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.

脓毒症和严重的手术条件导致肠上皮细胞增殖的显著减少， 地穴这种病理生理反应破坏了肠上皮细胞的稳态，这被认为有助于 脓毒症引起的免疫功能障碍和多器官功能衰竭的发病机制。证据显示 脓毒症与干扰素γ（IFNγ）水平升高有关，IFN γ是一种已知抑制 肠上皮细胞（IEC）体外增殖。在初步的研究中，我们发现IFNγ在细胞内起着重要的作用。 在脓毒症中破坏隐窝IEC增殖中的作用。肠腺中的增殖细胞由 表达Lgr 5的隐窝基底柱状细胞（Lgr 5 +-CBCs，一组肠干细胞[ISCs]）和转运 扩增细胞（TAC）。然而，脓毒症激活的IFNγ信号影响细胞增殖的机制尚不清楚。 这些细胞尚未被严格研究。此外，人们对如何维持核武器的扩散知之甚少。 Lgr 5 +-CBC和TAC在脓毒症中维持体内平衡。最近，我们发现脓毒症与de 肠内Lgr 5 +-CBCs和TAC中长链非编码RNA（lncRNA）分子H19的新生表达 地穴值得注意的是，我们发现H19 lncRNA在减弱脓毒症诱导的减少中起着关键作用。 促进肠上皮再生。从机制上讲，H19 lncRNA结合到 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在脓毒症中Lgr 5 +-CBC和TAC增殖的调节中起相反的作用。在 在本项目中，我们将检验脓毒症诱导的炎症激活H19从头表达的假设 lncRNA，其反过来拮抗IFNγ信号轴的有害作用，从而释放对IFN γ的抑制。 Lgr 5 +-CBC和TAC增殖和拯救肠粘膜的稳态。为了实现这一目标，我们 我们将执行三个互补的目标：（1）我们将研究脓毒性炎症是否以及如何失调 通过关注IFNγ信号传导的作用，观察Lgr 5 +-CBCs和TAC的增殖。我们将仔细研究 利用临床相关小鼠体内脓毒症对Lgr 5 +-CBCs和TAC增殖的有害影响 一种结合5-乙炔基-2 '-脱氧尿苷标记和多探针的新方法 荧光原位杂交分析和流式细胞术及细胞分选技术。此外，我们将 描述IFNγ信号传导如何导致脓毒症中Lgr 5 +-CBC和TAC增殖的抑制。(2)我们将 研究从头表达的H19 lncRNA拮抗抑制作用的分子机制。 脓毒症对Lgr 5 +-CBC和TAC增殖的影响，采用多学科的体内和体外方法， 结合了细胞生物学、类器官培养、分子生物学和小鼠基因工程技术。（三） 我们将研究脓毒性炎症如何诱导IEC中H19 lncRNA的从头表达。成功 这些目标的实现将填补脓毒症中肠上皮更新调控的知识空白 并最终导致新疗法的发展。