Mayo Score) and observed a strong correlation between the two indexes, indicating that apoptotic cell population in colon tissue correlates with UC severity. The findings of our study reveal a novel role for NRBF2 in regulating apoptotic cell clearance to restrict intestinal inflammation.

Abbreviation: ANOVA: analysis of variance; ATG14: autophagy related 14; ATG16L1: autophagy related 16-like 1 (S. cerevisiae); BMDM: bone marrow-derived macrophage; BSA: bovine serum albumin; CD: Crohn disease; CD68: CD68 molecule; CFP: cyan fluorescent protein; CMFDA: 5-chloromethylfluorescein diacetate; Co-IP, co-immunoprecipitation; CPR: C-reactive protein; Cy7: cyanine 7 maleimide; DAB: diaminobezidine 3; DAI: disease activity indexes; DAPI: 4ʹ6-diamidino-2-phenylindole; DMEM: dulbecco’s modified eagle’s medium; DMSO: dimethyl sulfoxide; DOC: sodium deoxycholate; DSS: dextran sulfate sodium; EDTA: ethylenediaminetetraacetic acid; EGTA: ethylenebis (oxyethylenenitrilo) tetraacetic acid; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; FRET: Förster resonance energy transfer; GDP: guanine dinucleotide phosphate; GEF: guanine nucleotide exchange factor; GFP: green fluorescent protein; GTP: guanine trinucleotide phosphate; GWAS: genome-wide association studies; HEK293: human embryonic kidney 293 cells; HRP: horseradish peroxidase; IBD: inflammatory bowel disease; IgG: immunoglobin G; IL1B/IL-1β: interleukin 1 beta; IL6: interleukin 6; IRGM: immunity related GTPase M; ITGAM/CD11b: integrin subunit alpha M; KO: knockout; LRRK2: leucine rich repeat kinase 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MPO: myeloperoxidase; NaCl: sodium chloride; NEU: neutrophil; NOD2: nucleotide binding oligomerization domain containing 2; NP40: nonidet-P40; NRBF2: nuclear receptor binding factor 2; PBS: phosphate buffer saline; PCR: polymerase chain reaction; PE: P-phycoerythrin; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PTPRC/CD45: protein tyrosine phosphatase receptor type C; SDS-PAGE: sodium dodecylsulphate-polyacrylamide gel electrophoresis; TBST: tris-buffered saline Tween-20; Tris-HCl: trihydroxymethyl aminomethane hydrochloride; TUNEL: TdT-mediated dUTP nick-end labeling; UC: ulcerative colitis; ULK1: unc-51 like autophagy activating kinase 1; WB: western blotting; WT: wild type; YFP: yellow fluorescent protein.

NRBF2 是 ATG14-BECN1/Beclin 1-PIK3C3/VPS34 复合物的调节亚基，正向调节巨自噬/自噬。在这项研究中，我们报告说，NRBF2 是清除小鼠结肠炎期间凋亡细胞和减轻炎症所必需的。NRBF2缺陷小鼠在给予溃疡性结肠炎诱导剂硫酸葡聚糖钠盐（DSS）后表现出更严重的结肠炎症状，并伴有明显的肠道炎症和凋亡细胞积聚。有趣的是，我们发现nrbf2 ^-/-小鼠和巨噬细胞表现出受损的凋亡细胞清除能力，而nrbf2 ^+/+巨噬细胞过继转移至nrbf2 ^-/-小鼠可减轻DSS 诱导的结肠炎病变。从机制上讲，NRBF2 是生成 RAB7 活性形式所必需的，通过与 MON1-CCZ1 复合物相互作用并调节复合物的鸟嘌呤核苷酸交换因子 (GEF) 活性，促进含有吞噬凋亡细胞的吞噬体与溶酶体之间的融合。来自临床样本的证据进一步揭示了NRBF2在维持肠道稳态中的生理作用。在 UC 患者结肠活检中，我们观察到结肠巨噬细胞中 NRBF2 上调，并且 NRBF2 阳性细胞吞噬凋亡细胞，表明 NRBF2 在 UC 中具有潜在的保护作用。为了证实凋亡细胞清除率与IBD发展之间的关系，我们将UC中TUNEL染色的细胞计数与UC严重程度（Mayo评分）进行比较，并观察到这两个指标之间存在很强的相关性，表明结肠组织中的凋亡细胞群与UC相关严重程度。我们的研究结果揭示了 NRBF2 在调节凋亡细胞清除以限制肠道炎症方面的新作用。

缩写： ANOVA：方差分析；ATG14：自噬相关14；ATG16L1：自噬相关 16-like 1（酿酒酵母）；BMDM：骨髓源性巨噬细胞；BSA：牛血清白蛋白；CD：克罗恩病；CD68：CD68分子；CFP：青色荧光蛋白；CMFDA：5-氯甲基荧光素二乙酸酯；Co-IP，免疫共沉淀；CPR：C反应蛋白；Cy7：花青7马来酰亚胺；DAB：二氨基联苯胺3；DAI：疾病活动指数；DAPI：4ʹ6-二脒基-2-苯基吲哚；DMEM：杜尔贝科改良鹰培养基；DMSO：二甲亚砜；DOC：脱氧胆酸钠；DSS：葡聚糖硫酸钠；EDTA：乙二胺四乙酸；EGTA：亚乙基双(氧亚乙基次氮基)四乙酸；FBS：胎牛血清；FITC：异硫氰酸荧光素；FRET：福斯特共振能量转移；GDP：鸟嘌呤二核苷酸磷酸；GEF：鸟嘌呤核苷酸交换因子；GFP：绿色荧光蛋白；GTP：鸟嘌呤三核苷酸磷酸；GWAS：全基因组关联研究；HEK293：人胚肾293细胞；HRP：辣根过氧化物酶；IBD：炎症性肠病；IgG：免疫球蛋白G；IL1B/IL-1β：白细胞介素1β；IL6：白细胞介素6；IRGM：免疫相关GTPase M；ITGAM/CD11b：整合素亚基 α M；KO：淘汰赛；LRRK2：富含亮氨酸的重复激酶2；MAP1LC3/LC3：微管相关蛋白1轻链3；MOI：感染复数；MPO：髓过氧化物酶；氯化钠：氯化钠；NEU：中性粒细胞；NOD2：含有2个的核苷酸结合寡聚化结构域；NP40：nonidet-P40；NRBF2：核受体结合因子2；PBS：磷酸盐缓冲液；PCR：聚合酶链式反应；PE：P-藻红蛋白；PIK3C3/VPS34：磷脂酰肌醇3-激酶催化亚基3型；PtdIns3P：3-磷酸磷脂酰肌醇；PTPRC/CD45：C型蛋白酪氨酸磷酸酶受体；SDS-PAGE：十二烷基硫酸钠-聚丙烯酰胺凝胶电泳；TBST：tris缓冲盐水Tween-20；Tris-HCl：三羟甲基氨基甲烷盐酸盐；TUNEL：TdT 介导的 dUTP 缺口末端标记；UC：溃疡性结肠炎；ULK1：unc-51 样自噬激活激酶 1；WB：蛋白质印迹；WT：野生型；YFP：黄色荧光蛋白。