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The Journal of Immunology, 2004, 172: 6894-6901.
Copyright © 2004 by The American Association of Immunologists

Production of IFN-{gamma} by CD4 T Cells Is Essential for Resolving Ehrlichia Infection1

Constantine Bitsaktsis*, Jennifer Huntington* and Gary Winslow2,*,{dagger}

* Wadsworth Center, New York State Department of Health, Albany, NY 12201; and {dagger} Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, NY 12201


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


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Host defense against facultative and obligate monocytotropic intracellular bacteria requires cellular immunity (1). A central role for Th1 CD4 cells has been demonstrated in a number of such infections, including Mycobacterium tuberculosis (2), Salmonella typhimurium (3), and Legionella pneumophila (4). Th1 cells are required for the production of type 1 cytokines such as IFN-{gamma} and TNF-{alpha}, which in turn are responsible for triggering microbiocidal activities in infected phagocytes (2, 4, 5, 6, 7, 8, 9). The extent to which what are regarded now as classical mechanisms of cellular immunity apply to the Anaplasmataceae, a family which includes the human pathogens Anaplasma phagocytophila, the agent of human granulocytic ehrlichiosis, and Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, is not fully understood. A role for IFN-{gamma} in the activation of ehrlichia-infected macrophages has been demonstrated in vitro (10, 11), and other studies have suggested an essential role for IFN-{gamma} and CD4 T cells during E. chaffeensis infection in vivo (12, 13, 14, 15), but the basic cellular immune mechanisms involved have not been characterized in depth. This has been due in part to the lack of a suitable mouse model of ehrlichiosis, as immunocompetent mice are refractory to fatal ehrlichia infection (16, 13, 17).

Recent studies have described an as yet unclassified ehrlichia that was isolated from an Ixodes ovatus tick in Japan (18, 19). Six closely related isolates were described (19), and one (HF565) has also been referred to as Ixodes ovatus ehrlichia (IOE)3 (20). IOE is closely related to E. chaffeensis, exhibiting 98.2% identity at the 16S ribosomal DNA gene (19) In contrast to E. chaffeensis, IOE causes fatal disease in immunocompetent mice. Moreover, the histopathological lesions that have been described in the mouse bear a strong similarity to those found during human ehrlichiosis (19, 20). The availability of this new animal model of ehrlichiosis has made it possible to assess the importance of cellular immunity in an immunocompetent mouse model. In this study, we have addressed the requirements for particular host cells and host cell factors during IOE infection. Our data suggest that classical immune mechanisms are at least in part required for ehrlichial immunity, in a manner that is likely to be similar to what has been described for other intracellular bacterial pathogens that reside within host phagocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

The mice used in these studies were obtained from The Jackson Laboratory (Bar Harbor, ME) or were bred in the Animal Care Facility at the Wadsworth Center under microisolater conditions in accordance with institutional guidelines for animal welfare. Inbred strains included the following: C57/BL6, BALB/c, BALB/c-scid, (BALB/cByJSmn-Prkdcscid/J), and C57BL/6-scid (B6.CB17-Prkdcscid/SzJ). The following gene-targeted strains were also used: CD4-deficient (B6.129S2-Cd4tm1Mak), CD8-deficient (B6.129S2-Cd8atm1Ma), IL-12p40-deficient (B6.129S1-Il12atm1Jm), IFN-{gamma}-deficient (C.129S7(B6)-Ifngtm1s), IFN-{gamma} receptor-deficient (B6.129-Ifngrtm1Agt), TNF-{alpha}-deficient (B6.129S6-Tnfftm1Gk1), TNF-receptor-deficient (B6.129S-Tnfrsf1{alpha}tm1ImxTnfrs1{beta}tm1Imx), and IL-4-deficient (C57BL/6-Il4tm1Nnt) mice. Mice were sex matched for each experiment and were 6–12 wk in age.

Antibodies

The following Abs used for flow cytometry were obtained from BD Biosciences (Franklin Lake, NJ): CD4 (GK1.5), CD44 (Pgp-1), and CD45.1 (A20). IFN-{gamma} was neutralized using the rat IgG1 which was produced by the hybridoma XMG1.2 and purified by protein A affinity chromatography. Biotinylated polyclonal rat anti-mouse Ig and biotinylated-anti-Lyt2 (anti-CD8) Abs were generated as previously described (21) and were kindly provided by Dr. W. Lee (Wadsworth Center).

Bacterial infections

The ehrlichia used in this study was obtained as a generous gift from Dr. M. Kawahara (Nagoya Public Health Institute, Nagoya, Japan). The isolate was described as HF565 in the original study (19). To establish the initial infection, bacteria from infected cryopreserved liver tissue were used to inoculate C57BL/6 mice. The IOE was subsequently passaged in mice by serial transfer of disaggregated spleen tissue. Experimental inoculations were performed using aliquots of spleen mononuclear cells that had been stored at –80°C in sucrose-phosphate-glutamate buffer (0.0038 M KH2PO4/0.0072 M K2HPO4/0.0049 M L-glutamate/0.218 M sucrose (pH 7.2)). Mice were inoculated via the peritoneum. Institutional standards for animal welfare did not permit the use of death as an end point in the infection experiments; therefore, mice were routinely sacrificed when deemed moribund. Quantitative PCR was used to determine the bacterial copy number in the frozen aliquots, as described previously for E. chaffeensis (13). This was possible because the IOE and E. chaffeensis 16S rDNA sequences were identical in the regions used for PCR amplification. Appropriate dilutions were made to allow administration of the desired number of bacterial copies. The quantitative PCR (QPCR) assay can conservatively detect 10 organisms in the 10 ng of genomic DNA used in the standard assays. This amount of mouse genomic DNA is equivalent to ~2040 cells (based on a diploid genome size of 4.9 pg), so the limit of detection of the PCR assay is approximately one organism within 200 host cells. No products were amplified from uninfected mouse tissues.

IOE lysate preparation

Spleens from infected animals were harvested, and the erythrocytes were lysed by a brief hypotonic lysis. The cells were centrifuged at 1700 x g for 20 min. The pellet was disrupted with a Braun-Sonic 2000 sonicator (40 W for 30 s twice, on ice), and the resulting lysate was layered onto a discontinuous sodium diatrizoate gradient (42%/36%/30%; Sigma-Aldrich, St. Louis, MO) followed by centrifugation at 80,000 x g for 60 min as described previously (22). The ehrlichiae in the heavy and light bands were collected, washed by centrifugation in sucrose-phosphate-glutamate buffer, resuspended in sucrose-phosphate-glutamate buffer, and stored at –80°C.

Hematology analyses

Blood samples were analyzed using a Sysmex SE-9000 TOA Medical Electronics (Kobe, Japan) automated hematology analyzer for total and differential lymphocyte counts, RBC counts, and platelet counts.

T cell purification

Spleens were harvested, the erythrocytes were lysed, as described above, and the mononuclear cells were centrifuged, washed in HBSS, and resuspended in complete tumor medium (CTM; Eagle’s medium, dextrose, essential amino acids (50x), nonessential amino acids (100x), and sodium pyruvate (100x), pH 7.0). Biotinylated polyclonal rat anti-mouse Ig and biotinylated-anti-Lyt2 (anti-CD8) Abs were added to the cell suspension at a concentration of 5 µg/ml, and the cells were incubated for 15 min on ice. The cells were then passed through a sterilized 100-µm nylon mesh, washed, and resuspended in CTM at concentrations of 108 cells/ml. Anti-NK cell and antibiotin magnetic microbeads (BD Biosciences) were added to the cell suspensions at a concentration of 1.0 µl and 7.0 µl per 1 x 107 cells, respectively. Following a 15-min incubation at 4°C, the cells were resuspended in degassed CTM at 500 µl/108 cells and were separated using an AutoMACS (BD Biosciences) cell sorter. The negative fraction, containing the CD4 T cells, was collected. Flow cytometry analyses revealed that the CD4 T cell preparation was typically 90% pure. Contaminating cells were largely B cells. For T cell adoptive transfer, CD4 and CD8 T cells purified by negative selection were resuspended in HBSS at a concentration of 2 x 106/ml and transferred to recipient mice by tail vein injection.

T cell proliferation assays

CD4 T cells were purified from the spleens of infected mice, as described above, and were cultured in CTM with the IOE lysate in the presence of mitomycin C-treated autologous spleen APC from uninfected animals. After 4 days, 0.5 µCi of [3H]thymidine (New England Nuclear, Boston, MA) was added to the cultures, and 24 h later the cells were harvested and [3H]thymidine incorporation was measured using a 1205 beta plate counter (Wallac, Gaithersburg, MD).

Cytokine assays

Purified CD4 T cells were cultured in CTM with IOE Ags as described for the proliferation assays. Culture supernatants were collected 4 days later, and cytokine concentrations in the supernatants were measured using the cytometric bead array kit (BD Biosciences) following the instructions of the manufacturer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose-dependent susceptibility to fatal IOE infection in C57BL/6 and BALB/c mice

To establish IOE infection in immunocompetent animals, C57BL/6 and BALB/c mice were infected via the peritoneum with standardized inocula of IOE obtained from cryopreserved infected mouse splenocytes. Because assays to quantitate viable bacteria are not available, we quantified bacterial copy number in the inocula using quantitative PCR as described previously (13). Institutional animal welfare standards do not permit the use of death as an experimental end point; therefore, the infected mice were monitored regularly and were sacrificed when deemed moribund, as indicated by ruffled coat, immobility, and hunched posture. Such signs of fatal disease were apparent in infected C57BL/6 mice within 10–12 days following administration of 1 x 103 or 1 x 104 bacteria (Fig. 1a). In contrast, mice infected with a dose of 1 x 102 bacteria showed no obvious signs of disease after 2 wk postinfection and did not develop disease when monitored as long as 30 days (data not shown). Bacteria colonization of spleen tissue, determined using quantitative PCR, appeared uncontrolled in mice that received a lethal dose of 1 x 103 bacteria, but reached a maximum within 8 days in mice that received a dose of 1 x 102 bacteria (Fig. 1b). By day 12 postinfection, bacteria were below the limits of detection in C57BL/6 mice. The limit of detection using PCR has been estimated to be one organism per 200 mouse cells (see Materials and Methods). Very similar data were obtained in similar studies using BALB/c mice, indicating that there were no significant differences in susceptibility between these strains (Fig. 1, c and d). In both C57BL/6 and BALB/c mice, the peak bacterial colonization in the spleen, in animals given a sublethal inoculum, occurred on or about day 7 postinfection.



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  		FIGURE 1. Dose-dependent susceptibility to IOE infection in immunocompetent and SCID mice. Wild-type C57BL/6 or BALB/c (a–d) or SCID mice (e–g) were injected via the peritoneum with the indicated number of bacteria, and the mice were monitored for morbidity (a, c, e, and g) or for bacterial colonization in the spleen (b, d, f, and h) using QPCR. Morbid mice were those that were judged to be incapable of surviving the infection, and were sacrificed in the interest of animal welfare. The QPCR data indicate the mean copy number of the IOE 16S rDNA gene in 10 ng of DNA recovered from spleens. Five mice were used per group, and the data are representative of four experiments.

 
Susceptibility to fatal IOE infection was also examined in C57BL/6-scid and in BALB/c-scid mice. Both strains exhibited a 10- to 100-fold greater susceptibility, which suggested that the fatally infected BALB/c and C57BL/6 mice were able to provide partial control of infection (Fig. 1, e and g). These data were supported by PCR analyses of bacterial colonization in the spleen of the SCID mice, which demonstrated high levels of infection within 8 days of administration of 1 x 102 bacteria (Fig. 1, f and h). Morbidity in mice that received an inoculum of 10 or fewer bacteria was slightly delayed, and was accompanied by lower levels of bacterial colonization, even though all of the mice eventually succumbed. The data indicated that SCID mice are susceptible to inoculation with very low numbers of bacteria, suggesting that most of the bacteria detected in the inoculum by PCR were infectious.

Lack of persistent IOE infection after low-dose IOE inoculation

SCID mice were highly susceptible to infection so we used them to test whether bacteria persisted at levels that were undetectable by QPCR in C57BL/6 mice that survived low-dose IOE infection. In these experiments C57BL/6-scid mice were inoculated with 2 x 107 splenocytes pooled from three infected C57BL/6 mice on days 8, 12, 21, and 30 postinfection. All of the SCID mice that received splenocytes from day 8 postinfection donor mice succumbed, as expected (Fig. 2). Morbidity was <50% in the SCID mice that received splenocytes from day 12- and day 21-infected mice, while all mice that received day 30 splenocytes survived, suggesting that persistent infection does not occur in C57BL/6 mice.



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  		FIGURE 2. Lack of persistent IOE infection. C57BL/6 mice were infected with a sublethal inoculum (1 x 102 bacteria), splenocytes were harvested on 8, 12, 21, and 30 days postinfection, erythrocytes were depleted, and 2 x 107 cells pooled from three mice were transferred via the peritoneum to groups of five C57BL/6-scid mice. Morbidity in the SCID mice was monitored on the indicated days following cell transfer.

 
Systemic infection in C57BL/6 mice

C57BL/6 mice infected with a high inoculum exhibited systemic bacterial colonization. Relative levels of infection were highest in the spleen, although infection was detected in all tissues examined, including, liver, lung, brain, and heart, as well as in peripheral blood and peritoneal exudate cells (Fig. 3). The PCR assay did not yield amplicons from uninfected mice (data not shown; Ref. 13). The findings are consistent with previous studies of E. chaffeensis infection, which indicated that the ehrlichiae were harbored in tissue-resident macrophages, and with findings of previous published reports of IOE-infected mice (13, 20, 23). Preliminary studies have also revealed the presence of bacteria in the host cell-free plasma of infected mice, as was also observed during E. chaffeensis infection (Ref. 24 ; data not shown).



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  		FIGURE 3. Systemic IOE infection in C57BL/6 mice. Two mice were inoculated with a lethal bacterial dose (1 x 103) via the peritoneum, and bacterial colonization in the indicated tissues was determined on day 8 postinfection. The data indicate the mean copy number of the 16S rDNA gene in 10 ng of DNA recovered from each of the tissues.

 
Thrombocytopenia, anemia, and lymphopenia

Thrombocytopenia is characteristic of ehrlichiosis (25, 26); therefore, platelet numbers were monitored in the blood of C57BL/6 mice that had been administered either the sublethal or lethal IOE inoculum. Lethally infected mice exhibited pronounced thrombocytopenia, which was first evident within 3 days postinfection, before the onset of other disease symptoms (Fig. 4a). Mice infected with the sublethal dose exhibited transient thrombocytopenia, which reached a maximum at day 7 postinfection. Platelet numbers in the mice returned to normal levels within 10 days postinfection. Anemia, also observed during severe ehrlichiosis, was observed only in the lethally infected mice after day 7 postinfection. (Fig. 4b). The IOE-infected mice also exhibited marked lymphopenia within 7 days postinfection (Fig. 4c). Mice that received the sublethal dose exhibited only transient lymphopenia, whereas lymphocyte numbers never recovered in the lethally infected mice. These data together indicate that the IOE mouse model recapitulates several hematological features of human ehrlichiosis.



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  		FIGURE 4. Thrombocytopenia, anemia, and lymphopenia in IOE-infected mice. C57/B6 mice were inoculated with 1 x 102 or 1 x 103 bacteria, and hematology analyses were performed on groups of three mice on the indicated days postinfection. Uninfected C57BL/6 mice were used as controls (Ctrl). The data are representative of three experiments.

 
The CD4 coreceptor and type 1 cytokines are critical for host defense

The requirement and role for particular T cell subsets and cytokines during ehrlichial infection has not yet been resolved. This was first addressed using gene-targeted mice. Although the targeted mutations were conducted on the C57BL/6 or mixed B6.129 genetic backgrounds (both H-2b), available evidence had not suggested any critical strain dependence for IOE susceptibility. To determine the possible involvement of CD4 and CD8 cells in host defense, we infected CD4- and CD8-deficient mice with 1 x 102 bacteria and monitored infection and disease. The CD4-, but not the CD8-, deficient mice were susceptible to sublethal infection, indicating a critical requirement for this coreceptor in host defense (Fig. 5a). QPCR analyses revealed high levels of bacterial colonization in the spleens of the CD4-deficient mice within 12 days postinfection, when the mice exhibited visible signs of morbidity (Fig. 5b). Histopathology analyses indicated that the CD4-deficient mice exhibited similar disease manifestations as the lethally infected C57BL/6 mice (data not shown).



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  		FIGURE 5. The CD4 coreceptor and proinflammatory cytokines were essential for host defense. Mice deficient for the CD4 and CD8 coreceptors (a), IL-12p40, IFN-{gamma}, TNF-{alpha} (c), TNFRI/TNFRII, the IFN-{gamma}R (IFNR) (e), and IL-4 (f), were inoculated with 1 x 102 bacteria, and morbidity was monitored at the specified times thereafter. In separate experiments, bacterial colonization in spleen tissue was monitored in several of the gene-targeted strains as indicated in b and d. C57BL/6 mice were used as controls in all experiments. Five mice were used per group, and the data are representative of three independent experiments.

 
The requirement for the CD4 coreceptor suggested a role for Th1 cytokine-producing CD4 T cells. To address the involvement of Th1-associated cytokines in host defense, we infected several cytokine- and cytokine receptor-deficient mice to determine whether they exhibited increased susceptibility. Mice deficient for IL-12p40, IFN-{gamma}, TNF-{alpha}, the IFN-{gamma}R, and TNFR I and II doubly deficient mice, were all susceptible to fatal disease after receiving a dose of 1 x 102 bacteria (Fig. 5, c and e). QPCR analyses revealed high levels of bacterial infection in the cytokine-deficient mice (Fig. 5d). In contrast, IL-4-deficient mice did not exhibit any increased susceptibility to fatal infection when compared with normal controls (Fig. 5f), indicating that this cytokine was not essential for immunity. Together, these data support the hypothesis that Th1 CD4 T cells are critically important in host defense during IOE infection.

Ag-specific CD4 T cell responses are elicited during IOE infection

To determine whether CD4 T cells underwent clonal expansion in response to IOE Ags, we performed a T cell proliferation assay using CD4 T cells obtained from C57BL/6 mice infected with a sublethal dose of IOE. The CD4 T cells were purified by magnetic bead negative selection (85–90% purity) and were cultured in vitro with either infected spleen APC or uninfected spleen APC plus a lysate of IOE-infected spleen cells. Four days after [3H]thymidine was added to the cultures, and radionucleotide incorporation was measured 18 h later. The purified CD4 T cells proliferated in response to the IOE-infected, but not the uninfected APC, and the IOE lysate (Fig. 6). The proliferative response to the IOE lysate was significantly above background levels of proliferation (p < 0.01). This observation that CD4 T cells undergo clonal expansion in response to ehrlichial Ags supports the involvement of these cells in host defense.



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  		FIGURE 6. CD4 T cell proliferation in vitro in response to IOE Ags. CD4 T cells were purified by negative selection from C57/B6 mice that had been inoculated 7 days before with 1 x 102 bacteria and were cultured for 4 days in the presence of mitomycin-treated splenocyte APC from uninfected or IOE-infected mice, alone or with the addition of a crude IOE Ag preparation (at the dilution indicated). Four days later, proliferation was measured by monitoring [3H]thymidine incorporation. The mean ± SD are shown for triplicate cultures.

 
A Th1-type response is generated during IOE infection

The data generated using the genetically deficient mice suggested a requirement for a type 1 cytokine response by CD4 T cells. For direct evaluation of cytokine production, purified CD4 T cells were obtained from C57BL/6 mice infected with a sublethal dose of IOE on day 7 postinfection and were cultured in vitro with uninfected spleen APC in the presence of the IOE lysate. The supernatants were assayed 4 days later for T cell-derived cytokines. The cytokine profiles observed were indicative of a Th1-like response, because high levels of IFN-{gamma} and TNF-{alpha} and low levels of IL-4 were observed (Table I). These data indicated that type 1 CD4 T cell responses are elicited during IOE infection.


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  		Table I. Cytokine Production by CD4 T cells

 
IFN-{gamma} production by CD4 T cells rescued susceptible mice from fatal infection

IFN-{gamma} is produced by a number of cell types, including CD4 and CD8 T cells, NK cells, and dendritic cells (7, 27, 28, 29). Given the apparent requirement for both CD4 T cells and IFN-{gamma}, we next addressed whether production of IFN-{gamma} by CD4 T cells was sufficient to rescue IFN-{gamma}-deficient mice from fatal infection. CD4 T cells were purified by negative selection from CD45-congenic C57BL/6 mice (B6.SJL-PtprcaPep3b/BoyJ) that had received a sublethal inoculum 21 days before and were transferred by i.v. injection to IFN-{gamma}-deficient recipients. Mice that received CD4 cells, but not vehicle-injected mice, were protected from infection with 1 x 102 bacteria, an inoculum that was fatal to the IFN-{gamma}-deficient controls (Fig. 7a). The data suggested that IFN-{gamma} production by CD4 T cells was sufficient to complement the defect in IFN-{gamma} production in the gene-targeted mice.



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  		FIGURE 7. Production of IFN-{gamma} by CD4 T cells rescued IFN-{gamma}-deficient mice from fatal infection. CD4 T cells were purified by negative selection from previously infected CD45.1-congenic C57BL/6 mice and were transferred by i.v. injection to IFN-{gamma}-deficient recipients. One day after, the recipient mice and mice that had received only HBSS (Control) were inoculated with 1 x 102 bacteria, and morbidity was monitored (a). To detect the donor T cells in the recipient mice, splenocytes were isolated from recipients 18 days later and CD44 expression was monitored on the donor (CD45.1+) CD4 T cells, b. Percentages of CD4 T cells in each of the quadrants are indicated. The flow cytometry data are representative of four mice. c, The experiment in a was repeated with the addition of a group of IFN-{gamma}-deficient mice that received, in addition, 0.5 mg of the anti-IFN-{gamma} Ab XMG1.2 on 1 day before and on days 1 and 4 after infection.

 
To address whether donor CD4 T cells could be identified in the recipient mice, we analyzed recipient CD4 T cells by flow cytometry for expression of the CD45.1 donor allotype marker and the T cell activation marker CD44 12 days after infection of the IFN-{gamma} recipient mice. Donor CD4 T cells were readily detected in the spleen and represented ~5% (4.1–6.8 ± 1.7%) of the total CD4 T cells (Fig. 7b). Most of the donor cells expressed high levels of CD44, a characteristic of effector/memory T cells, suggesting that the donor T cells likely underwent activation and expansion in response to IOE infection in the recipient mice.

Although CD4 T cell-derived IFN-{gamma} was likely required for the protection observed in the recipient mice, it was possible that other factors produced by, or activities of, the transferred wild-type CD4 T cells could have been responsible. To determine whether the production of IFN-{gamma} production by the donor CD4 T cells was essential, wild-type CD4 T cells were transferred to IFN-{gamma}-deficient donor mice that received, in addition, three doses of anti-IFN-{gamma} Ab. Neutralization of IFN-{gamma} blocked the ability of the donor CD4 T cells to transfer protection (Fig. 7c). These data demonstrate that IFN-{gamma} production by CD4 T cells was sufficient to resolve IOE infection.

IFN-{gamma} triggered microbiocidal activity in IOE-infected peritoneal macrophages

IFN-{gamma} is well known to induce microbiocidal activities in host macrophages during intracellular bacterial infection; therefore, the effect of IFN-{gamma} administration on IOE-infected peritoneal macrophages was investigated. Macrophages treated with 1000 ng/ml rIFN-{gamma} for 18 h exhibited an approximate 4-fold reduction in bacterial load (Fig. 8). These data suggest that IFN-{gamma} production by CD4 T cells directly induces killing of IOE residing in host macrophages.



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  		FIGURE 8. IFN-{gamma}-mediated induction of microbiocidal activity in IOE-infected peritoneal macrophages. Peritoneal macrophages were isolated from infected C57BL/6 mice on day 8 postinfection and were seeded in microculture wells in the absence (Ctrl) or presence of rIFN-{gamma} for 18 h. Bacterial copy number was measured in the cells by QPCR. Values of p were <0.05 (*) or <0.01 (**).

 
Vaccinated mice were not protected upon rechallenge

A hallmark of adaptive immunity is the ability to provide effective resistance against rechallenge. To address whether previous infection protected against lethal challenge, C57BL/6 mice were infected with a sublethal inoculum (1 x 102 bacteria) and were allowed to recover for 3 wk before challenge with a 10-fold higher inoculum. Although previous infection resulted in an approximate 3-day delay in the onset of morbidity after rechallenge, the mice failed to generate long-term immunity (Fig. 9a). The delayed onset of morbidity was accompanied by a decreased bacterial burden on day 8 postinfection, relative to lethally infected naive mice, but the end point bacterial load in the spleens of the infected animals was similar in both groups of challenged mice (Fig. 9b). Several other strategies also failed to provide protection against lethal challenge, including infection with E. chaffeensis and vaccination with heat-killed IOE lysate in CFA (data not shown). The data suggest that either IOE infection does not elicit efficient memory responses, or that secondary immune responses induce fatal immunopathology.



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  		FIGURE 9. Sublethal IOE infection failed to induce immunity upon rechallenge. C57BL/6 mice were inoculated with 1 x 102 bacteria (immunized), followed by challenge with a lethal inoculum (1x 103 bacteria) 21 days later. Control mice received only the challenge inoculum. Morbidity (a) and bacterial colonization in the spleen (b) were monitored on the indicated days postinfection. Five mice per group were analyzed, and the data are representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our understanding of host immunity to ehrlichial infection has been hampered by the lack of an immunocompetent animal model suitable for the detailed study of cellular immune responses. This problem has been resolved in part by the discovery of the IOE agent and development of this new model of ehrlichiosis (18, 19, 20), which has allowed us to begin to address the role of cellular immunity in host defense. In the present study, we have demonstrated that both C57BL/6 and BALB/c mice exhibit a dose-dependent susceptibility to IOE infection. Immuncompetent mice were fully susceptible to as few as 1000 bacteria, but were resistant to a 10-fold lower inoculum. In contrast, SCID mice were susceptible to as few as 1–10 bacteria. Resistance to fatal infection in immunocompetent C57BL/6 mice was associated with apparently sterile bacterial clearance, as no evidence was found for infection after transfer of a 10% spleen homogenate from C57BL/6 mice infected 30 days before susceptible SCID mice. Thus, persistence does not appear to be characteristic of IOE infection in the mouse.

Quantitation of the ehrlichiae was only possible using PCR, a method that does not discriminate between viable and nonviable bacteria. However, SCID mice were fully susceptible to as few as 10 bacteria, as quantitated by PCR, which suggested that most of the bacteria in the inocula used in the studies were viable. These data were similar to those obtained after inoculation of SCID mice with limiting numbers of E. chaffeensis (24). Thus, data from both models suggest that SCID mice offer little or no resistance to ehrlichia infection. The adaptive immune response provides protection to at most a two log greater challenge inoculum, beyond which mice are fully susceptible to fatal infection. The number of ehrlichiae delivered during tick feeding is not yet known, but the relatively low numbers of bacteria used to inoculate mice in this study are likely to be within the range that would be encountered during tick-borne infection. This study used i.p. infection, so the effect of inoculation via other routes, or by the natural vector, Amblyomma americanum, is not yet known. One study indicated that the route of administration did not significantly affect the outcome of ehrlichial infection (30). Thus, the IOE infection model described here and in previous studies is likely a highly relevant model of ehlichiosis. In this regard, infection in C57BL/6 mice caused marked thrombocytopenia, anemia, and lymphopenia, all of which have been described during human ehrlichioses (25, 26).

The IOE mouse model has also provided an opportunity to address the role of cellular immunity in host defense. A critical role was discovered for the CD4 coreceptor, but not for CD8. The CD4 coreceptor is expressed on cells other than T cells, and compensatory mechanisms can occur in mice that lack particular cell subsets; therefore, some caution is in order with respect to the interpretation of the data from the gene-targeted mouse studies. With this caveat, it is still very likely that CD4 T cells play an essential role in immunity against IOE. We demonstrated that CD4 T cells from IOE-infected mice proliferated in response to IOE Ags and secreted type I cytokines that were essential for host defense. The conclusion that CD4 T cells play an essential role during IOE infection is supported by data from other studies that have demonstrated a requirement for CD4 T cells during ehrlichial infection (12, 14, 31, 32) as well as other intracellular bacterial infections (1, 33, 34, 35, 36). One likely role of CD4 T cells during IOE infection is to induce, via inflammatory cytokines, production of reactive nitrogen and/or oxygen species that may be responsible for the killing of intracellular ehrlichiae within macrophages (11). Such an interpretation is consistent with our finding that IFN-{gamma} was able to induce destruction of IOE in infected peritoneal macrophages, although the exact mechanism whereby this occurs is not yet known. CD4 T cells likely provide a critical source of IFN-{gamma}, because CD4 T cells from wild-type mice were able to complement the IFN-{gamma} deficiency in the gene-targeted mice, and the complementation was IFN-{gamma} dependent.

In turn, Th1 cell development is likely promoted by IL-12p40-dependent cytokines produced by innate immune cells during infection. The identity of the IL-12p40-dependent cytokine involved in IOE immunity is not yet known, but may include IL-12 or IL-23 (37). The mechanism(s) involved in the induction of IL-12p40 synthesis by cells such as dendritic cells and macrophages are not yet understood. Although other bacteria express factors that trigger inflammatory cytokine production by innate immune cells, the ehrlichia components that trigger innate immune responses have not yet been identified. This topic is of some interest, because the ehrlichiae are not thought to contain LPS (38). One study has reported short-term bacterial persistence and decreased NO production from peritoneal macrophages in E. chaffeensis-infected Toll-like receptor 4 (TLR4)-deficient mice (14). TLR4 is required for LPS responses (39); therefore, it is possible that E. chaffeensis and perhaps IOE contain other components that trigger innate immunity and IL-12 production via TLR4 or other innate recognition receptors.

Although the current studies have indicated a critical role for cellular immunity during IOE infection, an additional role for humoral immune responses should nevertheless still be considered. Our previous studies have indicated that Abs can be effective during E. chaffeensis infection, and our preliminary data have suggested an important role for B cells during IOE infection (C.B. and G.W., unpublished data). Therefore, an additional or alternative role for CD4 T cells may be to provide help for Ab production by B cells.

Although Ag-specific CD4 T cells appear to be elicited during infection and likely play a critical role in protection, we were unable to generate protective immunity against a 10-fold higher rechallenge dose after either sublethal IOE infection or vaccination with a crude preparation of IOE Ags in CFA or after infection with E. chaffeensis. However, it has not yet been demonstrated formally that memory CD4 T cells were responsible for the rescue of the IFN-{gamma}-deficient mice. It is not known whether the mouse is a natural host for IOE, but it is possible that the bacterium has devised ways in which to limit memory responses to facilitate multiple rounds of infection of its vertebrate hosts. An alternative explanation for the failure to induce immunity is that the challenge dose induced significant immunopathology due to robust memory CD4 or CD8 T cell responses in wild-type hosts.

The findings from this study provide a foundation for further investigations of cellular immunity during ehrlichial infection. Our studies suggest that classical immune mechanisms involving macrophage activation by Th1 CD4 T cells are relevant during this infection. Additional studies of the roles of particular T cell subsets and the identification of T cell Ags, the role of the innate immune recognition, and the possible contribution of B cells will be important for a full understanding of host defense during this infection.


   Acknowledgments
 
We thank Dr. M. Kawahara of the Nagoya Public Health Research Institute for his generous gift of IOE. We also thank Ken Class and the Wadsworth Center Immunology Core Facility, Candace Ross and the Wadsworth Center Hematology Core, the Wadsworth Center Animal Care Facility, and Dr. Timothy Sellati of Albany Medical College for his assistance with the cytometric bead array assays.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grant R01 AI47963. Back

2 Address correspondence and reprint requests to Dr. Gary Winslow, Wadsworth Center, 120 New Scotland Avenue, Albany, NY 12208. E-mail address: gary.winslow{at}wadsworth.org Back

3 Abbreviations used in this paper: IOE, Ixodes ovatus ehrlichia; CTM, complete tumor medium; QPCR, quantitative PCR; TLR4, Toll-like receptor 4. Back

Received for publication December 3, 2003. Accepted for publication March 22, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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