Nahed Ismail

Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections

CD1d-restricted natural killer T (NKT) cells are innate-like lymphocytes that express a conserved T-cell receptor and contribute to host defence against various microbial pathogens. However, their target lipid antigens have remained elusive. Here we report evidence for microbial, antigen-specific activation of NKT cells against Gram-negative, lipopolysaccharide (LPS)-negative alpha-Proteobacteria such as Ehrlichia muris and Sphingomonas capsulata. We have identified glycosylceramides from the cell wall of Sphingomonas that serve as direct targets for mouse and human NKT cells, controlling both septic shock reaction and bacterial clearance in infected mice. In contrast, Gram-negative, LPS-positive Salmonella typhimurium activates NKT cells through the recognition of an endogenous lysosomal glycosphingolipid, iGb3, presented by LPS-activated dendritic cells. These findings identify two novel antigenic targets of NKT cells in antimicrobial defence, and show that glycosylceramides are an alternative to LPS for innate recognition of the Gram-negative, LPS-negative bacterial cell wall.

Emerging and re-emerging rickettsioses: endothelial cell infection and early disease events.

Rickettsiae cause some of the most severe human infections, including epidemic typhus and Rocky Mountain spotted fever. Substantial progress has been made in research into the genomics, vector relationships, pathogenesis and immunity of these obligate, intracellular, arthropod-transmitted bacteria. This Review summarizes our understanding of the early and late events in pathogenesis and immunity, modulation of the host response to rickettsial infection by the vector, host defence, virulence mechanisms and rickettsial manipulation of host cells.

Human Ehrlichiosis and Anaplasmosis

Human ehrlichiosis and anaplasmosis are acute febrile tick-borne diseases caused by various members of the genera Ehrlichia and Anaplasma (Anaplasmataceae). Human monocytotropic ehrlichiosis has become one of the most prevalent life-threatening tick-borne disease in the United States. Ehrlichiosis and anaplasmosis are becoming more frequently diagnosed as the cause of human infections, as animal reservoirs and tick vectors have increased in number and humans have inhabited areas where reservoir and tick populations are high. Ehrlichia chaffeensis, the etiologic agent of human monocytotropic ehrlichiosis (HME), is an emerging zoonosis that causes clinical manifestations ranging from a mild febrile illness to a fulminant disease characterized by multiorgan system failure. Anaplasma phagocytophilum causes human granulocytotropic anaplasmosis (HGA), previously known as human granulocytotropic ehrlichiosis. This article reviews recent advances in the understanding of ehrlichial diseases related to microbiology, epidemiology, diagnosis, pathogenesis, immunity, and treatment of the 2 prevalent tick-borne diseases found in the United States, HME and HGA.

Overproduction of TNF-α by CD8+ Type 1 Cells and Down-Regulation of IFN-γ Production by CD4+ Th1 Cells Contribute to Toxic Shock-Like Syndrome in an Animal Model of Fatal Monocytotropic Ehrlichiosis

Human monocytotropic ehrlichiosis (HME) is an emerging, life-threatening, infectious disease caused by Ehrlichia chaffeensis, an obligate intracellular bacterium that lacks cell wall LPS. We have previously developed an animal model of severe HME using a strain of Ehrlichia isolated from Ixodes ovatus ticks (IOE). To understand the basis of susceptibility to severe monocytotropic ehrlichiosis, we compared low and high doses of the highly virulent IOE strain and the less virulent Ehrlichia muris strain that are closely related to E. chaffeensis in C57BL/6 mice. Lethal infections caused by high or low doses of IOE were accompanied by extensive liver damage, extremely elevated levels of TNF-α in the serum, high frequency of Ehrlichia-specific, TNF-α-producing CD8+ T cells in the spleen, decreased Ehrlicha-specific CD4+ T cell proliferation, low IL-12 levels in the spleen, and a 40-fold decrease in the number of IFN-γ-producing CD4+ Th1 cells. All groups contained negligible numbers of IL-4-producing cells in the spleen. Transfer of Ehrlichia-specific polyclonal Abs and IFN-γ-producing Ehrlichia-specific CD4+ and CD8+ type 1 cells protected naive mice against lethal IOE challenge. Interestingly, infection with high dose E. muris provided protection against rechallenge with a lethal dose of IOE. Cross-protection was associated with substantial expansion of IFN-γ-producing CD4+ and CD8+ cells, but not TNF-α-producing CD8+ T cells, a high titer of IgG2a, and a low serum level of TNF-α. In conclusion, uncontrolled TNF-α production by CD8+ T cells together with a weak CD4+ Th1 cell response are associated with immunopathology and failure to clear IOE in the fatal model of HME.

Host- and Microbe-Related Risk Factors for and Pathophysiology of Fatal Rickettsia conorii Infection in Portuguese Patients

BACKGROUND The pathophysiologic mechanisms that determine the severity of Mediterranean spotted fever (MSF) and the host-related and microbe-related risk factors for a fatal outcome are incompletely understood. METHODS This prospective study used univariate and multivariate analyses to determine the risk factors for a fatal outcome for 140 patients with Rickettsia conorii infection admitted to 13 Portuguese hospitals during 1994-2006 with documented identification of the rickettsial strain causing their infection. RESULTS A total of 71 patients (51%) were infected with the Malish strain of Rickettsia conorii, and 69 (49%) were infected with the Israeli spotted fever (ISF) strain. Patients were admitted to the intensive care unit (40 [29%]), hospitalized as routine inpatients (95[67%]), or managed as outpatients (5[4%]). Death occurred in 29 adults (21%). A fatal outcome was significantly more likely for patients infected with the ISF strain, and alcoholism was a risk factor. The pathophysiology of a fatal outcome involved significantly greater incidence of petechial rash, gastrointestinal symptoms, obtundation and/or confusion, dehydration, tachypnea, hepatomegaly, leukocytosis, coagulopathy, azotemia, hyperbilirubinemia, and elevated levels of hepatic enzymes and creatine kinase. Some, but not all, of these findings were observed more often in ISF strain-infected patients. CONCLUSIONS Although fatalities and similar clinical manifestations occurred among both groups of patients, the ISF strain was more virulent than the Malish strain. Multivariate analysis revealed that acute renal failure and hyperbilirubinemia were most strongly associated with a fatal outcome.

Role of Tumor Necrosis Factor Alpha (TNF-α) and Interleukin-10 in the Pathogenesis of Severe Murine Monocytotropic Ehrlichiosis: Increased Resistance of TNF Receptor p55- and p75-Deficient Mice to Fatal Ehrlichial Infection

ABSTRACT Intraperitoneal (i.p.) infection with a high dose of a highly virulent Ehrlichia strain (IOE) results in a toxic shock-like syndrome characterized by severe liver injury and systemic overproduction of tumor necrosis factor alpha (TNF-α) by CD8+ T cells. We examined the role of TNF-α and TNF receptors in high-dose-IOE-induced shock/liver injury. TNF receptor (TNFR) I/II−/− mice lacking both the p55 and p75 receptors for this cytokine were more resistant to IOE-induced liver injury than their wild-type background controls. TNFR I/II−/− mice survived longer, dying between 15 and 18 days, with evidence of mild liver necrosis/apoptosis. In contrast, wild-type mice were not rescued from the lethal effect of IOE by TNF-α neutralization. TNF-α-depleted mice developed severe liver injury and succumbed to disease between days 9 and 11 postinfection, similar to sham-treated, infected wild-type mice. Although IFN-γ production in the spleens of IOE-infected TNFR I/II−/− and TNF-α-depleted mice was higher than that detected in wild-type controls, these mice had higher bacterial burdens than infected controls. Following high-dose IOE challenge, TNFR I/II−/− and TNF-α-depleted mice have an early increase in IL-10 levels in sera and spleens, which was produced mainly by adherent spleen cells. In contrast, a late burst of interleukin-10 (IL-10) was observed in control mice. Nonadherent spleen cells were the major source of IL-10 in IOE-infected wild-type mice. We conclude that TNFR I/II and TNF-α participate in Ehrlichia-induced shock and host defense by regulating liver injury and controlling ehrlichial burden. Our data suggest that fatal ehrlichiosis could be a multistep process, where TNF-α is not solely responsible for mortality.

An Intradermal Environment Promotes a Protective Type-1 Response against Lethal Systemic Monocytotropic Ehrlichial Infection

ABSTRACT Immune responses against monocytotropic ehrlichiosis during infection with a strain of Ehrlichia from Ixodes ovatus (IOE) were evaluated using a model that closely reproduces the pathology and immunity associated with tick-transmitted human monocytotropic ehrlichiosis. C57BL/6 mice were inoculated intradermally or intraperitoneally with high-dose highly virulent IOE or intraperitoneally with mildly virulent Ehrlichia muris. Intradermal (i.d.) infection with IOE established mild, self-limited disease associated with minimal hepatic apoptosis, and all mice survived past 30 days. Intraperitoneal (i.p.) infection with IOE resulted in acute, severe toxic shock-like syndrome and severe multifocal hepatic apoptosis and necrosis, and all mice succumbed to disease. Compared to i.p. infection with IOE, intradermally infected mice had a 100- to 1,000-fold lower bacterial load in the spleen with limited dissemination. Compared to mice infected intraperitoneally with IOE, i.d. infection stimulated a stronger protective type-1 cell-mediated response on day 7 of infection, characterized by increased percentages of both CD4+ and CD8+ splenic T cells, generation of a greater number of IOE-specific, gamma interferon-producing CD4+ Th1 cells, and higher levels of tumor necrosis factor (TNF-α) in the spleen but lower concentrations of serum TNF-α and interleukin-10. These data suggest that under the conditions of natural route of challenge (i.e., i.d. inoculation), the immune response has the capacity to confer complete protection against monocytotropic ehrlichiosis, which is associated with a strong cell-mediated type-1 response and decreased systemic production of pro- and anti-inflammatory cytokines.

Intralesional Expression of mRNA of Interferon-γ, Tumor Necrosis Factor-α, Interleukin-10, Nitric Oxide Synthase, Indoleamine-2,3-Dioxygenase, and RANTES Is a Major Immune Effector in Mediterranean Spotted Fever Rickettsiosis

BACKGROUND The mechanisms of immunity to Rickettsia conorii that have been elucidated in mouse models have not been evaluated in human tissues. METHODS In this study, quantitative real-time polymerase chain reaction was used to determine the levels of expression of inflammatory and immune mediators in skin-biopsy samples collected from 23 untreated patients with Mediterranean spotted fever (MSF). RESULTS In all 23 patients, the levels of intralesional expression of mRNA of tumor necrosis factor (TNF)- alpha , interferon (IFN)- gamma , interleukin (IL)-10, RANTES, and indoleamine-2,3-dioxygenase (IDO), an enzyme involved in limiting rickettsial growth by tryptophan degradation, were higher than those in control subjects; 6 of the 23 patients had high levels of inducible nitric oxide synthase (iNOS), a source of microbicidal nitric oxide. Positive correlations between TNF- alpha , IFN- gamma , iNOS, IDO, and mild/moderate MSF suggest that type 1 polarization plays a protective role. Significantly higher levels of intralesional expression of IL-10 mRNA were inversely correlated with levels of intralesional expression of IFN- gamma mRNA and TNF- alpha mRNA. The mRNA-expression level of the chemokine RANTES was significantly higher in patients with severe MSF. CONCLUSION Mild/moderate MSF is associated with a strong and balanced intralesional proinflammatory and anti-inflammatory response, with a dominant type 1 immunity, whereas severe MSF is associated with increased expression of chemokine mRNA. Whether these factors are simply correlates of mild and severe MSF or contribute to antirickettsial immunity and pathogenesis remains to be determined.

Differential Interaction of Dendritic Cells with Rickettsia conorii: Impact on Host Susceptibility to Murine Spotted Fever Rickettsiosis

ABSTRACT Spotted fever group rickettsioses are emerging and reemerging infectious diseases, some of which are life-threatening. In order to understand how dendritic cells (DCs) contribute to the host resistance or susceptibility to rickettsial diseases, we first characterized the in vitro interaction of rickettsiae with bone marrow-derived DCs (BMDCs) from resistant C57BL/6 (B6) and susceptible C3H/HeN (C3H) mice. In contrast to the exclusively cytosolic localization within endothelial cells, rickettsiae efficiently entered and localized in both phagosomes and cytosol of BMDCs from both mouse strains. Rickettsia conorii-infected BMDCs from resistant mice harbored higher bacterial loads compared to C3H mice. R. conorii infection induced maturation of BMDCs from both mouse strains as judged by upregulated expression of classical major histocompatibility complex (MHC) and costimulatory molecules. Compared to C3H counterparts, B6 BMDCs exhibited higher expression levels of MHC class II and higher interleukin-12 (IL-12) p40 production upon rickettsial infection and were more potent in priming naïve CD4+ T cells to produce gamma interferon. In vitro DC infection and T-cell priming studies suggested a delayed CD4+ T-cell activation and suppressed Th1/Th2 cell development in C3H mice. The suppressive CD4+ T-cell responses seen in C3H mice were associated with a high frequency of Foxp3+ T regulatory cells promoted by syngeneic R. conorii-infected BMDCs in the presence of IL-2. These data suggest that rickettsiae can target DCs to stimulate a protective type 1 response in resistant hosts but suppressive adaptive immunity in susceptible hosts.

Regulatory Roles of CD1d-Restricted NKT Cells in the Induction of Toxic Shock-Like Syndrome in an Animal Model of Fatal Ehrlichiosis

ABSTRACT CD1d-restricted NKT cells are key players in host defense against various microbial infections. Using a murine model of fatal ehrlichiosis, we investigated the role of CD1d-restricted NKT cells in induction of toxic shock-like syndrome caused by gram-negative, lipopolysaccharide-lacking, monocytotropic Ehrlichia. Our previous studies showed that intraperitoneal infection of wild-type (WT) mice with virulent Ehrlichia (Ixodes ovatus Ehrlichia [IOE]) results in CD8+ T-cell-mediated fatal toxic shock-like syndrome marked by apoptosis of CD4+ T cells, a weak CD4+ Th1 response, overproduction of tumor necrosis factor alpha and interleukin-10, and severe liver injury. Although CD1d−/− mice succumbed to high-dose IOE infection similar to WT mice, they did not develop signs of toxic shock, as shown by elevated bacterial burdens, low serum levels of tumor necrosis factor, normal serum levels of liver enzymes, and the presence of few apoptotic hepatic cells. An absence of NKT cells restored the percentages and absolute numbers of CD4+ and CD8+ T cells and CD11b+ cells in the spleen compared to WT mice and was also associated with decreased expression of Fas on splenic CD4+ lymphocytes and granzyme B in hepatic CD8+ lymphocytes. Furthermore, our data show that NKT cells promote apoptosis of macrophages and up-regulation of the costimulatory molecule CD40 on antigen-presenting cells, including dendritic cells, B cells, and macrophages, which may contribute to the induction of pathogenic T-cell responses. In conclusion, our data suggest that NKT cells mediate Ehrlichia-induced T-cell-mediated toxic shock-like syndrome, most likely via cognate and noncognate interactions with antigen-presenting cells.