Infection with Burkholderia pseudomallei or B. thailandensis triggers activation of the NLRP3 and NLRC4 inflammasomes leading to release of IL-1β and IL-18 and death of infected macrophages by pyroptosis, respectively. The non-canonical inflammasome composed of caspase-11 is also activated by these bacteria and provides protection through induction of pyroptosis. The recent generation of bona fide caspase-1-deficient mice allowed us to reexamine in a mouse model of pneumonic melioidosis the role of caspase-1 independently of caspase-11 (that was also absent in previously generated Casp1-/- mice). Mice lacking either caspase-1 or caspase-11 were significantly more susceptible than wild type mice to intranasal infection with B. thailandensis. Absence of caspase-1 completely abolished production of IL-1β and IL-18 as well as pyroptosis of infected macrophages. In contrast, in mice lacking caspase-11 IL-1β and IL-18 were produced at normal level and macrophages pyroptosis was only marginally affected. Adoptive transfer of bone marrow indicated that caspase-11 exerted its protective action both in myeloid cells and in radio-resistant cell types. B. thailandensis was shown to readily infect mouse lung epithelial cells triggering pyroptosis in a caspase-11-dependent way in vitro and in vivo. Importantly, we show that lung epithelial cells do not express inflammasomes components or caspase-1 suggesting that this cell type relies exclusively on caspase-11 for undergoing cell death in response to bacterial infection. Finally, we show that IL-18’s protective action in melioidosis was completely dependent on its ability to induce IFNγ production. In turn, protection conferred by IFNγ against melioidosis was dependent on generation of ROS through the NADPH oxidase but independent of induction of caspase-11. Altogether, our results identify two non-redundant protective roles for caspase-1 and caspase-11 in melioidosis: Caspase-1 primarily controls pyroptosis of infected macrophages and production of IL-18. In contrast, caspase-11 mediates pyroptosis of infected lung epithelial cells.

Gasdermin D (GSDMD) cleavage by caspase-1 or caspase-11 inflammasomes triggers pyroptosis, a lytic form of cell death protective against intracellular bacteria. In this study, we examine the role of GSDMD in a mouse model of melioidosis. Gsdmd 2/2 mice were more susceptible than wild-type mice to intranasal infection with Burkholderia thailandensis. Production of IL-18, but not IL-1b, was decreased in Gsdmd 2/2 infected mice. Despite lower IL-18, IFN-g was produced in similar amounts in wild-type and Gsdmd 2/2 mice. In vitro, secretion of both IL-1b and IL-18 by macrophages or dendritic cells infected with B. thailandensis was dependent on GSDMD. Surprisingly, wild-type or GSDMD-deficient neutrophils secreted similar amounts of IL-1b, suggesting these cells may be the source of the GSDMD-independent IL-1b detected in vivo. Recombinant GSDMD was able to directly kill B. thailandensis in vitro upon processing by active caspase-1. Moreover, bacteria harvested from wild-type, but not Gsdmd 2/2 , macrophages were more susceptible to the microbicidal effect of hydrogen peroxide or human b-defensin-3. Finally, we provide evidence that pyroptosis of in vitro infected macrophages is directly microbicidal. Taken together, these results indicate that the protective action of GSDMD in melioidosis is primarily due to induction of pyroptosis and direct killing of bacteria rather than production of cytokines.

Francisella tularensis is a zoonotic, facultative intracellular bacterial pathogen that replicates in a variety of cell types during infection. Following entry into the cell and phagosome escape, the bacterium replicates rapidly in the cytoplasm. F. tularensis intracellular growth depends on the availability of metabolizable essential nutrients to support replication.

Merocytophagy is a phagocytic process characterized by the transfer of cytosolic content between macrophages and the formation of a double-membraned vesical within recipient cells. Studies conducted in our laboratory have demonstrated that proteins and intracellular pathogens can be transferred between cells by this contact-dependent mechanism. In particular, Francisella tularensis, a highly infectious pathogen that spreads from cell to cell by hijacking the phagocytic pathway in macrophages, has been shown to replicate within cells that receive cargo via merocytophagy. While these observations may point to an alternative route of F. tularensis dissemination in vivo, it is also possible that this mechanism of bacterial spread could amplify immune responses. Ongoing studies aim to characterize molecular and cellular players in merocytophagy to gain a better understanding of this phenomenon and to develop strategies to assess merocytophagy in vivo in the future. In cell-to-cell contact studies, inhibition of spleen tyrosine kinase (SYK) expression and activity showed decreased cargo transfer by merocytophagy. Pharmacologic inhibition of SYK was also associated with decreased surface expression of several integrins, giving evidence that cell-to-cell contact is stabilized by integrins during merocytophagic transfer in macrophages. Supported by grants from NIH and NIGMS (R56 AI139476 and T32 GM008336).