Burkholderia pseudomallei triggers canonical inflammasome activation in a human primary macrophage-based infection model

Author summary Considering the constantly emerging antibiotic resistance of pathogens, comprehensive analyses of immune response mechanisms against infections are urgently needed to provide the basis for novel therapeutic strategies. Studies based on primary murine cells and cell lines of murine and human origin led to advances in the understanding of immune defense mechanisms against bacterial infections including B. pseudomallei. Nevertheless, results relying on these cell types are not always transferrable to primary human cells due to e.g. pathway alterations. We established and validated a macrophage-based model system derived from human peripheral blood monocytes, which yields high amounts of genetically identical cells more closely resembling cells found in the human host. This model system provides the basis for studying the pathogenesis of B. pseudomallei in primary human macrophages and for developing new human host directed therapies avoiding pitfalls from cell lines. Using our newly established model we demonstrate, that restriction of B. pseudomallei by primary human macrophages is mediated by T3SS-3 dependent canonical inflammasome activation and IFN-gamma induced intracellular bacterial restriction. Most of the current knowledge on Burkholderia pseudomallei-induced inflammasome activation and cell death in macrophages is derived from murine systems. Little is known about the involved bacterial structures and mechanisms in primary human macrophages. This is of particular relevance since murine and human macrophages as well as primary cells and cell lines differ in many aspects of inflammasome activation, including the proteins involved in the recognition of bacterial patterns. In this study, we therefore aimed (i) to establish an in vitro B. pseudomallei infection model with human monocyte-derived primary macrophages from single donors as these cells more closely resemble macrophages in the human host and (ii) to analyze B. pseudomallei-triggered cell death and bacterial elimination in those cells. Our results show that B. pseudomallei-infected primary human macrophages not only release the inflammasome-independent pro-inflammatory cytokines IL-8 and TNF-alpha, but are also engaged in canonical inflammasome activation as evidenced by caspase-1 and gasdermin D processing. Absence of the B. pseudomallei T3SS-3 needle protein BsaL, a potent activator of the canonical inflammasome, abolished lytic cell death, reduced IL-1 beta release, and caspas