Characterization of CCL28,CXCL14, and CXCL17, three mucosal chemokines

描述(申请人提供)：粘膜部位包括口腔、气管和支气管、消化系统和女性生殖系统。在这些部位形成的免疫反应对于抵御潜在的病原体非常重要。趋化因子是一种小的分泌蛋白，调节免疫系统细胞的迁移。我们已经确定了三种趋化因子(CXCL14、CXCL17和CCL28)在粘膜组织中表达最高。我们推测，它们在这些粘膜部位一定有重要的功能。在特定的目标1中，我们将利用研究这些趋化因子的遗传缺陷的小鼠的粘膜免疫系统来表征它们的功能。我们已经获得了一只CXCL14(-/-)小鼠，我们可以获得一只现有的CCL28(-/-)小鼠(可从Deltagen获得)，我们将从现有的胚胎干细胞中生产出CXCL17(-/-)，其中CXCL17基因已通过同源重组失活(可通过TIGM获得)。我们还将用两种细菌病原体(衣原体和沙门氏菌)感染这些小鼠，以研究这些趋化因子是否在针对这些病原体的免疫中发挥作用。我们已经观察到，在CXCL14(-/-)小鼠中，肠道中的IgA水平严重降低。因此，我们假设CXCL14是介导产生IgA的细胞归巢到肠道的主要趋化因子。在具体目标2中，我们将研究CXCL14控制肠道中IgA产生的机制。我们将研究这些小鼠肠道中正常存在的产生IgA的细胞是否存在。我们还将研究M细胞以及与肠道IgA反应有关的其他蛋白质的表达(如RANKL)，以及小鼠肠道中是否存在已知的其他免疫系统细胞(Th17，Tregs)。我们还将研究外源性给予CXCL14是否可以逆转CXCL14(-/-)小鼠肠道IgA的缺乏。其中两个趋化因子(CXCL14和CXCL17)没有已知的受体。因此，在特定的目标3中，我们将在两种细胞类型(未成熟树突状细胞和单核细胞)中表达的各种孤儿G蛋白偶联受体中寻找它们的受体，这些受体对这些趋化因子有反应。我们将通过将GPCRs导入适当的宿主细胞并检测趋化因子特异性诱导的钙离子通量来进行筛选。重要的是，通过这些实验，我们将识别一种新的趋化因子受体。其他趋化因子受体与人类疾病有关，是药物开发的潜在靶点。这些结果将导致对这些尚未被认识的粘膜趋化因子在粘膜免疫反应发展中所起作用的新的见解。 公共卫生相关性：这项建议旨在了解三种趋化因子(CXCL14、CXCL17、CCL28)的功能，这三种趋化因子在人体粘膜组织中高表达。这包括寻找其中两种趋化因子的受体，研究它们的杀微生物活性和在免疫反应中的作用，特别是那些在肠道和其他粘膜组织中发育的免疫反应。

DESCRIPTION (provided by applicant): The mucosal sites include the oral cavity, trachea and bronchi, the digestive system and the female reproductive system. The immune responses that develop in these sites are important to fend off potential pathogens. The chemokines are small secreted proteins that regulate the migration of cells of the immune system. We have identified three chemokines (CXCL14, CXCL17 and CCL28) as the most highly expressed in mucosal tissues. We hypothesize that they must have important functions in these mucosal sites. In Specific Aim 1, we will use study the mucosal immune system of mice genetically deficient for each of these chemokines in order to characterize their functions. We already have access to a CXCL14 (-/-) mouse, we can obtain an existing CCL28 (-/-) mouse (available from Deltagen) and we will produce a CXCL17 (-/-) from existing embryonal stem cells where the CXCL17 gene has been inactivated by homologous recombination (available through TIGM). We will also infect these mice with two bacterial pathogens (Chlamydia and Salmonella) to investigate whether these chemokines play a role in immunity against these pathogens. We have already observed that in the CXCL14 (-/-) mouse, the levels of IgA in the gut are severely reduced. Therefore, we hypothesize that CXCL14 is the main chemokine mediating the homing of IgA producing cells to the gut. In Specific Aim 2, we will study the mechanism through which CXCL14 controls IgA production in the gut. We will study whether the IgA-producing cells normally present in the gut exist in these mice. We will also study the M cells as well as the expression of other proteins that have been implicated in gut IgA responses (such as RANKL) as well as for the presence of other immune system cells known to exist in the mouse gut (Th17, Tregs). We will also study whether exogenously administered CXCL14 can reverse the lack of gut IgA in CXCL14 (-/-) mice. Two of these chemokines (CXCL14 and CXCL17) have no known receptors. Therefore, In Specific Aim 3, we will search for their receptors among various orphan G-Protein coupled receptors expressed in two cell types (immature dendritic cells and monocytes) that respond to these chemokines. We will screen by transfecting the GPCRs in appropriate host cells and detecting calcium fluxes specifically induced by the chemokines. Importantly, through these experiments we will identify a new chemokine receptor. Other chemokine receptors have been implicated in human diseases, and represent potential targets for pharmaceutical development. These results will lead to new insights into the role that these as yet unrecognized mucosal chemokines play in the development of mucosal immune responses. PUBLIC HEALTH RELEVANCE: This proposal aims to understand the function of three chemokines (CXCL14, CXCL17, CCL28) that are highly expressed in human mucosal tissues. This includes looking for the receptor of two of these chemokines, studying their microbicidal properties and roles in immune responses, particularly those that develop in the gut and other mucosal tissues.