The developing intestinal immune system in Yersinia enterocolitica infection

描述（由申请人提供）：绝大多数感染因子在粘膜表面遇到。了解免疫细胞和感染性微生物在这些位点的动态界面是微生物学和免疫学领域最大的挑战之一。这些相互作用在新生儿生命中尤其重要，因为这是第一次遇到微生物抗原。为了研究新生儿期免疫细胞/细菌的相互作用，我们建立了一种新的小鼠小肠结肠炎耶尔森菌细胞外感染模型。引人注目的是，新生儿对这种肠道病原体的抵抗力明显强于成年人，这是所有其他研究中新生儿-病原体相互作用的独特观察结果。该系统提供了一个前所未有的机会来了解如何在新生儿肠道中实现充分的保护性免疫。因此，本应用的一个主要目标是确定导致新生儿肠道暴露于小肠结肠炎耶氏菌的真核免疫机制。该应用程序的第二个主要目标是应用小肠结肠炎杆菌产品来调节新生儿肠道免疫的健康和疾病。这些明确的目标将为我们的长期目标(a)了解肠道免疫在个体发育过程中如何发展，(b)获得高效的儿科粘膜疫苗，以及(c)开发治疗早期病理性肠道炎症的新策略提供主要的新基石。特异性目标1将定义适应性免疫机制，保护新生儿免受口服小肠结肠炎感染。中和的效果？IFN和/或IL-17对炎症细胞浸润、菌血症和与死亡率相关的相对体重减轻的反应将被检查。B细胞反应的重要性将通过遗传和急性B细胞消融以及B细胞充足的新生儿无法建立细菌特异性抗体反应来测试。最后，将通过将成人Treg过继转移给受感染的新生儿和体内Treg室的耗竭来分析天然Treg （CD4+CD25+）的贡献。特异性目的2将利用小肠结肠炎耶氏菌效应蛋白YopP（其存在或不存在）来操纵新生儿免疫反应，并在体内识别关键的靶免疫细胞类型。自从？YopP突变株在新生儿肠道中引起严重炎症，我们将测试一种改良的？YopP突变株作为黏膜免疫的有效佐剂。表达卵清蛋白的？将创建YopP菌株，并监测卵白蛋白特异性抗体和Th反应，以及卵白蛋白特异性TCR转基因T细胞的体内激活。其次，将测试YopP本身是否可以下调新生儿致病性肠道炎症。仅表达YopP的细菌（小肠结肠炎和沙门氏菌）将被用于调节dss诱导的新生儿结肠炎的炎症。此外，对小肠结肠炎发病至关重要的细胞类型将通过评估NF？B激活肠细胞感染后？YopP压力。儿童极易感染通过受污染食物传播的微生物。这个应用程序有两个主要目标。首先，我们将发现生命早期肠道免疫系统如何产生针对微生物的保护性反应；其次，我们将了解微生物本身的产物如何用于增强儿童疫苗反应和治疗肠道炎症，如儿童克罗恩病。

DESCRIPTION (provided by applicant): The vast majority of infectious agents are encountered at mucosal surfaces. Understanding the dynamic interface of immune cells and infectious microbes at these sites is one of the greatest challenges in the fields of microbiology and immunology. These interactions are especially important in neonatal life when microbial antigens are encountered for the first time. To study immune cell/bacterial interactions during the neonatal period, we have developed a novel murine model of orogastric infection with the extracellular bacterium Yersinia enterocolitica. Strikingly, neonates are markedly more resistant than adults to this enteropathogen, an observation unique among all other studied neonate-pathogen interactions. This system provides an unprecedented opportunity for learning how fully protective immunity is achieved in the neonatal intestine. Thus, a major goal of this application is to identify the eukaryotic immune mechanisms leading to resistance of neonates to intestinal exposure to Y. enterocolitica. A second major goal of this application is to apply Y. enterocolitica products to the regulation of neonatal intestinal immunity in health and disease. These defined goals will provide major new building blocks for our long term goals of (a) understanding how intestinal immunity develops during ontogeny, (b) achieving highly effective pediatric mucosal vaccines, and (c) developing novel strategies for the treatment of pathological intestinal inflammation in early life. Specific Aim 1 will define adaptive immune mechanisms underlying protection of neonates from oral Y. enterocolitica infection. The effects of neutralization of ?IFN and/or IL-17 responses on inflammatory cell infiltration, bacteremia, and relative weight loss associated with mortality will be examined. The importance of B cell responses will be tested by genetic and acute B cell ablation and in B cell-sufficient neonates unable to mount bacterial-specific antibody responses. Lastly, the contribution of natural Treg (CD4+CD25+) will be analyzed by adoptive transfer of adult Treg to infected neonates and by the in vivo depletion of the Treg compartment. Specific Aim 2 will exploit the Y. enterocolitica effector protein YopP (its presence or its absence) to manipulate neonatal immune responses and identify key target immune cell types in vivo. Since the ?YopP mutant strain induces profound inflammation in the neonatal intestine, we will test the capacity of a modified ?YopP mutant strain to act as a potent adjuvant for mucosal immunization. An ovalbumin - expressing ?YopP strain will be created and ovalbumin-specific antibody and Th responses will be monitored, as well as the in vivo activation of ova-specific TCR transgenic T cells. Second, the possibility that YopP, by itself, can downregulate pathogenic intestinal inflammation in neonates will be tested. Bacteria (Y. enterocolitica and Salmonella) engineered to express only YopP will be applied to modulate inflammation in DSS-induced colitis in neonates. In addition, cell types critical for Y. enterocolitica pathogenesis will be identified by assessing NF?B activation in intestinal cells following infection with the ?YopP strain. Children are highly susceptible to microbes transmitted through contaminated food. This application has two main objectives. First, we will discover how the intestinal immune system in early life develops protective responses against microbes; second, we will learn how products of the microbes themselves can be used to enhance childhood vaccine responses and treat intestinal inflammation, such as pediatric Crohn's disease.