Control of Inflammatory Acidity in Mucosal Inflammation

在胃肠道内，粘膜表面的主要功能是提供一种选择性屏障。 到外面去。人们普遍认为，由于以下原因，胃肠道处于低度炎症状态 管腔抗原性材料的持续加工。胃肠道是数以万亿计的共生细菌的家园 对胃肠道动态平衡有显著贡献，但也可以启动和推动粘膜疾病的进展。 急性肠炎包括中性粒细胞(PMN)的早期积聚，然后是消退或 进展为慢性炎症。如果在渗入部位没有有效的PMN清除，PMN可能 积累并导致慢性炎症性疾病，包括炎症性肠病。这个 该方案中正在进行的研究表明，PMN跨上皮迁移(TEM)会导致 细胞外pH显著下降，被认为是炎症性酸化。这些研究的扩展有 表明这种酸性微环境影响肠道上皮细胞的屏障功能和基因转录。 细胞(IEC)。在体外低pH条件下无偏的IEC RNA序列和体内组织代谢组学的指导下， 我们确定了一个基因特征，包括诱导肿瘤坏死因子以及DUSP和NR4A的成员 家人。我们还确定了IEC GPR31在感知细胞外pH和微生物来源的损失方面的作用。 活动性炎症时的吲哚。扩展这些研究，我们观察到IEC的显著改善 GPR31基因敲除的T84细胞在酸性条件下的屏障功能。此外，我们 观察到吲哚治疗显著减少了PMN后的炎性酸化。 K12大肠杆菌在酸性条件下可诱导产生吲哚。基于这些观察结果，我们假设 炎性酸化促进了IEC的炎症信号，微生物区系衍生的吲哚 在促进炎症部位的pH动态平衡方面有显著作用。体外功能分析将 明确细胞外酸中毒和pH感知对PMN的影响、IEC屏障功能和IEC 对慢性细胞外酸中毒的适应。这些研究将扩展到考察 微生物/宿主相互作用对IEC胞外酸化反应的影响。最后，利用体内小鼠结肠炎 和回肠炎模型，我们将描述炎性酸化。我们会将这些研究扩展至调查 微生物组和微生物组衍生代谢物对炎症相关性酸中毒的影响。职业生涯 此申请中概述的发展和培训计划旨在指导和促进我过渡到 独立成为退伍军人管理局资助的研究人员。建议将授课课程、专业课程和 会议和指导委员会的设计是为了加强我对粘膜的知识 免疫学和粘膜炎症。从技术上讲，我将在小鼠肠道的生成方面获得经验 肠类，肠类的遗传修饰，化学性和自发性回肠炎小鼠模型 和结肠炎，并扩大我对微生物/宿主反应的知识和操纵 微生物区系。完成这项建议后，我将做好充分准备，使自己成为PH值方面的专家 调节和炎症相关的酸化。此外，我们希望这项研究的大纲是 该提案将为确定IBD新的治疗途径奠定基础，直接受益于 退伍军人群体。

Within the gastrointestinal (GI) tract the primary function of the mucosal surface is to provide a selective barrier to the outside. It is widely understood that the GI tract exists in a state of low-grade inflammation, as a result of constant processing of luminal antigenic material. The GI tract is home to trillions of commensal bacteria which contribute significantly to GI homeostasis, but can also initiate and drive the progression of mucosal diseases. Acute intestinal inflammation involves early accumulation of neutrophils (PMN) followed by either resolution or progression to chronic inflammation. Without efficient PMN clearance at sites of infiltration, PMN can accumulate and contribute to chronic inflammatory conditions, including inflammatory bowel disease. The ongoing studies outline in this proposal have revealed that PMN transepithelial migration (TEM) results in a significant decrease in extracellular pH, deemed inflammatory acidification. Extensions of these studies have shown that this acidic microenvironment impacts barrier function and gene transcription in intestinal epithelial cells (IEC). Guided by an unbiased RNAseq of IEC exposed to low pH in vitro and tissue metabolomics in vivo, we identified a gene signature that includes induction of both TNF and members of the DUSP and NR4A families. We also identified a role of IEC GPR31 in sensing extracellular pH and the loss of microbial-derived indoles during active inflammation. Extending these studies, we observed significant improvement in IEC barrier function under acidic conditions in T84 cells which had GPR31 knocked down. Additionally, we observed that indole treatment significantly decreased inflammatory acidification following PMN TEM and in K12 E. coli acidic conditions induce the production of indole. Based on these observations, we hypothesize that inflammatory acidification promotes inflammatory signaling in IEC and that the microbiota-derived indoles have a significant role in promoting pH homeostasis at sites of inflammation. In vitro functional assays will define the impact of extracellular acidosis and pH sensing on PMN TEM, IEC barrier function, and IEC adaptation to chronic extracellular acidosis. These studies will be extended to examine the influence of microbial/host interactions on IEC responses to extracellular acidification. Finally, utilizing in vivo murine colitis and ileitis models we will profile inflammatory acidification. We will extend these studies to investigate the impact of the microbiota and microbiota-derived metabolites on inflammation-associated acidosis. The career development and training plan outlined in this application is designed to guide and promote my transition to independence as a VA funded researcher. The proposed combination of didactic courses, professional conferences, and mentorship committee have been designed to strengthen my knowledge of mucosal immunology and mucosal inflammation. Technically, I will gain experience in the generation of murine intestinal enteroids, genetic modification of enteroids, both chemically induced and spontaneous murine models of ileitis and colitis, and expand my knowledge of microbial/host response and technical experience in manipulating the microbiota. Upon completion of this proposal I will be well prepared to establish myself as an expert in pH regulation and inflammation-associated acidification. Furthermore, it is our hope that the studies outline in this proposal will lay the groundwork for the identification of novel therapeutic avenues in IBD, directly benefiting the veteran population.