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.
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.
