Selective Activation of Macrophage Innate Signaling Pathways and Inflammatory Responses to Orientia tsutsugamushi Karp and Gilliam Strains

Orientia tsutsugamushi (Ot) is an obligatory intracellular bacterium that can cause scrub typhus, an emerging but severely neglected disease with high mortality rates. Ot Karp and Gilliam strains account for most reported cases in Southeast Asia. Our group has reported that Karp-infected outbred and inbred mice exhibit more severe disease outcomes than their Gilliam-infected counterparts, likely due to their excessive inflammation and tissue injury. Macrophages (M{Phi}s) serve as the main target cells for Ot replication and host defense against the infection; they also are key players in immune modulation and cytokine/chemokine production. However, it remains unclear as to how M{Phi}-Ot interactions impact disease outcomes. In this study, we focused on RNAseq from C57BL/6 mouse-derived M{Phi}s to reveal Ot infection- and strain-related immune signatures. While Ot infection modulated several common canonical pathways/genes, we identified unique pathway/gene signatures that were highly selective for Karp or Gilliam strain, respectively. Karp infection uniquely upregulated proinflammatory signaling and pattern recognition receptors, including C-type lectin receptors (CLRs), including Mincle/Clec4e and Dectin-2/Clec4n. In contrast, Gilliam infection enhanced M{Phi} proliferation and DNA replication, and Gilliam strain grew better in M0-like M{Phi}s than Karp strain. In IFN-primed (M1-like) M{Phi}s, however, Karp exhibited significantly greater resistance against host killing than Gilliam, suggesting its superior ability to evade host immune responses. Overall, Karp strain preferentially upregulated CLRs and activated type 1-skewed inflammatory responses, but it is also relatively resistant to IFN-mediated killing. This study provides new insights into potential mechanisms underlying Ot strain-associated immune responses and disease outcomes. Author SummaryOrientia tsutsugamushi (Ot), the causative agent of scrub typhus, is an understudied, life-threatening pathogen endemic to Southeast Asia. Among its various pathogenic strains, Karp and Gilliam are clinically dominant and exhibit distinct disease severity. Building on previous reports that Karp-infected mice display more severe disease than those infected with Gilliam, we explored how strain-specific interactions with macrophages (M{Phi}s), a central cell type in both Ot replication and host immunity, may contribute to these outcomes. Using RNAseq of primary M{Phi}s from C57BL/6 mice, we characterized transcriptional profiles of both strains. Karp infection was uniquely associated with increased proinflammatory signaling and the selective induction of bacterial-sensing molecules. In contrast, Gilliam infection promoted host cell proliferation and supported more efficient bacterial replication in naive M{Phi}s. Notably, Karp demonstrated increased resistance to host killing in stimulated (proinflammatory) M{Phi}s compared to Gilliam. These findings suggest that the Karp strain can drive strong inflammatory responses but is adept at evading IFN-mediated immune defenses. Our study uncovers key immunological distinctions between Ot strains and offers novel insights into the mechanisms of strain-specific pathogenesis in scrub typhus, with potential to uncover biomarkers for disease severity.