Received 9 September 2005/Returned for modification 14 October 2005/Accepted 20 December 2005

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1 INFECTION AND IMMUNITY, Mar. 2006, p Vol. 74, No /06/$ doi: /iai Copyright 2006, American Society for Microbiology. All Rights Reserved. Role of Tumor Necrosis Factor Alpha (TNF- ) 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 Nahed Ismail, 1,2,3,4 Heather L. Stevenson, 1 and David H. Walker 1,3,4 * Department of Pathology, 1 Department of Microbiology and Immunology, 2 Sealy Center for Vaccine Development, 3 and Center for Biodefense and Emerging Infectious Diseases, 4 University of Texas Medical Branch, Galveston, Texas Received 9 September 2005/Returned for modification 14 October 2005/Accepted 20 December 2005 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- ) bycd8 T cells. We examined the role of TNF- 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- neutralization. TNF- -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- production in the spleens of IOE-infected TNFR I/II / and TNF- -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- -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- 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- is not solely responsible for mortality. Monocytotropic Ehrlichia species are obligately intracellular bacteria that infect phagocytic bone marrow-derived cells in mammalian hosts (40, 56). Human monocytotropic ehrlichiosis is an emerging, potentially fatal, tick-transmitted disease caused by Ehrlichia chaffeensis (40, 11). Monocytotropic Ehrlichia spp. including E. chaffeensis as well as other related species such as Anaplasma phagocytophilum are gram-negative bacteria that lack lipopolysaccaride (LPS) (31, 53). Unlike infection with LPS-positive gram-negative bacteria, infection of mice with A. phagocytophilum, the agent of human granulocytic ehrlichiosis, is controlled by specific lymphocyte immunity even in the absence of Toll-like receptor 2 (TLR2), TLR4, and the TLR adaptor protein MyD88 (6, 55). In a murine model of severe ehrlichiosis, we have recently shown that immunocompetent mice infected with a highly virulent monocytotropic Ehrlichia strain, known as IOE, develop a toxic shock-like syndrome characterized by the presence of a high level of serum tumor necrosis factor alpha (TNF- ), generation of a high number of TNF- -producing CD8 T cells, and a weak Ehrlichia-specific CD4 Th1 immune response (24). The hallmarks of this weak Th1 immune response include a decreased total number of CD4 T cells, a low frequency of antigen (Ag)-specific gamma interferon (IFN- )-producing CD4 Th1 cells with no bias * Corresponding author. Mailing address: University of Texas Medical Branch, 301 University Blvd., Galveston, TX Phone: (409) Fax: (409) dwalker@utmb.edu. towards a Th2 response, decreased IOE-specific CD4 T-cell proliferation, and downregulation of interleukin-12 (IL-12). A study by Bitsaktsis et al. showed that resistance of C57BL/6 or BALB/c mice to a sublethal dose of IOE is dependent on CD4 T cells and IFN- production (2). Furthermore, exogenous IFN- was capable of inducing microbiocidal activity in infected macrophages (2). Other studies also suggested that cell-mediated immune mechanisms involving CD4 cells and type 1 cytokines, together with the immunoglobulin G2a (IgG2a) antibody (Ab) isotype, are responsible for macrophage activation and for elimination of this intracellular Ehrlichia (14, 18, 29, 30, 58, 59). We have previously found a strong correlation between increased levels of systemic TNF- and mortality in mice infected with a high dose of IOE (24). Based on these data, we hypothesize that LPS-negative-IOE-induced toxic shock is due to two interrelated mechanisms, namely, a weak Ag-specific CD4 Th1 response and a substantial expansion of TNF- producing, pathogenic Ag-specific CD8 T cells. The purpose of this study was to directly examine the roles played by TNF- in the host response to Ehrlichia and pathogenesis of ehrlichiosis. TNF-, a potent proinflammatory cytokine released primarily from stimulated macrophages, has a dual effect. TNF- is a key mediator of inflammatory responses. Many aspects of tissue damage following acute or chronic inflammatory reactions can be directly attributed to TNF- release, and these data provide the therapeutic ratio- 1846

2 VOL. 74, 2006 ROLE OF TNF- AND TNF RECEPTORS IN EHRLICHIA INFECTION 1847 nale for developing TNF- antagonists (19, 38, 57). TNF- also plays a pivotal role in host defense mechanisms. A requirement for TNF- in innate resistance to intracellular pathogens is well documented (3, 42, 49). The biologic activities of TNF- are mediated by two structurally related, but functionally distinct, receptors, p55 and p75, whose genes belong to the TNF receptor (TNFR) gene family (41). Experiments using receptor-specific Abs, receptor-specific ligands, and mice genetically deficient in either p55 or p75 indicate that p55 is the primary signaling receptor on most cell types through which the majority of inflammatory responses and host defense classically attributed to TNF- occur (50, 57). In contrast, TNF- -mediated apoptosis of activated mature T lymphocytes is mediated by p75 (7, 46). TNF- associated with the cell surface is biologically active and is superior to soluble TNF- in triggering p75. Additionally, surface-associated p75 is postulated to enhance a p55-dependent response, which involves overlapping intracellular signaling events triggered by p55 and p75 (7). The production of TNF- is regulated by the anti-inflammatory effect of IL-10 (9). TNF- and IL-10 play important but opposite roles during infections with many intracellular pathogens such as Mycobacterium tuberculosis (1, 16, 43, 44). In the presence of IL-10, both T-cell proliferation and IFN- production are inhibited, and the action of IL-10 has been linked to its down-regulation of macrophage activation (8, 10, 15, 26). IL-10 can inhibit TNF- and NO secretion (16, 26) and downregulate the expression of costimulatory molecules and major histocompatibility complex (MHC) class II (4, 10); therefore, it compromises both microbicidal mechanisms and Ag presentation by macrophages. In addition, TNF- and IL-10 have opposing roles in the induction of programmed cell death (23, 37, 43). To directly address the roles played by TNF- in the host response to Ehrlichia, we have studied the infection in mice which are deficient in the two known receptors (TNFR p55 and p75) for this cytokine. The effects of TNF- neutralization on the outcome of ehrlichial infection and the host response against highly virulent IOE were also examined. Our data reveal that TNFR I/II expression is a critical requisite for high-dose-ioe-induced shock/liver injury, and these receptors are important for controlling ehrlichial replication. Furthermore, our data suggest that the balance between TNF- and IL-10 produced by either macrophages or T cells in response to infection with Ehrlichia may modulate the induction of apoptosis during the infection. Finally, this study provides indirect evidence that IL-10 can function as a proinflammatory or antiinflammatory cytokine depending on the kinetics of its production and the type of cells producing IL-10 following infection with monocytotropic Ehrlichia. MATERIALS AND METHODS Mice. Normal sex- and age-matched C57BL/6 mice and mice lacking both the p55 (Tnfrsf1a, tumor necrosis factor receptor superfamily member 1a) and p75 chains (Tnfrsf1b, tumor necrosis factor receptor superfamily member 1b) of the TNF receptor (Tnfrsf1a tm1imx and Tnfrsf1b tm1imx ) were purchased from the Jackson Laboratory (Bar Harbor, ME). Genotyping was performed using PCR analyses. The double-tnf receptor-knockout (KO) mice (referred to here as TNFR p55/p75 / or TNFR I/II/ / ) used throughout these studies were of B6, 129 background and have been described previously (41). Animals were sacrificed and selected organs were studied as indicated below. Mouse handling and experimental procedures were conducted in accordance with the American Association of Accreditation of Laboratory Animal Care guidelines for animal care and use. Ehrlichia infections. Mice were inoculated intraperitoneally (i.p.) with 1 ml of either a high dose (10 2 dilution) of highly virulent IOE or a high dose (10 1 dilution) of mildly virulent Ehrlichia muris. Quantitative real-time PCR determined that the 10 2 dilution of the IOE inoculum contained bacterial genomes whereas the 10 1 dilution of the E. muris inoculum contained bacterial genomes. Control mice were inoculated with 1 ml of a 10 1 or 10 2 dilution of a spleen homogenate from naive C57BL/6 mice. On the days of infection mice were sacrificed and immune responses were assessed. Selected organs were harvested for histology, immunohistochemistry, and determination of bacterial load by real-time PCR and culture. Preparation of host cell-free Ehrlichia. For preparation of IOE antigens, IOE-infected spleens and livers were harvested from mice on day 7 postinfection (p.i.) as previously described (24). Mildly virulent E. muris was cultivated in P388D1 cells at 37 C in minimal essential medium supplemented with 5% bovine calf serum. Ehrlichiae were harvested when approximately 90 to 100% of the cells were infected, and cell-free ehrlichiae were prepared as previously described (24, 39). The total protein concentrations of the resulting bacterial preparations were determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL), and the preparations were used as Ag in the enzymelinked immunospot (ELISPOT) assays and enzyme-linked immunosorbent assays (ELISA) employed for the detection of Ag-specific-cytokine-producing cells and cytokine production in the culture supernatant, respectively. A mixture of spleen and liver from naive mice (mock Ag) was used as a negative control in all experiments employing cell-free IOE Ags, while uninfected-cell culture lysate (mock Ag) was used as a negative control in all experiments using E. muris Ags. Ehrlichial load determination by quantitative real-time PCR. The ehrlichial load in tissues was determined by real-time PCR (with SYBR Green) targeting the Ehrlichia dsb gene, which encodes a disulfide bond formation protein of E. muris and IOE (GenBank accession no. AY and AY236485). Primer sequences and quantification methods have been described previously (24). PCR analyses were considered negative for ehrlichial DNA if the critical threshold values exceeded 40 cycles. Expression of the ehrlichial load was normalized relative to the number of copies of GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Neutralization of TNF-. Groups of C57BL/6 mice received either 20 g per mouse per day of neutralizing anti-tnf- IgG1 monoclonal Ab (mab; R&D Systems, Minneapolis, MN) or equivalent amounts of isotype-matched irrelevant control antibodies (R&D Systems) 1 day before IOE infection and on days 2, 5, and 7 following IOE infection. Histology and immunohistochemistry. Samples of liver, spleen, and lung were processed for histopathological examination as described previously (24, 39, 51). For immunohistochemistry, sections of the organs were incubated for 45 min at 37 C with rabbit anti-e. muris polyclonal Ab, which detects E. muris and crossreacts with IOE Ags, at a dilution of 1/10,000. Slides were incubated for 30 min with biotinylated goat anti-rabbit IgG (heavy plus light chain) Ab at a 1/800 dilution (Vector Laboratories, Burlingame, CA) and then washed and incubated with avidin-horseradish peroxidase conjugate for 20 min at 37 C. This was followed by incubation with substrate containing 3-amino-9-ethylcarbazole (Vector Laboratories) for 8 min at 37 C. Sections were counterstained with hematoxylin. Normal rabbit serum was used as a negative control. TUNEL stain. The tissue sections were deparaffinized and processed for the terminal deoxynucleotidyltransferase-mediated dutp-biotin nick end labeling (TUNEL) assay as described before (51). Positive controls were generated by digesting the tissue sections with DNase I (10 g/ml, final concentration) in Tris-buffered saline containing 1 mmol/liter MgCl 2 for 20 min. Terminal deoxynucleotidyltransferase was omitted in slides used as negative controls. ELISPOT assays for Ag-specific Th1 or Th2 cytokine-producing T cells. Single-cell suspensions were obtained from the spleens of control and infected mice. CD4 and CD8 T cells were isolated by negative selection using mouse CD4 or CD8 subset enrichment columns (R&D Systems), and the purity ranged from 80 to 90% as determined by flow-cytometric analysis. Splenocytes and purified T-cell subsets were assessed via ELISPOT for cytokine production, as described previously (24). Briefly, 96-well nitrocellulose plates (Millipore, Bedford, MA) were coated at 4 C overnight with mabs (1.25 g/ml; 100 l/well) that are specific for murine IFN-, IL-4, IL-10, or TNF- (BD PharMingen, San Diego, CA). Twofold dilutions of spleen cells were added to wells starting at 10 6 to cells/well in the presence of an additional spleen cells from naive, unimmunized mice. The addition of normal spleen cells was necessary to ensure that the number of Ag-dependent spots observed was proportional to the number of immune spleen cells plated and that the response was linear. Immune spleen cells or purified T cells were stimulated with either the specific IOE Ag at

3 1848 ISMAIL ET AL. INFECT. IMMUN. FIG. 1. Increased resistance of TNFR I/II / mice to IOE-induced toxic shock. (A) Survival rates of various mouse groups following challenge with a high dose of highly virulent IOE or mildly virulent E. muris. Six- to 8-week-old C57BL/6 control (n 12) mice and mice treated with anti-tnf- mab (n 12) were infected i.p. with a high dose of IOE or E. muris 1 day after the initiation of TNF- neutralization. TNFR I/II / mice (n 15) were infected i.p. with the same dose of IOE. Mice were monitored daily for disease manifestations and death. The data shown represent one of three independent experiments. WT, wild type. (B and C) Higher bacterial burdens in different organs in anti-tnf- Ab-treated mice (B) and TNFR I/II / mice (C) than wild-type controls on day 8 following i.p. infection with IOE, as determined by real-time PCR. Significance was determined by Student s two-tailed t test (symbols above bars indicate P for IOE-infected anti-tnf- Ab-treated mice versus IOE-infected wild-type mice [B] or for IOE-infected TNFR I/II / KO mice versus IOE-infected wild-type mice [C]). Data represent the averages and standard deviations of triplicate amplifications with three mice per group. Similar results were observed in three independent experiments. a concentration of 10 g/well or with nonspecific Ag such as SRBC. Positive and negative controls contained 5 g/ml concanavalin A or medium, respectively. In all experiments, Ag-specific spots were determined by subtraction of the background spots (spots detected in the Ag-negative wells) from spots detected in the Ag-containing wells. Lymphocyte culture and cytokine detection. For in vitro cytokine measurements, spleens were removed aseptically and single-cell suspensions were prepared. Splenocytes were plated in 24-well tissue culture plates at a final concentration of cells/ml in RPMI 1640 supplemented with 2 mm glutamine, 25 mm HEPES, 10% fetal bovine serum, 50 M 2-mercaptoethanol, penicillin, and streptomycin. Cultures were incubated at 37 C in an atmosphere of 5% CO 2. Cells were stimulated with concanavalin A (5 g/ml), relevant cell-free E. muris or IOE antigens, or medium alone. Supernatant fluids were harvested at 72 h and assayed for cytokine production. TNF- and IL-10 levels were measured by specific capture Quantikine ELISA kits (R&D Systems, Minneapolis, MN) following the manufacturer s instructions. Cytokine levels were calculated using standard curves constructed with recombinant murine cytokines. The detection limits were 150 pg/ml and 5 pg/ml for IL-10 and TNF-, respectively. Serum levels of TNF- and IL-10 were measured using the same cytokine ELISA. Cell separation experiments. To identify the cell types predominantly responsible for IL-10 production, spleen cells were separated by adherence. Cells were resuspended in RPMI 1640 to a concentration of /ml, and 4 ml was dispensed into glass petri dishes. After incubation for 2 h, nonadherent cells were removed by three washings with RPMI Adherent cells were washed three times with RPMI 1640, exposed to 2 mm EDTA for 15 min at 4 C, and gently resuspended with a scraper. Once recovered, adherent and nonadherent cells were resuspended to a concentration of /ml. Cells were then cultured in 1-ml volumes in 24-well plates for 24 h in the presence of the relevant antigen as described above. Adherent cells from either control or TNF- depleted mice were 90% macrophages as determined by CD11b staining and fluorescenceactivated cell sorter analysis. Statistics. Values for secreted Ag-specific cytokine proteins, quantitative realtime PCR, and serum cytokine levels were compared. When comparing data in only two groups, Student s two-tailed t test was used. Comparisons across three or more groups were statistically compiled using analysis of variance (ANOVA). For repeated measures of IL-10 concentrations in the serum from the same mice in different groups at different time points, the data were analyzed by ANOVA for repeated measures. The Bonferroni method was used to adjust for multiple comparisons. A P value 0.05 was regarded as significant, and a P value 0.01 was considered highly significant. RESULTS Prolonged survival of TNFR I/II / mice following lethal Ehrlichia infection. To test the hypothesis that severe pathology and fatal ehrlichiosis are mediated by TNF- overproduction and to determine the role of TNF- in host defense and immunopathogenesis in ehrlichial infection, we studied mild and severe ehrlichiosis in TNFR I/II / mice and mice injected with neutralizing antibodies against TNF-, isotype-matched control IgG Ab, or phosphate-buffered saline (PBS) at 1, 2, 5, and 7 days of infection. All mice were infected i.p. with the same high dose of highly virulent IOE or mildly virulent E. muris known to cause severe and mild diseases in immunocompetent hosts, respectively. Since E. muris infection induces a strong protective cell-mediated immunity, the type and level of immune response in all mouse groups challenged with lethal IOE were compared to those generated in mice challenged with nonlethal E. muris. Disease progression and the immune response were examined. The outcomes of infection and host defense in wild-type C57BL/6 mice injected with PBS or with isotype-

4 VOL. 74, 2006 ROLE OF TNF- AND TNF RECEPTORS IN EHRLICHIA INFECTION 1849 matched control Ab were similar. Therefore, only the results for wild-type mice injected with isotype-matched control Ig will be described here. TNF- neutralization had no effect on the survival of IOE-infected mice, with 0% survival following lethal IOE challenge (Fig. 1A). Sham-treated wild-type and anti- TNF- -treated animals both succumbed to IOE infection on days 9 to 11 p.i. TNF- neutralization alone also did not have a significant effect on mice infected with the mildly virulent E. muris species (Fig. 1A). In contrast, TNFR I/II / C57BL/6 mice had prolonged survival following lethal IOE challenge, where mice survived until 18 days postinfection (Fig. 1A). TNF- is important for controlling infection with monocytotropic Ehrlichia. Quantitative measurement of bacterial burdens by real-time PCR showed that anti-tnf- -treated mice and TNFR I/II / KO mice infected with IOE harbored substantially higher bacterial burdens in the spleen, lung, and liver than those detected in infected sham-treated wild-type mice on day 8 p.i. (Fig. 1B and C). Confirming the quantitative realtime PCR measurement of the relative ehrlichial burden, immunohistochemistry showed that both anti-tnf- -treated mice and TNFR I/II / mice harbored a higher burden of organisms than sham-treated mice (Fig. 2A and B). The distribution of morulae also differed in the different groups of mice. In TNF- -depleted (data not shown) and TNFR I/II / mice (Fig. 2B), many ehrlichiae were detected in endothelial Kupffer cells and hepatocytes (arrowhead). In contrast, in the wild-type untreated control mice, ehrlichiae were localized mainly in sinusoidal-lining cells, including endothelial cells and Kupffer cells (arrow), with very few organisms present in the hepatocytes (arrowhead) (Fig. 2A). These data suggest that TNF- and TNFR I/II play protective roles in host defense against Ehrlichia by controlling bacterial replication. TNFR I/II / mice are resistant to highly virulent-ioeinduced liver and lung pathology. Mice were challenged i.p. with a high dose of IOE, and liver tissues were prepared at different time points thereafter. Pathology and apoptosis were analyzed by hematoxylin and eosin (H&E) and TUNEL staining, respectively. No measurable alterations were found in liver sections from wild-type mice on day 4 postinfection (data not shown). However, on day 7 postinfection, liver sections from infected wild-type mice displayed characteristic features of hepatocyte destruction, including extensive partially confluent foci of necrosis/apoptosis of contiguous hepatocytes, as well as extensive parenchymal hemorrhage (Fig. 3A). TUNEL assays showed that the livers of IOE-infected wild-type mice (Fig. 3E) contained more than 30 apoptotic cells per 20 high-power fields. Although most of the apoptotic cells were located lining the sinusoids (Kupffer cells, endothelial cells, and lymphocytes), a large number of scattered apoptotic hepatocytes were also present. In anti-tnf- treated mice, hepatocyte destruction was minimal 4 days after challenge, but severe hepatocyte destruction was found by day 7 postinfection, which was comparable to that in wild-type, isotype control mice at the same time point following IOE challenge (Fig. 3B). In contrast, only scant liver necrosis/apoptosis was detected in TNFR I/II / mice on day 7 after IOE challenge (Fig. 3C) and no exacerbation was found thereafter (data not shown) until the time of death. Interestingly, the hepatocytes in TNFR I/II / KO mice showed widespread randomly distributed middle- and large-sized-droplet FIG. 2. Immunohistochemical (IHC) detection of IOE in the livers of mice infected with a high dose of IOE. (A) Isotype control wild-type mice; (B) TNFR I/II / mice. IHC detection in livers from TNFR I/II / mice demonstrated numerous morulae in sinusoidal and blood vessel lining cells (Kupffer cells and endothelial cells) as well as in hepatocytes (arrowhead). In contrast, IHC detection in livers of IOEinfected wild-type control mice demonstrated fewer morulae, which were detected mainly in sinusoidal-lining cells, including endothelial and Kupffer cells (arrow). A horseradish peroxidase (diaminobenzidine)/hematoxylin stain was used. Original magnification, 400. fatty change (Fig. 3D), which was not detected in wild-type mice following lethal IOE infection. Lung pathology in all groups showed similar levels of interstitial pneumonia characterized by extensive infiltration with inflammatory cells and thickened alveolar septa (data not shown). We concluded that signaling via TNFR I/II, at least in part, mediates liver injury/apoptosis in severe ehrlichiosis.

5 1850 ISMAIL ET AL. INFECT. IMMUN. FIG. 3. Markedly decreased liver injury/apoptosis in TNFR I/II / mice. (A) Wild-type mice treated with isotype control; (B) anti-tnf- -treated mice; (C) TNFR I/II / mice. All mice were infected i.p. with a high dose of IOE, and livers were collected at 8 days postinfection and stained with H&E (A to D). Extensive necrosis was observed in wild-type and anti-tnf- -treated mice (arrows), while few apoptotic cells were detected in TNFR I/II / mice (arrowhead). H&E staining is shown. Original magnifications, 200 (A and D), 100 (C), and 400 (B). (D) A high-power view of the livers of TNFR I/II / treated mice showed steatosis with middle- (arrow) and large-sized-droplet fatty change. Anti-TNF- -treated mice had similar fatty change (not shown). (E) TUNEL assays using horseradish peroxidase (diaminobenzidine)/azure A stain of livers of wild-type mice showed extensive apoptosis of hepatocytes (arrow) and cells lining hepatic sinusoids such as Kupffer cells and endothelial cells (arrow). Original magnification, 400. Elevated serum levels of TNF- and IL-12 in TNFR I/II / mice following challenge with highly virulent Ehrlichia. Our previous data showed a correlation between a high serum level of TNF-, a low level of IL-12, and the development of LPSnegative IOE-induced toxic shock-like syndrome (24). We therefore compared serum levels of TNF- in wild-type mice, anti-tnf- -neutralized mice, and TNFR I/II / mice following challenge with high-dose IOE. Since a low level of IFN- occurs in LPS-negative IOE-induced toxic shock-like syndrome (24) and this cytokine is induced by IL-12 (12, 27, 42), serum levels of IL-12 (p40) and IL-12 (p70) were analyzed as well. Serum levels of IL-12 (p40), IL-12 (p70), and TNF- in wild-type mice peaked at 3, 3, and 7 days, respectively, after either IOE or E. muris challenge (data not shown). We therefore compared cytokine levels in the sera of IOE-infected anti-tnf- -treated, TNFR I/II /, and wild-type mice at the respective time points to that produced in wild-type resistant E. muris-infected control mice. We previously demonstrated that infection of wild-type mice with a high dose of mildly

6 VOL. 74, 2006 ROLE OF TNF- AND TNF RECEPTORS IN EHRLICHIA INFECTION 1851 FIG. 4. Serum levels of TNF- and IL-12 and frequencies of Agspecific IFN- - and IL-4-producing cells in the spleens of TNF- depleted, TNFR I/II /, and wild-type mice treated with isotype control following challenge with either IOE or E. muris. Mice were infected i.p. with either a high dose of IOE or E. muris (A to C). Sera were collected at 7 days for TNF- (A) and at 3 days [for IL-12 (p40) and Il-12 (p70) (B and C, respectively)] after challenge, and serum levels of these cytokines were determined by ELISA. The serum level of TNF- was lower in IOE-infected anti-tnf- -treated mice versus infected wild-type controls (solid star, P 0.001), but serum TNF- was higher in TNFR I/II / KO versus wild-type controls (solid diamond, P 0.005). Serum levels of IL-12 (p40) and IL-12 (p70) were slightly higher in TNFR I/II / KO mice versus wild-type controls [open star, P 0.05 for both IL-12 (p40) and IL-12 (p70)]. Serum levels of IL-12 (p40) and IL-12 (p70) were significantly lower in TNFR I/II / KO mice versus E. muris-infected control mice (four diamonds, P 0.001). Before Ehrlichia challenge, the above cytokines were virtually undetectable in the sera. (D) Spleens were collected at day 7 postinfection, and splenocytes were prepared thereafter and stimulated in vitro with the relevant antigen. The frequencies of IFN- and IL-4 were determined by ELISPOT assay. Data represent the averages and standard deviations of four mice per group. Similar results were observed in three independent experiments, with a total of 12 mice/ group. virulent E. muris resulted in mild disease and protective immunity characterized by substantial expansion of CD4 Th1 cells. Therefore, in this study, we employed E. muris infection to determine whether the cytokine levels and immunity generated in IOE-infected wild-type, TNFRI/II /, and anti-tnf- -treated mice are comparable to the protective levels observed in E. muris-infected wild-type mice. Serum levels of TNF- were significantly higher in TNFR I/II / mice than in wild-type or anti-tnf- -treated mice after challenge with IOE (Fig. 4A). Slight increases in the serum levels of IL-12 (p40) and IL-12 (p70) were detected in these mice following IOE challenge. Although the levels of Il-12 (p40) and IL-12 (p70) were higher in TNFR I/II / and anti-tnf- -treated mice than in IOE-infected wild-type mice, they were significantly lower than the protective levels of IL-12 (p40) and IL-12 (p70) in E. muris-infected wild-type mice, respectively (Fig. 4B and C). Thus, incomplete resistance of TNFR I/II / mice against LPS-negative-IOE-induced toxic shock was associated with an elevated serum level of TNF- and lower serum level of IL-12 than that detected in resistant mice infected with a high dose of E. muris. Elevated serum levels of TNF- and low serum levels of IL-12 in TNFR I/II / mice correlate with a slightly increased frequency of Ehrlichia-specific IFN- -producing cells. We demonstrated previously that LPS-negative-IOE-induced toxic shock is associated with a weak CD4 Th1 response when compared to the generation of a substantial number of CD4 Th1 cells in resistant mice following challenge with a high dose of mildly virulent E. muris. In this study, we compared numbers of IFN- - and IL-4-producing cells in the spleens of TNFR I/II /, TNF- -depleted, and wild-type mice on day 7 following high-dose-ioe or E. muris challenge using ELISPOT assays. Before challenge, IFN- - and IL-4-producing cells were few in all mouse groups, and no significant difference was found among these mice (data not shown). The frequencies of Ehrlichia-specific IFN- -producing cells among splenocytes were slightly increased in IOE-infected TNF- -depleted mice compared to IOE-infected wild-type mice (P 0.05). In contrast, the frequencies of Ehrlichia-specific IFN- -producing cells were significantly higher in infected TNFR I/II / mice than in IOE-infected wild-type mice (P 0.05; Fig. 4D). However, the frequencies of IOE-specific IFN- -producing cells among splenocytes of TNF- -depleted and TNFR I/II / mice following IOE challenge were significantly lower than that detected in splenocytes of wild-type mice at day 7 after infection with E. muris (P 0.001; Fig. 4D). In all mouse groups, the frequencies of Ehrlichia-specific IL-4-producing cells following either IOE or E. muris challenge were low (Fig. 4D). These results clearly demonstrated that an increase in the frequency of Ehrlichia-specific IFN- -producing cells in TNFR I/II / mice could be partly responsible for prolonged survival of these mice following IOE challenge. However, the inability of IOE-infected TNF- -depleted and TNR I/II / mice to generate a substantial number of protective IFN- -producing cells comparable to that generated in E. muris-infected mice may partially account for their susceptibility to LPS-negative- IOE-induced toxic shock.

7 1852 ISMAIL ET AL. INFECT. IMMUN. FIG. 5. Levels of IL-10 in the sera and spleen and frequencies of IL-10-producing spleen cells in TNF- -depleted, TNFR I/II /, and wild-type mice following lethal or nonlethal challenge with IOE or E. muris, respectively. Mice were infected i.p. with either a high dose of IOE or E. muris. (A) Sera were collected at 3, 8, and 9 days after challenge, and serum levels of IL-10 were determined by ELISA. Before Ehrlichia challenge, the above cytokines were virtually undetectable in the sera. (B) Spleens were collected at day 8 postinfection, and splenocytes were prepared thereafter and stimulated in vitro with the relevant antigen. The frequencies of Ag-specific IL-10-producing cells were determined by ELISPOT assay. The numbers of Ag-specific IL-10-producing cells were comparable among all IOE-infected mice in different groups (P 0.05) but were significantly higher than that detected in E. muris-infected mice (P ). Data represent the averages and standard deviations of four mice per group. Similar results were observed in three independent experiments, with a total of nine mice/group. Early elevated serum levels of IL-10 in TNFR I/II / mice following LPS-negative-IOE challenge. Several studies have demonstrated that under certain conditions IL-10 can function as an anti-inflammatory or proinflammatory cytokine (15, 25, 28, 43). TNF- -induced release of IL-10 occurs during LPSmediated gram-negative endotoxemia (47, 52), where IL-10 plays a protective anti-inflammatory role. To determine whether IL-10 participates in the susceptibility of mice to Ehrlichiainduced toxic shock, serum levels of IL-10 were compared in TNF- -depleted, TNFR I/II /, and wild-type mice following challenge with lethal or nonlethal IOE and E. muris, respectively. In all mouse groups, IL-10 was undetectable in sera before IOE or E. muris challenge (data not shown). On days 3 and 8 after infection with IOE, serum levels of IL-10 were significantly higher in TNFR I/II / and TNF- -depleted mice than in wild-type mice (P 0.05; Fig. 5A). On day 9 postinfection, very high serum levels of IL-10 were detected in all groups and wild-type mice had significantly higher levels of IL-10 than TNFR I/II / mice (P 0.05; Fig. 5A). Infection of wild-type mice with mildly virulent E. muris resulted in significantly lower levels of serum IL-10 on days 8 and 9 postinfection than were found in all IOE-infected wild-type, TNF- depleted, and TNFR I/II / mice (P 0.005; Fig. 5A). Elevated levels of IL-10 in TNFR I/II / mice correlates with increased frequency of IL-10-producing spleen cells. We compared numbers of IL-10-producing cells in the spleens of wild-type, TNF- -depleted, and TNFR I/II / mice following lethal IOE or nonlethal E. muris challenge on day 8 postinfection by ELISPOT. Ag-specific IL-10-producing cells were virtually undetectable in all mouse groups before challenge (data not shown). In contrast, the frequencies of Ag-specific IL-10- producing spleen cells among splenocytes were markedly increased in wild-type, TNF- -depleted, and TNFR I/II / mice following IOE challenge, and they were comparable among different groups (P 0.05; Fig. 5B). However, the frequency of Ag-specific IL-10 producers was significantly higher in all IOEinfected mice in different groups than in E. muris-infected animals (P ). Thus, higher serum levels of IL-10 in wild-type, TNF- -depleted, and TNFR I/II / mice following IOE challenge correlate with higher numbers of IL-10-producing spleen cells. Differential sources of elevated IL-10 levels in wild-type mice and TNFR I/II / mice following lethal LPS-negative- IOE challenge. Although IL-10 serum concentrations were elevated in wild-type and TNFR I/II / mice following lethal IOE challenge, the kinetics of its production were different. Our previous data showed that Ehrlichia-specific CD8 T cells were responsible for overproduction of TNF- in wild-type C57BL/6 mice following lethal LPS-negative-IOE-induced toxic shock. To analyze the source of IL-10 in different groups of mice, splenocytes harvested on day 8 postinfection were separated into two populations on the basis of glass adherence. The concentrations of IL-10 present in the supernatant of adherent and nonadherent cell cultures were measured after 48 h following in vitro IOE Ag stimulation by ELISA. In wild-type mice, the majority of IL-10-producing splenocytes were nonadherent cells (Fig. 6). Production of a high level of IL-10 by these cells was antigen specific. In contrast, most of the IL-10-producing cells in TNFRI/II / and TNF- -depleted mice were adherent cells (Fig. 6). Although adherent cells were 90% macrophages, as determined by CD11b staining and fluorescence-activated cell sorter analysis (data not shown), our data did not completely exclude the possibility that other cell types might also have contributed to the IL-10 production. Taken together, these data indicate that adherent cells, mainly macrophages, are major producers of IL-10 in the IOE-infected TNFR I/II / mice and TNF- -depleted mice. In contrast, in IOE-infected wild-type mice, IL-10 is mainly produced by antigen-specific nonadherent lymphocytes. DISCUSSION This work established that the TNFR I/II have dual effects that include a protective role in controlling ehrlichial replication and a detrimental role in triggering liver injury during LPS-negative Ehrlichia-induced toxic shock. TNFR I/II /

8 VOL. 74, 2006 ROLE OF TNF- AND TNF RECEPTORS IN EHRLICHIA INFECTION 1853 FIG. 6. IL-10 production by total unseparated, nonadherent and adherent spleen cells from wild-type, TNFR I/II /, and anti-tnf- treated mice after in vitro stimulation with IOE. All mice were infected with IOE, and spleens were recovered on day 8 postinfection. Supernatant was collected at 48 h, and the IL-10 level was measured by ELISA. In wild-type mice, IL-10 was secreted mainly by adherent cells in an antigen-dependent manner, while in TNFR I/II / and anti- TNF- -treated mice, IL-10 was mainly produced by adherent cells. Solid star, four diamonds, P for IL-10 produced by adherent or nonadherent cells from IOE-infected wild-type versus TNFR I/II / KO mice; solid diamond, open star, P for IL-10 produced by adherent or nonadherent cells from IOE-infected wild-type versus anti-tnf- -depleted mice. Bars represent means standard deviations of three independent observations conducted in duplicate. mice exhibited prolonged survival following infection with IOE but had impaired clearance of monocytotropic Ehrlichia, as well as reduced IFN- production in response to infection with highly virulent Ehrlichia. Although these animals displayed a marked reduction in liver injury and apoptosis, they still succumb to lethal IOE infection, which can be attributed to decreased production of IL-12 and IFN- and early increased IL-10 production. TNF- is associated with IOE-induced toxic shock (24) and is a ligand for TNFR (5, 32, 50). Thus, the elevated serum levels of TNF- in moderately susceptible TNFR I/II / mice compared with wild-type controls imply that a factor(s) downstream of TNF- signaling participates in the increased resistance of TNFR I/II / mice to IOE. A possible mechanism that may explain the susceptibility of TNFR I/II / mice to IOE-induced toxic shock is that a mediator other than TNF- produced during IOE infection is responsible for death in these mice. The only known ligands that bind to these receptors are TNF- and its close relative, secreted homodimeric lymphotoxin (LT ) (13, 36, 45). However, the extent to which functional redundancy between endogenously produced TNF- and LT is physiologically relevant remains unclear. This issue is further complicated by the presence of a related, membranebound cytokine, LT, that interacts with LT (5, 13, 36). The predominantly expressed heteromeric complex of LT and LT ( 1 2 ) does not bind either of the known TNFRs but does bind to the LT receptor (LT R). Therefore, the lethality of IOE infection in TNFR I/II / mice could be attributed to a pathway that does not involve the TNF receptors. Nevertheless, our data confirmed a critical role of TNF- and TNFR p55/p75 (TNFR I/II) in the innate response against intracellular Ehrlichia. Compared to wild-type mice infected with a high dose of IOE, infected TNFR I/II / and TNF- depleted mice had higher bacterial burdens at early time points postinfection (Fig. 1B and C). Previous studies showed that administration of neutralizing antibodies to either TNF- or p55, but not antibodies against p75, renders mice extremely susceptible to Listeria monocytogenes and results in inefficient elimination of bacteria (13, 60). More importantly, our data showed that, although TNFR I/II / mice remain susceptible to lethal IOE infection, they exhibit a marked decrease in liver injury manifested by decreased number of apoptotic hepatocytes and sinusoidal-lining cells (Fig. 3E). Generally most TNF- -induced apoptosis of liver cells and activated mature T lymphocytes is mediated by p75 (7, 25). It is worth noting that cell surface-associated TNF- is biologically active and is superior to soluble TNF- in triggering p75 (20, 21, 37). Therefore, one potential explanation for discrepant results in this study between TNF- -neutralized mice and TNFR I/II / mice is that neutralization of TNF- may have been less effective in abrogating the apoptotic signaling of the cell membrane form of TNF- through TNFR p75. In support of this possibility is our observation that TNF- -depleted mice developed severe liver necrosis and apoptosis similar to wild-type mice. We cannot exclude the possibility that the mab treatment failed to block TNF- with a ligand(s) hidden within tissue or incomplete neutralization of the membrane form of TNF-. Therefore, TNF- neutralization employed in this study does not formally exclude the possibility that susceptibility to infection with a high dose of highly virulent IOE is due to interactions between TNFR and its physiological ligands. It was found in this study that, relative to wild-type mice, TNFR I/II / and TNF- -depleted mice have markedly increased IL-10 serum levels early in the course of infection (Fig. 5A). This difference was associated with inability to localize the infection and resulting increased bacterial burden (Fig. 1B and C). Several mechanisms may account for the detrimental effects of IL-10 in ehrlichiosis. IL-10 potently inhibits the microbicidal and Ag-presenting functions of macrophages (8, 10). In vitro infection of DH82 macrophages with Ehrlichia canis, a monocytotropic Ehrlichia that is genetically related to IOE and the causative agent of canine ehrlichiosis, down-regulates MHC class II receptors (22). Whether the downregulation of MHC class II by Ehrlichia is IL-10 dependent has yet to be examined. Although IL-10 is considered a potent anti-inflammatory cytokine, recent studies have suggested that IL-10 also possesses an immunostimulatory effect. It could be speculated that, unlike what occurs during exposure to LPS or infection with facultative intracellular pathogens, IL-10 could exert proinflammatory effects in vivo during infection with Ehrlichia as an obligatory intracellular pathogen. Although direct proof of this proinflammatory effect has not been obtained in this study, three pieces of evidence support this hypothesis. First, in murine infection with LPS-positive gram-negative bacteria causing endotoxic shock, an inverse relationship exists between TNF- and IL-10 production, suggesting that IL-10 acts as an anti-inflammatory cytokine. On the other hand, in LPS-negative-Ehrlichia-mediated toxic shock-like syndrome, the upregulation of IL-10 following high-dose-ioe infection in wild-type

9 1854 ISMAIL ET AL. INFECT. IMMUN. mice not only failed to down-regulate TNF- overproduction but was associated with a high serum level of TNF-. Second, early high production of IL-10 in TNFR I/II / and TNF- depleted mice was associated with higher serum and splenic IFN- and TNF- levels. Third, in wild-type mice, the late overproduction of IL-10 together with TNF- is associated with enhancement of liver apoptosis/necrosis, suggestive of a proinflammatory effect. A recent study has shown that IL-10 treatment enhanced activation of human cytotoxic T lymphocytes and NK cells after LPS injection, as reflected by increased levels of soluble granzymes (28). In support of proinflammatory role of IL-10 is the finding that high-dose- IL-10 therapy in patients with inflammatory disorders is ineffective (28). In the present study, adherent macrophages were predominantly responsible for IL-10 production by spleen cells from TNFR I/II / and anti-tnf- -treated mice in response to IOE. In contrast, high IL-10 production in wild-type mice was detected in the nonadherent cell populations, including B and T lymphocytes. Previously, we have found that TNF- overproduction following lethal IOE infection is mediated by antigen-specific CD8 T cells. We have not determined in this study whether T or B cells are responsible for systemic IL-10 overproduction in wild-type mice. However, our preliminary data suggest that these are T cells (N. Ismail et al., unpublished observation). Why different cell subsets are responsible for overproduction of IL-10 in different groups of mice following the same high dose of IOE and what regulates their IL-10 production remain to be determined. In several experimental systems, TNF- and IL-10 have opposite roles in the induction of programmed cell death (21, 54). Signals transduced through TNFR (p75) can induce an activation of proteases (17), including cysteine proteases (caspases), which are recognized mediators of apoptosis. TNF- also increases synthesis of nitric oxide in various cells, and this molecule has been extensively associated with induction of DNA damage and apoptosis (3, 33). On the other hand, the effects of IL-10 on cell survival have been associated with increased expression of the antiapoptotic factor Bcl-2. In humans, IL-10 prevents lymphocyte activation-induced apoptosis (23, 48) by induction of the Bcl-2 proteins. Conversely, when lymphocytes are grown in the presence of neutralizing anti-il-10, there is an increase in apoptosis. Furthermore IL-10 down-regulates apoptosis in human alveolar macrophages infected with M. tuberculosis by inducing the release of TNFR II leading to the formation of nonactive TNF- TNFR II complexes (13, 54). Taking these results together, we envisage that significantly decreased liver apoptosis and prolonged survival of TNFR I/II / mice may be attributed partly to an early production of IL-10 by adherent macrophages, which down-regulated apoptosis in liver cells, thus resulting in prolonged survival. In contrast, late overproduction of IL-10 by nonadherent T cells in wild-type mice is a detrimental process, in which IL-10 functions as a proapoptotic or proinflammatory cytokine as described above. In support of this hypothesis, our preliminary data demonstrated that infection of IL-10 KO mice with a high dose of IOE resulted in a substantially decreased number of apoptotic liver cells compared to wild-type mice (Ismail et al., unpublished observation). Therefore, the differential secretion of IL-10 by different cell types, which influences the kinetics of IL-10 production in TNFR I/II / and wild-type mice, would determine whether IL-10 acts as pro- or antiapoptotic/inflammatory cytokine. Furthermore, as suggested by other studies (9, 10, 26), IL-10 produced by macrophages can inhibit macrophage function, resulting in enhanced bacterial intracellular growth. This conclusion is consistent with our findings that an early production of IL-10 in TNFR I/II / and TNF- -depleted mice was associated with higher bacterial burdens than in wild-type mice. Our observation that severe ehrlichiosis and mortality in TNFR I/II / mice are associated with an overwhelming infection (Fig. 1C) compared to wild-type mice following lethal IOE infection is similar to findings concerning severe and fatal human monocytotropic ehrlichiosis, where an overwhelming infection with high bacterial loads is detected only in immunocompromised individuals (11, 56). These data support our previous conclusion that severe ehrlichiosis in immunocompetent mice is attributed to immune-mediated mechanisms. In support of this conclusion, immunohistochemistry and TUNEL assays in this study demonstrated that, although ehrlichial morulae were present mainly in cells lining blood vessels and sinusoids (i.e., monocytes, Kupffer cells, and endothelial cells), apoptotic events involve both these cells and uninfected hepatocytes (Fig. 3E). Our data are consistent with studies using A. phagocytophilum, previously called Ehrlichia phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), indicating that histopathological lesions in the HGE murine model do not result from direct Ehrlichia-mediated injury but from immunopathological mechanisms initiated by ehrlichial infection (34, 35). Finally, our previous data showed that toxic shock-like syndrome in IOE-infected mice is associated with a concomitant decreased frequency of Ag-specific CD4 Th1 cells and expansion of Ag-specific TNF- - and IFN- -producing CD8 T cells. We hypothesize that CD8 T cells play a pathogenic role in severe ehrlichiosis, via cytotoxic killing of infected target cells, uninfected host cells, and/or activated Ag-specific CD4 T cells. The cytotoxic effects of CD8 T cells are known to be mediated via several pathways including TNF- /TNFR, perforin/granzyme, and Fas/FasL. Therefore, the mortality of IOEinfected TNFR I/II / or anti-tnf- -neutralized mice may have been caused by other antigen-specific CD8 T-cell-mediated cytotoxic killing mechanisms of host cells. Further studies investigating these factors will better clarify the relative contribution of TNF- in the pathogenesis of severe and fatal ehrlichiosis. In conclusion, this study has shed light on the previously uncharacterized dual roles of TNF- and IL-10 in the pathogenesis of severe ehrlichiosis that resembles toxic shock-like syndrome. It is interesting that TNF- neutralization and the absence of TNFR signaling resulted in a higher bacterial load, an altered histopathological response to IOE infection, and a significant early burst of the immunosuppressive IL-10 cytokine compared to findings for wild-type mice. This study suggests that alterations in the balance of TNF- and IL-10 production may influence accessory and effector macrophage functions as well as the induction of apoptosis and cell survival following Ehrlichia infection. Direct examination of the impact of IL-10 on severe ehrlichiosis and the relevance of apoptosis/ necrosis in TNF- -depleted and TNF single- or double-receptor-deficient mice, as well as characterization of the mechanisms by which IL-10 and TNF- mediate tissue damage,