Visualization of CXCR3 allelic usage in vivo

描述(申请人提供)：CXC趋化因子受体3(CXCR3)表达于：浆细胞样树突状细胞(PDCs)、嗜酸性粒细胞、NK细胞、自然调节T细胞、CD4和CD8T细胞。该受体的三个主要配体是CXC趋化因子CXCL9(Mig)、CXCL10(IP-10)和CXCL11(I-TAC)，它们在炎症过程中高水平产生。CXCR3通过调节效应器T细胞的募集和功能来介导对病原体如大利什曼病的免疫。然而，CXCR3也通过促进致病免疫细胞的募集，在同种异体移植排斥反应和许多自身免疫性疾病的发病机制中发挥作用。CXCR3基因被定位在X染色体上，因此在女性中通过随机X染色体失活(XCI)进行单等位基因的表达。然而，有充分的证据表明，许多X连锁基因“逃脱”了X染色体的失活，并在两条X染色体上表达。如果CXCR3是免疫细胞中逃脱X失活的基因之一，那么性别之间的CXCR3等位基因表达就会有数量上的差异。这可能会导致各种疾病的性别差异。对传染病和自身免疫性疾病易感性的性别差异已经有了很好的记录。女性比男性对利什曼原虫等细胞内病原体引起的感染更具抵抗力，但她们更容易患上类风湿性关节炎等自身免疫性疾病。我们的实验室正在研究CXCR3在不同形式的利什曼病中的作用，并有兴趣确定它在女性对这种疾病的性别相关抵抗力中的贡献。我们感兴趣的是在体内可视化不同免疫细胞使用的CXCR3等位基因，以确定CXCR3基因是经历X失活还是逃脱X失活。如果我们能够追踪小鼠体内表达CXCR3的细胞，同时可视化导致CXCR3表达的等位基因，我们在体内研究这一复杂过程的能力将大大增强。这个R03应用程序的目标是产生一个CXCR3-EFGP/RFP(CXCR3EGFP/RFP)双报告小鼠，携带分别与EGFP和RFP连锁的CXCR3等位基因(目标1)，然后使用实验性的大型乳杆菌感染模型(目标2)跟踪免疫细胞中CXCR3等位基因的使用(目标2)。我们最近培育了一只CXCR3-双顺反子EGFP报告基因(CIBER)小鼠，它通过病毒IRES元件表达与增强型绿色荧光蛋白(EGFP)相连的CXCR3。我们的初步数据显示，通过流式细胞仪和显微镜可以很容易地在CIBER小鼠体内追踪到CXCR3表达的细胞。在本项目中，我们将制备一只CXCR3-红色荧光蛋白(RFP)报告小鼠，然后将其与CIBER小鼠杂交，获得CXCR3EGFP/RFP小鼠。这种双重报告小鼠将携带一个与EGFP连锁的CXCR3等位基因，另一个与RFP连锁的CXCR3等位基因。我们的初步数据和CIBER小鼠的可用性证明了在两年内实现该项目目标的可行性。CXCR3 EGFP/RFP报告小鼠将使我们能够直观地看到CXCR3等位基因在感染期间在各种免疫细胞类型中的差异表达。这些小鼠也将是其他研究人员研究CXCR3在感染性、炎症性和肿瘤性疾病中的体内作用的宝贵工具。 公共卫生相关性：CXC趋化因子受体3对感染和自身免疫性疾病相关的白细胞趋化和炎症起关键作用。该项目将开发一种新的小鼠品系，可以显示CXCR3表达细胞，并研究活体动物在炎症和感染过程中CXCR3基因的调节。这些信息将对开发治疗炎症性和感染性疾病的免疫治疗方法非常重要。

DESCRIPTION (provided by applicant): The CXC chemokine receptor 3 (CXCR3) is expressed on: plasmacytoid dendritic cells (pDCs), eosinophils, NK cells, natural regulatory T cells, CD4+ and CD8+ T cells. The three main ligands of this receptor are the CXC chemokines CXCL9 (Mig), CXCL10 (IP-10) and CXCL11 (I-TAC), which are produced in high levels during inflammation. CXCR3 mediates immunity against pathogens such as Leishmania major by regulating the recruitment and function of effector T cells. However, CXCR3 also contributes to the pathogenesis of allograft rejection and many autoimmune diseases by promoting recruitment of pathogenic immune cells. The CXCR3 gene is mapped to chromosome X, and is therefore subject to monoallelic expression in females through random X chromosome inactivation (XCI). However, it is well documented that a number of X-linked genes "escape" X-chromosome inactivation and are expressed from both X chromosomes. If CXCR3 is amongst the genes which escape X-inactivation in immune cells, there will be quantitative differences in CXCR3 allele expression between the sexes. This could contribute to sex-associated differences in a variety of diseases. Gender differences in susceptibility to infectious and autoimmune diseases are well documented. Females are more resistant than males to infections caused by intracellular pathogens such as Leishmania, but they are more prone to autoimmune diseases such as rheumatoid arthritis. Our laboratory is studying the role of CXCR3 in different forms of leishmaniasis and is interested in determining its contribution to gender- associated resistance of females to this disease. We are interested in visualizing CXCR3 alleles that are used by different immune cells in females in vivo to determine whether the CXCR3 gene undergoes X-inactivation or escapes it. Our ability to study this complex process in vivo will be greatly enhanced if we are able to track CXCR3 expressing cells in mice and simultaneously visualize the alleles responsible for CXCR3 expression. The goals of this RO3 application are to generate a CXCR3-EFGP/RFP (CXCR3EGFP/RFP) dual reporter mouse carrying CXCR3 alleles which are linked to EGFP and RFP, respectively (Aim 1), and then to track the use of CXCR3 alleles in immune cells using an experimental L. major infection model (Aim 2). We have recently generated a CXCR3-bicistronic EGFP reporter (CIBER) mouse which expresses CXCR3 linked to enhanced green fluorescent protein (EGFP) via a viral IRES element. Our preliminary data show that CXCR3 expressing cells can be easily tracked in CIBER mice by flow cytometry and microscopy. In this project, we will generate a CXCR3-red fluorescent protein (RFP) reporter mouse and then cross it with a CIBER mouse to obtain a CXCR3EGFP/RFP mouse. This dual reporter mouse will carry one CXCR3 allele linked to EGFP and the other to RFP. Our preliminary data and the availability of CIBER mice demonstrate the feasibility of accomplishing the goals of this project within 2 years. CXCR3 EGFP/RFP reporter mice will allow us to visualize the differential expression of CXCR3 alleles in various immune cell types during infection. These mice will also be invaluable tools for other investigators to study the in vivo role of CXCR3 in infectious, inflammatory and neoplastic diseases. PUBLIC HEALTH RELEVANCE: The CXC chemokine receptor 3 is critical of leukocyte chemotaxis and inflammation associated with infection and autoimmune diseases. This project will develop a novel mouse strain which will allow visualization of CXCR3 expressing cells and study regulation of CXCR3 gene in living animal during inflammation and infection. This information will be important for developing immunotherapeutic approaches to treat inflammatory and infectious diseases.