Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel; Immunology Department, Weizmann Institute of Science, Rehovot, Israel.
Immunology Department, Weizmann Institute of Science, Rehovot, Israel; Department of Gastroenterology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
Gastroenterology. 2020 Nov;159(5):1807-1823. doi: 10.1053/j.gastro.2020.07.003. Epub 2020 Jul 9.
BACKGROUND & AIMS: The intestinal barrier protects intestinal cells from microbes and antigens in the lumen-breaches can alter the composition of the intestinal microbiota, the enteric immune system, and metabolism. We performed a screen to identify molecules that disrupt and support the intestinal epithelial barrier and tested their effects in mice.
We performed an imaging-based, quantitative, high-throughput screen (using CaCo-2 and T84 cells incubated with lipopolysaccharide; tumor necrosis factor; histamine; receptor antagonists; and libraries of secreted proteins, microbial metabolites, and drugs) to identify molecules that altered epithelial tight junction (TJ) and focal adhesion morphology. We then tested the effects of TJ stabilizers on these changes. Molecules we found to disrupt or stabilize TJs were administered mice with dextran sodium sulfate-induced colitis or Citrobacter rodentium-induced intestinal inflammation. Colon tissues were collected and analyzed by histology, fluorescence microscopy, and RNA sequencing.
The screen identified numerous compounds that disrupted or stabilized (after disruption) TJs and monolayers of epithelial cells. We associated distinct morphologic alterations with changes in barrier function, and identified a variety of cytokines, metabolites, and drugs (including inhibitors of actomyosin contractility) that prevent disruption of TJs and restore TJ integrity. One of these disruptors (putrescine) disrupted TJ integrity in ex vivo mouse colon tissues; administration to mice exacerbated colon inflammation, increased gut permeability, reduced colon transepithelial electrical resistance, increased pattern recognition receptor ligands in mesenteric lymph nodes, and decreased colon length and survival times. Putrescine also increased intestine levels and fecal shedding of viable C rodentium, increased bacterial attachment to the colonic epithelium, and increased levels of inflammatory cytokines in colon tissues. Colonic epithelial cells from mice given putrescine increased expression of genes that regulate metal binding, oxidative stress, and cytoskeletal organization and contractility. Co-administration of taurine with putrescine blocked disruption of TJs and the exacerbated inflammation.
We identified molecules that disrupt and stabilize intestinal epithelial TJs and barrier function and affect development of colon inflammation in mice. These agents might be developed for treatment of barrier intestinal impairment-associated and inflammatory disorders in patients, or avoided to prevent inflammation.
肠道屏障保护肠道细胞免受腔道中微生物和抗原的侵害——屏障的破坏会改变肠道微生物群、肠内免疫系统和代谢。我们进行了一项筛选，以确定破坏和支持肠道上皮屏障的分子，并在小鼠中测试它们的效果。
我们进行了基于成像的、定量的、高通量筛选（使用孵育脂多糖、肿瘤坏死因子、组氨酸、受体拮抗剂以及分泌蛋白、微生物代谢物和药物文库的 CaCo-2 和 T84 细胞），以鉴定改变上皮紧密连接（TJ）和焦点附着形态的分子。然后，我们测试了 TJ 稳定剂对这些变化的影响。我们发现的破坏或稳定 TJ 的分子用于给予葡聚糖硫酸钠诱导的结肠炎或柠檬酸杆菌诱导的肠道炎症的小鼠。收集结肠组织并通过组织学、荧光显微镜和 RNA 测序进行分析。
该筛选确定了许多破坏或稳定（破坏后）TJ 和上皮细胞单层的化合物。我们将不同的形态改变与屏障功能的变化相关联，并确定了多种细胞因子、代谢物和药物（包括肌动球蛋白收缩性抑制剂），这些物质可防止 TJ 破坏并恢复 TJ 完整性。其中一种破坏剂（腐胺）破坏了离体小鼠结肠组织中的 TJ 完整性；给予小鼠可加重结肠炎症、增加肠道通透性、降低结肠跨上皮电阻、增加肠系膜淋巴结中模式识别受体配体、减少结肠长度和存活时间。腐胺还增加了肠道水平和粪便中存活的 C rodentium 的脱落，增加了细菌对结肠上皮的附着，并增加了结肠组织中炎症细胞因子的水平。给予腐胺的小鼠结肠上皮细胞增加了调节金属结合、氧化应激和细胞骨架组织和收缩性的基因表达。腐胺与牛磺酸共同给药可阻断 TJ 的破坏和炎症的加剧。
我们确定了破坏和稳定肠道上皮 TJ 和屏障功能的分子，并影响了小鼠结肠炎症的发展。这些药物可能被开发用于治疗患者的肠道屏障损伤相关和炎症性疾病，或者避免使用以防止炎症。
