泄漏的人类结肠模型揭示早期Clostridioides difficile毒素暴露中的非耦合性顶端/基底细胞毒性

Clostridioides difficile（C. difficile）毒素A（TcdA）和B（TcdB）通过破坏上皮屏障功能，部分引起抗生素相关性结肠炎。准确的体外模型对于检测早期毒性动力学、研究疾病发病机制及开发新疗法的临床前模型至关重要。癌细胞系和类器官的固有特性限制了这些研究。我们开发了成人干细胞来源的分化成人结肠上皮单层（hCE），具有屏障功能，研究了毒素对单层的顶端/基底方面的影响，并评估了渗漏上皮屏障是否增强毒性。单细胞RNA测序（scRNAseq）将与C. difficile相关的基因映射到人类细胞谱系。转录组学比较了hCE与Caco-2细胞，指导体外干细胞分化的时间，并揭示了对TcdA的转录反应。跨上皮电阻（TEER）和荧光渗透性检测评估细胞毒性。在二氯苯胺引发的渗漏肠道模型中评估了TcdB毒性的贡献。scRNAseq显示受体表达广泛且变化多样。体内观察到的吸收性结肠细胞表现出较高的毒素受体、Rho GTP酶和细胞连接基因表达。先进的TcdA毒性反应通常降低细胞因子/趋化因子表达，增加紧密连接蛋白和死亡受体基因表达。分化的Caco-2细胞保持不成熟，而hCE单层类似成熟的结肠细胞。基底暴露TcdA/B引起的毒性和凋亡大于顶端暴露。顶端暴露毒素通过二氯苯胺增强。顶端/基底毒性表现出非耦合，基底毒素暴露后反应更快且程度更大。渗漏连接增强了顶端TcdB暴露的毒性。hCE单层代表一种生理相关且敏感的系统，用于评估微生物毒素对肠道上皮的影响。新颖的人结肠细胞单层培养系统，通过转录组学验证其生理相关性，能早期检测Clostridioides difficile毒素TcdA和TcdB的细胞病变影响。利用原代人结肠细胞中的荧光ZO-1报告器追踪TcdA引起的上皮屏障破坏。基底TcdA/B暴露通常比顶端暴露更早引发细胞毒性。转录组学显示TcdA暴露后紧密连接、趋化因子及细胞因子受体基因表达发生变化。二氯苯胺引发的渗漏上皮增强了顶端暴露的毒性。

Clostridioides difficile (C. difficile) toxins A (TcdA) and B (TcdB) cause antibiotic-associated colitis in part by disrupting epithelial barrier function. Accurate in vitro models are necessary to detect early toxicity kinetics, investigate disease etiology, and develop preclinical models for new therapies. Properties of cancer cell lines and organoids inherently limit these efforts. We developed adult stem cell-derived monolayers of differentiated human colonic epithelium (hCE) with barrier function, investigated the impact of toxins on apical/basal aspects of monolayers, and evaluated whether a leaky epithelial barrier enhances toxicity. Single-cell RNA-sequencing (scRNAseq) mapped C. difficile-relevant genes to human lineages. Transcriptomics compared hCE to Caco-2, informed timing of in vitro stem cell differentiation, and revealed transcriptional responses to TcdA. Transepithelial electrical resistance (TEER) and fluorescent permeability assays measured cytotoxicity. Contribution of TcdB toxicity was evaluated in a diclofenac-induced leaky gut model. scRNAseq demonstrated broad and variable toxin receptor expression. Absorptive colonocytes in vivo displayed increased toxin receptor, Rho GTPase, and cell junction gene expression. Advanced TcdA toxicity generally decreased cytokine/chemokine and increased tight junction and death receptor genes. Differentiated Caco-2 cells remained immature whereas hCE monolayers were similar to mature colonocytes in vivo. Basal exposure of TcdA/B caused greater toxicity and apoptosis than apical exposure. Apical exposure to toxins was enhanced by diclofenac. Apical/basal toxicities are uncoupled with more rapid onset and increased magnitude postbasal toxin exposure. Leaky junctions enhance toxicity of apical TcdB exposure. hCE monolayers represent a physiologically relevant and sensitive system to evaluate the impact of microbial toxins on gut epithelium.NEW & NOTEWORTHY Novel human colonocyte monolayer cultures, benchmarked by transcriptomics for physiological relevance, detect early cytopathic impacts of Clostridioides difficile toxins TcdA and TcdB. A fluorescent ZO-1 reporter in primary human colonocytes is used to track epithelial barrier disruption in response to TcdA. Basal TcdA/B exposure generally caused more rapid onset and cytotoxicity than apical exposure. Transcriptomics demonstrate changes in tight junction, chemokine, and cytokine receptor gene expression post-TcdA exposure. Diclofenac-induced leaky epithelium enhanced apical exposure toxicity.