Macrophage activation in the presence of Burkholderia pseudomallei

Abstract

Melioidosis is a potentially fatal disease caused by the soil dwelling bacterium Burkholderia pseudomallei. The disease is endemic in tropical and subtropical regions of the world but is primarily located in southeast Asia and in northern Australia. Acute melioidosis is characterised by a fulminating septicaemia that can result in death within days of exposure. The generation of sepsis results from a prolonged and or exaggerated stimulation of host immune cells by pathogens which culminates in the hyperproduction of inflammatory mediators. Stimulation of host cells by pathogens is facilitated by pattern recognition receptors which bind to microbial structures and initiate downstream signalling cascades. The most well studied pattern recognition receptors are those in the Toll-like receptor family (TLRs). Over the past decade a tremendous amount of work has been conducted to identify TLR specific ligands, TLR structures, associated signalling proteins and the cytokines produced by TLR activation. Understanding the nature of TLR mediated signalling in host cells is fundamental to elucidating the pathogenesis of sepsis and the improvement of clinical management. Up until 2007, no extensive publications had surfaced regarding TLR recognition of B. pseudomallei and the role of TLRs still remains an area of intense study. Therefore, the major focus of the research outlined within this thesis was the characterisation of TLR activation by B. pseudomallei during acute infection. This was achieved using infection studies in murine and human cell lines as well as in primary cells isolated from a previously characterised murine model of acute melioidosis. Primary cells were also isolated from partially resistant murine hosts and used in infection studies. To ascertain the degree of TLR activation by 26 B. pseudomallei isolates with varying levels of virulence, standard antibiotic protection assays were performed on RAW 264.7 macrophages and peritoneal exudate cells (PEC) challenged with B. pseudomallei. Reverse transcriptase-polymerase chain reaction (RT PCR) was performed to determine TLR2, TLR4, TLR5, and TLR9 expression. Internalisation and killing of bacteria were determined at the early stages of infection. ELISAs were performed to determine total protein levels of tumor necrosis factor alpha (TNF-α) from cultured supernatants. Griess assays were used to assess nitrite production by macrophages as a measure of cytotoxic activity. Up to 2 h post infection B. pseudomallei failed to significantly increase TLR4, TLR5 and TLR9 expression in both cell types. However, TLR2 expression was increased, irrespective of isolate virulence in RAW 264.7 macrophages. Levels of TNF-α and nitrite were significantly attenuated in RAW 264.7 macrophages and no correlation was found between the level of virulence of the infecting strain and TLR expression, bacterial uptake or killing. The ability of B. pseudomallei to evade detection by macrophages may be in part, due to possible signal dampening of TLR receptors at the early stages of infection. Susceptibility to B. pseudomallei infection is determined by host immunocompetence as well as bacterial virulence. During acute melioidosis, excessive levels of pro-inflammatory cytokines are found systemically and lead to fatal septicaemias. Using RT PCR analysis, we found that B. pseudomallei can induce TLR4, TLR5, and TLR9 expression in peritoneal exudate cells (PECs) derived from susceptible and partially resistant mice. Induction of TLR4, TLR5, and TLR9 expression, in addition to TNF-α and interleukin 12, p40 subunit (IL-12p40), was greater in susceptible hosts. These results indicate the importance of genetic factors in regards to TLR recognition and response to B. pseudomallei and indicate more pronounced TLR activation in susceptible hosts. To determine if macrophage activation is ubiquitous in the presence of B. pseudomallei isolates of different origin, we examined the role of TLR2 and TLR4 in the recognition of clinical isolates of high and low virulence. Using quantitative real time polymerase chain reaction (qRT PCR) analysis, transfection assays and ELISA, we determined transcription profiles of TLRs and cytokine secretion in macrophages co-cultured with B. pseudomallei. Our findings demonstrate that there are differences in TLR transcription profiles between isolates and that contrary to previous reports the degree of TLR2 and TLR4 mediated NF-κB activation may be dependant on the individual B. pseudomallei isolate. In summary, the results in the present study have provided a basic understanding of TLR involvement in B. pseudomallei recognition. They provide supporting evidence regarding the role of TLRs during melioidosis and the establishment of different TLR transcription in hosts with different susceptibilities to infection. These results also suggest that B. pseudomallei activation of TLRs may be different between isolates.

Key Biomarkers