Molecular basis of host-microbiota signaling in the zebrafish gut

描述（由申请人提供）：在对题为“斑马鱼肠道中宿主微生物群信号传导的分子基础”的母授权5 R 01 DK 75549 -3的拟议修订中，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发挥作用，以防止对驻留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是否在脊椎动物中是保守的。我们对IAP缺陷小鼠的初步研究表明，这种酶的作用与在斑马鱼中一样，可以预防肠道炎症。我们假设宿主肠道对LPS的反应是平衡的，以提供对革兰氏阴性病原体感染的保护，同时防止对常驻肠道细菌的慢性炎症反应造成的损害。因此，我们预测IAP活性的抑制将增加宿主对一种特征明确的革兰氏阴性肠道病原体S的抗性。鼠伤寒（以增加炎症为代价）。虽然可以选择IAP活性的正常设定点以优化肠道的长期健康，但IAP的短期抑制可能被证明是增强对革兰氏阴性病原体的抗性的有效预防方法。我们将通过具体测试以下假设来验证或反驳我们的模型：1. IAP可防止小鼠肠道中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由TLR信号传导调节，作为对LPS的初级先天免疫应答的一部分。我们将在存在和不存在1）细菌LPS和2）TLR 4介导的LPS传感的情况下测量IAP的转录调节。3.通过解毒LPS，IAP减少肠道对沙门氏菌的炎症反应。我们将检测肠道炎症对S.在野生型和IAP缺陷型小鼠中的鼠伤寒沙门氏菌感染。4.通过对LPS解毒，IAP使肠上皮更容易受到沙门氏菌的渗透。我们将测量S。口服感染野生型和IAP缺陷型小鼠后，伤寒沙门氏菌易位至肠系膜淋巴结。 公共卫生相关性：本研究拟以斑马鱼和小鼠两种模式脊椎动物为宿主，研究肠道碱性磷酸酶（IAP）在宿主-细菌相互作用中的作用。我们在这些系统中了解到的关于IAP如何促进对常驻肠道细菌的耐受性或改变对病原菌的易感性的知识将适用于人类，并将告知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.