GSBS Dissertations and Theses

Approval Date

10-26-2010

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Molecular Genetics and Microbiology

Subjects

Toll-Like Receptor 9; Aspergillus fumigatus; Aspergillosis; Dendritic Cells; Dissertations, UMMS

Abstract

The opportunistic fungus, Aspergillus fumigatus, is a leading cause of morbidity and mortality among the immunocompromised population. Experimental and clinical findings have established that phagocytic defenses are critical in the recognition and clearance of A. fumigatus. Previous studies found that Toll-like receptors (TLRs), specifically TLR2 and TLR4, were essential in the detection of the mold. Furthermore, one study found that mice deficient in TLR9 lived longer than their wild-type counterparts following challenge with A. fumigatus. We sought to determine the role of TLR9 during A. fumigatus infection. Our results show that A. fumigatus contains unmethylated CpG DNA, the natural ligand of TLR9. Furthermore, A. fumigatus DNA stimulates a potent pro-inflammatory response in mouse bone marrow derived dendritic cells (BMDCs) and human plasmacytoid dendritic cells (pDCs). A genome wide analysis showed that A. fumigatus DNA contains 87 human and 23 mouse putative immunostimulatory motifs. The response to A. fumigatus DNA is TLR9-dependent, as BMDCs from TLR9-/- mice were unresponsive to the fungal DNA. In addition, HEK293 cells cotransfected with human TLR9 and NFκB driven Luciferase conferred responsiveness to A. fumigatus CpG-rich sequences found in the fungal DNA. Our results show that TLR9 detects A. fumigatus DNA, resulting in the secretion of proinflammatory cytokines.

While pDCs secrete IFNα in response to A. fumigatus DNA, these cells have been mainly described to play critical roles in the antiviral responses. The role of pDCs during fungal infections remains to be elucidated. Our data show that CD304+ peripheral blood pDCs challenged with A. fumigatus hyphae secrete large concentrations of IFNα and TNFα in response to infection. Furthermore, the response appears to be TLR9- independent. However, pDCs spread over the hyphae and inhibit fungal growth. Furthermore, pDCs undergo cell lysis upon incubation with A. fumigatus. The antifungal activity of the pDCs was retained in the cell lysates, suggesting that this response was mediated by an intracellular factor. Addition of exogenous Zn2+, but not Fe3+, partially restores hyphal growth. In addition, western blot of pDC lysates show that these cells have the Zn2+-binding protein calprotectin.

Over 60% cell death is observed in the pDC population following a 2 hour incubation with A. fumigatus. The observed pDC cell death can be partially attributed to gliotoxin, as pDCs challenged with A. fumigatus stains deficient in production of the mycotoxin result in decreased pDC cytotoxicity. Furthermore, pDC cell death occurs independent of contact with the mold, confirming that pDC cell death is mediated by a secreted fungal factor. In addition, our results show that pDCs are required for the host response against A. fumigatus. Mice depleted of their pDCs are more susceptible to A. fumigatus infection than the control counterparts, suggesting that pDCs play a role in the antifungal response. Also, we observe a 5-fold increase in the pDC population in the lungs of infected mice. Therefore, the possibility of these cells playing a role in recruiting and communicating with other immune cells cannot be eliminated.

Upon maturation, pDCs acquire characteristics of conventional DCs (cDCs) such as upregulation of major histocompatability complex (MHC) and becoming more phagocytic. Whether mature pDCs are involved in the detection of and responses against fungal pathogens remains to be determined. Here we show that mature pDC secrete IFNα and TNFα in response to A. fumigatus conidia as early as 6 hours post-challenge. While cytokine secretion of mature pDCs against A. fumigatus does not require opsonization, it requires for A. fumigatus being alive and growing. Furthermore, supernatants from conidial growth induced cytokine secretion by the mature pDCs.

The work presented in this thesis establishes that the nucleic acids in A. fumigatus serve as a pathogen associated molecular pattern (PAMP) that can induce a TLR9- dependent response. Furthermore, I show that pDCs secrete cytokines and induce an antifungal response against A. fumigatus conidia and hyphae. While the pDC population in the blood appears to be small, our work shows that these cells could be intimately involved in the antifungal responses against A. fumigatus.

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