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*
Department of Molecular Microbiology and
Division of Comparative Medicine, Washington University School of Medicine, St. Louis, MO 63110; and
Division of Infectious Diseases, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-
, IL-12, and IL-18.
Extensive liver damage and lymphoid degeneration accompanied the
elevated levels of cytokines produced during lethal infection.
Increased time of survival following lethal infection with the RH
strain was provided by neutralization of IL-18, but not TNF- or
IFN-. Nonlethal infections with a low dose of type II PTG strain
parasites were characterized by a modest induction of Th1 cytokines
that led to control of infection and minimal damage to host tissues.
Our findings establish that overstimulation of immune responses that
are normally necessary for protection is an important feature of acute
toxoplasmosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dependent, cell-mediated
immune response. Chronic infection is a consequence of parasite
differentiation into slow-growing cysts that are capable of persisting
for the life of the host (3). In immunocompromised
individuals, Toxoplasma is an important opportunistic
pathogen due to fulminate re-emergence of chronic infections
(4). Acute infections acquired during pregnancy are also
capable of causing severe birth defects, including hydrocephaly,
calcification, neurological defects, and chorioretinitis, which may be
recurrent (5).
The outcome of toxoplasmosis in the mouse model is strongly
dependent on the strain of T. gondii and can be predicted
based on the parasite genotype (6, 7). Although T.
gondii strains are genetically very similar, they comprise three
distinct clonal lineages (6, 8). Strains of the type I
genotype are highly virulent in mice, and regardless of the
genetic background of the mouse host, they have a lethal dose of a
single viable parasite (6, 7). In contrast, type II and
III strains have a 50% lethal dose of 
103
parasites, and the outcome of infection is dependent on the genotype of
the host (9, 10, 11, 12).
Type II strains are the most prevalent in animals and are most commonly associated with human toxoplasmosis (8, 13). Despite an overall lower prevalence, type I strains are often associated with severe congenital toxoplasmosis (14, 15) and ocular toxoplasmosis (16), suggesting that they are also more pathogenic in humans. T. gondii is capable of infecting sites of immune privilege, including the retina, CNS, and placenta. In these tissues, cellular immunity is important for control of infection; however, the extent of tissue inflammation is often disproportionate to the presence of parasites, suggesting that the resulting pathology is partially immune-mediated (2, 17, 18, 19, 20). Furthermore, in cases of disseminated toxoplasmosis, which occur primarily in immunocompromised patients, systemic infections are characterized by a septic shock-like syndrome leading to death (21).
Control of toxoplasmosis during nonlethal infection with
nonvirulent strains has been extensively studied in the mouse model
where a vigorous IFN--dependent, Th1 cytokine response controls
infection. IL-12 is released early in infection and potentiates the
production of IFN- by NK cells and T cells (22, 23, 24).
IFN- is absolutely essential to the control of both acute and
chronic infections (25, 26, 27). The innate immune response is
implicated in the early induction of proinflammatory cytokines during
infection of naive animals, and studies have shown that dendritic cells
(28), neutrophils (29), and macrophages
(30) respond directly to parasite Ags by producing IL-12
and TNF-. TNF- is also important for controlling resistance to
acute (31, 32) and chronic (33, 34)
infections by T. gondii.
While nonvirulent strains are generally well controlled by the immune response, type I strains are uniformly lethal in mice, suggesting that they induce inadequate immune control or are endowed with destructive properties that directly cause pathology. To determine the basis of the remarkable difference in virulence of T. gondii strains, we examined the response of immunocompetent outbred mice to infection by type I and type II strains of T. gondii. Surprisingly, our findings indicate that lethal infections are associated with massive overstimulation of Th1 cytokines, which probably contribute to pathology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD1 mice were obtained from Charles River Laboratories (Wilmington, MA). Double-knockout mice lacking both TNFR I (p55) and TNFR II (p75; strain name, B6;129S-Tnfrsf1atm1ImxTnfrsf1bm1Imx), referred to as TNFR knockout (KO)4 mice, and control mice representing a cross between C57BL/6J and 129 (strain name, B6129SF2/J), referred to as B6/129 controls, were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice with normal responsiveness to LPS (strain name, C3H/HeOuJ MMTV-) and LPS-hyporesponsive mice (strain name, C3H/HeJ MMTV-) were obtained from The Jackson Laboratory. Fas-deficient mice (B6.MRL-Tnfrs6lpr), referred to as B6-lpr, and control C57BL/6J mice were obtained from The Jackson Laboratory. Animals were housed under specific pathogen-free conditions at Washington University School of Medicine (St. Louis, MO). Female mice between 8 and 12 wk of age were used for experiments.
Parasite growth
T. gondii strains were maintained by serial 2-day passage of tachyzoite stages in human foreskin fibroblast monolayers as described previously (35). The RH strain (36) was used as a representative of the type I lineage. A cloned line of the ME49 strain, called PTG (37), was used as a representative of the type II lineage. All parasite strains were tested for Mycoplasma contamination using the GenProbe kit (Fisher Scientific, Pittsburgh, PA) and remained negative throughout the experiments.
Parasite numbers in tissue and blood were determined every other day starting on day 2 throughout the acute stage of infection. Mice were sacrificed, and lungs, hearts, livers, spleens, mesenteric lymph nodes, peripheral lymph nodes, ileums, kidneys, peritoneal fluid, and brains were isolated. Tissue samples were homogenized using a saw-tooth generator at low speed (PowerGen 125, Fisher Scientific) under conditions that did not affect parasite viability (data not shown). The number of viable parasites in tissues was determined by plaque formation on fibroblast monolayers (duplicate 12-well cultures per sample) (38).
Histopathology
Tissues were fixed in 4% buffered formaldehyde and embedded in paraffin wax, and 5-µm serial sections were stained with H&E or periodic acid-Schiff (PAS) according to standard procedures. Serial sections were deparaffinized, rehydrated, and stained with specific primary Abs followed by use of the Vectastain Elite ABC HRP kit and Vector DAB or Vector Red substrates (Vector Laboratories, Burlingame, CA). Liver sections were screened for lipid accumulation by staining frozen sections with Oil-Red-O. H&E-stained liver sections were blindly scored for pathology.
Demonstration of apoptosis in situ
Apopotosis was detected by TUNEL labeling using the In Situ Cell
Death Detection Kit AP (Roche, Indianapolis, IN). Formalin-fixed,
paraffin-embedded sections of spleen or liver from control and infected
mice were labeled by TUNEL reaction and visualized using Vector Fast
Red, which provides colorimetric and fluorescence signals (Vector
Laboratories). Apoptosis was quantified by counting the number of
apoptotic cells per total nucleated cells within lymphoid follicles.
Five random fields at x20, consisting of 400 cells/field, were
counted from three or four mice per group, and the values are reported
as the mean ± SE.
For FACS-based analysis of apoptosis, cells were stained with Alexa 488-conjugated annexin V, and syntaxin green was used to identify dead cells (Molecular Probes, Eugene, OR). Stained cells were analyzed on a FACScan and evaluated using CellQuest software (Becton Dickinson, Cockeysville, MD).
Serum liver enzymes and cytokines
Blood was collected by cardiac puncture, and serum was isolated
using separator tubes (Sarstedt, Numbrecht, Germany). Serum enzyme
levels of alanine aminotransferase (ALT) and aspartate aminotransferase
(AST) were quantified using a commercial kit (Sigma, St. Louis, MO)
that was adapted for use in 96-well plates. Serum levels of IFN-,
IL-18, TNF-, IL-12 p40, IL-10, and IL-1
were assayed using
OptEIA, two-site ELISA sets from BD PharMingen (San Diego, CA). The
sensitivities of detection in the ELISAs were 31.3 pg/ml for IFN-
and IL-12 p40, and 62.5 pg/ml for IL-18, TNF-, IL-10, and
IL-1.
Antibodies
The following cell markers were detected using biotin-conjugated
Abs obtained from BD PharMingen (La Jolla, CA): Ly-6G (Gr-1) was
detected with mAb RB6-8C5, NK cells were detected using mAb DX5, CD8
cells were detected with mAb 53-6.7, CD4-positive T cells were detected
with mAb GK1.5, B cells were detected using mAb RA3-6B2 specific for
CD45R/B220, and TCR was detected with mAb H57-597. PE-conjugated mAb
GL3 to 
TCR was obtained from BD PharMingen. Unlabeled rabbit
polyclonal antiserum against murine inducible NO synthase (iNOS) was
obtained from BD Transduction Laboratories (Franklin Lakes, NJ). mAb
FA-11 to CD68 was obtained from Serotec (Oxford, U.K.). The F4/80
hybridoma was obtained from American Type Culture Collection
(Manassas, VA).
Ab neutralization of cytokines
For IL-18 neutralization, female CD1 outbred mice were given 200
µl i.p. of rabbit anti-IL-18 antiserum (39), rabbit
anti-GST (40) as an irrelevant control antisera, or
sterile pyrogen-free PBS. A hamster mAb H22, (41) against
murine IFN- was used to neutralize IFN-. A hamster mAb TN3-19.12
against murine TNF- was used to neutralize TNF-. Purified hamster
IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and sterile
pyrogen-free PBS were used as controls for neutralization with hamster
Abs. Five mice per group were used for each time of death study and
three to five additional mice per group were sacrificed on day 9
postinfection for determination of pathology and serum concentrations
of cytokines and liver transaminases.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have established that a single viable
type I strain parasite is sufficient to cause death in the mouse
(7, 8). To establish a model for examining acute
virulence, mice were challenged i.p. with low doses of the type I RH or
type II PTG strain of T. gondii. Challenge with 100
organisms of the RH strain resulted in 100% mortality within 8–9
days. In contrast, 80% of mice given a similar dose of the PTG strain
survived beyond 20 days (Fig. 1
A). Challenge with a high
dose of PTG (105 parasites) resulted in 80%
mortality (Fig. 1A). Differences in virulence in vivo were
not a result of inherent differences in viability, as shown by the
similar capability of the strains to cause plaques on host cell
monolayers in vitro (data not shown). This model, which discriminates
between strains that are virulent regardless of dose (type I) from
those which show a dose-dependent lethality (type II), was used to
evaluate the acute virulence of toxoplasmosis.
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We reasoned that the acute virulence of type I T.
gondii strains might be due to a greater ability to disseminate
and/or replicate in vivo relative to type II strains. To examine this
possibility, mice were challenged with a low dose (100) of RH or PTG
strain parasites, and the number of viable parasites in blood and
tissues was determined at intervals postinfection by plaque formation
in vitro. RH strain (Fig. 1
B) and PTG strain (Fig. 1C) parasites were equally capable of dissemination in vivo
and did not show tissue tropism despite a slight delay in dissemination
to the brain compared with other tissues. RH strain parasites did have
a slight replication/survival advantage in vivo, and peak parasite
numbers were achieved in 6 days compared with 8 days with PTG
infection. Importantly, the initial growth rate was similar, and peak
numbers of viable parasites achieved by the two strains differed by
only approximately 10- to 20-fold.
To determine whether lethal infection with PTG strain
parasites induced significantly higher tissue burdens than nonlethal
infections, mice were challenged with a low dose (100) or a high dose
(105) of PTG strain parasites, and parasite
burdens were determined (Fig. 1, C and D). Peak
parasite numbers were achieved in 4 days after high dose challenge with
PTG strain parasites, although the maximum levels were less than a log
greater than with low dose challenge (Fig. 1, C and
D). Collectively, these results indicate that a threshold
number of parasites is associated with mortality and that the timing in
reaching this tissue burden is critical to survival.
Lethal toxoplasmosis is associated with liver and lymphoid damage
To explore the pathology induced during acute toxoplasmosis, tissues were examined at intervals postinfection. Significant pathology during lethal RH strain infections was restricted to the liver and lymphoid tissue and was accompanied by peritonitis and accumulation of plural fluid. Lethal infections with a high dose of PTG strain parasites generally induced similar pathology as that with RH strain parasites, although to a lesser extent. In contrast, low dose challenge with PTG strain parasites induced little tissue pathology during acute infection.
To quantify liver damage during infection, the levels of
hepatocyte cytoplasmic enzymes released into the serum were determined.
Levels of AST were elevated 10–15 times over control levels during
lethal infections with either RH or PTG strain parasites (Fig. 2B and Table I). In contrast, AST levels were only
modestly elevated during nonlethal infections with a low dose of PTG
strain parasites (Fig. 2B and Table I). The level of the
liver-associated enzyme ALT was 10–12 times higher in RH-infected mice
compared with control mice and 5 times higher than that in mice
infected with either high or low doses of PTG strain parasites when
sampled on days 8–9 (Fig. 2A and Table I).
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C). Bilirubin and
-glutamyl transpeptidase levels were also unaltered relative to
control values, indicating that liver damage was not a consequence of
bile duct obstruction or hepatobiliary damage, respectively (Fig. 2C). Liver damage is independent of hepatocyte apoptosis and intracellular infection
Histopathological examination of the liver in mice
infected with RH strain parasites revealed generalized hepatocyte
enlargement, cytoplasmic vacuolization, and loss of sinusoid
architecture along with regions of cellular infiltration and foci of
coagulative necrosis (Fig. 3A,
Table I, and data not shown). Parasites were restricted to regions of
cellular infiltration, but were seldom detected within hepatocytes,
indicating that liver pathology was independent of cellular infection
(Fig. 3A). Immunohistochemical staining identified the
cellular infiltrate to consist largely of F4/80+
macrophages; however, only a subset of the macrophage infiltrate
appeared fully activated, as evidenced by low levels of iNOS expression
(Fig. 3A).
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A and Table I).
Macrophage infiltration and iNOS production in the liver occurred to a
similar extent and with a similar pattern regardless of the strain or
dose of infecting parasite (Fig. 3A). Collectively, these
findings indicate that the liver damage associated with lethal
infections by the RH strain was widespread and probably due to a
soluble mediator.
To evaluate whether hepatocyte apoptosis contributed to liver
damage during lethal infection, liver sections from RH strain-infected
mice were labeled by TUNEL staining. Very few TUNEL-positive cells were
detected, indicating that liver damage was not a consequence of
hepatocyte apoptosis (Fig. 3B).
To identify the mechanism of hepatocyte vacuolization during
lethal infection, liver sections from RH strain-infected mice were
stained histochemically to distinguish glycogen deposition (PAS
positive) from fatty liver degeneration (Oil Red O positive), or
hydropic degeneration (PAS and Oil Red O negative). The staining
pattern shown in Fig. 3B indicated that cytoplasmic
vacuolization was due to a combination of fatty liver degeneration and
hydropic degeneration and was not a consequence of glycogen
deposition.
Destruction of lymphoid follicles accompanies lethal infection
Lethal T. gondii infections led to a marked
acellularity in lymphoid compartments and loss of tissue architecture
in the spleen, peripheral lymph nodes, mesenteric lymph nodes, and
Peyer’s patches (Fig. 4A and
data not shown). Following lethal infection with either a low dose of
RH strain or a high dose of PTG strain parasites, loss of cells in the
spleen occurred in clusters that were concentrated in follicle regions
(Fig. 4A and data not shown). TUNEL staining of spleen
sections indicated that cell loss was due to extensive apoptosis or
late stage necrosis (Fig. 4A). Staining serial spleen
sections with TUNEL vs anti-T. gondii Ab revealed that
cell death did not occur in infected cells but, rather, occurred
primarily at sites distal from parasite replication. To identify the
cell types that were deleted during RH strain infection, spleen cells
were doubly stained with annexin V and cell type-specific Abs and were
analyzed by FACS. Increased levels of cell death in the spleen were
largely confined to T cells and NK cells (Fig. 4B).
In contrast, infection with a low dose (100) of PTG strain parasites
induced considerably less destruction and stimulated germinal center
formation in the spleen (Fig. 4A and data not shown).
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C). Significantly
less apoptosis was observed in RH strain-infected TNFR KO mice compared
with B6/129 controls (Fig. 4C); however, there was no change
in survival (Fig. 4D) or serum cytokine levels (data not
shown). Apoptosis in control C57BL/6J mice infected with RH strain
parasites was elevated to approximately 14% vs 1–2% in noninfected
mice. The level of apoptosis was not appreciably changed in
Fas-deficient mice (B6-lpr; 11%) infected with RH strain
parasites (Fig. 4C), and there was no difference in survival
(Fig. 4D). Therefore, the apoptosis of lymphocytes
associated with infection by RH strain parasites was predominantly
mediated through TNFRs and was independent of Fas.
High serum levels of IL-18 and IFN- correlate with lethality
The systemic nature of the liver and lymphoid pathology
during lethal infections with RH strain suggested that tissue damage
might be a consequence of overinduction of inflammatory cytokines. To
determine whether lethal infections with T. gondii induced
greater systemic levels of inflammatory cytokines than nonlethal
infections, serum levels of IL-12, IFN-, IL-18, IL-1, and TNF-
were determined throughout acute infection. Extremely high serum levels
of IL-18, IFN-, TNF-, and IL-12 were induced during lethal
infections caused by a low dose (100) of RH strain or a high dose
(105) of PTG strain parasites (Fig. 5, A–D). During lethal
infections, serum levels of IL-18, IFN-, and TNF- increased until
death, whereas IL-12 levels peaked and then decreased (Fig. 5, A–D). During RH infection, IL-1 increased until death,
whereas during high dose PTG infection, levels of IL-1 declined
following an initial rise (Fig. 5E). In contrast, nonlethal
infection with a low dose of PTG strain parasites induced almost no
IL-18 or IL-1 and substantially less IFN- (100 ng/ml; Fig. 5, A–E). TNF- and IL-12 were elevated to similar levels
during both lethal and nonlethal infections, although the increases
occurred later in nonlethal infections (Fig. 5, C and
D).
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F). Therefore, overproduction of IL-18 and IFN- during
toxoplasmosis is unlikely to be a consequence of inadequate regulation
by IL-10. Disease progression is independent of LPS or bacterial sepsis
Although there was minimal pathology in the small
intestine during lethal toxoplasmosis, mice did develop pronounced
peritonitis. Thus, a possible trivial explanation for death of mice
during late stage toxoplasmosis could be a breach in the intestinal
barrier resulting in bacterial sepsis. Consequently, we examined
whether LPS contributed to the virulence of RH strain parasites by
infecting mice that were genetically unresponsive (C3H/HeJ) or fully
responsive (C3H/HeOuJ) to LPS. C3H/HeJ mice displayed no difference in
survival, serum liver enzymes, or serum cytokine levels compared with
LPS-responsive C3H/HeOuJ mice (Fig. 6A and data not shown).
Furthermore, when mice were challenged with RH strain parasites by the
s.c. route, they developed similar liver and lymphoid pathology that
was associated with elevated cytokines and liver damage (data not
shown). Collectively, these results indicate that liver damage,
proinflammatory cytokine induction, and lethal infections caused by RH
strain were not due to endotoxemia or bacterial sepsis.
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The high levels of Th1 cytokines produced during lethal
infection suggest that cytokine-induced shock was responsible for
death. Therefore, we attempted to mitigate the severity of disease by
administering neutralizing Abs to IFN-, TNF-, or IL-18 during
infection with RH strain parasites. Neutralization of IL-18, but not
control antiserum (anti-GST) or PBS, enhanced the survival of
infected mice by up to 2 days, a result that was observed consistently
in three separate experiments (p 
0.01, by
Student’s t test; Fig. 6B). Prolonged survival
was not accompanied by decreased serum levels of IFN- and TNF-
(data not shown) or a decrease in liver or lymphoid pathology (Table I). Moreover, treatment with anti-IL-18 did not alter parasite
tissue burdens (data not shown). These results suggest that the
beneficial effects of neutralizing IL-18 are largely independent of
high levels of TNF- or IFN-.
To directly test the role of elevated IFN-, RH
strain-infected mice were given 0.5 mg of a neutralizing Ab against
IFN- (mAb H22) or control hamster IgG on day 5 postinfection. Ab
neutralization was successful in causing more than a 10-fold reduction
in serum IFN- levels, resulting in a lower level (
5 ng/ml) in
treated mice than that induced by low dose, nonlethal PTG strain
infection (70 ng/ml). However, no change was evident in the numbers
of parasites in the tissues or in survival or liver damage in mice
receiving anti-IFN- Ab relative to controls (Fig. 6C
and Table I). When a higher dose of mAb H22 was used (1 mg given on day
5 postinfection), a 5-fold increase in parasite numbers in the tissues
was observed, and there was no change in survival (data not shown).
While treatment with the lower dose of mAb H22 did not affect liver
pathology, there was a significant decrease in hepatocyte vacuolization
with the higher dose of neutralizing Ab (Table I). Neutralization of
TNF- during RH strain infection using mAb TN3-19.12 (0.25 mg given
on day 5 postinfection), also had no effect on survival relative to
that of mice receiving control hamster IgG (Fig. 6C).
Furthermore, in TNFR KO mice, similar liver damage occurred, indicating
that pathology during lethal infection was not dependent on signaling
through TNFRs (Table I). Collectively these results indicate that IL-18
contributes to pathology during toxoplasmosis and suggest that its
detrimental effects are independent of IFN- or TNF-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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parasites/g tissue). In contrast, low dose infections with the type II
PTG strain were delayed in reaching this threshold, and lethality was
only observed with a higher initial inoculum. Unexpectedly, lethal
infections were associated with overinduction of inflammatory cytokines
rather than an insufficient immune response. Lethality was associated
with excessive levels of Th1 cytokines, particularly IL-18 and IFN-,
in the serum. Our findings indicate that acute virulence in T.
gondii is associated with overstimulation of Th1 cytokines, which
paradoxically are also required for protection.
Following primary infection, type I and type II strains of
T. gondii did not differ substantially in their ability to
disseminate in vivo or to reach similar tissues. However, a key
property of the RH strain of T. gondii is the dramatic
increase in serum cytokine levels after low dose challenge, whereas the
PTG strain required a much higher challenge dose
(105 parasites). This difference may be due to
the ability of a low dose of RH strain parasites to rapidly reach
tissue levels of 106 parasites/g tissue
(within 4 days), whereas PTG strain parasites only reached this level
at 6–8 days postinfection. This difference was overcome by
administering a high initial dose of PTG strain parasite, resulting in
a faster increase in tissue burdens and a lethal outcome. Collectively,
these results suggest that the early interaction of the parasite with
the innate immune response is critical to triggering the lethal cascade
of cytokines. The basis for the dramatic difference in virulence
between type I (RH) and II (PTG) strains of T. gondii is
presently unknown, but is probably due to the 1–2% underlying genetic
differences between these strain types (6, 8).
The major site of tissue pathology during lethal toxoplasmosis was the liver. Damage was not directly attributable to intracellular parasite replication or apoptosis. Instead, lethal infections with RH strain induced widespread alterations in hepatocytes, including enlargement, cytoplasmic vacuolization, and release of liver enzymes. These results suggest that liver damage is mediated through a soluble parasite-derived factor(s) or an induced host factor(s) that reaches toxic levels during lethal infections. Liver pathology is probably due in part to elevated cytokine levels; however, additional factors may also contribute, as mice infected with lethal doses of RH strain vs PTG strain showed similar elevated cytokines, but developed different levels of tissue damage.
Lethal toxoplasmosis also induced extensive necrosis/apoptosis
of noninfected cells within lymphoid tissues. Apoptosis was selective
for T cells and NK cells, and was predominantly mediated through
TNF-, as shown by the significant reduction in apoptotic cells in
TNFR KO mice challenged with RH strain T. gondii. It has
previously been reported that direct infection by T. gondii
prevents activation of apoptosis within the parasitized host cell
(42). Nonetheless, splenic CD4+ T
cells isolated from mice challenged with high doses of ME49 (PTG)
strain parasites undergo apoptosis in vitro, resulting in diminished
immune responsiveness (43). Collectively, these studies
indicate that infection by T. gondii protects the resident
cell from apoptosis, but cell death is efficiently activated in
noninfected cells. Bacterial sepsis is also frequently accompanied by
profound depletion of lymphocytes associated with apoptosis in the
white pulp of the spleen, and this response is detrimental to host
survival (44, 45). In contrast, during acute
toxoplasmosis, apoptosis occurred only late in infection. Combined with
the similar outcome of infection in TNFR KO mice, which succumb at the
same rate despite exhibiting less apoptosis, these findings indicate
that apoptosis is probably a secondary consequence of pathogenesis
during toxoplasmosis rather than causal.
Previous studies have shown that infection with T.
gondii drives a potent Th1 response that is necessary for control
of infection in the mouse. Induction of IL-12 (22, 23, 24),
which drives production of IFN- (26, 30), is essential
to control parasite replication and prevent death due to toxoplasmosis
in the murine system. Consistent with this, infection of mice with a
low dose of type II strain (PTG) parasites resulted in an immune
response characterized by moderate levels of IFN-, IL-12, and
TNF- and leading to eventual control of parasite replication and
minimal tissue pathology. In marked contrast, lethal infections were
associated with systemic induction of extremely high levels of IL-18,
IFN-, and IL-12.
The finding that pathology during acute toxoplasmosis was associated with an overstimulation of the immune system was surprising, as previous models have suggested that lethality of type I strains was due to the inherent virulence of the parasite. However, this finding does have precedent in previous studies using C57BL/6 mice, which are uniquely susceptible to infection by normally nonlethal doses of the type II strain ME49 when administered orally (46, 47). Importantly, pathology in this previously described model differs substantially from the results of studies reported here using the RH strain, where 1) damage is systemic rather than localized to the gut, 2) expression is independent of host genotype rather than highly specific, 3) and disease is independent of route of challenge. Thus, our findings indicate that rather than being a limited case, overinduction of Th1 cytokines may be an important general mediator of pathology in toxoplasmosis, especially during infection by type I strains.
Although Th1 cytokines are essential for parasite control,
this response must be tightly regulated to prevent lethal
immunopathology. Evidence for this is provided by IL-10 knockout mice,
in which liver damage and death occur when mice are challenged with
normally nonlethal type II strain parasites as a consequence of
overinduction of IFN-, IL-12, and TNF- (48). In the
present study high serum levels of Th1 cytokines during lethal
toxoplasmosis in wild-type mice was not due to an absence of
down-regulatory IL-10, since this cytokine was also substantially
elevated in serum during lethal infection. Recent studies indicate
IL-10 can be proinflammatory during human endotoxemia
(49), suggesting that during lethal toxoplasmosis in mice,
IL-10 could act to further enhance the induction of inflammatory
cytokines.
In contrast to other Th1 cytokines, high levels of IL-18 were
only observed in mice destined to succumb to infection, and in the case
of mice infected with RH strain parasites, neutralization of this
cytokine prolonged survival. This effect, while only transiently
protective, is nonetheless highly significant given the extreme
virulence of the RH strain, which causes death with a lethal dose of a
single viable organism. Despite prolonging survival, anti-IL-18 was
not able to protect mice against liver damage, and these mice
ultimately died, suggesting that the cumulative effects of high levels
of other inflammatory mediators also contributed to death. Indeed, the
high levels of IFN- and IL-12 would be expected to result in
considerable pathology, and previous studies have shown that
coadministration of IL-12 and IL-18 is capable of causing death in mice
(50, 51).
IL-18 is related to IL-1 and shares biological properties with
IL-12, including induction of IFN- and enhancement of Th1 responses
(52, 53). IL-18 and IL-12 synergize to promote high levels
of IFN- production, and this leads to a more effective immune
response against certain intracellular pathogens (54, 55).
However, overproduction of IL-18, as occurs in murine models of
endotoxemia, results in lethal liver damage (56) and
splenocyte apoptosis (50, 56). IL-18 exerts its toxic
activity through overproduction of IFN- and through
IFN--independent mechanisms (50, 56). In the present
study neutralization of toxic levels of IL-18 that occurred during
lethal toxoplasmosis did not result in decreased IFN- or reduction
of hepatic damage, suggesting that the detrimental effects of IL-18 in
toxoplasmosis are independent of IFN-.
The elevated levels of cytokines preceding death due to acute
toxoplasmosis resemble systemic infections with Gram-negative bacterial
pathogens in that both stimulate high levels of IL-1, TNF-, and
IL-18. However, T. gondii differs from these bacterial
pathogens in several important traits. First, it does not contain LPS,
and the innate immune response to parasites such as T.
gondii is not mediated by Toll-like receptor 4, the classical
pattern recognition receptor for bacterial LPS. This conclusion is
supported by the similar outcome of T. gondii infection in
C3H/HEJ mice, which are genetically unresponsive to LPS due to a mutant
allele of the Toll-like receptor 4 (57). Second, the
production of IFN- that is necessary for control of infection by
T. gondii relies primarily on IL-12, but only minimally on
IL-18 (58). In contrast, IL-18 is required for induction
of an inflammatory pathway that results in control of shigellosis
(59). Third, endotoxemia leads to liver damage that is
typically associated with apoptosis mediated by Fas and TNFR
(60), while the hepatic damage in toxoplasmosis was
independent of these mediators.
The potent induction of Th1 cytokines during toxoplasmosis suggests that T. gondii contains a substance that is a potent inflammatory mediator. Importantly, our results cannot be ascribed to contamination with Mycoplasma lipopeptide, which has potent immunostimulatory properties (61), as we took extreme care to avoid contamination throughout this study. There are two possible models to explain the massive inflammatory response that occurs during toxoplasmosis: 1) the host responds to a substance produced by the parasite, analogous to the inflammatory response generated to bacterial LPS (yet clearly distinct); or 2) pathology is a secondary effect due to release of endogenous triggers from infected and/or damaged host cells, as has been suggested previously (62). Further studies to elucidate the mechanism of immune damage during acute toxoplasmosis could have important implications for the treatment of parasitic infections.
Note added in proof.
Independently, another group has recently reported that
RH strain infection induces splenocyte apoptosis and high levels of
IFN- (63).
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Acknowledgments |
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and TNF-. We are grateful to Robert
Schreiber and Richard Hotchkiss for critical review of the manuscript,
and to Stacie Gooch for expert technical assistance. |
Footnotes |
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2 Current address: Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011.
3 Address correspondence and reprint requests to Dr. L. David Sibley, Department of Molecular Microbiology, Box 8230, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: sibley{at}borcim.wustl.edu
4 Abbreviations used in this paper: KO, knockout; ALT, alanine aminotransferase; AST, aspartate aminotransferase; iNOS, inducible NO synthase; MLN, mesenteric lymph nodes; PAS, periodic acid-Schiff; PLN, peripheral lymph nodes.
Received for publication June 15, 2001. Accepted for publication August 9, 2001.
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