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 chemoattractant 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 FY11 we reported discoveries in the following areas: 1. West Nile virus pathogenesis;2. atherosclerosis;3. immunodeficiency and 4. behavior;5. candidiasis;6. identifying chemoattractant receptor genes;and 7. MRSA infection. 1.) We found evidence that the monocyte chemokine receptor Ccr2 is critical for survival and for selective inflammatory monocyte recruitment to brain in a mouse model of WNV encephalitis. WNV induces a selective monocytosis in the model that is completely Ccr2 dependent. We found that monocyte trafficking into the brain appears to involve two Ccr2-dependent processes: monocyte trafficking from bone marrow to blood and from blood to brain. This extends our previous work in this area that identified CCR5 and OAS1 as protective factors for symptomatic WNV infection. The recent work is important because it provides new insight into the mechanism by which CCR2 complements CCR5 in control of symptomatic WNV disease. The new data suggest that blocking CCR2 with a drug may increase the risk of symptomatic WNV disease should treated patients become infected with WNV. 2.) We found evidence that Ccr6 positively regulates atherogenesis in a mouse model based on diet and ApoE deficiency. The contribution of Ccr6 was strong compared to other established chemokine receptors in this model. The mechanism may involve Ccr6 control of monocytosis in ApoE deficient mice and Ccr6 regulation of monocyte chemotaxis. We showed that primary mouse monocytes express functional Ccr6. Bone marrow transfer studies established that Ccr6 is operating on a hematopoietic cell in the model, but the work left open the possibility that Ccr6 may function in the model at the level of other types of leukocytes besides monocytes. The importance of the finding is in identifying Ccr6 as a potential target for drug development in atherosclerotic cardiovascular disease. 3.) We identified two sibling children with a newly discovered Mendelian disease called G6PC3 deficiency. We showed that myelokathexis is a feature of this disease, which may explain in part the severe neutropenia and increased susceptibility to infection by bacterial pathogens. We found that the myelokathexis may result from increased expression on neutrophils of CXCR4 a known bone marrow home receptor for neutrophils. We also reported that the FDA-approved drug Mozobil (AMD3100) is fully effective as neutrophil mobilizing agent in mice with G6pc3 deficiency, and that it is also fully active at the mutant gain of function form of CXCR4 found in most patients with WHIM syndrome, a disease also characterized by myelokathexis. We have an approved open Phase 1 protocol to study the safety and efficacy of Mozobil in patients with WHIM syndrome. 4.) We found that mice lacking the chemoattractant receptor Fpr1 are hyperactive. The mice appear to have impaired fear memory and anxiety like behavior, and this may be due to abnormal regulation of the HPA axis resulting in increased corticosterone levels, a known modifier of anxiety like behavior in mice. This is the first work describing a functional role of a leukocyte chemoattractant receptor in behavior, and suggests that these receptors multitask outside of the immune system. 5.) We reported that the response of the immune system to systemic candida challenge is highly organ specific, with kidney alone unable to control the infection and the immunopathology. Other organs receiving an equal candida burden become infiltrated by neutrophils but in a transient, non-pathologic and highly idiosyncratic manner. 6.) We characterized a novel and atypical member of the FPR family of chemoattractant receptor genes. The gene named Fpr-rs8 is a duplication of Fpr-rs2, but is markedly truncated. We describe distinct evolutionary histories of the two genes, and argue that Fpr-rs8 is not a pseudogene, because it is transcribed and regulated, the protein appears to be made, and mice lacking it have a 30% shortened life-span. Fpr-rs8 appears to be restricted to the intracellular space and probably does not signal. 7.) We also discovered a mechanism by which a pathogen exploits a host defense factor to induce its own virulence factor. In particular, S. aureus is able to bind specific host chemokines which induce release of the virulence factor Staph protein A. This was defined in vitro but we have data attesting to its biological relevance in mouse models of S. aureus abscess formation and patient samples. 8.) Lastly, we published a preliminary report identifying self-administered hyperventilation as a quick-acting, free, easy to implement, safe and highly effective treatment for patients suffering from severe exercise-associated muscle cramping awakening them from sleep. The technique may also work for other forms of cramping.
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