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*
Department of Biochemistry and Immunology, Biological Sciences Institute, Federal University of Minas Gerais and Centro de Pesquisas René Rachou, Oswaldo Cruz Foundation, Belo Horizonte, Brazil;
Department of Parasitology, University of Sao Paulo, Sao Paulo, Brazil;
Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan;
Unit for Experimental Oncology, Federal University of Sao Paulo, Sao Paulo, Brazil; and
¶ Boston University School of Medicine, Boston, MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
NO by GPI anchors derived from T. cruzi trypomastigotes.
Thus, highly purified GPI anchors from T. cruzi
parasites are potent activators of TLR-2 from both mouse and human
origin. The activation of TLR-2 may initiate host innate defense
mechanisms and inflammatory response during protozoan infection, and
may provide new strategies for immune intervention during protozoan
infections. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
two mediators that appear to be important for the initiation of IFN-
synthesis by NK cells (8). Recognition of bacterial
glycolipids by IFN--exposed phagocytic leukocytes is also
responsible for the rapid triggering of a variety of protective immune
processes, including the synthesis of reactive oxygen and nitrogen
intermediates (9). Animals that have not yet developed
adaptive immune responses to pathogens depend upon these processes for
survival. Similar cytokine circuits and effector mechanisms appear to
be involved in resistance to early infection by various protozoan
parasites (8, 10), which are common etiologic agents of
medical and veterinary diseases. Glycosylphosphatidylinositol (GPI) anchors are abundant molecules in the membrane of parasitic protozoa (11). Recent studies have documented the immunostimulatory and regulatory activity of protozoan-derived GPI anchors and related structures (12). Among the biological properties of these protozoan GPI anchors is their ability to elicit the synthesis of pro-inflammatory cytokines as well as NO by host macrophages (12, 13, 14, 15, 16, 17, 18, 19), similar in many respects to the LPS of Gram-negative bacteria. In contrast, purified glycoinositolphospholipids (GIPLs) and lipophosphoglycan from protozoan parasites suppress several functions of the host immune system, especially at higher concentrations (20, 21, 22, 23, 24).
Trypanosoma cruzi is the causative agent of Chagas’ disease, a parasitic infection of enormous importance in Central and South America. To better understand the early stimulation of the innate immune system by parasitic protozoa, we have focused on the identification and chemical characterization of T. cruzi products that trigger the pro-inflammatory and effector functions of macrophages (17, 19). These studies indicate that GPI anchors purified from mucin-like glycoproteins (tGPI-mucin) of T. cruzi trypomastigotes (tGPI) play an essential role in triggering various macrophage functions. In addition, our data show that the potent pro-inflammatory activity of tGPI is dependent on its fine structure, which apart from its longer glycan core includes an unsaturated fatty acid at the sn-2 position of the alkylacylglycerolipid component (17, 19).
Despite the growing evidence implicating GPI structures from parasitic
protozoa in the induction of cytokine synthesis as well as in effector
functions by macrophages, not much is known about the receptor(s) and
signaling pathways that are triggered by these GPI anchors.
Nevertheless, different studies indicate a similarity of signaling
pathways, gene expression, and functions displayed by macrophages
exposed to either purified tGPI anchors or tGPI-mucin (12, 17, 18, 19, 25) and bacterial glycolipids/lipopeptides
(4, 5, 6, 7). Recent studies demonstrate that TLR-2 or TLR-4 are
responsible for triggering various functions in macrophages exposed to
Gram-positive, Mycoplasma spp. and M.
tuberculosis (5, 6, 7, 26) or Gram-negative bacteria
(4, 26), respectively. Here, we tested the ability of
T. cruzi-derived GPI anchors and GIPLs to trigger TLR-2 and
TLR-4, and studied the role of such receptors in the ability of
parasitic glycolipids to elicit IL-12, TNF-, and NO by murine
macrophages. Our results show that protozoan-derived GPI anchors and
GIPLs preferentially activate TLR-2, which is largely responsible for
the initiation of the various macrophage functions induced by these
protozoan-derived glycolipids.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Green monkey kidney-derived LLC-MK2 cells
(American Type Culture Collection, Manassas, VA) were grown at 37°C
in DMEM (Life Technologies, Grand Island, NY) containing 5%
heat-inactivated FBS, 40 µg of gentamicin sulfate/ml, 0.3% sodium
bicarbonate in a 5% CO2 atmosphere.
Trypomastigote forms of T. cruzi (Y strain) were obtained
from the supernatant of LLC-MK2 cells 6–8 days
after infection with 10 trypomastigotes per cell. Epimastigote forms,
from either Y or CL strain, were grown at 28°C in cell-free liver
infusion tryptose medium supplemented with 10% FBS. Live parasites
were collected from either tissue or liquid culture, washed three times
in ice-cold PBS, and used in the assays with transfected Chinese
hamster ovary (CHO) cells or kept at -70°C for purification of
GPI-mucins, GPI anchors, or GIPLs. The epimastigote- and
trypomastigote-derived GPI-mucins and GIPLs were purified by sequential
organic solvent extraction and hydrophobic interaction chromatography
as described elsewhere (19, 27). The following glycolipids
were purified from T. cruzi parasites and used in the
experiments presented here: tGPI-mucin, derived from tissue culture
trypomastigotes of Y strain of T. cruzi containing a
1-O-(C16:O)-2-O-alkyl-(C18:1 or C18:2)
acylglycerol; eGPI-mucin, derived from epimastigotes of Y strain of
T. cruzi containing a
1-O-(C16:O)-2-O-alkyl-(C16:0) acylglycerol; eGIPL
Y CER, derived from epimastigotes of Y strain of T. cruzi
containing a ceramide (C24:0) fatty acid-(C18:0) sphinganine; eGIPL CL
AAG, derived from epimastigotes of CL strain of T. cruzi
containing a (C16:O)-2-O-alkyl-(C16:0) acylglycerol; and
eGIPL CL CER, derived from epimastigotes of CL strain of T.
cruzi containing a ceramide (C24:0) fatty acid-(C18:0)
sphinganine. These GPI-anchored molecules also vary according to their
glycan core composition and ethanolaminephosphate and
ethanolaminephosphonate head groups. Detailed compositional and
structural analyses of each of the GPI-anchored preparations are
described in Almeida et al. (19). Nitrous acid deamination
was used to produce the phosphatidylinositol (PI) and glycan core of
tGPI or tGPI-mucins, as previously described (19). Edman
microsequencing (five cycles) and positive-ion mode matrix-assisted
laser desorption ionization time-of-flight mass spectrometry
(19), positive-ion mode electrospray mass spectrometry,
and Mycoplasma spp. detection ELISA tests were used to
exclude the possibility that T. cruzi-derived glycolipids
were contaminated with Mycoplasma spp.-derived
lipopeptides. These results indicated that T. cruzi-derived
glycolipids were free of Mycoplasma spp. lipopeptides.
Furthermore, N-acetylation abolished NO-, TNF--, and
IL-12-inducing activity of mycoplasma macrophage-activating
lipopeptide (MALP), but had no significant effect on bioactivity of the
tGPI-mucin (Ref. 28 ; data not shown).
Bacteria and bacteria-derived glycolipids/lipopeptides
The UV killing of Staphylococcus aureus (ATCC 12.692) and Escherichia coli (HB101) followed the same procedure. Bacteria were grown overnight in 200 ml of Luria Broth, centrifuged for 30 min at 1200 x g, and resuspended in 20 ml PBS. Bacterial density was resolved by limiting dilution of washed bacteria, determining the CFUs. A UV germicide lamp (G15T8; General Electric, Fairfield, CT) was used at 10 cm of an open petri dish with 2 ml of bacteria for 20 min, and the bacteria were stored at -20°C until use. LPS from E. coli serotype O55:B5 prepared by the Westphal method was obtained from Sigma (St. Louis, MO). Outer surface protein A (Osp A) was purified from B. burgdorferi (5), and synthetic MALP from M. fermentans was obtained as previously described (29).
CHO cell lines
The CHO reporter cell lines (CHO/CD14, CHO/CD14/TLR-2, and
CHO/CD14/TLR-4) (5) were maintained as adherent monolayers
in Ham’s F-12/DMEM supplemented with 5% FBS, at 37°C, 5%
CO2, and antibiotics. All of the cell lines are
derived from clone 3E10, a CHO/CD14 cell line that has been stably
transfected with a reporter construct containing the structural gene
for CD25 under the control of the human E-selectin promoter. This
promoter contains a NF-
B binding site; CD25 expression is completely
dependent upon NF-B translocation to the cell nucleus
(30). Cells expressing TLRs were constructed by stable
transfection of the CHO/CD14 reporter cell line with the cDNA for human
TLR-2 or TLR-4 as described (5). In addition to the LPS
responsive cell lines described above, we also tested an LPS
nonresponder cell line (30) designated clone 7.19 as well
as a clonal line derived from this mutant that was transfected with
CD14 and TLR-2 (7.19/CD14/TLR-2). This cell line was derived from 3E10,
and reports NF-B activation via the surface expression of CD25,
similarly to the other CHO lines described. The LPS nonresponsive
phenotype of the 7.19 cell lines appears to be due to a mutation in the
MD-2 gene, and thus is defective in signaling via
TLR-4 (30).
Flow cytometry analysis
CHO reporter cells were plated at a density of 1 x 105 cells/well in 24-well tissue culture dishes. The following day, either bacteria, live protozoa, or purified glycolipids were added as indicated, in a total volume of 0.25 ml of medium/well, for 18 h. The cells were then harvested with trypsin-EDTA and washed once with medium and again with PBS. Subsequently, the cells were counted and 1 x 105 cells stained with PE-labeled anti-CD25 (mouse mAb to human CD25, R-PE conjugate; Caltag Laboratories, Burlingame, CA) 1:200 in PBS, on ice, in the dark, for 30 min. After labeling, the cells were washed twice with 1 mM sodium azide in PBS, resuspended in 1 mM sodium azide in PBS, and examined by flow cytometry (BD Biosciences, San Jose, CA) for the expression of surface CD25 as described (5). Analyses were performed using CellQuest software (BD Biosciences).
Inflammatory and bone marrow macrophages
Wild-type, TLR-2 knockout (KO), and TLR-4 KO mice (4, 26) were inoculated i.p. with 2 ml of 3% thioglycollate and, 4 days later, the elicited peritoneal exudate cells were harvested in cold serum-free DMEM. The medium used in the macrophage cultures (MacMed) consisted of DMEM supplemented with 40 µg of gentamicin/ml and 5% heat-inactivated FCS. Bone marrow was washed from the femur of 8- to 12-wk-old wild-type or TLR-2 KO mice with DMEM supplemented with gentamicin. Cells were washed, resuspended in marrow culture medium, and plated at 10–15 x 106 cells/10 ml/plate on 100-mm nontissue culture-treated petri dishes. Marrow culture medium consisted of DMEM supplemented with 10% FCS and gentamicin, plus 30% supernatant from confluent cultures of L929 fibroblasts, as a source of macrophage-CSF. After 5–7 days of culture at 37°C, 5% CO2, plates were gently washed to remove nonadherent cells. Plates contained 3–5 x 106 macrophages, >95% pure.
Peritoneal and bone marrow-derived macrophages were resuspended in
MacMed at 2 x 106/ml, and 100-µl aliquots
were dispensed into wells of a 96-well plate. Cells were allowed to
adhere at 37°C and 5% CO2 for 3 h, and
were then washed once with serum-free DMEM and 150 µl of MacMed was
added to each well in the presence or absence of 50 U/ml IFN- and
incubated overnight at 37°C and 5%
CO2. Different macrophage-stimulating
preparations were added to the macrophage cultures in a final volume of
200 µl/well. Aliquots of the supernatant (50 and 100 µl) were
collected after 24 and 48 h of culture for nitrite, TNF-, and
IL-12 (p70 or p40, as indicated) measurements, respectively (17, 19). The concentration of nitrite was determined by the Griess
reaction (31). Levels of TNF- and IL-12 (p70 or
p40, as indicated) in the supernatants were measured by a
commercially available ELISA kit (Duoset; R&D Systems,
Minneapolis, MN).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-dependent expression of CD25 in CHO cells transfected with
TLR-2
CHO reporter cell lines that were stably transfected with
CD14 alone (CHO/CD14), TLR-2 and CD14 (CHO/CD14/TLR-2), or TLR-4 and
CD14 (CHO/CD14/TLR-4) were exposed for 24 h to live T.
cruzi trypomastigotes (17), and NF-B activation
was assessed by measuring the expression of CD25 by flow cytometry
(5, 30). No increase in the induction of CD25 expression
by CHO/CD14 cells exposed to T. cruzi parasites was observed
(Fig. 1
A). In contrast, live
T. cruzi trypomastigotes enhanced the CD25 expression in the
CHO/CD14/TLR-2 cell line (Fig. 1B), indicating that TLR-2
expression had led to the activation of NF-B. Activation of NF-B
was dependent on the parasite-to-CHO/CD14/TLR-2 cell ratio (Fig. 1C), the maximum effect being reached with 10 parasites per
cell. This concentration of parasites saturated the response; we
observed similar levels of activation when the CHO/CD14/TLR-2 cells
were exposed to 50 trypomastigotes per cell (data not shown). Mock
parasite pellets obtained from LLC-MK2 cell
supernatants were unable to trigger CD25 expression on the
CHO/CD14/TLR-2 cell line (data not shown). As positive controls, we
stimulated CHO/CD14 and CHO/CD14/TLR-2 cells with either UV-killed
E. coli or S. aureus (5). Data in
Fig. 1, D and E, show that E. coli
induced expression of CD25 in both CHO/CD14 and CHO/CD14/TLR-2 cells.
In contrast, S. aureus induced expression of CD25 only on
the surface of CHO/CD14/TLR-2 (Fig. 1E), but not on CHO/CD14
cells (Fig. 1D). The maximal CD25 expression on CHO cells
was obtained at a bacteria-to-cell ratio of 500:1 (Fig. 1F)
and 5000:1 (Fig. 1G) for E. coli and
S. aureus, respectively. We conclude that live T.
cruzi trypomastigotes were capable of triggering TLR-2.
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The CHO cells transfected with human CD14 alone or human CD14 and
TLR-2 were also exposed to tGPI-mucin (17, 19) or LPS, and
NF-B activation was evaluated by measuring the expression of the
CD25 reporter transgene by flow cytometry (5, 30). CD25
expression was enhanced in both CHO/CD14 (Fig. 2A) and CHO/CD14/TLR-2 (Fig. 2B) exposed to LPS, because both lines of CHO cells express
endogenous TLR-4. In contrast, tGPI-mucin induced expression of CD25 on
CHO/CD14/TLR-2 (Fig. 2B), but not on CHO/CD14 (Fig. 2A) cells. Fig. 2C shows the titration of LPS
activity in CHO/CD14 cells. It is noteworthy that maximal expression of
CD25 in CHO/CD14/TLR-2 cells exposed to tGPI-mucin (Fig. 2D)
was observed at 10 nM, but even at subnanomolar concentrations (i.e.,
0.1 nM), a significant increase in the expression of CD25 was achieved.
These data indicate that the tGPI-mucin is among the most potent TLR-2
agonists thus far described (5, 6, 7).
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, A and B, we confirmed that the ability of
tGPI-mucin to activate NF-B via TLR-2 was mostly recovered using a
highly purified tGPI preparation. Removal of the lipid moiety from the
tGPI (Fig. 3B) or tGPI-mucin (data not shown) by nitrous
acid deamination (19) completely abolished the activity of
tGPI anchors in CHO/CD14/TLR-2 cells. The resulting PI also failed to
elicit expression of the CD25 reporter transgene in these cells, even
at 100 nM (Fig. 3B). These results indicate that both the
lipid tail and the glycan moiety of the tGPI are necessary for
triggering TLR-2, which is in agreement with our previous studies
indicating that the cytokine and/or NO-inducing activity could not be
dissociated (19).
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Recently, we compared the bioactivity of 12 different preparations
of GPI anchors or GIPLs derived from either trypomastigote or
epimastigote developmental stages of T. cruzi. All these
preparations were highly purified and had defined structures as
determined by electrospray mass spectrometry and electrospray-mass
spectrometry-collision-induced dissociation/mass spectrometry
(ES-MS-(CID)-/MS) (19). We found that the
unsaturated fatty acids at the sn-2 position of the alkylacylglycerol
is likely to be essential for the extremely potent bioactivity of
tGPI-mucin or tGPI on murine inflammatory macrophages. We also tested
different GPI-mucins (eGPI-mucins) and GIPLs (eGIPLs) from the
epimastigote stage of T. cruzi for their ability to induce
the expression of CD25 by CHO/CD14/TLR-2. The eGPI-mucins and eGIPLs
differ in their fine structure from tGPI in that they contain a lipid
moiety with either ceramide or alkylacylglycerol containing only
saturated fatty acid chains. Furthermore, they contain a shorter glycan
moiety when compared with the tGPI one (19). As shown in
Fig. 3
C, eGPI-mucin or eGIPL activated
NF-B-mediated CD25 expression in CHO/CD14/TLR-2 cells, but only at
micromolar concentrations. The bioactivity of eGPI-mucin and eGIPLs on
CHO/CD14/TLR-2 cells was higher than that previously observed in murine
inflammatory macrophages (Refs. 17, 18, 19 and Fig. 3C). This could be explained in part by the high degree of
expression of TLR-2 in the transfected CHO cells, which may be higher
than macrophages. Together, these findings are consistent with our
previous study (17, 18, 19) and indicate that in general
eGPI-mucin or eGIPL are at least 100- to 1000-fold less active in
triggering the TLR-2 than the tGPI-mucin or tGPI.
GPI anchors and GIPLs derived from both trypomastigotes and
epimastigotes trigger NF-B-dependent expression of CD25 in CHO cells
transfected with TLR-2, but containing defective TLR-4 signaling
CHO cells express endogenous TLR-4, and low levels of activation
were observed in CHO cells transfected with CD14, but not with TLR-2,
when T. cruzi-derived tGPI anchors were used at high
concentrations (i.e., 0.1–1 µM). It was important to test the
ability of different GPI/GIPL preparations in triggering CD25
expression in CHO cells with a defective TLR-4 signaling complex (clone
7.19, a mutant LPS nonresponder cell line that is defective in the
expression of MD-2; Ref. 30) because of the possibility
that small amounts of contaminating endotoxin in our preparations might
confound interpretation of the results. As shown in Fig. 4, A-E, clones of 7.19
transfected with CD14 only or CD14 plus TLR-2 were highly refractory to
LPS stimulation. These findings are in agreement with previous studies
showing that TLR-4, but not TLR-2, is responsible for the CHO cell
activation by highly purified LPS (4, 33). In contrast,
the 7.19 transfected with CD14 and TLR-2, but not with CD14 only, were
responsive to tGPI-mucins, eGIPLs, or to the lipopeptide, OspA
(5). These results show that TLR-2 is sufficient for
responsiveness to these lipids, and does not require a functional
TLR-4/MD-2 signal transduction complex to mediate NF-B
activation in transfected CHO cells exposed to GPI anchors or GIPLs
derived from T. cruzi parasites. Consistent with the results
presented in Fig. 3, tGPI-mucin was more active than eGPI-mucin (data
not shown) or GIPLs in activating 7.19/CD14/TLR-2.
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We have shown previously that tGPI-mucin or tGPI induces high
levels of NO and IL-12 in IFN--primed inflammatory macrophages
(17, 19). In contrast, the induction of high TNF-
levels by protozoan glycolipids occurs in either IFN--primed or
unprimed inflammatory macrophages. Our previous studies also show that
although the receptors triggered by tGPI-mucin and LPS are functionally
similar (12, 25), they are not identical, because
macrophages from TLR-4 mutant C3H/HeJ mice can mount a major cytokine
and NO response upon stimulation with tGPI-mucin or tGPI
(17, 18, 19). Therefore, we tested the involvement of TLR-2
and TLR-4 on cytokine induction by tGPI-mucin and tGPI in murine
inflammatory macrophages. Macrophages from TLR-4 KO or TLR-2 KO mice
were exposed to the prototypical TLR-4 and TLR-2 "ligands" LPS
(4) and MALP (7), respectively. The data
presented in Fig. 5 confirm the results
obtained with CHO/CD14/TLR-2 cells, showing that IFN--primed
inflammatory macrophages from TLR-2 KO mice did not produce NO,
TNF-, and IL-12 upon stimulation with either tGPI-mucin or tGPI. The
inflammatory macrophages from TLR-4 KO were still responsive to the
T. cruzi glycolipids, producing levels of NO, TNF-, or
IL-12 comparable to macrophages from wild-type mice. In contrast,
macrophages from TLR-4 KO mice, but not from TLR-2 KO mice, were
unresponsive to LPS. As previously reported (17, 18, 19),
eGPI-mucin and eGIPLs at 100 nM were unable to elicit NO, TNF-, or
IL-12 synthesis by macrophages from either wild-type or TLR-2 KO mice.
Consistently, eGPI-mucins and eGIPLs (17, 18, 19) as well as
GPI anchors from T. brucei (16) and P.
falciparum (13) elicited NO and TNF- synthesis by
murine inflammatory macrophages only at micromolar concentrations. As
shown in Table I, although less
responsive than inflammatory macrophages, bone marrow macrophages also
produced significant amounts of NO, TNF-, and IL-12 upon stimulation
with tGPI-mucin. We observed TNF- synthesis by either unprimed or
IFN--primed macrophages exposed to tGPI-mucin. In essence, the
results with macrophages derived from TLR-2 KO and TLR-4 KO mice show
that TLR-2 is not only a major receptor triggered by GPI anchors from
parasitic protozoa, but is also essential for the macrophage response
to tGPI or tGPI-mucin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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signaling
pathway that also enhances host cell susceptibility to infection
(36). In contrast, induction of a NF-B-dependent
mechanism by the parasite results in enhancement of resistance to
T. cruzi infection in nonimmune host cells
(37). Independent studies have shown the ability of
parasite molecules to trigger different activities in cells from host
innate immune system, which are also important determinants of host
resistance to T. cruzi infection (38, 39, 40). In
fact, an early study demonstrated that host resistance/susceptibility
to infection is, at least in part, determined at the very early stages
of infection, before the development of acquired immunity
(41). Therefore, the combinatory effect of these different
signaling pathways triggered by T. cruzi parasites in
nonimmune cells and/or cells from innate immune system may have
important consequences in different aspects of T. cruzi
infection such as load of tissue parasitism, tissue tropism, and the
pathogenesis of Chagas’ disease.
Studies using infection with different parasitic protozoa, including
T. cruzi, have demonstrated the importance on the early
IL-12-induced T cell-independent IFN- synthesis on host protection,
before the development of parasite-specific immune responses (8, 10, 40). The nature of protozoan-stimulatory molecule(s) that
trigger the cells from host innate immune system is still not entirely
resolved, although recent reports (17, 18, 19, 42, 43, 44, 45) have
reflected serious efforts to address this question. The GPI anchors and
GIPLs are excellent candidates for important parasitic
molecules that initiate the recognition of protozoan parasites by the
host innate immune system (13, 19, 21, 25, 42, 43).
Several studies indicate that GPI anchors derived from T.
cruzi trypomastigotes have the ability to trigger the synthesis of
pro-inflammatory cytokines by cells of monocytic lineage (17, 19), as previously reported for P. falciparum
(13) and T. brucei (14, 16).
Despite the growing evidence implicating GPI structures from parasitic
protozoa in the induction of cytokine synthesis by macrophages, not
much is known about the counterpart receptor(s) and signaling pathways
that are triggered by protozoan GPI-anchors. Our recent study
(46) demonstrated that like LPS, tGPI-mucin or tGPI are
capable of triggering phosphorylation of extracellular
signal-related kinases-1 and -2, stress-activated protein kinase
kinase-1/mitogen-activated protein kinase kinase-4, and
stress-activated protein kinase-2. Furthermore, using different
specific inhibitors for mitogen-activated protein kinase and
NF-B, we found that similar IC50 values are
required to inhibit cytokine synthesis induced by LPS and tGPI-mucins
(or tGPI). However, macrophages from LPS-hyporesponsive mice were still
responsive to tGPI-mucins. Together, these results suggest that
although functionally similar, the receptors used by LPS and tGPI-mucin
are not the same.
In this study, we investigated the ability of T. cruzi
parasites to trigger TLR-2 and TLR-4. Our results show that different
GPI anchors and GIPLs derived from trypomastigote and epimastigote
stages of T. cruzi present variable potency in activating
NF-B-dependent CD25 expression in CHO cells transfected with both
human CD14 and TLR-2. As shown here, most protozoan-derived GPI
anchors had the ability to trigger TLR-2 function in the range of
0.1–1 µM. In addition, our results show that tGPI anchors containing
extra galactose residues in the glycan core and unsaturated fatty acids
in the sn-2 position of the alkylacylglycerolipid component present
maximal activity in the range of 1–10 nM. This activity was shown to
be independent of TLR-4 and essential for induction of the
pro-inflammatory cytokines (i.e., TNF- and IL-12) as well as NO by
murine macrophages.
The GPI-linked proteins are also ubiquitous on the plasma membrane of higher eukaryotic cells. Despite their diversity, all GPI anchors share a common core structure (11). Thus, our initial findings indicating that parasite GPI anchors are important in initiating host immune responses lead to the important question as to why mammalian GPI anchors do not ordinarily induce unrestrained autoimmunity. Mammalian cells typically express 105 copies of GPI anchors per cell, whereas parasitic protozoa express up to 1–10 million copies of GPI anchors (and related structures) per cell (11). In addition, as shown here, subtle changes in the GPI structure may confer an extreme potency in triggering TLR-2. Consequently, both the amounts as well as the fine structure of protozoan-derived GPI anchors may be important aspects in determining the activation of innate defense mechanisms in the vertebrate host.
It is well established that T. cruzi parasites are potent
nonspecific stimulators of the host innate immune system
(38, 39, 40). The data presented here suggest that tGPI
activation of TLR-2 may directly initiate IL-12, TNF-, and NO
production in vivo, thus fostering host resistance during early
infection with this parasite. Inflammation elicited by the parasite is
thought to play a role on the genesis of cardiac and/or esophageal
pathology observed in Chagas’ disease (40, 47, 48, 49). In
addition to TLR-2 and TLR-4, many other members of this receptor family
have been identified (1, 2, 3). The distribution of these
different members belonging to the family of TLRs appears to vary among
immune and nonimmune host cells (50, 51, 52). Thus, it is
possible that other TLRs may be triggered by parasite-derived
molecules, and not only be important on macrophage parasite
interaction, but also be involved in the interplay of parasites and
nonimmune host cells.
Finally, TLRs appear to be highly conserved, being ancient receptors that confer a certain degree of specificity to the innate immune system in both insects and mammals (1, 2, 3). Therefore, it seems reasonable to argue that the interaction of TLRs and GPI anchors from parasitic protozoa may have the important role of determining the fate of parasitism both in invertebrate and vertebrate hosts, thus being a significant element in the protozoan life cycle. Further understanding of the interaction of GPI anchors and related structures with receptors from TLR family, both from invertebrate and vertebrate hosts, may be helpful to develop new prophylactic and therapeutic means to fight the debilitating and often fatal diseases caused by distinct protozoan parasites.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ricardo T. Gazzinelli, Laboratory of Immunopathology, Centro de Pesquisas René Rachou, FIOCRUZ, Avenida Augusto de Lima 1715, Barro Preto, 30190-002, Belo Horizonte, MG, Brazil. E-mail address: ritoga{at}dedalus.lcc.ufmg.br
3 Abbreviations used in this paper: TLR, Toll-like receptor; CHO, Chinese hamster ovary; eGIPL, epimastigote derived glycoinositolphospholipids; eGPI-mucin, GPI-anchored mucin-like glycoproteins derived from T. cruzi epimastigotes; GIPLs, glycoinositolphospholipids; GPI, glycosylphosphatidylinositol; MALP, macrophage-activating lipopeptide; tGPI, GPI anchor purified from tGPI-mucin; tGPI-mucin, GPI-anchored mucin-like glycoproteins derived from T. cruzi trypomastigotes; KO, knockout; PI, phosphatidylinositol; CER, ceramide; AAG, alkylacylglycerol; Osp A, outer surface protein A.
Received for publication January 4, 2001. Accepted for publication April 23, 2001.
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References |
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N. Delgado, J. Xue, J.-J. Yu, C.-Y. Hung, and G. T. Cole A Recombinant {beta}-1,3-Glucanosyltransferase Homolog of Coccidioides posadasii Protects Mice against Coccidioidomycosis Infect. Immun., June 1, 2003; 71(6): 3010 - 3019. [Abstract] [Full Text] [PDF]
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 N. L. Bernasconi, N. Onai, and A. Lanzavecchia A role for Toll-like receptors in acquired immunity: up-regulation of TLR9 by BCR triggering in naive B cells and constitutive expression in memory B cells Blood, June 1, 2003; 101(11): 4500 - 4504. [Abstract] [Full Text] [PDF]
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T. K. Means, F. Hayashi, K. D. Smith, A. Aderem, and A. D. Luster The Toll-Like Receptor 5 Stimulus Bacterial Flagellin Induces Maturation and Chemokine Production in Human Dendritic Cells J. Immunol., May 15, 2003; 170(10): 5165 - 5175. [Abstract] [Full Text] [PDF]
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 A. Dunne and L. A. J. O'Neill The Interleukin-1 Receptor/Toll-Like Receptor Superfamily: Signal Transduction During Inflammation and Host Defense Sci. Signal., February 25, 2003; 2003(171): re3 - re3. [Abstract] [Full Text] [PDF]
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L. R. P. Ferreira, E. F. Abrantes, C. V. Rodrigues, B. Caetano, G. C. Cerqueira, A. C. Salim, L. F. L. Reis, and R. T. Gazzinelli Identification and characterization of a novel mouse gene encoding a Ras-associated guanine nucleotide exchange factor: expression in macrophages and myocarditis elicited by Trypanosoma cruzi parasites J. Leukoc. Biol., December 1, 2002; 72(6): 1215 - 1227. [Abstract] [Full Text] [PDF]
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D. O. Procopio, I. C. Almeida, A. C. T. Torrecilhas, J. E. Cardoso, L. Teyton, L. R. Travassos, A. Bendelac, and R. T. Gazzinelli Glycosylphosphatidylinositol-Anchored Mucin-Like Glycoproteins from Trypanosoma cruzi Bind to CD1d but Do Not Elicit Dominant Innate or Adaptive Immune Responses Via the CD1d/NKT Cell Pathway J. Immunol., October 1, 2002; 169(7): 3926 - 3933. [Abstract] [Full Text] [PDF]
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K. Artavanis-Tsakonas and E. M. Riley Innate Immune Response to Malaria: Rapid Induction of IFN-{gamma} from Human NK Cells by Live Plasmodium falciparum-Infected Erythrocytes J. Immunol., September 15, 2002; 169(6): 2956 - 2963. [Abstract] [Full Text] [PDF]
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J. B. de Souza, J. Todd, G. Krishegowda, D. C. Gowda, D. Kwiatkowski, and E. M. Riley Prevalence and Boosting of Antibodies to Plasmodium falciparum Glycosylphosphatidylinositols and Evaluation of Their Association with Protection from Mild and Severe Clinical Malaria Infect. Immun., September 1, 2002; 70(9): 5045 - 5051. [Abstract] [Full Text] [PDF]
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 S. E. Applequist, R. P. A. Wallin, and H.-G. Ljunggren Variable expression of Toll-like receptor in murine innate and adaptive immune cell lines Int. Immunol., September 1, 2002; 14(9): 1065 - 1074. [Abstract] [Full Text] [PDF]
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J. Caamano and C. A. Hunter NF-{kappa}B Family of Transcription Factors: Central Regulators of Innate and Adaptive Immune Functions Clin. Microbiol. Rev., July 1, 2002; 15(3): 414 - 429. [Abstract] [Full Text] [PDF]
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C. Brodskyn, J. Patricio, R. Oliveira, L. Lobo, A. Arnholdt, L. Mendonca-Previato, A. Barral, and M. Barral-Netto Glycoinositolphospholipids from Trypanosoma cruzi Interfere with Macrophages and Dendritic Cell Responses Infect. Immun., July 1, 2002; 70(7): 3736 - 3743. [Abstract] [Full Text] [PDF]
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O. Takeuchi, S. Sato, T. Horiuchi, K. Hoshino, K. Takeda, Z. Dong, R. L. Modlin, and S. Akira Cutting Edge: Role of Toll-Like Receptor 1 in Mediating Immune Response to Microbial Lipoproteins J. Immunol., July 1, 2002; 169(1): 10 - 14. [Abstract] [Full Text] [PDF]
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C. A. Scanga, J. Aliberti, D. Jankovic, F. Tilloy, S. Bennouna, E. Y. Denkers, R. Medzhitov, and A. Sher Cutting Edge: MyD88 Is Required for Resistance to Toxoplasma gondii Infection and Regulates Parasite-Induced IL-12 Production by Dendritic Cells J. Immunol., June 15, 2002; 168(12): 5997 - 6001. [Abstract] [Full Text] [PDF]
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A. Ouaissi, E. Guilvard, Y. Delneste, G. Caron, G. Magistrelli, N. Herbault, N. Thieblemont, and P. Jeannin The Trypanosoma cruzi Tc52-Released Protein Induces Human Dendritic Cell Maturation, Signals Via Toll-Like Receptor 2, and Confers Protection Against Lethal Infection J. Immunol., June 15, 2002; 168(12): 6366 - 6374. [Abstract] [Full Text] [PDF]
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P. S. Coelho, A. Klein, A. Talvani, S. F. Coutinho, O. Takeuchi, S. Akira, J. S. Silva, H. Canizzaro, R. T. Gazzinelli, and M. M. Teixeira Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi trypomastigotes induce in vivo leukocyte recruitment dependent on MCP-1 production by IFN-{gamma}-primed-macrophages J. Leukoc. Biol., May 1, 2002; 71(5): 837 - 844. [Abstract] [Full Text] [PDF]
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A. Fox-Marsh and L. C. Harrison Emerging evidence that molecules expressed by mammalian tissue grafts are recognized by the innate immune system J. Leukoc. Biol., March 1, 2002; 71(3): 401 - 409. [Abstract] [Full Text] [PDF]
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K. Adachi, H. Tsutsui, S.-I. Kashiwamura, E. Seki, H. Nakano, O. Takeuchi, K. Takeda, K. Okumura, L. Van Kaer, H. Okamura, et al. Plasmodiumberghei Infection in Mice Induces Liver Injury by an IL-12- and Toll-Like Receptor/Myeloid Differentiation Factor 88-Dependent Mechanism J. Immunol., November 15, 2001; 167(10): 5928 - 5934. [Abstract] [Full Text] [PDF]
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I. C. Almeida and R. T. Gazzinelli Proinflammatory activity of glycosylphosphatidylinositol anchors derived from Trypanosoma cruzi: structural and functional analyses J. Leukoc. Biol., October 1, 2001; 70(4): 467 - 477. [Abstract] [Full Text] [PDF]
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) and
on CHO/CD14/TLR-2 cells (
). The expression of the reporter transgene
(surface CD25) was measured by flow cytometry. The fold increase on
expression of CD25 was calculated by dividing the median fluorescence
from stimulated by the median fluorescence from corresponding
unstimulated control cells. The data presented reflect at least three
independent experiments.
), eGIPL Y CER (
),
eGIPL CL AAG (•), and eGIPL CL CER (
). Expression of CD25 was
evaluated 18 h later. The y-axis of both
B and C indicated fold increase obtained
by dividing the median fluorescence of activated cells by median
fluorescence of unstimulated cells. The data presented here reflect the
results of three independent experiments.
), or TLR-4 KO
mice (