Table 1_Enterotoxigenic Escherichia coli–induced intestinal epithelial necroptosis drives lamina propria immune cell pyroptosis and mucosal injury in piglets.docx

Necroptosis is an inflammatory programmed cell death pathway linked to diverse physiological and pathological disorders, yet its role in Enterotoxigenic Escherichia coli (ETEC)−induced intestinal inflammation and mucosal injury remains poorly understood. This study aimed to elucidate the contribution of intestinal epithelial cell necroptosis to the development of intestinal inflammation and injury induced by ETEC infection in piglets. Following ETEC challenge in piglets, key proteins involved in necroptosis, including phosphorylated receptor-interacting protein kinase 3 (p-RIP3) and high-mobility group box 1 (HMGB1), were upregulated in jejunal crypt epithelial cells, which are primarily composed of Paneth cells and stem cells, in a time-dependent manner. In addition, ETEC challenge triggered time−dependent pyroptosis in jejunal lamina propria lymphocytes, a population that includes macrophages, as demonstrated by elevated levels of NLRP3, Caspase−1, GSDMD−N, and Cleaved -IL−1β (p17) proteins in lamina propria lymphocytes. Necroptosis of jejunal crypt epithelial cells occurred prior to pyroptosis of lamina propria lymphocytes, indicating that epithelial cell necroptosis may contribute to the induction of pyroptosis in lamina propria lymphocytes. ETEC challenge induced progressive TNF−α and IL−1β upregulation in plasma, jejunal crypt epithelial cells, and lamina propria lymphocytes of piglets. These changes coincided with intestinal injury and barrier loss, which were indicated by increased plasma i−FABP and decreased jejunal ZO−1 and Occludin. Notably, Nec−1 pretreatment mitigates ETEC−induced intestinal inflammation and tissue damage in piglets by inhibition of crypt epithelial cells necroptosis and the ensuing pyroptosis of lymphocytes. These results indicate that targeting upstream epithelial-cell necroptosis is an important strategy to attenuate inflammation and preserve barrier integrity.

坏死性凋亡（Necroptosis）是一类炎症性程序性细胞死亡通路，与多种生理及病理紊乱密切相关，但其在产肠毒素大肠杆菌（Enterotoxigenic Escherichia coli, ETEC）诱导的肠道炎症与黏膜损伤中的作用仍不甚明确。本研究旨在阐明肠道上皮细胞坏死性凋亡在仔猪ETEC感染诱导的肠道炎症及损伤发生发展中的作用。研究人员对仔猪进行ETEC攻毒后发现，以潘氏细胞（Paneth cells）与干细胞为主要组分的空肠隐窝上皮细胞中，参与坏死性凋亡的关键蛋白——磷酸化受体相互作用蛋白激酶3（phosphorylated receptor-interacting protein kinase 3, p-RIP3）与高迁移率族蛋白B1（high-mobility group box 1, HMGB1）的表达呈时间依赖性上调。此外，ETEC攻毒可触发空肠固有层淋巴细胞（该细胞群包含巨噬细胞）发生时间依赖性焦亡（pyroptosis），具体表现为固有层淋巴细胞中NLRP3、半胱氨酸天冬氨酸蛋白酶-1（Caspase-1）、GSDMD-N以及切割型IL-1β（Cleaved-IL-1β, p17）的蛋白水平显著升高。空肠隐窝上皮细胞的坏死性凋亡早于固有层淋巴细胞的焦亡，提示上皮细胞坏死性凋亡可能参与诱导固有层淋巴细胞的焦亡过程。ETEC攻毒可诱导仔猪血浆、空肠隐窝上皮细胞及固有层淋巴细胞中TNF-α与IL-1β的表达呈进行性上调。上述变化与肠道损伤及肠屏障功能丧失相契合，具体表现为血浆肠型脂肪酸结合蛋白（intestinal fatty acid-binding protein, i-FABP）水平升高，以及空肠黏膜紧密连接蛋白ZO-1与Occludin的表达水平降低。值得注意的是，坏死性凋亡抑制剂Nec-1预处理可通过抑制隐窝上皮细胞坏死性凋亡及其后续引发的淋巴细胞焦亡，减轻ETEC诱导的仔猪肠道炎症与组织损伤。本研究结果表明，靶向上游上皮细胞坏死性凋亡是减轻肠道炎症、维持肠屏障完整性的重要策略。