A leaky human colon model reveals uncoupled apical/basal cytotoxicity in early Clostridioides difficile toxin exposure
A leaky human colon model reveals uncoupled apical/basal cytotoxicity in early Clostridioides difficile toxin exposure
复制标题
渗漏的人类结肠模型揭示了早期艰难梭菌毒素暴露中不偶联的顶端/基底细胞毒性
DOI：
10.1152/ajpgi.00251.2022
复制
发表时间：
2023
影响因子：
4.5
通讯作者：
Magness, Scott T.
中科院分区：
文献类型：
--
作者：
Ok, Meryem T.;Liu, Jintong;Bliton, R. Jarrett;Hinesley, Caroline M.;San Pedro, Ekaterina Ellyce;Breau, Keith A.;Gomez-Martinez, Ismael;Burclaff, Joseph;Magness, Scott T.
艰难梭菌（C.艰难梭菌）毒素A（TcdA）和B（TcdB）部分通过破坏上皮屏障功能而引起结肠炎相关性结肠炎。准确的体外模型对于检测早期毒性动力学、研究疾病病因和开发新疗法的临床前模型是必要的。癌细胞系和类器官的性质固有地限制了这些努力。我们开发了具有屏障功能的分化的人结肠上皮（hCE）的成体干细胞衍生单层，研究了毒素对单层顶端/基底面的影响，并评估了泄漏的上皮屏障是否会增强毒性。单细胞RNA测序（scRNAseq）定位C.与人类谱系相关的艰难基因。转录组学比较hCE与Caco-2，告知体外干细胞分化的时间，并揭示对TcdA的转录应答。跨上皮电阻（TEER）和荧光渗透性测定测量细胞毒性。在双氯芬酸诱导的肠漏模型中评估了TcdB毒性的贡献。scRNAseq证明了广泛和可变的毒素受体表达。体内吸收的结肠细胞显示毒素受体、Rho GT3和细胞连接基因表达增加。晚期TcdA毒性通常降低细胞因子/趋化因子，增加紧密连接和死亡受体基因。分化的Caco-2细胞保持不成熟，而hCE单层在体内类似于成熟的结肠细胞。TcdA/B基底暴露比顶暴露引起更大的毒性和细胞凋亡。双氯芬酸可增强根尖对毒素的暴露。顶端/基础毒性与更快速的发作和基础毒素暴露后增加的幅度脱钩。渗漏连接增强顶端TcdB暴露的毒性。hCE单层代表了一个生理相关的和敏感的系统，以评估微生物毒素对肠道上皮的影响。新&值得注意的新的人类结肠细胞单层培养，以转录组学为基准，用于生理相关性，检测艰难梭菌毒素TcdA和TcdB的早期细胞病变影响。原代人结肠细胞中的荧光ZO-1报告基因用于跟踪响应于TcdA的上皮屏障破坏。基底TcdA/B暴露通常比顶端暴露引起更快的发作和细胞毒性。转录组学显示TcdA暴露后紧密连接、趋化因子和细胞因子受体基因表达的变化。Diclethac-induced渗漏上皮增强根尖暴露毒性。
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.