Ixodes Ovatus Ehrlichia Research Articles

IL-18R signaling impairs hematopoietic recovery after severe, shock-like infection

Abstract Severe infection dramatically alters blood production, but the mechanisms impacting hematopoietic stem and progenitor cell (HSC/HSPC) recovery have not been carefully investigated. Using Ixodes ovatus Ehrlichia (IOE), a pathogen that causes severe shock-like illness and bone marrow (BM) aplasia, we explored the kinetics and mechanisms of hematopoietic recovery. Doxycycline treatment is necessary to prevent IOE-induced death and control bacterial burden in mice, but is not sufficient to prevent HSC/HSPC loss. During recovery from acute infection (12dpi) we saw increased cellularity of LT-HSCs and MPPs, accompanied by diminished function measured by decreased colony forming units (CFUs) in methocult assays. Reduced CFUs indicate a deficiency in BM myelopoietic function during recovery phase post- IOE. We previously showed that IL-18R-deficient mice exhibited improved CFUs at 12dpi, suggesting IL-18R signaling is detrimental to recovery from shock-like IOE. Utilizing a mixed BM chimeric mouse model (50:50 WT:IL-18R−/−), we observed that during recovery at 15dpi, IL-18R−/− cellularity predominates the LT-HSC and MPP2 populations, demonstrating an intrinsic role for IL-18R in limiting recovery of these populations. Additionally, BM chimeras at 15dpi showed increased levels of cell death (Annexin V+ and 7AAD+) in WT HSCs compared with IL-18R−/− HSCs. Transplantation of whole BM from mixed BM chimeric mice revealed a pronounced bias for IL-18R−/− cells, indicating that IL-18R−/− cells had a competitive advantage and improved function after IOE infection. Together, these data support the conclusion that IL-18R signaling impairs HSC function during recovery from severe, shock-like infection via decreased HSC viability. This work was supported by National Institutes of Health funding from NIGMS (R35GM131842) to KCM.

IL-18R-mediated HSC quiescence and MLKL-dependent cell death limit hematopoiesis during infection-induced shock

SummarySevere infection can dramatically alter blood production, but the mechanisms driving hematopoietic stem and progenitor cell (HSC/HSPC) loss have not been clearly defined. Using Ixodes ovatus Ehrlichia (IOE), a tick-borne pathogen that causes severe shock-like illness and bone marrow (BM) aplasia, type I and II interferons (IFNs) promoted loss of HSPCs via increased cell death and enforced quiescence. IFN-αβ were required for increased interleukin 18 (IL-18) expression during infection, correlating with ST-HSC loss. IL-18 deficiency prevented BM aplasia and increased HSC/HSPCs. IL-18R signaling was intrinsically required for ST-HSC quiescence, but not for HSPC cell death. To elucidate cell death mechanisms, MLKL- or gasdermin D-deficient mice were infected; whereas Mlkl−/− mice exhibited protected HSC/HSPCs, no such protection was observed in Gsdmd−/− mice during infection. MLKL deficiency intrinsically protected HSCs during infection and improved hematopoietic output upon recovery. These studies define MLKL and IL-18R signaling in HSC loss and suppressed hematopoietic function in shock-like infection.

IFNs induce prolonged hematopoietic suppression via IL-18-induced cell death during shock-like Ehrlichial infection

Abstract The long-term effects of shock-like infection on hematopoietic stem and progenitor cells (HSC/HSPC) has not been carefully investigated. Using Ixodes ovatus ehrlichia (IOE), a tick-born pathogen that causes severe shock-like illness and bone marrow (BM) aplasia, we explored mechanisms that cause hematopoietic suppression. During IOE infection, type I IFNs promote loss of HSPCs via direct (cell death) and indirect mechanisms (quiescence). Mice lacking IFNαβ receptor (IFNαR−/−) had improved survival relative to WT mice. IFNγR−/− mice had profound BM aplasia and mortality, like WT mice, however mice lacking both type I and II IFN receptors (IFNαR;IFNγR−/−) had protected HSPCs and improved survival, even relative to IFNαR−/− mice. WT and IFNγR−/− mice had reduced HSPCs and increased levels of IL-18 relative to IFNαR−/− and IFNαR;IFNγR−/− mice, while IL-18R expression induced during IOE required IFNγ signaling. IL-18-deficiency prevented IOE-induced BM aplasia and reduced HSC quiescence and necrotic (7AAD+) cells. To test whether IL-18R-signaling acted on HSPCs directly, we generated WT: IL-18R−/− mixed BM chimeras. IL-18R−/− cells had an advantage over WT cells and IL-18R was intrinsically required for increased HSPC cell death. In mixed BM chimeras given antibiotics to allow recovery from acute infection, we found that two weeks post-infection IL-18R−/− HSPCs were present at higher numbers and IL-18R−/− cells had greater functional output in colony-forming unit assays. Our data support IFN driven hematopoietic suppression through IFNαR-dependent induction of IL-18 and IFNγR-dependent IL-18R expression, and that direct effects of IL-18 on HSPCs during infection-induced shock may have long-lasting effects on HSCs.

Interferon Type I Regulates Inflammasome Activation and High Mobility Group Box 1 Translocation in Hepatocytes During Ehrlichia-Induced Acute Liver Injury.

Inflammasomes are an important innate immune host defense against intracellular microbial infection. Activation of inflammasomes by microbial or host ligands results in cleavage of caspase-1 (canonical pathway) or caspase-11 (noncanonical pathway), release of interleukin (IL)-1β, IL-18, high mobility group box 1 (HMGB1), and inflammatory cell death known as pyroptosis. Ehrlichia are obligate, intracellular, gram-negative bacteria that lack lipopolysaccharide but cause potentially life-threatening monocytic ehrlichiosis in humans and mice that is characterized by liver injury followed by sepsis and multiorgan failure. Employing murine models of mild and fatal ehrlichiosis caused by infection with mildly and highly virulent Ehrlichia muris (EM) and Ixodes ovatus Ehrlichia (IOE), respectively, we have previously shown that IOE infection triggers type I interferon (IFN-I) response and deleterious caspase-11 activation in liver tissues, which promotes liver injury and sepsis. In this study, we examined the contribution of IFN-I signaling in hepatocytes (HCs) to Ehrlichia-induced liver injury. Compared to EM infection, we found that IOE enter and replicate in vitro cultured primary murine HCs and induce secretion of IFNβ and several chemokines, including regulated upon activation, normal T-cell expressed, and secreted (RANTES), monocyte chemoattractant protein 1 (MCP1), monokine induced by gamma (MIG)/chemokine (C-X-C motif) ligand 9 (CXCL9), macrophage inflammatory protein 1 alpha (MIP1α), keratinocyte-derived chemokine (KC), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Notably, in vitro stimulation of uninfected and Ehrlichia-infected HCs with recombinant IFNβ triggered activation of caspase-1/11, cytosolic translocation of HMGB1, and enhanced autophagy and intracellular bacterial replication. Secretion of HMGB1 by IOE-infected HCs was dependent on caspase-11. Primary HCs from IOE- but not EM-infected mice also expressed active caspase-1/11. Conclusion: HC-specific IFN-I signaling may exacerbate liver pathology during infection with obligate intracellular Ehrlichia by promoting bacterial replication and detrimental caspase-11-mediated inflammasome activation.

MTORC1-mediated macrophage polarization into M1 Contributes to Ehrlichia-induced sepsis

Abstract Macrophages heterogeneity that undergo dynamic changes in phenotype and function as well as reprograming of immune response to multiple intracellular bacterial infections is an important key to understand the host responses to infection. Ehrlichia is an obligate Gram-negative intracellular bacterium that cause potentially fatal human monocytotropic ehrlichiosis (HME). We utilized murine models of mild and fatal ehrlichiosis caused by Ehrlichia muris and Ixodes Ovatus Ehrlichia (IOE), respectively, to evaluate the macrophages polarization to inflammatory (M1) and regenerative (M2). Our data show that in vivo and in vitro infections of macrophages with IOE significantly polarize macrophages towards M1, while EM infection enhances M2 polarization. M1 polarization of macrophages in IOE-infected mice was associated with recruitment and/or expansion of granzyme B+ neutrophils, NK cells, IL-17+ NKT cells, and IL-17+ CD4 Th17 cells compared to EM infection. We previously identified mTORC1 (mammalian target of rapamycin complex-1), a negative regulator of autophagy and the key mediator for immunopathology during IOE infection. We found that M1 polarization in the livers of IOE-infected mice is dependent on mTORC1 activation. Blockade of mTORC1 activation with rapamycin decreased frequency of Th17, and enhanced autophagy, which in turn decreased inflammation as well as the pathogenic immune response during lethal IOE infection. Together, these data reveal a key role of mTORC1 in M1 polarization of macrophages, which likely promotes Ehrlichia-induced sepsis. Understanding the dynamic changes of macrophage polarization and how it affects immune responses to infection is crucial for development novel therapeutic strategies.

Interplay of Wnt β-catenin pathway and mTORC1 in pathogenesis of Ehrlichia-induced sepsis

Abstract The Wnt/β-catenin pathway has been evolved in regulating cell proliferation, survival, and inflammation in cancer development. However, the role of β-catenin during infections remains elusive. Ehrlichia is obligate intracellular bacteria that cause life-threatening sepsis, characterized by liver injury and dysregulated inflammatory response with no effective therapy available. We examined here the contribution of Wnt pathway to the pathogenesis of Ehrlichia-induced sepsis employing murine models of mild and fatal ehrlichiosis caused by intraperitoneal infection with avirulent Ehrlichia muris (EM) and virulent Ixodes Ovatus Ehrlichia (IOE), respectively. We show that EM infection induces higher activation of the hepatic Wnt/β-catenin pathway and activates Wnt target genes; C-Myc and Cyclin D1 on day 7 post infection compared to uninfected controls. Notably, IOE infection attenuated Wnt/β-catenin pathway in liver tissue, which is associated with inflammasome activation, inhibition of autophagy in macrophages, host cell death, and increased number of replicating bacteria, suggesting that Wnt pathway is a host-protective mechanism that also enhances cell survival and attenuate inflammation during Ehrlichia infection. Notably, pharmacologic or genetic inhibition of mTORC1 activation in IOE-infected mice by rapamycin or MyD88 deficiency, respectively, enhanced Wnt signaling, induced autophagy, and decreased inflammation, suggesting that suppression of Wnt pathway during IOE infection is mediated by mTORC1. These novel findings unravel that virulent Ehrlichia may cause liver injury via mTORC1-mediated inhibition of Wnt pathway. Targeting of mTORC1 could provide a novel therapy for Ehrlichia-induced sepsis.

MyD88-dependent inflammasome activation and autophagy inhibition contributes to Ehrlichia-induced liver injury and toxic shock.

Severe hepatic inflammation is a common cause of acute liver injury following systemic infection with Ehrlichia, obligate Gram-negative intracellular bacteria that lack lipopolysaccharide (LPS). We have previously shown that type I IFN (IFN-I) and inflammasome activation are key host-pathogenic mediators that promote excessive inflammation and liver damage following fatal Ehrlichia infection. However, the underlying signals and mechanisms that regulate protective immunity and immunopathology during Ehrlichia infection are not well understood. To address this issue, we compared susceptibility to lethal Ixodes ovatus Ehrlichia (IOE) infection between wild type (WT) and MyD88-deficient (MyD88-/-) mice. We show here that MyD88-/- mice exhibited decreased inflammasome activation, attenuated liver injury, and were more resistant to lethal infection than WT mice, despite suppressed protective immunity and increased bacterial burden in the liver. MyD88-dependent inflammasome activation was also dependent on activation of the metabolic checkpoint kinase mammalian target of rapamycin complex 1 (mTORC1), inhibition of autophagic flux, and defective mitophagy in macrophages. Blocking mTORC1 signaling in infected WT mice and primary macrophages enhanced bacterial replication and attenuated inflammasome activation, suggesting autophagy promotes bacterial replication while inhibiting inflammasome activation. Finally, our data suggest TLR9 and IFN-I are upstream signaling mechanisms triggering MyD88-mediated mTORC1 and inflammasome activation in macrophages following Ehrlichia infection. This study reveals that Ehrlichia-induced liver injury and toxic shock are mediated by MyD88-dependent inflammasome activation and autophagy inhibition.

MyD88 suppresses IL-10 and IL-17 production in response to obligate intracellular Ehrlichia infection

Abstract Ehrlichia is an obligate intracellular gram negative bacterium that target macrophages, and causes the potentially fatal human monocytic ehrlichiosis (HME). In murine models of fatal ehrlichiosis (FE), Ixodes ovatus Ehrlichia (IOE) inhibits autophagy, suppresses protective acquired immunity, and causes immunopathology and toxic shock syndrome. Innate Ehrlichia detection by TLR2 mediates host resistance, while NOD2 signaling confers host susceptibility to FE. Recently, we have shown that deficiency of type I interferon receptor (IFNaR) phenocopy NOD2 deficiency in IOE-infected mice, implying a contribution of both NOD2-IFNaR pathways to susceptibility to FE. However, how TLR2 mediates protective function against Ehrlichia is not clearly defined. Here, we investigated the role of MyD88, the main adaptor molecule for the TLRs in host response to virulent IOE using MyD88 knockout mice (MyD88−/−). Our data showed that, MyD88−/− mice have an impaired bacterial clearance, increased liver injury, and a heightened mortality when compared to infected wild type (WT) mice. Increased bacterial load in MYD88−/− mice correlates with decreased levels of TNF-α in both in vivo and in vitro {serum and IOE- infected bone marrow-derived macrophages (BMM)}, and increased frequency of splenic CD4+ T cells producing IL-10 and IL-17. Protective anti-Ehrlichia immunity and minimal pathology in WT mice infected with low virulent Ehrlichia species correlates with a high Th1 response, but a low frequency of IL-10 and IL-17 producing splenic T cells, suggesting that IL-10 and IL-17 may play pathogenic roles in host defense against Ehrlichia. Together these data suggest that MYD88 plays a protective role in ehrlichiosis via suppression of IL-10 and IL-17.

NK Cell-Mediated Regulation of Protective Memory Responses against Intracellular Ehrlichial Pathogens.

Ehrlichiae are gram-negative obligate intracellular bacteria that cause potentially fatal human monocytic ehrlichiosis. We previously showed that natural killer (NK) cells play a critical role in host defense against Ehrlichia during primary infection. However, the contribution of NK cells to the memory response against Ehrlichia remains elusive. Primary infection of C57BL/6 mice with Ehrlichia muris provides long-term protection against a second challenge with the highly virulent Ixodes ovatus Ehrlichia (IOE), which ordinarily causes fatal disease in naïve mice. Here, we show that the depletion of NK cells in E. muris-primed mice abrogates the protective memory response against IOE. Approximately, 80% of NK cell-depleted E. muris-primed mice succumbed to lethal IOE infection on days 8–10 after IOE infection, similar to naïve mice infected with the same dose of IOE. The lack of a recall response in NK cell-depleted mice correlated with an increased bacterial burden, extensive liver injury, decreased frequency of Ehrlichia-specific IFN-γ-producing memory CD4+ and CD8+ T-cells, and a low titer of Ehrlichia-specific antibodies. Intraperitoneal infection of mice with E. muris resulted in the production of IL-15, IL-12, and IFN-γ as well as an expansion of activated NKG2D+ NK cells. The adoptive transfer of purified E. muris-primed hepatic and splenic NK cells into Rag2-/-Il2rg-/- recipient mice provided protective immunity against challenge with E. muris. Together, these data suggest that E. muris-induced memory-like NK cells, which contribute to the protective, recall response against Ehrlichia.

The Interaction between IL-18 and IL-18 Receptor Limits the Magnitude of Protective Immunity and Enhances Pathogenic Responses following Infection with Intracellular Bacteria

The binding of IL-18 to IL-18Rα induces both proinflammatory and protective functions during infection, depending on the context in which it occurs. IL-18 is highly expressed in the liver of wild-type (WT) C57BL/6 mice following lethal infection with highly virulent Ixodes ovatus ehrlichia (IOE), an obligate intracellular bacterium that causes acute fatal toxic shock-like syndrome. In this study, we found that IOE infection of IL-18Rα(-/-) mice resulted in significantly less host cell apoptosis, decreased hepatic leukocyte recruitment, enhanced bacterial clearance, and prolonged survival compared with infected WT mice, suggesting a pathogenic role for IL-18/IL-18Rα in Ehrlichia-induced toxic shock. Although lack of IL-18R decreased the magnitude of IFN-γ producing type-1 immune response, enhanced resistance of IL-18Rα(-/-) mice against Ehrlichia correlated with increased proinflammatory cytokines at sites of infection, decreased systemic IL-10 production, increased frequency of protective NKT cells producing TNF-α and IFN-γ, and decreased frequency of pathogenic TNF-α-producing CD8(+) T cells. Adoptive transfer of immune WT CD8(+) T cells increased bacterial burden in IL-18Rα(-/-) mice following IOE infection. Furthermore, rIL-18 treatment of WT mice infected with mildly virulent Ehrlichia muris impaired bacterial clearance and enhanced liver injury. Finally, lack of IL-18R signal reduced dendritic cell maturation and their TNF-α production, suggesting that IL-18 might promote the adaptive pathogenic immune responses against Ehrlichia by influencing T cell priming functions of dendritic cells. Together, these results suggested that the presence or absence of IL-18R signals governs the pathogenic versus protective immunity in a model of Ehrlichia-induced immunopathology.

Exit Mechanisms of the Intracellular Bacterium Ehrlichia

BackgroundThe obligately intracellular bacterium Ehrlichia chaffeensis that resides in mononuclear phagocytes is the causative agent of human monocytotropic ehrlichiosis. Ehrlichia muris and Ixodes ovatus Ehrlichia (IOE) are agents of mouse models of ehrlichiosis. The mechanism by which Ehrlichia are transported from an infected host cell to a non-infected cell has not been demonstrated.Methodology/Principal FindingsUsing fluorescence microscopy and transmission and scanning electron microscopy, we demonstrated that Ehrlichia was transported through the filopodia of macrophages during early stages of infection. If host cells were not present in the vicinity of an Ehrlichia-infected cell, the leading edge of the filopodium formed a fan-shaped structure filled with the pathogen. Formation of filopodia in the host macrophages was inhibited by cytochalasin D and ehrlichial transport were prevented due to the absence of filopodia formation. At late stages of infection the host cell membrane was ruptured, and the bacteria were released.Conclusions/Significance Ehrlichia are transported through the host cell filopodium during initial stages of infection, but are released by host cell membrane rupture during later stages of infection.

T-cell receptor repertoire diversity during persistent and fatal ehrlichial infections (40.17)

Abstract Human monocytotropic ehrlichiosis (HME), an emerging tick-transmitted bacterial disease, is caused by Ehrlichia chaffeensis. HME is modeled in C57BL/6 mice using Ehrlichia muris, which causes persistent infection, and Ixodes ovatus Ehrlichia (IOE), which is either acutely lethal or sub-lethal depending on the dose and route of inoculation. To better define the roles of T cells in protection and pathogenesis, we analyzed TCR Vβ usage in activated T cells from mice infected with E. muris or IOE by TCR-CDR3 spectratyping. Our data indicated that E. muris, but not IOE, induced oligoclonal expansion of CD4+ T cells within the several Vβ families (Vβ1, Vβ2, Vβ8.1, Vβ8.2, Vβ12, Vβ16 and Vβ19) early during the effector phase of T cell response. Further, oligoclonal expansion of CD8+ T cells within the Vβ8.2, Vβ4 and Vβ19 families was observed during E. muris infection compared to oligoclonal expansion of Vβ16 and Vβ19 CD8+ T cells in mice infected with IOE. Functional analysis of T cell subsets by flow cytometry indicated that E. muris induced a strong Th1 response involving several Vβ families compared to a weak Th1 response induced by IOE. The data suggested that unlike IOE, E. muris induces a strong Th1 response which tends to focus on certain Vβ families early during the effector phase of T cell response. Future studies will focus on functional characterization of memory T cell subsets induced following infection with E. muris.

Differential contribution of IL-18 receptor beta to protective immunity and immunopathology during infection with intracellular bacteria (134.18)

Abstract IL-18 has been shown to play a critical role in cytokine-induced organ failure during endotoxic shock. Human monocytotropic ehrlichiosis (HME) is a tick-borne infectious disease caused by E. chaffeenesis. HME is modeled in B6 mice using Ehrlichia muris (EM) that cause mild disease, and Ixodes ovatus Ehrlichia (IOE) that cause fatal shock-like syndrome. As Ehrlichia-induced septic shock and tissue injury is associated with increased IL-18 production, we examined the effect of blocking IL-18 signaling via IL-18Rβ on host defense & immune responses following sublethal (EM) and lethal (IOE) infection. Compared to IOE-infected isotype controls, in vivo IL-18Rβ neutralization in IOE-infected mice decreased bacterial burden, increased frequency of IFN-γ producing NK and NKT cells and enhanced production of TNF-α by T and non T cells on day 2 post-infection. On day 7 post-infection, blocking IL-18Rβ in lethally infected mice did not significantly influence the acquired pathogenic immune responses compared to lethally-infected isotype controls. Interestingly, blocking IL-18Rβ in the EM-infected mice increased bacterial burden, which was associated with suppressed early production of TNF-α and IFN-γ by different cell subsets compared to EM-infected isotype controls. These data suggest that IL-18Rβ may play dual protective and pathogenic roles in sublethal and lethal ehrlichial infection, respectively, through regulation of IFN-γ and TNF-α production by different innate cell subsets.

Antigenic protein modifications inEhrlichia

To develop effective vaccination strategies againstEhrlichia, we have previously reported developing an animal model of cross-protection in which C57BL/6 mice primed withE. muris were resistant to lethal infection withIxodes ovatus ehrlichia (IOE). Polyclonal antibody produced in mice after priming withE. muris and later injected with IOE-detected antigenic proteins inE. muris and IOE cell lysates. Cross-reaction of antigenic proteins was observed when we probed both theE. muris and IOE cell lysates with IOE andE. muris-specific polyclonal antibody. Analysis of the total proteins ofE. muris and IOE by two dimensional electrophoresis showed that bothE. muris and IOE have the same antigenic proteins. Finally, studies on post-translational protein modifications using a novel technique, Eastern blotting, showed thatE. muris proteins are more lipoylated and glycosylated than those of IOE.

NK Cells Dominate the Immunopathogenesis of Fatal Ehrlichiosis

Human monocytic ehrlichiosis (HME) is a potentially lethal, tick‐transmitted infection caused by the obligately intracellular bacterium Ehrlichia chaffeensis. C57BL/6 mice model severe HME when infected with Ixodes ovatus Ehrlichia (IOE); they succumb to a toxic‐shock like syndrome mediated in part by TNF and CD8+ T cells and characterized by lymphopenia and by liver apoptosis and necrosis. CD4+ T cells mediate a protective IFN‐γresponse. The aim of this study was to compare the contribution of NK cells to that of T cells in the pathogenesis of ehrlichiosis. Flow cytometry was used to track splenic NK cells (NK1.1+, CD3−), NKT cells (NK1.1+, CD3+), T cells (CD3+, NK1.1−) and monocytes (CD11b+, NK1.1−) over the course of disease; these populations failed to expand but rather contracted before the animals’ death. Furthermore, splenic NK cells produced more IFN‐γ, TNF, granzyme B and Fas than did NKT or T cells. We depleted NK cells with anti‐asialo GM1 immune serum and observed reduced serum concentrations of IFN‐γ, TNF, and IL‐10 by ELISA, lower bacterial burdens in the spleen, liver, and lung by real‐time PCR, and attenuated liver injury by histopathology. Our data indicate that NK cells, rather than T cells, are the dominant contributors to the pathogenic immune response against IOE infection.

Fatal Recall Responses Mediated by CD8 T cells during Intracellular Bacterial Challenge Infection

The roles(s) of CD8 T cells during infections by intracellular bacteria that reside in host cell endocytic compartments are not well understood. Our previous studies in a mouse model of human monocytotropic ehrlichiosis indicated that CD8 T cells are not essential for immunity. However, we have observed an unexpected role for these cells during challenge infection. Although immunocompetent mice cleared a primary low-dose (nonfatal) Ixodes ovatus ehrlichia infection, a secondary low-dose challenge infection resulted in fatal disease and loss of control of infection. The outcome was CD8-dependent, because CD8-deficient mice survived secondary low-dose challenge infection. Moreover, effector and/or memory phenotype CD8 T cells were responsible, because adoptive transfer of purified CD44(high) CD8 T cells to naive mice induced fatal responses following a primary low-dose infection. The fatal responses were perforin- and Fas ligand-independent, and were associated with high serum concentrations of TNF-alpha and CCL2, and low levels of IL-10. Accordingly, blockade of either TNF-alpha or CCL2 ameliorated fatal recall responses, and in vitro coculture of memory CD8 T cells and Ixodes ovatus ehrlichia-infected peritoneal exudate cells resulted in substantial increases in TNF-alpha and CCL2. Thus, during monocytotropic ehrlichiosis, inflammatory cytokine production, by CD8 T cells and/or other host cells, can trigger chemokine-dependent disease. These findings highlight a novel role for CD8 T cells, and reveal that live vaccines for intracellular bacteria can, under some conditions, induce undesirable consequences.

Susceptibility and Resistance to Monocytic Ehrlichiosis in the Mouse

To address the role of cellular immunity during ehrlichia infection, we have utilized a model of monocytic ehrlichiosis that results from infection of mice by Ixodes ovatus ehrlichia (IOE). Although ehrlichiosis in humans is largely a disease of immunocompromised individuals, the use of the IOE model has allowed us to identify factors required for host defense in normal mice. Using a low-dose infection C57BL/6 mouse model, we have demonstrated that host defense requires immune mechanisms involving CD4 T cell-mediated, TNF-alpha-, IL-12-, and IFN-gamma-dependent, macrophage activation. We have also provided formal evidence that IFN-gamma produced by CD4 Th1 cells is sufficient for protective immunity. Our recent studies have demonstrated, in addition, an essential role for IL-10, which is probably important in inhibiting immunopathological responses, and for inducible nitric oxide synthase. The latter observation establishes an important role for reactive nitrogen intermediates in bacterial elimination in vivo. In contrast, evaluation of mice carrying wild-type and mutant alleles of Nramp1 revealed at most a modest role for this gene in resistance to fatal IOE infection. Other studies in low-dose infected mice have indicated that the generation of immunological memory may be impaired during low-dose IOE infection, possibly due to bacterial immune subversion. These studies highlight the utility of the IOE mouse model in identifying important parameters of the immune response during ehrlichiosis.

Production of IFN-gamma by CD4 T cells is essential for resolving ehrlichia infection.

To address the role of cellular immunity during ehrlichia infection, we have used a newly described model of monocytic ehrlichiosis that results from infection of mice by an ehrlichia that was isolated from an Ixodes ovatus tick (Ixodes ovatus ehrlichia, IOE). Immunocompetent C57BL/6 and BALB/c mice exhibited a dose-dependent susceptibility to IOE infection. Mice infected with a high dose inoculum ( approximately 1000 organisms) exhibited pronounced thrombocytopenia, lymphopenia, anemia, and morbidity within 12 days postinfection. Infection was associated with bacterial colonization of a number of tissues. In contrast, mice infected with a low dose inoculum ( approximately 100 organisms) exhibited only transient disease and were able to resolve the infection. SCID mice were highly susceptible to low-dose infection, indicating that adaptive immunity was required. Resistance to sublethal challenge in both C57BL/6 and BALB/c mice was CD4-, but not CD8-, dependent and required IL-12p40-dependent cytokines, IFN-gamma, and TNF-alpha, but not IL-4. CD4 T cells purified from infected mice proliferated in vitro in response to IOE Ags. T cell proliferation was associated with production of IFN-gamma, and the production of this cytokine by CD4 T cells rescued IFN-gamma-deficient mice from fatal infection. Exogenous IFN-gamma was capable of inducing microbiocidal activity in infected macrophages. The data suggest that classical immune mechanisms involving CD4 cells and type 1 cytokines are responsible for macrophage activation and for elimination of this intracellular bacterial pathogen.