Orientia tsutsugamushi is an obligately intracellular bacterium and the etiological agent of scrub typhus. The lung is a major target organ of infection, displaying type 1-skewed proinflammatory responses. Lung injury and acute respiratory distress syndrome are common complications of severe scrub typhus; yet, their underlying mechanisms remain unclear. In this study, we investigated whether the C-type lectin receptor (CLR) Mincle contributes to immune recognition and dysregulation. Following lethal infection in mice, we performed pulmonary differential expression analysis with NanoString. Of 671 genes examined, we found 312 significantly expressed genes at the terminal phase of disease. Mincle (Clec4e) was among the top 5 greatest up-regulated genes, accompanied with its signaling partners, type 1-skewing chemokines (Cxcr3, Ccr5, and their ligands), as well as Il27. To validate the role of Mincle in scrub typhus, we exposed murine bone marrow-derived macrophages (MΦ) to live or inactivated O. tsutsugamushi and analyzed a panel of CLRs and proinflammatory markers via qRT-PCR. We found that while heat-killed bacteria stimulated transitory Mincle expression, live bacteria generated a robust response in MΦ, which was validated by indirect immunofluorescence and western blot. Notably, infection had limited impact on other tested CLRs or TLRs. Sustained proinflammatory gene expression in MΦ (Cxcl9, Ccl2, Ccl5, Nos2, Il27) was induced by live, but not inactivated, bacteria; infected Mincle-/- MΦ significantly reduced proinflammatory responses compared with WT cells. Together, this study provides the first evidence for a selective expression of Mincle in sensing O. tsutsugamushi and suggests a potential role of Mincle- and IL-27-related pathways in host responses to severe infection. Additionally, it provides novel insight into innate immune recognition of this poorly studied bacterium.

The arenaviruses Lassa virus (LASV), Junín virus (JUNV), and Machupo virus (MACV) can cause severe and fatal diseases in humans. Although these pathogens are closely related, the host immune responses to these virus infections differ remarkably, with direct implications for viral pathogenesis. LASV infection is immunosuppressive, with a very low-level interferon response. In contrast, JUNV and MACV infections stimulate a robust interferon (IFN) response in a retinoic acid-inducible gene I (RIG-I)-dependent manner and readily activate protein kinase R (PKR), a known host double-stranded RNA (dsRNA) sensor. In response to infection with RNA viruses, host nonself RNA sensors recognize virus-derived dsRNA as danger signals and initiate innate immune responses. Arenavirus nucleoproteins (NPs) contain a highly conserved exoribonuclease (ExoN) motif, through which LASV NP has been shown to degrade virus-derived immunostimulatory dsRNA in biochemical assays. In this study, we for the first time present evidence that LASV restricts dsRNA accumulation during infection. Although JUNV and MACV NPs also have the ExoN motif, dsRNA readily accumulated in infected cells and often colocalized with dsRNA sensors. Moreover, LASV coinfection diminished the accumulation of dsRNA and the IFN response in JUNV-infected cells. The disruption of LASV NP ExoN with a mutation led to dsRNA accumulation and impaired LASV replication in minigenome systems. Importantly, both LASV NP and RNA polymerase L protein were required to diminish the accumulation of dsRNA and the IFN response in JUNV infection. For the first time, we discovered a collaboration between LASV NP ExoN and L protein in limiting dsRNA accumulation. Our new findings provide mechanistic insights into the differential host innate immune responses to highly pathogenic arenavirus infections. IMPORTANCE Arenavirus NPs contain a highly conserved DEDDh ExoN motif, through which LASV NP degrades virus-derived, immunostimulatory dsRNA in biochemical assays to eliminate the danger signal and inhibit the innate immune response. Nevertheless, the function of NP ExoN in arenavirus infection remains to be defined. In this study, we discovered that LASV potently restricts dsRNA accumulation during infection and minigenome replication. In contrast, although the NPs of JUNV and MACV also harbor the ExoN motif, dsRNA readily formed during JUNV and MACV infections, accompanied by IFN and PKR responses. Interestingly, LASV NP alone was not sufficient to limit dsRNA accumulation. Instead, both LASV NP and L protein were required to restrict immunostimulatory dsRNA accumulation. Our findings provide novel and important insights into the mechanism for the distinct innate immune response to these highly pathogenic arenaviruses and open new directions for future studies.

Scrub typhus and spotted fever rickettsioses (SFR) are understudied, vector-borne diseases of global significance. Over 1 billion individuals are at risk for scrub typhus alone in an endemic region, spanning across eastern and southern Asia to Northern Australia. While highly treatable, diagnostic challenges make timely antibiotic intervention difficult for these diseases. Delayed therapy may lead to severe outcomes affecting multiple organs, including the central nervous system (CNS), where infection and associated neuroinflammation may be lethal or lead to lasting sequelae. Meningitis and encephalitis are prevalent in both scrub typhus and SFR. Additionally, case reports detailing focal neurological deficits have come to light, with attention to both acute and chronic sequelae of infection. Despite the increasing number of clinical reports outlining neurologic consequences of these diseases, relatively little research has examined underlying mechanisms of neuroinflammation. Animal models of scrub typhus have identified cerebral T-cell infiltration and vascular damage associated with endothelial infection and neuropathogenesis. Differential gene expression analysis of brain tissues during murine scrub typhus have revealed selective increases in CXCR3 ligands, proinflammatory and type-1 cytokines and chemokines, and cytotoxicity molecules, as well as alterations in the complement pathway. In SFR, microglial expansion and macrophage infiltration contribute to neurological disease progression. This narrative Review highlights clinical neurologic features of scrub typhus and SFR and evaluates our current understanding of basic research into neuroinflammation for both diseases in animal models. Further investigation into key mediators of neuropathogenesis may yield prognostic markers and treatment regimens for severe patients.

Infection with Orientia tsutsugamushi, an obligate intracellular bacterium, can cause mild or severe scrub typhus. Some patients develop acute lung injury, multi-organ failure, and fatal infection; however, little is known regarding key immune mediators that mediate infection control or disease pathogenesis. Using murine models of scrub typhus, we demonstrated in this study the requirement of TNF-TNFR signaling in protective immunity against this infection. Mice lacking both TNF receptors (TNFR1 and TNFR2) were highly susceptible to O. tsutsugamushi infection, displaying significantly increased tissue bacterial burdens and succumbing to infection by day 9, while most wild-type mice survived through day 20. This increased susceptibility correlated with poor activation of cellular immunity in inflamed tissues. Flow cytometry of lung- and spleen-derived cells revealed profound deficiencies in total numbers and activation status of NK cells, neutrophils, and macrophages, as well as CD4 and CD8 T cells. To define the role of individual receptors in O. tsutsugamushi infection, we used mice lacking either TNFR1 or TNFR2. While deficiency in either receptor alone was sufficient to increase host susceptibility to the infection, TNFR1 and TNFR2 played a distinct role in cellular responses. TNF signaling through TNFR1 promoted inflammatory responses and effector T cell expansion, while TNFR2 signaling was associated with anti-inflammatory action and tissue homeostasis. Moreover, TNFRs played an intrinsic role in CD8+ T cell activation, revealing an indispensable role of TNF in protective immunity against O. tsutsugamushi infection.

O. tsutsugamushi is an obligate intracellular bacterium and etiologic agent of scrub typhus, a life-threatening neglected disease endemic to the South-Pacific. The lungs harbor the greatest burden of infection and display strong type 1 proinflammatory responses. Here, we investigated whether the C-type lectin receptor Mincle contributes to immune recognition and inflammation. Following lethal infection in C57BL/6 mice, we performed pulmonary differential gene expression analysis via NanoString. We found 312 significantly induced genes (adj. p < 0.05) at the terminal phase of disease, of which Mincle (Clec4e) was the 4th most upregulated gene. Mincle signaling partners (Fcgrs), Cxcr3, Ccr5, and their ligands, as well as Il27, were also highly upregulated. To define a role of Mincle in immune recognition, we exposed WT or Mincle−/− bone marrow-derived macrophages (MΦ) to live or inactivated bacteria and analyzed inflammatory markers via qRT-PCR. We observed infectious dose-dependent increases in Mincle transcription along with Cxcl1, Ccl2, Tnf, and Nos2 early in infection. Mincle−/− cells had abrogated responses of Cxcl1 and Ccl2. Blocking TNF signaling prior to infection markedly reduced Mincle, Ccl2, Cxcl1, Cxcl10, and Il27 transcripts. MΦ treated with rTNF-α prior to infection had greater levels of Mincle, Il27, Cxcl10, and Ccl2 expression than treatment with TNF or Orientia alone, suggesting a synergistic effect. Our study provides the first evidence for Mincle sensing O. tsutsugamushi and a role of Mincle-, TNFα-, and IL-27-related crosstalk during infection. Since there are no effective vaccines for scrub typhus, our results help understand innate immune recognition of this understudied bacterium and its disease. Supported by grants from NIH (R01 AI132674 to LS, T32 AI007526-20 to LS, and R21 AI156536-01 to LS). JF was a recipient of a NIAID Emerging and Tropical Infectious Diseases T32 fellowship.

Scrub typhus is a poorly studied, but life-threatening, disease due to uncontrolled growth of Orientia tsutsugamushi bacteria within endothelial cells and phagocytes, in addition to dysregulated Th1 immune responses and inflammation. Cellular and humoral immunity in infected patients and experimental animals are short-lived, and wane within 1–2 years; however, the underlying mechanism of this transient immunity is unclear. No reports have examined splenic germinal center (GC) or B cell activation during this infection. The study objective is to fill this knowledge gap by using endothelium-targeted C57BL/6 mouse models that mimic pathological features of human scrub typhus. Through NanoString, qRT-PCR, and flow cytometry, we found significant repression of key markers crucial for B cell development (CCR7, BAFFR, PAX5) and GC formation (SLAMF1, ICOS-L, CXCR5, BCL-6) in both mild and severe disease outcomes. The widespread impairments in B and T follicular helper (Tfh) cell activation correlated with excessive Th1 cell activation during disease progression. Immunofluorescence staining (B220, CD3, GL7) revealed evidence of GC formation with sublethal infection, but the near absence of organized GCs at late stages of lethal infection, which was consistent with results from our splenic multi-color flow cytometry and serum antigen-specific ELISA assays for IgG isotypes. Ongoing RNA-seq studies for splenic B cells isolated from sub-lethally or lethally infected mice will provide in-depth, B cell-specific insight during infection. In conclusion, this is the first study to show profound alterations in B cell activation and GC formation in murine models, which helps understand “poor” humoral immunity seen with human scrub typhus. Supported by grants from NIH (R01 EY028773 to JS, R01 AI132674 to LS, R21 AI156536 to LS)

Scrub typhus is an emerging tropical disease caused by obligate intracellular bacteria Orientia tsutsugamushi, with over one million cases reported yearly. Patients may develop acute lung injury and multi-organ failure; the worldwide average mortality rate is 6%; however, little is known regarding key immune mediators during infection. Using murine scrub typhus models, we demonstrated the essential role of TNF signals in bacterial control and host protection. Mice lacking both TNF receptors (TNFR1/R2−/−) or receiving anti-TNF-α treatment were highly susceptible to infection with significantly increased tissue bacterial loads, inflammatory responses, and mortality. Their lung- and spleen-derived leukocytes displayed profound defects in total cell numbers and activation status for both innate immunity (NK cells, neutrophils, and macrophages) and adaptive immunity (CD4+ and CD8+ T cells). Infection of TNFR1−/− or TNFR2−/− mice confirmed the requirement of both receptors in efficient control of disease progression, as lacking either one receptor greatly reduced activated CD4+ and CD8+ T effector cells. Together, this study demonstrates a critical and indispensable role of TNF signals in host protective immunity against O. tsutsugamushi infection and severe scrub typhus. Supported by grants from NIH (R01 AI132674, R21 AI156536 and R21 AI153586).

Orientia tsutsugamushi is a Gram- and LPS-negative bacterium that can cause severe scrub typhus in the Asia-Pacific region. While bacteria replicate freely in endothelial cell (EC) and phagocyte cytosol, little is known regarding pathogen recognition and cellular responses. Our newly developed O. tsutsugamushi infection mouse models are valuable for mechanistic studies. Here, we show that a hallmark of lethal infection is Th1-skewed, but Th2-impaired, immune responses, accompanied by severe endothelial damage and acute tissue injury. While tissue bacteria peak at disease onset (D6), neutrophil and CD8 T cell influx/activation reach peaks around D10 (prior to mouse death). We further show the pathological characteristics of vascular activation and tissue damage: 1) a significant increase of ICAM-1, VEGFR2, and angiopoietin-2 proteins in infected tissues and lung-derived ECs, 2) a loss of endothelial quiescent/junction proteins (angiopoietin-1, VE-cadherin), and 3) a functional loss of Tie2 receptor. Pulmonary macrophages predominantly display an M1-like (CD80+CD64+CD11b+) phenotype at D6 and D9, which correlated with the significant loss of CD31+CD45− ECs and M2-like (CD206+CD64+CD11b+−) cells. NanoString analyses of infected lung and spleen tissues and in vitro studies suggested a role for Clec4e in pathogen recognition and the IL-12/IL-27 axis in driving proinflammatory immune responses and cellular injury. 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.