Mechanisms underlying regulation of intestinal epithelial homeostasis in sepsis

脓毒症和严重的手术条件导致肠道上皮细胞增殖显著减少 地窖。这种病理生理反应破坏了肠道上皮细胞的动态平衡，这被认为是导致 对脓毒症所致免疫功能障碍和多器官功能衰竭的发病机制有重要意义。有证据表明， 脓毒症与干扰素γ(干扰素γ)水平升高有关，干扰素是一种已知的抑制炎症的促炎细胞因子 肠上皮细胞(IEC)体外增殖。在初步研究中，我们发现干扰素γ在 脓毒症时隐窝内皮细胞增殖中断的作用。肠隐窝中的增殖细胞由 表达Lgr5的隐窝基柱状细胞(Lgr5-CBCs，一组肠道干细胞[ISCs])和转运体 放大细胞(TAC)。然而，脓毒症激活的干扰素γ信号影响细胞增殖的机制 这些细胞还没有得到严格的研究。此外，人们对如何维持细菌的扩散知之甚少 LGR5-CBCs和TACs在脓毒症中维持动态平衡。最近，我们发现败血症与糖尿病相关。 长非编码RNA(LncRNA)分子H19在肠道Lgr5-CBCs和TAC中的新表达 地窖。值得注意的是，我们发现h19 lncrna在减轻脓毒症诱导的减少中起着关键作用。 抑制隐窝内皮细胞增殖，促进肠上皮再生。从机制上讲，H19 lncRNA与 抑制细胞增殖并减弱其活性的分子。总的来说，这些数据表明干扰素γ 在脓毒症中，信号转导和H19lncRNA在调节Lgr5-CBC和TAC增殖中起着相反的作用。在……里面 在这个项目中，我们将检验脓毒症诱导的炎症激活H19从头表达的假设 ，从而拮抗干扰素γ信号轴的有害作用，从而释放对 LGr5-CBC和TAC的增殖和解救肠粘膜的动态平衡。为了实现这一目标，我们 将执行三个相辅相成的目标：(1)我们将检查脓毒症炎症是否以及如何失调调节 关注干扰素γ信号在LGR5-CBCs和TAC增殖中的作用。我们会精准地研究 脓毒症对临床相关小鼠体内Lgr5-CBCs和TACs增殖的有害影响 多菌败血症模型--5-乙炔-2‘-脱氧尿嘧啶核苷标记与多探针结合的新方法 荧光原位杂交技术、流式细胞术和细胞分选技术。此外，我们还将 阐明干扰素γ信号如何在脓毒症中抑制LGR5-CBC和Tac的增殖。(2)我们会 探讨从头表达的H19lncRNA拮抗抑制作用的分子机制 脓毒症对Lgr5-CBC和TAC增殖的影响，采用多学科的体内和体外方法 融合了细胞生物学、器官培养、分子生物学和小鼠基因工程技术。(3) 我们将研究脓毒症炎症如何诱导IECS中H19 lncRNA的从头表达。成功 这些目标的实现将填补脓毒症肠道上皮细胞更新调控方面的知识空白 并最终导致新疗法的开发。

Sepsis and severe surgical conditions cause profound decrease in epithelial cell proliferation in the intestinal crypts. This pathophysiological response disrupts intestinal epithelial homeostasis, which is thought to contribute to the pathogenesis of sepsis-induced immune dysfunction and multiple organ failure. Evidence shows that sepsis is associated with increased levels of interferon γ (IFNγ), a proinflammatory cytokine known to inhibit intestinal epithelial cell (IEC) proliferation in vitro. In preliminary studies, we found that IFNγ plays an important role in disruption of crypt IEC proliferation in sepsis. The proliferative cells in the intestinal crypts are composed of Lgr5-expressing crypt base columnar cells (Lgr5+-CBCs, a group of intestinal stem cells [ISCs]) and transit amplifying cells (TACs). However, the mechanism by which sepsis-activated IFNγ signal affects proliferation of these cells has not been rigorously studied. Furthermore, little is known about how to sustain proliferation of Lgr5+-CBCs and TACs to maintain homeostasis in sepsis. Recently, we found that sepsis is associated with de novo expression of a long noncoding RNA (lncRNA) molecule, H19, in Lgr5+-CBCs and TACs in the intestinal crypts. Remarkably, we discovered that H19 lncRNA plays a critical role in attenuating sepsis-induced reduction of crypt IEC proliferation and promoting intestinal epithelial regeneration. Mechanistically, H19 lncRNA binds to molecules that inhibit cell proliferation and attenuates their activity. Collectively, these data suggest that IFNγ signaling and H19 lncRNA play opposing roles in the regulation of Lgr5+-CBC and TAC proliferation in sepsis. In this project, we will test the hypothesis that sepsis-induced inflammation activates de novo expression of H19 lncRNA, which in turn antagonizes the deleterious effect of the IFNγ signal axis, thereby releasing inhibition of Lgr5+-CBC and TAC proliferation and rescuing homeostasis of the intestinal mucosa. To achieve this goal, we will execute three complementary aims: (1) We will examine whether and how septic inflammation dysregulates proliferation of Lgr5+-CBCs and TACs by focusing on the role of IFNγ signaling. We will precisely study the harmful effect of sepsis on proliferation of Lgr5+-CBCs and TACs in vivo utilizing a clinically relevant mouse model of polymicrobial sepsis, a novel method that combines 5-ethynyl-2'-deoxyuridine labeling with multi-probe fluorescence in situ hybridization assay, and flow cytometry and cell sorting technology. Furthermore, we will delineate how IFNγ signaling leads to inhibition of Lgr5+-CBC and TAC proliferation in sepsis. (2) We will investigate the molecular mechanisms by which de novo expressed H19 lncRNA antagonizes the inhibitory effect of sepsis on Lgr5+-CBC and TAC proliferation, taking a multidisciplinary in vivo and in vitro approach that incorporates cell biology, organoid culture, molecular biology, and mouse genetic engineering techniques. (3) We will study how septic inflammation induces de novo expression of H19 lncRNA in IECs. Successful achievement of these aims will fill gaps in knowledge about the regulation of intestinal epithelial renewal in sepsis and ultimately lead to development of new therapies.