Mincle and STING Activation in Proinflammatory Responses to Orientia Infection

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Project Summary/Abstract Scrub typhus is a life-threatening disease caused by Orientia tsutsugamushi, an LPS-negative bacterium. This bacterium is highly infectious, replicating preferentially in phagocytes and endothelial cells, leaving one third of world population at risk of infection. Scrub typhus vaccines are not yet available; information on host immune recognition and disease pathogenesis is limited. Our recently developed mouse models of O. tsutsugamushi Karp strain infection, which mimic severe infection in human patients, have revealed that excessive type 1, but deficient type 2, immune responses result in acute vascular damage in multiple organs. Using the NanoString technology, we examined more than 1,200 immune genes in inflamed tissues (lung, spleen, brain). We found a significant/selective increase in transcripts of Mincle/Clec4e (a C-type lectin receptor) and STING (a DNA sensor) during acute/lethal infection in mice and in infected primary phagocyte cultures. The underlying hypothesis is that Mincle/STING pathway activation in O. tsutsugamushi-infected monocytes are critical for effector T cell priming, and dysregulated Mincle/STING activation can lead to excessive type 1 inflammation and acute tissue damage. Aim 1 will test whether sustained bacterial replication can initiate Mincle and STING activation and trigger their downstream signaling pathways. Both mouse and human primary macrophages and monocytes will be used for in vitro infection under various conditions with normal or attenuated expression of Mincle or STING (via siRNA, pharmacological inhibitors, or genetic knockouts). Aim 2 will determine whether Mincle/STING activation in dendritic cells can effectively prime/activate T effector cells and if Mincle/STING contribute to dysregulated T-cell responses during Orientia infection. Dendritic cells from Mincle-/- and STING-/- C57BL/6J mice will be infected and examined for priming naïve CD4 T cells in vitro and in vivo; knockout mice will receive a non-lethal or lethal Orientia infection. The spectrum of innate and adaptive immune responses will be evaluated at the single-cell and molecular levels and integrated with those from bacteriologic/pathologic studies. While Mincle and STING ligands from certain human pathogens or damaged self-components are reported, the roles of Mincle or STING during Orientia infection have never been explored. This study will reveal, for the first time, how these innate sensors collectively contribute to host responses to an obligate intracellular bacterium, which is poorly studied but has public health impact. The feasibility is justified by our research expertise and BSL3 facilities. Discovery of the nature of initial immune activation/dysfunction will help reveal signature host profiles for scrub typhus prognosis and potential avenues for pharmacological intervention. This timely study will have broad implications for other intracellular pathogens.