Degree Type

Dissertation

Date of Award

2010

Degree Name

Doctor of Philosophy

Department

Veterinary Pathology

First Advisor

Douglas E. Jones

Abstract

Leishmaniasis is a vector-borne zoonotic disease caused by obligate intracellular protozoan parasites of the genus Leishmania. Infection of C3HeB/FeJ and C57BL/6 mice with Leishmania major stimulates a healing cell-mediated immune response, while Leishmania amazonensis infection leads to chronic disease. Here we show C3HeB/FeJ mice co-infected with both species of Leishmania heal, while co-infected C57BL/6 mice do not. Using an in vitro killing assay we determined B cells from infected C57BL/6 mice are ineffective in promoting parasite killing compared to B cells from infected C3HeB/FeJ mice. Furthermore, infected C57BL/6 mice produce less antigen-specific antibodies as compared to infected C3HeB/FeJ mice. The mechanism behind the inability of C57Bl/6 mice to heal L. amazonensis is not known. Here we describe for the first time a difference in the draining lymph node germinal center B cell response between co-infected C3H and B6 mice. There are more germinal center B cells, more antibody isotype-switched germinal center B cells, more memory B cells and more antigen-specific antibody-producing cells in co-infected C3H mice compared to B6 mice as early as 2 weeks post-infection. We also show that IL-21 production in both mouse strains is similar at 2 weeks, suggesting the difference in these mouse strains is due to intrinsic B cell differences, rather than a difference in IL-21 production within germinal centers.

Mice infected with L. amazonensis have a non-polarized T helper cell response and non-healing, chronic lesions. In vitro, a productive response to this pathogen has been recapitulated through macrophage production of both nitric oxide and superoxide. We show FcγR and cytochrome b558 are necessary for superoxide production during an established infection. We demonstrate NADPH oxidase assembly of gp91phox and p67phox occurs by day 1 during the in vitro infection and is localized directly adjacent to the parasite. However, measurable superoxide production was only detectable at day 5 in vitro, indicating that assembly of these subunits was not sufficient to trigger superoxide production. Using wortmannin inhibition of PI3K, we show inhibition of superoxide production at day 5 and indicating that PI3K is critical for superoxide production at this late stage of infection. These data establish that the FcγR-NADPH oxidase activation pathway is required to kill intracellular L. amazonensis. We propose that this novel pathway requires L. major antigen-specific B cell production of antibodies which bind stimulatory Fcγ receptors to produce superoxide through PI3K-mediated activation of assembled NADPH oxidase complexes that are associated with intracellular amastigote parasites. Understanding the role of this pathway in controlling non-healing cutaneous leishmaniasis caused by L. amazonensis may be critical in determining specific immunomodulation to successfully treat this disease.

Copyright Owner

Katherine Nicole Gibson-corley

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

126 pages

Share

COinS