1. The Role of Interleukin-10 in Burkholderia Infection of Human Mononuclear Cells Amy K. Dickey MD MSc, Johanna D. Robertson BS, T. Eoin West MD MPH Department of Medicine, University of Washington, and Harborview Medical Center, Seattle, WA, USA The flagellated bacterium and potential bioterrorism agent Burkholderia pseudomallei is responsible for melioidosis, a common cause of pneumonia and sepsis in Southeast Asia and tropical Australia. Because use of live B. pseudomallei is restricted, a similar but less pathogenic bacteria, B. thailandensis, is a commonly used instead as a model organism. A common TLR5 polymorphism, TLR5 1174C>T, results in a truncated TLR5 protein that does not signal in response to flagellin. Individuals carrying TLR5 1174T are protected against death after infection with melioidosis. In healthy carriers of the TLR5 1174T polymorphism, stimulation of whole blood with flagellin or B. pseudomallei results in lower IL-10 release as compared to wild-type individuals. Interferon gamma (IFN-γ) has bactericidal activity against intracellular bacteria such as B. pseudomallei, and is known to be down-regulated by IL-10.  We hypothesize that in primary blood mononuclear cells infected with B. pseudomallei, suppression of IL-10 increases both IFN-γ and intracellular bactericidal activity. IL-10 is released in response to heat-killed B. pseudomallei but not live B. thailandensis, limiting the ability to use B. thailandensis as a model organism for the study of the role of IL-10 in melioidosis infection. IL-10 inhibition does not affect IFN-γ release or intracellular bacterial survival in PBMCs infected with live B. thailandensis. Our data suggests that IL-10 inhibition may increase the survival of PBMCs infected with live B. thailandensis. Studies using live and not heat-killed B. pseudomallei are required to conclusively support or reject the hypothesis that in B. pseudomallei infection of PBMCs, suppression of IL-10 increases both IFN-γ and intracellular bactericidal activity. Primary blood mononuclear cells (PBMCs) were isolated from the blood of healthy individuals, treated with or without IL-10 monoclonal antibody inhibition, and stimulated with either heat-killed B. pseudomallei (BP) or live Burkholderia thailandensis (BT), both at 100 MOI, for the indicated length of time or 6 hours if not indicated. IL-10 and IFN-γ expression were measured in cell supernatants by ELISA. PBMC survival was determined using the Guava ViaCount Assay. The number of intracellular bacteria was assessed by washing and lysing cells, followed by plating cell lysates on LB agar at various dilutions. Colony forming units were counted after 24 hours. Background Methods Results Conclusions B. pseudomallei stimulation but not B. thailandensis infection induces IL-10 release B. pseudomallei stimulation or B. thailandensis infection induces minimal IFN-γ release, even with IL-10 inhibition Il-10 inhibition does not affect intracellular survival of B. thailandensis IL-10 inhibition may improve survival of PBMCs infected with B. thailandensis Funding for this project was provided by R01 HL113382. Contact adickey@uw.edu for more informationadickey@uw.edu B. pseudomallei (1026b and K9) and Salmonella typhimurium flagellin (Flag) induce lower IL-10 levels in carriers of TLR5 1174T. West TE et al. J Immunol. 2013; 190:3373-3379