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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
5I01BX000656-03
Application #
8259074
Study Section
Respiration (PULM)
Project Start
2010-04-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
3
Fiscal Year
2012
Total Cost
Indirect Cost
Name
Veterans Health Administration
Department
Type
DUNS #
096318480
City
Ann Arbor
State
MI
Country
United States
Zip Code
48105
Teitz-Tennenbaum, Seagal; Viglianti, Steven P; Roussey, Jonathan A et al. (2018) Autocrine IL-10 Signaling Promotes Dendritic Cell Type-2 Activation and Persistence of Murine Cryptococcal Lung Infection. J Immunol 201:2004-2015
So, Yee-Seul; Jang, Juyeong; Park, Goun et al. (2018) Sho1 and Msb2 Play Complementary but Distinct Roles in Stress Responses, Sexual Differentiation, and Pathogenicity of Cryptococcus neoformans. Front Microbiol 9:2958
Wang, Shiwen; Yang, Feng; Li, Dong et al. (2018) Clinical application of a multiplex genetic pathogen detection system remaps the aetiology of diarrhoeal infections in Shanghai. Gut Pathog 10:37
Neal, Lori M; Xing, Enze; Xu, Jintao et al. (2017) CD4+ T Cells Orchestrate Lethal Immune Pathology despite Fungal Clearance during Cryptococcus neoformans Meningoencephalitis. MBio 8:
Xu, Jintao; Flaczyk, Adam; Neal, Lori M et al. (2017) Scavenger Receptor MARCO Orchestrates Early Defenses and Contributes to Fungal Containment during Cryptococcal Infection. J Immunol 198:3548-3557
Roussey, Jonathan A; Viglianti, Steven P; Teitz-Tennenbaum, Seagal et al. (2017) Anti-PD-1 Antibody Treatment Promotes Clearance of Persistent Cryptococcal Lung Infection in Mice. J Immunol 199:3535-3546
Xu, Jintao; Flaczyk, Adam; Neal, Lori M et al. (2017) Exploitation of Scavenger Receptor, Macrophage Receptor with Collagenous Structure, by Cryptococcus neoformans Promotes Alternative Activation of Pulmonary Lymph Node CD11b+ Conventional Dendritic Cells and Non-Protective Th2 Bias. Front Immunol 8:1231
Neal, Lori M; Qiu, Yafeng; Chung, Jooho et al. (2017) T Cell-Restricted Notch Signaling Contributes to Pulmonary Th1 and Th2 Immunity during Cryptococcus neoformans Infection. J Immunol 199:643-655
Malachowski, Antoni N; Yosri, Mohamed; Park, Goun et al. (2016) Systemic Approach to Virulence Gene Network Analysis for Gaining New Insight into Cryptococcal Virulence. Front Microbiol 7:1652
Zhang, Yanmei; Wang, Shiwen; Hu, Binjie et al. (2016) Direct detection of Helicobacter pylori in biopsy specimens using a high-throughput multiple genetic detection system. Future Microbiol 11:1521-1534

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