Olszewski M.A.: Modulation of Pulmonary Defenses in Pathobiology of Chronic Infections Objectives: Macrophages (M) are crucial for either clearance or persistence of the opportunistic yeast C. neoformans. When properly activated, M generate fungicidal nitric oxide from L-arginine to destroy ingested cryptococci. In unfavorable clinical circumstances, M become alternatively activated and induce Arginase (ARG1), an intracellular enzyme that consumes L-arginine but does not induce nitric oxide. The proposed studies will focus on dysregulation of M function by C. neoformans, and the role of recently defined virulence factor, heat shock protein Ssa1 in this porcess. Our objective is to test the hypothesis that Ssa1, induces the alternative activation of M and in this fashion promotes rapid growth of C. neoformans in the lungs. We plan to determine, if Ssa1-induced effects are mediated through Ssa1-binding to the scavenger receptor A (SRA) on M and through subsequent upregulation of ARG1 in these cells. Research Plan: The proposed research is presented as three interrelated specific aims: 1) To determine if cryptococcal Ssa1 promotes alternative activation of MF. We propose a series of experiments with cultures of isolated murine M to analyze the effects of recombinants Ssa1 protein and the effects of Ssa1 expression by C. neoformans on M activation status. These in vitro studies will be validated by in vivo infections with Ssa1 producing and Ssa1-deleted mutants of C. neoformans in mice and M phenotype analysis. 2) To determine if the mechanism alternative activation of MF by Ssa1 is induced via the SRA signaling. In this aim, we will determine if SRA signaling is required for Ssa1-induced effects and for the changes in M activation status. 3) To determine if the effects of Ssa1 gene expression by C. neoformans on MF function can be reversed by ARG1 inhibition or manipulations with cytokine environment.
Our final aim will explore, if preventing the alternative activation of M by gamma- interferon and/or blocking Arginase1 enzyme would prevent the Ssa1-mediated virulence and lead to improved killing of C. neoformans by M. Methods: Models of murine C. neoformans infections and the isolated mouse M cell cultures, which yielded significant insights into the host-pathogen interaction and understanding mechanisms of anticryptococcal protection, will be used. Ssa1 producing wild type strain of C. neoformans: H99;Ssa1 knockout strain derived from H99;and the revertant strain ssa1::SSA1 with restored SSA1 gene will be used in these studies to induce pulmonary infections in mice or to treat the primary M cultures. Cryptococcal recombinant Ssa1 protein will be also used to treat these cells. Biology of M, cell phenotypes and cytokines produced by these cells will be evaluated in vivo and in vitro, utilizing: 6-color flow cytometry, qPCR and ELISA. Production of fungicidal nitric oxide by M will be evaluated using flow-ctometry based DAF-DT assay. We will use the knockout mice with disrupted SRA gene to determine if SRA signaling contributes to alternative activation of M by C. neoformans. Clinical Relevance: Pulmonary infectious diseases are significant source of morbidity in patients with HIV, organ transplant recipients, alcoholics, IV substance abusers and patients with lymphoproliferative malignancies. Treatment of fungal infections with conventional anti-microbial agents in these patients has been disappointing. Our studies reveal how a major opportunistic fungal pathogen C. neoformans may exploit the loopholes in M signaling to establish intracellular parasitism and propose a treatment strategy to prevent its occurrence.
Relevance to the Veterans Health: Pulmonary infectious diseases are a major source of morbidity and mortality in VA patients with compromised immune systems as a result of AIDS, substance abuse, or stemming from treatment of malignancy, auto-immune disease, or organ transplantation. Antibiotic therapy is often inadequate;thus knowledge of pulmonary host defense is of central importance to the development of new therapies Our studies reveal how a major opportunistic fungal pathogen C. neoformans may exploit the loopholes in the immune system defenses to establish intracellular parasitism and propose a treatment strategy to prevent its occurrence. Demonstrating importance of these pathways may become a cornerstone for novel therapeutic strategies for treatment of cryptococcosis in both immunocompromised and immunocompetent patients.
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