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and TNF Are Crucial for Control of Toxoplasma gondii in the Central Nervous System1
* Institut für Medizinische Mikrobiologie und Hygiene and
Zentrum für Medizinische Forschung, Universitätsklinikum Mannheim, Universität Heidelberg, Mannheim, Germany;
Abteilung für Neuropathologie, Universität zu Köln, Köln, Germany; and
Interdisziplinäres Zentrum für Medizinische Forschung, Universität Erlangen, Erlangen, Germany
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(LT), is still unknown. Upon oral infection with T. gondii, TNF-/-, LT-/-, and TNF/LT-/- mice failed to control intracerebral T. gondii and succumbed to an acute necrotizing Toxoplasma encephalitis, whereas wild-type (WT) mice survived. Intracerebral inducible NO synthase expression and–early after infection–splenic NO levels were reduced. Additionally, peritoneal macrophages produced reduced levels of NO upon infection with T. gondii and had significantly reduced toxoplasmastatic activity in TNF-/-, LT-/-, and TNF/LT-/- mice as compared with WT animals. Frequencies of parasite-specific IFN-
-producing T cells, intracerebral and splenic IFN- production, and T. gondii-specific IgM and IgG titers in LT-/- and TNF/LT-/- mice were reduced only early after infection. In contrast, intracerebral IL-10 and IL-12p40 mRNA expression and splenic IL-2, IL-4, and IL-12 production were identical in all genotypes. In addition, TNF-/-, LT-/-, and TNF/LT-/-, but not WT, mice succumbed to infection with the highly attenuated ts-4 strain of T. gondii or to a subsequent challenge infection with virulent RH toxoplasms, although they had identical frequencies of IFN--producing T cells as compared with WT mice. Generation and infection of bone marrow reconstitution chimeras demonstrated an exclusive role of hematogeneously produced TNF and LT for survival of toxoplasmosis. These findings demonstrate the crucial role of both LT and TNF for control of intracerebral toxoplasms. |
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-producing CD4 and CD8 T cells, which are recruited to the brain (3, 4, 5). In the absence of T cells, IFN- or IFN- receptors, mice are unable to control both acute and chronic toxoplasmosis (4, 6, 7, 8, 9).
The critical importance of T cells and IFN- for the control of T. gondii is further illustrated in the ts-4 vaccination model of toxoplasmosis. The ts-4 clone is an attenuated mutant derived from the highly virulent T. gondii RH strain (10). Vaccination of mice with ts-4 T. gondii results in an asymptomatic infection without parasite persistence. Repeated exposure to ts-4 T. gondii induces a CD4 and CD8 T cell response, which protects mice against an otherwise lethal challenge infection with the RH strain (9). However, T cell- and IFN--deficient mice already succumb to the infection with ts-4 parasites (11) illustrating that even this highly attenuated T. gondii strain requires control by T cells and IFN- to ensure host survival.
The induction of T. gondii-specific T cells is dependent on IL-12 produced by dendritic cells (12). Although the T cell response in toxoplasmosis is characterized by the production of IL-2 and IFN- and, thus, TH1-type orientated, TH2 cytokines including IL-4 are additionally produced and B cells also confer protection (13, 14).
In murine toxoplasmosis, one major function of IFN- is the induction of TNF, which is produced by many cell populations including macrophages, microglial cells, and astrocytes in the CNS and which is of critical importance for the in vivo control of T. gondii and survival of acute and chronic murine toxoplasmosis (5, 6, 15, 16, 17). In fact, the IFN- induced toxoplasmastatic activity of cells of the macrophage lineage including macrophages and microglial cells depends on TNF (18, 19). The endogenous production of TNF by these cells is required for the induction of NO, which limits growth of T. gondii in most murine cells. In addition to NO, IFN--induced production of indoleamine 2,3-dioxygenase (IDO), which degrades tryptophan, may contribute to the intracellular control of T. gondii. This has been demonstrated predominantly for human cells, but there is evidence that at least in some murine cells the control of T. gondii may also be mediated by IDO (20, 21, 22, 23).
In toxoplasmosis, TNF exerts its protective function exclusively via TNFR1 but not via TNFR2 (24, 25). At present, it is yet unknown whether other ligands of TNFR1 are also protective in murine toxoplasmosis. In addition to TNF, the secreted lymphotoxin (LT)- homotrimer (LT3) binds to TNFR1 with similar affinities (26). LT also forms a heterotrimer together with LT
(LT1LT2), which binds a distinct receptor, the LTR (27). In general, the function of LT is less well-characterized in infectious diseases as compared with TNF, but recent studies revealed that the function of LT is largely different depending on the underlying pathogen. In murine tuberculosis, LT is important for pathogen control (28), whereas in murine Trypanosoma brucei infection parasite control is improved in the absence of LT (29), and in murine cerebral malaria immunopathology is caused by LT (30).
To analyze the functional role of LT and to compare LT to TNF-mediated immune reactions in murine toxoplasmosis, we infected TNF-/-, LT-/-, TNF/LT-/- mice that had been generated directly in the C57BL/6 background and wild-type (WT) mice of an identical genetic background with T. gondii. These experiments revealed that in addition to TNF, LT is essential for control of T. gondii in the brain and host survival. Compared with WT mice, deficiency of TNF and/or LT resulted in reduced production of inducible NO synthase (iNOS), a failure to control T. gondii in the brain, and impaired toxoplasmastatic activity of macrophages.
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Materials and Methods |
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Age- and sex-matched C57BL/6 WT as well as TNF-/-, LT-/-, TNF/LT-/- mice, all prepared on a C57BL/6 background (31, 32), were used. NMRI mice were obtained from Harlan-Winkelman (Borchen, Germany). All animals were housed under conventional conditions in an isolation facility throughout the experiments.
Parasites and T. gondii infection
RH toxoplasms were grown in vitro in L929 fibroblasts in DMEM (PAA Laboratories, Cölbe, Germany) supplemented with 10% FCS (PAA Laboratories), 100 U/ml penicillin (Sigma-Aldrich, Deisenhofen, Germany), and 100 µg/ml streptomycin (Sigma-Aldrich) at 37°C and 5% CO2. Toxoplasms were harvested from freshly lysed fibroblasts. To separate parasites from cellular debris, the tissue culture medium was centrifuged at 50 x g and the supernatant was passed through a 5-µm syringe filter. Thereafter, RH toxoplasms were washed three times in 0.1 M PBS (20 min, 400 x g), counted microscopically, and heat-killed at 65°C for 20 min. Heat-killed toxoplasms (HKT) were stored at -80°C until use.
The ts-4 mutant of the RH strain was grown in human foreskin fibroblasts in DMEM supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 33°C and 5% CO2. For the infection of mice, ts-4 mutant was harvested from freshly lysed fibroblasts, purified as described for the RH strain, and adjusted to the respective concentration with sterile 0.1 M PBS (pH 7.4). The ts-4 mutant was i.p. injected into eight mice of each experimental group three times every 2 wk. The dose of the first two injections was 2 x 104 ts-4 parasites followed by 2 x 105 ts-4 parasites/mouse. Two weeks later, ts-4-vaccinated mice of all mouse strains and nonvaccinated WT animals were challenged i.p. with the RH strain (5 x 102/mouse).
To infect mice with the low-virulent DX strain of T. gondii, cysts were harvested from the brains of chronically infected NMRI mice. Brain tissue of these animals was dispersed in 0.1 M PBS (pH 7.4). The final concentration of the infectious agent was adjusted to a dose of 5 cysts/0.5 ml, which was administered orally to the experimental animals by gavage.
Immunohistochemistry
Uninfected and T. gondii-infected mice were perfused intracardially with 0.9% saline in deep Metofane (Janssen Pharmaceutica, Neuss, Germany) anesthesia at the indicated days postinfection (p.i.). For immunohistochemistry on 10-µm frozen sections, spleens, livers, hearts, lungs, and brains of three animals per group were dissected and blocks were mounted on thick filter paper with Tissue-Tek OTC Compound (Miles Scientific, Naperville, IL), snap-frozen in isopentane (Fluka, Neu-Ulm, Germany) precooled on dry ice, and stored at -80°C. Immunohistochemistry was performed as described previously (2). In brief, T. gondii was demonstrated by incubating sections with a polyclonal rabbit anti-T. gondii antiserum (BioGenix, Duiven, The Netherlands) followed by peroxidase-labeled goat anti-rabbit IgG F(ab')2 (Dianova, Hamburg, Germany). To detect iNOS immunoreactivity, sections were incubated with a polyclonal rabbit anti-mouse iNOS antiserum (Alexis, Grünberg, Germany) followed by goat anti-rabbit biotinylated IgG F(ab')2 (Vector Laboratories, Burlingame, CA) and a peroxidase-conjugated avidin-biotin complex (Vector Laboratories). In addition, sections were stained by an indirect immunoperoxidase protocol using rat anti-mouse CD45 (clone M1/9.3.4.HL.2), rat anti-mouse CD4 (clone G.K.1.5.), rat anti-mouse CD8 (clone 2.43), rat anti-mouse B220 (clone RA3-3A1/6.1), and rat anti-mouse Ly6-G (clone RB6-8C5) as primary Abs and peroxidase-linked goat anti-rat IgG F(ab')2 (Pharmacia, Freiburg, Germany) as secondary Ab. In addition, the avidin-biotin complex technique using rat anti-mouse F4/80 (clone F4/80), rat anti-mouse MHC class I (clone M1/42.3.9.8HLK), rat anti-mouse MHC class II (I-Ab,d,q, clone M5/114.15.2), rat anti-mouse ICAM-1 (CD54, clone YN1/1.7.4), and rat anti-mouse VCAM-1 (CD106, clone M/K-2.7) as primary Abs, biotinylated mouse serum-preadsorbed mouse anti-rat IgG F(ab')2 (Dianova) as secondary Ab, and the HRP-conjugated streptavidin-biotin complex (DAKO, Hamburg, Germany) was used. Peroxidase reaction products were visualized using 3,3'-diaminobenzidine (Sigma-Aldrich) and H2O2 as cosubstrate. Sections were lightly counterstained with hemalum.
Determination of the intracerebral parasitic load
At the indicated time points of infection, the number of ic parasites was determined microscopically on anti-T. gondii-stained frozen sections. One hundred high power fields were randomly selected and the number of parasites in representative areas were analyzed from three mice per experimental group and time point.
Isolation of splenic leukocytes, cerebral leukocytes, and peritoneal macrophages
Splenic and cerebral leukocytes were isolated as described before (33). In brief, animals were anesthetized and intracardially perfused with 0.9% NaCl to remove contaminating intravascular leukocytes from the brain at the indicated days p.i. Splenic leukocytes were isolated by passing spleens through a cell strainer (BD Biosciences, Heidelberg, Germany), and erythrocytes were lysed with ammonium chloride. Cerebral leukocytes were isolated by mincing brain tissue through a cell strainer, and leukocytes were separated by Percoll gradient centrifugation (Pharmacia).
Resident peritoneal cells were isolated by flushing the peritoneal cavity with 8 ml of HBSS (PAA Laboratories) supplemented with 3% FCS. Cells were adjusted to a concentration of 1 x 106/ml in DMEM with 10% FCS and seeded in a 96-well plate (100 µl/well). Plates were incubated with 5% CO2 at 37°C. After 2 h, nonadherent cells were removed by washing the plate two times with DMEM supplemented with 10% FCS. Adherent macrophages were immediately used for additional experiments.
Cytokine ELISAs and NO determination
Spleen cells were cultivated in 96-well flat-bottom plates (4 x 105 cells/well) in MEM supplemented with 10% FCS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C and 5% CO2 and were restimulated with either medium or HKT (two parasites per five leukocytes). After 24 or 48 h, the supernatant was harvested and IL-2, IL-4, IL-12, TNF, IFN-, and NO were determined. In addition, TNF and NO were determined in the supernatant of cultivated peritoneal macrophages (1 x 105 cells in 100 µl of DMEM supplemented with 10% FCS/well), which were infected with the RH strain of T. gondii isolated from freshly lysed fibroblast cultures (2 x 105 parasites/well). T. gondii-infected macrophages were additionally stimulated with 100 or 1000 U IFN-/ml or left unstimulated. Following either 24 or 48 h of incubation (5% CO2, 37°C), the supernatant was harvested. IL-2, IL-4, IL-12, TNF, and IFN- were determined by ELISA. The following capture and biotinylated detection Abs were used (all from BD Biosciences, Heidelberg, Germany): anti-IL-2 (clone JES6-1A12), biotinylated anti-IL-2 (clone JES6-5H4), anti-IL-4 (clone 11B11), biotinylated anti-IL-4 (clone BVD6-24G2), anti-IL-12p40 (clone C15.6), biotinylated anti-IL-12 (clone C17.8), anti-TNF (clone G281-2626), biotinylated anti-TNF (clone MP6-XT3), anti-IFN- (clone R4-6A2), and biotinylated IFN- (clone XMG1.2). ELISA plates were coated with the respective capture Ab (1–4 µg/ml) overnight at 4°C, washed, incubated with samples and standards (recombinant cytokines were obtained from BD Biosciences) for 4 h at room temperature, washed, and incubated with alkaline phosphatase-conjugated streptavidin (Dianova) for 30 min at room temperature. 4-methylumbellipheryl phosphate in glycine buffer was added, and fluorescence was measured with a fluorometer (Labsystems, Helsinki, Finland).
NO synthesis was assessed by measuring the accumulation of nitrite in cell culture supernatant as detected by the Griess reaction (1% sulfanilamide and 0.1% naphtylethylethylenediamide in 2.5% phosphoric acid). Absorption was measured at 550 nm, and nitrite concentrations were calculated by comparison with OD of NaNO2 standards.
Infection and enumeration of T. gondii in peritoneal macrophages
Peritoneal macrophages were seeded on sterile glass cover slips placed in 24-well microtiter plates (1 x 106 cells/ml) in DMEM supplemented with 10% FCS. Cells were stimulated with 100 U/ml or 1000 U/ml IFN- or left unstimulated. After 24 h, macrophages were infected with the RH strain of T. gondii (3 x 106 parasites/ml) isolated from freshly lysed fibroblast cultures. Thirty minutes thereafter, macrophage cultures were washed with sterile 0.1 M PBS (pH 7.4) three times. Coverslips from each experimental group were randomly chosen and fixed with 4% formaldehyde (Sigma-Aldrich) to determine the rate of infected cells 30 min after addition of parasites. The remaining macrophages attached on glass coverslips were incubated with DMEM with 10% FCS and IFN- treatment was continued for IFN--pretreated peritoneal macrophages. Eighteen to 24 h after infection, macrophage cultures were washed with 0.1 M PBS, fixed with 4% formaldehyde, and washed in PBS. To visualize T. gondii in macrophages, coverslips were stained with 4,6-diamidino-2-phenylindole. The number of T. gondii per parasitophorous vacuole (PV) was determined microscopically in 100 infected cells per experimental group.
ELISPOT assay
The frequency of T. gondii-specific T cells was determined in an IFN--specific ELISPOT assay as described in detail before (34). At the indicated days p.i., leukocytes were isolated from brain or spleen. Freshly isolated leukocytes (2 x 104/well) were cocultured in nitrocellulose-baked 96-well microtiter plates coated with rat anti-mouse IFN- mAb (BioSource International, Camarillo, CA) with spleen cells of uninfected mice, which served as APCs in the presence of HKT (two parasites per five effector leukocytes). All ELISPOT plates were incubated overnight and developed with biotin-labeled rat anti-mouse IFN- (BD Biosciences), alkaline phosphatase-conjugated streptavidin and amino-ethylcarbazole dye solution (Sigma-Aldrich). The frequency of Ag-specific, IFN--producing cells was calculated as the number of spots per leukocytes seeded.
Determination of anti-T. gondii IgM, IgG, and IgA production
Blood was obtained from uninfected mice and mice infected with T. gondii cysts by puncture of the retro-orbital plexus. Anti-T. gondii-specific IgM, IgG, and IgA Abs were determined in 10-fold serially diluted sera by incubation in 96-well microtiter plates coated with T. gondii Ag (Dade Behring, Marburg, Germany). After incubation for 2 h at 37°C, microtiter plates were washed with PBS four times and incubated with biotinylated goat anti-mouse IgM, IgG, and IgA, respectively (all from Sigma-Aldrich), for 1.5 h at room temperature. Plates were washed with PBS six times, and incubated with alkaline phosphatase-conjugated ExtrAvidin (Sigma-Aldrich) for 60 min at 37°C. Thereafter, plates were washed with PBS six times, and AttoPhos substrate (Roche, Mannheim, Germany) was added and fluorescence was measured with a fluorometer (Labsystems). The highest positive dilution of sera from T. gondii-infected mice was determined, and values were considered as positive if they were 3-fold above the corresponding dilution of uninfected mice of the same mouse strain.
Flow cytometry
Brain- and spleen-derived leukocytes were analyzed by double immunofluorescence staining followed by flow cytometry. CD4 and CD8 T lymphocytes were stained with PE-labeled rat anti-mouse-CD4 (clone GK1.5) and rat anti-mouse CD8-FITC (clone 53-6.7). Control staining included incubation of leukocytes with unlabeled or fluorochrome-labeled isotype-matched control Abs. All Abs were from BD Biosciences. Flow cytometry was performed on a FACScan (BD Biosciences), and the data were analyzed with Cell Quest Software (BD Biosciences).
RT-PCR
IFN-, TNF, LT, iNOS, IDO, IL-10, and IL-12p40 mRNA transcripts and hydroxyphosphoribosyltransferase (HPRT) mRNA expression were analyzed in brain tissue homogenates following a protocol described in detail before (35). Primer and probe sequences for cytokines and HPRT were as published before (5, 35, 36). In brief, RNA was extracted from tissue homogenates using an RNA extraction kit (Dianova). After reverse transcription of mRNA using the Superscript RT kit (Life Technologies, Eggenstein, Germany), PCRs were conducted in a volume of 30 µl. PCR conditions were optimized for each set of primers. PCR was performed at different cycle numbers to ensure that amplification occurred in the linear range. PCR products were electrophoresed through an agarose gel and the DNA was transferred to a nylon membrane (Pharmacia). Blots were hybridized using specific oligonucleotide probes, which were 3'-end labeled with digoxigenin (DIG) by use of a DIG oligonucleotide 3'-end labeling kit (Roche). A DIG luminescent kit (Roche) was used to visualize the hybridization products. Quantitation of RNA was performed with an imaging densitometer and Quantity One software (version 4.3.0, Bio-Rad, München, Germany). The intensity of each mRNA band was determined and related to the intensity of the respective autoradiogram band obtained for the internal control, HPRT. The results were expressed as x-fold increase over the respective RNA levels in uninfected animals of the same strain.
Bone marrow chimeras
Bone marrow chimeras were generated as described previously (37). In brief, WT mice were irradiated with 1000 rad and were i.v. reconstituted with 1–2 x 107 bone marrow cells isolated from the tibia and femur of TNF-/-, LT-/-, TNF/LT-/-, or WT mice. Ten weeks after bone marrow transplantation, chimeras and normal WT control mice were i.p. infected with T. gondii cysts of the DX strain. Survival of chimeras was monitored.
Statistical evaluation
For statistical evaluation of the experimental data, WINKS software (Texasoft, Cedar Hill, TX) was used. Survival analysis was performed with the Mantel-Haenszel log-rank test. The Student t and Wilcoxon rank sum tests were used to analyze differences between WT and TNF- and/or LT-deficient mice. Values of p < 0.05 were accepted as significant.
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-/-, and TNF/LT-/- mice succumb to acute TE, but WT mice survive
After application of T. gondii cysts, both TNF-/- and LT-/- mice succumbed to the infection within 28 days, whereas WT mice were still alive at day 60 p.i. (p < 0.001 for both TNF-/- and LT-/- vs WT mice; Fig. 1). The kinetics of the mortality for TNF-/- and LT-/- mice were not significantly different. TNF/LT-/- mice succumbed significantly earlier reaching 100% mortality up to day 20 p.i. as compared with TNF-/- and LT-/- mice (p < 0.05 for both TNF/LT-/- mice vs TNF-/- mice and TNF/LT-/- mice vs LT-/- mice, respectively).
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ultimately died of a severe necrotizing encephalitis due to failure of parasite control in the brain (Figs. 2 and 3). The differential susceptibility to the parasite and the mortality rates were paralleled by the number of ic parasites and the degree of tissue necrosis (Figs. 2 and 3). From days 12 to 19 p.i., TNF-/-, LT-/-, and TNF/LT-/- mice developed progressive encephalitis with parasite-induced tissue necrosis, which was most prominent in TNF/LT-/- mice, followed by TNF-/- mice, while encephalitis was less severe in LT-/- mice, which, however, were more severely affected than WT animals (Figs. 3 and 4). At day 19 p.i., in addition to T. gondii cysts, free tachyzoites were detectable in the brains of TNF/LT-/- and TNF-/- mice (Fig. 3). In contrast to strains lacking TNF and/or LT, WT animals showed regression of encephalitis with declining numbers of ic parasites up to day 28 p.i., whereas LT-/- mice, which were critically ill at this stage of infection, had developed severe tissue necrosis due to unrestricted parasite replication. In all strains of mice, ic parasites were associated with inflammatory infiltrates consisting predominantly of CD4 and CD8 T cells as well as F4/80+ macrophages and activated microglia. Although microglia and capillary endothelial cells were equally activated in all strains as evidenced by a marked induction of MHC class I and II molecules as well as ICAM-1 and, in the case of endothelium, VCAM-1 (data not shown), there were differences with respect to the number of iNOS+ leukocytes in the various strains of mice: at day 12 p.i., iNOS+ leukocytes were part of inflammatory infiltrates in WT mice, whereas iNOS+ leukocytes were absent from the brains of TNF/LT-/- mice (Fig. 5, a and d). At this stage of the infection, single iNOS+ cells were detectable in the brains of TNF-/- mice, while iNOS+ cells were restricted to the meninges in LT-/- animals (Fig. 5, b and c). From days 12 to 19 p.i., the amount of iNOS+ inflammatory foci increased in all strains of mice, and iNOS+ cells were associated with T. gondii cysts and also resided in the meninges. However, in TNF/LT-/-, TNF-/-, and LT-/- mice, the size of iNOS+ foci was reduced, in particular taking into account that these strains had recruited increased numbers of leukocytes to their brains in parallel to the more severe activity of TE (Fig. 4). In WT mice, microglia were also iNOS+. Occasional iNOS+ microglial cells were detectable in the brains of LT-/- mice, whereas in TNF-/- and TNF/LT-/- mice the microglia was consistently iNOS-negative.
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Because cytokines play a pivotal role for the control of ic toxoplasms, TNF, LT, IFN-, iNOS, IDO, IL-10, and IL-12 mRNA levels were determined in the brains of uninfected and T. gondii-infected mice.
In accordance with previous data, TNF mRNA was variably expressed in the brain of uninfected WT mice (Fig. 6). In TE, WT mice rapidly up-regulated TNF mRNA levels and produced equal amounts at days 12 and 19 p.i. In LT-/- mice, some uninfected mice also expressed low amounts of TNF mRNA. In TE, LT-/- mice up-regulated TNF mRNA expression, however, at days 12 and 19 p.i., they expressed reduced amounts of TNF mRNA as compared with WT animals. As expected, both TNF-/- and TNF/LT-/- mice did not transcribe TNF mRNA. Uninfected WT mice expressed only occasionally faint signals of LT mRNA. At day 12 p.i., WT mice strongly up-regulated LT mRNA expression with a further slight increase up to day 19 p.i. As compared with WT animals, TNF-/- mice produced lower amounts of LT mRNA at days 12 and 19 p.i., respectively. Both LT-/- and TNF/LT-/- did not express LT mRNA. WT mice showed a de novo expression of IFN- mRNA at day 12 p.i. and continued to produce similar amounts of IFN- mRNA at day 19 p.i. However, at day 12 p.i., LT-/-, TNF-/-, and–more pronounced–TNF/LT-/- mice produced lower amounts of IFN- mRNA as did WT animals. At day 19 p.i., all mouse strains expressed similar amounts of IFN- mRNA. Uninfected mice of either strain did not exhibit signals for iNOS mRNA. WT animals strongly induced iNOS mRNA at days 12 and 19 p.i. In parallel to the immunohistochemical observation, LT-/-, TNF-/-, and TNF/LT-/- mice did not express iNOS mRNA before day 19 p.i., and at this stage of infection iNOS mRNA expression was still reduced in these mouse strains as compared with WT animals. Faint signals for IDO mRNA were detectable in individual normal mice of the various strains. At day 12 pi., WT mice had markedly induced IDO mRNA, which persisted at high levels up to day 19 p.i. In contrast, TNF-/- and TNF/LT-/- mice marginally up-regulated IDO mRNA at day 12 p.i., and LT-/- mice mounted slightly reduced levels as compared with WT animals at this stage of the infection. From day 12 to 19 p.i., all mutant strains increased their IDO transcription and expressed similar amounts of IDO mRNA. In addition, IL-10 and IL-12p40 mRNA levels were analyzed in the brains of the various experimental groups and no significant differences were observed in the mRNA levels during the induction of TE (data not shown).
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-/-, and TNF/LT-/- mice
To further characterize the kinetics and magnitude of cytokine production in the various mouse strains, the splenic cytokine response of T. gondii-infected mice was studied. In these experiments, we focused on cytokines involved in the generation of T cell responses (IL-12, IL-2), a TH1/TH2 polarization (IL-12, IL-2, IL-4, IFN-), and parasite control (NO, TNF, IFN-).
Restimulation of splenocytes by HKT resulted in the induction of IL-12 in all strains of mice (Fig. 7). Maximal levels were observed as early as day 5 p.i. IL-12 levels did not differ between the various groups of TNF- and/or LT-deficient mice as compared with WT animals, except for day 12 p.i. when TNF-/- mice had significantly elevated IL-12 levels (p < 0.05 for WT vs TNF-/- mice).
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At day 5 p.i., the induction of IFN- was delayed in LT-/- and TNF/LT-/- mice (p < 0.005 for both WT vs LT-/- and WT vs TNF/LT-/- mice). At day 12 p.i., however, IFN- production was identical in all groups of HKT-restimulated splenocytes.
In parallel to the RT-PCR data obtained for the brain, spleen cells of TNF-/-, LT-/-, and TNF/LT-/- mice had a delayed induction of NO (day 5 p.i., p < 0.05 for WT vs TNF-/-, p < 0.005 for WT vs LT-/- and TNF/LT-/- mice), but ultimately mounted comparable levels of NO as compared with WT mice at day 12 p.i.
Interestingly, LT-/- mice had significantly reduced TNF production at days 5 and 12 p.i. as compared with WT mice (p < 0.05 at day 5 p.i., p < 0.005 at day 12 p.i.).
In vitro responses of T. gondii-infected peritoneal macrophages
To analyze whether TNF and/or LT deficiency alters the ability of macrophages to control T. gondii, peritoneal macrophages of WT, TNF-/-, LT-/-, and TNF/LT-/- mice were isolated and infected with T. gondii in the presence or absence of IFN-. In WT animals, IFN--stimulated macrophages contained significantly reduced numbers of parasites per PV as compared with unstimulated macrophages (Fig. 8a). In contrast, IFN- treatment did not induce a significant toxoplasmastatic effect in macrophages derived from TNF-/-, LT-/-, and TNF/LT-/- mice. The same results were obtained when macrophages were activated with higher amounts of IFN-, i.e., 1000 U/ml (data not shown).
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, macrophages of WT mice produced NO (Fig. 8b). IFN--stimulated macrophages of TNF-/- and LT-/- mice produced reduced amounts of NO after infection with T. gondii (p < 0.01 for WT vs TNF-/-). Combined deficiency for TNF and LT resulted in a further decline of NO production after T. gondii infection in combination with IFN- stimulation (p < 0.005 for WT vs TNF/LT-/- mice).
Because LT-/- mice had reduced ic TNF mRNA transcription in TE as well as reduced TNF production by spleen cells, TNF production of peritoneal macrophages was analyzed in vitro. In WT animals, T. gondii infection of macrophages resulted in production of TNF (Fig. 8c). Costimulation with IFN- resulted in further up-regulation of TNF levels in both infected as well as uninfected macrophages of WT mice. In addition, macrophages of LT-/- mice produced significantly reduced amounts of TNF as compared with WT macrophages (p < 0.01 for WT macrophages vs the corresponding group of LT-/- macrophages). As expected, macrophages of both TNF-/- and TNF/LT-/- mice did not produce TNF.
Thus, in vitro, macrophages require both TNF and LT for parasite control and optimal production of NO after infection with T. gondii.
Delayed induction of T. gondii-specific T cell responses in LT-/- and TNF/LT-/- mice
Because T cells are essential to control T. gondii, the frequency of splenic and ic T. gondii-specific T cells was determined by IFN- ELISPOT. T. gondii-specific T cells were already detectable at day 7 p.i. in the spleen of WT and TNF-/- mice (Fig. 9a). At this stage of infection, LT-/- and TNF/LT-/- mice had significantly reduced numbers of parasite-specific T cells as compared with WT mice (p < 0.05 for both WT vs LT-/- and WT vs TNF/LT-/- mice). At day 12 p.i., the number of splenic T. gondii-specific T cells was increased in all groups of mice. Whereas WT and TNF-/- mice had comparable numbers of parasite-specific T cells (p > 0.05), LT-/- and TNF/LT-/- mice had drastically reduced numbers of T. gondii-specific T cells (p < 0.01 for both WT vs LT-/- and WT vs TNF/LT-/- mice). At day 19 p.i., the numbers of splenic parasite-specific T cells further increased in all groups of mice. Whereas the frequency of parasite-specific IFN--producing T cells was still significantly reduced in LT-/- and TNF/LT-/- mice as compared with WT (p < 0.05 for both WT vs LT-/- and WT vs TNF/LT-/- mice), the frequency of T. gondii-specific IFN--producing T cells did not differ between WT and TNF-/- mice at day 19 p.i.
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-/- and TNF/LT-/- mice as compared with WT mice at day 12 p.i. (p < 0.05 for both WT vs LT-/- and WT vs TNF/LT-/- mice). In contrast to LT-/- and TNF/LT-/- animals, TNF-/- mice had similar numbers of parasite-specific T cells as compared with WT animals at this stage of infection (p > 0.05). At day 19 p.i., both TNF-/- and LT-/- mice had increased numbers of ic T. gondii-specific T cells (p < 0.05 for both WT vs TNF-/- and WT vs LT-/- mice), whereas TNF/LT-/- mice had similar numbers of parasite-specific T cells as compared with WT mice (p > 0.05).
A further analysis of the kinetics of ic T cells revealed that the relative and absolute numbers of ic CD4 and CD8 T cells increased in TE in all mouse strains. However, at day 12 p.i. the relative and absolute numbers of both T cell subsets were reduced in LT-/- and TNF/LT-/- mice as compared with WT and TNF-/- mice (p < 0.01 for both parameters of LT-/- and TNF/LT-/- mice as compared with WT and TNF-/- mice, data not shown). At day 19 p.i., all groups of mice had comparable relative and absolute numbers of ic CD4 and CD8 T cells.
Thus, all mouse strains mount T. gondii-specific T cells and recruit these cells to the brain, but the kinetics of these Ag-specific T cells is delayed in LT-/- and TNF/LT-/- mice.
Delayed anti-T. gondii-specific Ab responses in LT-/- and TNF/LT-/- mice
At day 12 p.i., all mouse strains had developed a T. gondii-specific IgM, IgG, and IgA response (Fig. 10). Whereas TNF-/- mice had comparable Ab titers to WT animals at day 12 p.i., LT-/- had reduced IgM and IgG titers (p < 0.05 for IgM, p < 0.01 for IgG) and TNF/LT-/- had reduced IgG titers (p < 0.05) as compared with WT mice. At day 19 p.i., none of the cytokine gene-deficient mice had reduced T. gondii-specific IgM, IgG, or IgA titers as compared with WT mice. In contrast, at this stage of infection TNF/LT-/- mice had significantly elevated IgM and IgA titers as compared with WT animals (p < 0.05 for IgM and IgA).
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, but not of TNF, the induction of T. gondii-specific IgM and IgG Abs was delayed at day 12 p.i., but not fundamentally impaired because TNF/LT-/- mice, i.e., mice with the highest ic parasite burden, had the highest IgM and IgA titers at day 19 p.i.
TNF-/- 
WT, LT-/- WT, and TNF/LT-/- WT, but not WT WT chimeras succumb to toxoplasmosis
To prove whether the expression of TNF and or LT by nonhematogenous cells including microglia and astrocytes is sufficient for survival of toxoplasmosis and whether the defective anlage of lymph nodes causes death of T. gondii-infected LT-/- and TNF/LT-/- mice, bone marrow chimeras between TNF-/-, LT-/-, and TNF/LT-/- donor mice and WT recipient animals were generated and infected with T. gondii cysts. Whereas normal WT mice and WT WT control chimeras survived the infection for at least 8 wk, TNF-/- WT, LT-/- WT, and TNF/LT-/- WT chimeras succumbed within 3–5 wk to the infection (Table I).
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by nonhematogenous cells is insufficient to control toxoplasmosis and that the normal lymph node anlage in WT mice reconstituted with LT-/- bone marrow does not rescue mice from fatal toxoplasmosis.
ts-4 vaccination does not protect TNF-/-, LT-/-, and TNF/LT-/- mice from lethal toxoplasmosis
To analyze whether an established T cell immunity protects TNF-/-, LT-/-, and TNF/LT-/- mice from toxoplasmosis, we used the ts-4 vaccination model. Vaccination of mice with the ts-4 mutant induces a parasite-specific T cell response, which protects these animals against an otherwise lethal challenge infection with highly virulent RH toxoplasms.
In accordance with these data, ts-4-vaccinated WT mice survived a challenge infection with the RH strain, whereas nonvaccinated WT mice rapidly succumbed to the challenge infection (data not shown). In contrast to WT mice, 20–25% of TNF-/- and LT-/- mice died after the third ts-4 vaccination and all surviving mice succumbed to the challenge infection. TNF/LT-/- mice were even more susceptible and 20% of the mice died after the second vaccination, with remaining mice dying upon the third injection of ts-4 parasites.
In parallel to the survival experiment, ELISPOT assays were performed to analyze the frequency of T. gondii-specific T cells in the spleen. At day 14 p.i., i.e., after the first ts-4 vaccination, WT, TNF-/-, LT-/-, and TNF/LT-/- mice had developed a T. gondii-specific T cell response (Fig. 11). As observed for infection with T. gondii cysts, induction of a parasite-specific T cell response was delayed in LT-/- and TNF/LT-/- animals, and these mouse strains had a reduced frequency of T. gondii-specific T cells at day 14 p.i. (p < 0.01 for both WT vs LT-/- and WT vs TNF/LT-/- mice). After the second ts-4 vaccination (day 28 p.i.), WT, TNF-/-, LT-/- and TNF/LT-/- mice had comparable numbers of T. gondii-specific T cells in their spleens. These findings illustrate that 1) TNF/LT-/- succumbed to vaccination despite their normal frequency of IFN--producing parasite-specific T cells, and that 2) TNF-/- and LT-/- mice succumbed to the RH challenge infection although they had established a T. gondii-specific T cell response with similar frequencies as compared with WT animals.
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plays a crucial role for survival of murine toxoplasmosis. Control of T. gondii in the brain, but not in other organs, was critically dependent on both LT and TNF, which could not compensate each other for parasite control. Because mice deficient in both TNF and LT succumbed significantly earlier than mice deficient in either TNF or LT, these two cytokines seem to act synergistically to control T. gondii.
Both LT and TNF mRNA expression were strongly up-regulated in the brain of WT mice during encephalitis. Interestingly, TNF mRNA expression in LT-/- mice and LT mRNA expression in TNF-/- mice were reduced indicating a cross-regulation of these molecules. This assumption is further supported by reduced TNF production of Toxoplasma Ag-stimulated spleen cells and T. gondii-infected macrophages of LT-/- mice. Thus, the susceptibility of TNF-/- and LT-/- mice may not only be caused by the absence of the deleted gene and its product, but also–at least partially–by the reduced production of the corresponding protein. The mechanism of this cross-regulation which has also been observed by Alexopoulou et al. (38) is yet unknown, but the genes for TNF and LT are located closely together on the same chromosome, and targeting of one of these genes may effect the other gene.
Common to TNF-/-, LT-/-, and TNF/LT-/- mice was their failure to control ic parasites and the lack of a toxoplasmastatic activity of their IFN--stimulated macrophages. The importance of TNF for the control of T. gondii in macrophages has been shown previously (18). In addition, TNF has been shown to be essential for the in vivo control of T. gondii (15, 16, 17). Our study illustrates for the first time that LT is also required for toxoplasmastatic activity of macrophages. The importance of LT for pathogen control has also been demonstrated in murine tuberculosis (28), whereas in T. brucei infection the absence of LT resulted in improved parasite control which was linked to an increased production of parasite-specific Abs (29). These findings imply that the function of LT in infectious diseases is largely determined by the underlying pathogen, which is further supported by the absence of immunopathology in cerebral malaria of LT-/- mice (30).
In our experiments, the recruitment of all leukocyte subsets to the brain was unimpaired in all strains of mice, and the reduced relative and absolute numbers of CD4 and CD8 T cells in LT and TNF/LT-/- mice at an early stage of TE (day 12 p.i.) is most probably explained by the delayed induction of parasite-specific T cells in the spleen of these animals. However, TNF and LT were required to induce an optimal production of NO, which is of importance for parasite control in vivo and in vitro (18, 39). Reduced ic levels of iNOS mRNA and reduced levels of NO produced by T. gondii Ag-stimulated spleen cells were observed early after infection. In contrast, at later stages ic iNOS mRNA remained reduced in TNF-/-, LT-/-, and TNF/LT-/- mice, whereas NO production of spleen cells was identical in all strains of mice. These findings illustrate that NO production is delayed in the absence of TNF and LT in both brain and spleen. However, at later stages of infection NO production in the brain, but not in the spleen, is TNF- and LT-dependent. This finding may also explain why the control of T. gondii in peripheral organs of TNF-/-, LT-/-, and TNF/LT-/- mice was–in contrast to the brain–unimpaired. These findings were supported by the immunohistochemical observation that TNF-/- and TNF/LT-/- microglial cells, the resident ic macrophage population, did not express iNOS protein, and that only few microglial cells of LT-/- mice were iNOS+ as compared with WT mice. In addition, foci of iNOS+ leukocytes in T. gondii-associated infiltrates of all strains of immunodeficient mice were smaller despite their increased ic parasitic load. These findings further argue for insufficient ic iNOS production at the site of parasite replication in TNF-/-, LT-/-, and TNF/LT-/- mice, an observation which has also been made in murine leishmaniasis of TNF-/- mice (40).
This assumption is further supported by the reduced in vitro NO production of T. gondii-infected macrophages of TNF-/-, LT-/-, and, even more pronounced, of TNF/LT-/- mice. Because the rapid production of NO after infection of macrophages is important to restrict growth of T. gondii (18), reduced NO levels of macrophages in vitro as well as the delayed induction and disorganized expression of NO in vivo may well contribute to the failure of TNF and LT to control T. gondii. This is further demonstrated by the correlation of the shortest survival time and the lowest NO levels of macrophages of TNF/LT-/- mice.
Expression of IDO, another anti-T. gondii effector molecule which may contribute to control of T. gondii in vivo, was also up-regulated in the brains of T. gondii-infected WT mice. Whereas LT-/- mice had similar kinetics of IDO mRNA expression as compared with WT mice and expressed only slightly reduced IDO mRNA levels, the induction of IDO mRNA was strongly delayed in TNF-/- and TNF/LT-/- mice indicating that TNF, but not LT, is involved in the induction of ic IDO mRNA expression. However, at later stages of disease (day 19 p.i.), IDO mRNA expression was similar in all strains of mice, and, thus, expression of IDO mRNA was only temporarily delayed. Recently, it has also been demonstrated that IFN- is important for the induction of IDO mRNA expression in TE (36).
Because IFN--producing T cells are of pivotal importance for control of T. gondii and both TNF and LT play an important role in the development of lymphatic tissues (31, 32, 41, 42, 43, 44), we analyzed the frequency of IFN--producing T cells in both brain and spleen of T. gondii-infected TNF-/-, LT-/-, and TNF/LT-/- in comparison to WT mice. TNF-/- mice had a normal frequency of splenic and ic IFN--producing T cells at all stages of the infection indicating that a disturbed T cell response does not account for the increased susceptibility of these animals. In addition, spleen cells of TNF-/- mice restimulated with T. gondii Ag produced similar amounts of IL-2 and IL-4 as compared with WT mice illustrating that the balance between TH1 and TH2 T cells is not affected by TNF deficiency. In contrast, LT-/- and TNF/LT-/- mice had a reduced frequency of splenic and ic parasite-specific T cells early after infection (day 12 p.i.), although they produced normal amounts of IL-12, which is required for optimal induction of T. gondii-specific T cells (45). At later stages of the disease (day 19 p.i.), LT-/- also had a normal frequency of ic parasite-specific T cells. In combination with the reduced TNF production of LT-/- mice, this delayed induction of parasite-specific T cells may well contribute to the insufficient control of T. gondii in the brain.
In addition, the production of T. gondii-specific IgM in LT-/- and of IgG Abs in LT- and TNF/LT-deficient mice were reduced as compared with WT animals at day 12, but not at day 19 p.i., when these immunodeficient mouse strains mounted a partially increased anti-parasitic Ab response. In the complete absence of Abs, mice of the same genetic background succumb between 3 and 4 wk after infection (14), and, thus, the delayed B cell response in LT- and TNF/LT-deficient mice may act in concert with the delayed T cell response in these animals and contribute to the fatal course of TE.
The delayed induction of T and B cell responses in LT-/- and TNF/LT-/- mice is most probably caused by the disturbed lymphoid architecture of LT- and TNF/LT-deficient mice (31, 32, 46, 47, 48). To prove whether this factor is indeed of key importance, we generated bone marrow chimeras between WT and TNF-/-, LT-/-, and TNF/LT-/- mice. In these experiments, WT mice served as recipients and immunodeficient mice as donors resulting in a normal amount and distribution of lymph nodes (32, 49), a normal expression of TNF and/or LT by radioresistant host cells (e.g., microglia), and a default expression of TNF and/or LT by hematogenous donor cells. From the observation that WT mice reconstituted with either TNF-/-, LT-/-, or TNF/LT-/- bone marrow succumbed within 3–5 wk after i.p. infection with T. gondii cysts and that irradiated WT mice reconstituted with WT bone marrow survived throughout the entire observation period (8 wk), it can be concluded that 1) the disturbed architecture of the lymphoid system does not account for death of LT-/- and TNF/LT-/- mice, and 2) that production of TNF and LT by radioresistant nonhematogenous cells is insufficient for survival of toxoplasmosis. This latter observation extents results in reciprocal bone marrow chimeras of WT- and TNFR-deficient mice showing that expression of TNFRs on both hematogenous and nonhematogenous cells is important for optimal control of T. gondii (50).
To further analyze the importance of the delayed induction of T. gondii-specific T cells for the death of LT-/- mice and whether the increased susceptibility of TNF-/-, LT-/-, and LT-/- mice to toxoplasmosis is also regulated by the experimental conditions, mice were infected with the attenuated ts-4 strain of T. gondii and challenged with highly virulent RH toxoplasms. In accordance to previous studies (10), WT mice survived ts-4 vaccination and the subsequent challenge infection with highly virulent RH T. gondii, whereas nonvaccinated WT mice succumbed to the RH challenge infection. In contrast, all TNF/LT-/- mice died either shortly before or after the third ts-4 application, and TNF-/- and LT-/- mice also succumbed to the vaccination or died rapidly after the RH challenge infection. Application of ts-4 parasites induced similar frequencies of IFN--producing T. gondii-specific T cells, which are important for survival of infection with both the ts-4 and the RH strain of T. gondii (9, 11), in all strains of mice at the time of death or even several weeks before. These findings indicate that differences in T cell responses most probably do not account for the increased susceptibility of TNF and LT gene-targeted mice, but argue for a more important role of the reduced toxoplasmastatic activity of infected cells for the increased susceptibility of TNF-/-, LT-/-, and TNF/LT-/- mice. Moreover, the earlier death of TNF/LT-/- mice as compared with TNF-/- and LT-/- after infection with ts-4 T. gondii reflects the increased susceptibility of TNF/LT-/- mice to oral infection with T. gondii cysts and further indicates that TNF and LT exert—at least partially—different protective mechanisms. This assumption is supported by the observation that LT-/- mice (51), which lack the LTR ligand LT1LT2, also succumbed within 35 days to oral infection with T. gondii cysts (D. Schlüter, unpublished data). Thus, mice deficient for TNF, LT, or TNFR1, to which LT3 and TNF bind, all succumb to an acute necrotizing TE, but the LT1LT2 heterotrimer is also required for survival of toxoplasmosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dirk Schlüter, Institut für Medizinische Mikrobiologie und Hygiene, Universitätsklinikum Mannheim, Universität Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim, Germany. E-mail address: dirk.schlueter{at}imh.ma.uni-heidelberg.de
3 Abbreviations used in this paper: TE, Toxoplasma encephalitis; ic, intracerebral; IDO, indoleamine 2,3-dioxygenase; LT, lymphotoxin; WT, wild type; iNOS, inducible NO synthase; HKT, heat-killed Toxoplasma; p.i., postinfection; PV, parasitophorous vacuole; HPRT, hydroxyphosphoribosyltransferase; DIG, digoxigenin.
Received for publication September 30, 2002. Accepted for publication April 4, 2003.
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