Objectives: [Cryptococcus neoformans (Cneo) is the leading cause of fatal fungal infection in AIDS patients and the second most common fungal infection in transplant patients. In surviving patients, failure to eradicate Cneo, or similar pulmonary pathogens, often results in persistent infection associated with chronic lung damage. Current therapies are lengthy, have limited efficacy, and are often toxic. Impaired or aberrant host immune responses are a prerequisite for failed pathogen clearance and contribute to the lung damage associated with persistent infections. Yet these responses are poorly understood.] The present studies are designed to determine how immunomodulatory monocyte-derived lung dendritic cells (i-moDC) orchestrate the cellular and molecular networks which impair Cneo clearance and promote persistent pulmonary infections and chronic lung inflammation. [Improved understanding of these aberrant immune responses may yield novel therapies for the treatment of pulmonary infections caused by Cneo or related pathogens.] Hypothesis: [Susceptibility and persistence in response to cryptococcal lung infection] results from the aberrant development of i-moDC signaling through pathways involving IL-10, alternative activation gene products, and PD-1/PD-L1. The following aims have been constructed to rigorously test this hypothesis.
Aim 1 : To determine whether autocrine monocyte-derived DC IL-10 production is necessary and sufficient to up-regulate expression of alternatively-activated genes and PD-L1/PD-L2 in response to cryptococcal antigens (in vitro) or active fungal lung infection (in vivo).
Aim 2 : To determine whether pulmonary i-moDC promote the polarization and expansion of T regulatory cells [in susceptible mice that develop persistent lung infection.] Aim 3: To determine whether blockade of IL-10 or the PD-1/PDL-1 signaling pathways [improves fungal clearance and] prevents persistent cryptococcal lung infection. Research Plan and Methods: Our proposal utilizes a well-established murine model of cryptococcal lung infection in susceptible C57BL/6 mice characterized by an accumulation of immunomodulatory dendritic cells, an expansion in T regulatory and Th2 cells, and persistent infection associated with immune-mediated lung damage. The contribution of lung dendritic cells to [failed fungal clearance and] the persistently-infected phenotype will be determined using the following experimental techniques: 1) comparative flow cytometric analysis and cell sorting, 2) qPCR performed on isolated lung leukocytes, 3) fungal CFU analysis, 4) histology, 5) in vitro cell culture and co-culture, and 6) in vivo adoptive cell transfers. Our investigations will be enhanced by our selected use of: a) IL-10 deficient mice, b) IL-10 reporter mice (Vert-X mice), c) F1 generation mice (obtained from breeding C57BL/6 mice and BALB/c mice), d) transgenic mice containing the diphtheria toxin receptor permitting cell-specific depletion following diphtheria toxin administration, e) transgenic mice containing a floxed IL-10 gene permitting cell specific IL-10 deficiency, and f) administration of anti-IL-10 and anti-PD-L1 receptor blocking antibodies to disrupt immunomodulatory signaling networks [and improve fungal clearance.]

Public Health Relevance

Fungal lung infections are a major source of morbidity and mortality in veterans with compromised immune systems as a result of AIDS, substance abuse, or stemming from treatment of malignancy, auto-immune disease, or organ transplantation. Persistent fungal lung infections in immunocompetent or mildly immunocompromised veterans results in chronic lung disease. Antibiotic therapy is often inadequate;thus knowledge of pulmonary host defense is of central importance to the development of new therapies or vaccine strategies to treat this patient population. The proposed studies will use mouse models of fungal lung infections to advance our basic understanding of pulmonary host defense;our results will shed insight into other inflammatory conditions within the lung including COPD, asthma, and possibly pulmonary fibrosis.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Veterans Health Administration
Ann Arbor
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Xu, Jintao; Eastman, Alison J; Flaczyk, Adam et al. (2018) Erratum for Xu et al., ""Disruption of Early Tumor Necrosis Factor Alpha Signaling Prevents Classical Activation of Dendritic Cells in Lung-Associated Lymph Nodes and Development of Protective Immunity against Cryptococcal Infection"". MBio 9:
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
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
Chen, Gwo-Hsiao; Teitz-Tennenbaum, Seagal; Neal, Lori M et al. (2016) Local GM-CSF-Dependent Differentiation and Activation of Pulmonary Dendritic Cells and Macrophages Protect against Progressive Cryptococcal Lung Infection in Mice. J Immunol 196:1810-21
Xu, Jintao; Eastman, Alison J; Flaczyk, Adam et al. (2016) Disruption of Early Tumor Necrosis Factor Alpha Signaling Prevents Classical Activation of Dendritic Cells in Lung-Associated Lymph Nodes and Development of Protective Immunity against Cryptococcal Infection. MBio 7:
Davis, Michael J; Eastman, Alison J; Qiu, Yafeng et al. (2015) Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence. J Immunol 194:2219-31

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