Lack of galectin-3 alters the balance of innateimmune cytokines and confers resistance toRhodococcus equi infection
Luciana C. Ferraz�1, Emerson S. Bernardes�1, Aline F. Oliveira1, Luciana P.
Ruas1, Marise L. Fermino1, Sandro G. Soares1, Adriano M. Loyola2,
Constance Oliver1, Maria C. Jamur1, Daniel K. Hsu3, Fu-Tong Liu3,
Roger Chammas4 and Maria-Cristina Roque-Barreira1
1 Departamento de Biologia Celular e Molecular e Bioagentes Patogenicos, Faculdade de
Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto-SP, Brazil2 Laboratorio de Patologia Bucal, Universidade Federal de Uberlandia, Uberlandia-MG, Brazil3 Department of Dermatology, School of Medicine, University of California-Davis, Sacramento,
CA, USA4 Laboratorio de Oncologia Experimental, Faculdade de Medicina, Universidade de Sao Paulo,
Sao Paulo-SP, Brazil
Galectin-3 is a b-galactoside-binding lectin implicated in the fine-tuning of innate
immunity. Rhodococcus equi, a facultative intracellular bacterium of macrophages, causes
severe granulomatous bronchopneumonia in young horses and immunocompromised
humans. The aim of this study is to investigate the role of galectin-3 in the innate resis-
tance mechanism against R. equi infection. The bacterial challenge of galectin-3-deficient
mice (gal3�/�) and their wild-type counterpart (gal31/1) revealed that the LD50 for the
gal3�/� mice was about seven times higher than that for the gal31/1 mice. When chal-
lenged with a sublethal dose, gal3�/� mice showed lower bacteria counts and higher
production of IL-12 and IFN-c production, besides exhibiting a delayed although increased
inflammatory reaction. Gal3�/� macrophages exhibited a decreased frequency of bacterial
replication and survival, and higher transcript levels of IL-1b, IL-6, IL-10, TLR2 and MyD88.
R. equi-infected gal31/1 macrophages showed decreased expression of TLR2, whereas
R. equi-infected gal3�/� macrophages showed enhanced expression of this receptor.
Furthermore, galectin-3 deficiency in macrophages may be responsible for the higher
IL-1b serum levels detected in infected gal3�/� mice. Therefore galectin-3 may exert a
regulatory role in innate immunity by diminishing IL-1b production and thus affecting
resistance to R. equi infection.
Key words: Bacterial infections . Galectin-3 . IL-1b . Innate immunity . Toll-like receptor
Introduction
Activation of resident macrophages is one of the earliest events
in the cellular host response to microbial invasion, and macro-
phage-derived cytokines play a key role in the initiation and
amplification of the inflammatory process as well as in the
regulation of the immune response. On the basis of its capacity to
recognize carbohydrates and its abundant expression in activated
macrophages [1, 2], galectin-3 has been considered an important
factor in the interaction of host cells with microorganisms [3].
Extracellular galectin-3 is able to activate cells [4–9], mediate
�These authors contributed equally to this work.Correspondence: Professor Maria-Cristina Roque-Barreirae-mail: [email protected]
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DOI 10.1002/eji.200737986 Eur. J. Immunol. 2008. 38: 2762–2775Luciana C. Ferraz et al.2762
cell–cell and cell–extracellular matrix interactions [10–12], and
induce phagocyte migration [13]. However, galectin-3 also
functions inside the cells and can contribute to macrophage
functions that are essential in the cellular response during the
infectious process, such as cell survival [14] and phagocytosis [15].
As a result of its ability to recognize glycans containing
b-galactoside, galectin-3 binds to glycoconjugates synthesized
by several pathogens such as Mycobacterium tuberculosis [16],
Leishmania major [17], Trypanosoma cruzi [18], Schistosoma
mansoni [19] and Candida albicans [20]. Recently, galectin-3
and TLR2 have been found to be associated in C. albicans-infected
differentiated macrophages, an association that has been
considered essential for TLR2-dependent cytokine production
in response to the fungal infection [21]. Therefore, galectin-3 has
been considered as a novel pattern recognition receptor, acting
either alone or in concert with Toll-like receptors (TLR).
Rhodococcus equi, a gram-positive bacteria originally isolated
by Magnusson in 1923, has been recognized as a pathogen
causing purulent pneumonia and enteritis in domesticated
livestock, especially foals [22]. Since the first human case
was reported in 1967 [23], R. equi has been considered an
opportunistic organism in patients receiving immunosuppressant
therapy or with AIDS, in whom, similarly to horses, it causes a
severe pulmonary disease [24]. R. equi is a facultative intracel-
lular pathogen and its propensity to infect and persist in mono-
nuclear phagocytes is central to its success in causing and
maintaining the disease. Its ability to resist clearance and
phagocytosis by macrophages [25] is associated with the
presence of 85- or 90-kb plasmids, which encode a highly
immunogenic 15- to 17-kDa surface-expressed virulence-asso-
ciated protein (VapA) [26]. Recently, it has been shown that
VapA can interact with TLR2 and trigger one of the major
mechanisms by which macrophages respond to R. equi, culmi-
nating in NF-kB activation. Furthermore, in the absence of TLR2,
but not TLR4, in vivo clearance of R. equi is impaired [27].
The present work was undertaken to examine the role of
galectin-3 in the innate immune defense mechanism during the
early course of R. equi infection. We show that the gal3�/� mice
are more resistant to R. equi infection and this resistance is
associated with higher production of cytokines by the gal3�/�
macrophages. We also show that galectin-3 may modulate innate
immunity, by interfering with macrophage IL-1b production. Our
data revealed novel mechanisms by which galectin-3 regulates
the innate response and how it influences the intensity of the
immune response to infectious agents.
Results
Gal3�/� mice are more resistant to R. equi infectionthan gal31/1 mice
We compared the outcome of R. equi infection in gal3�/� mice
and gal31/1 mice, both with C57BL/6 genetic background. The
LD50 for the gal31/1 and gal3�/� mice was estimated following
the intravenous inoculation of a virulent R. equi strain, ranging
from 106 to 109 viable bacteria, and monitoring the survival of
the infected mice. The highest inoculated doses (109 and 108)
provoked 100% mortality in both groups, whereas the lowest
dose (106) was not associated with animal death (data not
shown). Surprisingly, after inoculation of 107 bacteria, 100% of
the gal31/1 mice died within 10 days postinfection. In contrast,
80% of the gal3�/� mice survived for more than 15 days (Fig. 1).
Accordingly, the calculated LD50 for the gal3�/� mice (2� 107)
was about seven times higher than that for the gal31/1 mice
(3�106), suggesting that the absence of galectin-3 was
associated with an enhanced resistance to R. equi infection.
Gal3�/�mice show lower bacterial burden than gal31/1
mice
In order to investigate whether differences in the bacterial burden
could explain the enhanced survival to infection exhibited by
gal3�/� mice, we challenged mice with 1�106 of a virulent
R. equi strain. Organ burdens from the gal31/1 and gal3�/� mice
were determined at 0 and 4 days following intravenous infection
(Table 1). At day 0, the colony forming units (CFU) counts
revealed a similar bacteria organ burden in both the gal31/1 and
gal3�/� mice. On day 4, the CFU recovered from the liver and
spleen of the gal31/1 mice was approximately twofold higher
than those recovered from the organs of the gal3�/� mice
(Table 1). Thus, the increased mortality observed in gal31/1
mice corresponded to an increase in the bacterial burdens in their
visceral organs. These results indicate that the bacterial clearance
in the early course of R. equi infection was more efficient in
gal3�/� mice compared with gal31/1 mice.
Figure 1. Survival of gal31/1 and gal3�/� mice after R. equi infection.Mice were intravenously inoculated with 1� 107 viable bacteria, andsurvival was monitored. Data are representative of three experiments,each performed with five mice per group, yielding similar results. Theresults are expressed as the percentage of live animals during thecourse of infection.
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Gal3�/� mice develop a delayed but increased inflam-matory response compared with gal31/1 mice
It was previously shown that galectin-3 expression is upregulated
during allergic airway inflammation [28] and Toxoplasma gondii
infection [29]. Here, we found an increase in galectin-3 staining
in the liver and spleen from the gal31/1 mice that had been
intravenously inoculated with R. equi (Fig. 2A–D), mostly
associated with infiltrating neutrophils and mononuclear cells.
As expected, no appreciable staining was noted when tissue
sections from the gal3�/� mice were stained by the same anti-
galectin-3 antibody (Fig. 2E and F). In order to investigate the
role exerted by galectin-3 in the inflammatory reaction associated
with R. equi infection, gal31/1 and gal3�/� mice were
intravenously inoculated with 1� 106 of a virulent R. equi strain,
and the time course of inflammatory response in the liver was
analyzed. During the early phase (6 h and 1 day), inflammatory
foci of neutrophils were more prominent in the gal31/1 (Fig. 3A,
C and I) than in gal3�/� tissue (Fig. 3B, D and I), totaling
an average of 7.7571.73 and 4.1970.15 foci per mm2 of
tissue, respectively. At day 1 postinfection, the inflammatory
reaction was accompanied by hepatocyte necrosis, which
was more extensive in the gal31/1 (Fig. 3C) than in the gal3�/�
tissue (Fig. 3D). At the late phase (5 and 15 days), hepatocyte
necrosis was also observed on day 5 postinfection, but it
was more extensive in gal3�/� (Fig. 3F) than in gal31/1 tissue
(Fig. 3E), which is consistent with the higher number
of granulomatous inflammation found in gal3�/� than in gal31/1
tissue (on an average of 15.2370.47 and 8.4072.98
granulomas/mm2 of tissue, respectively) (Fig. 3I). On day 15
postinfection, while granulomas could still be observed in the
tissue of gal31/1 mice (Fig. 3G), the liver of the gal3�/�
mice appeared essentially normal (Fig. 3H), and presented
lower number of granulomas/mm2 than the liver tissue of
gal31/1 mice (Fig. 3I). Taken together, these results indicate
that gal3�/� mice exhibited an increased, although delayed,
inflammatory response, which may promote a more efficient
bacterial clearance and enhanced survival of gal3�/� mice
to R. equi infection.
Gal3�/� mice produce higher IL-12 levels than gal31/1
mice
Based on the previous demonstration that galectin-3 regulates
IL-12 production by APCs [29] and that R. equi is an efficient
inducer of IL-12 and TNF-a production by macrophages [27], we
measured the serum levels of both cytokines in gal31/1 and
Table 1. Kinetics of R. equi ATCC33701 recovering from liver and spleen of gal31/1 and gal3�/� mice
Days after infection Liver Spleen
gal31/1 gal3�/� gal31/1 gal3�/�
CFU/g7SD CFU/g7SD CFU/g7SD CFU/g7SD
0 276571709 33437910 24177716 26977948
4 44327943 19417915 55 47675134� 30 85478932��
Bacterial burden in gal31/1 and gal3�/� mice intravenously inoculated with R. equi (1�106). Bacterial burden was assessed ondays 0 and 4 postinfection. Results are expressed as the mean CFU7SD per gram of organ for groups of three mice. Similarresults were obtained in two independent experiments. �po0.0001 (between 0 and 4 days); ��po0.05 (between gal31/1 andgal3�/� mice).
Figure 2. Immunohistochemical staining for galectin-3 in organs ofR. equi-infected wild-type mice. Gal31/1 and gal3�/� mice wereintravenously inoculated with R. equi (1� 106), and splenic and liverimmunohistochemical studies were performed at several intervalspostinfection. The photomicrographs depict galectin-3 immunostain-ing (brown color) with hematoxylin counterstain. Galectin-3 isexpressed by inflammatory cells infiltrating the liver (A and C) andthe spleen (B and D) of R. equi-infected gal31/1 mice. As expected, noappreciable staining was noted when liver (E) and spleen (F) sectionsfrom R. equi-infected gal3�/� mice were stained using the same anti-galectin-3 antibody. Photomicrographs (A–D) magnification� 60 and(E and F)� 20. Circular area of brown staining indicates inflammatoryfoci, and arrows point to galectin-3-expressing cells.
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gal3�/� mice following R. equi inoculation. As early as 6 h
postinfection, a significantly higher IL-12 production was
detected in the gal3�/� mice compared with the gal31/1 mice
(Fig. 4A). There was no statistically significant difference in the
TNF-a levels between the gal31/1 and the gal3�/� mice during
all the periods of infection analyzed (Fig. 4B). We also measured
cytokine production by the spleen cells from the R. equi-infected
mice 8 days postinfection. As shown in Fig. 4 (C and D),
after 48 h stimulation with acetone precipitate containing
surface proteins of R. equi (APTX) (a VapA-enriched antigen
preparation), the supernatants of spleen cell cultures derived
from the gal3�/� mice contained more IL-12 (panel C)
and interferon-gamma (IFN-g) (panel D) than those obtained
from the gal31/1 mice, indicating that galectin-3 interferes
with the levels of cytokines produced in response to
R. equi infection.
Figure 3. Necrosis and inflammatory response intissues of R. equi-infected gal31/1 and gal3�/� mice.Mice were intravenously inoculated with R. equi(1� 106), and liver histological studies wereperformed at several intervals after infection. Asearly as 6 h (A and B) and 1 day (C and D)postinfection, the livers of gal31/1 mice (A and C)exhibited a greater number of inflammatory fociand more extensive hepatocyte necrosis than thatobserved in the liver of gal3�/� (B and D) mice. Onday 5 postinfection, the liver of gal3�/� miceexhibited a greater number of granulomas (F),which resulted in more extensive hepatocytenecrosis than that observed in gal31/1 (E) mice.On day 15 postinfection, granulomas could still beobserved in the liver of gal31/1 mice (G), while theliver of gal3�/� (H) mice appeared normal. H and E;A–D, magnification�20; E–H magnification� 10objective. Arrows show inflammatory foci, andasterisks (�) points to areas of necrosis. (I):Inflammatory foci count in the liver tissue ofgal31/1 and gal3�/� mice at the indicated timepoints postinfection. Asterisks indicate that differ-ences are statistically significant (po0.05) from gal31/1 mice by Student’s t test. The results representthe mean7SD of five mice per group from arepresentative experiment of three assays.
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Gal3�/� macrophages are more resistant to R. equiinfection than gal31/1 macrophages
Since galectin-3 has been shown to affect macrophage phagocy-
tosis [15] and R. equi is an intracellular bacterium that resides
primarily, if not exclusively, within macrophages [30], we
performed in vitro infection studies with peritoneal macrophages
from gal31/1 and gal3�/� mice. To evaluate the R. equi
internalization and replication, gal31/1 and gal3�/� macro-
phages were exposed to a multiplicity of infection (MOI) of five
bacteria per macrophage. At 1 h after infection, there was no
difference in the phagocytic rate between the gal31/1 and the
gal3�/� macrophages, as judged by the mean number of bacteria
found inside them (Table 2). On the other hand, the R. equi
replication was analyzed by counting bacteria within the
macrophage 4 and 12 h after infection. At both periods, bacteria
counts were significantly higher in the gal31/1 macrophages
than in the gal3�/� macrophages (Table 2). In order to provide
further support to our data, we also checked the ability of
thioglycollate-elicited gal31/1 and gal3�/� macrophages in
controlling the growth of intracellular R. equi. One hour
postinfection, we did not detect any difference in the CFU
recovery between gal31/1 and gal3�/� macrophages (Fig. 5). In
contrast to resident macrophages, thioglycollate-elicited macro-
phages were able to restrain the bacteria replication, and the
bacteria were eliminated faster from gal3�/� macrophages than
from gal31/1 macrophages (Fig. 5). Therefore, it is possible that
galectin-3 favors the replication and/or survival of R. equi inside
macrophages.
Gal3�/� macrophages show higher transcript levels forTLR2 and MyD88 than gal31/1 macrophages
TLR2 signaling has been shown to be dependent on MyD88, a
critical adaptor protein that couples TLR ligation to the activation
of NF-kB [31]. A recent study has identified TLR2 as the main
receptor mediating the innate immune response of macrophages
to R. equi [27]. Therefore, we investigated the expression of TLR2
and MyD88 mRNA in gal31/1 and gal3�/� peritoneal macro-
phages. Remarkably, we found higher levels of TLR2 mRNA in
gal3�/� peritoneal macrophages than in gal31/1 macrophages,
even in the absence of stimulation. Four hours after stimulation
with R. equi surface antigen (APTX), up to threefold higher TLR2
Figure 4. Cytokine levels in the serum and produced by activated spleen cells from R. equi-infected gal31/1 and gal3�/� mice. Serum samples wereharvested at the indicated time points postinfection, and IL-12p40 (A) and TNF-a (B) levels were measured by ELISA. The results represent themean7SD of five mice per group from a representative experiment of three assays. Levels of IL-12p40 (C) and IFN-g (D) produced by spleen cellsharvested on day 8 postinfection and restimulated in vitro with APTX (10 mg/mL) or left unstimulated (medium). Asterisks indicate that differencesare statistically significant (po0.05) from gal31/1 mice by Student’s t test. The results represent the mean7SD of triplicate samples in arepresentative experiment of three assays.
Table 2. Bacteria count inside gal31/1 and gal3–/– macro-phages in vitro infected with R. equi ATCC33701
Hours Mean number of bacteria/macrophage
gal31/1 gal3�/�
1 8.678.7 7.275.7
4 15.2712.9 11.0710.3�
12 20.4715.0 14.6712.0�
Data show the mean number of bacteria per macropha-ges7SD. A total number of 200 macrophages were countedand the bacteria visualized using Wright–Giemsa stain asdescribed in the Materials and methods. �po0.001 (betweengal31/1 and gal3�/� macrophage), Mann–Whitney test.
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transcript levels were found in the gal3�/� macrophages,
compared with the gal31/1 macrophages (Fig. 6A). Interestingly,
R. equi infection resulted in a significant decrease in TLR2 mRNA
transcript levels, which was even more pronounced in gal31/1
macrophages compared with gal3�/� counterparts (Fig. 6A). In
addition, R. equi infection evoked higher MyD88 mRNA expres-
sion levels in gal3�/� macrophages compared with gal31/1 cells
(Fig. 6A). Flow cytometry analysis of cell surface TLR2 expression
was consistent with mRNA expression in R. equi-infected gal3�/�
macrophages in comparison with infected gal31/1 macrophages
(Fig. 6B and C). Therefore, these data suggest that galectin-3 may
regulate TLR2 and MyD88 expression during R. equi infection.
Gal3�/� macrophages show higher cytokine transcriptlevels than gal31/1 macrophages
To further investigate the mechanism by which the lack of
galectin-3 favors host resistance against R. equi, infected gal31/1
and gal3�/� peritoneal macrophages (MOI 5 5) were analyzed
regarding the mRNA transcript levels of IL-12, IL-1b IL-6, IL-10
and TNF-a by quantitative real-time PCR. Interestingly, we found
that the mRNA levels of IL-1b, IL-6 and IL-10 were also enhanced
in the R. equi-infected gal3�/� macrophages compared with the
gal31/1 macrophages (Fig. 7A). It is worth mentioning that
mRNA levels of TNF-a were also analyzed and no difference was
found between the two genotypes (Fig. 7A). Even though the
expression of IL-12p40 mRNA was higher in the gal3�/�
macrophages compared with macrophages from gal31/1 mice,
no significant increase in IL-12p40 above that seen in the
uninfected control was found for macrophages infected with
R. equi (Fig. 7A). In addition, we were not able to detect IL-12p70
protein in the culture supernatants of infected macrophages. Our
data suggest that the pattern of cytokine production in response
to R. equi infection is altered in the absence of galectin-3, a fact
that might interfere in the bacterial growth and/or survival
within gal3�/� macrophages.
Gal3�/� mice produce higher IL-1 beta levels thangal31/1 mice
Because IL-1 has been shown to play an important role in host
defense against intracellular pathogens [32–34], we sought to
confirm whether R. equi-infected gal3�/� macrophages might
release increased IL-1b levels into the culture supernatants. As
shown in Fig. 7B, at 24 h postinfection up to fivefold higher IL-1blevels were detected in the supernatants of R. equi-infected gal3�/�
macrophages compared with those of R. equi-infected gal31/1
macrophages. This is consistent with the observation that higher
IL-1b mRNA expression was detected in the R. equi-infected
gal3�/� macrophages (Fig. 7A). To address whether higher IL-1bproduction might account for the increased resistance of gal3�/�
mice to R. equi infection, we evaluated the IL-1b levels in the
spleens of gal31/1 and gal3�/� infected mice. Confirming our
in vitro results, 24 h after infection, the IL-1b levels were up to
fivefold higher in the spleens of the gal3�/� mice than in the
spleens of gal31/1 mice (Fig. 7C). Thus, we hypothesize that the
IL-1b upregulation seen in the absence of galectin-3 might
account for the increased resistance to R. equi infection in
gal3�/� mice.
Discussion
Galectin-3 has long been described as a positive regulator of
inflammation and, in general, a positive regulator of immunity
[35]. On the other hand, data showing galectin-3 as a negative
regulator of adaptive immune response came from studies with
galectin-3-deficient mice, which developed a heightened Th1
response in two different experimental models [28, 29]. Indeed,
we have previously reported that galectin-3 might influence the
interface of innate and adaptive immunity by suppressing IL-12
production by dendritic cells [29]. Herein, the lack of galectin-3
was demonstrated to be associated with enhanced resistance to
R. equi infection, as indicated by the initial observation of higher
survival rate of gal3�/� mice to the bacterial infection. The full
characterization of such a phenotype was performed by
comparison with gal31/1 mice and showed that: (i) gal3�/�
mice display a decreased bacterial tissue burden, and a delayed
but increased inflammatory response in the liver; (ii) gal3�/�
macrophages are less permissive for R. equi replication and
survival; (iii) gal3�/� macrophages exhibit an mRNA upregula-
tion for TLR2 and MyD88, and for the proinflammatory cytokines
IL-6 and IL-1b; (iv) R. equi-infected gal3�/� macrophages show
enhanced surface expression of TLR2; and (v) R. equi-infected
gal3�/� mice and R. equi-infected gal3�/� macrophages present
Figure 5. R. equi survival within gal31/1 and gal3�/� macrophages.Thioglycollate-elicited peritoneal macrophages from gal31/1 andgal3�/� mice were incubated with R. equi (MOI of 5:1), as described inMaterials and methods. Results represent mean7SD of bacterial CFUobtained from duplicate wells at the indicated time points after in vitroinfection. Asterisks indicate significant differences (po0.05) fromgal31/1 macrophages by nonparametric Mann–Whitney test. Theresults shown were pooled from three independent experiments.
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Figure 6. R. equi infection induces higher transcription and protein levels of TLR2 in gal3�/� thioglycollate-elicited macrophages. (A) shows datafrom real-time PCR for TLR2 and MyD88 using RNA from macrophages either unstimulated, treated for 4 h with the R. equi antigen APTX (10 mg/mL)or infected with R. equi. cDNA contents were normalized on the basis of predetermined levels of b-actin. R. equi infection or stimulation with APTXinduced higher transcript levels of TLR2 in gal3�/� than gal31/1 macrophages. Asterisks indicate that differences are statistically significant(po0.05) from gal31/1 macrophages by Student0s t test. The results represent the mean7SD of triplicate samples in one of the three similarexperiments. (B and C) macrophages were either untreated (medium), infected with R. equi or stimulated with R. equi-derived APTX and cultured invitro for an additional 24 h. TLR2 expression was analyzed by flow cytometry using an anti-TLR2 antibody, and the numbers represent the meanpercentage of TLR2-positive macrophages; control cells (stained with IgG-PE) are also shown. TLR2 surface expression was higher in R. equi-infected gal3�/� macrophages (white histograms) in comparison with infected gal31/1 macrophages (black histograms). Results are representativeof three independent experiments with similar results.
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increased levels of IL-1b protein expression. Therefore, by
suppressing the production of innate cytokines, galectin-3 may
act as a ‘‘downregulator’’ of innate immune response even though
it exerts proinflammatory activities, such as inducing leukocyte
extravasation and activation.
It has been difficult to address the exact mechanism under-
lying the proinflammatory role of galectin-3. Several authors
have reported that the absence of galectin-3 affects the migration
of macrophages, eosinophils, neutrophils and lymphocytes in a
different way, depending on the experimental conditions [14, 28]
and the organ examined [29]. In the present study, gal3�/� mice
also exhibited an attenuated inflammatory response in the liver
during the first 24–48 h postinfection, which was not associated
with higher bacterial burdens. However, 5 days postinfection the
intensity of the inflammatory response was even higher in the
liver of gal3�/� mice. Recently, Nieminen and colleagues repor-
ted [36] that the neutrophil recruitment was impaired in Strep-
tococcus pneumoniae-infected gal3�/� mice, whereas a larger
number of neutrophils were detected in the BAL fluids of
Escherichia coli-infected gal3�/� mice, when compared with
gal31/1 mice. In our study, the enhanced inflammatory response
that occurred at later times postinfection did not corroborate the
previously reported proinflammatory role of galectin-3 and may
reflect a differential role of galectin-3 on the various cell types
that compose the granuloma.
Since the bacterial burden was similar in both gal31/1 or
gal3�/�mice on day 0, the lower number of bacteria found in the
organs of gal3�/� mice 4 days postinfection is not attributable to
differential tissue distribution and/or early elimination by innate
defense mechanisms present in the serum. However, it may be
attributed to bacterial elimination by tissue resident and/or
emigrated cells. It has long been known that R. equi attachment
to cells of a non-sensitized mammalian host is dependent on
complement and is restricted to cells expressing Mac-1, which
may explain why R. equi cellular invasion in vivo is limited to
macrophages [37]. Therefore, we investigated the interaction of
R. equi with host macrophages from gal31/1 and gal3�/� mice,
and we found that gal3�/� macrophages are less permissive for
R. equi replication and survival. No qualitative alteration of
phagocytosis of R. equi by macrophages from either gal31/1 and
gal3�/� was observed, even though it has been reported
that gal3�/� cells exhibited reduced phagocytosis of IgG-opson-
ized erythrocytes and apoptotic thymocytes in vitro [15].
These data suggest that the absence of galectin-3 impairs
bacterial replication and survival without altering bacterial
entry. The absence of a direct binding of galectin-3 to the surface
of R. equi (data not shown) may explain at least in part why
bacterial entry is not altered in gal3�/�macrophages. Although it
may not be the only mechanism, the higher production of
macrophage-derived cytokines by R. equi-infected gal3�/�
Figure 7. R. equi infection induces higher cytokine levels in gal3�/� mice. Thioglycollate-elicited macrophages from gal31/1 and gal3�/� mice wereinfected in vitro with R. equi and, IL-12p40, IL-1b, IL-6, IL-10 and TNF-a mRNA expression was quantified by real-time quantitative real-time PCR 4 hafter infection (A). The results represent the mean7SD of duplicate wells and indicate the fold change in gene expression relative to theuninfected control. The experiment was performed twice with similar results. Levels of IL-1b in the macrophage culture supernatants (B) or in thespleens from gal31/1 and gal3�/� mice (C) were also measured by ELISA, 24 h postinfection. The results represent the mean7SD of five mice pergroup. �po0.05 in comparison with gal31/1 by Student’s t test. The experiment was performed twice with similar results.
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macrophages may contribute to the lower rate of R. equi repli-
cation within those macrophages.
Darrah et al. [27] reported that R. equi-infected macrophages
produce a variety of proinflammatory mediators, including IL-12.
In our study, the higher IL-12p40 mRNA levels found in gal3�/�
macrophages did not lead to detectable IL-12p70 protein levels
in the culture supernatants of infected macrophages. We believe
that the contradictory result between Darrah et al. [27] and
our study is based on the fact they used IFN-gamma-primed
bone-marrow-derived R. equi-infected macrophages. Further-
more, our results do not exclude the possibility that galectin-3
regulates the production of IL-23. IL-23 is another cytokine,
closely related in structure to IL-12, one subunit composed of
identical to the p40 subunit of IL-12 and a second smaller
subunit, p19 [38].
We showed here that the absence of galectin-3 affects the
expression levels of TLR2 on the surface of R. equi-infected
macrophages. Indeed other lectins have been shown to cooperate
with TLR-driven pathways, culminating in enhanced cytokine
production [39, 40]. Recently, galectin-3 was found to be
coprecipitated with TLR2 in THP-1 cells after incubation with
C. albicans blastoconidia, suggesting that galectin-3 may be
associated with TLR2 at the membrane and following interaction
with the yeast [21]. However, it was also shown that gal3�/�
macrophages produced lower amounts of TNF-a after incubation
with C. albicans, compared with gal31/1 macrophages. In the
R. equi experimental model of infection, the lack of galectin-3 was
associated with a tendency for lower TNF-a levels, but they were
not statistically significantly different from those detected in the
presence of galectin-3. Notably, several cytokine and receptor
genes involved in proinflammatory responses were upregulated
in gal3�/� macrophages infected in vitro with R. equi. The
enhancement of IL-1b, IL-6, IL-10, TLR2 and MyD88 mRNA in
R. equi-infected gal3�/�macrophages suggests that TLR2/MyD88
signaling pathway might be altered in the absence of galectin-3.
Since TLR2 has four N-glycans linked to its leucine rich-extra-
cellular domains [41], it is possible that extracellular galectin-
3 molecules can interact with N-glycans present in the TLR and
form multivalent lattices able to interfere with TLR signaling, as
proposed for other cell surface glycosylated receptors, such as
TCR [42]. However, based on the fact that uninfected gal3�/�
macrophages were not more sensitive to TLR2 agonist
(Pam3CSK4) than gal31/1 macrophages (data not shown), we
believe that higher cytokine production (especially IL-1b) found
in R. equi-infected gal3�/� macrophages may not only be related
to the higher TLR2 expression. Furthermore, when gal31/1 and
gal3�/� peritoneal macrophages were pre-treated with lactose at
50 mM (to elute off galectin-3 from the cell surface) before
stimulation with APTX, we did not detect any difference in
cytokine production. Since galectin-3 is located in the nucleus
and cytoplasm besides on the cell surface, we hypothesized that
intracellular galectin-3 may play a major role in controlling
cytokine production
It has been recently shown that bacterial molecules can
interact with the host cell to activate caspase-1 independently
of TLR [43], which culminates with IL-1b processing and secre-
tion. It has been widely accepted that an increase in gene
expression or synthesis of the IL-1b precursor does not auto-
matically result in increased IL-1b activity [44]. The inactive IL-
1b precursor requires enzymatic cleavage to an active cytokine by
the intracellular cysteine protease, caspase-1. Caspase-1 repre-
sents the central effector protein of the inflammasome, a cytosolic
multi-protein complex that functions as molecular scaffold for
caspase activation [45]. Even though it has been widely demon-
strated that galectin-3 induces proinflammatory reactions, no
relationship between this molecule and the inflammasome
complex has been demonstrated so far. In our study we have
found that pro- IL-1b mRNA levels are increased in the absence of
galectin-3; however, the question of whether galectin-3 is able to
affect the activation of the IL-1b-processing inflammasome awaits
further investigation.
In the past few years, several authors have demonstrated
a role for IL-1 in host resistance to a number of pathogens,
including Listeria monocytogenes [32], M. tuberculosis [33],
L. major [34], Salmonella typhimurium [46], C. albicans [47]
and Pneumocystis carinii [48] exerted through mechanisms
that have not yet been elucidated. So far, a direct effect of IL-1
on macrophage antimicrobial activity has not been demonstrated.
The increased IL-1b levels produced by gal3�/� mice may
account for their enhanced resistance to R. equi infection.
This involves a mechanism almost certainly different from
the direct stimulation of macrophage antimicrobial activity
by bacteria. Lymphocytes are likely to mediate the protective
effect of IL-1 [34], since IFN-g production was found to be
absent in IL-1-deficient splenocytes and endogenous IL-1
was demonstrated to be important for the IFN-g production
induced by C. albicans stimulation [49]. In addition to
IL-1, higher IL-12 and IFN-g serum levels were produced
by gal3�/� mice, which may also contribute to the higher
resistance of these mice to R. equi infection, compared with
gal31/1 mice.
IL-1 has been found to be required for the regulation of
Th1 and/or Th2 responses [34]. Indeed, it has been demon-
strated that the major role of IL-1 is to downregulate Th2-like
responses in general and IL-4 expression in particular, by
inhibiting the IL-4 gene expression in T cells [34]. Interestingly,
it has been suggested that galectin-3 regulates both Th1 and
Th2 response because gal3�/� mice developed a lower Th2
response but a higher Th1 response in a murine model of
asthma [28]. In fact, we have previously suggested that galectin-3
may act in the regulatory loops controlling Th1 cytokine
production, especially by influencing IL-12 production by
dendritic cells [29]. Here, we add another piece of evidence that
galectin-3 is a tuner of the immune system, being able to adjust
the intensity of the immune response by regulating the innate
immune response.
In conclusion, we have shown in this study that galectin-3
controls the balance of innate immunity pathways by interfering
with macrophage IL-1b production, thus altering the suscept-
ibility of mice to R. equi.
Eur. J. Immunol. 2008. 38: 2762–2775Luciana C. Ferraz et al.2770
& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
Materials and methods
Experimental animals
Gal3�/� mice were generated as previously described, and
backcrossed to C57BL/6J for at least nine generations [14]. Age-
matched gal31/1 mice obtained from breeding gal31/– animals
were maintained in parallel with gal3�/� mice and were used as
controls in all the experiments. Mice were housed under approved
conditions of the Animal Research Facilities in the Medical School
of Ribeirao Preto-USP. All animals used for the experiments were
female, at 6 to 8 wk of age. The Ethics Committee on Animal
Research of the University of Sao Paulo approved all the
procedures performed in the studies described here.
Bacterial strain and growth conditions
The virulent strain of R. equi ATCC33701 was kindly provided by
Dr. Shinji Takai (University of Kitasato, Towada, Japan). R. equi
was inoculated into 250 mL of brain heart infusion broth (BHI,
Oxoid, Hampshire, England). Cultures were incubated in a rotary
shaker at 100 rpm, 371C, for 60 h (optical density, OD600 5 1.3).
For inoculation into mice, bacterial cultures were washed and re-
suspended in phosphate-buffered saline (PBS); actual numbers of
inoculated bacteria were confirmed by plating serial dilutions on
BHI agar plates at the injection time.
Experimental procedure and tissue processing
Gal31/1 and gal3�/� mice were infected intravenously with a
100mL volume of PBS containing 1� 106 viable bacteria. Groups
of three mice were euthanized at 6 h, 1, 3, 5, 10 and 15 days after
the experimental infection. Their livers and spleens were
collected, fixed in 10% neutral buffered formalin in PBS for
histopathological studies or in 2% paraformaldehyde in PBS
(Sigma, St. Louis, MO, USA) for immunocytochemical analysis.
The tissues were embedded in paraffin, sectioned (5 mm thick-
ness) and then stained with hematoxylin–eosin. The stained
sections were observed using brightfield microscopy with a Nikon
Eclipse 800 (Nikon, Melville, NY, USA).
Immunohistochemistry for detection of galectin-3 ininfected and uninfected animals
To detect galectin-3 expression by immunohistochemistry, depar-
affinized sections were incubated in a solution of 3% hydrogen
peroxide in methanol, for 30 min, to inhibit endogenous peroxidase
activity. The slides were then incubated with Fc Block (Pharmin-
gen, San Diego, CA, USA, 0.5 mg/mL) for 30 min, followed by rat
anti-mouse galectin-3 mAb (M3/38) [50] diluted in PBS containing
1% BSA for 1 h. After rinsing in PBS the samples were incubated in
secondary antibodies conjugated with horseradish peroxidase
(Sigma). The reaction was visualized by incubating the sections
with the chromogenic substrate 3,30-diaminobenzidine tetrahy-
drochloride (Pierce, Rockford, IL, USA) in PBS1H2O2 for 30 min. In
the control slides, normal rat IgG replaced the primary antibody.
The slides were viewed by bright microscopy using an Olympus
BX50 microscope (Olympus Instruments, Melville, NY, USA)
equipped with a digital camera Nikon DMX 1200 (Nikon). All
steps were performed at room temperature.
Granuloma and necrosis area measurement
Using an image processing software KS400/V2.0 (Carl Zeiss),
pictures were captured by a JVC camera (TK 1270), coupled to an
IBM-PC computer and an optical microscope (Axiophot II, Zeiss).
Ten random fields of the liver section to be analyzed were
photographed using a 20� objective. Each field was processed
and the number of granulomas and inflammatory foci, granu-
loma areas, the maximum and minimum diameter, the granu-
loma volume and density per mm2 were measured.
The hepatocyte necrosis areas were determined in two sepa-
rate sections per animal (three mice per group). The area occu-
pied by necrosis was calculated and expressed as a percentage of
the total liver area per mm2.
Estimation of median lethal dose
Groups of five gal31/1 and gal3�/� mice were intravenously
infected with four different doses (106, 107, 108 and 109) of
virulent R. equi. The mice were monitored for 15 days for the
mortality rate. The median lethal dose (LD50) was analyzed by
the MAI method [51].
Estimation of bacteria in the organs
The number of virulent R. equi recovered from the liver and
spleen following intravenous inoculation was estimated on days 0
(approximately 6 h after inoculation) and 4 postinfection. Briefly,
groups of three mice were infected with a sublethal dose
(1�106) of virulent R. equi ATCC33701 and euthanized by
cervical dislocation. Spleen and liver were collected, aseptically
weighed, homogenized and serially diluted in PBS to determine
the number of CFU. Aliquots of 100 mL of homogenates were
plated onto BHI agar in duplicates. The plates were incubated at
371C for 36 h, for bacterial counting. Results were expressed as
mean CFU7standard deviation (SD) per gram of organ for each
group of three mice.
Macrophage culture
Unless otherwise indicated, peritoneal macrophages were elicited
by injecting 1 mL of 3% thioglycollate (BBL Microbiology
Eur. J. Immunol. 2008. 38: 2762–2775 Innate immunity 2771
& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
Systems, Cockeysyville, MD) intraperitoneally and harvested 4
days later. Macrophages were plated in multiwell plates after
suspension in RPMI 1640 containing 5% fetal bovine serum
(FBS), and supplemented with penicillin G, streptomycin and
amphotericin B (1� 104 U/mL, 1� 104 mg/mL, and 25 mg/mL,
respectively). Resident macrophages were obtained from the
peritoneal cavities of the gal31/1 and gal3�/� mice after washing
the cavities with ice-cold PBS. A total of 5� 105 cells/mL were
placed on 13-mm-diameter glass coverslips in 24-well plates
(Nunc, Naperville, IL, USA). The cells were allowed to adhere for
15 min at 371C in a humidified atmosphere containing 5% CO2.
Non-adherent cells were removed by washing the slides three
times with warm RPMI 1640, and the adherent cells were
incubated for an additional 24 h in RPMI 1640 containing 5%
FBS, and supplemented with antibiotic–antimycotic solution.
Approximately 95% of the adherent cells were macrophages, as
determined by Wright–Giemsa stain.
Bacterial intracellular survival assay
R. equi cultures at a density of approximately 109 CFU/mL were
washed with PBS and resuspended in RPMI 1640 with 7% FBS.
Resident macrophages were washed once with warm RPMI 1640,
and the medium was replaced with RPMI 1640 supplemented with
7% FBS. Fresh normal mouse serum, as a source of the
complement, was added to the wells at a final concentration of
5%. Bacteria were added to the macrophage monolayers at an MOI
of five bacteria per macrophage, as described previously [30].
Briefly, bacteria were incubated with macrophages for 30 min at
371C, and then the monolayers were washed with RPMI 1640 to
remove unbound bacteria. Following an additional 30-min
incubation, the monolayers were washed again, and the medium
was replaced with RPMI 1640 supplemented with 7% FBS. Finally,
10mg of gentamicin sulfate per mL was added to kill any remaining
extracellular bacteria in order to prevent extracellular bacterial
growth with the possibility of continuous macrophage infection. At
1, 4 and 12 h postinfection, monolayers were fixed and stained
with Wright–Giemsa stain to enumerate R. equi organisms. The
number of bacteria associated with 200 macrophages was
determined. Moreover, because of the difficulty in achieving a
reliable quantification of large bacterial numbers within an
individual macrophage, an estimated number of 50 bacteria per
macrophage was assumed when high infection rates caused
bacterial numbers to be uncountable. This allowed the inclusion
of these data in the statistical analysis of the average number of
bacteria per macrophage (non-parametric test of Mann–Whitney).
Determination of bacterial burden (intracellular CFU)in macrophages
Thioglycollate-elicited peritoneal macrophages from gal31/1 and
gal3�/� mice were infected with virulent R. equi strain ATCC33701.
Bacterial cultures at a density of approximately 109 CFU/mL were
washed with PBS and resuspended in RPMI 1640 with 7% FBS.
Macrophage monolayers (2� 106/well) were washed once with
warm RPMI 1640, and the medium was replaced with RPMI 1640
supplemented with 7% FBS. Fresh normal mouse serum, a source of
complement, was added to the wells at a final concentration of 5%.
Bacteria were added to the macrophage monolayers at an MOI of
five bacteria per macrophage (1�107 CFU/mL), as described
previously [30]. Briefly, the bacteria were incubated with macro-
phages for 30 min at 371C, and then the monolayers were washed
with RPMI 1640 to remove unbound bacteria. After that, 10mg/mL
of gentamycin was added to kill any remaining extracellular bacteria
and to prevent extracellular growth with continuous reinfection of
macrophages. Following an additional 30 min incubation, the
monolayers were washed again, and the medium was replaced
with RPMI 1640 supplemented with 7% FBS. At 1, 4, 8 and 24 h
after infection, monolayers were lysed by addition of milli-Q water
and aliquots of 100mL were plated onto BHI agar in duplicates. The
plates were incubated at 371C for 24 h, for bacterial counting.
Flow cytometry analysis
Thioglycollate-elicited macrophages were cultured in vitro with
APTX (10mg/mL), R. equi (MOI 5 5:1) or medium alone for 24 h.
The cells were harvested and washed with ice-cold PBS and
incubated for 30 min with CD16/CD32 mAb (Fc block, clone 2.4G2,
BD Pharmingen), followed by the addition of 0.5mg per 1� 106 cells
of the PE-anti-TLR2 antibody or non-related control antibody
(E-bioscience, San Diego, CA). The cells were washed with PBS,
fixed and analyzed on a FACScan flow cytometer (BD Biosciences).
Real-time quantitative PCR analysis
Total RNA was isolated from 2� 106 cells using the TRIzol
Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA),
following the manufacturer’s instructions. cDNA synthesis was
performed in a final volume of 20 mL, using ImProm-II Reverse
Transcriptase (Promega Corporation, Madison, WI, USA). The
reaction mixture contained 4 mg of total RNA, 20 pmol of oligo dT
primer (Invitrogen Life Technologies), 40 U of RNasin, 500 mM of
dNTP mix and 1 U of reverse transcriptase in 1X reverse
transcriptase buffer. The cDNA was treated with 10 mg of RNase
(Gibco, Carlsbad, CA, USA) and then immediately used or stored
at �201C. PCR amplification and analysis were achieved using an
ABI Prism 7500 sequence detector (Applied Biosystems, Foster
City, CA, USA). All the reactions were performed with
SYBR Green Master Mix (Applied Biosystems) using a 25 mL
volume in each reaction, which contained 2mL of template cDNA,
5 pmol of each primer and 12.5mL of SYBR Green. The primers
used for PCR amplification were b-actin (forward:
50-AGCTGCGTTTTACACCCTTT-30, reverse: 50-AAGCCATGCCAA
TGTTGTCT-30); TLR2 (forward: 50-CTCTGACCCGCCCTTTAAGC-
30, reverse: 50-TTTTGTGGCTCTTTTCGATGG-30); IL-1b (forward:
50-AAATACCTGTGGCCTTGGGC-30; reverse, 50-CTTGGGATCCA
Eur. J. Immunol. 2008. 38: 2762–2775Luciana C. Ferraz et al.2772
& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
CACTCTCCAG-30); IL-6 (forward: 50-TCAATTCCAGAAACCGC
TATGA-30; reverse, 50-GAAGTAGGGAAGGCCGTGGT-30); MyD88
(forward: 50-CAGGAGATGATCCGGCAACT-30, reverse: 50-CTGG
CAATGGACCAGACACA-30); IL-10 (forward: 50-TGACTGGCAT-
GAGGATCAGC-30, reverse: 50-AGTCCGCAGCTCTAGGAGCA-30);
TNF-a (forward: 50-GACGTGGAACTGGCAGAAGAG-30, reverse:
50-GCCACAAGCAGGAATGAGAAG-30); and IL-12p40 (forward:
50-AACCATCTCCTGGTTTGCCA-30, reverse: 50-CGGGAGTC-
CAGTCCACCTC-30). The relative expressions of each gene were
obtained using the Comparative CT Method, and they were
normalized using b-actin as an endogenous control. For the
infected samples, evaluation of 2-DD CT indicates the fold change
in gene expression relative to the uninfected control.
Cytokine detection in spleen homogenates and serum
Spleens of R. equi-infected gal31/1 and gal3�/� mice, harvested
24 h postinfection, were homogenized in 1 mL of complete protease
inhibitor cocktail buffer (Calbiochem/Merck Biosciences, Notting-
ham, UK) using a tissue homogenizer. The samples were centrifuged
at 5000� g for 10 min, and the supernatants were stored at �201C
until assay. Serum from R. equi-infected gal31/1 and gal3�/� mice
were collected at 6, 24, 48 and 120 h after infection. The levels of
TNF-a, IL-1b, IL-12 and IFN-g in the spleen and serum were
measured by sandwich ELISA using commercial kits (BD Pharmin-
gen), according to the manufacturer’s instructions.
Cytokine detection in culture supernatants
The R. equi-infected gal31/1 and gal3�/� mice were euthanized 8
days postinfection and their spleens were removed. The spleen cell
suspensions were washed in RPMI medium and treated for 2 min
with erythrocyte lysing buffer (9 volumes of 0.16 M NH4Cl and 1
volume of 0.17 M Tris-HCl, pH 7.5). The erythrocyte-free cells were
then washed three times and adjusted to 2�106 cells/mL in RPMI
1640 supplemented with 10% heat-inactivated FBS. The cell
suspension (1 mL) was distributed in a 24-well tissue culture plate
(Corning), at 371C, in a humidified 5% CO2 incubator, with APTX
(10mg/mL), LPS (Sigma) (5mg/mL) plus IFN-g (BD Pharmingen)
(1.5 ng/mL) or medium alone. The culture supernatants were
collected after 48 h for determination of IL-12p40 and IFN-g, using a
sandwich ELISA, according to the manufacturer’s instructions (BD
Pharmingen). Cytokine concentrations were determined by compar-
ison with standard curves constructed with known amounts of
respective mouse recombinant cytokines.
Statistical analysis
Statistical determinations of the difference between the means
of the experimental groups for the quantification of cytokine
levels and bacteria clearance were performed using an
unpaired two-tailed Student’s t test. Levels of significance
between the groups of mice were determined by using the non-
parametric Mann–Whitney test for the average number of
bacteria by macrophages. A p value less than 0.05 was considered
significant.
Acknowledgements: This work was partially supported by
Fundac- ao de Amparo a Pesquisa do Estado de Sao Paulo
(FAPESP) and Conselho Nacional de Pesquisa Cientıfica
e Tecnologica (CNPq). We thank Vani M. A. Correa, Sandra
M. O. Thomaz and Patricia E. Vendruscolo for their excellent
technical assistance; Ms. Geraldo C. Reis for statistical analyses;
and Julio A. Siqueira, Savio H. F. Miranda and Ednelson
A. Mazzotto for expert animal care. We also thank Dr. Amilton
A. Barreira and Eng. Antonio Renato Meirelles Silva, chief
technician of the Laboratory of Applied and Experimental
Neurology of the Faculty of Medicine of Ribeirao Preto-USP, for
access to and assistance with the image analysis system. We are
grateful to Dr. Leandro Licursi de Oliveira and Dr. Michel Farchi
Guiraldelli for the fruitful discussions.
Conflict of interest: These authors declare no financial or
commercial conflicts of interest.
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Abbreviations: APTX: acetone precipitate containing surface proteins of
R. equi � VapA: virulence-associated protein
Full correspondence: Professor Maria Cristina Roque-Barreira,
Departamento de Biologia Celular e Molecular e Bioagentes
Patogenicos, Faculdade de Medicina de Ribeirao Preto-USP, Av. dos
Bandeirantes, 3900, Ribeirao Preto-SP, CEP 14049-900, Brazil
Fax: 155-16-3633-6631
e-mail: [email protected]
Received: 8/11/2007
Revised: 19/6/2008
Accepted: 22/7/2008
Eur. J. Immunol. 2008. 38: 2762–2775 Innate immunity 2775
& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu
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