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B1
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Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; and
Eukaryotic Transcriptional Regulation Section, Regulation of Cell Growth Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B-dependent proinflammatory cytokines such as IL-12 and TNF-
.
Activation of this important transcription factor is driven by
phosphorylation-dependent degradation of the inhibitory IB molecule,
an event which enables NF-B translocation from the cytoplasm to the
nucleus. In this study, we show that intracellular infection of
macrophages with the protozoan parasite Toxoplasma
gondii induces rapid IB phosphorylation and degradation.
Nevertheless, NF-B failed to translocate to the nucleus, enabling
the parasite to invade cells without triggering proinflammatory
cytokine induction. Infected cells subsequently subjected to LPS
triggering were severely crippled in IL-12 and TNF- production, a
result of tachyzoite-induced blockade of NF-B nuclear translocation.
Our results are the first to demonstrate the ability of an
intracellular protozoan to actively interfere with the NF-B
activation pathway in macrophages, an activity that may enable parasite
survival within the host. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)3 are well known
as important effectors of the innate immune system, and their ability
to produce IL-12 indicates that they possess the potential of directing
acquired immunity toward a Th1-biased response (1, 2). In
addition to producing proinflammatory cytokines such as IL-12, TNF-,
and IL-1
, which are important in initiation of the inflammatory
response to infection, M are a source of the microbicidal effector
molecule NO and are capable of phagocytosing pathogens and degrading
them within the phagolysosome. Because of these combined attributes,
M appear to be well armed to combat microbial infection through a
combination of cytokine release and direct microbicidal attack.
Nevertheless, several intracellular microbes use M as host cells
within which to establish infection. Such organisms have adopted
different strategies to survive within M. For example,
Listeria monocytogenes and Trypanosoma cruzi
escape the phagolysosome and reside within the cytoplasm (3, 4). In contrast, Leishmania major and
Mycobacterium tuberculosis, through distinct molecular
strategies, are able to survive within modified phagosomes
(5, 6, 7). In addition, L. major promastigotes
have been shown to selectively suppress M IL-12 production, although
the molecular details of this phenomenon remain enigmatic
(8, 9, 10).
The intracellular protozoan Toxoplasma gondii is a major
opportunistic infection in immunocompromised populations and is a
strong inducer of type 1 cytokines. The latter are essential to control
infection (11, 12, 13). The tachyzoite form of the
parasite invades a variety of cells, where it resides within a
specialized parasitophorous vacuole that is created during initial
parasite entry into the host cell. (14, 15, 16). In M, the
parasitophorous vacuole resists acidification and lysosomal
fusion (17), and the tachyzoites replicate, eventually
lysing the host cell. In contrast, when M are preactivated by
treatment in vitro with IFN-
and TNF-, the cells display strong
NO-dependent microbicidal activity toward T. gondii
(18, 19). However, the significance of M-derived NO
during in vivo infection has been called into question by the finding
that inducible NO synthase (iNOS; NOS2) knockout mice, which are unable
to produce inducible NO, are capable of surviving early infection
though eventually succumbing during the chronic stage
(20).
Infection with T. gondii induces high levels of the
cytokines IL-12 and TNF-, which are typically associated with M
function. Although these cytokines are required for protection against
the parasite, in certain situations they mediate host pathology
(21, 22, 23, 24, 25, 26, 27). The source of these cytokines during infection
has been the subject of intense investigation. Although M were
initially regarded as the major IL-12 source in vivo (1),
in recent years dendritic cells (DC) have emerged as essential
producers of this cytokine (28, 29). For example, studies
with Leishmania donovani indicate that DC, and not M,
produce IL-12 immediately after infection (30). In
addition, splenic DC are able to rapidly produce IL-12 after i.v.
injection of T. gondii Ag (31). Human DC appear
to respond similarly in vitro to tachyzoites, although in this case
optimal responses require T lymphocytes and CD40-CD40 ligand
interaction (32).
In parallel, our laboratory has found that both human and mouse
neutrophils serve as an early IL-12 source during parasite infection
(33, 34, 35). In the course of our studies on mouse responses
to infection, we found that i.p. tachyzoite inoculation induced a rapid
influx of IL-12-positive PMN. However, we were surprised to find that
in the same population neither infected nor noninfected M displayed
detectable IL-12 (33). We now formally show that
tachyzoites invade host M without inducing IL-12 or TNF- and
without inducing nuclear translocation of NF-B p65, a major
transcriptional activator of inflammatory cytokine genes. More
importantly, we demonstrate that upon host cell invasion, tachyzoites
rapidly induce a state of nonresponsiveness to subsequent stimulation
with bacterial LPS, and that nuclear translocation of NF-B is
inhibited in the presence of Toxoplasma. Despite the failure
in NF-B nuclear translocation, the parasite induced rapid IB
phosphorylation and degradation. This previously unrecognized
phenomenon might represent a parasite defense strategy to delay M
inflammatory response initiation, allowing the parasite to establish
itself within the host.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mAb to NF- p65 was obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). Abs directed against phosphorylated forms of
extracellular signal-regulated kinase 1/2, stress-activated
protein kinase/Janus kinase, p38, IB, as well as
nonphosphorylated IB, were purchased from New England Biolabs
(Beverly, MA). Anti-TNF and anti-p30 (SAG-1) mAbs were obtained
from BD PharMingen (San Diego, CA) and BioGenex Laboratories (San
Ramon, CA), respectively. Antisera against NF-B p50 and p65, used in
electromobility supershift assays were generously provided by Dr. N.
Rice (National Cancer Institute, Frederick, MD). Donkey
anti-goat FITC, goat anti-rabbit HRP, and goat anti-mouse
Texas Red secondary Abs were obtained from Jackson Immunoresearch (West
Grove, PA).
Parasites
Tachyzoites of the virulent RH strain were maintained in vitro by infection of human foreskin fibroblasts and biweekly passage in complete medium (CM) consisting of DMEM (Life Technologies, Gaithersburg, MD) supplemented with 1% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). Tachyzoites from freshly lysed fibroblast cultures were washed once with PBS and resuspended in CM for in vitro assays.
M and cell lines
Female C57BL/6 mice (8–12 wk of age) were injected i.p. with
1.5 ml 3% thioglycolate. Four days after injection, peritoneal exudate
cells were collected by lavage with 10 ml ice-cold PBS. Cells were
washed once in PBS and resuspended in CM. Cultures were incubated
overnight (37°C, 5% CO2), nonadherent cells
were removed, and fresh medium was added before each experiment. M
cell lines (RAW 264.7, J774, THP-1) were obtained from American Type
Culture Collection (Manassas, VA) and cultured in DMEM or in the case
of THP-1, RPMI 1640, supplemented with 10% FBS, penicillin (100
U/ml), and streptomycin (100 µg/ml).
In vitro infections
Parasites were added to M (3:1 ratio) and plates were briefly
centrifuged (500 x g, 1 min) to initiate parasite:cell
contact. At time points indicated in the figure legends, supernatants
were removed and fresh medium was added. Where indicated, cells were
stimulated for 30 min with 100 ng/ml LPS derived from Escherichia
coli strain 026:B6 (Sigma, St. Louis, MO). After LPS pulsing,
cells were washed and fresh medium was added. After 6 h,
supernatants were harvested for cytokine ELISA. For Western blotting,
ribonuclease protection assay (RPA), and EMSA analyses cells
were collected 3 h after LPS stimulation. In some experiments,
cycloheximide (10 µg/ml; Sigma) was added 15 min before infection or
LPS stimulation. In other experiments, MG-132 (50 µM; Calbiochem, San
Diego, CA) was added 60 min before exposure to LPS or tachyzoites.
In vivo infections
Tachyzoites (2 x 106) were injected i. p., then at varying times after infection, cells were collected by PBS lavage and subjected to confocal fluorescence microscopy.
Confocal microscopy
M (2 x 106) were plated onto
12-mm circular coverslips in 24-well plates and cultured overnight.
After the experiment was completed, coverslips were washed twice in
PBS, then fixed with 3% formaldehyde in PBS for 20 min at room
temperature (RT). Following fixation, coverslips were washed and cells
were incubated (45 min, RT) with the indicated primary Ab diluted in
0.075% saponin/PBS. After washing, coverslips were incubated (45 min,
RT) with fluorescein-conjugated secondary Ab diluted in 0.075%
saponin/PBS containing 5% serum. Propidium iodide (1 µg/ml) was
added during the final 10 min of incubation to stain nuclei. Finally,
coverslips were washed three times with saponin/PBS, then twice with
PBS before mounting with Pro-Long Antifade (Molecular Probes,
Pitchford, OR). Confocal images were collected with an Olympus Fluoview
confocal microscope system using Fluoview software (version 2.1.39;
Olympus, Melville, NY).
PAGE and Western blotting
Cell lysates or nuclear extracts (5 µg) were electrophoresed through 10% SDS-polyacrylamide minigels and proteins were subsequently electroblotted onto nitrocellulose. Membranes were blocked (1 h, RT) with 5% nonfat dry milk in PBS containing 0.05% Tween (PBST) then incubated with primary Ab (10 µg/ml PBST) overnight at 4°C. Membranes were washed three times with PBST, incubated with goat anti-rabbit HRP (1 h, RT), washed and developed with ECL substrate (Amersham, Arlington Heights, IL), and exposed to Biomax MS autoradiography film (Kodak, Rochester, NY).
Cytokine ELISA
Production of TNF- was measured with the murine TNF DuoSet
(Research Diagnostics, Flanders, NJ) according to the
manufacturer’s instructions. IL-12 p40 was measured as described
previously (35). Unless otherwise specified, cytokine
assays were performed on 6-h supernatants.
Ribonuclease protection assay (RPA)
Total RNA was isolated from 2 x 107
cells with RNA-Stat60 according to the manufacturer’s protocol and
subjected to RPA using the Riboquant assay system (BD PharMingen) with
the mCK2b, MCK3, and a custom multiprobe template. The MCK2b template
contains probes for IL-12 p35, IL-12 p40, IL-10, IL-1, IL-1,
IL-1R antagonist (IL-1Ra), IL-18, IL-6, IFN-, migration
inhibitory factor, and the housekeeping genes L32 and
GAPDH. The MCK3 template contains probes for TNF-,
lymphotoxin , TNF-, IL-6, IFN-, IFN-, TGF-1/2,
L32, and GAPDH. Dr. E. Pearce kindly provided the custom template
containing iNOS, IL-12 p35, IL-12 p40, TNF-, IL-1, IL-1Ra, IL-18,
IL-2R, L32, and GAPDH.
Nuclear extracts
Nuclear extracts were prepared from 2 x 107 cells essentially as described previously (36). Cells were scraped into ice-cold PBS and washed twice. Pellets were resuspended in 1 ml cold PBS and microfuged at 2000 rpm for 5 min. Pellets were resuspended in 200 µl lysis buffer (20 mM HEPES (pH 7.9) containing 10 mM NaCl, 3 mM MgCl2, 0.1% Nonidet P-40, 10% glycerol, 0.2 mM EDTA, 1 mM DTT, 0.4 mM PMSF, and 1 µg/ml leupeptin) and incubated on ice for 15 min, then centrifuged at 2000 rpm for 5 min. Pellets were washed once in 20 mM HEPES (pH 7.9) containing 20% glycerol, 0.2 mM EDTA, 1 mM DTT, 0.4 mM PMSF, and 1 µg/ml leupeptin. Pellets were then resuspended in 200 µl wash buffer containing 400 mM NaCl and incubated on ice for 45 min. Finally, suspensions were centrifuged (13,000 rpm, 15 min, 4°C) and supernatants were collected. Protein concentration was estimated according to the Bradford method (37), and the supernatants were stored in aliquots at -70°C.
EMSA
The oligonucleotide probe used for the EMSA consisted of the
B consensus site for the Ig L chain gene promoter, which binds
both p65:p50 heterodimers and p50:50 homodimers, and contained the
following sequence:
3'-GATCCTCCCTGGGACTTTCCAGGCTAGAGGGACCCCTGAAAGGTCCGATCTAG-5'. Probes
were labeled with Klenow polymerase (Promega, Madison, WI) and
[-32P]dCTP (New England Nuclear, Boston,
MA), according to supplier’s instructions. DNA binding reactions
contained 10 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, 6% glycerol,
1% BSA, 1 µg poly(dI-dC) (Amersham Pharmacia Biotech, Piscataway,
NJ), 200 ng labeled probe, and 4 µg nuclear extract in 15 µl total
volume. Reactions were conducted for 20 min at RT. Where supershift
assays were performed, 1 µl anti-p65 or anti-p50 antiserum
was added to nuclear extracts (30 min, on ice) before addition of DNA
probe. Samples were immediately loaded onto a 5%
acrylamide:bis-acrylamide (29:1) gel and electrophoresed in 0.5x TBE
(Tris-borate-EDTA) at 160 V for 2.5 h with cooling. Gels
were placed onto filter paper, dried under vacuum (80°C, 1 h),
and exposed to Kodak X-AR film for 18–24 h.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or IL-12
We have previously reported that, in contrast to neutrophils,
peritoneal macrophages derived from mice infected i.p. with tachyzoites
do not express detectable IL-12 p40 (33). To examine the
general capacity of M to produce and secrete IL-12 and TNF-,
thioglycolate-elicited peritoneal M (PM) and several macrophage
cell lines were subjected to tachyzoite infection with RH strain
tachyzoites (2 h) or to stimulation with LPS (30 min). After treatment,
supernatants were replaced with fresh medium, incubated for an
additional 6 h, and then assayed for TNF- and IL-12. As shown
in Fig. 1
, LPS was highly effective at
inducing both TNF- and IL-12 (p40), although absolute levels varied
from cell type to cell type. Contrasting with this result, tachyzoite
infection triggered little or no production of these proinflammatory
cytokines (Fig. 1), even with prior IFN- priming (data not shown).
There was a lack of response to T. gondii when the
tachyzoite:cell ratio was varied (10:1–0.1:1), and when treatment time
ranged from 30 min to 24 h (data not shown). In addition, this
result was not a peculiarity of the RH parasite strain (which is known
for its high virulence in mice), because an identical lack of response
was found using tachyzoites of the low virulence ME49 T.
gondii strain (data not shown).
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does not activate
nuclear translocation of NF-B
The M’s apparent unresponsiveness to Toxoplasma
infection as measured by cytokine production led us to examine the
activation of NF- in LPS-treated and
Toxoplasma-infected cells, because this transcription factor
is essential for the production of many proinflammatory cytokines,
particularly TNF- and IL-12 (38, 39). To examine the
activation state of NF-B in PM, we cultured cells on
coverslips, subjected them to LPS or Toxoplasma treatment
for various time periods, performed indirect immunofluorescence
staining for the p65 subunit of NF-B, and analyzed nuclear
localization of the transcription factor by confocal microscopy. As
shown in Fig. 2, when M were
stimulated with LPS, NF-B translocated to the nucleus within 15 min
of treatment. Nuclear NF-B was apparent throughout the LPS time
course, although levels appeared to wane after 2-h stimulation (Fig. 2). In contrast, the transcription factor remained in the cytoplasm of
both untreated (data not shown) and tachyzoite-infected M at all
time points (Fig. 2, bottom panels). The lack of NF-B
activation in tachyzoite-infected cells is consistent with and at least
partially explains why TNF- and IL-12 are not produced in response
to the parasite.
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We further investigated the apparent "silent entry" of
tachyzoites into M by testing whether infection stimulated
phosphorylation of MAP kinases involved in proinflammatory responses.
As shown in Fig. 3, tachyzoites induced
rapid phosphorylation of stress-activated protein kinase/Janus kinase,
extracellular signal-regulated kinase1/2, and p38 MAP kinases,
appearing as early as 15 min after parasite exposure. The finding that
MAP kinases are phosphorylated as soon as 15 min after parasite
addition suggests that the response is triggered by a signal provided
during initial parasite invasion, or possibly that extracellular
tachyzoites secrete MAP kinase-activating molecules.
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and IL-12 production
We next asked whether tachyzoites might actively suppress M
inflammatory cytokine induction. To address this issue, we tested the
effect of preinfection on the ability of PM to produce cytokines in
response to LPS stimulation. As shown in Fig. 4, 2
-h preinfection of PM with
parasites inhibited LPS-stimulated TNF- and IL-12 production in a
dose-dependent fashion. In Fig. 5, we
examined the rapidity with which T. gondii induced M
nonresponsiveness. As shown, within 30 min of tachyzoite infection
PM were refractory to LPS stimulation, as measured by TNF-
production. Therefore, the inhibitory effect of tachyzoites on M
cannot simply be explained by cytolytic effects of the parasite as it
replicates within the host cell, because the tachyzoite generation time
is 
6–8 h (40).
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released a suppressive molecule in response to the parasite, we
used a low parasite:PM ratio (0.5:1) which resulted in 30%
infection after 2 h. As shown in Fig. 6 (left panel),
tachyzoite infection alone did not stimulate TNF- production,
whereas 30-min LPS stimulation resulted in high levels of intracellular
cytokine (center panel). When LPS was added after 2-h
preinfection with tachyzoites, uninfected cells produced TNF- (Fig. 6, right panel), but infected PM did not produce cytokine
in response to LPS stimulation (arrow). This experiment strongly argues
that infected cells do not release inhibitory factors that
down-regulate cytokine production in LPS-stimulated, uninfected PM,
and that tachyzoite-triggered inhibition operates at an intracellular
level. Related to this issue, we also found identical parasite-induced
suppression in PM derived from IL-10 knockout mice, ruling out a
role for the latter anti-inflammatory cytokine in the results we
observed (data not shown).
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We next performed RPAs on RNA purified from PM to determine
whether production of other proinflammatory mediators was blocked by
T. gondii preinfection. As shown in Fig. 7, A–C, tachyzoite infection
induced little or no up-regulation of a broad range of proinflammatory
cytokine genes. In contrast, LPS induced up-regulation of mRNA for
IL-12(p40/35), TNF-, IL-1, IL-1, IL-1Ra, IL-18, IL-6,
migration inhibitory factor, IL-2Ra, and iNOS. Preinfection with
Toxoplasma resulted in a clear decrease in steady-state
levels of IL-12(p40/35), TNF-, IL-6, and iNOS mRNA (Fig. 7, A, B, and D). Although there was not
an obvious decrease in steady-state levels of mRNA for IL-1,
IL-1, and IL-18, densitometric analysis relative to GAPDH suggested
that these cytokine genes were also down-regulated by preinfection
(Fig. 7D).
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B
Because tachyzoite infection appeared to inhibit LPS-induced
transcription of several NF-B-dependent genes, we sought to
determine whether infection inhibited NF-B activation in response to
LPS. To answer this question, we stained infected, LPS-stimulated PM
for NF-B p65 and analyzed the cells by confocal microscopy. As shown
in Fig. 8, untreated and
tachyzoite-infected cells did not exhibit nuclear NF-B (A
and B, respectively), in contrast to LPS-stimulated PM
(C). Interestingly, although preinfected PM exhibited
evidence for some nuclear translocation of NF-B after LPS
stimulation, levels of this transcription factor appeared lower in
infected cells (compare C and D, Fig. 8).
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chain gene which binds both
p65:p50 heterodimer and the nontransactivating p50:p50 homodimer. As
shown in Fig. 9, nuclear extracts
prepared from PM (M) and tachyzoite-infected cells (Tg) contained
low amounts of NF-B species. In contrast, extracts derived from
LPS-stimulated (L) PM contained proteins that shifted the Ig
oligonucleotide. Confirming the results suggested by the confocal
microscopy (Fig. 8), relative levels of DNA binding proteins in
preinfected, LPS-treated nuclear extracts were lower than those in
LPS-only extracts. This result indicates that there is less active
NF-B in the nuclei of infected cells. To identify the NF-B
subunits in the complexes, antisera against p50 and p65 subunits were
used in supershift assays. As depicted in Fig. 9 (upper
panel), antiserum against the p65 subunit supershifted the higher
molecular mass band in the LPS and Tg/L lanes. These results indicate
that in the presence of LPS the upper band contains the p65 subunit and
is most likely the p65:p50 heterodimer. The bottom panel of
Fig. 9 shows the results of a supershift assay performed with
anti-p50 antiserum. The antiserum supershifted both upper and lower
complexes, suggesting that the bands contain p65:p50 and p50:p50
species, respectively. These results rule out induction of the p50:p50
homodimers as the basis of the inhibition reported here, findings which
are significant since the latter is thought to provide the molecular
basis for endotoxin tolerance (36, 41, 42).
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B
The blockade in NF-B translocation led us to examine whether
phosphorylation-driven IB degradation was defective in
parasite-infected cells. Fig. 10A shows an immunoblot
analysis using Ab specific for total IB and for the
phosphorylated form of this inhibitor molecule. Because the IB
gene itself can be induced by inflammatory stimuli such as LPS
(43, 44), the experiments were performed in the presence
and absence of cycloheximide (CHX) to directly examine phosphorylation
and degradation of IB.
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A, in the presence of CHX LPS
induces IB phosphorylation at 10 min after stimulation, which
is followed by rapid disappearance of both phosphorylated and
nonphosphorylated IB forms. In the absence of CHX, similar
phosphorylation and degradation occurred in response to LPS, but IB
reappeared at 60 min after stimulation. Despite failure of NF-B
to translocate in response to T. gondii, the parasite alone
induced IB phosphorylation and degradation at 20 min after
infection. In the absence of CHX, Toxoplasma also induced
resynthesis of IB at 60 min after infection.
We next assessed IB ubiquitination in response to LPS stimulation
or tachyzoite infection. Under normal conditions, IB degradation
rapidly follows ubiquitination, and it is therefore difficult to
visualize high molecular mass ubiquitinated IB species. This
problem can be circumvented by inclusion of the peptide aldehyde
MG-132, an inhibitor of the 26S proteasome (45). As shown
in Fig. 10B, inhibition of the proteasome results in
accumulation of high molecular mass IB species during both LPS
stimulation and tachyzoite infection, consistent with
polyubiquitination of this inhibitor molecule. This result is in accord
with the degradation of IB shown in Fig. 10A.
NF-B fails to activate during in vivo infection
Finally, we examined the effect of Toxoplasma on
NF-B translocation during in vivo infection. Mice were injected i.
p. with tachyzoites, then at varying times peritoneal cells were
collected and stained for NF-B p65 (Fig. 11). At no time examined was there
evidence for NF-B translocation in either M or neutrophils, the
major cell populations present in the peritoneal cavity of infected
mice. Although the PMN population was negative for p65 at early time
points, by 4 h, these cells were induced to cytoplasmically
express this transcription factor. Interestingly, we found that
cytoplasmic p65 levels in M appeared to be decreased in infected
cells at 2 and 3 h after infection. Furthermore, at 4 h p65
reappeared, although its expression continued to be restricted to the
cytosol.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are readily infected by T. gondii, yet at the same
time these phagocytic cells are capable of displaying potent
microbicidal activity through high level production of inflammatory
mediators. Here, we show that tachyzoites invade M without
activating NF-B and triggering TNF- and IL-12 production, two key
cytokines which display a strong dependency on this transcription
factor. More importantly, the parasite actively suppresses the M’s
ability to respond to LPS. The mechanistic basis for this effect
appears to be due, at least in part, to an ability of T.
gondii tachyzoites to prevent NF-B nuclear translocation,
despite triggering phosphorylation-dependent degradation of IB.
Our results are distinct from a recent report on Salmonella
infection in epithelial cells (45). In that system, as in
ours, both microbial pathogens induce IB phosphorylation, but
subsequent degradation necessary for NF-B activation is blocked
during Salmonella infection. The basis for
Salmonella-induced blockade of IB degradation was found
to be through prevention of IB ubiquitination, which normally
enables degradation by the 26S proteasome (46). For
Toxoplasma infection, IB ubiquitination and degradation
appear to be normal, placing the block downstream of these events.
We do not know why NF-B fails to translocate despite normal IB
degradation. Possibly, NF-B translocation in these cells is under
the control of other IB family members, such as IB or IkB
,
which would be blocked from degradation by Toxoplasma
infection. In addition, NF-B translocation requires association with
several proteins required for docking and transport across the nuclear
membrane (47). Phosphorylation of p65 has also been
implicated in both nuclear import and DNA binding activity (48, 49). We are currently examining whether any of these activities
are blocked by T. gondii infection.
Our results show that IB expression is reinduced during
tachyzoite infection, despite a failure to translocate NF-B p50:p65
heterodimers. Nevertheless, transcription of IB itself is well
known to be regulated by NF-B (43, 44). One possible
explanation for our results is that sufficiently small amounts of
NF-B translocate to enable IB synthesis. Another possibility
is that transcription is controlled by other NF-B family members or
possibly by factors independent of NF-B
Although our results clearly show a parasite-induced block in TNF-
production, up-regulation of mRNA was only partially blocked. Because
regulation of this cytokine is known to occur partly at the level of
transcription (50), it is possible that, in addition to
preventing IB degradation, T. gondii exerts an effect on
posttranscriptional regulation of TNF-. Related to this issue,
NF-B nuclear translocation in response to LPS was never completely
prevented by the parasite. This could result simply from incomplete
infection of the M populations, but may also indicate the presence
of other inhibitory mechanisms employed by the parasite to evade
inflammatory cytokine induction.
Host cell invasion by Toxoplasma is a tightly regulated
process, involving sequential discharge of microneme, rhoptry, and
dense granule contents. Release of the constituents of these complex
granules begins at the earliest stage of parasite entry and continues
over the course of the first 20 min of invasion (51). We
found that tachyzoites suppressed M responses to LPS within 30 min
of infection. Thus, it is possible that one or more such discharged
molecules are involved in inducing M nonresponsiveness. In this
regard, some Toxoplasma proteins, such as ROP-2 and GRA-5,
are known to insert into the parasitophorous vacuole membrane, thereby
exposing a portion of the parasite molecule to the host cell cytoplasm
(52, 53). Such proteins would be candidate molecules which
could potentially interfere with host cell signaling pathways.
Our results establish that T. gondii invasion of macrophages
is a silent event with regard to NF-B activation, and furthermore
that the activity of this transcription factor is suppressed by
preinfection. The NF-B transcription factor family is composed of
several members (NF-B1/p50: p65, NF-B2/p52: p65, RelA, RelB, and
c-Rel) which play critical roles in immunity (46).
Recently, several studies have used knockout mice to examine the role
of NF-B family members in resistance to toxoplasmosis. Thus,
NF-B2-/- and
Bcl-3-/- animals survive early infection but
display increased mortality during chronic infection, suggesting an
impaired ability to mount protective T cell responses (54, 55). In another study, RelB-/- mice were
found to display normal M responses, but defective production of NK
and T cell IFN-, and increased susceptibility to acute infection
(56). In the same study, NF-B1 knockout animals were
reported to display the phenotype of wild-type mice during T.
gondii infection. The latter result is consistent with our data
showing that NF-B1 is not activated during infection, and, indeed,
appears to be actively suppressed by the parasite. Because IL-12 and
TNF- are known to be induced during in vivo infection, we do not
exclude the possibility that cells such as fibroblasts and DC
translocate NF-B in response to infection. We are currently
examining the cell specificity of the inhibition reported
here.
Why would Toxoplasma selectively suppress proinflammatory
cytokine responses in infected M? There are two theoretical
possibilities. The parasite is an extremely strong activator of both
innate and acquired immunity. Thus, infection confers a high level of
resistance to reinfection, mainly through the activity of
CD4+ Th1 and CD8+ TC1
lymphocytes (57, 58, 59, 60). It may benefit the parasite to
temporarily delay or dampen the proinflammatory cytokine response to
allow initial rounds of tachyzoite division to increase parasite
numbers. Early expansion of the tachyzoite population may ensure that
enough parasites survive attack by the immune system so that cyst
formation and parasite persistence occurs within the host.
An alternate possibility is that Toxoplasma-induced
nonresponsiveness may represent a mechanism to avoid immunopathology
during infection. It is well established that the parasite can induce
type 1 cytokine overproduction, resulting in lethal pathology during
oral infection of certain inbred mouse strains as well as in IL-10
knockout mice (24, 25, 26). Actively inhibiting induction of
proinflammatory mediators in infected M may represent a way of
avoiding such pathology during normal infection. In this regard, it has
recently been shown that administration of soluble tachyzoite Ag
results in paralysis of splenic DC IL-12 production and that this can
prevent infection-induced immunopathology in IL-10 knockout mice
(61). Unlike the M nonresponsiveness reported here, DC
paralysis is not intrinsic to the cells, but rather is an effect of the
splenic microenvironment.
Toxoplasma infection is well known to induce high levels of
the inflammatory mediators which we now show are actively suppressed in
infected M. Originally, these cells were regarded as an important
early proinflammatory cytokine source during T. gondii
infection. It may be noteworthy that many of these earlier studies used
soluble parasite Ag rather than the live infection system used here.
Regardless, our studies reveal a hitherto unrecognized molecular
mechanism by which Toxoplasma evades proinflammatory
cytokine induction. This may enable the parasite to find temporary safe
harbor within M in a manner allowing parasite persistence and
long-term host survival.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eric Denkers, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. E-mail address: eyd1{at}cornell.edu
3 Abbreviations used in this paper: M, macrophage; iNOS, inducible NO synthase; DC, dendritic cell; CM, complete medium; PM, peritoneal M; RPA, ribonuclease protection assay; RT, room temperature; IL-1/2Ra, IL-1/2R antagonist; MAP, mitogen-activated protein; CHX, cycloheximide.
Received for publication December 20, 2000. Accepted for publication June 21, 2001.
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