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 FY12 we reported discoveries in the following areas: 1. WHIM syndrome;2. invasive candidiasis;3. Trypanosoma cruzi infection;4. the gastrointestinal tract microbiome. 1.) We published three papers related to clinical studies of the severe combined immunodeficiency disorder WHIM syndrome. a. The first described results from a Phase 1 study of the safety of plerixafor, a specific CXCR4 antagonist, in the treatment of 3 adult patients with WHIM syndrome, which is caused by gain of function mutations in CXCR4. We found in a dose escalation format that the drug was safe for 7 days up to the dose approved by the FDA for the original indication, stem cell mobilization in patients being transplanted for cancer. In addition, we found that the drug rapidly mobilized into the blood almost all subsets of leukocytes for which the patients were deficient. Mobilization was transient and followed the pharmacokinetics of the drug, and was dose-dependent throughout the 7 day trial. These results justify testing the drug for chronic safety and hematologic efficacy at the lowest effective dose and eventually for clinical efficacy (wart resolution, reduced incidence of infections). b. The second paper described the first missense mutation, E343K, that causes WHIM syndrome. All previous CXCR4 mutations have been C-tail truncating mutations, including E343X, which terminates at codon 343. We identified three patients with this mutation from a single family, and demonstrated increased signaling in leukocytes stimulated with CXCR4 agonist CXCL12. This finding suggests that all cases of WHIM syndrome due to CXCR4 mutations could be due to loss or mutation of E343. c. The third paper was done in collaboration with Alison McBride of LVD, and reported the tenth known polyomavirus, isolated from a wart sample biopsied from a patient with WHIM syndrome. This opened up new questions regarding the role of polyomavirus in collaboration with HPV in the pathogenesis of warts in WHIM syndrome. 2.) We reported that IL-10 is critical for control of T. cruzi pathogenesis in a mouse model. We identified IL-10 as a target by comparing the clinical, microbiological, immunological and molecular correlates of infection of a sensitive vs a resistant mouse strain to this pathogen. We then tested IL-10 ko mice and found uniform mortality and uncontrolled parasitemia related to T cell expression of IL-10. A non-T cell source was also implicated but not defined. 3.) We found that mouse gut content, both in the large and small bowel, contains potent and effective leukocyte chemotactic and activating factors. The activities appeared to be due to distinct factors in large and small bowel, and in case appeared not to be a protein. In particular, N-formylpeptides, which are produced by all bacteria and are powerful activating factors at specific receptors expressed on phagocytes, do not account for the activity as assessed in Fpr1 ko mice. This work is significant since the gut contains huge numbers of bacteria which produced factors that may interact with the immune system of the gut both in health and disease, particularly in inflammatory bowel disease. Knowledge about these factors could lead to new treatments in IBD. 4.) We defined organ specific immunity in a mouse model of invasive candidiasis in the model, 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. We found that fatal neutrophil-dependent immunopathology is dependent on chemokine receptor Ccr1, which however does not effect candida control. The effect appears to be due to direct action of Ccr1 agonists in the kidney at blood neutrophil Ccr1 which is expressed late in the model, at a time when Ccr1-dependent neutrophil recruitment is observed. The results highlight the delicate balance between the requirement for neutrophils to control Candida, and the harmful effects of uncontrolled neutrophil accumulation and activation on the kidney tissue. The work is significant because it identifies Ccr1 as a potential drug target in invasive candidiasis affecting the kidney.
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