Ehrlichial proliferation and acute hepatocellular necrosis in immunocompetent mice experimentally infected with the HF strain of Ehrlichia, closely related to Ehrlichia chaffeensis

Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, is closely related to the HF strain of Ehrlichia isolated from ticks in Japan. In this study, BALB/c mice inoculated intraperitoneally with the HF strain developed severe illness and died at about day 9 post-inoculation. At necropsy, diffuse liver necrosis was evident. Ehrlichial microcolonies were observed in endothelial cells, monocytes and macrophages of the liver, bone marrow, spleen, thymus, and large and small intestine. Immunocompetent mice infected with the HF strain would provide a useful model for studying pathogenesis and immunity in acute and severe ehrlichiosis caused by E. chaffeensis and related Ehrlichia spp. Copyright Harcourt Publishers Ltd.     

An intradermal environment promotes a protective type-1 response against lethal systemic monocytotropic ehrlichial infection

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-alpha) in the spleen but lower concentrations of serum TNF-alpha 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.     

Distribution of ehrlichiae in tissues as determined by in-situ hybridization

Specific identification of ehrlichiae in the tissues and determination of their distribution is difficult. In this study, an in-situ hybridization method was developed to detect ehrlichial 16S rRNA in tissue specimens from mice experimentally infected with the HF strain. This strain is closely related to Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis. HF strain-specific 16S rRNA was detected in endothelial cells and monocyte-macrophages in the liver, lungs, bone marrow, spleen, lymph nodes, and large and small intestinal tissues. The results suggest that the in-situ hybridization method with a digoxigenin-labelled RNA probe specific to ehrlichial 16S rRNA will be useful for post-mortem diagnosis and for the histopathological investigation of ehrlichial infection.     

Hepatic pathology in human monocytic ehrlichiosis. Ehrlichia chaffeensis infection

Ehrlichia chaffeensis causes human monocytic ehrlichiosis (HME) that usually includes fever, myalgias, and pancytopenia and, in 80% to 90% of patients, elevations in serum transaminase levels. Thus, the pathology of liver injury was studied in liver tissues from 7 patients with laboratory-confirmed HME. H&E and immunohistochemical stains for E chaffeensis and leukocyte markers were examined. Scattered lobular lymphohistiocytic foci and diffuse lymphohistiocytic infiltration and Kupffer cell hyperplasia with increased phagocytosis frequently were present. Various degrees of liver cell injury and death were observed. Cholestasis was evident in 6 cases, sometimes with bile duct epithelial injury. Rare to abundant E chaffeensis-infected mononuclear cells infiltrating lobules or portal regions or in Kupffer cells were observed in 5 patients. The inflammation was out of proportion to the infection in 6 cases. In the absence of infected hepatocytes or biliary epithelial cells, these findings suggest that host inflammatory or immune responses contribute to the liver injury seen in HME.     

Immunization with Ehrlichia P28 outer membrane proteins confers protection in a mouse model of ehrlichiosis

The obligately intracellular bacterium Ehrlichia chaffeensis that resides in mononuclear phagocytes is the etiologic agent of human monocytotropic ehrlichiosis (HME). HME is an emerging and often life-threatening, tick-transmitted infectious disease in the United States. Effective primary immune responses against Ehrlichia infection involve generation of Ehrlichia-specific gamma interferon (IFN-γ)-producing CD4(+) T cells and cytotoxic CD8(+) T cells, activation of macrophages by IFN-γ, and production of Ehrlichia-specific antibodies of the Th1 isotype. Currently, there are no vaccines available against HME. We evaluated the ability of 28-kDa outer membrane proteins (P28-OMP-1) of the closely related Ehrlichia muris to stimulate long-term protective memory T and B cell responses and confer protection in mice. The spleens of mice vaccinated with E. muris P28-9, P28-12, P28-19, or a mixture of these three P28 proteins (P28s) using a DNA prime-protein boost regimen and challenged with E. muris had significantly lower bacterial loads than the spleens of mock-vaccinated mice. Mice immunized with P28-9, P28-12, P28-19, or the mixture induced Ehrlichia-specific CD4(+) Th1 cells. Interestingly, mice immunized with P28-14, orthologs of which in E. chaffeensis and E. canis are primarily expressed in tick cells, failed to lower the ehrlichial burden in the spleen. Immunization with the recombinant P28-19 protein alone also significantly decreased the bacterial load in the spleen and liver compared to those of the controls. Our study reports, for the first time, the protective roles of the Ehrlichia P28-9 and P28-12 proteins in addition to confirming previous reports of the protective ability of P28-19. Partial protection induced by immunization with P28-9, P28-12, and P28-19 against Ehrlichia was associated with the generation of Ehrlichia-specific cell-mediated and humoral immune responses.     

Serological responses to Ehrlichia equi, Ehrlichia chaffeensis, and Borrelia burgdorferi in patients from New York State.

Serological testing at the New York State Department of Health for human granulocytic ehrlichiosis in the residents of Westchester County, N.Y., was performed with specimens from 176 patients by the indirect fluorescent-antibody (IFA) technique with Ehrlichia equi MRK-infected neutrophils. To understand whether human monocytotropic ehrlichiosis also occurs in this northeastern geographic region, specimens were also tested for antibodies to Ehrlichia chaffeensis Arkansas. Screening tests and immunoblots for Lyme disease (Borrelia burgdorferi infection) were also performed. Thirty-two patients had antibodies only to E. equi and 21 patients had antibodies to both E. equi and E. chaffeensis, whereas 12 patients had only E. chaffeensis antibodies by the IFA technique. The remaining patients did not have antibodies to either ehrlichia. Eighteen serum samples from 13 of these patients were coded and sent to the Ehrlichia Research Laboratory (Baltimore, Md.) for repeat analysis by the IFA test and for E. equi and E. chaffeensis immunoblots. Immunoblot analysis for E. equi in samples with positive IFA test results confirmed the results for eight of the nine specimens. Immunoblot analyses for E. chaffeensis were negative for all 18 serum samples. Borrelia-reactive antibodies were found in sera both from patients with granulocytic ehrlichiosis and from patients with monocytotropic ehrlichiosis from New York State. Our results suggest that E. equi antigen is an appropriate substrate for identifying human granulocytic ehrlichiosis. E. chaffeensis antigen lacks appropriate sensitivity to serve as a surrogate substrate for the detection of human granulocytic ehrlichiosis and should be used solely for the diagnosis of human monocytotropic ehrlichiosis. Heat shock proteins may, in some cases, cause cross-reactivity between B. burgdorferi and ehrlichiae.     

Regulatory roles of CD1d-restricted NKT cells in the induction of toxic shock-like syndrome in an animal model of fatal ehrlichiosis

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.     

Astrakhan scrub typhus: time course of infectious process according to electron microscopy findings

Structural changes in the liver and spleen of albino mice with Astrakhan scrub typhus were studied by electron microscopy. Rickettsia invasion and formation of granulomas induced structural (destructive) and metabolic changes in hepatocytes. Rickettsia were degraded in cytophagosomes and cytolysophagosomes of hepatic macrophages (Kupffer cells) and blood capillary endotheliocytes. In the spleen rickettsia were seen in the extracellular spaces and in various cell populations.     

Effect of macrophage source and activation on susceptibility in an age-dependent model of murine hepatitis caused by a phlebovirus,Punta Toro

Summary The Adames strain of a bunyavirus, Punta Toro virus (PTV), is an hepatotrophic virus that has been described to produce an age-dependent lethal hepatic necrosis in 3–4 week old C57BL/6 mice, but 8 week old mice survive with minimal necrosis. The course of PTV infection in vitro in macrophages derived from these mice served as a model to study the pathogenesis of phlebovirus infection. Peripheral blood monocytes, resident or elicited peritoneal macrophages, and Kupffer cell liver macrophages, as well as hepatocytes, were able to support replication of PTV in vitro to a variable extent. Kupffer cells were the only population of macrophages, however, that expressed an age-related ability to affect viral infection and replication in vitro, suggesting that liver macrophages may have a unique modulatory effect on the occurrence and severity of PTV-induced hepatitis in mice. Whereas PTV showed minimal replication in resident peritoneal macrophages, the virus could replicate effectively in peritoneal macrophages elicited by thioglycolate. Activation of peritoneal macrophages with endotoxin resulted in a significant inhibition of intrinsic PTV replication (p<0.001), and a modest extrinsic inhibitory effect on PTV replication in cocultured hepatocytes. Both effects persisted in the presence of antiinterferon. These results indicate that the source and state of activation of macrophage/monocyte populations can influence the course of infection in vitro by the phlebovirus, Punta Toro, and can modulate infection in cocultured target cells.     

Depletion of gamma interferon and tumor necrosis factor alpha in mice with Rickettsia conorii-infected endothelium: impairment of rickettsicidal nitric oxide production resulting in fatal, overwhelming rickettsial disease.

C3H/HeN mice infected intravenously with a dose of Rickettsia conorii (Malish 7 strain) that is sublethal for immunocompetent animals (1.1 x 10(3) PFU) developed disseminated infection of endothelial cells of the brain, lungs, heart, liver, kidney, testis, and testicular adnexa. In R. conorii-infected mice depleted of gamma interferon (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) by intravenous administration of neutralizing monoclonal antibodies on days 0, 2, and 4, the mortality rate was 100%. Death of the cytokine-depleted animals on days 5 and 6 was associated with overwhelming rickettsial infection documented by titration of rickettsial content in the brain and liver and by immunohistologic demonstration of massive quantities of R. conorii in endothelial cells of all organs examined, in macrophages of the liver and spleen, and in hepatocytes. Nondepleted, immunocompetent animals showed markedly reduced rickettsial content in the tissues on day 6, with rickettsial destruction in phagolysosomes not only in macrophages but also in endothelial cells and hepatocytes. All nondepleted, infected mice recovered and appeared completely healthy by day 9. Assay of liver infiltrated by lymphocytes and macrophages revealed mRNA of IFN-gamma and TNF-alpha, indicating that the host defenses were activated at the site of infection. Treatment of mice with an analog of L-arginine reduced the synthesis of nitric oxide and impaired rickettsial killing. Nitric oxide production was also impaired in cytokine-depleted infected mice. These observations support the hypothesis that IFN-gamma secreted by T lymphocytes and natural killer cells and TNF-alpha secreted by macrophages act in a synergistic, paracrine fashion on adjacent rickettsia-infected endothelial cells, hepatocytes, and macrophages to stimulate synthesis of nitric oxide, which kills intracellular R. conorii.     

Kupffer cell activation and hematopoiesis in the liver of autoimmune MRL-lpr/lpr mice

Hematopoiesis can be induced in the adult murine liver by the administration of macrophage activators. The proliferation of macrophages and extrathymic T cells is spontaneously induced in the liver of autoimmune MRL-lpr/lpr mice, and deeply involved in the development of disease. To study the role of Kupffer cell activation in the induction of hematopoiesis and lymphocyte proliferation in the liver, we histologically analysed the kinetic and spatial relationship between Kupffer cells and hematopoietic cells or lymphocytes. At 5 weeks of age before the onset of disease, there were no appreciable histological changes in the liver. At 7 weeks, Kupffer cells had slightly increased in number, while hematopoietic islands were not yet detected. When disease had fully developed at 14 weeks, Kupffer cells were considerably increased in number and size, and exhibited numerous lysosomes. Hematopoietic cells of erythroid and myeloid series frequently appeared in the sinusoid, and lay in close apposition to Kupffer cells. Promyelocytes further migrated into the space of Disse to cluster there, being surrounded by the stellate cells (or fat-storing cells) and hepatocytes. After maturation, metamyelocytes and mature granulocytes were released into the sinusoidal circulation. Mitotic figures were detected in the cells of both erythroid and myeloid series. Lymphocytes proliferated in various sites such as in the sinusoid lumen, the space of Disse, and interlobular connective tissue, whether associated or not with Kupffer cells. The present results indicate that erythropoiesis, granulopoiesis, and lymphocyte proliferation are induced in the liver of MRL-lpr/lpr mice and are closely associated with Kupffer cell activation.     

The role of Kupffer cells and regulation of neutrophil migration into the liver by macrophage inflammatory protein-2 in primary listeriosis in mice

Depletion of mouse Kupffer cells and splenic macrophages following intravenous administration of liposome-entrapped clodronate severely reduced host resistance to primary infection with Listeria monocytogenes. Infection of clodronate-treated mice with a sublethal dose of L. monocytogenes resulted in death of the mice within 3 days. The macrophage depletion resulted in marked increases in bacterial growth in the liver and spleen, but not in other tissues. The proliferation of L. monocytogenes was observed in a large number of hepatocytes that underwent apoptosis. Infiltration of neutrophils in the liver and rapid formation of microabscesses were observed in the control mice after L. monocytogenes infection. However, there was less accumulation of neutrophils in the liver of Kupffer cell-depleted mice than in the control mice. Expression of macrophage inflammatory protein-2 (MIP-2) was enhanced in the livers of both the control and Kupffer cell-depleted mice after L. monocytogenes infection. MIP-2 was also induced in a murine hepatocyte cell line following L. monocytogenes infection. The administration of neutralizing anti-interleukin-8 receptor homolog antibody severely abrogated neutrophil infiltration into the Listeria-infected mouse liver. Anti-MIP-2 antibody moderately reduced neutrophil infiltration and microabscess formation in the liver. These findings indicate that Kupffer cells protect hepatocytes from L. monocytogenes infection and the resultant apoptosis. Moreover, MIP-2 and its related molecules produced by the infected hepatocytes regulate neutrophil infiltration and microabscess formation in primary listeriosis.     

Effect of anti-tumor necrosis factor alpha antibodies on histopathology of primary Salmonella infections.

We reported that administration of anti-tumor necrosis factor alpha (anti-TNF-alpha) antibodies exacerbates the course of a Salmonella infection in both susceptible and resistant mice by preventing the suppression of bacterial growth in the reticuloendothelial system. In the present study, we evaluated the effect of in vivo neutralization of TNF-alpha on the histopathology of primary Salmonella infections. We show that in primary infections, the suppression of bacterial growth in the reticuloendothelial system coincides with granuloma formation in the spleen and liver. Administration of anti-TNF-alpha globulins on day -1 of salmonellosis affected neither the histological picture nor the course of the infection in the early stages of the disease (days 1 to 3), with splenic and hepatic lesions consisting mainly of polymorphonuclear leukocytes (PMNs); conversely, later in infection (days 3 to 7), the treatment inhibited the formation of granulomas. When the anti-TNF-alpha treatment was started well after the suppression of bacterial growth in the reticuloendothelial system and the formation of granulomatous lesions in the spleen and liver, a prompt relapse of the infection and regression of already established granulomas were seen. In anti-TNF-alpha-treated mice, salmonellae were found inside macrophages and PMNs and extracellularly in the necrotic tissue of the spleen, while in the liver the organisms were seen mainly in inflammatory mononuclear cells, resident Kupffer cells, and hepatocytes and occasionally in the extracellular compartment within necrotic lesions. The bacteria appeared most often in clusters, being morphologically intact when in the extracellular space or within hepatocytes, while undergoing various degrees of degeneration when inside phagocytes. The results suggest that TNF-alpha is required for granuloma formation in salmonellosis and that its neutralization does not completely abrogate the bactericidal activity of macrophages and PMNs. Salmonellae were observed to grow within both hepatocytes and phagocytes but were killed only in the latter.     

Repopulation of murine Kupffer cells after intravenous administration of liposome-encapsulated dichloromethylene diphosphonate.

Kupffer cells were selectively eliminated in mice by the intravenous administration of liposome-entrapped dichloromethylene diphosphonate. At 5 days, small peroxidase-negative and acid-phosphatase-weakly-positive macrophages appeared, increased in number, and differentiated into peroxidase- and acid-phosphatase-positive Kupffer cells. Repopulating small macrophages actively proliferated, and the number of Kupffer cells returned to the normal level by day 14. The numbers of macrophage precursors in the liver as detected by the monoclonal antibodies ER-MP20 and ER-MP58 increased after liposome-entrapped dichloromethylene diphosphonate injection. ER-MP58-positive cells proliferated and differentiated into ER-MP20-positive cells and eventually into BM8-positive Kupffer cells in the liver. Bone-marrow-derived ER-MP58-positive cells were also detectable in the liver and differentiated into ER-MP20-positive cells, but they did not become BM8-positive macrophages. Macrophage colony-stimulating factor mRNA expression was enhanced in the liver 1 day after injection. The administration of macrophage colony-stimulating factor did not shorten the period of Kupffer cell depletion but increased the number and the proliferative capacity of repopulating Kupffer cells. These findings implied that repopulating Kupffer cells are derived from a macrophage precursor pool in the liver rather than from bone-marrow-derived monocytes. Local production of macrophage colony-stimulating factor in the liver plays a crucial role in the differentiation, maturation, and proliferation of Kupffer cells.     

Supportive cellular elements for hepatic T cell differentiation: T cells expressing intermediate levels of the T cell receptor are cytotoxic against syngeneic hepatoma,and are lost after hepatocyte damage

Extrathymic T cells exist in the liver and are often seen in close contact with Kupffer cells in the hepatic sinusoids. Since selective depletion of Kupffer cells has become possible by using liposome-encapsulated clodronate, it was investigated whether elimination of Kupffer cells influences the level of extrathymic T cells in the liver. Extrathymic T cells were identified as interleukin-2 receptor β-chain (IL-2Rβ) intermediate TCR (TCR^int) cells by two-color staining for CD3 or T cell receptor (TCR) and IL-2Rβ. The elimination of Kupffer cells did not significantly affect levels of TCR^int cells up to 7 days after treatment. We then examined monocyte colony stimulating factor (M-CSF)-deficient op/op mice (low levels of Kupffer cells). Extrathymic T cells both in the liver and spleen of these mice were detected at a level comparable to that of control mice. Since extrathymic T cells in the liver are sometimes located in the parenchymal space, the relationship between extrathymic T cells and hepatocytes was then examined. Electron microscopy revealed that some hepatic T cells adhered directly to hepatocytes. When hepatocytes were damaged by a single injection of CCl₄, hepatocyte death and subsequent hepatic fibrosis were induced. Beginning 3 days after injection, CD3^int cells, but not other type of cells, decreased prominently. Purified CD3^int cells, as well as whole lymphocytes in the liver, were cytotoxic against syngeneic hepatoma. In parallel with the above-mentioned hepatic damage, the cytotoxic activity of lymphocytes against such targets was impaired in the liver. These results suggest that extrathymic generation of TCR^int cells and their acquisition of cytotoxic function are relatively independent of Kupffer cells, but are dependent on hepatocytes.     

Expression of macrophage colony-stimulating factor and its receptor in hepatic granulomas of Kupffer-cell-depleted mice.

In mice administered with liposome-entrapped dichloromethylene diphosphonate, which depletes Kupffer cells, the size and the number of zymosan-induced granulomas in the liver were smaller than in untreated mice. The number of macrophage precursors, as detected by the monoclonal antibodies for macrophage precursors, increased after zymosan injection in both groups of mice, proliferated, and differentiated into macrophages. Expression of macrophage colony-stimulating factor (M-CSF), interleukin-1, monocyte chemoattractant protein-1, tumor necrosis factor-alpha, and interferon-gamma mRNA was enhanced in the stage of granuloma formation in the control mouse liver, whereas it was suppressed in Kupffer-cell-depleted mice. However, M-CSF mRNA expression was increased in the Kupffer-cell-depleted mice to form granulomas in the late stages. In situ hybridization demonstrated the expression of M-CSF mRNA and c-fms mRNA in Kupffer cells and monocyte-derived macrophages in the sinusoid and granulomas. The concentration of M-CSF in serum of zymosan-injected control mice was within normal range, but that of zymosan-treated or untreated Kupffer-cell-depleted mice was markedly elevated at day 1. These findings imply that Kupffer cells are indispensable for granuloma formation and produce various cytokines including M-CSF. The local production and consumption of M-CSF in the liver may play a crucial role in granulomatous inflammation.     

Distribution and Localization of Monophosphoryl Lipid A in Selected Tissuef the Rat

The distribution and cellular localization of Monophosphoryl Lipid A in the kidney, liver, lung, spleen, and stomach of rat were investigated by immunohistochemistry on paraffin embedded tissue sections. Rats were sacrificed 24h or 48h following intraperitoneal administration of MPL at a dose of 5mg/Kg. The presence of MPL in selected tissues was indicated by a positive reaction to MPL antibody. In the kidneys, MPL was found in collecting tubules and distal convoluted tubules in the medulla, whereas the glomerulus was essentially free of it. Regarding liver, MPL was found to be abundant in hepatocytes but only occasionally present in Kupffer cells. In lungs, both alveolar and bronchiolar macrophages were positive, indicating the lung can also serve as a possible site for the elimination of MPL. In spleen, endothelial cells and macrophages were positive for MPL. In stomach, MPL was detected in the gastric mucosa and vascular endothelial cells.

In all the tissues studied the intensity of the peroxidase reaction was significantly weaker in 48h samples as compared to corresponding 24h samples indicating possible elimination of MPL from these tissues.     

Ehrlichia chaffeensis induces monocyte inflammatory responses through MyD88, ERK, and NF-κB but not through TRIF, interleukin-1 receptor 1 (IL-1R1)/IL-18R1, or toll-like receptors

Human monocytic ehrlichiosis, an influenza-like illness accompanied by signs of hepatitis, is caused by infection of monocytes/macrophages with a lipopolysaccharide-deficient bacterium, Ehrlichia chaffeensis. The E. chaffeensis strain Wakulla induces diffuse hepatitis with neutrophil infiltration in mice with severe combined immunodeficiency, which is accompanied by strong CXCL2 (mouse functional homolog of interleukin-8 [IL-8]) and tumor necrosis factor alpha (TNF-α) expression in the liver. In this study, we found that expression of IL-1β, CXCL2, and TNF-α was induced by strain Wakulla in mouse bone marrow-derived macrophages; this expression was dependent on MyD88, but not on TRIF, TLR2/4, IL-1R1/IL-18R1, or endosome acidification. When the human leukemia cell line THP-1 was exposed to E. chaffeensis, significant upregulation of IL-8, IL-1β, and TNF-α mRNA and extracellular regulated kinase 2 (ERK2) activation were detected. U0126 (inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 [MEK1/2] upstream of ERK), manumycin A (Ras inhibitor), BAY43-9006 (Raf-1 inhibitor), and NS-50 (inhibitor of NF-κB nuclear translocation) inhibited the cytokine gene expression. A luciferase reporter assay using HEK293 cells, which lack Toll-like receptors (TLRs), showed activation of both the IL-8 promoter and NF-κB by E. chaffeensis. Activation of the IL-8 promoter in transfected HEK293 cells was inhibited by manumycin A, BAY43-9006, U0126, and transfection with a dominant-negative Ras mutant. These results indicate that the E. chaffeensis Wakulla strain can induce inflammatory responses through MyD88-dependent NF-κB and ERK pathways, without the involvement of TRIF and TLRs.