Mechanisms underlying regulation of intestinal epithelial homeostasis in sepsis

脓毒症肠上皮稳态调节的机制

• 批准号: 10337285
• 负责人: Xiao-Di Tan
• 金额: $ 60万
• 依托单位: LURIE CHILDREN'S HOSPITAL OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-15 至 2022-11-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10337285
• 关键词: Abbreviations; Achievement; Address; Affect; American; Animal Model; Attenuated; Biological Assay; Bone Morphogenetic Proteins; Burn Trauma; Burn injury; Cell Proliferation; Cell Separation; Cells; Cellular biology; Columnar Cell; Columnar Epithelium; Data; Deoxyuridine; Development; Epithelial Cell Proliferation; Epithelial Cells; Equilibrium; Event; Flow Cytometry; Fluorescent in Situ Hybridization; Functional disorder; GTP-Binding Protein alpha Subunits, Gs; Genetic Engineering; Goals; Gut Mucosa; H19 gene; Homeostasis; Human; IFITM1 gene; Immune System Diseases; Immune system; Impairment; In Vitro; Infection; Inflammation; Inflammatory; Interferons; Interleukin-6; Intestinal Mucosa; Intestines; Knockout Mice; Knowledge; LGR5 gene; Label; Lead; Life; Lymphoid; Malnutrition; Mediating; Methods; Molecular; Molecular Biology; Molecular Target; Molecular and Cellular Biology; Multiple Organ Failure; Natural regeneration; Operative Surgical Procedures; Organ; Organoids; Pathogenesis; Patient-Focused Outcomes; Patients; Physiological; Play; Process; Proteins; RNA Binding; Regulation; Research; Role; Sepsis; Signal Transduction; Specimen; TP53 gene; Techniques; Technology; Testing; Traumatic injury; Untranslated RNA; Up-Regulation; Villus; WNT Signaling Pathway; Work; base; cecal ligation puncture; clinically relevant; cytokine; cytokine release syndrome; dysbiosis; epithelium regeneration; gastrointestinal epithelium; gut homeostasis; improved; in vivo; intestinal crypt; intestinal epithelium; intestinal homeostasis; microbiome; monocyte; mouse genetics; mouse model; multidisciplinary; new therapeutic target; novel; novel strategies; novel therapeutic intervention; novel therapeutics; polymicrobial sepsis; preservation; response; restoration; self-renewal; septic; septic patients; severe burns; stem cell proliferation; stem cells; systemic inflammatory response

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.

败血症和严重的手术状况导致肠道上皮细胞增殖的巨大下降 地下室。这种病理生理反应破坏了肠道上皮稳态，这被认为有助于 败血症引起的免疫功能障碍和多器官衰竭的发病机理。证据表明 败血症与干扰素γ（IFNγ）的水平升高，这是一种已知抑制的促炎细胞因子 体外肠上皮细胞（IEC）增殖。在初步研究中，我们发现IFNγ起重要 败血症中隐窝IEC增殖的破坏中的作用。肠道隐窝中的增殖细胞组成 表达LGR5的隐窝基柱细胞（LGR5+-CBC，一组肠干细胞[ISC]）和Transit 放大细胞（TAC）。但是，败血症激活IFNγ信号的机制会影响 这些细胞尚未进行严格研究。此外，关于如何维持的扩散知之甚少 LGR5+-CBC和TACS维持败血症的稳态。最近，我们发现败血症与DE有关 在肠道中，在LGR5+-CBC中的长无编码RNA（LNCRNA）分子的NOVO表达 地下室。值得注意的是，我们发现H19 lncRNA在减轻败血症诱导的还原方面起着关键作用 Crypt IEC增殖和促进肠上皮再生。从机械上讲，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IFNγ 信号传导和H19 lncRNA在败血症中的LGR5+-CBC和TAC增殖中起着相反的作用。在 这个项目，我们将检验以下假设：败血症诱导的注射激活H19的从头表达 lncRNA反过来拮抗IFNγ信号轴的有害作用，从而释放了抑制 LGR5+-CBC和TAC的增殖和营救肠粘膜的稳态。为了实现这一目标，我们 将执行三个完整的目标：（1）我们将检查败血症注射是否以及如何失调 LGR5+-CBC和TAC的增殖通过着重于IFNγ信号的作用。我们将精确研究 败血症对使用临床相关的小鼠在体内LGR5+-CBC和TAC的增殖的有害作用 多因素败血症的模型，一种新的方法，将5-乙基2'-脱氧尿苷标记与多探针结合 荧光原位杂交测定法，流式细胞仪和细胞分选技术。此外，我们会的 描述IFNγ信号如何导致败血症中LGR5+-CBC和TAC增殖的抑制作用。 （2）我们会的 研究从头表达H19 lncRNA拮抗抑制性的分子机制 败血症对LGR5+-CBC和TAC增殖的影响，采用多学科的体内和体外方法 结合细胞生物学，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在IEC中诱导H19 lncRNA的从头表达。成功的 这些目标的实现将填补有关脓毒症肠上皮更新的知识的空白 并最终导致新疗法的发展。