Mechanisms underlying regulation of intestinal epithelial homeostasis in sepsis

脓毒症肠上皮稳态调节的机制

基本信息

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
项目类别：
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

项目摘要

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道上皮细胞增殖显着下降这种病理生理反应破坏了肠上皮稳态，这被认为有助于有证据表明脓毒症引起的免疫功能障碍和多器官衰竭的发病机制。脓毒症与干扰素 γ (IFNγ) 水平升高有关，干扰素 γ 是一种促炎细胞因子，已知可抑制在体外肠上皮细胞（IEC）增殖中，我们发现IFNγ起着重要作用。在败血症中破坏隐窝 IEC 增殖中的作用肠隐窝中的增殖细胞由以下组成。表达 Lgr5 的隐窝基底柱状细胞（Lgr5+-CBC，一组肠干细胞 [ISC]）和运输然而，脓毒症激活的 IFNγ 信号影响扩增细胞增殖的机制。此外，人们对这些细胞如何维持增殖知之甚少。 Lgr5+-CBC 和 TAC 在脓毒症中维持稳态最近，我们发现脓毒症与 de 相关。长非编码 RNA (lncRNA) 分子 H19 在肠道 Lgr5+-CBC 和 TAC 中的新表达值得注意的是，我们发现 H19 lncRNA 在减轻脓毒症引起的减少中发挥着关键作用。 H19 lncRNA 与隐窝 IEC 增殖并促进肠上皮再生。总的来说，这些数据表明 IFNγ 抑制细胞增殖并减弱其活性。信号传导和 H19 lncRNA 在脓毒症中 Lgr5+-CBC 和 TAC 增殖的调节中发挥相反的作用。在这个项目中，我们将测试脓毒症诱导的炎症激活 H19 从头表达的假设 lncRNA，反过来拮抗 IFNγ 信号轴的有害作用，从而释放对 Lgr5+-CBC 和 TAC 增殖并挽救肠粘膜稳态为了实现这一目标，我们。将执行三个互补的目标：（1）我们将检查化脓性炎症是否以及如何失调我们将通过关注 IFNγ 信号传导的作用来精确研究 Lgr5+-CBC 和 TAC 的增殖。利用临床相关小鼠体内脓毒症对 Lgr5+-CBC 和 TAC 增殖的有害影响多微生物脓毒症模型，一种结合 5-乙炔基-2'-脱氧尿苷标记与多探针的新方法荧光原位杂交检测、流式细胞术和细胞分选技术。此外，我们将描述 IFNγ 信号传导如何抑制脓毒症中的 Lgr5+-CBC 和 TAC 增殖 (2) 我们将。研究从头表达的 H19 lncRNA 拮抗抑制的分子机制脓毒症对 Lgr5+-CBC 和 TAC 增殖的影响，采取体内和体外多学科方法 (3)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。我们将研究脓毒症炎症如何成功诱导 IEC 中 H19 lncRNA 的从头表达。这些目标的实现将填补脓毒症肠上皮更新调节的知识空白并最终导致新疗法的开发。