Proinflammatory properties of Paneth cells in intestinal inflammation

项目总结 克罗恩病是一种慢性炎症性疾病，最常影响回肠，但目前尚不清楚 多因素病因学。肿瘤坏死因子Δ/小鼠模型概括了回肠克罗恩病的特征，并由 由于全身性过度表达肿瘤坏死因子，一种促炎细胞因子，在克罗恩病患者中升高。这个 肠上皮细胞类型在肿瘤坏死因子诱导的慢性回肠炎症中的作用尚不清楚。帕内斯 细胞是一种肠道上皮细胞，它分泌抗菌素来保护上皮免受微生物的侵袭。 移居管腔隔室。据推测，由于Paneth的作用，抗菌屏障功能受损 细胞的缺陷和丢失，促进了克罗恩病的发展。然而，我们的初步数据表明 潘氏细胞在慢性回肠炎中可能具有促炎特性，如潘氏细胞消融术 逆转肿瘤坏死因子ΔARE/小鼠的回肠疾病。这一提议的假设是潘氏细胞具有促炎作用 在肿瘤坏死因子Δ中驱动回肠炎症的特性是克罗恩病的小鼠模型。程序性细胞死亡 和自噬是受到严格调控的细胞过程，允许组织管理老化或应激的细胞。 尽管有证据表明这些过程在克罗恩病中可能是失调的，但并没有机制 有证据表明促炎症潘氏细胞死亡是慢性回肠炎症的驱动因素。在AIM 1中，单细胞 将利用技术来研究潘氏细胞死亡的特定机制如何对回肠做出贡献 炎症的肿瘤坏死因子Δ是/模型。实验方法包括先进的Paneth细胞消融技术， 我们的多重免疫荧光(MxIF)成像流水线和透射电子显微镜。此外， 间充质细胞，如成纤维细胞，与潘氏细胞和上皮间充质密切相关。 串扰是肠道中的一种既定现象。潘氏细胞表达细胞因子，如肿瘤坏死因子和白介素2。 17，而且，肌成纤维细胞被上皮源性细胞因子激活，以促进炎症 卡斯卡德。然而，潘氏细胞衍生的因子，如细胞因子，与克罗恩病无关。 疾病，此外，潘氏细胞-间充质细胞的相互作用尚未建立。在目标2中，单一- 细胞技术将被用来识别潘氏细胞衍生的促炎细胞因子，并计算 方法将被用来确定潘氏细胞-间充质细胞的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成像流水线。我还将使用小鼠模型来驱动Paneth细胞中的肿瘤坏死因子过表达，并在 确定潘氏细胞来源的肿瘤坏死因子是否直接激活成纤维细胞的体外方法。启示性 潘氏细胞驱动回肠炎症的机制对于理解克罗恩病和意志是至关重要的 导致新的治疗靶点。

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.