Proinflammatory properties of Paneth cells in intestinal inflammation

项目摘要 克罗恩病是一种最常见的影响回肠的慢性炎症性疾病， 多因素病因学TnfΔARE/+小鼠模型重现了回肠克罗恩病的特征， 通过全身性过度表达TNF，一种在克罗恩病患者中升高的促炎细胞因子。的 肠上皮细胞类型对慢性TNF诱导的回肠炎症的作用尚不清楚。潘氏 细胞是一种肠上皮细胞，其分泌抗菌剂以保护上皮免受 在腔室中定植。据推测，由于Paneth， 细胞缺陷和损失，有助于克罗恩病的发展。然而我们的初步数据显示 潘氏细胞在慢性回肠炎症中可能具有促炎特性， 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症作用 在克罗恩病的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，其允许组织管理老化或应激细胞。 虽然有证据表明，这些过程可能是失调的克罗恩病，没有机制， 作为慢性回肠炎症驱动因素的促炎潘氏细胞死亡的证据。在目标1中，单细胞 技术将被用来研究潘氏细胞死亡的具体机制如何有助于回肠 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）成像管道，和透射电子显微镜。此外，委员会还认为， 间充质细胞，如成纤维细胞，与潘氏细胞和上皮-间充质细胞非常接近。 串话是肠内的一种既定现象。潘氏细胞表达细胞因子，如TNF和IL-1。 17，此外，肌成纤维细胞被上皮来源的细胞因子激活以促进炎症反应 级联。然而，潘氏细胞衍生因子，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，单一- 细胞技术将被用来鉴定潘氏细胞衍生的促炎细胞因子， 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 方法包括单细胞RNA测序，我们的计算细胞间通讯管道，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过表达， 体外方法来确定潘氏细胞衍生的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理解克罗恩病至关重要， 从而产生新的治疗靶点。

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.