Author ORCID Identifier

https://orcid.org/0000-0001-7551-2367

Date Available

9-21-2017

Year of Publication

2017

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Microbiology, Immunology, and Molecular Genetics

First Advisor

Dr. Beth Garvy

Abstract

Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts, including AIDS patients. Pneumocystis species have a biphasic life cycle consisting of single-nucleated trophic forms and ascus-like cysts. Both stages live within the host, and, thus, must contend with threats from the host immune system. The cyst cell wall β-glucans have been shown to stimulate immune responses in lung epithelial cells, dendritic cells and alveolar macrophages. Little is known about how the trophic life forms, which do not have a fungal cell wall, interact with immune cells. In this study, the immune response to the life cycle stages of Pneumocystis murina was evaluated.

Here, we report differences in the immune response of immunocompetent mice to the trophic and cystic life cycle stages of P. murina. Upon infection with purified trophic forms, wild-type adult mice developed a delayed innate and adaptive immune response compared to inoculation with the normal mixture of trophic forms and cysts. Cysts, but not trophic forms, stimulated Th1-type responses in the lungs of infected mice.

Surprisingly, trophic forms are sufficient to generate protective adaptive responses, leading to clearance in immunocompetent mice. We report that CD4+ T cells primed in the presence of trophic forms are sufficient to mediate clearance of trophic forms and cysts. In addition, primary infection with trophic forms is sufficient to prime B cell memory responses capable of clearing a secondary infection with Pneumocystis following CD4+ T cell depletion. While trophic forms are sufficient for initiation of adaptive immune responses in immunocompetent mice, infection of immunocompromised RAG2-/- mice with trophic forms in the absence of cysts does not lead to the severe weight loss and infiltration of innate immune cells associated with the development of Pneumocystis pneumonia.

Dendritic cells screen the alveolar spaces for pathogens, and are in a prime position to initiate the immune response against lung pathogens, including Pneumocystis. Our data demonstrate that trophic forms broadly dampen the ability of dendritic cells to respond to pathogen-associated molecular patterns. Bone marrow-derived dendritic cells were stimulated with trophic forms, a mixture of trophic forms and cysts, and various other inflammatory materials, including β-glucan. Trophic forms inhibited multiple components involved in antigen presentation by dendritic cells, including secretion of inflammatory cytokines and expression of MHC class II and costimulatory molecules on the cell surface. Furthermore, trophic forms suppressed or failed to induce the expression of multiple genes related to activation and maturation in dendritic cells. Dendritic cells silenced by trophic forms are unable to induce CD4+ T cell responses. These data suggest that immune evasion by trophic forms is dependent on the suppression of innate responses, and the development of adaptive immunity represents a “point of no return” at which the trophic forms are no longer able to escape clearance.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2017.402

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