The role of dendritic cells in the dissemination of Burkholderia pseudomallei infection

Burkholderia pseudomallei infection, known as melioidosis in humans and animals, is an important cause of community-acquired bacterial sepsis that is associated with high mortality rates in endemic regions including Northern Australia and Southeast Asia. In Thailand, melioidosis causes a significant number of fatalities, placing it as the third leading cause of deaths from an infectious disease behind AIDS and tuberculosis. In individuals exposed to B. pseudomallei-contaminated soil or water, the bacteria can rapidly disseminate to a variety of organs resulting in diverse manifestations that range in severity from asymptomatic to acute, chronic and latent infections. Irrespective of the initial presentation, bacteraemia and sepsis are common complications associated with melioidosis. Early diagnosis and provision of appropriate antibiotic therapy are crucial for preventing mortality but is hampered by the lack of an accurate, reliable and cost-effective diagnostic assay. Improved diagnosis, treatment and future vaccine development is dependent on understanding host-pathogen interactions and the mechanisms involved in the progression of melioidosis.\ud \ud The work presented in this thesis endeavoured to further our understanding of hostpathogen interactions between dendritic cells (DC) and B. pseudomallei and the potential use of B. pseudomallei-specific T cell assays for monitoring the development of protective immune responses in patients. The research presented is the first to investigate the capacity of specialised type I interferon (IFN)-producing plasmacytoid DC (pDC) to respond to B. pseudomallei. The current research also describes a novel mechanism for B. pseudomallei dissemination, whereby migrating DC traffic the bacterium to sites distal from the infection site. In addition, the work presented demonstrates the benefit of B. pseudomallei-specific T cell assays as an alternative method to current serological assays, such as the indirect haemagglutination assay (IHA), for detecting the development of adaptive immune responses. These assays could become useful tools for detecting exposure to B. pseudomallei and improve monitoring of patients with melioidosis.\ud \ud Plasmacytoid DC are a subset of DC that provide innate immune responses by rapidly producing large quantities of type I IFN (IFN-α and IFN-β) to modulate immune cell activation, such as NK cell cytolytic activity and cytokine production. Although the role of pDC during viral infections is well described, their functional responses to bacterial infections are underappreciated. The limited evidence available suggests that pDC and type I IFN may drive beneficial anti-bacterial responses or alternatively detrimental immune suppressive functions. Recently, leukocytes from patients with acute melioidosis were found to activate type I IFNmediated signalling pathways. However, the dominant cell type producing type I IFN and the significance of inflammatory signalling via type I IFN during B. pseudomallei infection is yet to be defined. Because type I IFN modulate a range of immune cell functions, excessive signalling via type I IFN during B. pseudomallei infection could potentially contribute to the mechanisms driving the development of septic shock in melioidosis. Therefore, the functional responses of pDC following in vitro exposure to B. pseudomallei were investigated. Human and murine pDC internalised and killed B. pseudomallei as efficiently as conventional DC. Interestingly, pDC generated from B. pseudomallei-susceptible BALB/c mice demonstrated significantly increased IFN-α production and were unable to kill intracellular B. pseudomallei in comparison to pDC generated from B. pseudomalleiresistant C57BL/6 mice. The findings indicate that pDC are an additional innate immune cell capable of responding to B. pseudomallei that potentially contribute to the excessive cytokine response and increased bacterial burden causing rapid mortality in BALB/c mice. The outcomes of this work provide the first evidence of pDC bactericidal activity against B. pseudomallei.\ud \ud Conventional dendritic cells (DC) are considered professional antigen presenting cells that link the innate and adaptive immune responses. In vitro studies have demonstrated that highly phagocytic, immature DC internalise and kill B. pseudomallei. These in vitro DC-B. pseudomallei interactions triggered DC maturation, a process whereby DC up-regulated expression of antigen presenting molecules (MHC class II) and T cell co-stimulatory molecules (CD80, CD86). Due to the rapid dissemination of disease in acute melioidosis, it was hypothesised that migrating DC may serve as a vehicle for B. pseudomallei dissemination. Using an in vitro migration assay, it was demonstrated that B. pseudomallei stimulated significantly increased migration of bone marrow derived DC (BMDC) compared to uninfected BMDC. Importantly, migrated BMDC were found to harbour live B. pseudomallei. Subsequent studies provided evidence that B. pseudomallei stimulated in vivo migration of fluorescently labelled BMDC from the footpad to the draining popliteal lymph node (pLN). Furthermore, in vivo migration of B. pseudomallei-infected BMDC facilitated dissemination of the bacterium to distal secondary lymphoid tissue and lungs of mice. DC-associated dissemination of B. pseudomallei corresponded with significantly increased bacterial burden in the spleen and lungs of mice in comparison to mice infected with B. pseudomallei alone. These novel findings demonstrate that B. pseudomallei is capable of persisting within migrating DC, which enhances the ability of the bacteria to rapidly disseminate and colonise lymphoid tissue.\ud \ud The DC migration assays used in vitro cultured BMDC exposed to B. pseudomallei to demonstrate that B. pseudomallei hijacks DC migration. To determine whether DC at the infection site also traffic B. pseudomallei, C57BL/6 mice with infected with B. pseudomallei via the footpad then the intracellular bacteria within tissueresident DC in the footpad injection site and also in the draining pLN, spleen and lung were enumerated. The findings presented are the first to provide evidence of B. pseudomallei internalisation by skin DC at the site of infection. In addition, the findings demonstrate that dissemination of B. pseudomallei was higher in tissueresident DC compared to non-DC in the pLN and spleen of infected mice. Of interest, spleen DC appear to facilitate intracellular persistence of B. pseudomallei. Collectively, the findings of the current investigation are the first to prove that the migratory response of DC to secondary lymphoid tissues for antigen presentation inadvertently facilitates the systemic spread of B. pseudomallei.\ud \ud Activation of adaptive cell-mediated immune (CMI) responses for elimination of intracellular pathogens including B. pseudomallei are important for protection against disease progression. B. pseudomallei-specific T cell responses are evident in patients who have recovered from melioidosis. Furthermore, studies suggest that a strong CMI may protect an individual from the development of clinical melioidosis. Although CMI-based assays are used to diagnose other diseases, such as the QuantiFERON assay for diagnosing tuberculosis, assessment of B. pseudomalleispecific T cell responses in clinical practice is underutilised. Instead, the IHA is typically used as an affordable, serological diagnostic aid despite its poor sensitivity and specificity. Furthermore, approximately 10 % of patients with culture-confirmed melioidosis remain persistently IHA negative (IHA-negative patients). Given the unreliability of the IHA, it was proposed that assessment of B. pseudomallei-specific T cell recall responses would be useful to demonstrate the development of adaptive immune responses in IHA-negative patients. B. pseudomallei-specific T cell responses were detected in peripheral blood mononuclear cells isolated from IHAnegative patients confirming both exposure and the development of an adaptive immune response to B. pseudomallei, despite negative IHA serology. This response was predominantly driven by CD4+ T cells, with a strong IFN-γ response. The findings of this study are the first to demonstrate B. pseudomallei-specific T cell responses in patients with culture-confirmed melioidosis who are persistently IHA negative and support the development of a CMI-based assay, possibly an IFN-γ detection assay akin to the tuberculosis QuantiFERON assay, which would be beneficial for monitoring the immune status of patients with melioidosis.\ud \ud In summary, the current study is the first to describe the innate response of pDC exposed to B. pseudomallei and demonstrate that increased IFN-α production and impaired bacterial killing by pDC may contribute to host susceptibility. Importantly, the current study provides novel data on the migratory capacity of DC exposed to B. pseudomallei, which demonstrates that DC migration to secondary lymphoid tissues is a mechanism that facilitates the rapid systemic dissemination of the bacterium. In addition, the current study confirmed the development of B. pseudomallei-specific T cell responses in patients who have recovered from melioidosis but have no detectable B. pseudomallei-specific antibody response as determined by the IHA. Collectively, the work described has contributed to significantly furthering our understanding of early host-pathogen interactions following infection with B. pseudomallei and have demonstrated that assessment of B. pseudomallei-specific T cell responses would be beneficial to determine exposure to this pathogen and for monitoring patients with melioidosis