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 FY14 we reported discoveries in the following areas: 1. invasive candidiasis;2. Behavior and 3. The primary immunodeficiency disease WHIM syndrome. 1.) In FY14, 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 FY14, 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 FY14, we published the first paper identifying Atypical Chemokine Receptor 1 (ACKR1), previously known as Duffy Antigen Receptor for Chemokines or DARC as a regulator of motor function and behavior in mice. ACKR1 stands out among chemokine receptors for high selective expression on cerebellar Purkinje neurons. Although ACKR1 ligands activate Purkinje cells in vitro, evidence for ACKR1 regulation of brain function in vivo had been lacking. We demonstrated that Ackr1 -/- mice have markedly impaired balance and ataxia on a rotating rod and increased tremor when injected with harmaline, which induces whole-body tremor by activating Purkinje cells. Ackr1 -/- mice also exhibited impaired exploratory behavior, increased anxiety-like behavior and frequent episodes of marked hypoactivity under low-stress conditions. Surprisingly, Ackr1 +/- had similar behavioral abnormalities, indicating pronounced haploinsufficiency. The behavioral phenotype of Ackr1 -/- mice was the opposite of mouse models of cerebellar degeneration, and the defects persisted when Ackr1 was deficient only on non-hematopoietic cells. Together, the results suggest that normal motor function and behavior may partly depend on negative regulation of Purkinje cell activity by Ackr1. 3. In FY14 we reported results of a Phase 1 clinical trial of plerixafor in patients with WHIM syndrome (Warts, hypogammaglobulinemia, infections, and myelokathexis syndrome), a rare immunodeficiency disorder caused by gain-of-function mutations in the G protein-coupled chemokine receptor CXCR4. The CXCR4 antagonist plerixafor, which is approved by the US Food and Drug Administration (FDA) for stem cell mobilization in cancer and administered for that indication at 0.24 mg/kg, has been shown in short-term (1- to 2-week) phase 1 dose-escalation studies to correct neutropenia and other cytopenias in WHIM syndrome. However, long-term safety and long-term hematologic and clinical efficacy data were lacking. Our results provided the first long-term clinical trial of plerixafor in any disease, in which 3 adults with WHIM syndrome self-injected 0.01 to 0.02 mg/kg (4% to 8% of the FDA-approved dose) subcutaneously twice daily for 6 months. Circulating leukocytes were durably increased throughout the trial in all patients, and this was associated with fewer infections and improvement in warts in combination with imiquimod. No drug-associated side effects were observed. These results provide preliminary evidence for the safety and clinical efficacy of long-term, low-dose plerixafor in WHIM syndrome and support its continued study as mechanism-based therapy in this disease. The ClinicalTrials.gov identifier for this study is NCT00967785. In FY14 we also made substantive contributions to collaborative work initiated by NIH colleagues leading to the discovery of a divergent variant of the 11th human polyomavirus species, Saint Louis polyomavirus (STLPyV), in a sanitized human skin wart specimen from a patient with WHIM syndrome. The result strongly suggests that STLPyV directly infects humans and is not simply a dietary contaminant. In FY14, we also organized, developed and published a major review on chemokine receptors including development of a new nomenclature for atypical chemokine receptors. We also filed a patent application describing CXCR4 haploinsufficiency as a method for enhancing HSC engraftment.
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