Molecular basis of host-microbiota signaling in the zebrafish gut

描述(由申请人提供)：在这项对5R01DK75549-3的拟议修订中，题为“斑马鱼肠道中宿主-微生物区系信号的分子基础”，我们建议将我们对肠道碱性磷酸酶功能的分析扩展到小鼠。使用斑马鱼模型，我们已经证明了IAP在抗炎先天免疫反应中的功能，以防止针对驻留的微生物群的过度肠道炎症。我们建议测试IAP在微生物-宿主相互作用中的功能和调节在脊椎动物中是否保守。我们对IAP缺陷小鼠的初步研究表明，这种酶具有预防肠道炎症的功能，就像对斑马鱼所做的那样。我们假设宿主肠道对内毒素的反应是平衡的，以提供对革兰氏阴性病原体感染的保护，同时防止慢性炎症反应对常驻肠道细菌的损害。因此，我们预测抑制IAP活性将增加宿主对具有良好特征的革兰氏阴性肠道病原体鼠伤寒沙门氏菌的抵抗力(以增加炎症为代价)。而选择IAP活性的正常设定值可以优化肠道的长期健康，短期抑制IAP可能被证明是一种有效的预防方法，以提高对革兰氏阴性菌的抵抗力。我们将通过专门测试以下假设来验证或驳斥我们的模型：1.IAP可防止小鼠肠道内细菌引起的过度炎症。我们将测量在存在或不存在微生物的情况下饲养的IAP缺陷小鼠的肠道炎症。2.IAP受TLR信号调节，是对内毒素的原始先天免疫反应的一部分。我们将测量在1)细菌内毒素存在和不存在的情况下IAP的转录调控，以及2)TLR4介导的内毒素感应。3.通过解毒内毒素，IAP可降低沙门氏菌引起的肠道炎症反应。我们将分析野生型和IAP缺陷小鼠对沙门氏菌感染的肠道炎症反应。4.通过解毒内毒素，IAP使肠道上皮更容易受到沙门氏菌的渗透。我们将测量野生型和IAP缺陷小鼠口服感染后鼠伤寒沙门氏菌向肠系膜淋巴结的移位。 公共卫生相关性：项目描述我们建议研究肠道碱性磷酸酶(IAP)在两种模式脊椎动物宿主斑马鱼和小鼠宿主-细菌相互作用中的作用。我们在这些系统中了解到的IAP如何促进对常驻肠道细菌的耐受性或改变对病原菌的敏感性将适用于人类，并将有助于我们对肠道病原体感染和炎症性肠病等疾病的理解。

DESCRIPTION (provided by applicant): In this proposed revision to the parent grant 5R01DK75549-3, entitled "Molecular basis of host-microbiota signaling in the zebrafish gut", we propose to expand our analysis of intestinal alkaline phosphatase function to mice. Using a zebrafish model, we have demonstrated that IAP functions in an anti-inflammatory innate immune response to prevent excessive intestinal inflammation against the resident microbiota. We propose to test whether the function and regulation of IAP in microbial-host interactions is conserved among vertebrates. Our preliminary studies with IAP deficient mice indicate that this enzyme functions to prevent intestinal inflammatory as it does in zebrafish. We hypothesize that the host intestinal responses to LPS are balanced to provide protection against infection by Gram-negative pathogens while at the same time preventing damage from chronic inflammatory responses to resident gut bacteria. Therefore we predict that inhibition of IAP activity should increase host resistance to a well- characterized Gram-negative intestinal pathogen S. typhimurium (at the cost of increased inflammation). Whereas the normal set point of IAP activity may be selected to optimize the long-term health of the intestine, short-term inhibition of IAP may prove to be an effective prophylactic method to boost resistance to Gram-negative pathogens. We will validate or refute our model by specifically testing the following hypotheses: 1. IAP prevents excessive inflammation in response to bacteria in the mouse intestine. We will measure intestinal inflammation in IAP deficient mice reared in the presence or absence of microbes. 2. IAP is regulated by TLR signaling as part of a primary innate immune response to LPS. We will measure transcriptional regulation of IAP in the presence and absence of 1) bacterial LPS and 2) TLR4 mediate LPS sensing. 3. By detoxifying LPS, IAP reduces intestinal inflammatory responses to Salmonella. We will assay intestinal inflammation in response to S. typhimrium infection in wild type and IAP deficient mice. 4. By detoxifying LPS, IAP renders the intestinal epithelium more susceptible to Salmonella penetration. We will measure S. typhimuium translocation to mesenteric lymph nodes after oral infection of wild type and IAP deficient mice. PUBLIC HEALTH RELEVANCE: Project narrative We propose to study the function of Intestinal Alkaline Phosphatase (IAP) in host- bacterial interactions in the two model vertebrate hosts zebrafish and mouse. What we learn in these systems about how IAP promote tolerance to resident gut bacteria or alter susceptibility to pathogenic bacteria will be applicable to humans and will inform our understanding of infections with enteric pathogens and conditions such as Inflammatory Bowel Diseases.