Barnes, J. L. (2004) Melioidis: an investigation of cellular immune responses. PhD thesis, James Cook University.

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Abstract

Melioidosis is a potentially fatal disease caused by the soil bacterium Burkholderia pseudomallei and is predominantly seen in southeast Asia and northern Australia. Protection from infections with other facultative intracellular bacteria such as Listeria monocytogenes and Legionella pneumophila, has been shown to be mediated largely by a cell-mediated immune (CMI) response of the host. B. pseudomallei is also a facultative intracellular bacterium but despite decades of research being conducted on this pathogen, few studies have focussed on the CMI response in melioidosis. The nature of a protective host immune response, and the conditions under which it is induced, are fundamental for improved clinical management of patients and vaccine development. Therefore, the major focus of the research outlined within this thesis was the characterisation of the CMI responses involved in the development of protective immunity in melioidosis. This was achieved using a previously characterised mouse model of acute and chronic melioidosis. Following intravenous (iv) inoculation, BALB/c mice are highly susceptible, and C57BL/6 mice relatively resistant to B. pseudomallei infection. Using the murine model, we compared the pathogenesis of B. pseudomallei infection following inoculation of the bacterium by iv, intraperitoneal (ip), intranasal (in), per os (po) and subcutaneous (sc) routes of infection. These studies emphasized that determination of bacterial virulence is highly dependent on the route of infection. BALB/c mice consistently demonstrated greater susceptibility toward B. pseudomallei, independent of the route of infection. Spleen and liver were the primary organs targeted following infections by all methods tested. Interestingly, following in and po inoculation of C57BL/6 mice, significant levels of bacteria were detected in the brain in the absence of septicemia. Reverse transcriptase-polymerisation chain reaction (RT-PCR) and histology were used to assess the increased expression of messenger (m) ribonucleic acid (RNA) for interferon-γ-inducible protein 10 (IP-10), monocyte interferon- γ -inducible protein (Mig), regulated upon activation, normal T-cell expressed and secreted chemokine (RANTES), monocyte chemoattractant protein-1 (MCP-1), cytokine-induced neutrophil chemoattractant (KC), macrophage inflammatory protein-2 (MIP-2), granulocyte colony-stimulating factor (G-CSF), macrophage (M)-CSF, granulocyte-macrophage (GM)-CSF during infection with a highly virulent strain of B. pseudomallei (NCTC 13178). Histological changes and bacterial loads were also monitored in the livers and spleens of C57BL/6 and BALB/c mice infected with NCTC 13178. Disparate expression of mRNA was demonstrated for IP-10, Mig, MCP-1, KC, MIP-2, GM-CSF and M-CSF. The magnitude of cellular responses observed in tissue correlated with increased levels of the chemokines and CSF investigated, as well as bacterial load. Compared with C57BL/6 mice, greater infiltration of neutrophils was observed in liver and spleen of BALB/c mice. In contrast, early lesions in C57BL/6 mice predominantly comprised macrophages. These results suggested that the inability of BALB/c mice to contain the infection at sites of inflammation may underlie the susceptible phenotype of this mouse strain toward B. pseudomallei infection. The nitric oxide (NO) secretory activity of C57BL/6 and BALB/c peritoneal macrophages were compared following in vitro stimulation with B. pseudomallei antigens. Comparable levels of NO were produced by peritoneal macrophages from both mouse strains, suggesting that other antimicrobial mechanisms may underlie differences in the in vitro intracellular killing capacities of peritoneal macrophages from C57BL/6 compared to BALB/c mice. However, the low numbers of macrophages observed histologically at sites of B. pseudomallei infection in BALB/c mice suggest that failure to recruit sufficient macrophages to these sites may also contribute to the susceptibility of this mouse strain. The present studies are the first to demonstrate the role of T cell responses in experimental and human melioidosis. Following exposure to a less virulent strain of B. pseudomallei (NCTC 13179), both C57BL/6 and BALB/c mice demonstrated delayed-type hypersensitivity (DTH) responses (P<0.05) and lymphocyte proliferation (P<0.05) towards B. pseudomallei antigens. Such a response indicated the generation of B. pseudomallei-specific lymphocytes following initial exposure to the bacterium. The strength of DTH and lymphocyte proliferation responses was dependent on the concentration of the primary inoculating dose. Adoptive transfer experiments were carried out using mononuclear leucocytes (MNL) or purified T cells from spleen of B. pseudomallei-immunised C57BL/6 mice. The transfer of B. pseudomallei-specific MNL to naïve C57BL/6 mice was demonstrated by a DTH response (P<0.05) to B. pseudomallei antigens. However, these mice were not protected from a subsequent lethal challenge with NCTC 13178. Similarly, recipient mice that received purified T cells were not protected from a subsequent lethal challenge. The results of this study suggested that a single exposure to B. pseudomallei is insufficient to induce a protective adaptive immune response. Therefore, we attempted to induce resistance in susceptible BALB/c mice using repetitive low-dose exposure to live B. pseudomallei NCTC 13179. Immune responses and resistance following sc immunisation with live B. pseudomallei was compared to exposure to B. pseudomallei antigens. Low-dose immunisation with live bacteria induced protection (P<0.01) to a subsequent lethal challenge with NCTC 13178. In comparison, mice immunised with B. pseudomallei antigens were not protected but demonstrated significantly increased levels of IgG2a (P<0.01) and IgG1 (P<0.01) in serum. These findings suggest that although exposure to B. pseudomallei antigens induced a DTH response and lymphocyte proliferation to B. pseudomallei antigens in vitro, the generation of a protective immune response following B. pseudomallei infection requires the presence of live bacteria. The present studies have also given the first evidence of the development of CMI responses to B. pseudomallei in patients who had recovered from melioidosis. Significantly higher lymphocyte proliferation, IFN-γ production and activation of CD4+ and CD8+ T cell subsets were observed in the patient group compared with control subjects after in vitro challenge of peripheral blood mononuclear leucocytes (PBML) cultured with B. pseudomallei antigens. It is tempting to reason that the survival of the patients included in this study was due to the development of a protective adaptive immune response to B. pseudomallei. Strong CMI responses were also demonstrated in subclinical melioidosis infection. Individuals who had been exposed to B. pseudomallei without any clinical manifestations displayed enhanced lymphocyte proliferation and IFN-γ production in response to stimulation with B. pseudomallei antigens in vitro, when compared to individuals who had recovered from clinical melioidosis. Such a response may be essential for determining protection following B. pseudomallei infection. In summary, the results of the present studies have provided basic data regarding the involvement of CMI responses during B. pseudomallei infection. They provide evidence supporting an essential role for T cells in the development of an effective adaptive immune response to experimental and human B. pseudomallei infection and suggest that differences in the development of T cell responses may influence the outcome of infection.

Item ID:	964
Item Type:	Thesis (PhD)
Keywords:	Burkholderia pseudomallei, Cell-mediated immune response, T cells, Bacterial virulence, Route of infection, Reverse transcriptase-polymerisation chain reaction, Histology, Messenger ribonucleic acid, mRNA, IP-10, RANTES, MCP-1, KC, MIP-2, G-CSF, (M)-CSF, (GM)-CSF, NCTC 13178, NCTC 13179, Delayed-type hypersensitivity responses, Lymphocyte proliferation, IFN-γ
Date Deposited:	25 Oct 2006
FoR Codes:	11 MEDICAL AND HEALTH SCIENCES > 1108 Medical Microbiology > 110802 Medical Infection Agents (incl Prions) @ 0% 11 MEDICAL AND HEALTH SCIENCES > 1107 Immunology > 110704 Cellular Immunology @ 0% 11 MEDICAL AND HEALTH SCIENCES > 1108 Medical Microbiology > 110801 Medical Bacteriology @ 0%
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