Biology subjects

Villacreses, D.

Publications and source records attributed to Villacreses, D..

3 recordsLinked to original sources

bioRxiv2026

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.

immunology↗

bioRxiv2025

A Humanized IFN-γ Mouse Model Reveals Skin Eschar Formation, Enhanced Susceptibility and Scrub Typhus Pathogenesis

Scrub typhus, caused by Orientia tsutsugamushi (Ot) bacteria, is a serious acute febrile illness associated with significant mortality. An estimated one million cases occur annually, with more than one billion people at risk. No effective vaccine is currently available, largely due to the complex Ot strain diversity and an incomplete understanding of protective immune mechanisms. To overcome these challenges, there is a critical need for a suitable animal model that mimics human disease through the natural route of infection via mites. Here, we report for the first time that a genetically engineered humanized mouse strain (with triple knockout/knock-in of IFN-{gamma} and its receptors), exhibits increased susceptibility to intradermal Ot infection compared to wild-type (WT) mice. This is evidenced by greater body weight loss, elevated bacterial burden, and reduced expression of interferon-stimulated genes (ISGs). Humanized mice exhibit pronounced biochemical abnormalities and tissue pathology accompanied by dysregulated T cell and neutrophil responses following infection. Notably, these immunocompetent mice develop skin eschar-like lesions resembling those observed in human patients. Overall, our study introduces a promising humanized mouse model to dissect the immunopathogenesis of scrub typhus and evaluate future vaccine candidates. Author SummaryScrub typhus is a serious disease caused by the obligately intracellular bacterium Ot that spreads to humans through the bite of larval mites called chiggers. It affects over a million people each year, primarily in Asia, and can lead to life-threatening complications. Unfortunately, we still lack a clear understanding of how this infection causes disease, partly because there is not a good laboratory model that closely reflects how humans respond to infection. Our recent reports have suggested an important role of IFN-{gamma} in host protection against Ot infection. In this study, we used a new genetically modified mouse strain that carries human IFN-{gamma} signaling in place of its mouse counterpart. We found that these humanized mice are more vulnerable to infection, develop skin eschar lesions like those in patients, and show signs of systemic inflammation and organ damage. Their immune response also resembled what has been observed in human patients. This new mouse model can help scientists better understand the mechanisms as to how this bacterial species causes severe disease outcomes in patients. Once those mechanisms are understood, this mouse model will serve a further purpose as a tool for testing new vaccines and treatments to aid humans at-risk for scrub typhus.

immunology↗

bioRxiv2025

Unique B Cell and Germinal Center Responses in Mice with Severe versus Mild Orientia tsutsugamushi Infection

The intracellular bacterium Orientia tsutsugamushi (Ot) is the causative agent of scrub typhus, an emerging and neglected tropical disease. Immunity against Ot infection in patients is known to be short-lived, as circulating antibodies can wane around one-year post-infection. However, its underlying mechanisms remain unclear; host immune signatures to clinically prevalent Ot strains are also undefined. In this study, we infected C57BL/6 mice with two clinically prevalent Ot strains with distinct virulence to examine splenic B cell and germinal center (GC) responses. While highly virulent Karp strain-infected mice had 50% mortality rates, intermediate virulent strain Gilliam-infected mice survived, gained body weight, and maintained relatively low bacterial burden levels during infection. Yet, Gilliam infection resulted in strong splenic B cell responses, as judged by absolute cell numbers of total splenic B cells, follicular B cells, and marginal zone (MZ) B cells alongside with significantly higher titers of total IgG and IgM antibodies at the late stage of infection. While altered splenic architecture with increased distance between white pulp regions was detectable in both strains, GC disorganization/collapse and MZ abrogation were only detected in Karp-infected spleens. To define the driving force of GC loss, we compared RNAseq profiles between spleens infected with the Karp or Gilliam strain, and found significantly upregulated inflammation biomarkers (Ccl2, Il33, Ifng) and severe scrub typhus-related inflammatory pathways (Il1, Il6, Tnf, and Ifng) in Karp infection at day 4 (D4, early stage of infection). IPA analysis further showed upregulated neutrophil degranulation and defense response pathways in Karp-infected spleens, while Gilliam-infected spleens showed upregulation of phagocytosis signaling pathways. Flow cytometry confirmed a marked influx of activated myeloid cell subsets (neutrophils, M1 macrophages, and inflammatory monocytes) in the spleen during Karp infection as compared to Gilliam infection, indicating excessive inflammatory infiltration in highly virulent Ot strain infection. Collectively, our findings establish several Ot strain-related immune signatures which help fill the knowledge gap of deficiencies in the host adaptive immune response during acute scrub typhus. Author SummaryThe intracellular bacterium O. tsutsugamushi (Ot) is the etiologic agent of the neglected tropical disease scrub typhus. It is well-known that immunity against Ot infection is short-lived, yet no in-depth studies have examined or compared adaptive immunity or deficiencies at cellular and molecular levels during acute Ot infection with clinically relevant Ot strains. In this study, we used two clinically prevalent Ot strains that cause differential diseases outcomes in the C57BL/6 murine model to examine B cell and germinal center (GC) responses. In sharp contrast to Karp infection, which caused 50% mortality rates by D12, Gilliam infection elicited self-limiting infection with low tissue splenic bacterial burdens. Yet, Gilliam-infected mice demonstrated robust humoral immune responses, as demonstrated by strong IgM and IgG antibody titers at D12 and absolute numbers of splenic B cell subsets at D8 and D12. While both Ot strains altered typical splenic white pulp architecture, only Karp infection abrogated GC and MZ structures. Comparison of RNAseq profiles of Karp-vs. Gilliam-infected spleens revealed several unique features, including Karp infection-associated upregulation of inflammation genes (Ccl2, Il33, Ifng) and inflammatory pathways (Il1, Il6, Tnf, Ifng) at D4, as well as upregulated leukocyte recruitment, neutrophil degranulation, and defense response pathways. In sharp contrast, Gilliam infection upregulated pathways involved in phagocytosis immunoregulatory interactions. Flow cytometry confirmed significant influx and activation of myeloid cell subsets (neutrophils, macrophages, M1 macrophages, monocytes) during Karp infection. Our study reveals unique patterns in the differential immune responses to two clinically prevalent Ot strains and helps understand potential mechanisms of immune alterations during severe scrub typhus.

immunology↗