ABSTRACT Scrub typhus is a life-threatening disease caused by Orientia tsutsugamushi (Ot), an LPS-negative bacterium that replicates preferentially in endothelial cells (EC) and phagocytes. While one million people are infected yearly, with about one third of world population at risk of infection, effective strategies for infection control are lacking. Information on disease pathogenesis and immune dysregulation is very limited. To address these challenges, we have developed four different mouse models of scrub typhus that recapitulate clinical outcomes in humans. This grant support has led to the first reports that i) Mincle (a phagocyte C-type lectin receptor, CLR) can sense Ot Karp strain and synergize with TNFα to drive M1/Th1-skewed responses; ii) sustained lung/brain/kidney inflammation and progressive loss of Tie2 (a key receptor for EC function and survival) are hallmarks of lethal Karp infection, correlating with activation of endogenous damage-associated molecular patterns (DAMPs); and iii) the concordance of “type 1-skewed/type 2-repressed immune signatures” is evident for severe scrub typhus in both inbred (C57BL/6) and outbred (CD-1) mouse models. In contrast, the Ot Gilliam- induced non-pathogenic outcome seems to be linked to distinct CLR and innate immune signatures. The objective of this study is to define pathogenic mechanisms of vascular dysfunction and shared markers or therapeutic targets for severe scrub typhus. Our central hypothesis is that innate immune programs induced by Ot Karp or Gilliam infection in target cells shape pathogenic or protective adaptive immunity, vascular function, and clinical outcomes. This hypothesis will be tested in two cohesive Specific Aims. Aim 1 will delineate mechanisms by which CLR-like, pathogen-associated molecular patterns (PAMPs) drive innate immune programs and distinct clinical outcomes in inbred mice. We will use two Ot strains to examine 1) innate immune signatures in inflamed lung/spleen/brain tissues, focusing on CLR- and TGFβ-regulated pathways; 2) infection- triggered M1/M2 polarization and their impact on EC activation/survival/death; 3) functional diversity of dendritic cell (DC) subsets during infection and T cell-priming. Aim 2 will examine whether/how differential alarmin responses, especially those regulated by the IL-33 and CD24 pathways, determine acute tissue injury, vascular dysfunction, and host mortality. 1) To define disease “shared” immune signatures and vascular markers beyond Tie2, we will assess transcriptomics of lung/brain/kidney during severe disease stages and use perfused lungs for 30-color spectrum flow cytometry. 2) To examine alarmin-associated regulation, we will use Karp/Gilliam- infected inbred mice, soluble CD24Fc (a known repressor for DAMP, but not PAMP, pathways), and knockout mice. 3) We will use peripheral blood samples from all four models for cell RNAseq and serum Olink proteomic profiling. Validation of blood- and tissue-derived immune signatu...