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The American Journal of Pathology logoLink to The American Journal of Pathology
. 2009 Jan;174(1):144–152. doi: 10.2353/ajpath.2009.080235

A C-Type Lectin MGL1/CD301a Plays an Anti-Inflammatory Role in Murine Experimental Colitis

Kengo Saba 1, Kaori Denda-Nagai 1, Tatsuro Irimura 1
PMCID: PMC2631327  PMID: 19095961

Abstract

Inflammatory bowel disease is caused by abnormal inflammatory and immune responses to harmless substances, such as commensal bacteria, in the large bowel. Such responses appear to be suppressed under healthy conditions, although the mechanism of such suppression is currently unclear. The present study aimed to reveal whether the recognition of bacterial surface carbohydrates by the macrophage galactose-type C-type lectin-1, MGL1/CD301a, induces both the production and secretion of interleukin (IL)-10. Dextran sulfate sodium salt (DSS) was orally administrated to mice that lacked MGL1/CD301a (Mgl1−/− mice) and their wild-type littermates. Mgl1−/− mice showed significantly more severe inflammation than wild-type mice after administration of DSS. MGL1-positive cells in the colonic lamina propria corresponded to macrophage-like cells with F4/80-high, CD11b-positive, and CD11c-intermediate expression. These cells in Mgl1−/− mice produced a lower level of IL-10 mRNA compared with wild-type mice after the administration of DSS for 2 days. Recombinant MGL1 was found to bind both Streptococcus sp. and Lactobacillus sp. among commensal bacteria isolated from mesenteric lymph nodes of DSS-treated mice. Heat-killed Streptococcus sp. induced an increase in IL-10 secretion by MGL1-positive colonic lamina propria macrophages, but not the macrophage population from Mgl1−/− mice. These results strongly suggest that MGL1/CD301a plays a protective role against colitis by effectively inducing IL-10 production by colonic lamina propria macrophages in response to invading commensal bacteria.

The gastrointestinal tract is continuously exposed to exogenous and endogenous antigens, and the immune response to these antigens is delicately regulated. Inflammatory bowel diseases, such as Crohn’s disease and ulcerative colitis, are known to be caused by an abnormal mucosal immune response to ordinarily harmless antigens. However, the mechanisms of the pathogenesis of inflammatory bowel diseases remains unclear and effective methods to prevent or treat these diseases are not established.

Oral administration of dextran sulfate sodium salt (DSS) is widely used as a model for ulcerative colitis. In this model, the colonic epithelial barrier has been shown to be disrupted and abnormal infiltration of commensal bacteria was observed.1 Germ-free mice were previously reported to have less severe inflammation in this model, indicating that interactions between the host and intestinal bacteria play an important role during the pathogenesis of colitis.2 Not all bacteria were harmful, however, and administration of several species of Lactobacilli ameliorated experimental colitis,3,4 although the mechanism of the anti-inflammatory effect of these beneficial commensal bacteria is unknown. Interleukin (IL)-10 is one candidate because IL-10-deficient mice have been reported to develop colitis spontaneously, indicating that this cytokine acts as a suppressing regulatory factor for experimental colitis.5 Indeed, therapeutic administration of the IL-10 gene has been shown to be effective to treat DSS-induced colitis,6 although which cells produce IL-10 and how IL-10 inhibits the pathogenesis of colitis remain unknown.

Macrophages and dendritic cells (DCs) are known to have distinct properties in the intestine compared with other organs, and serve as candidates for the identification of IL-10-producing cells. These cells have been shown to possess an immune suppressive function and were shown to monitor external antigens without any inflammatory responses. Furthermore, human intestinal macrophages were reported to uniquely induce inflammatory anergy, in which these cells showed unresponsiveness toward inflammatory stimuli but retained the ability to be phagocytic and bacteriocidal.7 Interestingly, DCs and macrophages in the intestinal tract have been reported to express a higher amount of IL-10 when responding to inflammatory stimuli than DCs from the spleen.8 Thus, the anti-inflammatory response should be considered a unique characteristic of intestinal macrophages and DCs.

We hypothesized that the anti-inflammatory response of intestinal macrophages and DCs was mediated by cell surface molecules recognizing bacterial components. Lectins, carbohydrate recognition proteins, therefore, serve as one of the candidates of these recognition molecules. In the present report, we tested the role of macrophage galactose-type C-type lectin (MGL/CD301), a type II transmembrane lectin that specifically recognizes terminal galactose (Gal) and N-acetylgalactosamine (GalNAc) residues as monosaccharides in a calcium-dependent manner.9,10,11 Although other C-type lectins on macrophages and DCs, such as macrophage mannose receptor and DC-specific intracellular adhesion molecules-3 grabbing nonintegrin (DC-SIGN), were previously proposed to play an important role in the elimination of invading pathogens through their function as endocytic receptors, signal transducers, or signal modulators,12,13,14 definite proofs of the role of these lectins in the intestinal protection in vivo with knockout mice has not yet been obtained.

Mgl1-deficient mice were used in the present study. The Mgl family is known to have two homologous genes in mice, Mgl1 and Mgl2, and these two lectins have distinct carbohydrate recognition specificities, although their distinct roles have not yet been defined.15,16 In our previous reports, we found that MGL1 and/or MGL2 were mainly expressed on macrophages and immature DCs, and that these cells were observed mainly in the connective tissue of various organs, especially in skin, large intestines, and lymph nodes.17 These lectins were found to be involved in the uptake of mucin-like GalNAc-conjugated polymers by murine bone marrow-derived and human monocyte-derived DCs,18,19 which was thought to be an important process of antigen processing. Mgl1-deficient mice did not show obvious defects in lymphoid and erythroid homeostasis.20 In an in vivo study with mouse embryos, MGL1 was shown to function as an endocytic receptor for X-irradiation-induced apoptotic cells, whereas Mgl1-deficient mice showed retarded clearance of apoptotic cells in neural tubes.21 It was also suggested that MGL1 regulated trafficking of MGL1-expressing cells from skin to lymph nodes.22,23 Antigen-induced inflammatory tissue formation in skin was abrogated in Mgl1-deficient mice,24 suggesting that MGL1 functioned under inflammatory conditions.

The present study strongly suggests that MGL1 expressed on intestinal lamina propria macrophages functions through its interaction with commensal bacteria by magnifying the IL-10 production by these cells. DSS-induced experimental colitis caused by infiltration of bacteria was more severe in Mgl1-deficient mice than in wild-type mice, probably because of insufficient suppression of inflammation by the shortage of IL-10.

Materials and Methods

Mice

Mgl1-deficient mice and littermate wild-type mice (C57BL/6J strain) were maintained under specific pathogen-free conditions at the Graduate School of Pharmaceutical Sciences of the University of Tokyo. They were fed and housed according to the guidelines of the Bioscience Committee of the University of Tokyo.

Induction and Assessment of Colitis

Colitis was induced in 6- to 8-week-old female mice by feeding them with water containing 2.5% (w/v) DSS (molecular weight, 35,000 to 44,000; ICN Biomedicals, Irvine, CA) for 7 days as previously described.1 Body weights were measured, and stools were collected daily. Stool blood was assessed by the use of guaiac reaction.25 Histological score was assessed by the criteria described previously.26,27 Two sections of the colon were assessed for each mouse.

Immunohistochemical Staining

MGL1-positive cells were immunohistochemically detected in 10-μm-thick cryostat sections of the large intestine, modified as previously described.17 Nonspecific bindings were blocked using phosphate-buffered saline (PBS) containing 2% normal goat serum and 3% bovine serum albumin (BSA). The sections were treated with the first antibodies at 4°C for 16 hours, and with alkaline phosphatase-conjugated goat anti-rat IgG (Invitrogen, Eugene, OR). Visualization was performed with Histomark RED (KPL, Gaithersburg, MD). For the staining of isolated cells, cells were attached on poly-l-lysine-coated glass slides on a Cytospin (Thermo Fisher Scientific, Waltham, MA). Cells were fixed with 4% paraformaldehyde for 5 minutes, and stained as described above, except for the use of Alexa-488 streptavidin (Invitrogen). Antibodies used in this study were anti-MGL1 monoclonal antibody (LOM-8.7), anti-CD11b (eBioscience, San Diego, CA), and anti-IL-10 monoclonal antibody (JES5-2A5).

Isolation of Lamina Propria Mononuclear Cells (LPMCs)

Dissected large intestines were cut into small pieces and washed with calcium- and magnesium-free Hanks’ balanced salt solution (CMF/HBSS). The epithelium was removed by two consecutive treatments with 5 mmol/L ethylenediaminetetraacetic acid (EDTA) in CMF/HBSS containing 10% fetal calf serum for 15 minutes at 37°C. Intestinal tissue pieces were digested with 1 mg/ml of collagenase (Wako, Tokyo, Japan) and 100 U/ml of DNase I (Roche, Basel, Switzerland) in RPMI 1640 medium containing 10% fetal calf serum at 37°C for 120 minutes. Cells were washed with CMF/HBSS containing 5 mmol/L EDTA and 10% fetal calf serum. LPMCs were further purified by Percoll (GE Health Care, Uppsala, Sweden) density gradient centrifugation.

Flow Cytometry and Cell Sorting

Cells were incubated with fluorescein isothiocyanate-conjugated anti-CD11b, CD11c, MHC class II, phycoerythrin-conjugated F4/80 (eBioscience), and biotin-conjugated anti-MGL1 mAb LOM-8.7 for 30 minutes on ice. Rat IgG2a and rat IgG2b were used as isotype controls. Allophycocyanin-conjugated streptavidin (eBioscience) was used for the detection of biotin-conjugated antibodies. As a marker of viable cells, 7-amino-actinomycin D (eBioscience) was used. All antibodies and streptavidin were diluted by PBS containing 0.1% (w/v) BSA and 0.1% (w/v) sodium azide. Analysis was performed by FACSAria (BD, Franklin Lakes, NJ) and analyzed with FlowJo software (Tree Star, Ashland, OR).

Incorporation of Latex Beads

Cells were incubated with fluorescent-labeled latex beads (0.1 μm; Sigma, St. Louis, MO) at 37°C in the CO2 incubator for 4 hours. Cells were washed with cold PBS and cyto-spun onto poly-l-lysine-coated glass slides. Cells were counterstained with TOTO-3 (Invitrogen), and observed on a confocal microscope, MRC1024 (Bio-Rad, Hercules, CA).

Esterase Staining

Cells were placed on poly-l-lysine-coated glass slides and dried at room temperature. Nonspecific esterase staining was performed by using 1-naphthylacetate in 2-methoxyethanol as a substrate. Cells were counterstained with methyl green and examined on a light microscope.

Conventional and Real-Time Polymerase Chain Reaction (PCR)

Total RNA was extracted from the sorted cells by using a RNeasy mini kit (Qiagen, Valencia, CA). Total RNA was reverse-transcribed into cDNA by Superscript II (Invitrogen). All procedures were performed according to the manufacturers’ instructions. Quantitative real-time PCR was performed on an ABI Prism 7700 (Applied Biosystems, Foster City, CA) using Power SYBR Green master mix (Applied Biosystems). The primers used for the reaction are listed in Table 1.

Table 1.

Primers Used for Conventional and Real-Time PCR

Gene Forward Reverse
Conventional PCR
 Mgl1 5′-TCTCTGAAAGTGGATGTGGAGG-3′ 5′-CACTACCCAGCTCAAACACAATCC-3′
 Mgl2 5′-TCTCTGAAAGTGGATGTGGAGG-3′ 5′-GCTATAAGTTGTGGGGAGTGGGC-3′
 Il-10 5′-ATGCAGGTCTTTAAGGGTTACTTGGGTT-3′ 5′-ATTTCGGAGAGAGGTACAAACGAGGTTT-3′
 β-Actin 5′-CTTCTACAATGAGCTGCGTGTGG-3′ 5′-TGATGACCTGGCCGTCAGGCA-3′
Real-time PCR
 Il-10 5′-AGGCGCTGTCATCGATTTCTC-3′ 5′-TGGCCTTGTAGACACCTTGGTC-3′
 β-Actin 5′-TCCAGCCTTCCTTCTTGGGTA-3′ 5′-CAGCACTGTGTTGGCATAGAGG-3′
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Culture of Infiltrated Intestinal Bacteria

Infiltrated intestinal bacteria were cultured from mesenteric lymph nodes of DSS-treated mice obtained on day 7. All procedures were conducted under sterile conditions. Mesenteric lymph nodes were homogenized, plated on MacConkey agar and sheep’s blood agar plates (BD Bioscience, San Jose, CA), and cultured at 37°C for 24 hours under aerobic or anaerobic conditions. Bacteria species were determined by Gram staining and selective media. For harvesting bacterial bodies, Streptococcus sp. and Lactobacillus sp. were plated on a sheep’s blood agar plate and an MRS agar plate (BD Bioscience), respectively, for 48 hours at 37°C under anaerobic conditions. Bacterial bodies were washed with PBS twice, and heat-killed bacteria were prepared by incubating them at 100°C for 20 minutes. For in vitro stimulation of cells, heat-killed bacteria were added into the cell culture at a concentration of 10 μg/ml, and incubated for 16 hours at 37°C.

Binding Assays

Recombinant MGL1 (rMGL1) was prepared as previously described10 and immobilized onto 96-well plates (Greiner, Frickenhausen, Germany) for 16 hours at 4°C. Inhibition of binding was performed with 100 mmol/L of Gal or mannose or 5 mmol/L EDTA by pre-incubation of immobilized rMGL1 with these carbohydrates at room temperature. Heat-killed bacteria were suspended in Dulbecco’s modified PBS (DPBS; containing 0.91 mmol/L CaCl2 and 0.49 mmol/L MgCl2), and incubated at room temperature for 1 hour. After mild washing with DPBS, bacteria were fixed with 0.25% glutaraldehyde (Wako) and stained with crystal violet. After washing with water, crystal violet was eluted with a mixture of water, ethanol, and methanol (5:4:1) and absorbance was measured at 550 nm. For the uptake assays, CHO cells stably expressing MGL1 were used.23 Heat-killed bacteria were labeled with the PKH-26 red fluorescent cell linker kit (Sigma) according to the manufacturer’s instructions. Cells were incubated with labeled bacteria for 60 minutes at 37°C and analyzed by flow cytometry on a FACSAria.

Preparation of Bacterial Cell Walls

Bacterial bodies were harvested and ultrasonicated for 30 minutes on ice. Residual cell pellets were removed by centrifugation at 5000 × g for 30 minutes at 4°C. Supernatants were collected and centrifuged at 18,000 × g for 30 minutes at 4°C. Precipitates were dissolved in 4% sodium dodecyl sulfate and boiled for 40 minutes. Cell walls were collected by centrifugation at 18,000 × g at 4°C for 30 minutes and washed three times with distilled water.

Enzyme-Linked Immunosorbent Assay

Cell walls were immobilized on a 96-well plate (Greiner) by loading cell wall solutions at a concentration of 10 μg/ml in PBS at 4°C for 16 hours. After blocking with 2% BSA in DPBS, biotinylated rMGL1 (brMGL1) that was pre-incubated with and without 1 mmol/L of Gal or mannose at 4°C for 1 hour was incubated with immobilized cell walls for 2 hours. brMGL1 was detected with horseradish peroxidase-conjugated streptavidin (1:1000, Invitrogen), and 1 mmol/L 2,2′-amino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ammonium solutions containing 0.34% H2O2 in 0.1 mol/L sodium citrate buffer (pH 4.3). The absorbance at 405 nm was measured.

Statistics

Data are presented as mean ± SD, where n represents the number of mice per study. Data were compared using either a Student’s t-test or a Mann-Whitney U-test, and the differences were statistically significant when P values were <0.05.

Results

Mgl1-Deficient Mice Showed More Severe Inflammation

To assess the difference in the sensitivity of Mgl1−/− and littermate wild-type mice to colitis formation, the mice were fed with water containing 2.5% DSS for 7 days and the body weights were monitored daily. A decrease in the body weight was observed earlier in Mgl1−/− mice than in wild-type mice, although the difference at each time point was not statistically significant between these two groups (Figure 1A). Stool blood level was assessed as an indicator of severity of the inflammation. An increase in blood in feces was observed with Mgl1−/− mice earlier than with Mgl1+/+ mice, and on day 4, Mgl1−/− mice (2.29 ± 0.76, n = 7) showed a significantly higher score of bleeding than Mgl1+/+ mice (1.43 ± 0.53, n = 7; P < 0.05) (Figure 1B). By histological assessment, marked cellular infiltrations and ulcer formations were observed in the large intestine of Mgl1−/− mice, whereas the epithelial structure remained intact in Mgl1+/+ mice on day 7 (Figure 1C). The severity of colitis was evaluated by histological scoring, and Mgl1−/− mice (3.13 ± 0.83, n = 8) showed a significantly higher score than Mgl1+/+ mice (2.2 ± 0.91, n = 10; P < 0.05) (Figure 1D).

Figure 1.

Figure 1

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DSS-induced colitis in wild-type mice and MglI-deficient mice. Mgl1-deficient mice and littermate wild-type mice were fed with 2.5% DSS for 7 days. A: Body weights of Mgl1+/+ (filled circle; n = 5) and Mgl1−/− (open circle; n = 5) mice. B: Blood contents in stools of Mgl1+/+ (filled circle; n = 7) and Mgl1−/− (open circle; n = 7) mice. C: H&E-stained sections of colon from Mgl1+/+ (left) and Mgl1−/− (right) mice. D: Histological scores of damage of colonic walls on day 7. Colon from Mgl1+/+ (filled circle) and Mgl1−/− (open circle) were compared. *P < 0.05. Data represent mean ± SD. Scale bar = 200 μm.

Properties of Lamina Propria MGL1-Positive Cells

To identify the cell populations that express MGL1 in the steady state, colonic LPMCs were prepared from untreated mice and analyzed by flow cytometry for the expression of cell surface markers using anti-MGL1 mAb LOM-8.7. Cells expressing MGL1 were shown to express CD11b, CD11c, a high level of F4/80, and MHC class II (Figure 2A). CD11b+ and F4/80-high cells expressed MGL1, whereas CD11b+and F4/80-intermediate cells were negative for MGL1 (Figure 2B).

Figure 2.

Figure 2

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Characterization of lamina propria macrophages. A: Lamina propria macrophages isolated from the large intestine were analyzed by flow cytometry with the anti-MGL1 mAb LOM-8.7 and anti-CD11c, anti-CD11b, anti-MHC class II, or anti-F4/80. B: Expression of MGL1 on CD11b-positive F4/80-high cells (R1) and CD11b-positive F4/80-intermediate cells (R2) was examined. C: Expression of Mgl1, Mgl2, and IL-10 in R1 and R2 cells was examined by RT-PCR. D: Incorporation of fluorescent-labeled latex beads (green) by CD11b-positive F4/80-high (R1) and CD11b-positive F4/80-intermediate (R2) cells was assessed under a confocal microscope. Nuclei were counterstained with TOTO-3 (blue). E: Nonspecific esterase staining was performed with R1 and R2 cells. Esterase-positive cells stained in dark red. Nucleus was counterstained with methyl green. F: Percentage of R1 fraction was examined in Mgl1+/+ (filled bar) and Mgl1−/− (open bar) mice. Data represent mean ± SD. Original magnifications, × 100.

Based on these results, cells with high levels of MGL1 expression in wild-type mice were sorted with CD11b and F4/80, and the presence of Mgl1 and Mgl2 mRNA was determined by RT-PCR. The mRNA was detected in CD11b+ F4/80-high cells but not in CD11b+ F4/80-intermediate cells (Figure 2C). Nearly a 100% of the sorted cells that expressed a combination of high levels of CD11b and F4/80 were shown to incorporate fluorescein isothiocyanate-labeled latex beads in vitro (Figure 2D). These cells were also positively stained for a nonspecific esterase (Figure 2E). Therefore, the predominant populations of LPMCs expressing MGL1 were considered to be macrophages, although they expressed CD11c.

Because many reports previously suggested that IL-10 plays a key role in the modulation of DSS-induced experimental colitis in mice as well as Crohn’s disease and ulcerative colitis in humans,28 the expression of IL-10 mRNA by MGL1-positive macrophage-like cells was investigated. CD11b+and F4/80-high cells, which consist of the MGL1-positive cells, were shown to contain significantly higher levels of IL-10 mRNA than F4/80-intermediate MGL1-negative cells (Figure 2C). These intestinal macrophage populations were likely to play an immune suppressive role through their IL-10 production, yet the number of these cells in lamina propria was not significantly different in Mgl1−/− mice (Figure 2F).

Expression of MGL1 Was Gradually Reduced in the Lamina Propria as Inflammation Proceeded

To further characterize the cells expressing MGL1, untreated or DSS-treated large intestines were stained with the anti-MGL1 mAb LOM-8.7. In untreated tissue, cells expressing MGL1 were observed in the lamina propria and the submucosa (Figure 3A) where immune cells, putatively macrophages, DCs, and lymphocytes, were present. After induction of colitis, cells expressing MGL1 were observed only in the edematous submucosa and were almost absent in the lamina propria of the severe ulcer region, where CD11b+and MGL1-negative cells were observed (Figure 3A).

Figure 3.

Figure 3

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Cells expressing MGL1 in healthy and inflamed colon. A: Distribution of cells expressing MGL1 was investigated by immunohistochemical staining with the specimen from DSS-untreated (day 0) and treated mice (day 2 and day 7). MGL1-positive or CD11b-positive cells were detected with mAb LOM-8.7 or anti-CD11b, respectively, and stained in red. Nuclei were counterstained with hematoxylin. B: Total RNA was isolated from CD11b-positive F4/80-high cells of DSS-treated mice on day 0 (left) and day 2 (right), and IL-10 mRNA was quantified by real-time PCR. IL-10 mRNA levels in the cells from Mgl1−/− (open bar) were significantly lower than Mgl1+/+ (filled bar). *P < 0.05. Data represent mean ± SD.

Colonic Lamina Propria Macrophages of Mgl1−/− Mice Expressed a Smaller Quantity of IL-10 mRNA than Those in Wild-Type Mice at the Early Phase of DSS-Induced Colitis

IL-10 mRNA expression levels were measured in colonic lamina propria macrophages from wild-type mice and the equivalent cells in Mgl1−/− mice. mRNA obtained from lamia propria macrophages was examined for the relative quantity of IL-10 by real-time PCR. The level of IL-10 in lamina propria macrophages was not significantly different on day 0. However, IL-10 from wild-type mice was 2.1-fold higher than that from Mgl1−/− mice on day 2 (n = 3) (Figure 3B). The results indicate that the cell population with MGL1 on the surface was capable of producing IL-10 and that the level was significantly lower when MGL1 was absent.

MGL1 Interacts with Colonic Commensal Bacteria Isolated from Mice

It is known that interaction between host cells and commensal bacteria plays a crucial role in the pathogenesis of DSS-induced colitis. We tested whether MGL1 recognized infiltrating bacteria during the experimental colitis. Because bacterial penetration through the intestinal wall from the lumen seemed to be important,26 commensal bacteria were isolated from mesenteric lymph nodes of DSS-treated mice on day 7. The bacteria obtained were identified as Escherichia coli, Enterococcus sp., Streptococcus sp., and Lactobacillus sp. according to morphological examination and growth characteristics in selection media. No bacteria were isolated from lymph nodes of healthy untreated wild-type mice. Binding of these bacteria to immobilized MGL1 is shown in Figure 4A with background binding to immobilized BSA. Of all bacteria isolated from the mice, heat-killed Streptococcus sp. and heat-killed E. coli bound to immobilized MGL1. The binding was significantly reduced by the addition of 100 mmol/L Gal but not mannose (Figure 4A). The binding was also abrogated by the addition of 5 mmol/L EDTA, indicating that the interaction between the bacteria and MGL1 was calcium-dependent (Figure 4B). To evaluate the interaction of bacteria with MGL1 on cell surfaces, uptake of fluorescent-labeled bacteria by CHO cells transfected with Mgl1 was examined. These cells engulfed Streptococcus sp., but not E. coli or Enterococcus sp. (Figure 4C), suggesting that Streptococcus sp. was one of the candidates of bacteria that interact with MGL1 during the pathogenesis of experimental colitis.

Figure 4.

Figure 4

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MGL1 binding to intestinal commensal bacteria. A: Commensal bacteria were isolated from mesenteric lymph nodes of DSS-treated mice on day 7. Heat-killed bacterial bodies were applied on microtiter plates immobilized with recombinant MGL1 or BSA. Bound bacteria were detected with crystal violet and measured by absorbance at 550 nm. Hatched bar, plates immobilized with BSA; open bar, plates immobilized with MGL1; filled bar, plates immobilized with MGL1 plus 100 mmol/L Gal; gray bar, plates immobilized with MGL1 plus 100 mmol/L mannose. B: Inhibition of binding of bacteria to immobilized MGL1 by incubations with EDTA solutions. Open bar, without EDTA; filled bar, with EDTA. C: Engulfment of fluorescent-labeled bacteria examined by flow cytometry. CHO cells transfected with mock (gray) or MGL1 (black line) were tested. D: Binding of MGL1 to bacterial cell walls measured by enzyme-linked immunosorbent assay. Cell walls from Streptococcus sp. and Lactobacillus sp. were immobilized, and reacted with biotinylated recombinant MGL1. *P < 0.05, **P < 0.005, n = 3. Data represent mean ± SD.

Because Lactobacillus sp. showed autoaggregation and could not be tested with the binding assays or the uptake assays, binding of MGL1 to these cell wall fractions was measured by enzyme-linked immunosorbent assay. The cell walls of Streptococcus sp. also bound MGL1, and this binding was blocked by 1 mmol/L Gal, suggesting that the cell wall fractions contained the MGL1-binding determinants. The cell wall of Lactobacillus sp. was also reactive with MGL1 in a carbohydrate-dependent manner (Figure 4D).

Production of IL-10 by Colonic Lamina Propria Macrophages Expressing MGL1

Colonic lamina propria macrophages in Mgl1−/− mice were shown to express a smaller amount of IL-10 mRNA than those in wild-type mice on day 2, as shown in Figure 3B. Therefore, we hypothesized that MGL1 modulates the response of lamina propria macrophages to MGL1-reactive commensal bacteria. Intestinal CD11b+and F4/80-high cells were prepared from LPMCs isolated from untreated wild-type or Mgl1−/− mice and considered as colonic lamina propria macrophages. These cells were cultured in the presence or absence of heat-killed Streptococcus sp. for 16 hours. IL-10 mRNA was compared by real-time PCR. Cells from wild-type mice co-incubated with heat-killed Streptococcus sp. showed higher levels of IL-10 expression than control cells (2.6-fold) (Figure 5A). Cells from Mgl1−/− mice showed only a slight increase in IL-10 mRNA (1.3-fold) (Figure 5A). To confirm that this mRNA up-regulation lead to an increase in IL-10 protein, colonic lamina propria macrophages were co-cultured with or without 10 μg/ml of heat-killed Streptococcus sp., and stained with anti-IL-10 monoclonal antibodies. The levels of IL-10 were significantly elevated when lamina propria macrophages were cultured with Streptococcus sp. (Figure 5B). Such elevation was not observed with the equivalent cells from Mgl1−/− mice.

Figure 5.

Figure 5

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Expression of IL-10 in colonic lamina propria macrophages. A: Isolated colonic lamina propria macrophages from the large intestine were incubated with or without heat-killed Streptococcus sp. for 16 hours. mRNA of cells from Mgl1+/+ and Mgl1−/− mice were isolated and IL-10 mRNA was measured by real-time PCR. Relative expression levels were normalized to the expression of β-actin, and fold increase of the cytokine expression was normalized to untreated cells. Open bar, medium (med); filled bar, heat-killed Streptococcus sp. (St.). B: Isolated colonic lamina propria macrophages were co-cultured with or without heat-killed Streptococcus, and the cells were stained with anti-IL-10 monoclonal antibody (green). Nuclei were counterstained with DAPI (blue). Scale bar = 10 μm.

Discussion

The hallmark of inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis, is abnormal inflammation of the gastrointestinal tract, but the pathogenesis has not been fully elucidated. In the present study, Mgl1-deficient mice exhibited a more severe inflammation than wild-type mice in an experimental model for ulcerative colitis. The mechanistic basis for this difference was determined to be a change in cytokine production in response to intestinal commensal bacteria. The predominant portion of cells expressing MGL1 in colonic lamina propria were found to be macrophages, and these cells were shown to produce IL-10 in response to the bacteria. IL-10 was previously considered a crucial cytokine for the maintenance of intestinal homeostasis because IL-10-deficient mice spontaneously developed colitis.5 The present study clearly shows that lamina propria macrophages lacking Mgl1 produced less IL-10 than these cells expressing MGL1.

IL-10 produced by a variety of cells, such as macrophages, DCs, and T cells, plays an important role in the pathogenesis of colitis. In macrophage-depleted mice, DSS-induced colitis was more severe, and IL-10 mRNA from the whole colon of these mice was decreased compared with sham-treated mice, indicating that colonic macrophages secrete IL-10 during colonic inflammation.29 However, the most important target of IL-10 remains unclear. IL-10 is likely to influence the functions of a wide range of immune cell populations by suppressing pro-inflammatory responses.29 For example, IL-10 is known to suppress production of IL-12, tumor necrosis factor-α, and reactive oxygen species by macrophages and DCs. It also inhibits Th1 and Th2 responses, which aggravate pathogenic inflammation. Colonic epithelial cells are not likely to act as the target of IL-10. IL-10 has been shown to inhibit MHC class II expression on epithelial cell lines, but it does not affect the chemokine secretion responsible for the recruitment of neutrophils and monocytes to injured sites.30,31

By immunohistochemical analysis, MGL1-expressing cells were observed in the lamina propria and in the submucosa, where many other types of immune cells were also observed. After the induction of colitis, cells expressing MGL1 were absent from this area. Although it is possible that cells expressing MGL1 migrated to other regions, it is likely that MGL1 expression was down-modulated on CD11b+cells, considering that CD11b+ cells with similar morphology were present in this ulcerated region. Furthermore, when isolated lamina propria macrophages were exposed to heat-killed Streptococcus sp., these cells significantly reduced the cell surface expression of MGL1, as indicated by the binding of monoclonal antibody LOM-8.7 (data not shown). Thus, the disappearance of MGL1 was likely to be attributable to its down-regulation, although the possibility that MGL1-positive macrophages migrated to other regions could not be eliminated. Similarly, expression of MGL on bone marrow-derived DCs was previously shown to be abrogated after maturation by lipopolysaccharide,19 and absence of MGL1 expression in the late phase of colitis might be a consequence of activation with several stimuli. The immunological significance of this down-modulation of MGL1 remains unclear.

To date, many reports have shown that C-type lectins interact with microorganisms and are involved in endocytosis, signal transduction, and opsonization.12 Pathogenic microorganisms have been shown to use these lectins for their infection,32 as observed with human MGL acting as an entry site for filovirus.33 MGL1 should be considered unique among the C-type lectins expressed on macrophages and DCs because of its distinct carbohydrate specificity. Mannose or glucose residues on the surface of pathogens have been found to be reactive with lectins, such as macrophage mannose receptor, DC-SIGN, and dectin-1, whereas MGL1 binds specifically to terminal Gal and GalNAc residues as a monosaccharide. Previously, a soluble lectin expressed in the intestine, intelectin, was shown to bind to Nocardia rubra and the binding was inhibited by the addition of Gal,34 although the biological consequence of the binding of bacteria to this lectin is unknown. The present report is the first to demonstrate the role of a Gal-type C-type lectin in the recognition of commensal bacteria.

The presence of the Gal/GalNAc residue in the cell wall polysaccharide of streptococci has been reported.35 Co-aggregation of Streptococcus viridans, a member of the oral flora, was inhibited by the addition of oligosaccharides containing Gal and/or GalNAc,36 strongly suggesting that Gal/GalNAc residues were exposed on the surfaces of Streptococci. Another study showed that Gal and GalNAc residues were present in an exo-polysaccharide or a capsular polysaccharide produced by Lactobacillus.37 Bacteria have been known to produce many types of glycoproteins. The carbohydrate structures are different depending on the species and environments,38 although it is still unclear which glycoprotein is reactive to MGL1.

Possible mechanisms of regulation of cytokine expression by MGL1 may be mediated by the YXXL motif in the cytoplasmic tail. MGL1, MGL2, Dectin-1, and DC-SIGN have been previously shown to contain similar motifs in the cytoplasmic domain, and IL-10 expression has been reported to be induced by the signals from the YXXL motif of Dectin-1.39,40 However, possible signal transduction through MGL1/2 was not previously reported. Alternatively, MGL1 might be involved with the modulation of signals through pathogen recognition receptors. Constitutive signals from commensal bacteria through TLRs and MyD88 were reported to be necessary to suppress colitis.26,41 Notably, a mutation in Nod-2 was shown to be a major genetic factor in determining the susceptibility to Crohn’s disease.42,43 DC-SIGN was also reported to modulate signals from bacterial components and to induce IL-10 expression, although the mechanism was YXXL motif-independent.44 Collectively, signaling through these endogenous lectins may be essential for the maintenance of intestinal homeostasis.

In the present study, we provide new insight into the role of a C-type lectin, MGL1, in the pathogenesis of colitis. MGL1 is expressed on lamina propria macrophages of colon and is responsible for the interaction of these cells with commensal bacteria. The received signals from bacterial carbohydrates enhance IL-10 production in these cells, resulting in the suppression of intestinal inflammation.

Acknowledgments

We thank Ms. Kyoko Sakai and Ms. Miki Noji for assistance in the preparation of this manuscript; Dr. Takashi Nishimura, Division of Immunoregulation, Research Section of Disease Control, Institute for Genetical Medicine, Hokkaido University for the kind gift of anti-IL-10 monoclonal antibody (JES5-2A5); and Dr. Kikuji Itoh, Laboratory of Veterinary Public Health, Department of Veterinary Medicine, Graduate School of Agricultural and Life Science, The University of Tokyo, for assisting in identifying the bacterial species.

Footnotes

Address reprint requests to Dr. Tatsuro Irimura, Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan. E-mail: irimura@mol.f.u-tokyo.ac.jp.

Supported by the Ministry of Education, Science, Sports, and Culture of Japan (grants in aid 11557180, 11672162, and 12307054); the Research Association for Biotechnology, and the Program for Promotion of Fundamental Studies in Health Sciences of the Pharmaceutical and Medical Device Agency.

References

  1. Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, Nakaya R. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology. 1990;98:694–702. doi: 10.1016/0016-5085(90)90290-h. [DOI] [PubMed] [Google Scholar]
  2. Kitajima S, Morimoto M, Sagara E, Shimizu C, Ikeda Y. Dextran sodium sulfate-induced colitis in germ-free IQI/Jic mice. Exp Anim. 2001;50:387–395. doi: 10.1538/expanim.50.387. [DOI] [PubMed] [Google Scholar]
  3. Castagliuolo I, Galeazzi F, Ferrari S, Elli M, Brun P, Cavaggioni A, Tormen D, Sturniolo GC, Morelli L, Palu G. Beneficial effect of auto-aggregating Lactobacillus crispatus on experimentally induced colitis in mice. FEMS Immunol Med Microbiol. 2005;43:197–204. doi: 10.1016/j.femsim.2004.08.011. [DOI] [PubMed] [Google Scholar]
  4. Herías MV, Koninkx JF, Vos JG, Huis in't Veld JH, van Dijk JE. Probiotic effects of Lactobacillus casei on DSS-induced ulcerative colitis in mice. Int J Food Microbiol. 2005;103:143–155. doi: 10.1016/j.ijfoodmicro.2004.11.032. [DOI] [PubMed] [Google Scholar]
  5. Kühn R, Lohler J, Rennick D, Rajewsky K, Muller W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell. 1993;75:263–274. doi: 10.1016/0092-8674(93)80068-p. [DOI] [PubMed] [Google Scholar]
  6. Lindsay JO, Sandison A, Cohen P, Brennan FM, Hodgson HJ. IL-10 gene therapy is therapeutic for dextran sodium sulfate-induced murine colitis. Dig Dis Sci. 2004;49:1327–1334. doi: 10.1023/b:ddas.0000037830.22065.71. [DOI] [PubMed] [Google Scholar]
  7. Smythies LE, Sellers M, Clements RH, Mosteller-Barnum M, Meng G, Benjamin WH, Orenstein JM, Smith PD. Human intestinal macrophages display profound inflammatory anergy despite avid phagocytic and bacteriocidal activity. J Clin Invest. 2005;115:66–75. doi: 10.1172/JCI19229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Iwasaki A, Kelsall BL. Unique functions of CD11b+, CD8 alpha+, and double-negative Peyer’s patch dendritic cells. J Immunol. 2001;166:4884–4890. doi: 10.4049/jimmunol.166.8.4884. [DOI] [PubMed] [Google Scholar]
  9. Imai Y, Irimura T. Quantitative measurement of carbohydrate binding activity of mouse macrophage lectin. J Immunol Methods. 1994;171:23–31. doi: 10.1016/0022-1759(94)90224-0. [DOI] [PubMed] [Google Scholar]
  10. Sato M, Kawakami K, Osawa T, Toyoshima S. Molecular cloning and expression of cDNA encoding a galactose/N-acetylgalactosamine-specific lectin on mouse tumoricidal macrophages. J Biochem (Tokyo) 1992;111:331–336. doi: 10.1093/oxfordjournals.jbchem.a123758. [DOI] [PubMed] [Google Scholar]
  11. Yamamoto K, Ishida C, Shinohara Y, Hasegawa Y, Konami Y, Osawa T, Irimura T. Interaction of immobilized recombinant mouse C-type macrophage lectin with glycopeptides and oligosaccharides. Biochemistry. 1994;33:8159–8166. doi: 10.1021/bi00192a021. [DOI] [PubMed] [Google Scholar]
  12. Cambi A, Koopman M, Figdor CG. How C-type lectins detect pathogens. Cell Microbiol. 2005;7:481–488. doi: 10.1111/j.1462-5822.2005.00506.x. [DOI] [PubMed] [Google Scholar]
  13. Geijtenbeek TB, Van Vliet SJ, Koppel EA, Sanchez-Hernandez M, Vandenbroucke-Grauls CM, Appelmelk B, Van Kooyk Y. Mycobacteria target DC-SIGN to suppress dendritic cell function. J Exp Med. 2003;197:7–17. doi: 10.1084/jem.20021229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Saijo S, Fujikado N, Furuta T, Chung SH, Kotaki H, Seki K, Sudo K, Akira S, Adachi Y, Ohno N, Kinjo T, Nakamura K, Kawakami K, Iwakura Y. Dectin-1 is required for host defense against Pneumocystis carinii but not against Candida albicans. Nat Immunol. 2007;8:39–46. doi: 10.1038/ni1425. [DOI] [PubMed] [Google Scholar]
  15. Tsuiji M, Fujimori M, Ohashi Y, Higashi N, Onami TM, Hedrick SM, Irimura T. Molecular cloning and characterization of a novel mouse macrophage C-type lectin, mMGL2, which has a distinct carbohydrate specificity from mMGL1. J Biol Chem. 2002;277:28892–28901. doi: 10.1074/jbc.M203774200. [DOI] [PubMed] [Google Scholar]
  16. Oo-Puthinan S, Maenuma K, Sakakura M, Denda-Nagai K, Tsuiji M, Shimada I, Nakamura-Tsuruta S, Hirabayashi J, Bovin NV, Irimura T. The amino acids involved in the distinct carbohydrate specificities between macrophage galactose-type C-type lectins 1 and 2 (CD301a and b) of mice. Biochim Biophys Acta. 2008;1780:89–100. doi: 10.1016/j.bbagen.2007.10.017. [DOI] [PubMed] [Google Scholar]
  17. Mizuochi S, Akimoto Y, Imai Y, Hirano H, Irimura T. Unique tissue distribution of a mouse macrophage C-type lectin. Glycobiology. 1997;7:137–146. doi: 10.1093/glycob/7.1.137. [DOI] [PubMed] [Google Scholar]
  18. Higashi N, Fujioka K, Denda-Nagai K, Hashimoto S, Nagai S, Sato T, Fujita Y, Morikawa A, Tsuiji M, Miyata-Takeuchi M, Sano Y, Suzuki N, Yamamoto K, Matsushima K, Irimura T. The macrophage C-type lectin specific for galactose/N-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells. J Biol Chem. 2002;277:20686–20693. doi: 10.1074/jbc.M202104200. [DOI] [PubMed] [Google Scholar]
  19. Denda-Nagai K, Kubota N, Tsuiji M, Kamata M, Irimura T. Macrophage C-type lectin on bone marrow-derived immature dendritic cells is involved in the internalization of glycosylated antigens. Glycobiology. 2002;12:443–450. doi: 10.1093/glycob/cwf061. [DOI] [PubMed] [Google Scholar]
  20. Onami TM, Lin MY, Page DM, Reynolds SA, Katayama CD, Marth JD, Irimura T, Varki A, Varki N, Hedrick SM. Generation of mice deficient for macrophage galactose- and N-acetylgalactosamine-specific lectin: limited role in lymphoid and erythroid homeostasis and evidence for multiple lectins. Mol Cell Biol. 2002;22:5173–5181. doi: 10.1128/MCB.22.14.5173-5181.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Yuita H, Tsuiji M, Tajika Y, Matsumoto Y, Hirano K, Suzuki N, Irimura T. Retardation of removal of radiation-induced apoptotic cells in developing neural tubes in macrophage galactose-type C-type lectin-1-deficient mouse embryos. Glycobiology. 2005;15:1368–1375. doi: 10.1093/glycob/cwj028. [DOI] [PubMed] [Google Scholar]
  22. Chun KH, Imai Y, Higashi N, Irimura T. Involvement of cytokines in the skin-to-lymph node trafficking of cells of the monocyte-macrophage lineage expressing a C-type lectin. Int Immunol. 2000;12:1695–1703. doi: 10.1093/intimm/12.12.1695. [DOI] [PubMed] [Google Scholar]
  23. Kumamoto Y, Higashi N, Denda-Nagai K, Tsuiji M, Sato K, Crocker PR, Irimura T. Identification of sialoadhesin as a dominant lymph node counter-receptor for mouse macrophage galactose-type C-type lectin 1. J Biol Chem. 2004;279:49274–49280. doi: 10.1074/jbc.M409300200. [DOI] [PubMed] [Google Scholar]
  24. Sato K, Imai Y, Higashi N, Kumamoto Y, Onami TM, Hedrick SM, Irimura T. Lack of antigen-specific tissue remodeling in mice deficient in the macrophage galactose-type calcium-type lectin 1/CD301a. Blood. 2005;106:207–215. doi: 10.1182/blood-2004-12-4943. [DOI] [PubMed] [Google Scholar]
  25. Qualls JE, Kaplan AM, van Rooijen N, Cohen DA. Suppression of experimental colitis by intestinal mononuclear phagocytes. J Leukoc Biol. 2006;80:802–815. doi: 10.1189/jlb.1205734. [DOI] [PubMed] [Google Scholar]
  26. Fukata M, Michelsen KS, Eri R, Thomas LS, Hu B, Lukasek K, Nast CC, Lechago J, Xu R, Naiki Y, Soliman A, Arditi M, Abreu MT. Toll-like receptor-4 is required for intestinal response to epithelial injury and limiting bacterial translocation in a murine model of acute colitis. Am J Physiol. 2005;288:G1055–G1065. doi: 10.1152/ajpgi.00328.2004. [DOI] [PubMed] [Google Scholar]
  27. Ohkawara T, Nishihira J, Takeda H, Hige S, Kato M, Sugiyama T, Iwanaga T, Nakamura H, Mizue Y, Asaka M. Amelioration of dextran sulfate sodium-induced colitis by anti-macrophage migration inhibitory factor antibody in mice. Gastroenterology. 2002;123:256–270. doi: 10.1053/gast.2002.34236. [DOI] [PubMed] [Google Scholar]
  28. Li MC, He SH. IL-10 and its related cytokines for treatment of inflammatory bowel disease. World J Gastroenterol. 2004;10:620–625. doi: 10.3748/wjg.v10.i5.620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Couper KN, Blount DG, Riley EM. IL-10: the master regulator of immunity to infection. J Immunol. 2008;180:5771–5777. doi: 10.4049/jimmunol.180.9.5771. [DOI] [PubMed] [Google Scholar]
  30. Denning TL, Campbell NA, Song F, Garofalo RP, Klimpel GR, Reyes VE, Ernst PB. Expression of IL-10 receptors on epithelial cells from the murine small and large intestine. Int Immunol. 2000;12:133–139. doi: 10.1093/intimm/12.2.133. [DOI] [PubMed] [Google Scholar]
  31. Song F, Ito K, Denning TL, Kuninger D, Papaconstantinou J, Gourley W, Klimpel G, Balish E, Hokanson J, Ernst PB. Expression of the neutrophil chemokine KC in the colon of mice with enterocolitis and by intestinal epithelial cell lines: effects of flora and proinflammatory cytokines. J Immunol. 1999;162:2275–2280. [PubMed] [Google Scholar]
  32. Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, Cornelissen IL, Nottet HS, KewalRamani VN, Littman DR, Figdor CG, van Kooyk Y. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell. 2000;100:587–597. doi: 10.1016/s0092-8674(00)80694-7. [DOI] [PubMed] [Google Scholar]
  33. Takada A, Fujioka K, Tsuiji M, Morikawa A, Higashi N, Ebihara H, Kobasa D, Feldmann H, Irimura T, Kawaoka Y. Human macrophage C-type lectin specific for galactose and N-acetylgalactosamine promotes filovirus entry. J Virol. 2004;78:2943–2947. doi: 10.1128/JVI.78.6.2943-2947.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tsuji S, Uehori J, Matsumoto M, Suzuki Y, Matsuhisa A, Toyoshima K, Seya T. Human intelectin is a novel soluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall. J Biol Chem. 2001;276:23456–23463. doi: 10.1074/jbc.M103162200. [DOI] [PubMed] [Google Scholar]
  35. Cisar JO, Sandberg AL, Abeygunawardana C, Reddy GP, Bush CA. Lectin recognition of host-like saccharide motifs in streptococcal cell wall polysaccharides. Glycobiology. 1995;5:655–662. doi: 10.1093/glycob/5.7.655. [DOI] [PubMed] [Google Scholar]
  36. Takahashi Y, Ruhl S, Yoon JW, Sandberg AL, Cisar JO. Adhesion of viridans group streptococci to sialic acid-, galactose- and N-acetylgalactosamine-containing receptors. Oral Microbiol Immunol. 2002;17:257–262. doi: 10.1034/j.1399-302x.2002.170409.x. [DOI] [PubMed] [Google Scholar]
  37. Tallon R, Bressollier P, Urdaci MC. Isolation and characterization of two exopolysaccharides produced by Lactobacillus plantarum EP56. Res Microbiol. 2003;154:705–712. doi: 10.1016/j.resmic.2003.09.006. [DOI] [PubMed] [Google Scholar]
  38. Upreti RK, Kumar M, Shankar V. Bacterial glycoproteins: functions, biosynthesis and applications. Proteomics. 2003;3:363–379. doi: 10.1002/pmic.200390052. [DOI] [PubMed] [Google Scholar]
  39. Rogers NC, Slack EC, Edwards AD, Nolte MA, Schulz O, Schweighoffer E, Williams DL, Gordon S, Tybulewicz VL, Brown GD, Reis e Sousa C. Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins. Immunity. 2005;22:507–517. doi: 10.1016/j.immuni.2005.03.004. [DOI] [PubMed] [Google Scholar]
  40. Hara H, Ishihara C, Takeuchi A, Imanishi T, Xue L, Morris SW, Inui M, Takai T, Shibuya A, Saijo S, Iwakura Y, Ohno N, Koseki H, Yoshida H, Penninger JM, Saito T. The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and Toll-like receptors. Nat Immunol. 2007;8:619–629. doi: 10.1038/ni1466. [DOI] [PubMed] [Google Scholar]
  41. Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell. 2004;118:229–241. doi: 10.1016/j.cell.2004.07.002. [DOI] [PubMed] [Google Scholar]
  42. Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche J, Almer S, Tysk C, O'Morain CA, Gassull M, Binder V, Finkel Y, Cortot A, Modigliani R, Laurent-Puig P, Gower-Rousseau C, Macry J, Colombel JF, Sahbatou M, Thomas G. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature. 2001;411:599–603. doi: 10.1038/35079107. [DOI] [PubMed] [Google Scholar]
  43. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, Britton H, Moran T, Karaliuskas R, Duerr RH, Achkar JP, Brant SR, Bayless TM, Kirschner BS, Hanauer SB, Nunez G, Cho JH. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature. 2001;411:603–606. doi: 10.1038/35079114. [DOI] [PubMed] [Google Scholar]
  44. Gringhuis SI, den Dunnen J, Litjens M, van Het Hof B, van Kooyk Y, Geijtenbeek TB. C-type lectin DC-SIGN modulates toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB. Immunity. 2007;26:605–616. doi: 10.1016/j.immuni.2007.03.012. [DOI] [PubMed] [Google Scholar]

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