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: mcrbarre@fmrp.usp.br

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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.

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& 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

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& 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.

References

1 Cherayil, B. J., Weiner, S. J. and Pillai, S., The Mac-2 antigen is a galactose-

specific lectin that binds IgE. J. Exp. Med. 1989. 70: 1959–1972.

2 Sato, S. and Hughes, R. C., Regulation of secretion and surface expression

of Mac-2, a galactoside-binding protein of macrophages. J. Biol. Chem.

1994. 269: 4424–4430.

3 Mandrell, R. E., Apicella, M. A., Lindstedt, R. and Leffler, H., Possible

interaction between animal lectins and bacterial carbohydrates. Methods

Enzymol. 1994. 236: 231–254.

4 Liu, F.-T., Hsu, D. K., Zuberi, R. I., Kuwabara, I., Chi, E. Y. and Henderson,

Jr., W. R., Expression and function of galectin-3, a beta galactoside-

binding lectin, in human monocytes and macrophages. Am. J. Pathol. 1995.

147: 1016–1029.

5 Frigeri, L. G., Zuberi, R. I. and Liu, F.-T., Epsilon BP, a betagalactoside-

binding animal lectin, recognizes IgE receptor (Fc epsilon RI) and activates

mast cells. Biochemistry 1993. 32: 7644–7649.

6 Zuberi, R. I., Frigeri, L. G., Liu, F.-T., Activation of rat basophilic leukemia

cells by epsilon BP, an IgE-binding endogenous lectin. Cell Immunol. 1994.

156: 1–12.

7 Yamaoka, A., Kuwabara, I., Frigeri, L. G. and Liu, F.-T., A human lectin,

galectin-3 (epsilon bp/Mac-2), stimulates superoxide production by

neutrophils. J. Immunol. 1995. 154: 3479–3487.

8 Hsu, D. K., Hammes, S. R., Kuwabara, I., Greene, W. C. and Liu, F.-T.,

Human T lymphotropic virus-1 infection of human T lymphocytes

induces expression of the beta-galactose-binding lectin, galectin-3. Am.

J. Pathol. 1996. 148: 1661–1670.

Eur. J. Immunol. 2008. 38: 2762–2775 Innate immunity 2773

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

9 Dong, S. and Hughes, R. C., Galectin-3 stimulates uptake of extracellular

Ca21 in human Jurkat T-cells. FEBS Lett. 1996. 395: 165–169.

10 Kuwabara, I. and Liu, F.-T., Galectin-3 promotes adhesion of human

neutrophils to laminin. J. Immunol. 1996. 156: 3939–3944.

11 Inohara, H., Akahani, S., Koths, K. and Raz, A., Interactions between

galectin-3 and Mac-2-binding protein mediate cell–cell adhesion. Cancer

Res. 1996. 56: 4530–4534.

12 Sato, S. and Hughes, R. C., Binding specificity of a baby hamster kidney

lectin for H type I and II chains, polylactosamine glycans, and

appropriately glycosylated forms of laminin and fibronectin. J. Biol. Chem.

1992. 267: 6983–6990.

13 Sano, H., Hsu, D. K., Yu, L., Apgar, J. R., Kuwabara, I., Yamanaka, T.,

Hirashima, M. and Liu, F.-T., Human galectin-3 is a novel chemoattractant

for monocytes and macrophages. J. Immunol. 2000. 165: 2156–2164.

14 Hsu, D. K., Yang, R. Y., Yu, Z. P. L., Salomon, D. R., Fung-Leung, W. P. and

Liu, F.-T., Targeted disruption of the galectin-3 gene results in

attenuated peritoneal inflammatory responses. Am. J. Pathol. 2000. 156:

1073–1083.

15 Sano, H., Hsu, D. K., Apgar, J. R., Yu, L., Sharma, B. B., Kuwabara, I.,

Izui, S. and Liu, F.-T., Critical role of galectin-3 in phagocytosis by

macrophages. J. Clin. Invest. 2003. 112: 389–397.

16 Beatty, W. L., Rhoades, E. R., Hsu, D. K., Liu, F.-T. and Russell, D. G.,

Association of a macrophage galactoside-binding protein with Mycobac-

terium-containing phagosomes. Cell Microbiol. 2002. 4: 167–176.

17 Pelletier, I. and Sato, S., Specific recognition and cleavage of galectin-3 by

Leishmania major through species-specific polygalactose epitope. J. Biol.

Chem. 2002. 277: 17663–17670.

18 Moody, T. N., Ochieng, J. and Villalta, F., Novel mechanism that

Trypanosoma cruzi uses to adhere to the extracellular matrix mediated

by human galectin-3. FEBS Lett. 2000. 470: 305–308.

19 van den Berg, T. K., Honing, H., Franke, N., van Remoortere, A.,

Schiphorst, W. E., Liu, F.-T., Deelder, A. M. et al., LacdiNAc-glycans

constitute a parasite pattern for galectin-3-mediated immune recogni-

tion. J. Immunol. 2004. 173: 1902–1907.

20 Fradin, C., Poulain, D. and Jouault, T., Beta-1,2-linked oligomannosides

from Candida albicans bind to a 32-kilodalton macrophage membrane

protein homologous to the mammalian lectin galectin-3. Infect. Immun.

2000. 68: 4391–4398.

21 Jouault, T., El Abed-El Behi, M., Martinez-Esparza, M., Breuilh, L., Trinel,

P. A., Chamaillard, M., Trottein, F. and Poulain, D., Specific recognition of

Candida albicans by macrophages requires galectin-3 to discriminate

Saccharomyces cerevisiae and needs association with TLR2 for signaling.

J. Immunol. 2006. 177: 4679–4687.

22 Prescott, J. F. and Hoffman, A. M., Rhodococcus equi. Vet. Clin. North Am.

Equine Pract. 1993. 9: 375–384.

23 Golub, B., Falk, G. and Spink, W. W., Lung abscess due to Coryne-

bacterium equi: report of first human infection. Ann. Intern. Med. 1967. 66:

1174–1177.

24 Takai, S., Sasaki, Y., Ikeda, T., Uchida, Y., Tsubaki, S. and Sekizaki, T.,

Virulence of Rhodococcus equi isolates from patients with and without

AIDS. J. Clin. Microbiol. 1994. 32: 457–460.

25 Takai, S., Michizoe, T., Matsumura, K., Nagai, M., Sato, H. and

Tsubaki, S., Correlation of in vitro properties of Rhodococcus (Corynebac-

terium) equi with virulence for mice. Microbiol. Immunol. 1985. 29:

1175–1184.

26 Sekizaki, T., Takai, S., Egawa, Y., Ikeda, T., Ito, H. and Tsubaki, S.,

Sequence of the Rhodococcus equi gene encoding the virulence-associated

15-17-kDa antigens. Gene 1995. 155: 135–136.

27 Darrah, P. A., Monaco, M. C., Jain, S., Hondalus, M. K., Golenbock, D. T.

and Mosser, D. M., Innate immune responses to Rhodococcus equi.

J. Immunol. 2004. 173: 1914–1924.

28 Zuberi, R. I., Hsu, D. K., Kalayci, O., Chen, H. V., Sheldon, H. K., Yu, L.,

Apgar, J. R. et al., Critical role for galectin-3 in airway inflammation and

bronchial hyperresponsiveness in a murine model of asthma. Am. J.

Pathol. 2004. 165: 2045–2053.

29 Bernardes, E. S., Silva, N. M., Ruas, L. P., Mineo, J. R., Loyola, A. M., Hsu, D.

K., Liu, F.-T. et al., Toxoplasma gondii infection reveals a novel regulatory

role for galectin-3 in the interface of innate and adaptive immunity. Am. J.

Pathol. 2006. 168: 1910–1920.

30 Hondalus, M. K. and Mosser, D. M., Survival and replication of

Rhodococcus equi in macrophages. Infect. Immun. 1994. 62: 4167–4175.

31 Takeda, K., Kaisho, T. and Akira, S., Toll-like receptors. Annu. Rev.

Immunol. 2003. 21: 335–376.

32 Havell, E. A., Moldawer, L. L., Helfgott, D., Kilian, P. L. and Sehgal, P. B.,

Type I IL-1 receptor blockade exacerbates murine listeriosis. J. Immunol.

1992. 148: 1486–1492.

33 Juffermans, N. P., Florquin, S., Camoglio, L., Verbon, A., Kolk, A. H.,

Speelman, P., van Deventer, S. J. and van Der Poll, T., Interleukin-1

signaling is essential for host defense during murine pulmonary

tuberculosis. J. Infect. Dis. 2000. 182: 902–908.

34 Satoskar, A. R., Okano, M., Connaughton, S., Raisanen-Sokolwski, A.,

David, J. R. and Labow, M., Enhanced Th2-like responses in IL-1 type 1

receptor-deficient mice. Eur. J. Immunol. 1998. 28: 2066–2074.

35 Rabinovich, G. A., Liu, F.-T., Hirashima, M. and Anderson, A., An

emerging role for galectins in tuning the immune response: lessons from

experimental models of inflammatory disease, autoimmunity and

cancer. Scand. J. Immunol. 2007. 66: 143–158.

36 Nieminen, J., St-Pierre, C., Bhaumik, P., Poirier, F. and Sato, S., Role of

galectin-3 in leukocyte recruitment in a murine model of lung infection

by Streptococcus pneumoniae. J. Immunol. 2008. 180: 2466–2473

37 Hondalus, M. K., Diamond, M. S., Rosenthal, L. A., Springer, T. A. and

Mosser, D. M., The intracellular bacterium Rhodococcus equi

requires Mac-1 to bind to mammalian cells. Infect. Immun. 1993. 61:

2919–2929.

38 Oppmann, B., Lesley, R., Blom, B., Timans, J. C., Xu, Y., Hunte, B., Vega, F.

et al., Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with

biological activities similar as well as distinct from IL-12. Immunity 2000.

13: 715–725.

39 Gringhuis, S. I., den Dunnen, J., Litjens, M., van Het Hof, B., van Kooyk, Y.

and Geijtenbeek, T. B., C-type lectin DC-SIGN modulates Toll-like

receptor signaling via Raf-1 kinase-dependent acetylation of transcrip-

tion factor NF-kappaB. Immunity 2007. 26: 605–616.

40 Gantner, B. N., Simmons, R. M., Canavera, S. J., Akira, S. and Underhill,

D. M., Collaborative induction of inflammatory responses by dectin-1 and

Toll-like receptor 2. J. Exp. Med. 2003. 197: 1107–1117.

41 Weber, A. N., Morse, M. A. and Gay, N. J., Four N-linked glycosylation sites

in human toll-like receptor 2 cooperate to direct efficient biosynthesis

and secretion. J. Biol. Chem. 2004. 279: 34589–34594.

42 Demetriou, M., Granovsky, M. and Quaggin, S., Negative regulation of

T-cell activation and autoimmunity by Mgat5 N-glycosylation. Nature

2001. 409: 733–739.

43 Kanneganti, T. D., Lamkanfi, M., Kim, Y. G., Chen, G.,

Park, J. H., Franchi, L., vandenabeele, P. and Nunez, G., Pannexin-1-

mediated recognition of bacterial molecules activates the cryopyrin

inflammasome independent of Toll-like receptor signaling. Immunity

2007. 26: 433–443.

Eur. J. Immunol. 2008. 38: 2762–2775Luciana C. Ferraz et al.2774

& 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

44 Dinarello, C. A., Biologic basis for interleukin-1 in disease. Blood 1996. 87:

2095–2147.

45 Martinon, F. and Tschopp, J., Inflammatory caspases and

inflammasomes: master switches of inflammation. Cell Death Differ.

2007. 14: 10–22.

46 Raupach, B., Peuschel, S. K. and Monack, D. M., Caspase-1-mediated

activation of interleukin-1beta (IL-1beta) and IL-18 contributes to innate

immune defenses against Salmonella enterica serovar Typhimurium infec-

tion. Infect. Immun. 2006. 74: 4922–4926.

47 Kullberg, B. J., van ’t Wout, J. W. and van Furth, R., Role of granulocytes in

increased host resistance to Candida albicans induced by recombinant

interleukin-1. Infect. Immun. 1990. 58: 3319–3324.

48 Chen, W., Havell, E. A., Moldawer, L. L., McIntyre, K. W., Chizzonite, R. A.

and Harmsen, A. G., Interleukin 1: an important mediator of

host resistance against Pneumocystis carinii. J. Exp. Med. 1992. 176:

713–718.

49 Netea, M. G., Stuyt, R. J., Kim, S. H., Van der Meer, J. W., Kullberg, B. J. and

Dinarello, C. A., The role of endogenous interleukin (IL)-18, IL-12,

IL-1beta, and tumor necrosis factor-alpha in the production of inter-

feron-gamma induced by Candida albicans in human whole-blood

cultures. J. Infect. Dis. 2002. 185: 963–970.

50 Ho, M. K. and Springer, T. A., Mac-2, a novel 32,000 Mr mouse

macrophage subpopulation-specific antigen defined by monoclonal

antibodies. J. Immunol. 1982. 128: 1221–1228.

51 Welkos, S. and O’Brien, A., Determination of median lethal and infectious

doses in animal model systems. Methods Enzymol. 1994. 235: 29–39.

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: mcrbarre@fmrp.usp.br

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