Proinflammatory properties of Paneth cells in intestinal inflammation

潘氏细胞在肠道炎症中的促炎特性

• 批准号: 10617327
• 负责人: Paige Nicole Vega
• 金额: $ 3.54万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10617327
• 关键词: Ablation; Affect; Apoptosis; Autophagocytosis; Biological Assay; Cell Communication; Cell Death; Cell Death Process; Cell physiology; Cell secretion; Cells; Cellular Morphology; Cellular Stress; Chronic; Computing Methodologies; Coupled; Crohn' s disease; Data; Defect; Development; Disease; Epithelium; Etiology; Fibroblasts; Genes; IL17 gene; Ileal Diseases; Ileitis; Image; Immunofluorescence Immunologic; Impairment; In Vitro; Induction of Apoptosis; Inflammation; Inflammatory; Intestines; Investigation; Mesenchymal; Microbe; Modeling; Mucous Membrane; Mus; Myofibroblast; Organoids; Paneth Cells; Pathogenesis; Pathway interactions; Patients; Play; Process; Property; Proteins; Role; Signal Pathway; Signal Transduction; TNF gene; Techniques; Testing; Therapeutic; Tissues; Transmission Electron Microscopy; Tumor Necrosis Factor Receptor; Western Blotting; Wild Type Mouse; aged; antimicrobial; cell type; computational pipelines; cytokine; experimental study; gain of function; gut inflammation; ileum; in vivo; innovation; intestinal epithelium; loss of function; microbiota; mouse model; new therapeutic target; novel; overexpression; prevent; programs; risk variant; single cell technology; single-cell RNA sequencing

PROJECT SUMMARY Crohn's disease, a chronic inflammatory condition that most commonly affects the ileum, has an unclear but multifactorial etiology. The TnfΔARE/+ mouse model recapitulates features of ileal Crohn's disease and is driven by systemic overexpression of TNF, a proinflammatory cytokine that is elevated in Crohn's disease patients. The contribution of intestinal epithelial cell types to chronic, TNF-induced ileal inflammation is not known. Paneth cells are a type of intestinal epithelial cell that secrete antimicrobials to protect the epithelium from microbes that colonize the luminal compartment. It is hypothesized that impaired antimicrobial barrier function, due to Paneth cell defects and loss, contributes to the development of Crohn's disease. However, our preliminary data suggests that Paneth cells may have proinflammatory properties in chronic ileal inflammation, as Paneth cell ablation reverses ileal disease in TnfΔARE/+ mice. The hypothesis of this proposal is that Paneth cells have proinflammatory properties that drive ileal inflammation in the TnfΔARE/+ mouse model of Crohn's disease. Programmed cell death and autophagy are tightly regulated cellular processes that allow the tissue to manage aged or stressed cells. Although there is evidence that these processes may be dysregulated in Crohn's disease, there is no mechanistic evidence of proinflammatory Paneth cell death as a driver of chronic ileal inflammation. In Aim 1, single-cell technologies will be leveraged to investigate how specific mechanisms of Paneth cell death contribute to ileal inflammation the TnfΔARE/+ model. Experimental approaches include advanced Paneth cell ablation techniques, our multiplex immunofluorescence (MxIF) imaging pipeline, and transmission electron microscopy. Furthermore, mesenchymal cells, such as fibroblasts, are in close proximity to Paneth cells and epithelial-mesenchymal crosstalk is an established phenomenon in the intestine. Paneth cells express cytokines, such as TNF and IL- 17, and moreover, myofibroblasts are activated by epithelial-derived cytokines to promote an inflammatory cascade. However, Paneth cell-derived factors, such as cytokines, have not been associated with Crohn's disease, and furthermore, Paneth cell-mesenchymal cell interactions have not been established. In Aim 2, single- cell techniques will be leveraged to identify Paneth cell-derived proinflammatory cytokines, and computational methods will be used to determine if Paneth cell-mesenchymal cell interactions promote ileal inflammation. My approaches include single-cell RNA-sequencing, our computational cell-cell communication pipeline, and our MxIF imaging pipeline. I will also use mouse models to drive TNF overexpression from Paneth cells, and use in vitro approaches to determine whether Paneth cell-derived TNF directly activates fibroblasts. Revealing mechanisms by which Paneth cells drive ileal inflammation is critical for understanding Crohn's disease and will lead to novel therapeutic targets.

项目概要 克罗恩病是一种最常影响回肠的慢性炎症性疾病，其发病机制尚不明确。 多因素病因学。 TnfΔARE/+小鼠模型概括了回肠克罗恩病的特征并被驱动 TNF 是一种促炎细胞因子，在克罗恩病患者中升高。这 肠上皮细胞类型对 TNF 诱导的慢性回肠炎症的影响尚不清楚。帕内斯 细胞是一种肠上皮细胞，可以分泌抗菌剂来保护上皮免受微生物的侵害 定植于腔室。据推测，由于潘氏菌，抗菌屏障功能受损 细胞缺陷和损失，导致克罗恩病的发展。然而，我们的初步数据表明 潘氏细胞可能在慢性回肠炎症中具有促炎特性，如潘氏细胞消融 逆转 TnfΔARE/+ 小鼠的回肠疾病。该提议的假设是潘氏细胞具有促炎作用 在克罗恩病 TnfΔARE/+ 小鼠模型中驱动回肠炎症的特性。程序性细胞死亡 和自噬是严格调节的细胞过程，使组织能够管理老化或应激的细胞。 尽管有证据表明克罗恩病中这些过程可能失调，但尚无机制。 促炎性潘氏细胞死亡是慢性回肠炎症驱动因素的证据。在目标 1 中，单细胞 将利用技术来研究潘氏细胞死亡的具体机制如何促进回肠 炎症 TnfΔARE/+ 模型。实验方法包括先进的潘氏细胞消融技术， 我们的多重免疫荧光 (MxIF) 成像流程和透射电子显微镜。此外， 间充质细胞，例如成纤维细胞，与潘氏细胞和上皮间充质细胞非常接近 串扰是肠道中的一种既定现象。潘氏细胞表达细胞因子，例如 TNF 和 IL- 17，此外，肌成纤维细胞被上皮源性细胞因子激活，促进炎症 级联。然而，潘氏细胞衍生的因子，例如细胞因子，与克罗恩病无关 疾病，此外，潘氏细胞-间充质细胞相互作用尚未建立。在目标 2 中，单 将利用细胞技术来识别潘氏细胞衍生的促炎细胞因子，并通过计算 方法将用于确定潘氏细胞-间充质细胞相互作用是否促进回肠炎症。我的 方法包括单细胞 RNA 测序、我们的计算细胞间通信管道以及我们的 MxIF 成像管道。我还将使用小鼠模型来驱动潘氏细胞中的 TNF 过度表达，并用于 体外方法确定潘氏细胞来源的 TNF 是否直接激活成纤维细胞。揭示 潘氏细胞驱动回肠炎症的机制对于了解克罗恩病至关重要 导致新的治疗靶点。