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Infection and Immunity, April 1999, p. 1887-1893, Vol. 67, No. 4
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Role of Gamma Interferon in Cellular Immune
Response against Murine Encephalitozoon cuniculi
Infection
Imtiaz A.
Khan,* and
Magali
Moretto
Department of Medicine and Microbiology,
Dartmouth Medical School, Lebanon, New Hampshire 03756
Received 14 September 1998/Returned for modification 28 October
1998/Accepted 8 January 1999
 |
ABSTRACT |
Microsporidia are obligate intracellular protozoan parasites that
cause a wide variety of opportunistic infection in patients with AIDS. Because it is able to grow in vitro, Encephalitozoon cuniculi is currently the best-studied microsporidian. T
cells mediate protective immunity against this parasite. Splenocytes obtained from infected mice proliferate in vitro in response to irradiated parasites. A transient state of hyporesponsiveness to
parasite antigen and mitogen was observed at day 17 postinfection. This
downregulatory response could be partially reversed by addition of
nitric oxide (NO) antagonist to the culture. Mice infected with
E. cuniculi secrete significant levels of
gamma interferon (IFN-
). Treatment with antibody to IFN- or
interleukin-2 (IL-12) was able to neutralize the resistance to the
parasite. Mutant animals lacking the IFN- or IL-12 gene were highly
susceptible to infection. However, mice unable to secrete NO withstood
high doses of parasite challenge, similar to normal wild-type
animals. These studies describe an IFN--mediated protection
against E. cuniculi infection that is
independent of NO production.
| |
INTRODUCTION |
Microsporidia are obligate
intracellular parasites that infect an extremely wide range of hosts in
the animal kingdom (4). They are distinct enough to be
placed in a separate phylum, Microspora (5), and
are characterized by the polar filament which is used to inject
sporoplasm into the host cell (51). Species of microsporidia that infect mammals are unicellular, gram-positive organisms with mature spores 0.5 to 2 by 1 to 4 µm in diameter (10).
Classification is based on size, nuclear arrangement, mode of division,
and association of proliferative forms within the host cell.
Most of what is known about the biology of microsporidia is based on
the microsporidian Encephalitozoon cuniculi,
which commonly infects rodents and has been found in humans as well
(54). Little is known regarding host immunity to
E. cuniculi. E. cuniculi
was the first mammalian microsporidian successfully grown in
vitro (43). It infects epithelial and endothelial cells,
fibroblasts, and macrophages in a variety of mammals, including
rabbits, rodents, carnivores, monkeys, and humans (6, 8,
19). In an experimental model, normal mice infected with
E. cuniculi usually express few clinical
signs of disease (35). Conversely, immunodeficient hosts,
such as athymic or SCID mice, develop lethal disease after experimental
infection (26, 41). The studies conducted have shown that T
cells are responsible for the prevention of lethal disease. Adoptive
transfer of sensitized syngeneic T-cell-enriched spleen cells protected
athymic or SCID mice against E. cuniculi challenges (18, 40). Transfer of naive lymphocytes or
hyperimmune serum fail to protect or prolong the survival of these
mice. Furthermore, E. cuniculi is
increasingly being recognized an opportunistic infection in the
individuals with AIDS (19, 50). Studies by Didier have shown
that cytokines released by sensitized T cells activate macrophages to
kill E. cuniculi in vitro (9).
However, there are no in vivo data demonstrating the mechanism of
T-cell-mediated protection against this emerging opportunistic pathogen.
The data herein demonstrate that E. cuniculi infection in the immunocompetent host induces
a strong cellular immune response characterized by the production of
gamma interferon (IFN-). Mice unable to produce this cytokine are
susceptible to infection. Thus, protective immunity induced in the
normal mice is dependent on Th1 type of immune response.
| |
MATERIALS AND METHODS |
Mice.
Maurice Gately (Hoffman-La Roche) kindly provided a
breeding pair of p40
/ mice on a C57BL/6 background.
These mice lack the gene for the p40 chain of interleukin-12 (IL-12)
heterodimer and thus are unable to produce IL-12 (30).
Inducible nitric oxide synthase-deficient (iNOS/) mice
on a C57BL/6 × 129 background were provided by John Mackmicking and Carl Nathan (Cornell University Medical School, Ithaca, N.Y.). These mice were backcrossed for five generations to wild-type C57BL/6
as previously described (25). The mice were bred under conditions approved by the Animal Research Facilities at Dartmouth Medical School. Mice deficient in the IFN- gene and wild-type C57BL/6 mice were obtained from The Jackson Laboratory, Bar Harbor, Maine.
Parasites and infection.
A rabbit isolate of E. cuniculi, kindly provided by Elizabeth Didier (Tulane
Medical Research Center), was used throughout the study. Parasites were
grown in rabbit kidney (RK-13) cells (American Type Culture Collection)
which were maintained in RPMI 1640 (Gibco BRL) containing 10% fetal
calf serum (HyClone Laboratories). Organisms were collected from the
culture medium and centrifuged at 325 × g for 10 min.
After two washes with phosphate-buffered saline the parasites were
resuspended and injected via the intraperitoneal (i.p.) route to mice.
Unless stated otherwise, mice were challenged with 107 parasites.
T-cell proliferation.
Following euthanasia, the spleens from
infected animals were removed and homogenized in a petri dish, and
contaminating erythrocytes were lysed in RBC lysis buffer (Sigma
Chemical Co., St. Louis, Mo.). Cells were suspended in RPMI 1640 with
10% fetal calf serum and centrifuged for 10 min at 500 × g. Cells were cultured at the concentration of 2 × 105/well in 96-well flat-bottomed plates in a 200-µl
volume with 5 µg of concanavalin A (ConA; Sigma Chemical Co.) per ml
or 5 × 103 irradiated spores. After 72 h at
37°C in 5% CO2, [3H]thymidine (0.5 µCi/well; Amersham, Arlington Heights, Ill.) was pulsed for 8 h
to determine DNA synthesis. An automated cell harvester was used to
harvest pulsed splenocytes onto glass filters. The filters were dried,
and incorporation of radioactive thymidine was determined by liquid scintillation.
Detection of cytokine mRNA by quantitative PCR.
Splenocytes
from E. cuniculi-infected animals were
collected at days 0, 10, 17, and 24 p.i. (post infection). RNA
from spleen cells was collected by using Trizol (Life Technologies
Inc., Gaithersburg, Md.) as instructed by the manufacturer. Reverse
transcription was performed with Moloney murine leukemia virus reverse
transcriptase (Life Technologies) and random hexamer primers (Promega,
Madison, Wis.). Expression of mRNAs for IFN-, IL-10, IL-4, and iNOS
was measured by quantitative PCR using the PQRS quantitative method (36). The splenocytes from uninfected mice were used to
establish a baseline value of 1.0, against which the level of message
for cytokine in the test mice was quantitated.
Cytokine detection in serum.
Serum was collected from the
blood of infected mice and tested for the presence of IFN- and IL-4
(Endogen, Cambridge, Mass.) by enzyme-linked immunosorbent assay
(ELISA) according to the manufacturer's instructions.
Measurement of cytokine production in spleen cell cultures.
Cytokine production in the culture supernatants of the ConA- and
parasite-stimulated spleen cell cultures was measured. Levels of
IFN- were determined by cytokine ELISA as described above. Nitrite
production in the culture supernatants was assayed by the Greiss
reaction (13). Briefly, 100 µl of the culture supernatant was added to a mixture of 1% sulfanilamide dihydrochloride in 2.5%
phosphoric acid and 0.1% napthylenediamine hydrochloride in 2.5%
H3PO4, then incubated for 10 min at room
temperature, and read with a spectrometer
(A570). Nitrite concentration was calculated
from a NaNO2 standard curve.
Cytokine depletion assays.
Rat anti-mouse IL-10 (Endogen)
was used at a concentration of 40 µg/ml. Control antibody was
isotype-specific rat immunoglobulin G (IgG; Sigma). For IFN-
depletion, mice were treated with rat anti-mouse IFN- (XMG6) (3 mg/mouse per week) i.p. starting 2 days prior to challenge. Control
mice received equal amount of rat IgG. Endogenous IL-12 was neutralized
by administering 0.5 mg of goat anti-mouse IL-12 (kindly provided by
Maurice Gately) beginning 2 days prior to infection. The antibody
treatment was continued twice weekly thereafter. Control mice were
treated with equal quantity of goat IgG (Sigma).
Statistical analysis.
Statistical analysis of the data was
performed by Student's t test (34).
| |
RESULTS |
Antigen-specific proliferation of splenocytes from E. cuniculi-infected mice.
A time course studying
antigen-specific T-cell proliferation was performed. Splenocytes from
E. cuniculi-infected mice were isolated at
days 10, 17, and 24 p.i., and the proliferative response to
antigenic stimulation was determined. Antigen-specific proliferation of
splenocytes from day 10-p.i. animals was significantly greater (P = 0.01) than for uninfected animals (Fig.
1A). Spleen cells from the infected mice
showed a normal ConA response (Fig. 1A). At day 17 p.i., the
splenocytes failed to proliferate in response to antigenic stimulation
(Fig. 1B). To determine whether this was an antigen-specific
downregulation, splenocytes from infected mice were stimulated with
mitogen. As with parasite antigen, these splenocytes failed to
proliferate with mitogen (Fig. 1B), possibly due to the generalized
immunosuppression that has been observed during acute infections in
other parasite infections (3, 24, 46). The immunosuppression
was ablated at day 24 p.i., and splenocytes from the infected
animals responded significantly (P = 0.001) to
antigenic stimulation (Fig. 1C). However, the ConA response at this
time point, although significantly improved (P = 0.01), was still significantly lower than for the uninfected controls (Fig.
1C) (P = 0.04).
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FIG. 1.
Proliferation of antigen (Ag)-specific splenocytes
following i.p. infection with 107 spores of E. cuniculi. Pooled splenocytes (n = 3)
were collected at days 10 (A), 17 (B), and 24 (C) postchallenge. Spleen
cells were cultured in quadruplicate in the presence of ConA or
irradiated spores in 96-well plates. After 72 h of incubation,
proliferation was measured by [3H]thymidine
incorporation. The data are representative of two separate experiments.
UI, uninfected; inf, infected.
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NO mediates a role in reduced lymphoproliferative response.
Nitric oxide (NO) is known to downregulate the host immune response in
wide range of infections. The role of this molecule in the inhibition
of lymphoproliferative response during E. cuniculi infection was studied. The NO synthase
antagonist L-NMMA was added to the day 17-p.i. splenocyte
cultures at concentrations known to antagonize NO. Addition of
L-NMMA to the spleen cell cultures significantly
neutralized the suppression in both mitogen (P = 0.001)- and antigen (P = 0.05)-stimulated cultures
(Fig. 2). Although IL-10 has been
reported to be involved in the immunosuppression in other models
(22, 52), no reversal was observed with antibody to IL-10 in
both the mitogen- and antigen-stimulated conditions. No difference was
observed when rat IgG was added to cultures (data not shown). Culture
supernatants obtained from splenocytes of infected mice at various time
points p.i. were assayed for the production of IFN- and nitrites. As
shown in Table 1, both IFN- and
nitrite production was greatest in the culture supernatant at day
17 p.i. Interestingly, this is the time point at which the
splenocytes from E. cuniculi-infected mice
are unable to proliferate in response to both antigen and mitogen.
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FIG. 2.
Reversal of E. cuniculi-mediated suppression by an NO synthase
antagonist. Pooled splenocytes (n = 3) from mice
infected 17 days earlier with E. cuniculi
were cultured in the presence of ConA (A) or irradiated spores (B) and
treated with either 0.5 mM L-NMMA or rat anti-murine IL-10
(40 µg/ml). After 72 h of incubation, lymphoproliferation was
assayed by [3H]thymidine incorporation. The data are
representative of two separate experiments. Abbreviations are as in
Fig. 1.
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|
Cytokine profile of E. cuniculi-infected animals.
Cytokine analysis of
splenocytes from infected animals was performed by quantitative PCR.
Message for IFN- and IL-10 was increased at day 10 p.i. (Fig.
3A and B). Both cytokines are known to
play an important immunoregulatory role in infectious diseases. The
message levels for these cytokines remained more or less unchanged at
day 24 p.i. The message for IL-4 could not be detected in the infected animals at any of the time points tested (data not shown). The
iNOS message was undetectable at day 10 p.i. However, at day 17 p.i., we observed severalfold increase in the message for this molecule, which continued up to day 24 p.i. (Fig. 3C).
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FIG. 3.
Cytokine mRNA expression of splenocytes following
E. cuniculi infection. Splenocytes from
E. cuniculi-infected mice (three per group)
were harvested at various time points (days 0, 10, 17, and 24) p.i.
mRNA expression for IFN- (A), IL-10 (B), and iNOS (C) was assayed by
reverse transcription-PCR. The differences in transcriptional levels
for the genes are expressed relative to day 0 (assigned as 1). The cDNA
concentration examined at each time point was standardized to the
HPRT mRNA level (not shown).
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Circulating IFN-
and IL-4 levels in the sera of infected
animals were determined. Pooled serum from three
E. cuniculi-infected
mice assayed for these cytokines.
The level of IFN-
in the serum
was elevated at day 10 p.i.
(245 ± 12 pg/ml). The cytokine level
peaked at day 17 p.i.
(1,850 ± 65 pg/ml) and began to taper off
by day 24 p.i.
(1,235 ± 30 pg/ml). In contrast, no IL-4 was detected
in sera
from the infected animals. Sera from control uninfected
animals lacked
any of these
cytokines.
In vivo role of IFN- in protection against E. cuniculi infection.
To determine whether the
increased level of IFN- was important in host immunity, a depletion
study was performed. Two days prior to infection, mice were depleted of
either IL-12 or IFN- by treatment with a monoclonal antibody.
Antibody treatment was continued throughout the study. The mice were
infected with parasites via the i.p. route and assessed daily for
evidence of morbidity (development of asciteis) and mortality. All mice
depleted of IFN- died by day 25 p.i. (Fig.
4); mice treated with anti-IL-12 antibody
died 4 days later. Depletion of IL-12 most likely resulted in decreased
levels of IFN-. The delay in time to death between the two different
test groups could be due to other immune modulators stimulating IFN-
production when IL-12 is depleted.
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FIG. 4.
Neutralization of IFN- and IL-12 in E. cuniculi-infected mice. (A) C57BL/6 mice (six per
group) were infected i.p. with 107 spores of E. cuniculi and treated with antibody to IFN- (3 mg/mouse) weekly at 2 days prior to infection. The control animals were
injected with an equal amount of rat IgG (RIgG). (B) C57BL/6 mice (six
per group) were treated with 0.5 mg of goat anti-mouse IL-12 twice
weekly beginning two days prior to infection or an equal volume of goat
IgG (GIgG). The animals were observed daily for morbidity or mortality.
The study was performed twice, with similar results.
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|
The importance of IFN-
in the protective immune response is reported
to be mediated by NO production (
14,
27,
32).
However,
recent studies by others and us have shown a limited
role for this
molecule in protection against
Toxoplasma gondii (
23,
39). To evaluate the importance of NO in protection against
E. cuniculi infection,
iNOS
/ mice were infected as previously described. None
of these animals
died or exhibited any signs or symptoms of disease
throughout
the course of experiment and appeared clinically indistinct
from
the wild-type controls (Fig.
5). In
contrast, mice lacking the
p40 or IFN-
gene succumbed to infection.
These animals died at
approximately the same time as the
antibody-depleted mice. These
results exclude a role of NO in
E. cuniculi immunity.
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FIG. 5.
Survival of gene knockout animals following
E. cuniculi challenge. Knockout mice on a
C57BL/6 background and the parental wild-type (WT) animals (six per
group) were infected i.p. with 107 spores of E. cuniculi (A), and mortality was monitored on a daily
basis (B). The study was performed twice, with similar findings.
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To determine if the susceptibility of the knockout mice was dose
dependent, mice were challenged with a fivefold-higher dose
of
parasites. While the IFN-
/ and p40
/
mice succumbed to infection earlier than those challenged with
lower
dose, the increase in the size of inoculum made no difference
in the
susceptibility of iNOS
/ and parental wild-type mice
(data not shown). These animals survive
for the duration of the
experiment. Gene knockout animals have
been shown to be able to
compensate for the loss by alternate
redundant mechanisms (
7,
29). Although less likely, it is
possible that
iNOS
/ mice are protected against
E. cuniculi infection by an IFN-
-independent
mechanism. To rule out this possibility, both mutant and parental
wild-type animals were infected with 10
7 parasites as
described earlier. The infected animals were treated
with
anti-IFN-
antibody starting 2 days prior to infection. Both
the
iNOS
/ and wild-type mice depleted of IFN-
succumbed
to infection at
almost the same time after challenge (Fig.
6). None of the mice
treated with control
antibody died during the period of experimentation.
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FIG. 6.
Depletion of IFN- in iNOS/ animals
infected with E. cuniculi. Parental
wild-type (WT) C57BL/6 (A) and iNOS/ (B) mice (six per
group) were treated with anti-IFN- antibody 2 days prior to
challenge as described for Fig. 5. The control animals were injected
with an equal amount of rat IgG. The animals were monitored daily for
survival.
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| |
DISCUSSION |
E. cuniculi, previously observed in
laboratory animals, is considered a zoonotic infection (8).
Complications due to E. cuniculi infection
have been found in immunocompromised patients (44). Human
immunodeficiency virus (HIV)-infected patients coinfected with
E. cuniculi have a wide range of organ
involvement, including liver failure, pneumonitis, sinusitis, and
granulomatous liver necrosis (6). E. cuniculi is closely related to other microsporidia, including Encephalitozoon intestinalis and
Encephalitozoon hellum, which are also associated with
disseminated infection in HIV-infected individuals (5).
The immune responses generated during natural E. cuniculi infection are not well studied. Currently
available literature suggests that T cells play a very important role
in protection against the parasite (18, 41). In this report,
induction of strong cellular immune response in E. cuniculi-infected host is demonstrated. T-cell
proliferation and production of immune cytokines p.i. characterize this response.
The role of T-cell immunity against E. cuniculi infection has been described by other
laboratories (26, 40). In the present study,
antigen-specific proliferation of splenocytes from mice infected 10 days prior was observed. This response was followed by a period of
transient immunosuppression, characterized by a low proliferative index
to both antigen and mitogen. Lymphocyte proliferation was restored by
day 24 postchallenge. Earlier studies by Didier and Shadduck have
demonstrated that spleen cells from mice infected 1 week earlier
express a significantly lower mitogenic response (11).
However, downregulation of both mitogenic and antigenic responses at
day 17 p.i. is reported here. The differences between our findings
and those of Didier and Shadduck could be attributed to the different
mouse strains used in these studies. Variation in immune response,
depending on the strain of mice, has been reported with other
infectious disease models (28, 33). Transient periods of
lymphocyte hyporesponsiveness have also been described for other
parasitic infections (22, 46). During murine T. gondii infection, the period of lymphocyte hyporesponsiveness persists from days 7 to 14 p.i. (17). Infection with
Neospora caninum results in immunosuppression which is
shorter in duration.
The principal mechanism for the downregulatory response appears to be
via induction of NO. Interestingly, maximal immunosuppression is observed at day 17 p.i., when levels of both IFN- and
nitrite production are highest in spleen cell cultures. This also
coincides with the high serum IFN- levels in infected animals. No
IL-4 was detected in sera of infected mice. Our findings are similar to
those reported recently for murine salmonellosis infection (42). Bacterial challenge was followed by NO-mediated
immunosuppression which was neutralized by IFN- depletion. NO
has also been demonstrated to be an important immunoregulatory
molecule in, for example, plasmodium (38), T. gondii (3, 22), N. caninum (24), and Trypanosoma brucei (46)
infections. Treatment of spleen cell cultures with L-NMMA,
an inhibitor of NO synthase, restores approximately 50% of the
proliferative response by day 17-p.i. mice. The reversal of
transient lymphocyte hyporesponsiveness was observed under both
mitogen- and antigen-stimulated conditions.
Apart from its downregulatory role in lymphoproliferation, the
microbicidal activity of NO against intracellular pathogens is well
documented (15, 21). The release of NO is mediated by a key
Th1-type cytokine, IFN- (20, 37). This cytokine plays a
critical role in protective immunity against wide variety of
intracellular pathogens, including viruses, bacteria, and parasites (47, 49, 53). In the present study, antibody depletion of IFN- or IL-12, the cytokine important for the induction of IFN-, resulted in mortality of E. cuniculi-infected mice. Studies have shown that
IFN- knockout mice challenged with E. intestinalis, a parasite closely related to E. cuniculi,
develop a chronic disease, whereas parental wild-type animals are able
to clear the infection (1, 12). The IFN--mediated
protection is believed to be primarily dependent on the cytokine's
ability to activate macrophages (52). Following a cascade of
complex molecular events, including costimulation with tumor necrosis
factor, activated macrophages release NO (14). In vitro
killing of E. cuniculi by IFN--activated macrophages has been recently reported by Didier (9).
Addition of L-NMMA to the cultures blocked the killing of
the parasites, suggesting that the effect was mediated by NO. Based on
these findings, it can be postulated that the absence of NO would
result in loss of protection against the parasite. However,
iNOS/ mice, which are unable to make NO through the
inducible pathway, survived a very high dose of challenge, similar to
the wild-type animals. In contrast, mice lacking the IFN- or IL-12
gene were unable to survive infection with a fivefold-lower dose.
The mechanism of host protection against E. cuniculi, as for other intracellular pathogens,
appears to be dependent on IL-12 and IFN-. However, while in
other infectious agents IFN--mediated immunity is dependent on the
production of NO, such does not seem to be the case with
E. cuniculi. These findings are similar to those presented in a recent report on murine toxoplasmosis, where vaccine-based immunity was found to be independent of NO
(23). Although macrophage activation and subsequent release
of NO are among the important parasiticidal effects of IFN-, other
considerations are the enhancement of class I and class II expression
on antigen-presenting cells, resulting in greater proliferation of T
cells (2, 31). A role of IFN- in the induction of
CD8+ T-cell responses has been reported for viral systems
(45, 48). It is likely that in the absence of this cytokine,
T-cell responses are compromised, leading to inappropriate protection
against infection. Further studies exploring the mechanism of T-cell
protection against the parasite are under way. Alternatively,
iNOS/ mice may be protected by an IFN--dependent
redundant pathway as has been recently described for influenza virus
(16). Identification of these pathways will be beneficial in
understanding the host immune response against this emerging
opportunistic infection in the HIV-infected population (6).
| |
ACKNOWLEDGMENTS |
We thank Julie Weiss for help with statistical analysis.
This work was supported by National Institutes of Health grant AI43693.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medicine and Microbiology, HB 7506, Dartmouth Medical School, Lebanon, NH 03756. Phone: (603) 650-8706. Fax: (603) 650-6841. E-mail: Imtiaz.Khan{at}dartmouth.edu.
Editor:
S. H. E. Kaufmann
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Infection and Immunity, April 1999, p. 1887-1893, Vol. 67, No. 4
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
