Molecular Analysis Of Leukocyte Activation By Chemoattractants

National Institute Of Allergy And Infectious Diseases

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Abstract

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 phenotypes in patients with mutations in human chemoattractant system genes. 1.) In FY22, we reported a case of severe neutropenia in a 6-year-old boy. The patient has a classic CXCR4 mutation consistent with WHIM syndrome. The case illustrates the heterogeneity of presentation in this disorder. 2.) In FY22, we reported the clinical and hematologic effects of endotoxin in WHIM Syndrome model mice. Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome immunodeficiency is caused by autosomal dominant gain-of-function CXCR4 mutations that promote severe panleukopenia caused by bone marrow retention of mature leukocytes. Consequently, WHIM patients develop recurrent bacterial infections; however, sepsis is uncommon. To study this clinical dichotomy, we challenged WHIM model mice with LPS. The LD50 was similar in WHIM and wild-type (WT) mice, and LPS induced acute lymphopenia in WT mice that was Cxcr4 independent. In contrast, in WHIM mice, LPS did not affect circulating T cell levels, but the B cell levels anomalously increased because of selective, cell-intrinsic, and Cxcr4 WHIM allele-dependent emergence of Cxcr4high late pre-B cells, a pattern that was phenocopied by Escherichia coli infection. In both WT and WHIM mice, the CXCR4 antagonist AMD3100 rapidly increased circulating lymphocyte levels that then rapidly contracted after subsequent LPS treatment. Thus, LPS-induced lymphopenia is CXCR4 independent, and a WHIM mutation does not increase clinical LPS sensitivity. Anomalous WT Cxcr4-independent, but Cxcr4 WHIM-dependent, promobilizing effects of LPS on late pre-B cell mobilization reveal a distinct signaling pathway for the variant receptor. 3.) In FY22, we reviewed a new discovery from the lab involving chemokine binding to phosphatidylserine for apoptotic cell clearance. This is a follow up to our 2021 paper in PLoS Biology. 4.) In FY22, we also contributed to collaborative work using genomics to identify novel phenotypes and diseases in immunodeficiency patients. In particular, as detailed in the FY21 annual report, this effort led to the discovery of SASH3 deficiency as a novel inborn error of immunity. In FY22, the section also advanced its work on T.cruzi pathogenesis, Mouse cytomegalovirus pathogenesis, novel treatment for WHIM syndrome, gene therapy for WHIM syndrome, a novel preclinical bone marrow conditioning protocol that allows universal transplantation, and new work on covid-19 pathogenesis; however, these projects are ongoing and did not result in published work in this reporting period.