Thomas R. Shelite

Unresolved Problems Related to Scrub Typhus: A Seriously Neglected Life-Threatening Disease

Ecology and epidemiology. Scrub typhus is a life-threatening infectious disease that presents as an acute undifferentiated febrile illness. Its agent,Orientia tsutsugamushi, is an obligately intracytosolic bacterium that is transmitted by feeding larval trombiculid mites, which are the reservoir of the agent and the only life stage that feeds on a vertebrate host. Nymphal and adult trombiculid mites live in the soil and feed on the eggs of insects. Mites maintain the organisms by transovarian transmission as well as transtadial transmission through the mite’s lifecycle. Although mites transmit O. tsutsugamushi to vertebrate hosts such as rodents, only a small proportion of uninfected mites acquire Orientia during feeding on infected animals, and the ingested Orientia do not establish disseminated infection in the mites and are not transmitted transovarially to the next generation. It seems that chigger cofeeding on rodents is more relevant for effective mouseto-mite transmission of Orientia than feeding on rickettsemic hosts. Thus, the true role of rodents as reservoirs requires additional investigation, because there may be marked genetic variability in chiggers with respect to the ability to acquire rickettsiae by feeding. Also, rodents may be better considered as dead-end hosts just as humans are rather than a reservoir. However, the evolutionary selection of tremendous antigenic diversity of the immunodominant major 56 kDa surface protein presents an enigma if immune variation plays no role in survival of O. tsutsugamushi. The potential immunomodulatory effects of the saliva of larval mites on the pathogenesis of and immunity to infection with O. tsutsugamushi remain undetermined. One million cases of scrub typhus occur each year with an estimated 10% case fatality rate unless treated appropriately, very likely resulting in more deaths than dengue. Currently, scrub typhus has predominantly been reported from an area extending from the Russian Far East and Korea in the north to northern Australia in the south and Afghanistan in the west, and it includes islands of the western Pacific and Indian Oceans, including Japan, Taiwan, Philippines, Papua New Guinea, Indonesia, and Sri Lanka. This geographical range may be an underrepresentation, because case reports have been published from Africa and South America. The recent isolation of a novel species O. chuto acquired by a patient in Dubai, the detection of another divergentOrientia transmitted to a patient in Chile, and serologic diagnoses of scrub typhus acquired in Africa indicate that a wider geographic distribution and genetic diversity of the genus should be investigated. The burden of disease in rural areas of Asia is large, with studies showing scrub typhus causing up to 20% of febrile hospital admissions, an incidence of infections of greater than 3% of the population monthly, seroprevalence over 50% of the population, despite a significant annual rate of reversion to seronegativity of 50% of cases, and a seroconversion rate of 484 per 1,000 person-years. In 1999, the World Health Organization (WHO) stated, “Scrub typhus is probably one of the most underdiagnosed and underreported febrile illnesses requiring hospitalization in the region.” This opinion remains valid today and could justifiably be adjusted to scrub typhus is probably the single most prevalent, under-recognized, neglected, and severe but easily treatable disease in the world. This ancient disease is currently undergoing increased awareness both because of re-emergence and rising incidence in previously unrecognized areas and improved diagnostic testing. It is apparent that scrub typhus has been recognized to occur frequently now in places where the illness was nearly forgotten, including India, Sri Lanka, the Maldives, and Micronesia. However, scrub typhus has also emerged in regions north of the Yangtse River in China, where it was not known previously. The emergence of scrub typhus should also take into consideration the expansion of farmlands that have generated ideal habitats for trombiculid mites. The potential relationship with global climate change is unclear. Greater recognition in some countries, such as Thailand, Laos, Taiwan, and Japan, may reflect increased medical investigations and application of new diagnostic methods. The critical unresolved issues regarding epidemiology emphasize the need to determine the reasons for emergence and re-emergence and the true incidence of this neglected disease, for which calculated days of disability-adjusted years of life lost (DALYs) have not been determined and to which appropriate attention has not been paid (Table 1). Clinical manifestations. Scrub typhus ranges from a mild to a fatal illness. The early clinical manifestations are an eschar, representing localized cutaneous necrosis at the site of mite feeding (which is not always present), and regional lymphadenopathy followed subsequently by fever, headache, myalgia, generalized lymphadenopathy, cough, gastrointestinal symptoms, transient hearing loss, and rash. Progression of severe scrub typhus may manifest as acute respiratory distress, meningoencephalitis, gastrointestinal bleeding, acute renal failure, hypotensive shock, and coagulopathy. Unresolved clinical issues include the reason for the wide range of occurrence of eschars (7–97%), the reason for rash and severity of illness, and the need for characterization and determination of the mechanisms of the coagulopathy, hemorrhages, interstitial pneumonia, and meningoencephalitis. There is currently no unified approach to assess and stratify *Address correspondence to David H. Walker, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609. E-mail: dwalker@utmb.edu

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Significance Our studies combining genetic and pharmacological manipulations provide convincing evidence that exchange protein directly activated by cAMP (Epac) 1 plays a critical role in fatal spotted fever group rickettsioses. Inhibition of Epac1 suppresses bacterial adhesion and/or invasion. Most importantly, we show that a small-molecule Epac inhibitor can prevent and suppress rickettsial infection. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses. This is significant from the biodefense viewpoint because it suggests that Epac1 inhibitor can be potentially used as a prophylaxis to thwart initial bacterial infection in the event of a bioterrorism threat. Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.

Amblyomma imitator ticks as vectors of Rickettsia rickettsii, Mexico.

Real-time PCR of Amblyomma imitator tick egg masses obtained in Nuevo Leon State, Mexico, identified a Rickettsia species. Sequence analyses of 17-kD common antigen and outer membrane protein A and B gene fragments showed to it to be R. rickettsii, which suggested a potential new vector for this bacterium.

Permissive and protective roles for neutrophils in leishmaniasis

Leishmania parasites are the causative agents of leishmaniasis, a neglected tropical disease that causes substantial morbidity and considerable mortality in many developing areas of the world. Recent estimates suggest that roughly 10 million people suffer from cutaneous leishmaniasis (CL), and approximately 76 000 are afflicted with visceral leishmaniasis (VL), which is universally fatal without treatment. Efforts to develop therapeutics and vaccines have been greatly hampered by an incomplete understanding of the parasites biology and a lack of clear protective correlates that must be met in order to achieve immunity. Although parasites grow and divide preferentially in macrophages, a number of other cell types interact with and internalize Leishmania parasites, including monocytes, dendritic cells and neutrophils. Neutrophils appear to be especially important shortly after parasites are introduced into the skin, and may serve a dual protective and permissive role during the establishment of infection. Curiously, neutrophil recruitment to the site of infection appears to continue into the chronic phase of disease, which may persist for many years. The immunological impact of these cells during chronic leishmaniasis is unclear at this time. In this review we discuss the ways in which neutrophils have been observed to prevent and promote the establishment of infection, examine the role of anti‐neutrophil antibodies in mouse models of leishmaniasis and consider recent findings that neutrophils may play a previously unrecognized role in influencing chronic parasite persistence.

A Hematogenously Disseminated Orientia tsutsugamsushi-Infected Murine Model of Scrub Typhus

Orientia tsutsugamushi, the etiologic agent of scrub typhus, is a mite-borne rickettsia transmitted by the parasitic larval stage of trombiculid mites. Approximately one-third of the worlds population is at risk of infection with Orientia tsutsugamushi, emphasizing its importance in global health. In order to study scrub typhus, Orientia tsutsugamushi Karp strain has been used extensively in mouse studies with various inoculation strategies and little success in inducing disease progression similar to that of human scrub typhus. The objective of this project was to develop a disease model with pathology and target cells similar to those of severe human scrub typhus. This study reports an intravenous infection model of scrub typhus in C57BL/6 mice. This mouse strain was susceptible to intravenous challenge, and lethal infection occurred after intravenous inoculation of 1.25×106 focus (FFU) forming units. Signs of illness in lethally infected mice appeared on day 6 with death occurring ∼6 days later. Immunohistochemical staining for Orientia antigens demonstrated extensive endothelial infection, most notably in the lungs and brain. Histopathological analysis revealed cerebral perivascular, lymphohistiocytic infiltrates, focal hemorrhages, meningoencephalitis, and interstitial pneumonia. Disseminated infection of endothelial cells with Orientia in C57BL/6 mice resulted in pathology resembling that of human scrub typhus. The use of this model will allow detailed characterization of the mechanisms of immunity to and pathogenesis of O. tsutsugamushi infection.

CD4+ CD25+ Foxp3- T-regulatory cells produce both gamma interferon and interleukin-10 during acute severe murine spotted fever rickettsiosis.

ABSTRACT Spotted fever group rickettsiae cause life-threatening human infections worldwide. Until now, the immune regulatory mechanisms involved in fatal rickettsial infection have been unknown. C3H/HeN mice infected with 3 × 105 PFU of Rickettsia conorii developed an acute progressive disease, and all mice succumbed to this infection. A sublethal infection induced protective immunity, and mice survived. Compared to splenic T cells from sublethally infected mice, splenic T cells from lethally infected mice produced significantly lower levels of interleukin-2 (IL-2) and gamma interferon (IFN-γ) and a higher level of IL-10, but not of IL-4 or transforming growth factor β, and there was markedly suppressed CD4+ T-cell proliferation in response to antigen-specific stimulation with R. conorii. Furthermore, lethal infection induced significant expansion of CD4+ CD25+ Foxp3− T cells in infected organs compared to the levels in naïve and sublethally infected mice. In a lethal infection, splenic CD4+ CD25+ Foxp3− T cells, which were CTLA-4high T-bet+ and secreted both IFN-γ and IL-10, suppressed the proliferation of and IL-2 production by splenic CD4+ CD25− Foxp3− T cells in vitro. Interestingly, depletion of CD25+ T cells in vivo did not change the disease progression, but it increased the bacterial load in the lung and liver, significantly reduced the number of IFN-γ-producing Th1 cells in the spleen, and increased the serum levels of IFN-γ. These results suggested that CD4+ CD25+ T cells generated in acute murine spotted fever rickettsiosis are Th1-cell-related adaptive T-regulatory cells, which substantially contribute to suppressing the systemic immune response, possibly by a mechanism involving IL-10 and/or cytotoxic T-lymphocyte antigen 4.

Strong type 1, but impaired type 2, immune responses contribute to Orientia tsutsugamushi-induced pathology in mice.

Scrub typhus is a neglected, but important, tropical disease, which puts one-third of the worlds population at risk. The disease is caused by Orientia tsutsugamushi, an obligately intracellular Gram-negative bacterium. Dysregulation in immune responses is known to contribute to disease pathogenesis; however, the nature and molecular basis of immune alterations are poorly defined. This study made use of a newly developed murine model of severe scrub typhus and focused on innate regulators and vascular growth factors in O. tsutsugamushi-infected liver, lungs and spleen. We found no activation or even reduction in base-line expression for multiple molecules (IL-7, IL-4, IL-13, GATA3, ROR-γt, and CXCL12) at 2, 6 and 10 days post-infection. This selective impairment in type 2-related immune responses correlated with a significant activation of the genes for IL-1β, IL-6, IL-10, TNF-α, IFN-γ, as well as CXCR3- and CXCR1-related chemokines in inflamed tissues. The elevated angiopoietin (Ang)-2 expression and Ang-2/Ang-1 ratios suggested excessive inflammation and the loss of endothelial integrity. These alterations, together with extensive recruitment of myeloperoxidase (MPO)-expressing neutrophils and the influx of CD3+ T cells, contributed to acute tissue damage and animal death. This is the first report of selective alterations in a panel of immune regulators during early O. tsutsugamushi infection in intravenously inoculated C57BL/6 mice. Our findings shed new light on the pathogenic mechanisms associated with severe scrub typhus and suggest potential targets for therapeutic investigation.

Systemic treatment with cpg-b after sublethal rickettsial infection induces mouse death through indoleamine 2,3-dioxygenase (ido)

Due to its strong immune stimulatory effects through TLR9, CpG-containing oligodeoxynucleotides (CpG ODN) have been tested in multiple clinical trials as vaccine adjuvant for infectious diseases and cancer. However, immune suppression induced by systemic administration of CpGs has been reported recently. In this study, we evaluated the impact of CpGs in an acute rickettsiosis model. We found that systemic treatment with type B CpG (CpG-B), but not type A CpG (CpG-A), at 2 days after sublethal R. australis infection induced mouse death. Although wild-type (WT) B6 and IDO−/− mice showed similar survival rates with three different doses of R. australis infection, treatment with CpG-B after sublethal infection consistently induced higher mortality with greater tissue bacterial loads in WT but not IDO−/− mice. Also, CpG-B treatment promoted the development of higher serum concentrations of proinflammatory cytokines/chemokines through IDO. Furthermore, while T cell-mediated immune responses enhanced by CpG-B were independent of IDO, treatment with CpG-B promoted T cell activation, PD-1 expression and cell apoptosis partially through IDO. A depletion study using anti-mPDCA-1 mAb indicated that plasmacytoid dendritic cells (pDC) were not required for CpG-B-induced death of R. australis-infected mice. Additionally, the results in iNOS−/− mice suggested that nitric oxide (NO) was partially involved in CpG-B-induced death of R. australis-infected mice. Surprisingly, pre-treatment with CpG-B before administration of a lethal dose of R. australis provided effective immunity in WT, IDO−/− and iNOS−/− mice. Taken together, our study provides evidence that CpGs exert complex immunological effects by both IDO-dependent and -independent mechanisms, and that systemic treatment with CpGs before or after infection has a significant and distinct impact on disease outcomes.

IL-33-Dependent Endothelial Activation Contributes to Apoptosis and Renal Injury in Orientia tsutsugamushi-Infected Mice

Endothelial cells (EC) are the main target for Orientia tsutsugamushi infection and EC dysfunction is a hallmark of severe scrub typhus in patients. However, the molecular basis of EC dysfunction and its impact on infection outcome are poorly understood. We found that C57BL/6 mice that received a lethal dose of O. tsutsugamushi Karp strain had a significant increase in the expression of IL-33 and its receptor ST2L in the kidneys and liver, but a rapid reduction of IL-33 in the lungs. We also found exacerbated EC stress and activation in the kidneys of infected mice, as evidenced by elevated angiopoietin (Ang) 2/Ang1 ratio, increased endothelin 1 (ET-1) and endothelial nitric oxide synthase (eNOS) expression. Such responses were significantly attenuated in the IL-33-/- mice. Importantly, IL-33-/- mice also had markedly attenuated disease due to reduced EC stress and cellular apoptosis. To confirm the biological role of IL-33, we challenged wild-type (WT) mice with a sub-lethal dose of O. tsutsugamushi and gave mice recombinant IL-33 (rIL-33) every 2 days for 10 days. Exogenous IL-33 significantly increased disease severity and lethality, which correlated with increased EC stress and activation, increased CXCL1 and CXCL2 chemokines, but decreased anti-apoptotic gene BCL-2 in the kidneys. To further examine the role of EC stress, we infected human umbilical vein endothelial cells (HUVEC) in vitro. We found an infection dose-dependent increase in the expression of IL-33, ST2L soluble ST2 (sST2), and the Ang2/Ang1 ratio at 24 and 48 hours post-infection. This study indicates a pathogenic role of alarmin IL-33 in a murine model of scrub typhus and highlights infection-triggered EC damage and IL-33-mediated pathological changes during the course of Orientia infection.

An Intradermal Inoculation Mouse Model for Immunological Investigations of Acute Scrub Typhus and Persistent Infection

Scrub typhus is a neglected tropical disease, caused by Orientia tsutsugamushi, a Gram-negative bacterium that is transmitted to mammalian hosts during feeding by Leptotrombidium mites and replicates predominantly within endothelial cells. Most studies of scrub typhus in animal models have utilized either intraperitoneal or intravenous inoculation; however, there is limited information on infection by the natural route in murine model skin or its related early host responses. Here, we developed an intradermal (i.d.) inoculation model of scrub typhus and focused on the kinetics of the host responses in the blood and major infected organs. Following ear inoculation with 6 x 104 O. tsutsugamushi, mice developed fever at 11–12 days post-infection (dpi), followed by marked hypothermia and body weight loss at 14–19 dpi. Bacteria in blood and tissues and histopathological changes were detected around 9 dpi and peaked around 14 dpi. Serum cytokine analyses revealed a mixed Th1/Th2 response, with marked elevations of MCP-1/CCL2, MIP-1α/CCL3 and IL-10 at 9 dpi, followed by increased concentrations of pro-inflammatory markers (IL-6, IL-12, IFN-γ, G-CSF, RANTES/CCL5, KC/CCL11, IL-1α/β, IL-2, TNF-α, GM-CSF), as well as modulatory cytokines (IL-9, IL-13). Cytokine levels in lungs had similar elevation patterns, except for a marked reduction of IL-9. The Orientia 47-kDa gene and infectious bacteria were detected in several organs for up to 84 dpi, indicating persistent infection. This is the first comprehensive report of acute scrub typhus and persistent infection in i.d.-inoculated C57BL/6 mice. This is a significant improvement over current murine models for Orientia infection and will permit detailed studies of host immune responses and infection control interventions.