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

Clostridioides difficile infection (CDI) is a common cause of nosocomial diarrhea. TcdB is a major C. difficile 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 in vitro. In mice, gavage with TcdB-hyperproducing C. difficile 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. Abbreviations: 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: C. difficile 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.

艰难梭菌感染（Clostridioides difficile infection, CDI）是医院获得性腹泻的常见病因。TcdB是艰难梭菌的主要外毒素，可激活巨噬细胞以促进炎症反应与上皮损伤。溶酶体功能受损是已知的炎症触发因素。本研究假设，在CDI病程中，TcdB可通过损伤巨噬细胞溶酶体功能来介导炎症反应。
本研究在体外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性SQSTM1/p62-核因子κB（NFKB）信号通路的影响，并考察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CTNNB1/β-连环蛋白（β-catenin）活性，下调黑素细胞诱导转录因子（MITF，melanocyte inducing transcription factor）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过程。由此引发的溶酶体功能障碍进而损伤自噬流，并激活SQSTM1/p62-NFKB信号通路，驱动白细胞介素1β（IL1B/IL-1β，interleukin 1 beta）、IL8及趋化因子（C-X-C基序）配体2（CXCL2，chemokine (C-X-C motif) ligand 2）的表达。
通过强制表达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性细胞因子的表达。
在小鼠体内，经口灌胃给予产TcdB能力强的艰难梭菌，或向结扎结肠段注射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CDI病程中，TcdB可通过抑制CTNNB1-MITF轴以削弱溶酶体酸化，并激活巨噬细胞内的下游SQSTM1/p62-NFKB信号通路。维生素D3与卡马西平可通过恢复小鼠体内MITF表达与溶酶体功能，从而对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（carbamazepine）；
CDI：艰难梭菌感染（Clostridioides difficile infection）；
CXCL：趋化因子C-X-X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（melanocyte inducing transcription factor）；
NFKB：核因子κB（nuclear factor kappa B）；
PMA：佛波醇12-肉豆蔻酸酯13-乙酸酯；
TcdA：梭菌毒素A（Clostridial toxin A）；
TcdB：梭菌毒素B（Clostridial toxin B）；
TFE3：转录因子E3；
TFEB：转录因子EB。