INFECTION AND IMMUNITY, Sept. 2007, p. 4608–4620 Vol. 75, No. 90019-9567/07/$08.00�0 doi:10.1128/IAI.00198-07Copyright © 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. Walker1,2,3

Department of Pathology,1 Department of Microbiology and Immunology,4 Sealy Center for Vaccine Development,2 and Center forBiodefense and Emerging Infectious Diseases,3 University of Texas Medical Branch, Galveston, Texas 77555-0609

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 syndromecharacterized by a decreased number of Ehrlichia-specific CD4� Th1 cells, the expansion of tumor necrosisfactor alpha (TNF-�)-producing CD8� T cells, and the systemic overproduction of interleukin-10 (IL-10) andTNF-�. Here, we investigated the role of CD4� and CD8� T cells in immunity to Ehrlichia and the pathogenesisof fatal ehrlichiosis caused by infection with low- and high-dose (103 and 105 bacterial genomes/mouse,respectively) ehrlichial inocula. The CD4� T-cell-deficient mice showed exacerbated susceptibility to a lethalhigh- 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. Theabsence 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 incomparison to infected WT mice. Although fatal disease is perforin independent, our data suggested thatperforin 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 serumconcentration 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 Ehrlichia-induced toxic shock, which is associated with IL-10 overproduction and CD4� T-cell apoptosis.

Human monocytotropic ehrlichiosis (HME) resemblestoxic shock syndrome and is caused by Ehrlichia chaffeensis,a gram-negative, obligately intracellular bacterium thatlacks lipopolysaccharide (LPS) and peptidoglycan (30, 37,49, 52). In mice, inoculation with a high dose of IOE (amonocytotropic Ehrlichia species isolated from Ixodes ova-tus ticks in Japan) (46) results in a fatal syndrome thatmimics severe HME (26, 48). IOE is genetically and anti-genically closely related to E. chaffeensis and Ehrlichiamuris, both of which cause only mild self-limited disease inmice (16, 17, 26, 50, 58). Severe and fatal infection of im-munocompetent mice with IOE, however, causes severe tis-sue damage to multiple organ systems in a setting of a lowbacterial burden (26). This observation mirrors what occursin immunocompetent patients who develop multisystem or-gan failure without an overwhelming infection (49). Ourprevious studies demonstrated that resistance to fatal dis-ease is mediated by gamma interferon (IFN-�) productionand CD4� Th1 cells, while tumor necrosis factor alpha(TNF-�) production from antigen-specific CD8� T cells iskey to the development of fatal ehrlichiosis (26). We con-firmed TNF-�’s pathogenic role using TNF receptor(TNFR) p55 and p75 double knockout mice. TNFR p55binds to soluble TNF-� and is critical for controlling intra-

cellular bacterial replication, while TNFR p75 interacts withtrans-membrane TNF-� to mediate apoptotic or necrotichost cell death. The absence of both TNF receptors in dou-ble knockout mice abrogates severe liver pathology anddelays mortality following infection with a high dose of IOEcompared to wild-type (WT) mice (25). We also showed thatinterleukin-10 (IL-10) production is associated with fataldisease (25). Thus, both pro- and anti-inflammatory cyto-kines are implicated in Ehrlichia-induced toxic shock.

In contrast to the well-known CD4� T-cell functions againstintracellular bacteria, particularly those organisms that residein endocytic compartments and are inaccessible to the endog-enous pathway of major histocompatibility complex (MHC)class I antigen presentation, the role of CD8� T cells is lessclearly defined. CD8� effector T cells are key mediators in theimmune response to several intracellular pathogens such asviruses (51) and intracytosolic bacteria including Listeriamonocytogenes (19, 27, 33) and Rickettsia (15, 53). CD8� Tcells appear to exert their function through two mechanisms.First, CD8� effector cells may act as cytotoxic lymphocytes tokill 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 arepotent producers of cytokines, particularly IFN-� and TNF-�,both of which play a critical role in limiting pathogen replica-tion (22, 56), while excess production induces immunopathol-ogy (13, 24, 35).

The present study assessed the relative contributions ofdifferent T-cell subsets to Ehrlichia-induced toxic shock. Inparticular, we focus here on CD8� T cells, soluble factors TNF-�

* Corresponding author. Mailing address: Department of Pathology,Center for Biodefense and Emerging Infectious Diseases, 301 Univer-sity Blvd., Galveston, TX 77555-0609. Phone: (409) 772-3111. Fax:(409) 772-5683. E-mail: naismail@utmb.edu.

� Published ahead of print on 11 June 2007.

4608

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Oct

ober

202

1 by

82.

102.

110.

121.

and IL-10, and perforin as critical components of cytotoxiclymphocyte-dependent cytotoxicity, a function that could beresponsible for either pathogen clearance or immunopathol-ogy. We describe a novel role of CD8� T cells as pathogenicmediators of Ehrlichia-induced toxic shock. We used a mousemodel of fatal ehrlichiosis to demonstrate that TNF-� andIL-10 overproduction, CD4� T-cell apoptosis, and the down-regulation of the Ehrlichia-specific CD4� Th1 response areassociated with mortality. Furthermore, we showed thatTNF-� production in Ehrlichia-induced toxic shock is perforindependent, while IL-10 production is perforin independent.

MATERIALS AND METHODS

Mice. The following gene-targeted strains were used: �2-microglobulin (�2m)-deficient (�2m�/�) strain B6.129P2-�2mtm1Unc/J, transporter associated with an-tigen processing (TAP)-deficient strain B6.129S2-Tap1tm1Arp/J, MHC class II-deficient (MHC class II�/�) strain B6.129S-H2dlAb1-Ea/J, and perforin-deficient(perforin�/�) strain C57BL/6-Prf1tm1Sdz/J. All mice were purchased from Jack-son Laboratories (Bar Harbor, ME). C57BL/6J mice were used as the controlbackground strain in all experiments involving knockout mice. Mice were gendermatched for each experiment and were 6 to 12 weeks old. Mouse handling andexperimental procedures were conducted in accordance with the University ofTexas Medical Branch Institutional Committee for Animal Care and Use.

Bacterial stocks and experimental design. Two monocytotropic ehrlichialstrains were used in this study, a highly virulent Ehrlichia sp. strain (designatedIOE) isolated from Ixodes ovatus ticks (a gift from M. Kawahara, Nagoya CityPublic Health Research Institute, Nagoya, Japan) and a mildly virulent E. murisstrain (provided by Y. Rikihisa, Ohio State University, Columbus, OH). IOE andE. muris stocks were produced as described previously (25, 26). Mice wereinfected intraperitoneally (i.p.) with 1 ml of a low dose (103 bacterial genomes/mouse) or a high dose (105 bacterial genomes/mouse) of fresh inoculum asdetermined by quantitative real-time PCR. For E. muris infection, mice wereinfected i.p. with a high (nonlethal) dose of E. muris (108 bacterial genomes/mouse). Control mice were given 1 ml of a 10�1 or 10�2 dilution of a spleenhomogenate from uninfected C57BL/6 mice. Mice were sacrificed on the indi-cated days postinfection, and selected organs were harvested for histology, im-munohistochemistry, cell culture, and determination of bacterial load by real-time PCR.

Preparation of host cell-free Ehrlichia. For preparation of host cell-free IOEantigen, IOE-infected spleens and livers were harvested from WT mice on day 7postinfection, and antigen was prepared as previously described (25, 26). Mockantigen was prepared from spleens and livers of uninfected WT mice for allexperiments. For the preparation of E. muris antigen, E. muris was cultivated inP388D1 cells grown in 5% bovine calf serum-supplemented modified Eagle’smedium 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 ly-sates served as a mock antigen for E. muris. The total protein concentration wasdetermined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL),and the ehrlichial genome copy number was measured by quantitative real-time PCR.

Histology and immunohistochemistry. Samples of liver, spleen, lung, andkidney were processed for histopathological examination as described previously(38). For immunohistochemistry, slides were incubated for 45 min at 37°C witha 1:10,000 dilution of rabbit anti-E. chaffeensis polyclonal antibody, which cross-reacts with E. muris and IOE (26, 48). Slides were then incubated for 30 min withbiotinylated goat anti-rabbit immunoglobulin G (IgG) (Vector Laboratories,Burlingame, CA). The slides were then washed and incubated with avidin-horseradish peroxidase conjugate for 20 min at 37°C, followed by incubation withsubstrate containing 3-amino-9-ethylcarbazole for 8 min at 37°C (Vector Labo-ratories). For control slides, normal canine serum was used in lieu of a primaryantibody to rule out nonspecific staining. Grading of liver lesions was performedas described previously (38). The number of foci of apoptotic/necrotic hepato-cytes and degree of inflammatory infiltrates in the liver were used for grading.Each parameter was scored as described previously (38). A total score wasobtained from data for each animal by adding all parameters and obtaining themean.

ELISPOT assays for antigen-specific cytokine-producing T cells. CD4� T cellswere isolated from splenic homogenates by negative selection using mouse CD4subset enrichment columns (R&D Systems, Minneapolis, MN), and the purityranged from 80 to 90% as determined by flow cytometry. Cytokine production

from splenocytes and enriched CD4� cells was assessed by an enzyme-linkedimmunospot (ELISPOT) assay as described previously (26, 41). Briefly, spleno-cytes were assayed at two dilutions, starting from 106 to 2 � 105 cells/well. Anadditional 1 � 106 naıve splenocytes/well were added to immune splenocytes toensure that the number of antigen-dependent spots was linearly proportional tothe number of immune spleen cells plated. Splenocytes or enriched CD4� cellswere stimulated with 10 �g/well of E. muris, IOE, or mock antigens. Positive andnegative controls contained 5 �g/ml concanavalin A or medium, respectively.Antigen-specific spots were determined by subtracting the number of spots in themock antigen wells from the number in the E. muris or IOE antigen-stimulatedwells.

Cytokine ELISA. Splenocytes were cultured at 5 � 106 cells/ml in Dulbecco’smodified 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 pres-ence or the absence of 50 �g/ml of E. muris, IOE, or mock antigen. Supernatantswere collected at 48 h and assayed by enzyme-linked immunosorbent assay(ELISA) (Quantikine; R&D Systems) for IL-10 and TNF-� according to themanufacturer’s instructions. The detection limits were 150 pg/ml for IL-10 and 5pg/ml for TNF-�. In some experiments, sera from infected mice were collectedand assayed for IL-10 and TNF-�.

Ehrlichial load determination by quantitative real-time PCR. The copy num-ber of ehrlichiae in the inoculum and the bacterial burden in different organswere determined by quantitative real-time PCR as described previously (25, 26).

Flow cytometry. Splenocytes were harvested, counted, and resuspended influorescence-activated cell sorter (FACS) buffer (Dulbecco’s phosphate-bufferedsaline 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-wellV-bottom culture plate (Costar, Corning, NY) at a concentration of 106 cells perwell. Fc�III/II receptor-blocking was followed by fluorescein isothiocyanate-conjugated anti-CD4 and phycoerythrin-conjugated anti-CD8� (BD Pharmin-gen). Isotype-matched monoclonal antibodies were used as controls. All incuba-tions were done for 15 min at 4°C followed by two consecutive washes in FACSbuffer. Apoptotic CD4� and CD8� lymphocytes were identified using annexinV-fluorescein isothiocyanate and phycoerythrin-conjugated anti-CD4 (L3T4,clone GK1.5) or anti-CD8 (Ly-2, clone 53-6.7) (BD Pharmingen) according tothe manufacturer’s instructions. Cells were run within 1 h on a FACSCalibur flowcytometer (BD Immunocytometry Systems, San Jose, CA) and analyzed withCell Quest (Immunocytometry Systems) or FlowJo (Tree Star Inc., Ashland,OR) software. Lymphocytes were gated based on forward and side scatter, andspecific staining was determined using isotype controls. The absolute number ofapoptotic CD4� and CD8� T cells was determined by multiplying the percentageof 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 anti-bodies and/or neutralizing rat anti-mouse TNF-� IgG1 monoclonal antibodies orequivalent 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 ofIOE. As a negative control, a group of mice was injected with phosphate-bufferedsaline.

Preparation of liver mononuclear cells. Liver mononuclear cells were isolatedas previously described (34) using the modified enzymatic dispersal protocol.Cells were centrifuged at low speed, 36 � g, for 1 min at room temperature toremove most of the hepatocytes, and the recovered mononuclear cells wereassayed by flow cytometry.

Statistical analysis. Student’s two-tailed t test was used when data from twogroups were compared. Comparisons across three or more groups were statisti-cally evaluated using analysis of variance. The Bonferroni method was used toadjust for multiple comparisons. A P value of �0.05 was regarded as beingsignificant, and a P value of �0.01 was considered to be highly significant.

RESULTS

�2m-deficient mice are highly resistant to IOE challenge. Toassess the roles of CD4� and CD8� T cells in the immuneresponse and pathogenesis of fatal monocytotropic ehrlichio-sis, we used MHC class II�/� mice that lack �� CD4� T cellsand �2m�/� mice that lack �� CD8� T cells. All mice wereinoculated i.p. with a high (105 organisms) or low (103 organ-isms) dose of IOE and scored for illness daily after infectionfor 30 days. Similar to our previous report (7), WT mice suc-

VOL. 75, 2007 ROLE OF CD8� T CELLS IN EHRLICHIA-INDUCED TOXIC SHOCK 4609

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Oct

ober

202

1 by

82.

102.

110.

121.

cumbed to a high dose of IOE on days 8 to 12, with a mediansurvival time (MST) of 9 days (Fig. 1A), while a low-doseinfection prolonged survival to days 14 to 17 postinfection(MST, 15 days) (Fig. 1B). MHC class II�/� mice were moresusceptible to lethal IOE infection than WT mice, succumbingon days 7 to 9 or days 11 to 15 to high- or low-dose IOEinfection, respectively (Fig. 1A and B) (MST, 7 and 14 days forhigh- and low-dose IOE infection, respectively). Interestingly,�2m�/� mice were resistant to a low dose of IOE (90% sur-vival) (Fig. 1B), although all succumbed to a high dose (Fig.1A). �2m�/� mice exhibited only mild signs of illness (ruffledfur, inactivity, and/or gaunt posture) and maintained normalweight and temperature (data not shown). We confirmed thedeficiency of CD8� T cells in spleens of �2m�/� mice after i.p.

infection with IOE: 9.7% 0.7% (mean standard error ofthe mean) of splenocytes from infected WT mice were CD8�,whereas only 1.5% 0.1% of splenocytes were CD8� in in-fected �2m�/� mice. Similar differences in percentages ofCD8� T cells were observed in uninfected mice (data notshown). These data show that CD8� T cells contribute signif-icantly to mortality from Ehrlichia-induced toxic shock.

Although it is our hypothesis that �2m�/� mice are resistantto IOE due to a lack of MHC class I-restricted CD8� T cells,NKT cells may also be implicated. This is because �2m is acomponent of a number of other antigen-presenting mole-cules, including CD1d, which selects NK1.1� CD3� (NKT)cells. However, MHC class I molecules are loaded with peptideantigens in a TAP-dependent fashion (8, 36, 47), whereas CD1molecules, including CD1d, bind lipid-based antigens indepen-dent of TAP (7, 11, 14, 47). We therefore compared TAP-deficient (TAP�/�) mice to �2m�/� mice to determine if NKTcells contribute to the IOE-resistant �2m�/� phenotype. TheTAP1 gene disruption causes a profound CD8� T-cell defi-ciency without affecting NKT cells, a phenotype that we con-firmed 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 infectedTAP�/� mice. Interestingly, TAP�/� mice also had highersurvival rates following infection with a low, but not a high,dose of IOE (70% survival up to day 30 postinfection, the timepoint at which all experiments were terminated) than did WTmice (0% [WT mice succumbed to infection on days 15 to 18postinfection, with an MST of 16 days]) (Fig. 1C). These datasuggest that a CD8� T-cell deficiency significantly contributesto host resistance against Ehrlichia, although the survival dif-ference between TAP�/� and �2m�/� mice indicates that NKTcells may also be involved.

CD8� T cells are not critical for controlling ehrlichial rep-lication in WT mice. We have previously shown that IOE-infected WT mice develop severe liver injury in the absence ofan overwhelming infection (25, 26). Since CD8� T cells arecritical for protection against many intracellular pathogens, weexamined the role of CD8� T cells in controlling the ehrlichialburden and systemic dissemination. We infected WT, �2m�/�,TAP�/�, and MHC class II�/� mice i.p. with a low dose of IOEand compared the bacterial burden in different organs on day8 postinfection. Ehrlichial burdens in the lungs and spleens ofinfected �2m�/� and TAP�/� mice were significantly lower(P 0.001 and 0.012, respectively) than those of infected WTand MHC class II�/� mice (Fig. 2). MHC class II�/� mice hadsubstantially higher bacterial burdens (P 0.002) in all organsthan did WT mice, suggesting that an overwhelming infectionoccurs in the absence of CD4� T cells. Interestingly, the ab-sence of CD8� T cells did not significantly (P 0.467) influ-ence the bacterial burden in the liver, a major site of ehrlichialtropism. These data suggest that CD4� T cells, but not CD8�

T cells, contribute to the control of Ehrlichia replication anddissemination.

CD8� T cells mediate severe immunopathology in fatal ehr-lichiosis. To examine the extent to which CD8� T cells medi-ate tissue damage, we examined the pathology in differentorgans of WT, �2m�/�, TAP�/�, and MHC class II�/� mice ondays 12 and 14 following i.p. infection with a lethal low dose ofIOE. WT and MHC class II�/� mice developed severe liver

FIG. 1. Resistance of �2m�/� and TAP�/� mice to a lethal chal-lenge with a virulent ehrlichial strain (IOE). WT, MHC class II�/�,and �2m�/� mice were challenged i.p. with either high (1 � 105) orlow (1 � 103) doses of IOE. All mice were monitored for 30 days,and the survival rate was determined following lethal infection witha 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 shownrepresent one of three independent experiments with a total of 18mice/group.

4610 ISMAIL ET AL. INFECT. IMMUN.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Oct

ober

202

1 by

82.

102.

110.

121.

injury marked by extensive apoptosis and necrosis and minimalinflammatory cell infiltration on day 12 postinfection (Fig. 3Aand B). Although both WT and MHC class II�/� mice hadsevere liver injury, the apoptotic and necrotic events weremore focal in WT mice than in MHC class II�/� mice, whichhad extensive, widespread liver damage. The latter could bedue to the presence of an overwhelming infection in the ab-sence of CD4� T cells. In contrast to WT and MHC class II�/�

mice, �2m�/� and TAP�/� mice developed mild liver pathol-ogy characterized by marked cellular infiltration and few fociof hepatic apoptosis on day 12 (Fig. 3C and D) and day 14(data not shown). These data suggest that CD8� T cells di-rectly or indirectly mediate severe liver damage, a character-istic feature of Ehrlichia-induced toxic shock.

Lymphoid tissue cellularity is preserved in the absence ofCD8� T cells. We then examined the gross and histologicalchanges that CD8� T cells exert on the lymphoid organs dur-ing IOE infection. The spleens of WT mice infected with a lowdose of IOE were small and pale on day 12 postinfection.However, the spleens of the �2m�/� mice infected with thesame dose of IOE became large and deep red, similar to thespleens of WT mice responding to low-virulence E. muris (25,26). Histological examination revealed massive tissue destruc-tion in the spleens (Fig. 4A) and lungs (data not shown) of WTand MHC class II�/� mice (data not shown). Numerous apop-totic and necrotic foci markedly disrupted the splenic follicularstructure. In contrast, �2m�/� mice maintained the integrity ofthe splenic structure (Fig. 4B) and had large splenic lympho-cyte populations (Table 1). Infected WT mice had approxi-mately one-half the total number of CD4� and CD8� T cellsof uninfected WT mice. In contrast, E. muris-infected WTmice, a model of mild HME, exhibited 1.4- to 2.0-fold-higher

numbers of total splenocytes, CD4� T cells, and CD8� T cellsthan uninfected WT mice. �2m�/� mice responded to IOEinfection by an expansion of total splenocytes and CD4� Tcells (Table 1). These data suggest that Ehrlichia-inducedCD8� T cells mediate a reduction in splenic cellularity, par-ticularly in the CD4� and CD8� T-cell populations, shortlybefore the death of the host.

CD4� T-cell apoptosis is significantly higher in IOE-in-fected mice than in E. muris-infected mice. Analysis of the totalnumbers of CD4� and CD8� T cells in the spleens of WT miceinfected with a low dose of IOE revealed a marked decrease inthe percentage and absolute number of both CD4� and CD8�

T cells in the spleen shortly before death (Table 1), similar towhat was observed previously with a high dose of IOE (26).Because the loss of splenic cellularity may have been due toeither T-cell apoptosis or the migration of T cells to peripheralorgans, we examined both possibilities. First, we compared thepercentage of apoptotic splenic CD4� and CD8� T cells be-tween IOE-infected and E. muris-infected WT mice. IOE-infected mice had a significantly (P 0.003) higher percentage(Fig. 5A) and absolute number (Fig. 5C) of apoptotic CD4� Tcells than did E. muris-infected mice Second, we examinedT-cell migration by comparing the numbers of CD4� andCD8� T cells in the livers of IOE- and E. muris-infected WTmice. Both infections resulted in increases in the total liverCD4� and CD8� T-cell populations compared to those inuninfected mice (Fig. 5D). However, E. muris-infected micehad significantly higher numbers of liver CD4� T cells (P 0.002) than did IOE-infected mice. There were no significantdifferences in the numbers of liver CD8� T cells between thetwo infected groups (Fig. 5C), indicating that CD8� T cells inboth models are equally capable of migration. The low number

FIG. 2. �2m�/� and TAP�/� mice have lower bacterial burdens than WT and MHC class II�/� mice. Organs were harvested from WT, �2m�/�,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-timePCR amplification of the dsb gene and normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). MHC class II�/� mice developed anoverwhelming infection compared to other groups. Data represent the averages and standard deviations of triplicate amplifications with three miceper group. Similar results were observed in three independent experiments. Ehrlichial burden in the lungs and spleens of �2m�/� and TAP�/� micewas significantly lower than that detected in WT mice (P 0.001 and 0.012, respectively). MHC class II�/� mice had a significantly higher bacterialburden in all examined organs than did WT, �2m�/�, and TAP�/� mice (P 0.002).

VOL. 75, 2007 ROLE OF CD8� T CELLS IN EHRLICHIA-INDUCED TOXIC SHOCK 4611

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Oct

ober

202

1 by

82.

102.

110.

121.

of CD4� T cells in the livers of IOE-infected mice resulted ina ratio of CD4� to CD8� T cells of 1:1, compared to 4:1 in E.muris-infected mice. Our data indicate that the decreasednumber of splenic CD4� T cells in IOE-infected WT mice,which coincides with the development of toxic shock, is notdue to the migration of effector Ehrlichia-specific CD4� Tcells to the liver but rather is due to apoptotic CD4� T-celldeath.

CD8 � T cells are responsible for the decreased frequency ofIFN-�-producing CD4� Th1 cells following lethal ehrlichialinfection. Next, we tested the possibility that CD8� T cells couldmediate fatal immunopathology by promoting the apop-tosis of protective CD4� Th1 cells (25, 26). Compared toIOE-infected WT mice, �2m�/� and TAP�/� mice had sig-nificantly (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 ofIOE. The levels of CD4� Th1 cells in the infected �2m�/�

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 classII�/� mice lack CD4� T cells, IFN-�-producing T cells arelikely to be CD8� T cells or NKT cells. However, the factthat the IFN-� production is antigen specific makes CD8� Tcells the more likely candidate. The latter finding suggeststhat the induction of IOE-specific CD8� T-cell responses isCD4� T-cell independent. More importantly, our resultsdemonstrate that CD8� T cells mediate the downregulationof protective CD4� Th1 cells in IOE-infected WT mice, ahallmark of the defective immune response in fatal murineehrlichiosis (26).

FIG. 3. CD8� T cells mediate extensive liver pathology following lethal ehrlichial infection. WT, �2m�/�, TAP�/�, and MHC class II�/� micewere infected with a low dose of IOE, and the livers were harvested on day 12 postinfection for paraffin embedding followed by hematoxylin andeosin staining. WT mice (A) and MHC class II�/� mice (B) developed extensive tissue damage (arrows). In contrast, �2m�/� (C) and TAP�/� mice(D) developed mild hepatic pathology characterized by few apoptotic foci (arrows) and marked cellular infiltration (arrowhead). Data arerepresentative of one experiment with three mice per group. Similar results were observed in three independent experiments.

4612 ISMAIL ET AL. INFECT. IMMUN.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Oct

ober

202

1 by

82.

102.

110.

121.

Antigen-stimulated splenocytes from �2m�/� mice producesignificantly less TNF-� and IL-10 than those from WT mice.To examine whether CD8� T-cell-mediated toxic shock islinked to dysregulated TNF-� and IL-10 production as sug-gested by previous studies (25, 26), we infected WT and�2m�/� mice with an ordinarily lethal low dose of IOE andcompared the TNF-� and IL-10 concentrations in the serumand in supernatants of cultured splenocytes. Antigen-stimu-lated splenocytes from �2m�/� mice produced significantly(P 0.005) less TNF-� and IL-10 than did those from WTmice on day 8 postinfection (Fig. 7A). The levels of TNF-� andIL-10 produced by IOE-stimulated WT splenocytes rose sig-nificantly (P 0.007) between day 8 and day 12 postinfection(Fig. 7A), a finding that correlated with the in vivo productionof high serum levels of TNF-� and IL-10 (Fig. 7B). In contrast,

on days 8 and 12 postinfection, �2m�/� mice produced signif-icantly (P 0.005) lower levels of TNF-� and IL-10 in vivo(serum) and in vitro by immune splenocytes stimulated withIOE antigen (Fig. 7A and B). These data suggest that CD8� Tcells mediate the dysregulated Ehrlichia-specific overproduc-tion of TNF-� and IL-10 in fatal murine ehrlichiosis that re-sembles toxic shock syndrome.

In vivo neutralization of IL-10 increased the numbers ofCD4� and CD8� T cells in IOE-infected WT mice. Our pre-vious data showed that lethal murine ehrlichiosis that resem-bles toxic shock is associated with the presence of high levels ofIL-10 throughout the course of disease (25). To explore therelative contributions of IL-10 to disease pathogenesis, wedepleted WT mice of IL-10 and infected them with a lethal lowdose of IOE. The percentages of splenic CD4� and CD8� Tcells were determined by flow cytometry on day 12 postinfec-tion, 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 ofWT mice treated with isotype control antibody with a lethaldose of IOE decreased the percentages (Fig. 8) of CD4� andCD8� T cells. Strikingly, the depletion of IL-10 resulted in asignificant expansion of CD4� and CD8� T cells in WT micefollowing infection with a lethal low dose of IOE (Fig. 8). Thisexpansion was associated with marked splenomegaly and anincrease in total viable splenocytes as determined by trypanblue staining (data not shown), suggesting that IL-10 depletionincreases splenic cellularity. These data demonstrate thatIL-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 theobserved tissue damage through a perforin-mediated cytotoxicmechanism. To test this hypothesis, we infected WT, �2m�/�,and perforin�/� mice with a low dose of IOE and monitoredsurvival. Both wild-type and perforin�/� C57BL/6 mice inoc-ulated with a low dose of IOE failed to control disease pro-gression, and both types of mice succumbed to infection (Fig.9A). However, perforin�/� mice had a significantly higher bac-terial burden in the livers (P 0.021) and lungs (P 0.034),but not in the spleens, than wild-type mice (Fig. 9B). Com-pared to infected WT mice that developed extensive partiallyconfluent foci of apoptosis or necrosis of contiguous hepato-cytes, similar to that shown in Fig. 3A, on day 12 postinfection,

FIG. 4. CD8� T cells mediate splenocyte apoptosis and destruc-tion of follicular structure following lethal ehrlichial infection. WTand �2m�/� mice were infected with a low dose of IOE, and paraffinsections of splenic tissues collected on day 12 postinfection werestained with hematoxylin and eosin. WT mice (A) developed mas-sive apoptosis in splenic lymphoid follicles and had numerous tin-gible body macrophages (arrows) within poorly distinguishable fol-licles. �2m�/� mice (B) maintained the integrity of the splenicstructure, and large populations of lymphocytes could clearly beidentified within the follicles. Tingible body macrophages were notidentified in these mice. Data are representative of one experimentwith three mice per group. Similar results were observed in twoindependent experiments.

TABLE 1. Changes in total numbers of splenocytes and CD4� andCD8� 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 (106) SD

Splenocytes CD4�

T cellsCD8�

T cells

WT uninfected 97.6 5.6 21.3 3.3 11.3 1.6WT � high dose of

E. muris (sublethal)115.8 17.5 41.4 3.6 23.2 2.9

WT � low dose of IOE 36.0 5.4 11.7 0.4 3.5 0.4�2m�/� uninfected 82 0.2 22.6 4.5 1.5 0.8�2m�/� � low dose of

IOE84 12.3 31.5 4.4 1.3 0.2

VOL. 75, 2007 ROLE OF CD8� T CELLS IN EHRLICHIA-INDUCED TOXIC SHOCK 4613

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Oct

ober

202

1 by

82.

102.

110.

121.

4614

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Oct

ober

202

1 by

82.

102.

110.

121.

perforin�/� mice had mild to moderate liver injury (Fig. 9C)marked by the presence of fewer foci of apoptotic or necrotichepatocytes. 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 datasuggest that CD8 T-cell-mediated toxic shock is a multistepprocess 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-cellpopulation 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). Thepercentages of apoptotic CD4� (A) and CD8� (B) T cells as well as their absolute numbers (C) in the spleens of infected mice and uninfectedcontrols (NEG) on day 12 postinfection were determined by annexin staining as described in Materials and Methods. The absolute numbers ofCD4� 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 uninfectedWT mice (D). These data represent the average of data from three mice per group. Similar results were observed in two independent experiments.

FIG. 6. �2m�/� and TAP�/� mice produce high numbers of antigen-specific, IFN-�-producing CD4� Th1 cells when infected with IOE.Splenocytes from WT, �2m�/�, or TAP�/� mice were harvested on day 12 postinfection with a low dose of IOE or a high dose of E. muris, andCD4� T cells were purified by positive selection. They were then stimulated with IOE or E. muris antigens, respectively, in the presence or absenceof naıve, syngeneic splenocytes (1 � 106 splenocytes/well). The number of antigen-specific, IFN-�-producing CD4� T cells or IL-4-producing CD4�

T cells per 106 cells was determined by ELISPOT assay (A). IOE-infected �2m�/� (F) and TAP�/� (�) mice have significantly higher quantitiesof IFN-�-producing CD4� T cells than do infected WT mice (P 0.003). A similar comparison was made between low-dose IOE-infected WTand 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. Datarepresent the averages and standard deviations of data from four mice per group. Similar results were observed in three independent experiments.

VOL. 75, 2007 ROLE OF CD8� T CELLS IN EHRLICHIA-INDUCED TOXIC SHOCK 4615

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Oct

ober

202

1 by

82.

102.

110.

121.

trolling bacterial infection and mediating tissue injury in fatalehrlichiosis.

Susceptibility of perforin�/� mice is associated with in-creased IL-10 production. To determine whether the suscep-tibility of perforin�/� mice to lethal ehrlichiosis is associatedwith a weak CD4� Th1 response and IL-10 overproductionsimilar to WT mice, we measured the serum level of IL-10 andthe 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, �2m�/�, and perforin�/� mice were har-vested, and the number of IFN-�-producing CD4� Th1 cellswas determined by ELISPOT assay. Compared to low-dose-infected �2m�/� mice, both perforin�/� and WT mice infectedwith the same dose of IOE had significantly lower frequenciesof IFN-�-producing CD4� Th1 cells (data not shown). More-over, IOE-infected perforin�/� and WT mice had a substan-tially elevated serum level of IL-10, which was significantly(P 0.004) higher than that detected in �2m�/� mice (Fig.9D). These data suggest that IL-10 overproduction and de-

creased CD4� Th1 cells associated with Ehrlichia-inducedtoxic shock are perforin independent.

DISCUSSION

This study was undertaken to examine the relative contribu-tions of different T-cell subsets, particularly CD8� T cells, in amodel of Ehrlichia-induced toxic shock. Our study revealed anovel role for CD8� T cells as mediators of fatal diseasefollowing infection with intracellular bacteria. The absence ofCD8� T cells in �2m�/� mice resulted in a significant reduc-tion in liver injury, decreased bacterial burden, restoration ofthe splenic CD4� T-cell numbers, increased frequency of Ehr-lichia-specific CD4� Th1 cells, and abrogation of the systemicand local overproduction of TNF-� and IL-10 in comparison tolethally infected WT mice. Although fatal disease is not de-pendent solely on perforin, our data reveal a critical role ofperforin in controlling bacterial replication and developmentof severe liver injury. Furthermore, we show here that thedecreased total number of spleen CD4� T cells in lethallyinfected WT mice is due to apoptotic cell death rather thanmigration to peripheral sites of infection.

Based on our findings here, we hypothesize that LPS-nega-tive Ehrlichia strains induce toxic shock by the uncontrolledactivation of pathogenic CD8� T cells, which mediate theapoptotic death of infected and uninfected host cells includingCD4� T cells. These findings offer insight into human andcanine ehrlichioses. Patients with severe HME manifested astoxic shock have elevated hepatic enzymes, thrombocytopenia,and leukopenia with lymphopenia (2, 37, 52). Similarly,marked lymphopenia and thrombocytopenia are found in

FIG. 7. CD8� T cells are critical for the local and systemic produc-tion of TNF-� and IL-10 following lethal ehrlichial infection. WT and�2m�/� mice were infected with a low dose of IOE, and the levels ofTNF-� and IL-10 in the serum and supernatants of cultured spleno-cytes were determined by ELISA on days 8 and 12 postinfection.Harvested splenocytes were stimulated in vitro with IOE antigen for48 h. Substantially higher levels of IL-10 and TNF-� were produced byantigen-stimulated splenocytes of WT mice than those of �2m�/� miceon day 8. The levels increased significantly on day 12 postinfection (A).Similar levels were detected in serum (B). No IL-10 or TNF-� wasdetected on day 0 (data not shown). Data represent the averages andstandard deviations of data from four mice per group. Similar resultswere observed in three independent experiments.

FIG. 8. Neutralization of IL-10 restores the total numbers ofsplenic CD4� and CD8� T cells in WT mice during IOE infection. WTmice were treated with anti-IL-10 or isotype control as described inMaterials and Methods. Mice were infected i.p. with a lethal low doseof IOE or a nonlethal high dose of E. muris, and splenocytes werecollected on day 12 postinfection for flow cytometric analysis. Lym-phocytes were gated based on size and granularity. The absolute num-bers of CD4� and CD8� T cells were higher in IL-10-depleted micethan 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-cellnumbers in E. muris-infected mice that develop mild and self-limiteddisease (P 0.556). Data represent the averages and standard devia-tions of data from three mice per group. Similar results were observedin two independent experiments.

4616 ISMAIL ET AL. INFECT. IMMUN.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Oct

ober

202

1 by

82.

102.

110.

121.

canine granulocytic anaplasmosis caused by Anaplasma phago-cytophilum (21, 40), which is in the same family, the Anaplas-mataceae, as Ehrlichia. Interestingly, an inverted CD4�-to-CD8� T-cell ratio was described previously for a case of canineehrlichiosis (23) caused by Ehrlichia canis, which is closelyrelated to E. chaffeensis. Those studies, together with our cur-rent data, suggest that an immune overactivation mechanism isthe cause of severe disease, similar to what occurs with someviral infections. In a lymphocytic choriomeningitis virus(LCMV) model, LCMV-specific CD8� T cells are stronglyactivated by proinflammatory stimuli, leading to a loss ofCD4� T-cell and B-cell populations, immunopathology, weightloss, and death (4, 12, 39). In simian immunodeficiency virus-infected rhesus monkeys, Mycobacterium bovis bacillusCalmette-Guerin (BCG) coinfection enhances viral pathoge-nicity and accelerates simian immunodeficiency virus-induceddisease (45). In that model, BCG coinfection not only en-hances the decline of CD4� T-cell counts in the peripheralblood but also increases viral replication, both of which corre-

late with the T-cell activation-related shock that develops inthese animals (45).

Our previous kinetic analysis showed a concomitant associ-ation between the expansion of CD8� T cells producingTNF-� and IFN-� and the substantial decrease in the numberof IFN-�-producing CD4� T cells in the spleens of WT miceinfected with a lethal dose of IOE (26). The current studyrevealed that the decrease in CD4� T-cell numbers was notdue to the migration of CD4� T cells to peripheral sites ofinfection such as the liver but rather was due to the dispropor-tional apoptosis of this population following lethal IOE infec-tion. This conclusion was supported by data from experimentscomparing lethal disease caused by low-dose IOE infection tononlethal disease caused by E. muris infection, which includesthe presence of (i) a significantly higher percentage and abso-lute number of apoptotic splenic CD4� T cells in IOE-infectedmice than in E. muris-infected mice (Fig. 5A and C) and (ii)significantly lower numbers of CD4� T cells in the livers ofIOE-infected mice than in E. muris-infected animals (Fig. 5D).

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 threeindependent experiments, each with four mice per group. Bacterial burdens in different organs of IOE-infected perforin�/� mice on day 8postinfection 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 groupand 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, �2m�/�, andperforin�/� mice on day 10 postinfection. Compared to �2m�/� 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 threemice per group.

VOL. 75, 2007 ROLE OF CD8� T CELLS IN EHRLICHIA-INDUCED TOXIC SHOCK 4617

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Oct

ober

202

1 by

82.

102.

110.

121.

Our previous kinetic analysis (26) excludes the possibility thatdecreased levels of CD4� T cells in the liver of IOE-infectedmice are due to a defective induction of CD4� Th1 cells inIOE-infected mice. In that study (26), we showed that miceinfected with a lethal low dose of IOE had significant expan-sion of CD4� Th1 cells on day 7 postinfection followed by adramatic decrease in the total number of CD4� T cells and thefrequency of CD4� Th1 cells in the spleen on day 14 postin-fection (i.e., 1 to 2 days before mice succumbed to infection).The decline in total CD4� T cells and decreased CD4� Th1response corresponded to a concomitant substantial expansionof TNF-�-producing CD8� T cells in the spleen. These data,together with the greater frequency of antigen-specific CD4�

Th1 cells in �2m�/� mice than in WT mice, indicate that CD8�

T cells mediate not only severe liver pathology in fatal ehr-lichiosis but also end-stage CD4� T-cell apoptosis. Althoughour study does not indicate whether apoptotic CD4� T cellsare Ehrlichia specific or include bystander T cells, the absenceof evidence of nonspecific immune suppression in patients withHME or in our animal model of fatal ehrlichiosis (data notshown) suggests that apoptotic mechanisms involve antigen-specific CD4� T cells. In support of this conclusion, previousstudies demonstrated that differentiated antigen-specificCD4� Th1 cells upregulate TNFR and Fas/FasL on their sur-faces, thus becoming more susceptible to activation-inducedcell death (5, 42, 59) than CD4� Th2 cells.

In regard to CD8� T-cell migration to the peripheral site ofinfection, our data show that the absolute numbers of CD8� Tcells in the spleens and livers of IOE-infected mice were com-parable to those present in E. muris-infected mice (Fig. 5C andD). However, our current and previous studies (16) reveal adifferential role for CD8� T cells in mild (E. muris infection)and fatal (IOE infection) murine ehrlichiosis, where they playprotective and pathogenic roles, respectively. The protectiverole of CD8� T cells is supported by our previous findings thatMHC class I-deficient mice lacking CD8� T cells and micedepleted of CD8� T cells are highly susceptible to E. murisinfection (16). The mechanisms by which CD8� T cells provideprotection in mild ehrlichiosis is dependent on IFN-� produc-tion and cytotoxic functions (16).

Our data also suggest that CD4� T cells are critical forprotective immunity against virulent Ehrlichia strains. MHCclass II�/� mice develop an overwhelming infection and suc-cumb to IOE (Fig. 1 and 2). Although IFN-� production byantigen-stimulated splenocytes, most likely by CD8� T cells,was detected in CD4� T-cell-deficient mice, the magnitude ofthe type 1 response in these mice was insufficient to containreplication of the highly virulent ehrlichial strain IOE (Fig. 2and 6B). These data confirm previous studies showing thatEhrlichia-specific CD4� Th1 cells are crucial for the elimina-tion of Ehrlichia-infected cells via IFN-� production and mac-rophage activation (9, 10, 16, 17, 18, 26, 54). It has also beenshown that CD4� T cells mediate the production of Ehrlichia-specific Th1 isotype (IgG2a) antibodies, which are able toreduce ehrlichial replication by bacterial opsonization andphagocytosis (26, 28, 54, 55). Whether antibodies are essentialfor protection against rapidly progressive fatal ehrlichiosis isnot yet clear.

Our data suggest that CD8� T-cell-mediated overproduc-tion of IL-10 plays a pivotal role in the pathogenesis of Ehr-

lichia-induced toxic shock. IL-10 production was elevated inperforin�/� mice and WT mice that succumbed to lethal dis-ease (Fig. 9D). In addition, resistant CD8� T-cell-deficientmice had significantly lower levels of IL-10 in their sera andspleens (Fig. 7A and B), which suggests that pathogenic CD8�

T cells mediate IL-10 overproduction. Our previous kineticanalysis showed that a late burst of IL-10 is usually preceded byvery high levels of TNF-� in serum and spleen of WT miceinfected with a lethal high dose of IOE (25, 26). As we dem-onstrated previously, CD8� T cells are responsible for TNF-�overproduction, while IL-10 was produced by an undeterminedsubset of T cells (25, 26). Although the mechanism by whichIL-10 causes fatal disease is not completely understood, ourdata suggest that IL-10 is responsible for the marked decreasein the total number of splenic CD4� and, to some extent,CD8� T cells in Ehrlichia-induced toxic shock. In vivo neutral-ization of IL-10 in IOE-infected mice restored the percentagesof splenic CD4� and CD8� T cells (Fig. 8). Since decreasedlevels of splenic T cells in IOE-infected mice were associatedwith significant percentages of apoptotic CD4� T cells (Fig. 5Ato C), it is possible that IL-10 could function as a proapoptotic/proinflammatory cytokine that mediates the apoptosis ofCD4� T cells and/or infected target cells. In support of thisconcept, recent studies revealed novel proinflammatory andproapoptotic functions of IL-10 under certain pathologicalconditions. In those studies, IL-10 mediates apoptotic celldeath via the upregulation of FasL and TNFR on host cells (3,20, 32, 44). Our finding that IL-10 overproduction is detrimen-tal in Ehrlichia-induced toxic shock has an important clinicalapplication, as it can explain the failure of TNF-� inhibitors toimprove disease outcome in patients with septic shock. In ad-dition, IL-10 overproduction can explain our previous resultsshowing that TNF-� neutralization is unable to protect IOE-infected mice against a lethal outcome (25). Therefore, the de-velopment of a method to directly suppress CD8� T cells mayprove to more effectively rebalance the dysregulated immuneresponse in patients with fatal ehrlichiosis due to toxic shock.

Finally, our data show that perforin�/� mice are equallysusceptible to lethal ehrlichiosis as WT mice (Fig. 9A). Al-though we have not measured the frequencies of TNF-�- andIFN-�-producing CD8� T cells in perforin�/� mice, the de-cline in the CD4� Th1 response, as well as the elevation inIL-10 levels (Fig. 9D), suggests that these mice succumb to thesame dysregulated immune response as do WT mice. However,these results do not exclude the role of perforin in the immu-nopathogenesis of CD8� T-cell-mediated toxic shock, as per-forin was critical for controlling bacterial replication (Fig. 9B)and mediating liver pathology (Fig. 9C). One possible mecha-nism that explains the susceptibility of perforin�/� mice tofatal disease is that perforin�/� mice fail to eliminate antigen-loaded dendritic cells or infected macrophages that furtherinduce the activation and proliferation and of pathogenicCD8� T cells. The escape of infected target cells from per-forin-mediated lysis could explain the higher bacterial burdenobserved in these mice (Fig. 9B). In addition, previous reportsdemonstrated that perforin�/� mice generate exaggerated T-cell responses to LCMV and Listeria and during graft-versus-host disease (1, 31, 57), suggesting that there is a failure toeliminate antigen-presenting cells, which stimulate pathogenicT cells, or that T-cell elimination is impaired. The other pos-

4618 ISMAIL ET AL. INFECT. IMMUN.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Oct

ober

202

1 by

82.

102.

110.

121.

sible mechanism that could account for the susceptibility ofperforin�/� mice is that IL-10 overproduction in these micemay mediate the upregulation of Fas/FasL on CD4� T cells asdiscussed above, which could enhance apoptotic cell death andthus decrease the frequency of CD4� Th1 cells.

In conclusion, the present study underscores the importanceof CD8� T cells in mediating toxic shock following infectionwith this obligately intracellular pathogen that lacks LPS. Im-portantly, our study indicates that fatal ehrlichiosis resemblingtoxic shock syndrome is a multistep process, which is depen-dent on several distinct cytotoxic effector mechanisms medi-ated by pathogenic CD8� T cells. Furthermore, the differentialsusceptibility of �2m�/� and TAP�/� mice to fatal ehrlichiosissuggests that other immune cells such as invariant NK1.1���TCR T (NKT) cells that are competent in the latter group ofknockout mice may also contribute to the pathogenesis of thedisease. This unique mouse model provides researchers withthe ability to explore the mechanisms of toxic shock that areindependent of LPS. Our findings provide pivotal informationthat should be considered in designing immunotherapy orvaccines against LPS-negative organisms that cause toxicshock-like syndromes so as to boost protective immunity whileavoiding immunopathology.

ACKNOWLEDGMENTS

We thank Sherrill Hebert and Doris Baker for their excellent sec-retarial assistance. We also thank the biostatistician, Alai Tan, for herhelp with statistical analysis.

This work was supported in part by an NIH grant from the NationalInstitute of Allergy and Infectious Diseases (AI31431).

REFERENCES

1. Badovinac, V. P., S. E. Hamilton, and J. T. Harty. 2003. Viral infectionresults in massive CD8� T cell expansion and mortality in vaccinated per-forin-deficient mice. Immunity 18:463–474.

2. Bakken, J. S., M. E. Aguero-Rosenfeld, R. L. Tilden, G. P. Wormser, H. W.Horowitz, J. T. Raffalli, M. Baluch, D. Riddell, J. J. Walls, and J. S. Dumler.2001. Serial measurements of hematologic counts during the active phase ofhuman granulocytic ehrlichiosis. Clin. Infect. Dis. 32:862–870.

3. Balasa, B., K. Van Gunst, N. Jung, D. Balakrishna, P. Santamaria, T.Hanafusa, N. Itoh, and N. Sarvetnick. 2000. Islet-specific expression of IL-10promotes diabetes in nonobese diabetic mice independent of Fas, perforin,TNF receptor-1, and TNF receptor-2 molecules. J. Immunol. 165:2841–2890.

4. Barchet, W., S. Oehen, P. Klenerman, D. Wodarz, G. Bocharov, A. L. Lloyd,M. A. Nowak, H. Hengartner, R. M. Zinkernagel, and S. Ehl. 2000. Directquantitation of rapid elimination of viral antigen-positive lymphocytes byantiviral CD8(�) T cells in vivo. Eur. J. Immunol. 30:1356–1363.

5. Barreiro, R., G. Luker, J. Herndon, and T. A. Ferguson. 2004. Terminationof antigen-specific immunity by CD95 ligand (Fas ligand) and IL-10. J. Im-munol. 173:1519–1525.

6. Barry, M., and R. C. Bleackley. 2002. Cytotoxic T lymphocytes: all roads leadto death. Nat. Rev. Immunol. 2:401–409.

7. Bendelac, A., M. N. Rivera, S. Park, and J. H. Roark. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu. Rev.Immunol. 15:535–562.

8. Bevan, M. J. 1995. Antigen presentation to cytotoxic T lymphocytes in vivo.J. Exp. Med. 182:639–641.

9. Bitsaktsis, C., J. Huntington, and G. Winslow. 2004. Production of IFN-gamma by CD4 T cells is essential for resolving Ehrlichia infection. J. Im-munol. 172:6894–6901.

10. Brown, W. C., K. A. Brayton, C. M. Styer, and G. H. Palmer. 2003. Thehypervariable region of Anaplasma marginale major surface protein 2(MSP2) contains multiple immunodominant CD4� T lymphocyte epitopesthat elicit variant-specific proliferative and IFN-gamma responses in MSP2vaccinates. J. Immunol. 170:3790–3798.

11. Chen, Y., N. M. Chiu, M. Mandal, N. Wang, and C. Wang. 1997. ImpairedNK1� T cell development and early IL-4 production in CD1-deficient mice.Immunity 6:459–467.

12. Ehl, S., J. Hombach, P. Aichele, T. Rulicke, B. Odermatt, H. Hengartner, R.Zinkernagel, and H. Pircher. 1998. Viral and bacterial infections interferewith peripheral tolerance induction and activate CD8� T cells to causeimmunopathology. J. Exp. Med. 187:763–774.

13. Elzey, B. D., T. S. Griffith, J. M. Herndon, R. Barreiro, J. Tschopp, and T. A.Ferguson. 2001. Regulation of Fas ligand-induced apoptosis by TNF. J. Im-munol. 167:3049–3056.

14. Emoto, M., Y. Emoto, and S. H. E. Kaufmann. 1995. IL-4 producing CD4�

TCR� �int liver lymphocytes: influence of thymus, �2-microglobulin andNK1.1 expression. Int. Immunol. 7:1729–1739.

15. Feng, H., V. L. Popov, G. Yuoh, and D. H. Walker. 1997. Role of T lympho-cyte subsets in immunity to spotted fever group rickettsiae. J. Immunol.158:5314–5320.

16. Feng, H. M., and D. H. Walker. 2004. Mechanisms of immunity to Ehrlichiamuris: a model of monocytotropic ehrlichiosis. Infect. Immun. 72:966–971.

17. Ganta, R. R., C. Cheng, M. J. Wilkerson, and S. K. Chapes. 2004. Delayedclearance of Ehrlichia chaffeensis infection in CD4� T-cell knockout mice.Infect. Immun. 72:159–167.

18. Ganta, R. R., M. J. Wilkerson, C. Cheng, A. M. Rokey, and S. K. Chapes.2002. Persistent Ehrlichia chaffeensis infection occurs in the absence of func-tional major histocompatibility complex class II genes. Infect. Immun. 70:380–388.

19. Geginat, G., S. Schenk, M. Skoberne, W. Goebel, and H. Hof. 2001. A novelapproach of direct ex vivo epitope mapping identifies dominant and sub-dominant CD4 and CD8 T cell epitopes from Listeria monocytogenes. J. Im-munol. 166:1877–1884.

20. Georgescu, L., R. K. Vakkalanka, K. B. Elkon, and M. K. Crow. 1997.Interleukin-10 promotes activation-induced cell death of SLE lymphocytesmediated by Fas ligand. J. Clin. Investig. 100:2622–2633.

21. Goldman, E. E., E. B. Breitschwerdt, C. B. Grindem, B. C. Hegarty, J. J.Walls, and J. S. Dumler. 1998. Granulocytic ehrlichiosis in dogs from NorthCarolina and Virginia. J. Vet. Intern. Med. 12:61–70.

22. Harty, J. T., A. R. Tvinnereim, and D. W. White. 2000. CD8� T cell effectormechanisms in resistance to infection. Annu. Rev. Immunol. 18:275–308.

23. Heeb, H. L., M. J. Wilkerson, R. Chun, and R. R. Ganta. 2003. Largegranular lymphocytosis, lymphocyte subset inversion, thrombocytopenia,dysproteinemia, and positive Ehrlichia serology in a dog. J. Am. Anim. Hosp.Assoc. 39:379–384.

24. Hussell, T., A. Pennycook, and P. J. Openshaw. 2001. Inhibition of tumornecrosis factor reduces the severity of virus-specific lung immunopathology.Eur. J. Immunol. 31:2566–2573.

25. Ismail, N., H. L. Stevenson, and D. H. Walker. 2006. Role of tumor necrosisfactor alpha (TNF-�) and interleukin-10 in the pathogenesis of severe mu-rine monocytotropic ehrlichiosis: increased resistance of TNF receptor p55-and p75-deficient mice to fatal ehrlichial infection. Infect. Immun. 74:1846–1856.

26. Ismail, N., L. Soong, J. W. McBride, G. Valbuena, J. P. Olano, H. M. Feng,and D. H. Walker. 2004. Overproduction of TNF-alpha by CD8� type 1 cellsand down-regulation of IFN-gamma production by CD4� Th1 cells contrib-ute to toxic shock-like syndrome in an animal model of fatal monocytotropicehrlichiosis. J. Immunol. 172:1786–1800.

27. Kursar, M., K. Bonhagen, A. Kohler, T. Kamradt, S. H. E. Kaufmann, andH.-W. Mittrucker. 2002. Organ-specific CD4� T cell response during Listeriamonocytogenes infection. J. Immunol. 168:6382–6387.

28. Li, J. S., E. Yager, M. Reilly, C. Freeman, G. R. Reddy, A. A. Reilly, F. K.Chu, and G. M. Winslow. 2001. Outer membrane protein-specific monoclo-nal antibodies protect SCID mice from fatal infection by the obligate intra-cellular bacterial pathogen Ehrlichia chaffeensis. J. Immunol. 166:1855–1862.

29. Lieberman, J. 2003. The ABCs of granule-mediated cytotoxicity: new weap-ons in the arsenal. Nat. Rev. Immunol. 3:361–370.

30. Lin, M., and Y. Rikihisa. 2003. Ehrlichia chaffeensis and Anaplasma phago-cytophilum lack genes for lipid A biosynthesis and incorporate cholesterol fortheir survival. Infect. Immun. 71:5324–5331.

31. Maeda, Y., R. B. Levy, P. Reddy, C. Liu, S. G. Clouthier, T. Teshima, andJ. L. Ferrara. 2005. Both perforin and Fas ligand are required for theregulation of alloreactive CD8� T cells during acute graft-versus-host dis-ease. Blood 105:2023–2027.

32. Marra, L. E., Z. X. Zhang, B. Joe, J. Campbell, G. A. Levy, J. Penninger, andL. Zhang. 2004. IL-10 induces regulatory T cell apoptosis by up-regulation ofthe membrane form of TNF-alpha. J. Immunol. 172:1028–1035.

33. Mittrucker, H.-W., A. Kohler, and S. H. E. Kaufmann. 2000. Substantial invivo proliferation of CD4� and CD8� T lymphocytes during secondaryListeria monocytogenes infection. Eur. J. Immunol. 30:1053–1059.

34. Morsy, M. A., P. J. Norman, R. Mitry, M. Rela, N. D. Heaton, and R. W.Vaughan. 2005. Isolation, purification and flow cytometric analysis of humanintrahepatic lymphocytes using an improved technique. Lab. Investig. 85:285–296.

35. Neuzil, K. M., Y. W. Tang, and B. S. Graham. 1996. Protective role of TNF-�in respiratory syncytial virus infection in vitro and in vivo. Am. J. Med. Sci.311:201–204.

36. Ohteki, T., and H. R. MacDonald. 1994. Major histocompatibility complexclass I related molecules control the development of CD4�8� and CD4�8�

subsets of natural killer 1.1� T cell receptor-�/�� cells in the liver of mice.J. Exp. Med. 180:699–704.

37. Olano, J. P., and D. H. Walker. 2002. Human ehrlichioses. Med. Clin. N.Am. 86:375–392.

VOL. 75, 2007 ROLE OF CD8� T CELLS IN EHRLICHIA-INDUCED TOXIC SHOCK 4619

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Oct

ober

202

1 by

82.

102.

110.

121.

38. Olano, J. P., G. Wen, H. M. Feng, W. McBride, and D. H. Walker. 2004.Histologic, serologic, and molecular analysis of persistent ehrlichiosis in amurine model. Am. J. Pathol. 165:997–1006.

39. Planz, O., S. Ehl, E. Furrer, E. Horvath, M. A. Brundler, H. Hengartner, andR. M. Zinkernagel. 1997. A critical role for neutralizing-antibody-producingB cells, CD4(�) T cells, and interferons in persistent and acute infections ofmice with lymphocytic choriomeningitis virus: implications for adoptive im-munotherapy of virus carriers. Proc. Natl. Acad. Sci. USA 94:6874–6879.

40. Poitout, F. M., J. K. Shinozaki, P. J. Stockwell, C. J. Holland, and S. K.Shukla. 2005. Genetic variants of Anaplasma phagocytophilum infecting dogsin western Washington State. J. Clin. Microbiol. 43:796–801.

41. Power, C. A., C. L. Grand, N. Ismail, N. C. Peters, D. P. Yurkowski, and P. A.Bretscher. 1999. A valid ELISPOT assay for enumeration of ex vivo, antigen-specific, IFN-�-producing T cells. J. Immunol. Methods 227:99–107.

42. Ramsdell, F., M. S. Seaman, R. E. Miller, K. S. Picha, M. K. Kennedy, andD. H. Lynch. 1994. Differential ability of Th1 and Th2 T cells to express Fasligand and to undergo activation-induced cell death. Int. Immunol. 6:1545–1553.

43. Russell, J. H., and T. J. Ley. 2002. Lymphocyte-mediated cytotoxicity. Annu.Rev. Immunol. 20:323–370.

44. Schmidt, M., N. Lugering, H. G. Pauels, K. Schulze-Osthoff, W. Domschke,and T. Kucharzik. 2000. IL-10 induces apoptosis in human monocytes in-volving the CD95 receptor/ligand pathway. Eur. J. Immunol. 30:1769–1777.

45. Shen, Y., D. Zhou, L. Chalifoux, L. Shen, M. Simon, X. Zeng, X. Lai, Y. Li,P. Sehgal, N. L. Letvin, and Z. W. Chen. 2002. Induction of an AIDSvirus-related tuberculosis-like disease in macaques: a model of simian im-munodeficiency virus-mycobacterium coinfection. Infect. Immun. 70:869–877.

46. Shibata, S., M. Kawahara, Y. Rikihisa, H. Fujita, Y. Watanabe, C. Suto, andT. Ito. 2000. New Ehrlichia species closely related to Ehrlichia chaffeensisisolated from Ixodes ovatus ticks in Japan. J. Clin. Microbiol. 38:1331–1335.

47. Sigal, L. J., and K. L. Rock. 2000. Bone marrow-derived antigen-presentingcells are required for the generation of cytotoxic T lymphocyte responses toviruses and use transporter associated with antigen presentation (TAP)-dependent and -independent pathways of antigen presentation. J. Exp. Med.192:1143–1150.

48. Sotomayor, E. A., V. L. Popov, H.-M. Feng, D. H. Walker, and J. P. Olano.

2001. Animal model of fatal human monocytotropic ehrlichiosis. Am. J.Pathol. 158:757–760.

49. Stone, J. H., K. Dierberg, G. Aram, and J. S. Dumler. 2004. Human mono-cytic ehrlichiosis. JAMA 292:2263–2270.

50. Sumner, J. W., G. A. Storch, R. S. Buller, A. M. Liddell, S. L. Stockham, Y.Rikihisa, S. Messenger, and C. D. Paddock. 2000. PCR amplification andphylogenetic analysis of groESL operon sequences from Ehrlichia ewingii andEhrlichia muris. J. Clin. Microbiol. 38:2746–2749.

51. van Lier, R. A., I. J. ten Berge, and L. E. Gamadia. 2003. Human CD8(�)T-cell differentiation in response to viruses. Nat. Rev. Immunol. 3:931–939.

52. Walker, D. H., and J. S. Dumler. 1997. Human monocytic and granulocyticehrlichioses: discovery and diagnosis of emerging tick-borne infections andthe critical role of the pathologist. Arch. Pathol. Lab. Med. 121:785–791.

53. Walker, D. H., J. P. Olano, and H. M. Feng. 2001. Critical role of cytotoxicT lymphocytes in immune clearance of rickettsial infection. Infect. Immun.69:1841–1846.

54. Winslow, G. M., and C. Bitsaktsis. 2005. Immunity to the ehrlichiae: newtools and recent developments. Curr. Opin. Infect. Dis. 18:217–221.

55. Winslow, G. M., E. Yager, K. Shilo, E. Volk, A. Reilly, and F. K. Chu. 2000.Antibody-mediated elimination of the obligate intracellular bacterial patho-gen Ehrlichia chaffeensis during active infection. Infect. Immun. 68:2187–2195.

56. Wong, P., and E. G. Pamer. 2003. CD8 T cell responses to infectious patho-gens. Annu. Rev. Immunol. 21:29–70.

57. Yang, J., S. P. Huck, R. S. McHugh, I. F. Hermans, and F. Ronchese. 2006.Perforin-dependent elimination of dendritic cells regulates the expansion ofantigen-specific CD8� T cells in vivo. Proc. Natl. Acad. Sci. USA 103:147–152.

58. Yu, X. J., X. F. Zhang, J. W. McBride, Y. Zhang, and D. H. Walker. 2001.Phylogenetic relationships of Anaplasma marginale and ‘Ehrlichia platys ’ toother Ehrlichia species determined by GroEL amino acid sequences. Int. J.Syst. Evol. Microbiol. 51:1143–1146.

59. Zhang, X., T. Brunner, L. Carter, R. W. Dutton, P. Rogers, L. Bradley, T.Sato, J. C. Reed, D. Green, and S. L. Swain. 1997. Unequal cell death in Thelper cell (Th1) and Th2 effectors: Th1, but not Th2, effectors undergorapid Fas/FasL-mediated apoptosis. J. Exp. Med. 10:1837–1849.

Editor: W. A. Petri, Jr.

4620 ISMAIL ET AL. INFECT. IMMUN.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/i

ai o

n 15

Oct

ober

202

1 by

82.

102.

110.

121.