Michael E. Woods

Identification of a Glycosylated Ehrlichia canis 19-Kilodalton Major Immunoreactive Protein with a Species-Specific Serine-Rich Glycopeptide Epitope

ABSTRACT Ehrlichia canis has a small subset of major immunoreactive proteins that includes a 19-kDa protein that elicits an early Ehrlichia-specific antibody response in infected dogs. We report herein the identification and molecular characterization of this highly conserved 19-kDa major immunoreactive glycoprotein (gp19) ortholog of the Ehrlichia chaffeensis variable-length PCR target (VLPT) protein. E. canis gp19 has substantial carboxyl-terminal amino acid homology (59%) with E. chaffeensis VLPT and the same chromosomal location; however, the E. chaffeensis VLPT gene (594 bp) has tandem repeats that are not present in the E. canis gp19 gene (414 bp). Consistent with other ehrlichial glycoproteins, the gp19 protein exhibited a larger-than-predicted mass (∼3 kDa), O-linked glycosylation sites were predicted in an amino-terminal serine/threonine/glutamate (STE)-rich patch (26 amino acids), carbohydrate was detected on the recombinant gp19 protein, and the neutral sugars glucose and galactose were detected on the recombinant amino-terminal polypeptide. E. canis gp19 composition consists of five predominant amino acids, cysteine, glutamate, tyrosine, serine, and threonine, concentrated in the STE-rich patch and a carboxyl-terminal domain predominated by cysteine and tyrosine (55%). The amino-terminal STE-rich patch contained a major species-specific antibody epitope strongly recognized by serum from an E. canis-infected dog. The recombinant glycopeptide epitope was substantially more reactive with antibody than the synthetic (nonglycosylated) peptide, and periodate treatment of the recombinant glycopeptide epitope reduced its immunoreactivity, demonstrating the importance of a carbohydrate immunodeterminant(s). The gp19 protein was present on reticulate and dense-cored cells, and it was found extracellularly in the fibrillar matrix and associated with the morula membrane, the host cell cytoplasm, and the nucleus.

The Absence of Toll-Like Receptor 4 Signaling in C3H/HeJ Mice Predisposes Them to Overwhelming Rickettsial Infection and Decreased Protective Th1 Responses

ABSTRACT The importance of toll-like receptor 4 (TLR4) in immunity to rickettsiae remains elusive. To investigate the role of TLR4 in protection against rickettsioses, we utilized C3H/HeJ mice, which are naturally defective in TLR4 signaling, and compared the responses of C3H/HeN and C3H/HeJ mice following intravenous inoculation with Rickettsia conorii. Mice genetically defective in TLR4 signaling developed overwhelming, fatal rickettsial infections when given an inoculum that was nonfatal for TLR4-competent mice. In addition, mice lacking the ability to signal through TLR4 had significantly greater rickettsial burdens in vivo. Moreover, we observed greater concentrations of the cytokines interleukin 6 (IL-6), tumor necrosis factor alpha, IL-12p40, IL-12p70, and IL-17 in the sera of mice with intact TLR4 function as well as significantly greater quantities of activated CD4+ and CD8+ T lymphocytes. Additionally, we also observed that Th17 cells were present only in TLR4-competent mice, suggesting an important role for TLR4 ligation in the activation of this subset. In agreement with these data, we also observed significantly greater percentages of immunosuppressive regulatory T cells in the spleen during infection in TLR4-defective mice. Together, these data demonstrate that, while rickettsiae do not contain endotoxic lipopolysaccharide, they nevertheless initiate TLR4-specific immune responses, and these responses are important in protection.

Host Defenses to Rickettsia rickettsii Infection Contribute to Increased Microvascular Permeability in Human Cerebral Endothelial Cells

Rickettsiae are arthropod-borne intracellular bacterial pathogens that primarily infect the microvascular endothelium leading to systemic spread of the organisms and the major pathophysiological effect, increased microvascular permeability, and edema in vital organs such as the lung and brain. Much work has been done on mechanisms of immunity to rickettsiae, as well as the responses of endothelial cells to rickettsial invasion. However, to date, no one has described the mechanisms of increased microvascular permeability during acute rickettsiosis. We sought to establish an in vitro model of human endothelial-target rickettsial infection using the etiological agent of Rocky Mountain spotted fever, Rickettsia rickettsii, and human cerebral microvascular endothelial cells. Endothelial cells infected with R. rickettsii exhibited a dose-dependent decrease in trans-endothelial electrical resistance, which translates into increased monolayer permeability. Additionally, we showed that the addition of pro-inflammatory stimuli essential to rickettsial immunity dramatically enhanced this effect. This increase in permeability correlates with dissociation of adherens junctions between endothelial cells and is not dependent on the presence of nitric oxide. Taken together, these results demonstrate for the first time that increased microvascular permeability associated with rickettsial infection is partly attributable to intracellular rickettsiae and partly attributable to the immune defenses that have evolved to protect the host from rickettsial spread.

Rickettsiae-Stimulated Dendritic Cells Mediate Protection against Lethal Rickettsial Challenge in an Animal Model of Spotted Fever Rickettsiosis

The role played by dendritic cells (DCs), initiators and orchestrators of the immune response, remains unclear in rickettsial infections. To investigate their importance in rickettsioses, we analyzed the responses of murine bone marrow-derived DCs (BMDCs) after rickettsial stimulation in vitro and their protective role in vivo. Rickettsia conorii stimulation of BMDCs caused significant maturation and production of proinflammatory cytokines. Transfer of rickettsiae-stimulated DCs protected mice from lethal rickettsial challenge by limiting rickettsial proliferation in vivo, whereas partial protection was observed in mice receiving lipopolysaccharide (LPS)-stimulated DCs. Immunity to R. conorii after transfer of DCs was associated with up-regulation of CD40, CD80, CD86, and major histocompatibility complex class II; with production of IL-2, IL-12, and IL-23; and with production of antigen-specific interferon- gamma in T cells. Taken together, our data suggest that a vigorous proinflammatory response in DCs is associated with protective immunity to rickettsiae and that generation of antigen-specific immunity is crucial to complete protection.

Rickettsiae stimulate dendritic cells through Toll-like receptor 4, leading to enhanced NK cell activation in vivo.

Adoptive transfer of Toll-like receptor (TLR) 4-stimulated dendritic cells (DCs) induces protective immunity against an ordinarily lethal rickettsial challenge, but the mechanism underlying this protection remains elusive. Therefore, we sought to determine the importance of TLR4 in early immunity to rickettsiae in vivo, particularly that conferred by TLR4-stimulated DCs. Rickettsial growth proceeded logarithmically in mice lacking TLR4 function, whereas in TLR4-competent mice rickettsial growth manifested a lag phase early, suggesting that TLR4 may initiate innate rickettsial immunity. TLR4-competent mice produced significant amounts of interferon (IFN)-gamma on day 1 of Rickettsia conorii infection, which was associated with significant expansion of the population of activated NK cells. Moreover, NK cells from TLR4-competent mice produced significantly higher levels of IFN-gamma and had greater cytotoxic activity than did those from TLR4-deficient mice. Last, adoptive transfer of rickettsiae-exposed, TLR4-stimulated DCs activated NK cells in vivo. Together, these data reveal an important role for DCs in recognizing rickettsiae through TLR4 and inducing early antirickettsial immunity.

Rickettsiae Stimulate Dentric Cells Through Toll-Like Receptor 4, Leading To Enhanced NK Cell Activation In Vivo

Adoptive transfer of Toll-like receptor (TLR) 4-stimulated dendritic cells (DCs) induces protective immunity against an ordinarily lethal rickettsial challenge, but the mechanism underlying this protection remains elusive. Therefore, we sought to determine the importance of TLR4 in early immunity to rickettsiae in vivo, particularly that conferred by TLR4-stimulated DCs. Rickettsial growth proceeded logarithmically in mice lacking TLR4 function, whereas in TLR4-competent mice rickettsial growth manifested a lag phase early, suggesting that TLR4 may initiate innate rickettsial immunity. TLR4-competent mice produced significant amounts of interferon (IFN)-gamma on day 1 of Rickettsia conorii infection, which was associated with significant expansion of the population of activated NK cells. Moreover, NK cells from TLR4-competent mice produced significantly higher levels of IFN-gamma and had greater cytotoxic activity than did those from TLR4-deficient mice. Last, adoptive transfer of rickettsiae-exposed, TLR4-stimulated DCs activated NK cells in vivo. Together, these data reveal an important role for DCs in recognizing rickettsiae through TLR4 and inducing early antirickettsial immunity.

Nitric oxide as a mediator of increased microvascular permeability during acute rickettsioses.

Abstract: Rickettsiae primarily infect the microvascular endothelium, leading to changes in microvascular permeability that result in potentially severe pulmonary and cerebral edema. The mechanisms responsible for these changes are not well understood. One potential mechanism of increased vascular permeability is the anti‐rickettsial nitric oxide response described by Walker and colleagues. We hypothesized that anti‐rickettsial levels of nitric oxide adversely affects microvascular permeability in vitro. To this end we sought to describe the effects of exogenous nitric oxide on the proliferation of intracellular rickettsiae while monitoring the transendothelial electrical resistance as a measure of endothelial barrier integrity. It was determined that the addition of the NO‐donor DETA NONOate at certain levels results in a dose‐dependent change in electrical resistance across the monolayer while effectively limiting the number of intracellular rickettsiae in human microvascular endothelial cells. The data presented support the idea that nitric oxide produced by infected endothelial cells may be contributing to the changes in vascular permeability that occur during acute rickettsioses. Future experiments aim to elaborate on these results in a model that more clearly depicts the in vivo response as well as to describe the changes that occur with respect to interendothelial junctions.

Rickettsiae stimulate dendritic cells through TLR4, leading to enhanced NK cell activation in vivo

Adoptive transfer of Toll-like receptor (TLR) 4-stimulated dendritic cells (DCs) induces protective immunity against an ordinarily lethal rickettsial challenge, but the mechanism underlying this protection remains elusive. Therefore, we sought to determine the importance of TLR4 in early immunity to rickettsiae in vivo, particularly that conferred by TLR4-stimulated DCs. Rickettsial growth proceeded logarithmically in mice lacking TLR4 function, whereas in TLR4-competent mice rickettsial growth manifested a lag phase early, suggesting that TLR4 may initiate innate rickettsial immunity. TLR4-competent mice produced significant amounts of interferon (IFN)-gamma on day 1 of Rickettsia conorii infection, which was associated with significant expansion of the population of activated NK cells. Moreover, NK cells from TLR4-competent mice produced significantly higher levels of IFN-gamma and had greater cytotoxic activity than did those from TLR4-deficient mice. Last, adoptive transfer of rickettsiae-exposed, TLR4-stimulated DCs activated NK cells in vivo. Together, these data reveal an important role for DCs in recognizing rickettsiae through TLR4 and inducing early antirickettsial immunity.