Cutting Edge: TLR9 and TLR2 Signaling Together Account
for MyD88-Dependent Control of Parasitemia in
Trypanosoma cruzi Infection1
Andre Bafica,2* Helton Costa Santiago,†Romina Goldszmid,* Catherine Ropert,2‡
Ricardo T. Gazzinelli,2,3†‡and Alan Sher3*
derived molecules such as GPI anchors and DNA induces
proinflammatory cytokine production and host defense
mechanisms. In this study, we demonstrate that DNA
TLR9-dependent manner and synergizes with parasite-
derived GPI anchor, a TLR2 agonist, in the induction of
cytokines by macrophages. Compared with wild-type an-
imals, T. cruzi-infected Tlr9?/?mice displayed elevated
parasitemia and decreased survival. Strikingly, infected
Tlr2?/?Tlr9?/?mice developed a parasitemia equiva-
lent to animals lacking MyD88, an essential signaling
molecule for most TLR, but did not show the acute mor-
tality displayed by MyD88?/?animals. The enhanced
susceptibility of Tlr9?/?and Tlr2?/?Tlr9?/?mice was
associated with decreased in vivo IL-12/IFN-? responses.
Our results reveal that TLR2 and TLR9 cooperate in the
control of parasite replication and that TLR9 has a pri-
mary role in the MyD88-dependent induction of IL-12/
IFN-? synthesis during infection with T. cruzi. The
Journal of Immunology, 2006, 177: 3515–3519.
known to sense distinct molecular structures on microbes and
trigger NF-?B-dependent gene functions leading to the pro-
duction of proinflammatory cytokines, costimulatory mole-
cules, and other mediators which both restrict early pathogen
3). Nevertheless, the mechanisms by which different pathogen-
induced signals are orchestrated in the generation of host resis-
tance are poorly understood.
ammalian TLR are a group of at least eleven struc-
turally related signaling molecules that are thought
to play a pivotal role in both innate immunity and
Trypanosoma cruzi, the causative agent of Chagas disease, is
parasite has been shown to depend on both humoral and cell-
mediated adaptive responses as well as elements of the innate
cruzi is supported by the observations that mice deficient in
MyD88, an adaptor molecule required for signaling events by
most TLR as well as IL-1R and IL-18R, show greatly enhanced
susceptibility to infection with this protozoan parasite (5). So
far, no single TLR has been shown to account for the acute sus-
ceptibility displayed by MyD88-deficient animals (5–9). For
this reason, we hypothesized that more than one TLR might
cooperate in controlling T. cruzi infection in vivo.
Mice lacking TLR2 have been shown to display slightly en-
hanced susceptibility to T. cruzi in vivo (5), whereas macro-
phages from the same animals present impaired proinflamma-
tory cytokine production when exposed to the live pathogen in
vitro (5). GPI anchors are a major class of T. cruzi molecules
recognized by TLR2 (6). In addition, other parasite structures
such as the T. cruzi-released protein Tc52 and a particular sub-
set of free GPI anchors containing ceramide (GIPL-ceramide)
tokine production via TLR2 (8) and TLR4 (7), respectively.
DNA preparations from both T. cruzi and Trypanosoma brucei
have been shown to stimulate cytokine responses from macro-
phages and dendritic cells (DCs) (9), suggesting that the ge-
nomes of these trypanosomatids contain sufficient CpG motifs
from T. brucei triggers macrophage cytokine production in a
TLR9-dependent manner and mice deficient in this TLR dis-
play impaired resistance to T. brucei infection (10). Neverthe-
its possible interaction with the parasite-induced TLR2 signal-
ing pathway described above has not been investigated.
In the present study, we show that mice lacking TLR2 and
TLR9 display enhanced susceptibility to T. cruzi infection,
*Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy
and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;†Departa-
mento de Bioquı ´mica e Imunologia, ICB, Universidade Federal de Minas Gerais, Belo
Minas Gerais, Brazil
Received for publication April 3, 2006. Accepted for publication July 14, 2006.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
1R.T.G. and C.R. are Conselho Nacional de Pesquisas (Brazil) fellowship recipients.
2Address correspondence and reprint requests to Dr. Andre ´ Ba ´fica, Immunobiology Sec-
tion, National Institute of Allergy and Infectious Diseases–Laboratory of Parasitic Dis-
eases, 50 NIH South Dr, Room 6146, Bethesda, MD 20892. E-mail address:
email@example.com or Dr. Ricardo T. Gazzinelli, Lab. de Imunopatologia, Centro de
Pequisas Rene Rachou–Fundac ¸ao Oswaldo Cruz, Av. Augusto de Lima 1715, B. Hori-
zonte, MG 30190-002. E-mail address: firstname.lastname@example.org
3R.T.G. and A.S. contributed equally to this study.
Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00
which recapitulates the high blood parasitemia observed in
responses in vivo. Our data thus reveal a role for TLR9 in host
resistance to T. cruzi and provide the first evidence for TLR
cooperation in the control of protozoan infection in vivo.
Materials and Methods
C57BL/6 mice were purchased from Taconic Farms. Breeding pairs of
B6.129P2-Myd88tmAki(Myd88?/?), B6.129P2-Tlr2tmAki(Tlr2?/?), and
B6.129P2-Tlr9tmAki(Tlr9?/?) mice were obtained from Dr. S. Akira (Osaka
University, Japan) via Dr. D. Golenbock (University of Massachusetts Medi-
cal School, MA) or Dr. R. Seder (Vaccine Research Center, National Institutes
of Health). Tlr2?/?Tlr9?/?mice were generated by mating TLR2?/?with
TLR9-deficient animals as previously described (11). Animals were bred and
maintained at an American Association of Laboratory Animal Care-accredited
facility at the National Institute of Allergy and Infectious Diseases, National
Institutes of Health. Mice of both sexes between 8 and 14 wk old were used in
T.cruzi culture-derived trypomastigotes and T.cruzi-derived
Trypomastigote forms of the Y strain of T. cruzi were grown in a human fibro-
blast cell line and purified by centrifugation as described previously (12). The
tGPI-mucin was isolated from tissue culture trypomastigotes as described else-
TLR agonists and other reagents
Ultra-pure LPS (Escherichia coli 0111:B4) and DNA calf thymus were pur-
chased from Invitrogen and Sigma-Aldrich, respectively. Purified genomic
DNA from T. cruzi was purchased from American Type Culture Collection
Bone marrow macrophages (BMM) and thioglycollate elicited peritoneal mac-
rophages were obtained from C57BL/6 and knockout (KO) mice as previously
described (6). Macrophages were cultured in supplemented DMEM (106cells/
ml) (Invitrogen Life Technologies) and stimulated with live trypomastigotes or
TLR ligands for 24 h to evaluate TNF and IL-12p40 production by ELISA
Experimental infection with T. cruzi
T. cruzi as previously described (5). Parasitemia levels were evaluated by count-
was evaluated by ELISA (1/2 sera dilution).
CD4?T cell recall response assay
Purified CD4?T spleen cells (106cells/ml) from T. cruzi-infected wild-type
row-derived dendritic cell (BMDC) (5 ? 105cells/ml) from WT animals for
72 h (a protocol used elsewhere). IFN-? (R&D Systems) levels in culture su-
pernatants were then determined by ELISA.
ANOVA was used to analyze the significance of differences in means between
test. Statistical significance was defined as p ? 0.05.
Results and Discussion
DNA from T. cruzi stimulates IL-12 and TNF in a TLR9-dependent
manner and synergizes with GPI anchors for proinflammatory cytokine
Brown and colleagues (9) have demonstrated that T. cruzi
DNA activates macrophages to produce cytokines and reactive
nitrogen intermediates and stimulates B lymphocyte prolifera-
otides. As indicated in Fig. 1A, purified DNA from T. cruzi
stimulated IL-12p40 as well as TNF (data not shown) produc-
tion by BMM in a TLR9-dependent manner. Similarly, T.
cruzi DNA induced IL-12p40 synthesis by CD11c?splenic
DCs through a MyD88- and TLR9-dependent pathway (data
and trigger the synthesis of various proinflammatory cytokines
and reactive nitrogen intermediates (12–14), we next investi-
gated whether GPI anchors and DNA derived from T. cruzi
cooperate in the induction of cytokine synthesis by macro-
phages. As shown in Fig. 1, B and C, GPI and DNA from T.
cruzi synergize in the induction of both IL-12p40 and TNF
production by macrophages. Thus, simultaneous stimulation
of TLR2 and TLR9 by T. cruzi components is important for
the induction of optimal innate immune responses against the
Proinflammatory cytokine responses by APC exposed to live T. cruzi in
vitro are regulated by both TLR2 and TLR9
We next investigated the role of TLR2 or TLR9 in T. cruzi-
induced proinflammatory cytokine production by APC in
4Abbreviations used in this paper: DC, dendritic cells, BMM, bone marrow-derived mac-
rophages; BMDC, bone marrow-derived dendritic cell; KO, knockout; WT, wild type.
matory cytokine responses by macrophages. A, BMM from WT or Tlr9?/?
mice were exposed to purified DNA from [DNATc] (10 ?g/ml), and 24 h
later, IL-12p40 was assayed in supernatants by ELISA. B and C, Peritoneal
macrophages from C57BL/6 or Myd88?/?(?) mice were stimulated with
different concentrations of DNATc (0.1–50 ?g/ml), GPI (1 or 10 ng/ml), or
both for 24 h. TNF (A) and IL-12p40 (B) production were measured in the
culture supernatants by ELISA. Results are means ? SE of triplicate mea-
surements. Experiments shown are representative of two performed. ?, p ?
0.05 between values from DNA plus GPI treatment vs DNA alone (10 or
50 ?g/ml) or GPI alone (10 ng/ml). No synergy in proinflammatory cyto-
kine production was observed when macrophages were treated with control
calf thymus DNA (50 ?g/ml) plus GPI (10 ng/ml) for 24 h.
Effects of DNA and GPI-mucin from T. cruzi on proinflam-
3516CUTTING EDGE: TLR9-TLR2 COOPERATION IN CONTROL OF T. CRUZI INFECTION
vitro. T. cruzi-exposed peritoneal macrophages from Tlr2?/?,
2A), suggesting that TLR2 and TLR9 act in concert in trigger-
ing IL-12. In contrast, the TNF response appeared to be con-
trolled primarily by TLR2 and not TLR9 (Fig. 2B).
TLR2 and TLR9 cooperate in the control of T. cruzi infection
Despite the fact that tGPI-mucins are potent TLR2 agonists,
previous studies (5) have shown that infected Tlr2?/?mice
mount an unimpaired proinflammatory cytokine response and
display no loss in host resistance. To assess the possible role of
fected with 1000 trypomastigotes of the Y strain T. cruzi and
Tlr9?/?animals presented elevated parasite numbers in the
blood in contrast to Tlr2?/?mice, which failed to display a
significant increase in parasitemia. Nevertheless, the Tlr2?/?/
Tlr9?/?mice were even more susceptible than the Tlr9?/?
mice, indicating that although TLR9 may have a dominant
role in innate recognition of the parasite, TLR2 also contrib-
utes to this process (Fig. 3A). The enhanced susceptibility to
T. cruzi infection was associated with decreased serum levels
of IL-12 as well as IFN-? in the Tlr9
mice (Fig. 3B).
We have previously shown that Myd88?/?mice display a
4-fold enhancement in parasitemia and accelerated mortality
tory cytokine responses by T. cruzi-exposed macrophages in vitro. Perito-
neal macrophages from WT, MyD88-, TLR2-, TLR9-, or TLR2/TLR9-
deficient mice were exposed to live T. cruzi (1 parasite: 1 cell), DNA from
T. cruzi (10 ?g/ml), or LPS (1 ?g/ml) for 20 h. IL-12p40 (A) and TNF (B)
were measured in culture supernatants by ELISA. Results are means ? SE
of triplicate measurements. Experiments shown are representative of two
performed. ?, p ? 0.05 between WT vs KO values.
Role of TLR2 and TLR9 in the regulation of proinflamma-
infection. A, WT, Myd88?/?, Tlr2?/?, Tlr9?/?, and Tlr2?/?Tlr9?/?mice
were infected with 103trypomastigotes of the Y strain of T. cruzi, para-
sitemia (left panel) was assessed daily and survival (right panel) was mon-
itored. Left panel, Statistical analysis conducted at day 10 postinfection
denotes differences in blood trypomastigotes between animal groups re-
vealed statistically significant differences between Tlr9KO vs WT or
Tlr2KO (?, p ? 0.05). When comparing parasitemia from Tlr2?/?Tlr9?/?
(or Myd88?/?) groups to WT (?) or Tlr2?/?mice (?), the symbols indi-
cate that differences are statistically significant (p ? 0.05). Right panel,
Statistical analysis revealed that the MyD88- and TLR2/TLR9-deficient
mice were significantly more susceptible (p ? 0.01) than WT animals and
that the mean survival curve of Tlr2?/?Tlr9?/?mice is significantly dif-
ferent from that of the Myd88?/?(p ? 0.01) animal group. The experiment
shown is representative of two to three performed. B, Animals described in
A were bled at 10 days postinfection and IL-12p40 (■) as well as IFN-?
(o) levels were measured in sera by ELISA. C, Purified splenic CD4?T
cells from mice as described in A were cocultured with syngeneic BMDC
infected with T. cruzi (multiplicity of infection ? 5) for 72 h. IFN-? was
assayed by ELISA in culture supernatants. The means ? SE of measure-
ments from triplicate wells are presented. The experiment shown was per-
formed twice with similar results. ?, Values significantly different (p ?
0.05) between WT and KO cells.
Interaction of TLR9 and TLR2 in host resistance to T. cruzi
3517The Journal of Immunology
when compared with WT mice challenged with T. cruzi (5).
Importantly, Tlr2?/?Tlr9?/?and Myd88?/?animals dis-
played similar numbers of circulating parasites in the blood,
suggesting that together TLR2 and TLR9 account for most of
the Myd88-dependent resistance to acute infection with T.
cruzi. However, whereas Myd88?/?mice succumbed by day
30 postinfection, only 40–50% of the Tlr2?/?Tlr9?/?ani-
mals died in a 50-day period (Fig. 3A), a finding that predicts
the involvement of additional MyD88-dependent TLR/IL-1R
family member(s) in the control of mortality.
In contrast to TLR2, TLR9 controls IFN-? recall responses by CD4?T
cells from T. cruzi-infected mice
Because IFN-? is a major mediator of resistance to T. cruzi and
our data indicate an important influence of TLR9 on parasite-
induced IFN-? in vivo, we asked whether defects in in
vivo IFN-? responses are also evident in Tlr9?/?as well as
Tlr2?/?Tlr9?/?mice. To do so, we tested the recall responses
of splenic CD4?T cells from 10 day infected mice using
BMDC from WT animals that had been infected in vitro with
live T. cruzi as APCs. As shown in Fig. 3C, major defects in T.
cruzi-specific IFN-? responses were observed in Myd88?/?,
Tlr9?/?, and Tlr2?/?Tlr9?/?mice, but not in the Tlr2?/?
tibility of Myd88?/?mice to T. cruzi infection to the role of a
single TLR (5, 7). Two possible explanations are: 1) that an as
yet-to-be-determined TLR accounts for the MyD88-depen-
dent resistance; or 2) that different TLR act in concert in deter-
mining pathogen control. In this study, we have demonstrated
that TLR9 is activated by T. cruzi genomic DNA and plays a
major role in the early recognition of this parasite contributing
adaptive immune response to the pathogen. In addition, we
provide evidence for the cooperation of TLR9 with a second
TLR (TLR2) in the induction of optimal host resistance to T.
TLR9 is known to participate in the control of T. brucei in-
fection and the Th1 responses it induces in mice (10). Never-
theless, the role of this receptor and its possible collaboration
with other TLR in host resistance to T. cruzi have never been
addressed. The findings presented herein confirm that T. cruzi
through TLR9 and more importantly establish a role for this
TLR in the IL-12 and IFN-?/Th1 responses to live T. cruzi in
significantly increased parasitemia compared with WT animals
and T. cruzi-infected Tlr9?/?but not Tlr2?/?mice showed
impaired CD4?T cell IFN-? recall responses. However, in
contrast to Tlr2?/?macrophages, Tlr9?/?cells did not show
defective TNF production in vitro, suggesting that TLR2 and
TLR9 control distinct arms of the immune response to the par-
tion and host resistance against T. cruzi. Because no defects in
IL-10 production by spleen cells were noted in the T. cruzi-
infection cannot be attributed to the absence of TLR2-depen-
dent triggering of this cytokine as described by others (15).
Although Tlr2?/?Tlr9?/?and Myd88?/?animals dis-
played similar peripheral blood parasite counts, the former
mice. This observation suggests that additional TLR/IL-1R
family members are involved in the pathogenesis of T. cruzi in-
fection in mice. A role for TLR4 has been suggested by the sig-
nificantly enhanced susceptibility of mice deficient in this re-
ceptor to T. cruzi challenge (7). Therefore, it will be of interest
to determine whether greater defects in resistance to T. cruzi
will be evident in mice with the appropriate triple receptor
(TLR2-TLR4-TLR9) deficiency. Although not evaluated in
the present study, the contribution of MyD88-independent
molecule TRIF should be considered. The participation of
MyD88-independent signaling pathways in host resistance to
T. cruzi is also suggested by the enhanced susceptibility to the
parasite of IFN-??/?mice relative to MyD88-deficient
Th1 responses have previously (1, 4) been demonstrated to
play a major role in control of T. cruzi infection. Importantly,
in the present study the decreased resistance of both Tlr9?/?
and Tlr2?/?Tlr9?/?mice was associated with impaired serum
IFN-? production as well as CD4?/IFN-? responses suggest-
studies in both T. brucei (10) and mycobacterial experimental
infection models (11). Indeed, in the latter report TLR2 and
TLR9 were observed to cooperate in resistance to Mycobacte-
rium tuberculosis with TLR2 playing a major role in controlling
TNF production by macrophages and lung pathology and
TLR9 regulating IFN-? synthesis by CD4?T cells (11). In the
present experiments, we were unable to detect alterations in
liver, spleen, or cardiac tissue pathology in Tlr2?/?mice (data
not shown). Instead TLR2 function was primarily detected
vitro. Because no effect of TLR2 deficiency on in vivo IL-12 or
IFN-? responses was detected and TNF is known to be crucial
for NO synthase 2 expression and host resistance to T. cruzi
(16–18) we speculate that TLR2 regulates control of the para-
that although asymptomatic in the single Tlr2?/?animals, the
effects of this TNF response defect become accentuated in
TLR9-dependent IFN-? response. Although TLR2/TLR9 in-
teraction has now been shown to regulate host resistance to
both M. tuberculosis and T. cruzi, the latter pathogen is distinct
from the former in that it rapidly escapes host endocytic vacu-
oles and dwells in the cytoplasm (19), which is thought to be
parasite within lysosome fused parasitophorus vacuoles follow-
ing invasion or through phagocytosis of trypomastigotes al-
ready killed through action of other innate defense mecha-
nisms. The cellular pathways leading to TLR9 activation in
underlying cooperation with TLR2 signaling are currently un-
der study in our laboratories.
3518CUTTING EDGE: TLR9-TLR2 COOPERATION IN CONTROL OF T. CRUZI INFECTION
Acknowledgments Download full-text
We thank Sara Hieny and Pat Casper for their expert technical assistance and
Drs. Carl Feng, Dragana Jankovic, and Georgio Trinchieri for their invaluable
suggestions and comments on this work.
The authors have no financial conflict of interest.
receptor signaling pathway in host resistance and pathogenesis during infection with
protozoan parasites. Immunol. Rev. 201: 9–25.
2. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev. Immunol.
3. Iwasaki, A., and R. Medzhitov. 2004. Toll-like receptor control of the adaptive im-
mune responses. Nat. Immunol. 5: 987–995.
4. Golgher, D., and R. T. Gazzinelli. 2004. Innate and acquired immunity in the patho-
genesis of Chagas disease. Autoimmunity 37: 399–409.
5. Campos, M. A., M. Closel, E. P. Valente, J. E. Cardoso, S. Akira, J. I. Alvarez-Leite,
C. Ropert, and R. T. Gazzinelli. 2004. Impaired production of proinflammatory cy-
tokines and host resistance to acute infection with Trypanosoma cruzi in mice lacking
functional myeloid differentiation factor 88. J. Immunol. 172: 1711–1718.
6. Campos, M. A., I. C. Almeida, O. Takeuchi, S. Akira, E. P. Valente, D. O. Procopio,
L R. Travassos, J. A. Smith, D. T. Golenbock, and R. T. Gazzinelli. 2001. Activation
of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan par-
asite. J. Immunol. 167: 416–423.
7. Oliveira, A. C., J. R. Peixoto, L. B. de Arruda, M. A. Campos, R. T. Gazzinelli,
D. T. Golenbock, S. Akira, J. O. Previato, L. Mendonca-Previato, A. Nobrega, and
ness to Trypanosoma cruzi glycoinositolphospholipids and higher resistance to infec-
tion with T. cruzi. J. Immunol. 173: 5688–5696.
8. Ouaissi, A., E. Guilvard, Y. Delneste, G. Caron, G. Magistrelli, N. Herbault,
N. Thieblemont, and P. P. Jeannin. 2002. The Trypanosoma cruzi Tc52-released pro-
tein induces human dendritic cell maturation, signals via Toll-like receptor 2, and
confers protection against lethal infection. J. Immunol. 168:6366–6374.
9. Shoda, L. K., K. A. Kegerreis, C. E. Suarez, I. Roditi, R. S. Corral, G. M. Bertot,
J. Norimine, and W. C. Brown. 2001. DNA from protozoan parasites Babesia bovis.
Trypanosoma cruzi, and T. brucei is mitogenic for B lymphocytes and stimulates mac-
Immun. 69: 2162–2171.
10. Drennan, M. B., B. Stijlemans, J. Van den Abbeele, V. J. Quesniaux, M. Barkhuizen,
F. Brombacher, P. De Baetselier, B. Ryffel, and S. Magez. 2005. The induction of a
type 1 immune response following a Trypanosoma brucei infection is MyD88 depen-
dent. J. Immunol. 175: 2501–2509.
11. Bafica, A., C. A. Scanga, C. Feng, L. Leifer, A. Cheever, and A. Sher. 2005. TLR9
regulates Th1 responses and cooperates with TLR2 in mediating optimal responses in
host resistance to Mycobacterium tuberculosis. J. Exp. Med. 202: 1715–1724.
12. Camargo, M. M., I. C. Almeida, M. E. Pereira, M. A. Ferguson, L. R. Travassos, and
R. T. Gazzinelli. 1997. Glycosylphosphatidylinositol-anchored mucin-like glycopro-
teins isolated from Trypanosoma cruzi trypomastigotes initiate the synthesis of proin-
flammatory cytokines by macrophages. J. Immunol. 158: 5890–5901.
13. Almeida, I. C., M. M. Camargo, D. O. Procopio, L. S. Silva, A. Mehlert,
L. R. Travassos, R.T. Gazzinelli, and M.A. Ferguson. 2000. Highly purified glyco-
sylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents.
EMBO J. 19: 1476–1485.
cies membranes trigger nitric oxide synthesis as well as microbicidal activity in IFN-
?-primed macrophages. J. Immunol. 159: 6131–6139.
15. Dillon, S., A. Agrawal, T. Van Dyke, G. Landreth, L. McCauley, A. Koh,
C. Maliszewski, S. Akira, and B. Pulendran. 2004. A Toll-like receptor 2 ligand stim-
ulates Th2 responses in vivo, via induction of extracellular signal-regulated kinase mi-
togen-activated protein kinase and c-Fos in dendritic cells. J. Immunol. 172:
necrosis factor ? mediates resistance to Trypanosoma cruzi infection in mice by induc-
mun. 63: 4862–4867.
17. Castanos-Velez, E., S. Maerlan, L. M. Osorio, F. Aberg, P. Biberfeld, A. Orn, and
M. E. Rottenberg. 1998. Trypanosoma cruzi infection in tumor necrosis factor recep-
tor p55-deficient mice. Infect. Immun. 66: 2960–2968.
18. Aliberti, J. C., J. T. Souto, A. P. Marino, J. Lannes-Vieira, M. M. Teixeira, J. Farber,
R. T. Gazzinelli, and J. S. Silva. 2001. Modulation of chemokine production and
inflammatory responses in interferon-?- and tumor necrosis factor-R1-deficient mice
during Trypanosoma cruzi infection. Am. J. Pathol. 158: 1433–1440.
19. Andrade, L. O., and N. W. Andrews. 2005. The Trypanosoma cruzi-host-cell inter-
play: location, invasion, retention. Nat. Rev. Microbiol. 3: 819–823.
20. Latz, E., A. Schoenemeyer, A. Visintin, K. A. Fitzgerald, B. G. Monks, C. F. Knetter,
E. Lien, N. J. Nilsen, T. Espevik, and D.T. Golenbock. 2004. TLR9 signals after
21. Leifer, C. A., M. N. Kennedy, A. Mazzoni, C. Lee, M. J. Kruhlak, and D. M. Segal.
2004. TLR9 is localized in the endoplasmic reticulum prior to stimulation. J. Immu-
nol. 173: 1179–1183.
3519The Journal of Immunology