Received 6 February 2007/Returned for modification 22 May 2007/Accepted 3 June 2007

Transcription

1 INFECTION AND IMMUNITY, Sept. 2007, p Vol. 75, No /07/$ doi: /iai Copyright 2007, American Society for Microbiology. All Rights Reserved. Relative Importance of T-Cell Subsets in Monocytotropic Ehrlichiosis: a Novel Effector Mechanism Involved in Ehrlichia-Induced Immunopathology in Murine Ehrlichiosis Nahed Ismail, 1,2,3 * Emily C. Crossley, 1,4 Heather L. Stevenson, 1 and David H. Walker 1,2,3 Department of Pathology, 1 Department of Microbiology and Immunology, 4 Sealy Center for Vaccine Development, 2 and Center for Biodefense and Emerging Infectious Diseases, 3 University of Texas Medical Branch, Galveston, Texas Received 6 February 2007/Returned for modification 22 May 2007/Accepted 3 June 2007 Infection with gram-negative monocytotropic Ehrlichia strains results in a fatal toxic shock-like syndrome characterized by a decreased number of Ehrlichia-specific CD4 Th1 cells, the expansion of tumor necrosis factor alpha (TNF- )-producing CD8 T cells, and the systemic overproduction of interleukin-10 (IL-10) and TNF-. Here, we investigated the role of CD4 and CD8 T cells in immunity to Ehrlichia and the pathogenesis of fatal ehrlichiosis caused by infection with low- and high-dose (10 3 and 10 5 bacterial genomes/mouse, respectively) ehrlichial inocula. The CD4 T-cell-deficient mice showed exacerbated susceptibility to a lethal high- or low-dose infection and harbored higher bacterial numbers than did wild-type (WT) mice. Interestingly, the CD8 T-cell-deficient mice were resistant to a low dose but succumbed to a high dose of Ehrlichia. The absence of CD8 T cells abrogated TNF- and IL-10 production, reduced tissue injury and bacterial burden, restored splenic CD4 T-cell numbers, and increased the frequency of Ehrlichia-specific CD4 Th1 cells in comparison to infected WT mice. Although fatal disease is perforin independent, our data suggested that perforin played a critical role in controlling bacterial burden and mediating liver injury. Similar to WT mice, mortality of infected perforin-deficient mice was associated with CD4 T-cell apoptosis and a high serum concentration of IL-10. Depletion of IL-10 restored the number of CD4 and CD8 T cells in infected WT mice. Our data demonstrate a novel mechanism of immunopathology in which CD8 T cells mediate Ehrlichiainduced toxic shock, which is associated with IL-10 overproduction and CD4 T-cell apoptosis. * Corresponding author. Mailing address: Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, 301 University Blvd., Galveston, TX Phone: (409) Fax: (409) naismail@utmb.edu. Published ahead of print on 11 June Human monocytotropic ehrlichiosis (HME) resembles toxic shock syndrome and is caused by Ehrlichia chaffeensis, a gram-negative, obligately intracellular bacterium that lacks lipopolysaccharide (LPS) and peptidoglycan (30, 37, 49, 52). In mice, inoculation with a high dose of IOE (a monocytotropic Ehrlichia species isolated from Ixodes ovatus ticks in Japan) (46) results in a fatal syndrome that mimics severe HME (26, 48). IOE is genetically and antigenically closely related to E. chaffeensis and Ehrlichia muris, both of which cause only mild self-limited disease in mice (16, 17, 26, 50, 58). Severe and fatal infection of immunocompetent mice with IOE, however, causes severe tissue damage to multiple organ systems in a setting of a low bacterial burden (26). This observation mirrors what occurs in immunocompetent patients who develop multisystem organ failure without an overwhelming infection (49). Our previous studies demonstrated that resistance to fatal disease is mediated by gamma interferon (IFN- ) production and CD4 Th1 cells, while tumor necrosis factor alpha (TNF- ) production from antigen-specific CD8 T cells is key to the development of fatal ehrlichiosis (26). We confirmed TNF- s pathogenic role using TNF receptor (TNFR) p55 and p75 double knockout mice. TNFR p55 binds to soluble TNF- and is critical for controlling intracellular bacterial replication, while TNFR p75 interacts with trans-membrane TNF- to mediate apoptotic or necrotic host cell death. The absence of both TNF receptors in double knockout mice abrogates severe liver pathology and delays mortality following infection with a high dose of IOE compared to wild-type (WT) mice (25). We also showed that interleukin-10 (IL-10) production is associated with fatal disease (25). Thus, both pro- and anti-inflammatory cytokines are implicated in Ehrlichia-induced toxic shock. In contrast to the well-known CD4 T-cell functions against intracellular bacteria, particularly those organisms that reside in endocytic compartments and are inaccessible to the endogenous pathway of major histocompatibility complex (MHC) class I antigen presentation, the role of CD8 T cells is less clearly defined. CD8 effector T cells are key mediators in the immune response to several intracellular pathogens such as viruses (51) and intracytosolic bacteria including Listeria monocytogenes (19, 27, 33) and Rickettsia (15, 53). CD8 T cells appear to exert their function through two mechanisms. First, CD8 effector cells may act as cytotoxic lymphocytes to kill pathogen-infected cells via perforin-dependent TNF/ TNFR pathways or by Fas/Fas ligand (FasL) interactions (6, 22, 29, 43, 56). Second, pathogen-specific CD8 T cells are potent producers of cytokines, particularly IFN- and TNF-, both of which play a critical role in limiting pathogen replication (22, 56), while excess production induces immunopathology (13, 24, 35). The present study assessed the relative contributions of different T-cell subsets to Ehrlichia-induced toxic shock. In particular, we focus here on CD8 T cells, soluble factors TNF- 4608

2 VOL. 75, 2007 ROLE OF CD8 T CELLS IN EHRLICHIA-INDUCED TOXIC SHOCK 4609 and IL-10, and perforin as critical components of cytotoxic lymphocyte-dependent cytotoxicity, a function that could be responsible for either pathogen clearance or immunopathology. We describe a novel role of CD8 T cells as pathogenic mediators of Ehrlichia-induced toxic shock. We used a mouse model of fatal ehrlichiosis to demonstrate that TNF- and IL-10 overproduction, CD4 T-cell apoptosis, and the downregulation of the Ehrlichia-specific CD4 Th1 response are associated with mortality. Furthermore, we showed that TNF- production in Ehrlichia-induced toxic shock is perforin dependent, while IL-10 production is perforin independent. MATERIALS AND METHODS Mice. The following gene-targeted strains were used: 2 -microglobulin ( 2 m)- deficient ( 2 m / ) strain B6.129P2-2 m tm1unc /J, transporter associated with antigen processing (TAP)-deficient strain B6.129S2-Tap1 tm1arp /J, MHC class IIdeficient (MHC class II / ) strain B6.129S-H2 dlab1-ea /J, and perforin-deficient (perforin / ) strain C57BL/6-Prf1 tm1sdz /J. All mice were purchased from Jackson Laboratories (Bar Harbor, ME). C57BL/6J mice were used as the control background strain in all experiments involving knockout mice. Mice were gender matched for each experiment and were 6 to 12 weeks old. Mouse handling and experimental procedures were conducted in accordance with the University of Texas Medical Branch Institutional Committee for Animal Care and Use. Bacterial stocks and experimental design. Two monocytotropic ehrlichial strains were used in this study, a highly virulent Ehrlichia sp. strain (designated IOE) isolated from Ixodes ovatus ticks (a gift from M. Kawahara, Nagoya City Public Health Research Institute, Nagoya, Japan) and a mildly virulent E. muris strain (provided by Y. Rikihisa, Ohio State University, Columbus, OH). IOE and E. muris stocks were produced as described previously (25, 26). Mice were infected intraperitoneally (i.p.) with 1 ml of a low dose (10 3 bacterial genomes/ mouse) or a high dose (10 5 bacterial genomes/mouse) of fresh inoculum as determined by quantitative real-time PCR. For E. muris infection, mice were infected i.p. with a high (nonlethal) dose of E. muris (10 8 bacterial genomes/ mouse). Control mice were given 1 ml of a 10 1 or 10 2 dilution of a spleen homogenate from uninfected C57BL/6 mice. Mice were sacrificed on the indicated days postinfection, and selected organs were harvested for histology, immunohistochemistry, cell culture, and determination of bacterial load by realtime PCR. Preparation of host cell-free Ehrlichia. For preparation of host cell-free IOE antigen, IOE-infected spleens and livers were harvested from WT mice on day 7 postinfection, and antigen was prepared as previously described (25, 26). Mock antigen was prepared from spleens and livers of uninfected WT mice for all experiments. For the preparation of E. muris antigen, E. muris was cultivated in P388D1 cells grown in 5% bovine calf serum-supplemented modified Eagle s medium at 37 C and harvested when 90 to 100% of the cells were infected. E. muris antigens were prepared as previously described (25). Uninfected cell lysates served as a mock antigen for E. muris. The total protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL), and the ehrlichial genome copy number was measured by quantitative realtime PCR. Histology and immunohistochemistry. Samples of liver, spleen, lung, and kidney were processed for histopathological examination as described previously (38). For immunohistochemistry, slides were incubated for 45 min at 37 C with a 1:10,000 dilution of rabbit anti-e. chaffeensis polyclonal antibody, which crossreacts with E. muris and IOE (26, 48). Slides were then incubated for 30 min with biotinylated goat anti-rabbit immunoglobulin G (IgG) (Vector Laboratories, Burlingame, CA). The slides were then washed and incubated with avidinhorseradish peroxidase conjugate for 20 min at 37 C, followed by incubation with substrate containing 3-amino-9-ethylcarbazole for 8 min at 37 C (Vector Laboratories). For control slides, normal canine serum was used in lieu of a primary antibody to rule out nonspecific staining. Grading of liver lesions was performed as described previously (38). The number of foci of apoptotic/necrotic hepatocytes and degree of inflammatory infiltrates in the liver were used for grading. Each parameter was scored as described previously (38). A total score was obtained from data for each animal by adding all parameters and obtaining the mean. ELISPOT assays for antigen-specific cytokine-producing T cells. CD4 T cells were isolated from splenic homogenates by negative selection using mouse CD4 subset enrichment columns (R&D Systems, Minneapolis, MN), and the purity ranged from 80 to 90% as determined by flow cytometry. Cytokine production from splenocytes and enriched CD4 cells was assessed by an enzyme-linked immunospot (ELISPOT) assay as described previously (26, 41). Briefly, splenocytes were assayed at two dilutions, starting from 10 6 to cells/well. An additional naïve splenocytes/well were added to immune splenocytes to ensure that the number of antigen-dependent spots was linearly proportional to the number of immune spleen cells plated. Splenocytes or enriched CD4 cells were stimulated with 10 g/well of E. muris, IOE, or mock antigens. Positive and negative controls contained 5 g/ml concanavalin A or medium, respectively. Antigen-specific spots were determined by subtracting the number of spots in the mock antigen wells from the number in the E. muris or IOE antigen-stimulated wells. Cytokine ELISA. Splenocytes were cultured at cells/ml in Dulbecco s modified Eagle s medium containing 10% fetal bovine serum, 2 mm glutamine, 100 U/ml penicillin G sodium, and 100 g/ml streptomycin sulfate in the presence or the absence of 50 g/ml of E. muris, IOE, or mock antigen. Supernatants were collected at 48 h and assayed by enzyme-linked immunosorbent assay (ELISA) (Quantikine; R&D Systems) for IL-10 and TNF- according to the manufacturer s instructions. The detection limits were 150 pg/ml for IL-10 and 5 pg/ml for TNF-. In some experiments, sera from infected mice were collected and assayed for IL-10 and TNF-. Ehrlichial load determination by quantitative real-time PCR. The copy number of ehrlichiae in the inoculum and the bacterial burden in different organs were determined by quantitative real-time PCR as described previously (25, 26). Flow cytometry. Splenocytes were harvested, counted, and resuspended in fluorescence-activated cell sorter (FACS) buffer (Dulbecco s phosphate-buffered saline containing 1% heat-inactivated fetal calf serum and 0.09% sodium azide, with the ph adjusted to 7.4 to 7.6). Splenocytes were aliquoted into a 96-well V-bottom culture plate (Costar, Corning, NY) at a concentration of 10 6 cells per well. Fc III/II receptor-blocking was followed by fluorescein isothiocyanateconjugated anti-cd4 and phycoerythrin-conjugated anti-cd8 (BD Pharmingen). Isotype-matched monoclonal antibodies were used as controls. All incubations were done for 15 min at 4 C followed by two consecutive washes in FACS buffer. Apoptotic CD4 and CD8 lymphocytes were identified using annexin V-fluorescein isothiocyanate and phycoerythrin-conjugated anti-cd4 (L3T4, clone GK1.5) or anti-cd8 (Ly-2, clone ) (BD Pharmingen) according to the manufacturer s instructions. Cells were run within 1 h on a FACSCalibur flow cytometer (BD Immunocytometry Systems, San Jose, CA) and analyzed with Cell Quest (Immunocytometry Systems) or FlowJo (Tree Star Inc., Ashland, OR) software. Lymphocytes were gated based on forward and side scatter, and specific staining was determined using isotype controls. The absolute number of apoptotic CD4 and CD8 T cells was determined by multiplying the percentage of apoptotic cells measured by annexin staining by the absolute number of CD4 and CD8 T cells, respectively, in the spleen. In vivo neutralization of TNF- and IL-10. Groups of WT mice received either 20 g per mouse of neutralizing rat anti-mouse IL-10 IgG1 monoclonal antibodies and/or neutralizing rat anti-mouse TNF- IgG1 monoclonal antibodies or equivalent amounts of isotype control antibodies (R&D Systems, Minneapolis, MN) on days 3, 5, and 7 postinfection. All mice were infected with a low dose of IOE. As a negative control, a group of mice was injected with phosphate-buffered saline. Preparation of liver mononuclear cells. Liver mononuclear cells were isolated as previously described (34) using the modified enzymatic dispersal protocol. Cells were centrifuged at low speed, 36 g, for 1 min at room temperature to remove most of the hepatocytes, and the recovered mononuclear cells were assayed by flow cytometry. Statistical analysis. Student s two-tailed t test was used when data from two groups were compared. Comparisons across three or more groups were statistically evaluated using analysis of variance. The Bonferroni method was used to adjust for multiple comparisons. A P value of 0.05 was regarded as being significant, and a P value of 0.01 was considered to be highly significant. RESULTS 2 m-deficient mice are highly resistant to IOE challenge. To assess the roles of CD4 and CD8 T cells in the immune response and pathogenesis of fatal monocytotropic ehrlichiosis, we used MHC class II / mice that lack CD4 T cells and 2 m / mice that lack CD8 T cells. All mice were inoculated i.p. with a high (10 5 organisms) or low (10 3 organisms) dose of IOE and scored for illness daily after infection for 30 days. Similar to our previous report (7), WT mice suc-

3 4610 ISMAIL ET AL. INFECT. IMMUN. FIG. 1. Resistance of 2 m / and TAP / mice to a lethal challenge with a virulent ehrlichial strain (IOE). WT, MHC class II /, and 2 m / mice were challenged i.p. with either high ( )or low ( ) doses of IOE. All mice were monitored for 30 days, and the survival rate was determined following lethal infection with a high (A) or low (B) dose of IOE. (C) Survival of WT and TAP / mice after i.p. infection with a low dose of IOE. The data shown represent one of three independent experiments with a total of 18 mice/group. cumbed to a high dose of IOE on days 8 to 12, with a median survival time (MST) of 9 days (Fig. 1A), while a low-dose infection prolonged survival to days 14 to 17 postinfection (MST, 15 days) (Fig. 1B). MHC class II / mice were more susceptible to lethal IOE infection than WT mice, succumbing on days 7 to 9 or days 11 to 15 to high- or low-dose IOE infection, respectively (Fig. 1A and B) (MST, 7 and 14 days for high- and low-dose IOE infection, respectively). Interestingly, 2 m / mice were resistant to a low dose of IOE (90% survival) (Fig. 1B), although all succumbed to a high dose (Fig. 1A). 2 m / mice exhibited only mild signs of illness (ruffled fur, inactivity, and/or gaunt posture) and maintained normal weight and temperature (data not shown). We confirmed the deficiency of CD8 T cells in spleens of 2 m / mice after i.p. infection with IOE: 9.7% 0.7% (mean standard error of the mean) of splenocytes from infected WT mice were CD8, whereas only 1.5% 0.1% of splenocytes were CD8 in infected 2 m / mice. Similar differences in percentages of CD8 T cells were observed in uninfected mice (data not shown). These data show that CD8 T cells contribute significantly to mortality from Ehrlichia-induced toxic shock. Although it is our hypothesis that 2 m / mice are resistant to IOE due to a lack of MHC class I-restricted CD8 T cells, NKT cells may also be implicated. This is because 2 misa component of a number of other antigen-presenting molecules, including CD1d, which selects NK1.1 CD3 (NKT) cells. However, MHC class I molecules are loaded with peptide antigens in a TAP-dependent fashion (8, 36, 47), whereas CD1 molecules, including CD1d, bind lipid-based antigens independent of TAP (7, 11, 14, 47). We therefore compared TAPdeficient (TAP / ) mice to 2 m / mice to determine if NKT cells contribute to the IOE-resistant 2 m / phenotype. The TAP1 gene disruption causes a profound CD8 T-cell deficiency without affecting NKT cells, a phenotype that we confirmed to hold true during a low-dose IOE infection: 8.0% 0.4% of splenocytes in IOE-infected WT mice were CD8 compared to only 1.2% 0.2% of splenocytes in infected TAP / mice. Interestingly, TAP / mice also had higher survival rates following infection with a low, but not a high, dose of IOE (70% survival up to day 30 postinfection, the time point at which all experiments were terminated) than did WT mice (0% [WT mice succumbed to infection on days 15 to 18 postinfection, with an MST of 16 days]) (Fig. 1C). These data suggest that a CD8 T-cell deficiency significantly contributes to host resistance against Ehrlichia, although the survival difference between TAP / and 2 m / mice indicates that NKT cells may also be involved. CD8 T cells are not critical for controlling ehrlichial replication in WT mice. We have previously shown that IOEinfected WT mice develop severe liver injury in the absence of an overwhelming infection (25, 26). Since CD8 T cells are critical for protection against many intracellular pathogens, we examined the role of CD8 T cells in controlling the ehrlichial burden and systemic dissemination. We infected WT, 2 m /, TAP /, and MHC class II / mice i.p. with a low dose of IOE and compared the bacterial burden in different organs on day 8 postinfection. Ehrlichial burdens in the lungs and spleens of infected 2 m / and TAP / mice were significantly lower (P and 0.012, respectively) than those of infected WT and MHC class II / mice (Fig. 2). MHC class II / mice had substantially higher bacterial burdens (P 0.002) in all organs than did WT mice, suggesting that an overwhelming infection occurs in the absence of CD4 T cells. Interestingly, the absence of CD8 T cells did not significantly (P 0.467) influence the bacterial burden in the liver, a major site of ehrlichial tropism. These data suggest that CD4 T cells, but not CD8 T cells, contribute to the control of Ehrlichia replication and dissemination. CD8 T cells mediate severe immunopathology in fatal ehrlichiosis. To examine the extent to which CD8 T cells mediate tissue damage, we examined the pathology in different organs of WT, 2 m /, TAP /, and MHC class II / mice on days 12 and 14 following i.p. infection with a lethal low dose of IOE. WT and MHC class II / mice developed severe liver

4 VOL. 75, 2007 ROLE OF CD8 T CELLS IN EHRLICHIA-INDUCED TOXIC SHOCK 4611 FIG m / and TAP / mice have lower bacterial burdens than WT and MHC class II / mice. Organs were harvested from WT, 2 m /, TAP /, and MHC class II / mice on day 8 postinfection with a low dose of IOE. Ehrlichial burden was determined on DNA isolates by real-time PCR amplification of the dsb gene and normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). MHC class II / mice developed an overwhelming infection compared to other groups. Data represent the averages and standard deviations of triplicate amplifications with three mice per group. Similar results were observed in three independent experiments. Ehrlichial burden in the lungs and spleens of 2 m / and TAP / mice was significantly lower than that detected in WT mice (P and 0.012, respectively). MHC class II / mice had a significantly higher bacterial burden in all examined organs than did WT, 2 m /, and TAP / mice (P 0.002). injury marked by extensive apoptosis and necrosis and minimal inflammatory cell infiltration on day 12 postinfection (Fig. 3A and B). Although both WT and MHC class II / mice had severe liver injury, the apoptotic and necrotic events were more focal in WT mice than in MHC class II / mice, which had extensive, widespread liver damage. The latter could be due to the presence of an overwhelming infection in the absence of CD4 T cells. In contrast to WT and MHC class II / mice, 2 m / and TAP / mice developed mild liver pathology characterized by marked cellular infiltration and few foci of hepatic apoptosis on day 12 (Fig. 3C and D) and day 14 (data not shown). These data suggest that CD8 T cells directly or indirectly mediate severe liver damage, a characteristic feature of Ehrlichia-induced toxic shock. Lymphoid tissue cellularity is preserved in the absence of CD8 T cells. We then examined the gross and histological changes that CD8 T cells exert on the lymphoid organs during IOE infection. The spleens of WT mice infected with a low dose of IOE were small and pale on day 12 postinfection. However, the spleens of the 2 m / mice infected with the same dose of IOE became large and deep red, similar to the spleens of WT mice responding to low-virulence E. muris (25, 26). Histological examination revealed massive tissue destruction in the spleens (Fig. 4A) and lungs (data not shown) of WT and MHC class II / mice (data not shown). Numerous apoptotic and necrotic foci markedly disrupted the splenic follicular structure. In contrast, 2 m / mice maintained the integrity of the splenic structure (Fig. 4B) and had large splenic lymphocyte populations (Table 1). Infected WT mice had approximately one-half the total number of CD4 and CD8 T cells of uninfected WT mice. In contrast, E. muris-infected WT mice, a model of mild HME, exhibited 1.4- to 2.0-fold-higher numbers of total splenocytes, CD4 T cells, and CD8 T cells than uninfected WT mice. 2 m / mice responded to IOE infection by an expansion of total splenocytes and CD4 T cells (Table 1). These data suggest that Ehrlichia-induced CD8 T cells mediate a reduction in splenic cellularity, particularly in the CD4 and CD8 T-cell populations, shortly before the death of the host. CD4 T-cell apoptosis is significantly higher in IOE-infected mice than in E. muris-infected mice. Analysis of the total numbers of CD4 and CD8 T cells in the spleens of WT mice infected with a low dose of IOE revealed a marked decrease in the percentage and absolute number of both CD4 and CD8 T cells in the spleen shortly before death (Table 1), similar to what was observed previously with a high dose of IOE (26). Because the loss of splenic cellularity may have been due to either T-cell apoptosis or the migration of T cells to peripheral organs, we examined both possibilities. First, we compared the percentage of apoptotic splenic CD4 and CD8 T cells between IOE-infected and E. muris-infected WT mice. IOEinfected mice had a significantly (P 0.003) higher percentage (Fig. 5A) and absolute number (Fig. 5C) of apoptotic CD4 T cells than did E. muris-infected mice Second, we examined T-cell migration by comparing the numbers of CD4 and CD8 T cells in the livers of IOE- and E. muris-infected WT mice. Both infections resulted in increases in the total liver CD4 and CD8 T-cell populations compared to those in uninfected mice (Fig. 5D). However, E. muris-infected mice had significantly higher numbers of liver CD4 T cells (P 0.002) than did IOE-infected mice. There were no significant differences in the numbers of liver CD8 T cells between the two infected groups (Fig. 5C), indicating that CD8 T cells in both models are equally capable of migration. The low number

5 4612 ISMAIL ET AL. INFECT. IMMUN. Downloaded from FIG. 3. CD8 T cells mediate extensive liver pathology following lethal ehrlichial infection. WT, 2 m /, TAP /, and MHC class II / mice were infected with a low dose of IOE, and the livers were harvested on day 12 postinfection for paraffin embedding followed by hematoxylin and eosin staining. WT mice (A) and MHC class II / mice (B) developed extensive tissue damage (arrows). In contrast, 2 m / (C) and TAP / mice (D) developed mild hepatic pathology characterized by few apoptotic foci (arrows) and marked cellular infiltration (arrowhead). Data are representative of one experiment with three mice per group. Similar results were observed in three independent experiments. of CD4 T cells in the livers of IOE-infected mice resulted in a ratio of CD4 to CD8 T cells of 1:1, compared to 4:1 in E. muris-infected mice. Our data indicate that the decreased number of splenic CD4 T cells in IOE-infected WT mice, which coincides with the development of toxic shock, is not due to the migration of effector Ehrlichia-specific CD4 T cells to the liver but rather is due to apoptotic CD4 T-cell death. CD8 T cells are responsible for the decreased frequency of IFN- -producing CD4 Th1 cells following lethal ehrlichial infection. Next, we tested the possibility that CD8 T cells could mediate fatal immunopathology by promoting the apoptosis of protective CD4 Th1 cells (25, 26). Compared to IOE-infected WT mice, 2 m / and TAP / mice had significantly (P 0.01) high numbers of IOE-specific IFN- producing CD4 Th1 cells in the spleen on days 8 (Fig. 6A) and 12 (data not shown) postinfection with a low dose of IOE. The levels of CD4 Th1 cells in the infected 2 m / and TAP / mice were similar (P 0.725) to those in E. muris-infected WT mice. Interestingly, MHC class II / mice were capable of generating Ehrlichia-specific IFN- producing T cells in the spleen (Fig. 6B). Since MHC class II / mice lack CD4 T cells, IFN- -producing T cells are likely to be CD8 T cells or NKT cells. However, the fact that the IFN- production is antigen specific makes CD8 T cells the more likely candidate. The latter finding suggests that the induction of IOE-specific CD8 T-cell responses is CD4 T-cell independent. More importantly, our results demonstrate that CD8 T cells mediate the downregulation of protective CD4 Th1 cells in IOE-infected WT mice, a hallmark of the defective immune response in fatal murine ehrlichiosis (26). on April 8, 2019 by guest

6 VOL. 75, 2007 ROLE OF CD8 T CELLS IN EHRLICHIA-INDUCED TOXIC SHOCK 4613 FIG. 4. CD8 T cells mediate splenocyte apoptosis and destruction of follicular structure following lethal ehrlichial infection. WT and 2 m / mice were infected with a low dose of IOE, and paraffin sections of splenic tissues collected on day 12 postinfection were stained with hematoxylin and eosin. WT mice (A) developed massive apoptosis in splenic lymphoid follicles and had numerous tingible body macrophages (arrows) within poorly distinguishable follicles. 2 m / mice (B) maintained the integrity of the splenic structure, and large populations of lymphocytes could clearly be identified within the follicles. Tingible body macrophages were not identified in these mice. Data are representative of one experiment with three mice per group. Similar results were observed in two independent experiments. Antigen-stimulated splenocytes from 2 m / mice produce significantly less TNF- and IL-10 than those from WT mice. To examine whether CD8 T-cell-mediated toxic shock is linked to dysregulated TNF- and IL-10 production as suggested by previous studies (25, 26), we infected WT and 2 m / mice with an ordinarily lethal low dose of IOE and compared the TNF- and IL-10 concentrations in the serum and in supernatants of cultured splenocytes. Antigen-stimulated splenocytes from 2 m / mice produced significantly (P 0.005) less TNF- and IL-10 than did those from WT mice on day 8 postinfection (Fig. 7A). The levels of TNF- and IL-10 produced by IOE-stimulated WT splenocytes rose significantly (P 0.007) between day 8 and day 12 postinfection (Fig. 7A), a finding that correlated with the in vivo production of high serum levels of TNF- and IL-10 (Fig. 7B). In contrast, on days 8 and 12 postinfection, 2 m / mice produced significantly (P 0.005) lower levels of TNF- and IL-10 in vivo (serum) and in vitro by immune splenocytes stimulated with IOE antigen (Fig. 7A and B). These data suggest that CD8 T cells mediate the dysregulated Ehrlichia-specific overproduction of TNF- and IL-10 in fatal murine ehrlichiosis that resembles toxic shock syndrome. In vivo neutralization of IL-10 increased the numbers of CD4 and CD8 T cells in IOE-infected WT mice. Our previous data showed that lethal murine ehrlichiosis that resembles toxic shock is associated with the presence of high levels of IL-10 throughout the course of disease (25). To explore the relative contributions of IL-10 to disease pathogenesis, we depleted WT mice of IL-10 and infected them with a lethal low dose of IOE. The percentages of splenic CD4 and CD8 T cells were determined by flow cytometry on day 12 postinfection, and we compared these mice to E. muris-infected mice, which underwent splenic CD4 and CD8 T-cell expansion (Fig. 8). Consistent with our previous data (26), infection of WT mice treated with isotype control antibody with a lethal dose of IOE decreased the percentages (Fig. 8) of CD4 and CD8 T cells. Strikingly, the depletion of IL-10 resulted in a significant expansion of CD4 and CD8 T cells in WT mice following infection with a lethal low dose of IOE (Fig. 8). This expansion was associated with marked splenomegaly and an increase in total viable splenocytes as determined by trypan blue staining (data not shown), suggesting that IL-10 depletion increases splenic cellularity. These data demonstrate that IL-10 is causally involved in the decline of CD4 and CD8 T-cell populations in Ehrlichia-induced toxic shock. CD8 T-cell-mediated toxic shock is not dependent solely on perforin. We hypothesized that CD8 T cells induce the observed tissue damage through a perforin-mediated cytotoxic mechanism. To test this hypothesis, we infected WT, 2 m /, and perforin / mice with a low dose of IOE and monitored survival. Both wild-type and perforin / C57BL/6 mice inoculated with a low dose of IOE failed to control disease progression, and both types of mice succumbed to infection (Fig. 9A). However, perforin / mice had a significantly higher bacterial burden in the livers (P 0.021) and lungs (P 0.034), but not in the spleens, than wild-type mice (Fig. 9B). Compared to infected WT mice that developed extensive partially confluent foci of apoptosis or necrosis of contiguous hepatocytes, similar to that shown in Fig. 3A, on day 12 postinfection, TABLE 1. Changes in total numbers of splenocytes and CD4 and CD8 T-cell populations on day 12 postinfection with a low dose of IOE or a high dose of E. muris Mouse group Avg total no. of cells (10 6 ) SD Splenocytes CD4 T cells CD8 T cells WT uninfected WT high dose of E. muris (sublethal) WT low dose of IOE m / uninfected m / low dose of IOE

7 4614

8 VOL. 75, 2007 ROLE OF CD8 T CELLS IN EHRLICHIA-INDUCED TOXIC SHOCK 4615 Downloaded from FIG m / and TAP / mice produce high numbers of antigen-specific, IFN- -producing CD4 Th1 cells when infected with IOE. Splenocytes from WT, 2 m /,ortap / mice were harvested on day 12 postinfection with a low dose of IOE or a high dose of E. muris, and CD4 T cells were purified by positive selection. They were then stimulated with IOE or E. muris antigens, respectively, in the presence or absence of naïve, syngeneic splenocytes ( splenocytes/well). The number of antigen-specific, IFN- -producing CD4 T cells or IL-4-producing CD4 T cells per 10 6 cells was determined by ELISPOT assay (A). IOE-infected 2 m / (F) and TAP / ( ) mice have significantly higher quantities of IFN- -producing CD4 T cells than do infected WT mice (P 0.003). A similar comparison was made between low-dose IOE-infected WT and MHC class II / mice, and the frequency of antigen-specific IFN- - or IL-4-producing T cells in the spleens of these mice is shown in B. Data represent the averages and standard deviations of data from four mice per group. Similar results were observed in three independent experiments. on April 8, 2019 by guest perforin / mice had mild to moderate liver injury (Fig. 9C) marked by the presence of fewer foci of apoptotic or necrotic hepatocytes. In addition, the livers of infected perforin / mice have more macrophage-rich inflammatory infiltrates than observed in the liver of WT mice (data not shown). Our data suggest that CD8 T-cell-mediated toxic shock is a multistep process that is not dependent solely on perforin. Furthermore, our data suggest that perforin plays an important role in con- FIG. 5. Lethal ehrlichial infection in WT mice results in the apoptosis of CD4 T cells in the spleen and is associated with a small CD4 T-cell population in the liver. WT mice were infected i.p. with either a lethal low dose of IOE or a high dose of mildly virulent E. muris (EM). The percentages of apoptotic CD4 (A) and CD8 (B) T cells as well as their absolute numbers (C) in the spleens of infected mice and uninfected controls (NEG) on day 12 postinfection were determined by annexin staining as described in Materials and Methods. The absolute numbers of CD4 and CD8 T cells in the livers of IOE- and E. muris-infected WT mice on day 12 postinfection were compared to those in livers of uninfected WT mice (D). These data represent the average of data from three mice per group. Similar results were observed in two independent experiments.

9 4616 ISMAIL ET AL. INFECT. IMMUN. FIG. 7. CD8 T cells are critical for the local and systemic production of TNF- and IL-10 following lethal ehrlichial infection. WT and 2 m / mice were infected with a low dose of IOE, and the levels of TNF- and IL-10 in the serum and supernatants of cultured splenocytes were determined by ELISA on days 8 and 12 postinfection. Harvested splenocytes were stimulated in vitro with IOE antigen for 48 h. Substantially higher levels of IL-10 and TNF- were produced by antigen-stimulated splenocytes of WT mice than those of 2 m / mice on day 8. The levels increased significantly on day 12 postinfection (A). Similar levels were detected in serum (B). No IL-10 or TNF- was detected on day 0 (data not shown). Data represent the averages and standard deviations of data from four mice per group. Similar results were observed in three independent experiments. FIG. 8. Neutralization of IL-10 restores the total numbers of splenic CD4 and CD8 T cells in WT mice during IOE infection. WT mice were treated with anti-il-10 or isotype control as described in Materials and Methods. Mice were infected i.p. with a lethal low dose of IOE or a nonlethal high dose of E. muris, and splenocytes were collected on day 12 postinfection for flow cytometric analysis. Lymphocytes were gated based on size and granularity. The absolute numbers of CD4 and CD8 T cells were higher in IL-10-depleted mice than in isotype control-treated mice infected with IOE (P 0.006), and the numbers of CD4 and CD8 T cells were similar to the T-cell numbers in E. muris-infected mice that develop mild and self-limited disease (P 0.556). Data represent the averages and standard deviations of data from three mice per group. Similar results were observed in two independent experiments. trolling bacterial infection and mediating tissue injury in fatal ehrlichiosis. Susceptibility of perforin / mice is associated with increased IL-10 production. To determine whether the susceptibility of perforin / mice to lethal ehrlichiosis is associated with a weak CD4 Th1 response and IL-10 overproduction similar to WT mice, we measured the serum level of IL-10 and the frequency of CD4 Th1 cells on day 10 postinfection, i.e., 1 to 2 days before mice succumbed to infection. Purified CD4 T cells from WT, 2 m /, and perforin / mice were harvested, and the number of IFN- -producing CD4 Th1 cells was determined by ELISPOT assay. Compared to low-doseinfected 2 m / mice, both perforin / and WT mice infected with the same dose of IOE had significantly lower frequencies of IFN- -producing CD4 Th1 cells (data not shown). Moreover, IOE-infected perforin / and WT mice had a substantially elevated serum level of IL-10, which was significantly (P 0.004) higher than that detected in 2 m / mice (Fig. 9D). These data suggest that IL-10 overproduction and decreased CD4 Th1 cells associated with Ehrlichia-induced toxic shock are perforin independent. DISCUSSION This study was undertaken to examine the relative contributions of different T-cell subsets, particularly CD8 T cells, in a model of Ehrlichia-induced toxic shock. Our study revealed a novel role for CD8 T cells as mediators of fatal disease following infection with intracellular bacteria. The absence of CD8 T cells in 2 m / mice resulted in a significant reduction in liver injury, decreased bacterial burden, restoration of the splenic CD4 T-cell numbers, increased frequency of Ehrlichia-specific CD4 Th1 cells, and abrogation of the systemic and local overproduction of TNF- and IL-10 in comparison to lethally infected WT mice. Although fatal disease is not dependent solely on perforin, our data reveal a critical role of perforin in controlling bacterial replication and development of severe liver injury. Furthermore, we show here that the decreased total number of spleen CD4 T cells in lethally infected WT mice is due to apoptotic cell death rather than migration to peripheral sites of infection. Based on our findings here, we hypothesize that LPS-negative Ehrlichia strains induce toxic shock by the uncontrolled activation of pathogenic CD8 T cells, which mediate the apoptotic death of infected and uninfected host cells including CD4 T cells. These findings offer insight into human and canine ehrlichioses. Patients with severe HME manifested as toxic shock have elevated hepatic enzymes, thrombocytopenia, and leukopenia with lymphopenia (2, 37, 52). Similarly, marked lymphopenia and thrombocytopenia are found in

10 VOL. 75, 2007 ROLE OF CD8 T CELLS IN EHRLICHIA-INDUCED TOXIC SHOCK 4617 FIG. 9. CD8 T-cell-mediated toxic shock and IL-10 production are perforin independent, while TNF- production is perforin dependent. Percent survival of WT and perforin / mice following challenge with a low dose of IOE is shown in A. The data shown represent one of three independent experiments, each with four mice per group. Bacterial burdens in different organs of IOE-infected perforin / mice on day 8 postinfection showed a significantly higher number of ehrlichiae in the liver (, P 0.021) and lung (F, P 0.034) and lower number in the spleen (}, P 0.05) of perforin / mice than in WT mice (B). The data show the mean bacterial burdens and standard deviations for four mice per group and represent one of three independent experiments. Liver sections from perforin / mice on day 12 postinfection show moderate liver pathology (arrow) characterized by only focal areas of apoptosis and necrosis (C). (D) Serum levels of IL-10, as determined by ELISA, in WT, 2 m /, and perforin / mice on day 10 postinfection. Compared to 2 m / mice, the serum level of IL-10 was significantly higher in perforin / and WT mice ( and }, P 0.02 and 0.023, respectively). Data represent the averages and standard deviations of three independent experiments, each with three mice per group. canine granulocytic anaplasmosis caused by Anaplasma phagocytophilum (21, 40), which is in the same family, the Anaplasmataceae, as Ehrlichia. Interestingly, an inverted CD4 -to- CD8 T-cell ratio was described previously for a case of canine ehrlichiosis (23) caused by Ehrlichia canis, which is closely related to E. chaffeensis. Those studies, together with our current data, suggest that an immune overactivation mechanism is the cause of severe disease, similar to what occurs with some viral infections. In a lymphocytic choriomeningitis virus (LCMV) model, LCMV-specific CD8 T cells are strongly activated by proinflammatory stimuli, leading to a loss of CD4 T-cell and B-cell populations, immunopathology, weight loss, and death (4, 12, 39). In simian immunodeficiency virusinfected rhesus monkeys, Mycobacterium bovis bacillus Calmette-Guérin (BCG) coinfection enhances viral pathogenicity and accelerates simian immunodeficiency virus-induced disease (45). In that model, BCG coinfection not only enhances the decline of CD4 T-cell counts in the peripheral blood but also increases viral replication, both of which correlate with the T-cell activation-related shock that develops in these animals (45). Our previous kinetic analysis showed a concomitant association between the expansion of CD8 T cells producing TNF- and IFN- and the substantial decrease in the number of IFN- -producing CD4 T cells in the spleens of WT mice infected with a lethal dose of IOE (26). The current study revealed that the decrease in CD4 T-cell numbers was not due to the migration of CD4 T cells to peripheral sites of infection such as the liver but rather was due to the disproportional apoptosis of this population following lethal IOE infection. This conclusion was supported by data from experiments comparing lethal disease caused by low-dose IOE infection to nonlethal disease caused by E. muris infection, which includes the presence of (i) a significantly higher percentage and absolute number of apoptotic splenic CD4 T cells in IOE-infected mice than in E. muris-infected mice (Fig. 5A and C) and (ii) significantly lower numbers of CD4 T cells in the livers of IOE-infected mice than in E. muris-infected animals (Fig. 5D).

11 4618 ISMAIL ET AL. INFECT. IMMUN. Our previous kinetic analysis (26) excludes the possibility that decreased levels of CD4 T cells in the liver of IOE-infected mice are due to a defective induction of CD4 Th1 cells in IOE-infected mice. In that study (26), we showed that mice infected with a lethal low dose of IOE had significant expansion of CD4 Th1 cells on day 7 postinfection followed by a dramatic decrease in the total number of CD4 T cells and the frequency of CD4 Th1 cells in the spleen on day 14 postinfection (i.e., 1 to 2 days before mice succumbed to infection). The decline in total CD4 T cells and decreased CD4 Th1 response corresponded to a concomitant substantial expansion of TNF- -producing CD8 T cells in the spleen. These data, together with the greater frequency of antigen-specific CD4 Th1 cells in 2 m / mice than in WT mice, indicate that CD8 T cells mediate not only severe liver pathology in fatal ehrlichiosis but also end-stage CD4 T-cell apoptosis. Although our study does not indicate whether apoptotic CD4 T cells are Ehrlichia specific or include bystander T cells, the absence of evidence of nonspecific immune suppression in patients with HME or in our animal model of fatal ehrlichiosis (data not shown) suggests that apoptotic mechanisms involve antigenspecific CD4 T cells. In support of this conclusion, previous studies demonstrated that differentiated antigen-specific CD4 Th1 cells upregulate TNFR and Fas/FasL on their surfaces, thus becoming more susceptible to activation-induced cell death (5, 42, 59) than CD4 Th2 cells. In regard to CD8 T-cell migration to the peripheral site of infection, our data show that the absolute numbers of CD8 T cells in the spleens and livers of IOE-infected mice were comparable to those present in E. muris-infected mice (Fig. 5C and D). However, our current and previous studies (16) reveal a differential role for CD8 T cells in mild (E. muris infection) and fatal (IOE infection) murine ehrlichiosis, where they play protective and pathogenic roles, respectively. The protective role of CD8 T cells is supported by our previous findings that MHC class I-deficient mice lacking CD8 T cells and mice depleted of CD8 T cells are highly susceptible to E. muris infection (16). The mechanisms by which CD8 T cells provide protection in mild ehrlichiosis is dependent on IFN- production and cytotoxic functions (16). Our data also suggest that CD4 T cells are critical for protective immunity against virulent Ehrlichia strains. MHC class II / mice develop an overwhelming infection and succumb to IOE (Fig. 1 and 2). Although IFN- production by antigen-stimulated splenocytes, most likely by CD8 T cells, was detected in CD4 T-cell-deficient mice, the magnitude of the type 1 response in these mice was insufficient to contain replication of the highly virulent ehrlichial strain IOE (Fig. 2 and 6B). These data confirm previous studies showing that Ehrlichia-specific CD4 Th1 cells are crucial for the elimination of Ehrlichia-infected cells via IFN- production and macrophage activation (9, 10, 16, 17, 18, 26, 54). It has also been shown that CD4 T cells mediate the production of Ehrlichiaspecific Th1 isotype (IgG2a) antibodies, which are able to reduce ehrlichial replication by bacterial opsonization and phagocytosis (26, 28, 54, 55). Whether antibodies are essential for protection against rapidly progressive fatal ehrlichiosis is not yet clear. Our data suggest that CD8 T-cell-mediated overproduction of IL-10 plays a pivotal role in the pathogenesis of Ehrlichia-induced toxic shock. IL-10 production was elevated in perforin / mice and WT mice that succumbed to lethal disease (Fig. 9D). In addition, resistant CD8 T-cell-deficient mice had significantly lower levels of IL-10 in their sera and spleens (Fig. 7A and B), which suggests that pathogenic CD8 T cells mediate IL-10 overproduction. Our previous kinetic analysis showed that a late burst of IL-10 is usually preceded by very high levels of TNF- in serum and spleen of WT mice infected with a lethal high dose of IOE (25, 26). As we demonstrated previously, CD8 T cells are responsible for TNF- overproduction, while IL-10 was produced by an undetermined subset of T cells (25, 26). Although the mechanism by which IL-10 causes fatal disease is not completely understood, our data suggest that IL-10 is responsible for the marked decrease in the total number of splenic CD4 and, to some extent, CD8 T cells in Ehrlichia-induced toxic shock. In vivo neutralization of IL-10 in IOE-infected mice restored the percentages of splenic CD4 and CD8 T cells (Fig. 8). Since decreased levels of splenic T cells in IOE-infected mice were associated with significant percentages of apoptotic CD4 T cells (Fig. 5A to C), it is possible that IL-10 could function as a proapoptotic/ proinflammatory cytokine that mediates the apoptosis of CD4 T cells and/or infected target cells. In support of this concept, recent studies revealed novel proinflammatory and proapoptotic functions of IL-10 under certain pathological conditions. In those studies, IL-10 mediates apoptotic cell death via the upregulation of FasL and TNFR on host cells (3, 20, 32, 44). Our finding that IL-10 overproduction is detrimental in Ehrlichia-induced toxic shock has an important clinical application, as it can explain the failure of TNF- inhibitors to improve disease outcome in patients with septic shock. In addition, IL-10 overproduction can explain our previous results showing that TNF- neutralization is unable to protect IOEinfected mice against a lethal outcome (25). Therefore, the development of a method to directly suppress CD8 T cells may prove to more effectively rebalance the dysregulated immune response in patients with fatal ehrlichiosis due to toxic shock. Finally, our data show that perforin / mice are equally susceptible to lethal ehrlichiosis as WT mice (Fig. 9A). Although we have not measured the frequencies of TNF- - and IFN- -producing CD8 T cells in perforin / mice, the decline in the CD4 Th1 response, as well as the elevation in IL-10 levels (Fig. 9D), suggests that these mice succumb to the same dysregulated immune response as do WT mice. However, these results do not exclude the role of perforin in the immunopathogenesis of CD8 T-cell-mediated toxic shock, as perforin was critical for controlling bacterial replication (Fig. 9B) and mediating liver pathology (Fig. 9C). One possible mechanism that explains the susceptibility of perforin / mice to fatal disease is that perforin / mice fail to eliminate antigenloaded dendritic cells or infected macrophages that further induce the activation and proliferation and of pathogenic CD8 T cells. The escape of infected target cells from perforin-mediated lysis could explain the higher bacterial burden observed in these mice (Fig. 9B). In addition, previous reports demonstrated that perforin / mice generate exaggerated T- cell responses to LCMV and Listeria and during graft-versushost disease (1, 31, 57), suggesting that there is a failure to eliminate antigen-presenting cells, which stimulate pathogenic T cells, or that T-cell elimination is impaired. The other pos-