Host and Pathogenic Features That Drive Cellular Immune Response to Leishmania spp. Infection

Leishmaniases is a group of vector borne parasitic diseases that affects approximately 12 million people worldwide and results in diverse clinical pathologies, the initiating species often dictates these disease outcomes. Visceral leishmaniasis, the fatal form of the disease, results from infection with Leishmania donovani parasites while cutaneous leishmaniasis generally develops following infection with Leishmania major parasites. In the majority of L. major cases, lesions heal within several months and provide life-long immunity pending re-introduction of the disease. The source of this protective immunity to leishmaniasis is largely influenced by host cellular events leading to a robust IL-12 driven T-helper 1 (Th1) mediated response that produces high levels of IFN-γ which ultimately promotes anti- microbicidal production of nitric oxide (NO) and reactive oxygen species (ROS) that destroy invading pathogens. Macrophages (MPs) and Dendritic Cells (DCs) are major producers of IL-12. In this work, the overall goal of my research dissertation was to investigate the role of both host cell and pathogenic features that contribute to the IL-12 response elicited during Leishmania infection. To this end in vitro MP and DC infection assays coupled with host cell microarray based technologies and molecular genetics approaches were employed to address the interplaying host and pathogen determinants influencing the IL-12 response. Interestingly, previous studies have demonstrated a general suppression of IL-12 production in Leishmania infected macrophages that is tightly controlled at the level of IL-12p40 transcription. In chapter 2, we demonstrated that L. major inhibited promoter activity in RAW264.7 MPs that stably express Ets binding sites in the presence of (IFN-γ) or lipopolysacchride (LPS) stimulation, suggesting L. major may be inhibiting macrophage production of IL-12 by specifically targeting the Ets promoter element.

Remarkably, human DCs exhibit a dynamic range in IL-12 production in response to Leishmania infection. IL-12 is generally enhanced during L. major infections, in contrast to, L. donovani infections which do not induce IL-12. In chapter 3, our microarray analysis of L. major infected human DCs (2, 4, 8, and 24 hrs) revealed a distinct type I IFN associated transcriptomic signature including upregulation of IRF2, IRF7, IRF9, and STAT1/2. L. major infected human DCs pre-treated with a neutralizing type I IFN pathway inhibitor, B18R, induced fewer IL-12p40 mRNA levels. Furthermore, in the context of L. donovani infections type I IFN genes IRF2, IRF7, and IFIT5 were not induced, a trend analogous to the IL-12p40 gene expression elicited by this species. These data indicated that the L. major induced IL-12 response is dependent upon a type I IFN response.

In chapter 4, we investigated the role of pathogenic surface molecules on the 'high- IL-12 inducing' L. major background strain FV1. Parasite lipophosphoglycan (LPG) was previously characterized as a virulence determinant associated with subverting host cell immune mechanisms including complement mediated lysis, phagolysosomal maturation, and leukocyte migration. LPG modulates cytokine secretion in host cells, however, the role of FV1 LPG on the human DC IL-12 response remained to be elucidated. Parasite mutants lacking LPG alone or all PGs were generated through the inactivation of the galactosylfuranose, LPG1 or the golgi GDP-Mannose nucleotide sugar transporter, LPG2, respectively. We demonstrated that infection with the LPG-null FV1 lpg1- mutant resulted in little to no IL-12p40 mRNA, indicating LPG is essential for stimulating IL-12. Curiously, infection with the PG-null lpg2-, led to an increase in IL-12p40 mRNA levels implicating PGs and other LPG2-depedent metabolites may suppress IL-12. Moreover, the effect of LPG was also observed for IRF8 and TNF-α, suggesting LPG targets IRF8 for transcriptional regulation of IL-12 and TNF-α. Together, these data reflect the ability of Leishmania molecules to differentially impact the host cell IL-12 immune response during infection. In summary, challenges still remain that hinder a complete understanding of the immunobiology of Leishmania infection. However, this work contributes information about potential targets in both the host cell and pathogen that will aid future investigations aimed towards development of broadly applicable drug and vaccine regimes for leishmaniasis and other related infectious diseases.