The aim of this project is to define the molecular mechanisms and biological contexts for blood leukocyte migration to specific tissue sites that are inflamed or infected. We have focused on chemoattractant proteins that mediate this process and have identified members of a large family of chemoattractant receptors that are deployed on the leukocyte cell surface. We have also identified members of a diverse group of chemoattractant and chemoattractant receptor mimics made by viruses, including herpesviruses, poxviruses and HIV. We use genomics, molecular biology, cell biology and epidemiology as the principle methods for analyzing these molecules. A major goal is to identify specific disease associations of individual chemoattractants and chemoattractant receptors, in order to identify potential new therapeutic targets. A key strategy is to analyze phenotypes of gene knockout mice in disease models as well as associations of loss of function mutations in the corresponding human genes in human disease cohorts. In FY13 we reported discoveries in the following areas: 1. Cataract pathogenesis;2. invasive candidiasis;3. atherosclerosis. 1.) In FY13, we continued our work published in FY11 and FY12 on organ specific immunity in a mouse model of invasive candidiasis, in which the all organs studied are infected to similar levels initially;however, only kidney cannot resolve the infection and is destroyed by a combination of uncontrolled pathogen growth and ineffective neutrophil accumulation, accounting for death in the model. In FY11, we published a descriptive paper about the immunopathology of the model and in FY12 a mechanistic paper defining an important role for chemokine receptor Ccr1 in mediating neutrophil-dependent immunopathology and death in the model. Now in FY13, we have identified Cx3cr1 as a critical chemokine receptor that promotes early fungal clearance and survival in the model by promoting accumulation of kidney macrophages. The main mechanism appears to be exerted through both hematopoietic and non-hematopoietic (or non-radio-resistant) cells, and monocytes and macrophages are the main hematopoietic cells in the kidney in the model that express Cx3cr1. We found that Cx3cr1 promotes survival of kidney macrophages in the model but does not appear to be important in killing capacity of the cell or their trafficking into the kidney. This is the first identification of a key molecular factor regulating the role of monocytes/macrophages in invasive candidiasis. Together our work to date in the model highlights the delicate balance between neutrophils and macrophages to control Candida mediated by distinct chemokine receptors, and the harmful effects that occur leading to mortality if either pathogen or immune cells are not regulated properly. The latest work is significant because it identifies humans with defective Cx3cr1 due to inheritance of the M280 allele as having increased risk of poor outcome in the context of invasive candidiasis. 2.) In FY13, we published the first paper identifying expression of chemoattractant Fpr1 on human lens epithelial cells. The work was undertaken because of our discovery of severe cataracts in Fpr1 knockout mice during ageing (not yet published). The phenotype was not related to inflammation or infection in the lens, so we asked whether lens cells could also express the receptor. The availability of lens epithelial cell lines facilitated the work, and the paper is basically a biochemical analysis of the receptor on these cells at the gene, RNA, protein and functional levels. Although we found evidence for the receptor at all levels, its properties were atypical compared to the properties of Fpr1 in leukocytes. In particular, expression is low and most of the binding sites appear to be non-specific. Nevertheless, surface expression by FACS appears high as does the ability of the cell to transduce signals in response to Fpr1 agonists. The significance of this finding is that it provides molecular plausibility for a direct role for Fpr1 in lens maintenance consistent with our unpublished cataract phenotype in Fpr1 ko mice. 3.) In FY13, we continued our long-term project on chemokine regulation of atherosclerosis. We found that the chemokine receptor Ccr7 is critical in control of this process in atherosusceptible mice lacking ApoE fed a high fat diet. We found that Ccr7 operated through expression on hematopoietic cells and regulated accumulation of T cells in the vessel wall. The significance of this finding is that it identifies a molecular factor important in the adaptive immune response in the vessel wall and identifies a potential target for modulation in the disease. In FY13 we also made substantive contributions to collaborative work initiated by NIH colleagues on the role of Fpr2 in colitis and colitis associated tumorigenesis, the role of Fpr1 in control of dysbiosis and mortality in T. gondii infection and in control of Listeria infection in the liver, and the lack of evidence that CCR5 deficiency modulates risk of severe polio infection. In FY13, we also organized and developed a major review on chemokine receptors including development of a new nomenclature for atypical chemokine receptors.
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