Vitamin D₃ and carbamazepine protect against <i>Clostridioides difficile</i> infection in mice by restoring macrophage lysosome acidification

<i>Clostridioides difficile</i> infection (CDI) is a common cause of nosocomial diarrhea. TcdB is a major <i>C. difficile</i> exotoxin that activates macrophages to promote inflammation and epithelial damage. Lysosome impairment is a known trigger for inflammation. Herein, we hypothesize that TcdB could impair macrophage lysosomal function to mediate inflammation during CDI. Effects of TcdB on lysosomal function and the downstream pro-inflammatory SQSTM1/p62-NFKB (nuclear factor kappa B) signaling were assessed in cultured macrophages and in a murine CDI model. Protective effects of two lysosome activators (i.e., vitamin D₃ and carbamazepine) were assessed. Results showed that TcdB inhibited CTNNB1/β-catenin activity to downregulate MITF (melanocyte inducing transcription factor) and its direct target genes encoding components of lysosomal membrane vacuolar-type ATPase, thereby suppressing lysosome acidification in macrophages. The resulting lysosomal dysfunction then impaired autophagic flux and activated SQSTM1-NFKB signaling to drive the expression of IL1B/IL-1β (interleukin 1 beta), IL8 and CXCL2 (chemokine (C-X-C motif) ligand 2). Restoring MITF function by enforced MITF expression or restoring lysosome acidification with 1α,25-dihydroxyvitamin D₃ or carbamazepine suppressed pro-inflammatory cytokine expression <i>in vitro</i>. In mice, gavage with TcdB-hyperproducing <i>C. difficile</i> or injection of TcdB into ligated colon segments caused prominent MITF downregulation in macrophages. Vitamin D₃ and carbamazepine lessened TcdB-induced lysosomal dysfunction, inflammation and histological damage. In conclusion, TcdB inhibits the CTNNB1-MITF axis to suppress lysosome acidification and activates the downstream SQSTM1-NFKB signaling in macrophages during CDI. Vitamin D₃ and carbamazepine protect against CDI by restoring MITF expression and lysosomal function in mice. <b>Abbreviations:</b> ATP6V0B: ATPase H+ transporting V0 subunit b; ATP6V0C: ATPase H+ transporting V0 subunit c; ATP6V0E1: ATPase H+ transporting V0 subunit e1; ATP6V1H: ATPase H+ transporting V1 subunit H; CBZ: carbamazepine; CDI: <i>C. difficile</i> infection; CXCL: chemokine C-X-X motif ligand; IL: interleukin; LAMP1: lysosomal-associated membrane protein 1; LC3: microtubule-associated protein 1 light chain 3; LEF: lymphoid enhancer binding factor 1; MITF: melanocyte inducing transcription factor; NFKB: nuclear factor kappa B; PMA: phorbol 12-myristate 13-acetate; TcdA: Clostridial toxin A; TcdB: Clostridial toxin B; TFE3: transcription factor E3; TFEB: transcription factor EB.艰难梭菌（Clostridioides difficile）感染（CDI）是医院获得性腹泻的常见病因。TcdB是艰难梭菌（C. difficile）的主要外毒素，可激活巨噬细胞以促进炎症反应与上皮损伤。溶酶体功能障碍是已知的炎症触发因素。本研究假设，在艰难梭菌感染进程中，TcdB可能通过损伤巨噬细胞溶酶体功能介导炎症反应。 本研究在培养的巨噬细胞与小鼠CDI模型中，评估了TcdB对溶酶体功能及下游促炎SQSTM1/p62-核因子κB（NFKB）信号通路的影响，并检测了两种溶酶体激活剂——维生素D₃与卡马西平的保护作用。 实验结果显示，TcdB可抑制β-连环蛋白（CTNNB1/β-catenin）活性，下调黑素细胞诱导转录因子（MITF）及其编码溶酶体膜空泡型ATP酶组分的直接靶基因，从而抑制巨噬细胞的溶酶体酸化。由此引发的溶酶体功能障碍进而损伤自噬流，并激活SQSTM1-NFKB信号通路，驱动白细胞介素1β（IL1B/IL-1β）、IL8及趋化因子C-X-C基序配体2（CXCL2）的表达。通过强制过表达MITF以恢复其功能，或使用1α,25-二羟基维生素D₃、卡马西平恢复溶酶体酸化，均可在体外抑制促炎细胞因子的表达。 在小鼠体内，灌胃产TcdB能力强的艰难梭菌，或向结扎的结肠段注射TcdB，均可导致巨噬细胞中MITF显著下调。维生素D₃与卡马西平可减轻TcdB诱导的溶酶体功能障碍、炎症反应与组织学损伤。 综上，在艰难梭菌感染进程中，TcdB通过抑制CTNNB1-MITF轴以削弱溶酶体酸化，并激活巨噬细胞内下游的SQSTM1-NFKB信号通路。维生素D₃与卡马西平可通过恢复小鼠体内的MITF表达与溶酶体功能，对CDI起到保护作用。 **缩写说明：** ATP6V0B：ATP酶H+转运V0亚基b；ATP6V0C：ATP酶H+转运V0亚基c；ATP6V0E1：ATP酶H+转运V0亚基e1；ATP6V1H：ATP酶H+转运V1亚基H；CBZ：卡马西平；CDI：艰难梭菌感染；CXCL：趋化因子C-X-C基序配体；IL：白细胞介素；LAMP1：溶酶体相关膜蛋白1；LC3：微管相关蛋白1轻链3；LEF：淋巴增强因子结合蛋白1；MITF：黑素细胞诱导转录因子；NFKB：核因子κB；PMA：佛波醇12-肉豆蔻酸酯13-乙酸酯；TcdA：梭菌毒素A；TcdB：梭菌毒素B；TFE3：转录因子E3；TFEB：转录因子EB。

搜集汇总

背景与挑战

背景概述

该数据集支持一项研究，探讨维生素D3和卡马西平如何通过恢复巨噬细胞溶酶体酸化来保护小鼠免受艰难梭菌感染。研究揭示了毒素TcdB通过抑制CTNNB1-MITF轴导致溶酶体功能障碍和炎症，而两种药物能逆转这一过程。数据集包含原始数据或补充材料，覆盖生物化学、微生物学等多个学科领域。