Biology subjects

Soong, L.

Publications and source records attributed to Soong, L..

14 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↗

bioRxiv2026

Inducible nitric oxide synthase (iNOS) regulates skin eschar lesions, bacterial persistence, and inflammatory resolution in mouse models of scrub typhus

Orientia tsutsugamushi (Ot) is an obligately intracellular bacterium that causes scrub typhus, a potentially severe infectious disease characterized by systemic inflammation and multiorgan dysfunction. We recently reported a protective role for IFN-{gamma} signaling in host defense against Ot infection; however, the underlying mechanisms remain obscure. Inducible nitric oxide synthase (iNOS, encoded by Nos2) is a key antimicrobial effector induced downstream of IFN-{gamma} signaling. Here, we used transgenic mouse models to further investigate the biological functions of iNOS. We first revealed the requirement of iNOS for the restriction of Ot growth in cultured bone marrow-derived macrophages. Using an intradermal mouse model, we found that while tissues of Nos2-/- and wild-type mice exhibited comparable bacterial burdens during acute infection phases, Nos2-/- mice developed eschar-like lesions similar to those observed in Ifngr1-/- mice, indicating a critical role for the IFN-{gamma}/iNOS axis in regulating skin pathology in scrub typhus. Notably, Nos2-/- mice displayed impaired bacterial clearance during the recovery phase (day 42), with persistent bacterial burdens in multiple organs accompanied by sustained immune activation and elevated inflammatory responses. Histopathological and biochemical analyses further revealed increased tissue damage and dysregulated physiological homeostasis in Nos2-/- mice during recovery. Mechanistically, iNOS deficiency resulted in heightened myeloid cell activation and prolonged expression of proinflammatory mediators, suggesting a dual contribution of iNOS in both antimicrobial defense and inflammation resolution. Collectively, these findings provide new insight into IFN-{gamma}-mediated defense mechanisms and imply the distinct roles of iNOS during different stages of scrub typhus. Author summaryScrub typhus is a potentially severe infectious disease caused by the bacterium Orientia tsutsugamushi (Ot), which is transmitted to humans through the bite of infected mites. Despite its global impact and expanding geographic distribution, the immune mechanisms that protect against this infection remain incompletely understood. In this study, we examined the role of inducible nitric oxide synthase (iNOS), an immune effector molecule that helps the host control infection. Using mouse models, we found that iNOS plays dual and stage-specific roles during Ot infection. Mice lacking iNOS developed dysregulated immune homeostasis during acute infection and exhibited skin lesions resembling the eschars observed in some patients with scrub typhus. In addition, these mice showed delayed bacterial clearance, prolonged inflammation, and increased tissue damage during the recovery phase. Our findings indicate that iNOS contributes not only to host antimicrobial defense but also to the control of excessive inflammation following infection. These results provide new insight into host defense mechanisms in scrub typhus and may help inform future therapeutic or preventive strategies.

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↗

bioRxiv2024

Lack of the IFN-γ signal leads to lethal Orientia tsutsugamushi infection in mice with skin eschar lesions

Scrub typhus is an acute febrile disease due to Orientia tsutsugamushi (Ot) infection and can be life-threatening with organ failure, hemorrhage, and fatality. Yet, little is known as to how the host reacts to Ot bacteria at early stages of infection; no reports have addressed the functional roles of type I versus type II interferon (IFN) responses in scrub typhus. In this study, we used comprehensive intradermal (i.d.) inoculation models and two clinically predominant Ot strains (Karp and Gilliam) to uncover early immune events. Karp infection induced sequential expression of Ifnb and Ifng in inflamed skin and draining lymph nodes at days 1 and 3 post-infection. Using double Ifnar1-/-Ifngr1-/- and Stat1-/- mice, we found that deficiency in IFN/STAT1 signaling resulted in lethal infection with profound pathology and skin eschar lesions, that resembled to human scrub typhus. Further analyses demonstrated that deficiency in IFN-{gamma}, but not IFN-I, resulted in impaired NK cell and macrophage activation and uncontrolled bacterial growth and dissemination, leading to metabolic dysregulation, excessive inflammatory cell infiltration, and exacerbated tissue damage. NK cells were found to be the major cellular source of early IFN-{gamma}, contributing to the initial Ot control. In vitro studies with dendritic cell cultures revealed a superior antibacterial effect offered by IFN-{gamma} than IFN-{beta}. Comparative in vivo studies with Karp- and Gilliam-infection revealed a crucial role of IFN-{gamma} signaling in protection against progression of eschar lesions and Ot infection lethality. Additionally, our i.d. mouse models of lethal infection with eschar lesions are promising tools for immunological study and vaccine development for scrub typhus. SummaryScrub typhus can lead to severe complications and even fatality if not treated properly; however, the early host immune responses to Ot bacterium infection remain unclear. This study focused on the functional roles of IFNs in i.d. inoculation mouse models of scrub typhus. We found that mice lacking IFN receptors were highly susceptible to Ot infection, which resulted in severe pathology and skin eschar lesions that resembled to human scrub typhus. Further investigation revealed that the lack of IFN-{gamma}, but not IFN-I, resulted in dysregulated innate immune responses, leading to uncontrolled bacterial burdens and tissue damage. Using IFN-{gamma} reporter mice and neutralizing antibody treatment, we confirmed that NK cells were the major source of early IFN-{gamma}, and thus played a key role in controlling Ot dissemination. Moreover, our comparative studies with two Ot strains revealed bacterium strain- and dose-dependent eschar formation and disease severity. In conclusion, our study highlights the crucial role of IFN-{gamma} signaling in ensuring host protection against Ot infection. Our mouse models resemble skin eschar lesions and lethal infections observed in human disease, offering potential for future immunological studies on scrub typhus.

immunology↗

bioRxiv2023

Differential cellular immune responses against Orientia tsutsugamushi Karp and Gilliam strains following acute infection in mice

Scrub typhus is the leading source of febrile illness in endemic countries due to infection with Orientia tsutsugamushi (Ot), a seriously understudied intracellular bacterium. Pulmonary complications in patients are common and can develop into life threatening conditions. The diverse antigenicity of Ot genotypes and inter-strain differences seem to be connected to varied virulence and clinical outcomes; however, detailed studies of strain-related pulmonary immune responses in human patients or experimental animals are lacking. In this study, we used two clinically prevalent bacterial strains, Karp and Gilliam, and revealed cellular immune responses in inflamed lungs and potential biomarkers of disease severity. We found that outbred CD-1 mice were highly susceptible to both Karp and Gilliam strains; however, C57BL/6 (B6) mice were susceptible to Karp, but resistant to Gilliam (with self-limiting infection), corresponding to their tissue bacterial burdens and lung pathological changes. Multicolor flow cytometric analyses of perfused B6 mouse lungs revealed robust and sustained influx and activation of innate immune cells (monocytes, macrophages, neutrophils, and NK cells), followed by those of CD4+ and CD8+ T cells, during Karp infection, but such responses were greatly attenuated during Gilliam infection. The robust cellular responses in Karp-infected B6 mice were positively correlated with significantly early and high levels of serum cytokine/chemokine protein levels (CXCL1, CCL2/3/5, and G-CSF), as well as pulmonary gene expression (CXCL1/2, CCL2/3/4, and IFN{gamma}). In vitro infection of B6 mouse-derived primary macrophages also revealed bacterial strain-dependent immune gene expression profiles. This study provided the first lines of evidence that highlighted differential tissue cellular responses against Karp vs. Gilliam infection, offering a framework for future investigation of Ot strain-related mechanisms of disease pathogenesis vs. infection control. Authors SummaryOrientia tsutsugamushi (Ot) infection-induced scrub typhus is a leading cause of febrile illness in endemic countries. Research on Ot strain-related disease outcomes or immune signatures in tissue and blood samples is very limited. Using two clinically prevalent strains (Karp and Gilliam), we examined host susceptibility in inbred and outbred mouse models and provided new evidence for the activation of pulmonary immune cell subsets during the acute stages of infection. While Gilliam-infected C57BL/6 (B6) mice developed self-limiting infection, mild cellular responses, and tissue injury, Karp infection led to a strong and sustained activation of innate immune cells, followed by extensive influx of activated T cells, which correlated to protein levels of inflammatory cytokines/chemokines in serum samples. We also provided in vitro evidence for Ot strain-dependent immune gene profiles, indicating differential macrophage responses to Karp versus Gilliam bacteria. This is the first comparison of different scrub typhus mouse models with in-depth analyses of cellular responses in inflamed lungs, offering novel insights into potential mechanisms of disease progression versus infection control related to Ot strains and laying the foundation for future investigations.

immunology↗

bioRxiv2023

Alterations in Germinal Center Formation and B Cell Activation during Severe Orientia tsutsugamushi Infection in Mice

Scrub typhus is a poorly studied but life-threatening disease caused by the intracellular bacterium Orientia tsutsugamushi (Ot). Cellular and humoral immunity in Ot-infected patients is not long-lasting, waning as early as one-year post-infection; however, its underlying mechanisms remain unclear. To date, no studies have examined germinal center (GC) or B cell responses in Ot-infected humans or experimental animals. This study was aimed at evaluating humoral immune responses at acute stages of severe Ot infection and possible mechanisms underlying B cell dysfunction. Following inoculation with Ot Karp, a clinically dominant strain known to cause lethal infection in C57BL/6 mice, we measured antigen-specific antibody titers, revealing IgG2c as the dominant isotype induced by infection. Splenic GC responses were evaluated by immunohistology, co-staining for B cells (B220), T cells (CD3), and GCs (GL-7). Organized GCs were evident at day 4 post-infection (D4), but they were nearly absent at D8, accompanied by scattered T cells throughout splenic tissues. Flow cytometry revealed comparable numbers of GC B cells and T follicular helper (Tfh) cells at D4 and D8, indicating that GC collapse was not due to excessive death of these cell subtypes at D8. B cell RNAseq analysis revealed significant differences in expression of genes associated with B cell adhesion and co-stimulation at D8 versus D4. The significant downregulation of S1PR2 (a GC-specific adhesion gene) was most evident at D8, correlating with disrupted GC formation. Signaling pathway analysis uncovered downregulation of 71% of B cell activation genes at D8, suggesting attenuation of B cell activation during severe infection. This is the first study showing the disruption of B/T cell microenvironment and dysregulation of B cell responses during Ot infection, which may help understand the transient immunity associated with scrub typhus. Author SummaryScrub typhus is an understudied disease caused by the intracellular bacterium O. tsutsugamushi. A hallmark of scrub typhus is the unexplained, non-durable immunity after infection. While cellular immune responses are known to be important for controlling this infection, our understanding of B cell and GC responses remains limited. In this study, we examined B cell activation and GC responses using our recently established murine model of severe scrub typhus. We measured bacterial antigen-specific antibody titers and identified IgG2c, an IFN-{gamma}-driven isotype, as the major IgG subtype. We also found that O. tsutsugamushi infection disrupted spleen morphology, exemplified by randomly dispersed T cells and lost GC structures. Transcriptomic analysis of purified splenic B cells demonstrated reduced expression of genes encoding critical adhesion and co-stimulation molecules, including GC-adhesion gene S1PR2, at severe stages of infection. Several humoral immune response pathways relevant to B cell receptor signaling, B cell activation and differentiation were significantly downregulated during infection. This study is the first report describing humoral immunity in a scrub typhus mouse model and provides detailed evidence that B cell and GC responses are impaired during acute infection.

immunology↗

bioRxiv2022

Host transcriptomic profiling of CD-1 outbred mice with severe clinical outcomes following infection with Orientia tsutsugamushi

Orientia tsutsugamushi is an obligately intracellular bacterium with endothelial tropism and can cause mild to lethal scrub typhus in humans. No vaccine is available for this reemerging and severely neglected infection. Previous scrub typhus studies have utilized inbred mice, yet such models have intrinsic limitations. Thus, the development of suitable mouse models that better mimic human diseases is in great need for immunologic investigation and future vaccine studies. This study is aimed at establishing scrub typhus in outbred CD-1 mice and defining immune biomarkers related to disease severity. CD-1 mice received O. tsutsugamushi Karp strain via the i.v. route; major organs were harvested at 2-12 days post-infection for kinetic analyses. We found that for our given infection doses, CD-1 mice were significantly more susceptible (90-100% lethal) than were inbred C57BL/6 mice (0-10% lethal). Gross pathology of infected CD-1 mouse organs revealed features that mimicked human scrub typhus, including pulmonary edema, interstitial pneumonia, perivascular lymphocytic infiltrates, and vasculitis. Alteration in angiopoietin/receptor expression in inflamed lungs implied endothelial dysfunction. Lung immune gene profiling using NanoString analysis displayed a Th1/CD8-skewed, but Th2 repressed profile, including novel biomarkers not previously investigated in other scrub typhus models. Bio-plex analysis revealed a robust inflammatory response in CD-1 mice as evidenced by increased serum cytokine and chemokine levels, correlating with immune cell recruitment during the severe stages of the disease. This study provides an important framework indicating a value of CD-1 mice for delineating host susceptibility to O. tsutsugamushi, immune dysregulation, and disease pathogenesis. This preclinical model is particularly useful for future translational and vaccine studies for severe scrub typhus. Author SummaryScrub typhus is a severely neglected and potentially fatal disease caused by Orientia tsutsugamushi, a genetically intractable, obligately intracellular bacterium that annually infects at least one million people worldwide. There is no vaccine available, and our current understanding of the host immunological response and mechanisms remains very limited. Appropriate animal models of infection that recapitulate the disease are essential to the development of effective therapeutics and vaccines. In this study, we characterized the immunologic responses by transcriptomics and Bio-plex assays in outbred CD-1 mice with lethal O. tsutsugamushi infection. We found that CD-1 mice were highly susceptible to infection and that the high mortality correlated with a Th1/CD8-skewed, but Th2 repressed, immune profile during the acute phase of disease. This proinflammatory state was further confirmed by elevated cytokine and chemokine levels in the sera. Collectively, this study established CD-1 mice as a practical, preclinical model to define pathogenic mechanisms underlying severe scrub typhus and for future immunologic and translational studies during O. tsutsugamushi infection.

microbiology↗

bioRxiv2022

Leishmania amazonensis infection induces PD-L1 expression on dendritic cells in an mTor-dependent manner and impairs Th1 responses in vitro and in vivo

Leishmania amazonensis is one of the etiological agents of diffuse cutaneous leishmaniasis in South America. In murine models of this infection, dysregulated expansion of effector T cells or exhaustion of Th1 responses are known to be related to pathogenesis, while the induction of regulatory T cells (Tregs) promotes lesion resolution. Recent research has identified several important co-stimulator/receptor pairs, including PD-1/PD-L1, for modulating Th1 responses and Treg induction. In this study, we examined the roles of these molecules in L. amazonensis-infected C57BL/6 mice. We found a significant and selective increase in the expression of PD-1 and PD-L1 (20-fold and 5-fold, respectively), in infected footpad tissues, which correlated with an increased percentage of PD-L1+CD11c+ dendritic cells (DCs) and PD-1+CD4+ T cells in the draining lymph nodes. To evaluate the mechanism of parasite-induced PD-L1 expression, we infected bone marrow-derived DCs (BMDCs) with promastigotes and amastigotes in vitro in the presence of small molecular inhibitors of critical signaling pathways. While L. amazonensis infection decreased PD-L2 expression on the surface of BMDCs, infection-mediated PD-L1 up-regulation was consistently detected, which was dependent on mTOR and partially dependent on STAT3, PI3K, and MAPK. Infected BMDCs also significantly inhibited the expansion of Th1 cells, but were more competent in inducing CD25+FoxP3+ Tregs in vitro than the non-infected BMDCs; such effects were PD-L1-dependent, as BMDCs from PD-L1-/- mice failed to do the same. In vivo experiments revealed that infected PD-L1-/- mouse tissues showed increased Th1 responses and IFN-{gamma} production, as well as reduced lesion sizes and parasite loads, without affecting Tregs cell expansion. Together, these results support a protective role for PD-1/PD-L1 signaling in regulating local immune responses during L. amazonensis infection. This study provides new insights on immune regulation in New World cutaneous leishmaniasis.

immunology↗

bioRxiv2021

Combinatorial mRNA vaccination enhances protection against SARS-CoV-2 delta variant

Emergence of SARS-CoV-2 variants of concern (VOC), including the highly transmissible delta strain, has posed challenges to current COVID-19 vaccines that principally target the viral spike protein (S). Here, we report a nucleoside-modified mRNA vaccine that expresses the more conserved viral nucleoprotein (mRNA-N). We show that mRNA-N alone was able to induce a modest but significant control of SARS-CoV-2 in mice and hamsters. Critically, by combining mRNA-N with the clinically approved S-expressing mRNA vaccine (mRNA-S-2P), we found that combinatorial mRNA vaccination (mRNA-S+N) led to markedly enhanced protection against the SARS-CoV-2 delta variant compared to mRNA-S. In a hamster model, we demonstrated that while mRNA-S alone elicited significant control of the delta strain in the lungs ([~]45-fold reduction in viral loads compared to un-vaccinated control), its effectiveness in the upper respiratory tract was weak, whereas combinatorial mRNA-S+N vaccination induced markedly more robust control of the delta variant infection in the lungs ([~]450-fold reduction) as well as in the upper respiratory tract ([~]20-fold reduction). Immune analyses indicated that induction of N-specific immunity as well as augmented S-specific T-cell response and neutralizing antibody activity were collectively associated the enhanced protection against SARS-CoV-2 delta strain by combinatorial mRNA vaccination. These findings suggest that the combined effects of protection in the lungs and upper respiratory tract could both reduce the risk of severe disease as well as of infection and transmission.

microbiology↗

bioRxiv2020

EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K/eNOS-dependent manner during inflammation

Coagulopathy is associated with both inflammation and infection, including infection with the novel SARS-CoV-2 (COVID-19). Endothelial cells (ECs) fine tune hemostasis via cAMP-mediated secretion of von Willebrand factor (vWF), which promote the process of clot formation. The exchange protein directly activated by cAMP (EPAC) is a ubiquitously expressed intracellular cAMP receptor that plays a key role in stabilizing ECs and suppressing inflammation. To assess whether EPAC could regulate vWF release during inflammation, we utilized our EPAC1-null mouse model and revealed an increased secretion of vWF in endotoxemic mice in the absence of the EPAC1 gene. Pharmacological inhibition of EPAC1 in vitro mimicked the EPAC1-/- phenotype. EPAC1 regulated TNF-triggered vWF secretion from human umbilical vein endothelial cells (HUVECs) in a phosphoinositide 3-kinases (PI3K)/endothelial nitric oxide synthase (eNOS)-dependent manner. Furthermore, EPAC1 activation reduced inflammation-triggered vWF release, both in vivo and in vitro. Our data delineate a novel regulatory role of EPAC1 in vWF secretion and shed light on potential development of new strategies to controlling thrombosis during inflammation. Key PointPI3K/eNOS pathway-mediated, inflammation-triggered vWF secretion is the target of the pharmacological manipulation of the cAMP-EPAC system.

cell biology↗

bioRxiv2019

Annexin A2 depletion exacerbates the intracerebral microhemorrhage induced by acute rickettsia and Ebola virus infections

Intracerebral microhemorrhages (CMHs) are small foci of hemorrhages in the cerebrum. Acute infections induced by some intracellular pathogens, including rickettsia, can result in CMHs. Annexin a2 (ANXA2) has been documented to play a functional role during intracellular bacterial adhesion. Here we report that ANXA2-knockout (KO) mice are more susceptible to CMHs in response to rickettsia and Ebola virus infections, suggesting an essential role of ANXA2 in protecting vascular integrity during these intracellular pathogen infections. Proteomic analysis via mass spectrometry of whole brain lysates and brain-derived endosomes from ANXA2-KO and wild-type (WT) mice post-infection with R. australis revealed that a variety of significant proteins were differentially expressed, and the follow-up function enrichment analysis had identified several relevant cell-cell junction functions. Immunohistology study confirmed that both infected WT and infected ANXA2-KO mice were subjected to adherens junctional protein (VE-cadherin) damages. However, key blood-brain barrier (BBB) components, tight junctional proteins ZO-1 and occludin, were disorganized in the brains from R. australis-infected ANXA2-KO mice, but not those of infected WT mice. Similar ANXA2-KO dependent CMHs and fragments of ZO-1 and occludin were also observed in Ebola virus-infected ANXA2-KO mice, but not found in infected WT mice. Overall, our study revealed a novel role of ANXA2 in the formation of CMHs during R. australis and Ebola virus infections; and the underlying mechanism is relevant to the role of ANXA2-regulated tight junctions and its role in stabilizing the BBB in these deadly infections. Author SummaryTraditionally, spontaneous intracerebral microhemorrhages (CMHs) were defined as small foci of intracerebral hemorrhages. Such atraumatic CMHs are due to the rupture of a weak blood vessel wall. Infections complicating cerebrovascular accidents have been extensively investigated. However, the role of CMHs complicating infections, in particularly acute systemic infections, has been poorly explored. Population-based retrospective cohort studies suggest there are potentially more undiscovered cases of CMHs accompanying acute systemic infections. Given both the lack of an animal model and cellular/molecular pathophysiology of CMHs following acute systemic infections, there is an urgent need to increase our comprehensive understanding of acute infection-induced CMHs. Overall, our study revealed a novel role of annexin a2 (ANXA2) in the formation of CMHs during R. australis and Ebola virus infections; and the underlying mechanism is relevant to the role of ANXA2-regulated endothelial tight junctions and its role in stabilizing the blood-brain barrier in these deadly infections.

pathology↗

bioRxiv2019

Polarized Lung Inflammation and Tie2/Angiopoietin-Mediated Endothelial Dysfunction during Severe Orientia tsutsugamushi Infection

Orientia tsutsugamushi infection can cause acute lung injury and high mortality in humans; however, the underlying mechanisms are unclear. Here, we tested a hypothesis that dysregulated pulmonary inflammation and Tie2-mediated endothelial malfunction contribute to lung damage. Using a murine model of lethal O. tsutsugamushi infection, we demonstrated pathological characteristics of vascular activation and tissue damage: 1) a significant increase of ICAM-1, VEGFR2, and angiopoietin-2 (Ang2) proteins in inflamed tissues and lung-derived endothelial cells (EC), 2) a progressive loss of endothelial quiescent and junction proteins (Ang1, VE-cadherin/CD144, occuludin), and 3) a profound impairment of Tie2 receptor at the transcriptional and functional levels. In vitro infection of primary human EC cultures and serum Ang2 proteins in scrub typhus patients support our animal studies, implying endothelial dysfunction in severe scrub typhus. Flow cytometric analyses of lung-recovered cells further revealed that pulmonary macrophages (M{Phi}) were polarized toward an M1-like phenotype (CD80+CD64+CD11b+Ly6G-) during the onset of disease and prior to host death, which correlated with the significant loss of CD31+CD45- ECs and M2-like (CD206+CD64+CD11b+Ly6G-) cells. In vitro studies indicated extensive bacterial replication in M2-type, but not M1-type, M{Phi}s, implying the protective and pathogenic roles of M1-skewed responses. This is the first detailed investigation of lung cellular immune responses during acute O. tsutsugamushi infection. It uncovers specific biomarkers for vascular dysfunction and M1-skewed inflammatory responses, highlighting future therapeutic research for the control of this neglected tropical disease.\n\nAuthor SummaryScrub typhus is a life-threatening disease, infecting an estimated one million people yearly. Acute lung injury is the most common clinical observation; however, its pathogenic biomarkers and mechanisms of progression remain unknown. Here, we used a lethal infection mouse model that parallels certain aspects of severe scrub typhus, primary human endothelial cell cultures, and patient sera to define pathogenic biomarkers following Orientia tsutsugamushi infections. We found a significant increase in the levels of endothelial activation/stress markers (angiopoietins and ICAM-1) in infected mouse lungs and in patient sera, but a progressive loss of endothelium-specific Tie2 receptor and junction proteins (VE-cadherin), at severe stages of disease. These signs of vasculature disruption positively correlated with the timing and magnitude of recruitment/activation of proinflammatory M{Phi} subsets in infected lungs. Bacterial growth in vitro was favored in M2-like, but not in M1-like, M{Phi}s. This study, for the first time, reveals endothelial malfunction and dysregulated inflammatory responses, suggesting potential therapeutic targets to ameliorate tissue damage and pathogenesis.

microbiology↗

bioRxiv2019

Protection induced by anti-PD-1 and anti-PD-L1 treatment in Leishmania amazonensis-infected BALB/c mice

Leishmaniasis is a neglected disease, for which current treatment presents numerous issues. Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The roles of the programmed death-1 (PD-1) receptor on lymphocytes and its ligand (PD-L1) on antigen-presenting cells have been well studied in tumor and other infection models; but little is known about their roles in non-healing cutaneous leishmaniasis. Our previous report of L. amazonensis-induced PD-L1 expression on dendritic cells, in combination with decreased IFN-{gamma} production by CD4+ T cells in C57BL/6 mice, led to a hypothesis that the formation of the PD-1/PD-L1 complex contributes to down-modulation of immune responses, especially T cell suppression, enabling parasite survival and persistence. In this study, we tested the therapeutic potential of anti-PD-1 and anti-PD-L1 monoclonal antibodies (MoAbs) against a non-healing L. amazonensis infection in BALB/c mice. We observed that L. amazonensis induced PD-1 expression on both CD4+ and CD8+ T cells, and that anti-PD-1 and anti-PD-L1 treatment significantly increased IFN-{gamma}-producing CD4+ and CD8+ T cells, respectively. Compared with infection controls, mice that received treatment with anti-PD-1 and anti-PD-L1, but not anti-PD-L2, displayed bigger lesions with significantly lower parasite loads. Treatment did not affect anti-Leishmania antibody or IL-10 production, but anti-PD-1 treatment reduced both IL-4 and TGF-{beta} production. Together, our results highlight the therapeutic potential of an anti-PD-1-based treatment in promoting the reinvigoration of T cells for the control of parasite burden.

immunology↗