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and Early Death in C57BL/6 Mice1
Departments of
*
Microbiology and Immunology and
Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and NO response relative to
animals infected with T. gondii alone. Plasma levels of
TNF-
and aspartate transaminase in double-infected mice were greatly
elevated relative to mice infected with either parasite alone.
Consistent with the latter observation, these animals exhibited severe
liver pathology, with regions of coagulative necrosis and hepatocyte
vacuolization unapparent in mice carrying either infection alone.
Interestingly, mean egg granuloma size was 
50% of that in mice with
S. mansoni infection alone. The exacerbated liver
pathology in coinfected mice did not appear to be a result of
uncontrolled tachyzoite replication, because both parasite-specific
RT-PCR analysis and immunohistochemical staining demonstrated a low
number of tachyzoites in the liver. We hypothesize that mortality in
these animals results from the high level of systemic TNF-, which
mediates a severe liver pathology culminating in death of the
animal. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and NO, most
likely resulting from absence of the anti-inflammatory activity of
IL-4 (1). To a large extent, host control of infection
appears to be directed toward preventing a massive inflammatory
response to the parasite and its products, rather than limiting
presence of the parasite itself. This situation contrasts with that occurring during infection with another even more widespread parasite, the intracellular protozoan, Toxoplasma gondii (Tg). This microbial pathogen is a major opportunistic infection in immunocompromised individuals, and congenital infection can lead to severe birth defects. Under normal conditions an asymptomatic, chronic infection is established that is associated with the presence of quiescent cysts located in tissues of the skeletal muscle and CNS. Nevertheless, the ability to survive toxoplasmosis requires an ongoing vigorous type 1 cytokine response and strong cell-mediated immunity (2). The cytokine IL-12 is crucial in initiating these responses to the parasite (3, 4, 5).
Control of toxoplasmosis is thought to involve activation of
macrophages or macrophage-related cells by parasites and the
inflammatory cytokines TNF- and IFN-. These activated cells
display potent microbicidal effector functions such as production of NO
(6, 7, 8, 9). Thus, the inability to mount a strong type 1
cytokine response, either through anti-cytokine mAb administration
or cytokine gene disruption, results in death associated with high
parasitemia and dissemination throughout host tissues
(10, 11, 12, 13, 14, 15, 16, 17). Nevertheless, in certain experimental situations
such as in IL-10 KO and D-galactosamine-sensitized mice, Tg
appears to induce a lethal inflammatory cytokine response
(18, 19, 20). Therefore, a successful host response to the
parasite requires induction of inflammatory cytokines, but the latter
must be tightly regulated to prevent host pathology associated with
overproduction of these mediators.
Type 1 and type 2 cytokines are well known for their cross-regulatory
properties (21). For example, IL-4 and IL-10 antagonize
IFN--induced macrophage activation, and IFN- itself displays
anti-proliferative activity toward Th2 cells (22).
However, less well understood is how these opposing responses interfere
with each other during the course of normal infections when the host
simultaneously harbors type 1- and type 2-inducing pathogens. This is
surprising because the worldwide prevalence of protozoan and helminth
infections in humans would predict a vast number of coinfected
individuals.
We have recently become interested in using murine models of Sm and Tg infection to address this important issue. In particular, we wished to determine how the murine host responds to orally administered Tg when the animals are already undergoing a type 2-dominated gut response to chronic Sm infection. Two opposite outcomes could be envisioned based upon previous studies. First, in an Sm-induced Th2 environment, Toxoplasma could fail to elicit protective Th1 effector cells, resulting in increased tachyzoite growth and host susceptibility. Second, an ongoing Sm egg-driven type 2 cytokine response in the intestinal mucosa could prevent Tg-induced inflammatory gut pathology which occurs in mice such as those of the C57BL/6 strain (23). This hypothesis predicts that Sm infection would confer resistance to the detrimental effects of Tg in the gut.
The results presented here provide evidence that underlying Sm
infection does indeed minimize Tg-induced inflammatory gut pathology
and prevent the appearance of serum IFN- and NO. Nevertheless,
TNF- levels were higher in the double-infected mice, and the animals
rapidly succumbed, showing signs of catastrophic liver damage.
Interestingly, the pathology of double-infected mice was not associated
with greater numbers of tachyzoites in the liver. Additionally, in
Sm-infected animals administered a low Tg dose, establishment of cysts
in the brain was equivalent to mice receiving Tg alone. These results
indicate that increased tachyzoite burden per se is not responsible for
increased susceptibility of the animals, and they suggest that a
secondary factor, possibly TNF-, mediates lethality.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 strain and outbred Swiss-Webster mice (6–8 wk of age) were obtained from Taconic Farms (Germantown, NY) and The Jackson Laboratory (Bar Harbor, ME), respectively. Animals were housed under specific pathogen-free conditions in the College of Veterinary Medicine animal facility at Cornell University.
Parasites and infections
The ME49 Tg strain was maintained by i.p. inoculation of Swiss-Webster mice with brain homegenate from mice that had been infected 6–8 wk earlier. Before injection, brain suspensions were adjusted to give a concentration of 20 cysts/mouse. For experimental infections, 250 µl of cyst-containing brain homogenate from infected mice was administered by gavage to ether-anesthetized mice.
Cercariae of Sm (NMRI strain) were maintained in Biomphalaria glabrata snails (kindly supplied by Dr. F. Lewis; Biomedical Research Institute, Rockville, MD). Cercariae were recovered by shedding, then were counted and immediately used for infection. For infection, C57BL/6 animals were ether-anesthetized, their abdomens shaved, and subsequently exposed to 70 cercariae.
Cytokine measurements
To measure plasma cytokines, blood was collected in EDTA-containing tubes from the tail vein or by cardiac puncture, centrifuged (12,000 x g, 10 min at 4°C), and the resulting plasma was stored at -70°C until day of assay.
IFN- was measured by a two-site ELISA using plate-bound mAb HB170
(anti-IFN-), a rabbit polyclonal anti-mouse IFN-, and
peroxidase-conjugated donkey anti-rabbit Ig (Jackson ImmunoResearch
Laboratories, West Grove, PA) as described in detail elsewhere
(14). Absorbances (405 nm) were measured on a Microplate
Bio Kinetics Reader (Bio-Tek Instruments, Winooski, VT) and compared
with known amounts of recombinant IFN- standard (Genzyme, Cambridge,
MA). TNF- levels were measured using a mouse-specific TNF- ELISA
kit according to the manufacturer’s instructions (Genzyme). The
sensitivities of detection in the ELISAs were 10 pg/ml (IL-12) and 15
pg/ml (TNF-).
Plasma NO
A modified Griess reaction was employed to measure serum NO levels as described elsewhere (20). Briefly, blood was collected, centrifuged at 12,800 x g for 5 min, and 100 µl of the resulting plasma was added to a suspension of E. coli in 1 M HEPES (Sigma, St. Louis, MO) with 3 M formate (Sigma). Bacteria were prepared in a nitrogen-rich environment to induce high levels of nitrate reductase activity, then resuspended in PBS and stored at -70°C. The bacteria-plasma suspension was incubated (1 h, 37°C), centrifuged to pellet bacteria (3 min, 12,800 x g), and the resulting supernatant was transferred to a 96-well plate with 100 µl of a 1:1 mixture of sulfanilamide (1% in 2.5% H3PO4) and napthlethylenediamine dihydrochloride (0.1% in 2.5% H3PO4). The absorbance was measured at 600 nm and compared with standard concentrations of NaNO2.
Plasma transaminase assay
To measure the liver-associated enzyme aspartate transaminase
(AST) in plasma, a protocol modified from a commercial kit (Sigma) was
employed (20). Briefly, 20 µl of plasma was added to 100
µl of 0.2 M DL-aspartate and 1.8 mM -ketoglutaric acid
in PBS, tubes were mixed and incubated (37°C, 1 h), then 100
µl 2,4-dinitrophenylhydrazine (DNP) was added, and the mixture
incubated a further 20 min at room temperature. The reaction was halted
with the addition of 1 ml 0.4 N NaOH, and sample absorbances were
measured at 490 nm after 5 min.
Serum IgE
Total plasma IgE levels were measured using a two-site ELISA as previously described (1). Briefly, plates coated with an anti-IgE mAb (EM-95; 50 ng/well) were incubated with plasma samples and a standard curve generated with known concentrations of purified mouse IgE mAb. Bound serum IgE was detected with a FITC-conjugated anti-mouse IgE mAb (PharMingen, San Diego, CA) followed by a HRP-conjugated sheep anti-FITC mAb (Amersham, Arlington Heights, IL) and 2,2-azinodi-(3-ethylbenzthiazoline-6-sulfonic) (ABTS) substrate. Absorbances were measured at 405 nm.
RT-PCR analysis
Livers from mice were collected and homogenized in RNA STAT 60 (Teltest, Friendswood, TX). RNA was isolated as described (8) and reverse transcribed using 3'-specific primers in a modification of a previously published protocol (24). Briefly, 7 µg RNA was incubated at 65°C for 5 min and then chilled on ice. The following were subsequently added to the sample: 5 µl 5x RT buffer (Life Technologies, Grand Island, NY), 2.5 µl dNTP (2.5 mM; Sigma), 1 µl RNasin (Promega, Madison, WI), 0.5 µl Superscript II (Life Technologies) and 1 µl 3' primers (100 µM; hypoxanthine phosphoribosyltransferase (HPRT) and SAG-2 primer sequences from Ref. 8). After 5 min incubation (at room temperature), the reaction mix was incubated at 42°C for 60 min followed by 52°C for 30 min on an automated thermocycler (MJ Research, Cambridge, MA). Amplification of HPRT and SAG-2 cDNA was conducted as described (8). The PCR products were resolved by electrophoresis in a 2% agarose gel, and DNA bands were visualized by staining with ethidium bromide. A 100-bp ladder was run with samples to confirm that PCR products were of the predicted length.
Histopathological analyses
Livers and gastrointestinal tracts were collected from mice following CO2 asphyxiation and fixed in 10% (w/v) buffered formaldehyde. Tissues were embedded in paraffin wax, cut into 6-µm sections, and stained with hematoxylin and eosin according to standard procedures. Liver sections were screened for lipid accumulation by staining formalin-fixed frozen sections with Oil-Red-O. Tissue sectioning and staining was performed by the Cornell University Department of Biomedical Sciences Histology Laboratory.
TUNEL staining
To detect the presence of apoptotic cells in the liver, tissue sections were subjected to TUNEL employing FITC-conjugated dUTP to visualize positive-staining cells. Reagents for this assay were obtained as a kit (Boehringer Mannheim, Indianapolis, IN) and used according to instructions supplied.
Granuloma volume measurement
The volume of liver granulomas was digitally measured (Scion Image, version 1.60, Sony progressive 3CCD color video camera, x0.45 projection lens; Nikon Optophot microscope, x10). Only granulomas with a single, centrally located egg were chosen for analysis. The diameter of each granuloma was measured six times at 45° angles and the average calculated. Diameter was then used to calculate area. Between 15 and 25 granulomas per mouse were measured.
Statistical analyses
Significant differences in granuloma volumes, AST, NO, and
cytokine levels were determined by Student’s t test or
ANOVA. When required, nonparametric analysis was conducted using the
Wilcoxon signed rank test. Probability values 
0.05 were considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Percutaneous infection with Sm cercariae results in onset of egg
deposition 35 days later, which is associated with an egg-induced
type 2 cytokine response. Tg infection results in an acute infection
7–10 days later, followed by establishment of chronic disease around 1
mo after infection. Control of both stages of murine toxoplasmosis is
associated with a strong type 1 cytokine response. As shown in Fig. 1
, our experimental set-up was to infect
mice with Sm, then 7 wk later initiate Tg infection by oral
administration of cysts of the low virulence ME49 strain.
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When C57BL/6 mice were orally infected with 200 ME49 cysts, 60%
succumbed during acute infection (Fig. 2A). Death in this group was
presumably due to the lethal inflammatory gut pathology previously
reported in this mouse strain (23). When C57BL/6 mice
carrying a chronic Sm infection were infected with the same dose of
ME49, the animals lost weight and all died within 10 days, correlating
with the peak of acute toxoplasmosis (Fig. 2, A and
B). At a 10-fold lower cyst dose, all Sm or Tg
single-infected animals survived, whereas 34% of the double-infected
animals succumbed (Fig. 2C). Both control and Sm-infected
mice displayed severe weight loss during the peak of acute Tg infection
(day 10 post-Tg infection), but double-infected animals continued to
lose weight at later time points, whereas mice with Tg alone regained a
significant amount of their original weight (Fig. 2D). We
conclude that superimposition of Tg infection on mice with chronic
schistosomiasis mansoni results in increased morbidity and
mortality.
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To ask whether Tg infection modulated ongoing Sm-induced cytokine
responses, we used serum IgE as a signature Ab isotype for measuring
the relative level of the underlying type 2 cytokine response. As shown
in Fig. 3, mice with Sm alone displayed
elevated serum IgE at day 9 post-Tg infection. Consistent with the
minimal level of IL-4 normally induced by Tg (14, 25),
infection with this parasite failed to elicit an IgE response. The
serum IgE levels in Sm-infected animals were not affected by subsequent
oral Tg infection.
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We next asked whether appearance of inflammatory mediators in the
serum, which normally accompanies acute toxoplasmosis, was altered by
the presence of an ongoing Sm infection. Fig. 4A shows that serum IFN-
levels are highly elevated in mice undergoing acute Tg infection,
whereas in animals with Sm alone, serum IFN- remained close to
background levels obtained from noninfected mice. When Sm-infected
animals were subsequently challenged with Tg, production of Tg-induced
IFN- was curtailed. This same pattern was repeated for serum NO
levels (Fig. 4B). Thus, pre-infection with Sm prevented the
dramatic increase in both IFN- and NO, which is normally associated
with acute Tg infection.
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levels (Fig. 4
C). While infection with Tg, but not Sm, induced serum
TNF-, the two infections in combination had a profoundly synergistic
effect on levels of this inflammatory cytokine. Thus, TNF- levels
were 4-fold higher in Tg + Sm animals relative to the Tg alone
group. Double-infected mice display improved gut pathology
At day 9 following oral Tg infection, immediately before
onset of death, intestines were removed and processed for
histopathological examination. For mice infected with Sm alone (Fig. 5A), granulomas were
distributed throughout the small intestine, characterized by a
heterogenous accumulation of lymphoid cells surrounding the parasite
egg. Basic gut architecture, in particular the villus-to-crypt ratio,
was essentially normal in these animals. This was not the case for
Tg-infected mice (Fig. 5B). These animals displayed loss of
crypt structure and severe blunting of the villi, a pathology which has
been reported to be associated with endogenous IFN- and is dependent
upon the presence of CD4+ T cells
(23).
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C),
we found evidence that the intestinal pathological changes normally
associated with Tg infection were much less severe, as we had predicted
might be the case. Although some blunting of the villi was evident, the
basic villus and crypt structure was preserved, unlike the case of
animals infected with Tg alone. These data provide evidence that patent
Sm infection does indeed alleviate gut pathology associated with Tg.
Nevertheless, as is clear from Fig. 2, double-infected animals display
a higher rate of morbidity and mortality relative to single-infected
animals, indicating that the partial alleviation of damage to the small
intestine is not sufficient to prevent death. Because the liver is a
major target of Sm egg-induced pathology, and because Tg has severe
pathological effects in the liver after
D-galactosamine sensitization (20),
we turned our attention to this organ. Sm and Tg synergize to promote severe liver damage
We first observed that mice with Sm infection displayed massive hepatomegaly and multiple egg granulomas, visible macroscopically as a white speckling covering the entire organ. In livers from animals with acute toxoplasmosis, hepatic discoloration was apparent, with little or no enlargement of the organ. Livers from double-infected animals displayed severe discoloration, and a reversal of the hepatomegaly normally induced by Sm. At this level, granulomas were also no longer visible (data not shown).
Histopathological examination of the livers of Sm-infected mice
revealed large multifocal eosinophilic granulomas, but in regions
removed from granulomas, liver structure appeared relatively normal
(Fig. 6A). Livers from
Tg-infected animals displayed occasional foci of inflammatory cells,
although the essential structure of this organ was preserved (Fig. 6B). However, in Tg + Sm-infected mice, large regions of
coagulative necrosis and extensive hepatocyte vacuolization were
apparent (Fig. 6C). The extensive vacuolization was
suggestive of fatty change in the liver, indicating an accumulation of
lipid as a result of disrupted metabolism. We confirmed that this was
the case, because cross-sections of livers from double-infected mice
stained strongly with the lipid-sensitive dye, Oil-Red-O, whereas
livers from Sm or Tg single-infected animals displayed very little of
this staining (Fig. 7,
A–C).
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. Thus, livers from coinfected mice
possessed large TUNEL-positive regions (Fig. 8A). This
staining, which is virtually identical with that obtained when normal
tissue sections were pre-incubated with DNase I before staining (Fig. 8B), suggests that heptatocytes within this area have
recently undergone programmed cell death. In situ TUNEL staining of
livers from single infections of Tg and Sm failed to reveal similar
apoptotic regions (data not shown).
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. In mice infected with either Sm or Tg
alone, the liver-associated enzyme AST is present in the serum at 2–3
fold greater levels than in noninfected animals. This result indicates
that hepatic damage normally accompanies both Tg and Sm infections, and
results in leakage into the serum of an enzyme normally confined to the
liver. Nevertheless, in combination, the serum AST level was
approximately seven times higher than background levels. Thus, Sm and
Tg together exert severe pathological effects on the liver.
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, A and C, Sm egg
granuloma size was smaller in double-infected mice, as predicted based
on the absence of visible gross lesions in the livers of these animals
(data not shown). We measured granuloma area in several cross-sections
and found that the lesions were 55% smaller in
double-infected mice (Fig. 10A). Despite the smaller
size of the granulomas, general cellular composition appeared
approximately equivalent. In both cases a broad mix of granulocytes,
lymphocytes, and macrophages were present in the lesions.
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The decreased systemic IFN- in double-infected animals might
suggest a generalized inability to control tachyzoites, but we failed
to find any evidence that this is the case. As shown in Fig. 6C, double infection precipitates major pathological changes
in the liver. Nevertheless, immunohistochemical staining does not show
significant numbers of tachyzoites in either Tg or Sm + Tg groups (data
not shown). Nevertheless, as shown in Fig. 11, RT-PCR amplification of transcripts
for the Tg tachyzoite surface protein SAG-2 (p22) (26)
revealed the presence of Toxoplasma transcripts in both Tg
alone and Tg + Sm-infected animals. Importantly, this analysis
indicated that there was no evidence that SAG-2 transcript levels were
higher in double-infected mice, despite the fact that the latter
display increased morbidity and mortality. Finally, Sm-infected and
control animals were infected with a low ME49 dose, and 30 days later
brains removed and cysts enumerated. As shown in Fig. 10B,
the number of cysts contained within brain tissue of Sm and control
mice was not significantly different.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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as well as IL-12 KO animals die when
infected with Tg (10, 11, 28). This dichotomy in host
response pattern suggests that mice infected with schistosomes might
display high susceptibility to subsequent Tg infection as a result of
Sm-induced Th2 cytokines, because the latter mediators possess
down-regulatory activity on development of Th1 responses
(21). A report several years ago (predating discovery of
Th1 and Th2 cells) indicated double-infected mice do, indeed, display
increased susceptibility (29). In addition, several more
recent reports indicate that Sm infection is capable of affecting
development of type 1 responses to unrelated Ag
(30, 31, 32, 33).
We decided to re-examine the issue of Sm-Tg coinfection in light of
recent data suggesting that C57BL/6 mice orally infected with Tg cysts
die from intestinal inflammation associated with high levels of IFN-
(23, 34). Based upon this model, Sm infection might be
predicted to confer resistance to Tg, because type 2 responses
associated with eggs passing across the intestinal wall would mitigate
IFN--mediated pathology. The latter hypothesis is supported in part
by our data showing that intestinal pathology in double-infected
C57BL/6 animals appears less severe than in those animals infected with
either parasite alone (Fig. 5). Nevertheless, oral inoculation of
Sm-infected mice with Tg resulted in severe morbidity and high
mortality. Death in these animals was accompanied by catastrophic liver
necrosis, suggesting that the primary site of Tg-induced pathology had
shifted from the intestine to the liver.
In many situations, overt type 1 cytokine responses are associated with serious intestinal inflammation (reviewed in Ref. 35). We have hypothesized elsewhere that this places evolutionary pressure on the host to respond to chronic intestinal damage, such as occurs during Sm infection, by mounting a type 2 response consisting of production of the anti-inflammatory cytokines IL-10 and IL-4 (1). Such an environment would tend to prevent the proinflammatory response induced by Toxoplasma, thereby reducing local inflammation, but this cytokine milieu might also be expected to increase local tachyzoite proliferation and promote parasite dissemination. Nevertheless, we failed to find evidence that the Tg burden in coinfected mice was higher, as measured by cysts establishing in the brains of animals given sublethal Tg doses.
To specifically determine whether increased susceptibility after Tg
infection correlated with decreased type 1 immune responses in
Sm-infected mice, we examined plasma levels of IFN-, TNF-, and
NO. As expected, mice infected with ME49 alone displayed elevated
levels of all three of these proinflammatory mediators. In contrast,
plasma levels of IFN- and NO in double-infected animals were no
greater than in mice infected with Sm alone. We do not as yet know the
precise mechanism underlying defective production of IFN- and
NO, but the most likely explanation is that Sm-induced type 2 cytokines
limit development and expression of the type 1 response normally
associated with Tg infection. Although plasma levels of type 1
mediators were reduced in dual-infected animals, it is unlikely that
Tg-specific responses are completely absent because, as mentioned
above, cyst burden in dual-infected mice were no greater than in mice
infected with Tg alone.
Plasma TNF- levels, which were high in mice infected with ME49
alone, were elevated even further in double-infected animals. The
presence of high levels of TNF- in the near absence of IFN- and
NO was unexpected. Nevertheless, it has previously been reported that
schistosome infection primes animals for massive TNF- production
when subsequently exposed to the appropriate triggering molecule
(36). Thus, serum from Sm-infected mice previously
injected i.v. with LPS displays potent tumoricidal activity as a result
of high amounts of TNF-. Because Tg is able to induce TNF- during
the normal course of infection, it seems likely that this pathogen
functions in a manner similar to LPS in inducing TNF- overproduction
in Sm-infected animals. Moreover, while IFN- activated macrophages
are clearly a major TNF- source, other cell types such as mast
cells, Th2 lymphocytes, and B cells can also produce this cytokine
(37). Because all of these cells are present in the
diseased liver of a schistosome-infected mouse (38), it is
possible that Tg triggers one of these cell types to synthesize
TNF-.
Mice dying from dual infection displayed severely diseased livers, with
evidence of steatosis and coagulative necrosis in areas adjacent to egg
granulomas. Although our data do not directly demonstrate that this
severe liver disease is responsible for death in the animals, this
would appear plausible given the severity of the pathology. A seemingly
likely possibility accounting for the exacerbated liver condition of
double-infected mice is that decreased NO and IFN- levels result in
high numbers of replicating tachyzoites and associated tissue
destruction in this organ. However, our results do not support this
hypothesis. Thus, RT-PCR analysis of Tg SAG-2 transcripts in the liver
did not provide evidence for higher tachyzoite numbers in
double-infected mice. In addition, immunohistopathological analysis
using Tg-specific antiserum failed to reveal elevated tachyzoite
numbers in the liver of double-infected mice (data not shown).
In some situations when the liver is stressed, TNF- possesses the
capability of inducing programmed cell death in hepatocytes (39, 40), and it is possible that the high level of this cytokine is
related to the liver damage we report here. We recently reported that
mice sensitized with the hepatotoxin, D-galactosamine, are
exquisitely sensitive to Tg-triggered, TNF--mediated liver
destruction (20). Furthermore, because NO has been
reported to protect against TNF--mediated liver pathology
(41), its absence in the double-infected mice could also
contribute to the severe disease observed. Indeed, nuclear
condensation, which is indicative of apoptosis, is apparent within a
subset of damaged hepatocytes in dual-infected mice. Furthermore,
TUNEL-positive areas of liver from coinfected animals are readily
demonstrable.
Related to this issue, it has recently been shown that inducible NO
synthase KO (iNOS-/-) mice display increased resistance
to Tg during acute infection (42). Thus, it is possible
that during Tg infection, NO production, by virtue of its
immunosuppressive activity, is detrimental to the host, but that in the
Tg-Sm animals its absence results in failure to protect against
TNF--mediated liver pathology. The severe cachexia afflicting
Tg-infected mice also indicates a role for TNF- in the pathology of
acute toxoplasmosis, and anti-TNF- mAb treatment of mice with
lethal acute Tg infection delays time to death (20). We
are currently further examining the role of this inflammatory cytokine
using TNF receptor KO mice infected with both parasites.
The liver pathology in mice undergoing concurrent Tg and Sm infection
is, in part, reminiscent of that occurring in schistosome-infected T
cell deficient mice (43, 44, 45). In the latter animals,
granulomas fail to form and the hepatocytes surrounding the eggs
accumulate lipids and take on a vacuolated appearance. Liver failure
and death rapidly follow. The damage to this organ is believed to be
due to the action of Sm egg-derived hepatotoxins, such as the
glycoprotein designated 
1 (46). In immunocompetent
animals, egg hepatotoxins would be sequestered within the granuloma,
and therefore liver damage is minimized. Thus, the egg granuloma is
believed to be a protective host response which contributes to the
continued functioning of the liver despite a large egg burden. In Sm
and Tg coinfected mice, granulomas were significantly smaller as has
been reported previously (47), and it is possible that
liver damage in these animals was the result of inadequate
sequestration of Sm egg-derived toxins. Why the granulomas should be
smaller in dual-infected mice is unclear, but it is possible that local
effects of a Tg-induced type 1 responses limit the extent of the
largely Th2-mediated granulomatous lesions (48).
Many regions of the world are endemic for severe helminth, protozoan, bacterial, and viral infections. Nevertheless, very little is known regarding immune responses elicited by pathogens simultaneously infecting one host, and the influence of such responses on each other. Similarly, few clinical studies have examined how the immune system responds to coinfection. The studies presented here clearly demonstrate that Tg and Sm, together within one host, synergize to promote a severe pathology that could not be predicted based upon studies with either parasite alone. The grossly dysregulated cytokine production in these animals strongly suggests an immune-mediated component of the lethal pathology. Our current efforts are focused on defining the molecular and cellular basis for the pathology induced by concurrent infection with these pathogens.
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Acknowledgments |
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Footnotes |
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2 A.J.M. and L.R.B. made equal contributions to this work.
3 Address correspondence and reprint requests to Dr. E. J. Pearce, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. E-mail address:
4 Abbreviations used in this paper: Sm, S. mansoni; Tg, T. gondii; AST, aspartate transaminase; KO, knockout; HPRT, hypoxanthine phosphoribosyltransferase.
Received for publication January 5, 1999. Accepted for publication June 7, 1999.
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K. R. Page, A. E. Jedlicka, B. Fakheri, G. S. Noland, A. K. Kesavan, A. L. Scott, N. Kumar, and Y. C. Manabe Mycobacterium-Induced Potentiation of Type 1 Immune Responses and Protection against Malaria Are Host Specific Infect. Immun., December 1, 2005; 73(12): 8369 - 8380. [Abstract] [Full Text] [PDF]
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C.-C. Chen, S. Louie, B. McCormick, W. A. Walker, and H. N. Shi Concurrent Infection with an Intestinal Helminth Parasite Impairs Host Resistance to Enteric Citrobacter rodentium and Enhances Citrobacter-Induced Colitis in Mice Infect. Immun., September 1, 2005; 73(9): 5468 - 5481. [Abstract] [Full Text] [PDF]
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 G L Radford-Smith Will worms really cure Crohn's disease? Gut, January 1, 2005; 54(1): 6 - 8. [Full Text] [PDF]
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O. Liesenfeld, I. R. Dunay, and K. J. Erb Infection with Toxoplasma gondii Reduces Established and Developing Th2 Responses Induced by Nippostrongylus brasiliensis Infection Infect. Immun., July 1, 2004; 72(7): 3812 - 3822. [Abstract] [Full Text] [PDF]
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 N. Ismail, L. Soong, J. W. McBride, G. Valbuena, J. P. Olano, H.-M. Feng, and D. H. Walker Overproduction of TNF-{alpha} by CD8+ Type 1 Cells and Down-Regulation of IFN-{gamma} Production by CD4+ Th1 Cells Contribute to Toxic Shock-Like Syndrome in an Animal Model of Fatal Monocytotropic Ehrlichiosis J. Immunol., February 1, 2004; 172(3): 1786 - 1800. [Abstract] [Full Text] [PDF]
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A. C. La Flamme, K. Ruddenklau, and B. T. Backstrom Schistosomiasis Decreases Central Nervous System Inflammation and Alters the Progression of Experimental Autoimmune Encephalomyelitis Infect. Immun., September 1, 2003; 71(9): 4996 - 5004. [Abstract] [Full Text] [PDF]
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 L. S. MANSFIELD, D. T. GAUTHIER, S. R. ABNER, K. M. JONES, S. R. WILDER, and J. F. URBAN ENHANCEMENT OF DISEASE AND PATHOLOGY BY SYNERGY OF TRICHURIS SUIS AND CAMPYLOBACTER JEJUNI IN THE COLON OF IMMUNOLOGICALLY NAIVE SWINE Am J Trop Med Hyg, March 1, 2003; 68(3): 0 - 0. [Abstract] [Full Text] [PDF]
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 D. Sewell, Z. Qing, E. Reinke, D. Elliot, J. Weinstock, M. Sandor, and Z. Fabry Immunomodulation of experimental autoimmune encephalomyelitis by helminth ova immunization Int. Immunol., January 1, 2003; 15(1): 59 - 69. [Abstract] [Full Text] [PDF]
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L. S. MANSFIELD, D. T. GAUTHIER, S. R. ABNER, K. M. JONES, S. R. WILDER, and J. F. URBAN ENHANCEMENT OF DISEASE AND PATHOLOGY BY SYNERGY OF TRICHURIS SUIS AND CAMPYLOBACTER JEJUNI IN THE COLON OF IMMUNOLOGICALLY NAIVE SWINE Am J Trop Med Hyg, January 1, 2003; 68(1): 70 - 80. [Abstract] [Full Text] [PDF]
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A. J. Buendia, L. Del Rio, N. Ortega, J. Sanchez, M. C. Gallego, M. R. Caro, J. A. Navarro, F. Cuello, and J. Salinas B-Cell-Deficient Mice Show an Exacerbated Inflammatory Response in a Model of Chlamydophila abortus Infection Infect. Immun., December 1, 2002; 70(12): 6911 - 6918. [Abstract] [Full Text] [PDF]
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A. S. MacDonald, M. I. Araujo, and E. J. Pearce Immunology of Parasitic Helminth Infections Infect. Immun., February 1, 2002; 70(2): 427 - 433. [Full Text] [PDF]
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S. K. Bliss, L. C. Gavrilescu, A. Alcaraz, and E. Y. Denkers Neutrophil Depletion during Toxoplasma gondii Infection Leads to Impaired Immunity and Lethal Systemic Pathology Infect. Immun., August 1, 2001; 69(8): 4898 - 4905. [Abstract] [Full Text] [PDF]
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V. Thomas, J. Anguita, S. W. Barthold, and E. Fikrig Coinfection with Borrelia burgdorferi and the Agent of Human Granulocytic Ehrlichiosis Alters Murine Immune Responses, Pathogen Burden, and Severity of Lyme Arthritis Infect. Immun., May 1, 2001; 69(5): 3359 - 3371. [Abstract] [Full Text] [PDF]
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M. I. Araujo, S. K. Bliss, Y. Suzuki, A. Alcaraz, E. Y. Denkers, and E. J. Pearce Interleukin-12 Promotes Pathologic Liver Changes and Death in Mice Coinfected with Schistosoma mansoni and Toxoplasma gondii Infect. Immun., March 1, 2001; 69(3): 1454 - 1462. [Abstract] [Full Text] [PDF]
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A. O. T. Lau, J. B. Sacci Jr., and A. F. Azad Host Responses to Plasmodium yoelii Hepatic Stages: A Paradigm in Host-Parasite Interaction J. Immunol., February 1, 2001; 166(3): 1945 - 1950. [Abstract] [Full Text] [PDF]
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K. F. Hoffmann, A. W. Cheever, and T. A. Wynn IL-10 and the Dangers of Immune Polarization: Excessive Type 1 and Type 2 Cytokine Responses Induce Distinct Forms of Lethal Immunopathology in Murine Schistosomiasis J. Immunol., June 15, 2000; 164(12): 6406 - 6416. [Abstract] [Full Text] [PDF]
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