Departments of 1 Physiology, 2 Microbiology, 3 Pathology, and 4 Internal Medicine, Faculty of Medicine, University of Geneva, 1211 Geneva 4, Switzerland; and 5 Laboratory of Nutrition/Infection 5, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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Injection of 10 cysts of
Toxoplasma
gondii (Me49 strain) into Swiss
Webster mice results in 1) an acute
phase of infection lasting for 2-3 wk, characterized by weight
loss, and 2) a chronic phase in
which surviving mice show either partial weight recovery (Gainers) or
persistent, although stable, cachexia (Nongainers). In response to a
second immunological stimulation with lipopolysaccharide (LPS) in the
chronic phase of the infection, it is shown that 1) the increase in energy
expenditure was more prolonged in both groups of infected mice than in
controls, 2) the intensity and duration of hypophagia were also differently affected with Nongainers > Gainers > controls, and 3) the
infected mice had higher serum levels of tumor necrosis factor-
(TNF-
) and interleukin (IL)-10 and a lower ratio of IL-10 to TNF-
than controls. In contrast, serum IL-4 increased to the same level in
all three groups. Evaluation of the permeability of the blood-brain
barrier by intravenous injection of Evans blue revealed a marked
staining in the brain of only the infected Nongainers. Taken together,
these results indicate that, in mice with chronic toxoplasmosis, a
second nonspecific challenge (with LPS) exacerbates the hypophagic and
hypermetabolic states, the latter being associated with
hyperresponsiveness in TNF-
and IL-10 production. Furthermore, the
greater exacerbation of the hypophagic state in mice showing persistent
cachexia may be due to a preexisting higher permeability of the
blood-brain barrier, which would allow a greater access of plasma-borne
cytokines and/or other neuroimmunologically active substances
to the central nervous system.
cachexia; thermogenesis; anorexia; starvation
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INTRODUCTION |
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INFECTION can lead to cachexia, and this has been associated with an unfavorable outcome in terms of survival, particularly when there is a drastic loss of body weight. A role for cytokines in inducing cachexia has been suggested by their ability to reduce appetite (15) and/or to increase energy expenditure (17), thus leading to a negative energy balance. In most published metabolic studies, however, injections of a single cytokine were performed, and it is difficult to separate pharmacological from physiological effects.
To gain insights into the physiological role of in vivo cytokine levels
and their interactions in infection-induced cachexia, we have recently
described an experimental murine model of chronic infection with
Toxoplasma
gondii to study the consequences of infection on long-term energy balance (1). Injection (ip) of 10 cysts
of T.
gondii (Me49 strain) into female Swiss
Webster mice was shown to induce a progressive and substantial loss in body weight within 2-3 wk, and this was associated with a
mortality rate of <25% during this acute phase of cachexia. In the
chronic phase of the infection, about one-half of the surviving mice
had persistent, although stable, cachexia (Nongainers), whereas the other half showed a partial weight regain (Gainers). This evolution into two distinct groups of surviving mice is a reproducible
phenomenon, since it was regularly observed. The Nongainers were
characterized by hypermetabolism, hypophagia, and elevated mRNA levels
of spleen and brain cytokines, particularly those of the TH2 profile,
i.e., those which are primarily involved in humoral immunity
[e.g., interleukin (IL)-4, IL-5, IL-10] rather than in
cellular immunity [e.g., interferon (IFN)-]. The
Gainers, on the other hand, although still hypophagic, were no longer
hypermetabolic, and their cytokine mRNA levels were no longer elevated,
except for tumor necrosis factor-
(TNF-
) and IL-10.
In this context of chronic infection, it was of interest to investigate
whether a second immunological challenge, such as intraperitoneal
injection of bacterial lipopolysaccharide (LPS) to Gainers and
Nongainers mice, would induce further changes in energy balance and to
compare their immune response by the determination of TNF-, IL-4,
and IL-10 production, three cytokines which have previously been found
to be highly expressed in the spleen and brain of these
T.
gondii chronically infected mice.
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MATERIALS AND METHODS |
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Mice and diets. Female Swiss Webster mice (BRL, Fullinsdorf, Switzerland) of ~1 mo of age were used and kept at room temperature with a 12:12-h light-dark cycle. The mice were housed in polycarbonate cages (4 mice/cage) and had free access to tap water and standard laboratory chow diet. These mice were injected intraperitoneally with 10 cysts of the Me49 strain of T. gondii, and after 7-10 days all lost weight. Subsequently, the infected mice were classified as Gainers if they showed a regain in body weight after infection-induced cachexia or Nongainers if they showed no regain after the acute phase of infection (Fig. 1). On day 90 postinfection, when both groups of infected mice had stable body weight and food intake, they were compared with age-matched noninfected mice fed ad libitum (the control group). All four separate experiments reported here used identical design as described above.
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Experiment 1: Food intake and energy expenditure. In the three groups (n = 12-15), food intake was determined from the difference between the amount of food given and that removed from the cage, after the amount of any food spilled was taken into account. Energy expenditure (EE) was measured using open-circuit indirect calorimetry with computerized equipment. The calorimeter includes six chambers maintained at 29°C. The values for O2 consumption were automatically and sequentially recorded every minute in each chamber. Mice were acclimatized to metabolic chambers for 2 days before the start of the experiment. Within each group, EE was measured in subgroups of four mice per chamber over 10-h periods. LPS of Salmonella (10 µg; Sigma, Buchs, Switzerland) dissolved in phosphate-buffered saline (PBS) (Boehringer Mannheim, Rotkreuz, Switzerland) was injected intraperitoneally after the mice had spent 4 h in the calorimeter. The first 15 min after injection were not included in EE determinations, and the last 6 h were used to compare the EE among groups. EE was calculated off-line using Weir's equation. The values were then averaged during 60 1-min sampling periods. Injection of the vehicle buffer (PBS) under the same conditions had no detectable effect on EE in the controls nor in the infected mice. The results are expressed either per mouse or normalized per kilogram body weight to the power 0.75 to adjust for the weight differences.
Experiment 2: Determination of circulating cytokines.
To assess the circulating levels of TNF-, IL-4, and IL-10 mice of
each group (n = 12) were divided into
two subgroups (of 6 mice each) and injected intraperitoneally either
with 10 µg of LPS in 250 ml of PBS or with the vehicle PBS buffer
alone. Blood was obtained by retro-orbital sinus puncture from animals at 90 min after injection, a time point at which serum TNF-
was found to be produced at its maximal rate in our previous studies (3).
Control mice received PBS alone. After centrifugation, serum samples
were immediately aliquoted and frozen at
20°C until analysis. Serum TNF-
titer was determined colorimetrically by its
toxicity on L929 cells in an in vitro assay (5), and positive serums
for TNF-
were confirmed by repeating the assay using rabbit polyclonal antiserum against murine TNF-
as a control. One cytotoxic unit was defined as the highest dilution of test material that caused
50% or more destruction of the monolayer of L929 cells and this
corresponded to 5 pg/ml of murine recombinant TNF-
(detection limit
of the assay), and the titer was expressed as picograms per milliliter
of serum. IL-4 and IL-10 were measured by the murine IL-4 and IL-10
Immunoassay kits (Quantikine M from R+D Systems; UK) using their
recommended protocol. All assays were conducted in duplicate.
Experiment 3: Determination of mRNA expression and tissue and serum levels of cytokines. An additional experiment in our model of chronic toxoplasmosis was conducted to assess, in the same study, these three cytokines at the levels of 1) tissue mRNA expression, 2) tissue protein, and 3) serum protein, in response to PBS or to LPS in each of the three groups (n = 3).
Blood was obtained by retroorbital sinus puncture from animals at 90 min after injection of LPS or PBS, and the animals were then immediately killed in order to remove spleen and brain. The cytokine mRNA expression in spleen and brain in all three groups was determined by Northern blot as detailed previously (1), the tissue cytokine proteins were extracted from the whole organ in 2 ml of 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in RPMI 1640 medium (Boehringer Mannheim) according to the method described by Nakane et al. (12), and both tissue and serum proteins were assayed using the immunoassay kits (Quantikine M from R&D Systems, UK) for IL-4 and IL-10, or the InnoBasics ELISA kit (Innogenetics, Ghent, Belgium) for TNF-Experiment 4: Blood-brain barrier permeability. To evaluate the permeability of the blood-brain barrier, mice from the control, Gainer, and Nongainer groups (n = 3) were injected with 200 µl of 1% Evans blue (10) in normal saline in the retro-orbital sinus. Animals were killed 120 min after injection, the organs were washed by infusion of saline into the left heart ventricle, and the brains were excised and rapidly put in Formalin 10%.
Data analysis. All data are presented as means ± SE. Statistical analysis was performed using one-way analysis of variance (ANOVA), with a post hoc Newman-Keuls multiple comparison test for pairwise comparison after ANOVA established significant differences among the groups.
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RESULTS |
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Experiment 1: Body weight, food intake and EE.
The profile of weight changes after the infection is presented in Fig.
1. After a delay of 9-11 days, infection with
T.
gondii resulted in substantial weight
loss, after which the surviving mice could be differentiated into
Gainers (i.e., those showing partial regain in body weight) or
Nongainers (those showing no weight regain). The body weight of
controls (n = 12), Gainers (n = 12), and Nongainers
(n = 15) on
day
90 after infection were 49.1 ± 3.5, 37.0 ± 2.9, and 24.8 ± 1.4 g, respectively, and 2 days
after the second challenge with LPS, the loss in body weight in
Nongainers (2.48 g) was significantly greater
(P < 0.02) than in Gainers
(
0.93 g) and controls (
1.22 g). Food intake after LPS
injection was compared with the average food intake before LPS. It was
found that both the duration and the magnitude of hypophagia differed
among groups in the following order: Nongainers > Gainers > controls (Fig. 2). Three days after LPS
injection, the food intake of the controls and Gainers was ~70% of
the level before LPS, whereas the food intake of Nongainers was still
markedly reduced, amounting to only 20% of that required for their
preinjection energy balance. Chronic infection with
T.
gondii therefore is associated with an
increased hypophagic response to LPS both in duration and intensity.
After LPS injection, an increase in the absolute level of EE was
observed in the three groups of mice (Fig.
3A),
which peaked ~1-2 h postinjection; however, the increase in EE
was sustained only in the infected groups. This differential thermogenic response between the infected groups and the noninfected controls is clearly evident when EE is expressed in watts per kilogram
to the power of 0.75 and as the ratio of postinjection to preinjection
values (Fig. 3B). It is found that
at 1 h after LPS, the increases in EE were not significantly different
among controls, Gainers, and Nongainers, but they were subsequently higher and more sustained in both groups of infected mice than in
controls. It is to be noted that on
day 2 postinjection, two out of the 12 Nongainers mice died, whereas no death
occurred in the two other groups.
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Experiment 2: Circulating cytokines.
The cytokine levels were determined 90 min after LPS injection, since
it was shown in previous studies (3) that TNF- concentration was
maximal at this time point. In each group, mice that were injected with
the vehicle buffer PBS had no detectable circulating cytokine levels.
After LPS injection, serum TNF-
levels increased to a greater extent
in the infected mice than in controls (Fig. 4); infected Gainers and Nongainers had
about the same level of circulating TNF-
. Hence, chronic infection
enhanced serum TNF-
level in response to LPS. Serum IL-4
concentrations increased to a similar level in the three groups,
indicating that chronic infection did not significantly modify the IL-4
response. By contrast, IL-10 response was significantly greater in
infected mice, and, in addition, the Gainers had a higher concentration
of IL-10 than Nongainers (P < 0.001). The ratio of IL-10 to TNF-
was found to be significantly
lower (P < 0.001) in the infected
mice (Nongainers: 0.06 and Gainers: 0.08) compared with the controls
(0.31), whereas these ratios for both groups of infected mice were not
statistically different.
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Experiment 3: mRNA expression and tissue and serum levels of
cytokines.
In our analysis of the data on circulating cytokines in the above
experiment, it became clear that in the absence of LPS few or no serum
levels of TNF-, IL-10, and IL-4 were detectable in mice chronically
infected with T.
gondii. These observations contrast with our previously reported findings in this same model that mRNA
expressions of these cytokines were markedly upregulated in spleen and
brain (1). Because of this apparent inconsistency between the cytokine
mRNA levels in our previous study and the circulating levels in the
present study, we have conducted an additional experiment in our model
of chronic toxoplasmosis in which we have assessed, in the same study,
these three cytokines at the levels of
1) tissue mRNA expression,
2) tissue protein, and
3) serum protein, in response to PBS
or to LPS in each of the three groups. The results are presented in
Fig. 5. First, they confirm the
above-mentioned discrepancy that, in the absence of LPS (i.e., in
PBS-injected mice), serum levels of TNF-
and IL-10 are almost
undetectable in the chronically infected mice, whereas the mRNA is
upregulated both in the spleen and the brain. The tissue protein levels
of these two cytokines (both in spleen and brain and expressed per ml
of tissue extract) were, however, found to increase in parallel to
their mRNA expressions in the infected animals. Similarly for IL-4,
despite the marked upregulation of its mRNA and its protein both in the
brain and spleen of the chronically cachexic mice (Nongainers), serum
IL-4 was not detectable in the absence of LPS.
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Experiment 4: Blood-brain barrier permeability. As can be seen in Fig. 6, only Nongainers showed a marked Evans blue accumulation in their brains (reflected by being darker), thereby indicating that the permeability of the blood-brain barrier of these mice was clearly increased, in contrast to mice in the control or Gainer groups.
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DISCUSSION |
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Using this murine model of infection with
T.
gondii, we have previously shown that,
during the chronic phase of infection, mice from the Gainers group were
no longer hypermetabolic, since their 24-h
EE/kg0.75 was similar to that of
controls, and that their lower body weight could be quantitatively
accounted for by their lower food intake (1). In contrast, the
Nongainers showed not only a reduction in food intake but also an
increase in EE, as judged by their higher 24-h
EE/kg0.75 relative to ad
libitum-fed controls or by their higher absolute 24-h EE (i.e., per
mouse) relative to underfed weight-matched controls, i.e., noninfected
mice underfed to lose the same amount of weight as the infected mice.
Marked differences between infected Gainers and Nongainers were also
observed in cytokine mRNA expressions in brain and spleen; namely, only
TNF- and IL-10 mRNA were expressed in Gainers, whereas in the
Nongainers all cytokines measured IL-1, IL-2, IL-4, IL-5, IL-6, IL-10,
TNF-
, and IFN-
mRNA were expressed, with values for IL-4, IL-5,
IL-6, and IL-10 being particularly elevated (1). Moreover, the
Nongainers had increased serum immunoglobulins and glomerulonephritis,
which are indications of an autoimmune state. These findings suggested
that the chronically cachexic mice (in the Nongainer group) showed a
reduced capability to resist T.
gondii infection, with a predominant
TH2-type cytokine expression pattern (i.e., primarily involved in
humoral immunity), and had a greater number of cysts in their brains
(1).
The study described here further demonstrates that chronic infection
with T.
gondii modifies the response to an LPS
challenge: 1) by prolonging the
hypermetabolic response and potentiating the hypophagic phase and
2) by enhancing the concentration of circulating TNF- and IL-10, which, unlike serum IL-4, were found to
be greater in the infected groups than in the control group in response
to LPS. To what extent other cytokines (e.g., IL-1, IL-2, IL-5, IL-6,
and so forth) might also be implicated in the hyperresponsiveness of
the infected animals to a second challenge cannot be disregarded and
would need to be addressed in future studies, but it is clear from the
data presented here that at least two cytokines (IL-10 and TNF-
) can
be associated with the anorectic and thermogenic hyperresponsiveness
after the second challenge with LPS.
It is to be noted that, despite the elevated cytokine mRNA level
previously observed on day
90 in the Nongainers, no detectable circulating TNF-, IL-4, and IL-10 were found before LPS injection in
this group. This again brought into focus the problems raised in our
previous work (1) in the attribution of specific cytokines to metabolic
responses and prompted us to conduct an additional experiment in which
these three cytokines were examined at the serum level as well as at
the levels of their mRNA expression and their protein product in the
spleen and in the brain. The results of this experiment confirm the
existence of this inconsistency between the upregulated cytokine mRNA
levels and the virtually undetectable serum levels of their protein
products but also reveal that the marked mRNA expression of these
cytokines in the spleen and brain of the chronically infected mice was
associated with parallel increases in the tissue level of their
respective protein products. On the basis of these findings, it is
therefore reasonable to attribute the inconsistency between mRNA
expression and lack of elevation in circulating cytokines to the
regulation of the secretory processes that underlie the release of
cytokines from the tissues into the circulation rather than to the
regulation of cytokines at the translational level. Because marked
increases in circulatory levels of these cytokines occurred only when
both the mRNA expression and tissue protein levels were enhanced in response to LPS, the possibility arises that either an acute stimulus is essential and/or a critical threshold in tissue cytokine
level needs to be reached before it can be released into the
circulation. There is clearly a need for further studies that would
address the mechanisms underlying cytokine secretion from the tissues into the circulation, on the one hand, and the relationship between tissue content of a given cytokine and its autocrine effect
(particularly in the brain) on the other.
It is nonetheless clear from the present study that, in response to a
second immunological challenge (with LPS), the chronically T.
gondii-infected mice (the Nongainers)
show a greater increase in serum levels of TNF- and IL-10 than the
controls. An increase in TNF-
response to LPS injection during
chronic infection has previously been reported in Swiss Webster (4) and
in C57BL/J (5) mice infected with T.
gondii (C56 strain). The same finding has also been reported in hamsters infected with
Leishmania
donovani, in which their macrophages
showed hyperproduction of TNF-
in the chronic phase (8th wk)
compared with the acute phase (2nd wk) of the infection (13). IL-10 has
been shown to have antagonistic effects on the pathophysiological
action of TNF-
in vivo (2). In support of this observation, we have
presently found that the severity of LPS-induced hypophagia in the
infected Nongainers and Gainers is associated with a lower ratio of
serum IL-10 to TNF-
than in control mice. However, little or no
difference in this ratio was observed between Gainers and Nongainers,
and hence cannot be invoked for the greater severity of the hypophagic
response in the Nongainers. A possible explanation for the prolonged
duration of LPS-induced hypophagia in Nongainers may reside from a
greater permeability of their blood-brain barrier, thus allowing a
greater access to the central nervous system for LPS-induced
circulating cytokines such as TNF-
and/or LPS itself, where
they would be acting locally (16). Furthermore, an autocrine role for
brain cytokines underlying the prolonged suppression of appetite is also a plausible explanation, particularly in light of our findings presented here and those of Chang et al. (5) in chronic murine T.
gondii encephalitis, that brain
cytokines are found to be markedly elevated in the absence of any
detectable increase in circulating (systemic) cytokine. To what extent
cytokine induction of leptin, a protein that has been demonstrated to
produce anorexia and hypermetabolism (8, 14), also plays a role in this
exaggerated hypophagic and thermogenic response to LPS in chronic
toxoplasmosis is currently unknown. This is certainly an interesting
future line of investigation, particularly in light of a recent report
(18) showing that 1) administration
of LPS to mice increased leptin gene expression and circulating leptin
levels and 2) the administration of
TNF-
and IL-1 produced a dose-dependent increase in serum leptin
levels.
In conclusion, the negative energy balance induced by an immunological
stress such as LPS injection is worsened in mice chronically infected
with T.
gondii compared with controls. The
more severe anorectic state of the chronically infected mice is
associated with a higher LPS-induced rise in the concentration of
circulating TNF- and IL-10 and a marked reduction in the ratio of
IL-10 to TNF-
. However, although a causative link between anorexia
and these cytokines cannot be ascertained, the severity of the
hypophagic response seems to depend on preexisting lesions of the
blood-brain barrier, associated with signs of autoimmunity. Given the
importance of secondary infections in the exacerbation of cachexia in
many pathological conditions, further studies are warranted to better define the role of cytokines in the exacerbation of anorexia and hypermetabolism. In this context, the murine model of chronic toxoplasmosis described here could be of value, particularly since its
metabolic responses to LPS reflect those observed in patients with
secondary infections. The enhanced thermogenic and anorectic effects
from secondary challenge in this murine model are reminiscent of the
marked anorexia, high resting EE, and rapid weight loss of acquired
immune deficiency syndrome (AIDS) patients with active secondary
opportunistic or bacterial infections such as
Mycobacterim avium complex (6, 7). Furthermore, as
in this model of chronic toxoplasmosis, in humans with chronic
infections associated with cachexia, serum cytokines are generally not
found to be higher than in controls (healthy individuals), but only in
response to secondary challenge, such as during sepsis or acute
parasitic infections (6). In the case of human immunodeficiency virus infection, for example, elevated levels of serum TNF-
in AIDS were
predominantly found in patients with secondary infections (6,
9).
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ACKNOWLEDGEMENTS |
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The excellent technical assistance of Angela Roatti and Claudette Duret is gratefully acknowledged. We would also like to thank Christian Versin as well as R. Schumacher and F. Strub for the illustrations.
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FOOTNOTES |
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This work was supported by Grants 31-47211.96 and 31-042275.94 from the Swiss National Science Foundation.
Address for reprint requests: A. G. Dulloo, Dept. of Physiology, Faculty of Medicine, Univ. of Geneva, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland.
Received 6 May 1997; accepted in final form 12 November 1997.
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