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Title: Immunoregulatory mechanisms involved in intracellular microbial infections
Author: Al-Ojali, Samia M. I.
Awarding Body: University of Ulster
Current Institution: Ulster University
Date of Award: 2011
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Murine infection with the natural mouse pathogen S. typhimurium is the most widely accepted model for studying immunity to typhoid fever. Attenuated strains of Salmonella have been developed for use as live vaccines, and there is an extensive body of evidence demonstrating their efficacy both in human and animal studies [1]. Several attenuated strains of S. typhimurium were engineered to express defined cytokines, including IL- 10, IL-2, IL-4, IL-6, TNF-o or IFN-γ. Moreover these recombinant strains have been shown to be efficient vectors for the delivery of cytokines in experimental disease models [2],[3], Thus targeted delivery of cytokines can potentially be used as a therapeutic approach in treatment of autoimmune conditions, infectious disease and cancer. The main aim of this research was to develop animal models to facilitate the investigation of the role of cytokines by assessing their potential of modulating the immune response to microbial infections, namely using an attenuated strain of S. typhimurium, which expresses IFN gamma (IFN-γ). For this purpose, two attenuated strains of S. typhimurium were compared for their efficacy in inducing protective immune responses in a number of different genetically-defined inbred mouse lines differing in their genetic susceptibility profile; mice expressing wild type allele of Nramp1 gene (Nramp1 n), but differing in their expression of TLR4 gene, key genes controlling mouse susceptibility to Salmonella infection, C3H/HeN (TLRn/Nrampn), and C3H/HeJ (TLRd/Nrampn) mice. Mice on C57BLl6 background with defective Nramp1 gene (TLRn/Nrampd), the other critical determinant of susceptibility to Salmonella infection In order to characterise further the potential mechanism of the enhanced influence of the IFNγ-expressing Salmonella strain, two additional recombinant mouse strains, on the C57BLl6 background with a second gene defect in CD154 (CD40L) or MyD88 protein, a critical adaptor molecule essential for signalling through most of the TLRs were also used. It was hypothesised that host immune responses may be amenable to manipulation by cytokine-expressing bacterial strains. The nature of the immune response to infection with a parental attenuated strain of S. typhimurium (designated BRD509) compared with its recombinant derivative that has been engineered to express IFN-γ (designated GIDIFN). The two bacterial strains used in this study were the aroA aroD double auxotrophic strain (designated BRD509,) and its recombinant derivative that has been genetically engineered to express murine IFN-γ (designated GIDIFN). The investigations focused on assessment of the immunoregulatory influence of bacteria-expressed IFN-γ on the Immune response to Salmonella infection by following in vivo bacterial survival in peritoneal cavity (site of inoculation). The role of IFN-γ as a mediator at the different stages of infection, including the early innate response, plateau clearance stages, as well as the developments of protective memory responses were defined. The level of bacterial load in the peritoneal cavity was assessed during the first 48 hrs post i.p infection with -1x106 BRDq09 or GIDIFN organisms per mouse. Rapid clearance of IFNγ-expressing Salmonella in vivo host target organs, mice were inoculated i.p. with -0.5-1 x 106 CFUs/mouse and at the indicated time points were sacrificed and the bacterial load in spleens and livers enumerated, as well as bacterial shedding in faeces. Outcome of the infection on animal morbidity and mortality were assessed, Production of proinflammatory cytokines (IL-6, IL-12p40, and TNF-a) and nitric oxide by ex vivo cultured splenocytes and peritoneal exudates cells, the levels of IFN-y in the serum was determined. Phenotype of cellular infiltrates in the peritoneal cavity was assessed using flow cytometry. While infiltrates in spleens and livers of infected mice were assessed by histology and immunohistochemistry at different time points after inoculation. The potential influence of bacteria-expressed IFN-γ on the production of systemic/mucosal Salmonella-specific antibodies (lgM, IgG and slgA isotypes was determined titre and isotype of anti-Salmonella antibodies up to 2 months following inoculation. Efficacy of BRD509 and GIDIFN strains in affording protection against virulent challenge using titrated doses for vaccination was assessed and efficacy of the Salmonella strains as vaccines was demonstrated through their capacity to protect against virulent challenge. The results of this investigation demonstrated that IFNγ-expressing Salmonella strain (GIDIFN) has immunopotentiating effects on the host's immune response, in comparison to non-IFNγ-expressing stain (BRD509). The data obtained demonstrated that the GIDIFN strain was able to induce more vigorous innate and adaptive immune responses and, hence, was cleared more rapidly from the infected host than the parental BRD509 strain in all mouse strains tested. The major experimental findings that GIDIFN strain enhanced the early, proinflammatory cytokine response to infection, including IL-6, IL-12, TNF-a and IFN-y, improved inflammatory cell recruitment into infected tissues/organs, increased bacterial shedding in the faeces and lead to a stronger mucosal IgA response. GIDIFN enhanced the Th1-driven Immunoglobulin isotype response in susceptible mice. The GIDIFN-induced immune responses resulted in significantly enhanced host survival following infection in susceptible mouse strains. Moreover, there was a small but significant enhancement in the survival of immunised animals following lethal challenge with virulent Salmonella. The present study demonstrates that IFNγ-expressing Salmonella strains has immunopotentiating effects on the host's immune response and provide direct evidence for the utility of IFNγ-expressing attenuated Salmonella in enhancement of immune responsiveness in immune-deficient hosts. Thus, the expression of IFN-y by attenuated S. typhimurium renders the recombinant strain safer and more immunogenic to use, particularly in immunocompromised hosts, and open the way for further fine-tuning of immune responses. These findings point to the possibility of constructing more efficacious vaccines for protection against typhoid fever in humans and in animals. Moreover, IFNγ-expressing Salmonella may well be useful as bacterial vectors that act as effectors to manipulate unwanted immune responses in chronic diseases such as cancer and autoimmune conditions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available