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Colonization of Mice by Candida albicans Is Promoted by Chemically Induced Colitis and Augments Inflammatory Responses through Galectin-3

  1. Samir Jawhara1,3,
  2. Xavier Thuru3,
  3. Annie Standaert-Vitse1,2,
  4. Thierry Jouault1,
  5. Serge Mordon2,
  6. Boualem Sendid1,5,
  7. Pierre Desreumaux3 and
  8. Daniel Poulain1,5
  1. 1Inserm U 799, Physiopathologie des Candidoses, Faculté de Médecine, Centre Hospitalier Régional Universitaire de Lille, Institut Fédératif de Recherche 114, Université Lille 2
  2. 2Laboratoire de Parasitologie, Faculté de Pharmacie, Avenue du Professeur Laguesse
  3. 3Inserm U 795, Physiopathologie des Maladies Inflammatoires Intestinales
  4. 4UPRES EA 2689, Détresses Respiratoires et Circulatoires, Pavillon Vancostenobel
  5. 5Laboratoire de Parasitologie-Mycologie, Pôle de Microbiologie, Centre Hospitalier Universitaire, LilleFrance
  1. Reprints or correspondence: Daniel Poulain, Inserm U799, Physiopathologie des Candidoses, Faculté de Médecine, 1, Place de Verdun, 59045 Lille Cedex, France (dpoulain{at}univ-lille2.fr)
 
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Abstract

BackgroundLittle is known about the relationship between colonic inflammation and Candida albicans colonization. Galectin-3 (Gal-3) is an intestinal lectin that binds to specific C. albicans glycans and is involved in inflammation

MethodsColitis was experimentally induced in wild-type and Gal3−/− mice using dextran sulfate sodium (DSS) before oral administration of C. albicans Yeast recovered from stools was quantified. The presence of yeast and inflammation were evaluated in sections of colon by histologic examination, quantification of myeloperoxidase (MPO) activity, and by gene expression for cytokines and innate immune receptors. Serum from mice was collected for determination of anti-yeast mannan antibodies, including anti–Saccharomyces cerevisiae antibodies (ASCA), which are biomarkers of an inflammatory bowel disease

ResultsInflammation strongly promoted C. albicans colonization. Conversely, C. albicans augmented inflammation induced by DSS, as assessed by histologic scores, MPO activity, and tumor necrosis factor (TNF)-α and Toll-like receptor (TLR)-2 expression. C. albicans colonization generated ASCA. The absence of Gal-3 reduced DSS inflammation and abolished the response of TLR-2 and TNF-α to C. albicans colonization

ConclusionsDSS-induced colitis provides a model for establishing C. albicans colonization in mice. This model reveals that C. albicans augments inflammation and confirms the role of Gal-3 in both inflammation and the control of host responses to C. albicans

Candida albicans colonizes the human gut, which is the primary source of yeast for invasive infections in hospitalized, immunocompromised patients [1, 2]. Recently, a link was established between C. albicans and inflammatory bowel diseases through demonstration that this endogenous yeast could induce anti-oligomannose antibodies (anti–Saccharomyces cerevisiae antibodies, or ASCA), which are markers of Crohn disease [3]

However, little is known about the molecular relationships between C. albicans and the host receptors that govern colonization, tolerance, inflammation, and invasion at the gut level [4]. Experimental studies in mice are limited by the fact that C. albicans is not a natural part of the murine digestive flora [5, 6]. Establishment of colonization therefore requires the use of either infant [5, 7] or antibiotic-treated adult mice [8–10]. In this study, we used dextran sulfate sodium (DSS)-induced colitis in mice, a model that is widely used to study the relationship between inflammation and the endoluminal microbiota [11], but which had never been adapted to C. albicans

With this model, we explored the effect of C. albicans colonization on inflammation, at both the macroscopic and molecular levels. We examined the involvement of innate immunity mediators and receptors, including galectin-3 (Gal-3), which is a pleiotropic lectin that participates in inflammation but has also been described as a specific receptor for C. albicans [12]. A recent study has confirmed that Gal-3 binds to C. albicans and that high levels of Gal-3 can be detected in human tissues infected by C. albicans [13]. Simultaneously, it was demonstrated that C. albicans promoted the association of Gal-3 with the innate receptor Toll-like receptor (TLR)-2 to induce a macrophage response, which included secretion of the proinflammatory cytokine tumor necrosis factor (TNF)-α [14]

Using wild-type (WT) and Gal3–deficient mice (hereafter, Gal3−/− mice), the objectives of this study were to investigate the following: (1) the effect of DSS-induced inflammation on C. albicans colonization; (2) the effect of C. albicans colonization on inflammation, as measured by histologic scores, neutrophil infiltration, gene expression of pathogen recognition receptors, and cytokines; and (3) the role of Gal-3 in the regulation of inflammation induced by C. albicans specifically in relation to TLR-2 and TNF-α secretion and ASCA generation

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Materials and Methods

AnimalsAll animal experiments conformed to the Ministère de l’Agriculture et de la Forêt Resolution on the use of animals in research and were approved by the Subcommittee on Research Animal Care of the Regional Hospital Center of Lille (protocol 2003–35). The production of Gal3−/− mice by use of gene-targeting technology has been described elsewhere [15]. As controls, age- and sex-matched WT (C57BL/6) littermates were used. Mice were maintained by Charles River Laboratories (France). Six- to 8-week-old female mice were used in this study. Animals were housed in groups and had free access to regular rodent chow and tap water

Induction of colitisC. albicansadministration, and experimental designColitis was experimentally induced in mice by administration of 5% DSS (molecular weight, 36–50 kDa; MP Biomedicals) in drinking water from day 1 to day 7. Mice were inoculated on day 3 by single gavage with 200 μL of PBS containing 107 live cells of C. albicans SC5314 reference strain [16]. No mortality was observed during the 7 days that DSS was administered

WT and Gal3−/− mice were each distributed into 1 control group and 3 experimental groups. A group of healthy mice was used as controls (CTL) (5 WT and 6 Gal3−/− mice). A second group of mice was gavaged orally with C. albicans without any other treatment (CaCTL) (5 WT and 8 Gal3−/− mice). A third group was treated with DSS (DSS) (5 WT and 7 Gal3−/− mice). A fourth group was treated with DSS and gavaged orally with C. albicans (CaDSS) (6 WT and 7 Gal3−/− mice)

At day 14, the animals were sacrificed by cervical dislocation. Blood was collected by cardiac puncture and serum samples were stored at −20°C until use. The entire colon from the cecum to the anus was removed, and different anatomic sections were stored at −80°C until use

Evaluation ofC. albicanscolonizationThe presence of yeast in the intestinal tract was evaluated by performing plate counts for cultures of feces collected from each animal on day 14. The fecal samples were suspended in 1 mL of PBS, ground in a glass tissue homogenizer and plated onto Sabouraud dextrose agar containing 500 mg/L amikacin sulfate. Colonies of C. albicans were counted after 48 h incubation at 37°C. The results were noted as cfu/10 μL, which corresponded to cfu/10 μg of feces

Detection of ASCASerum antibodies against C. albicans and S. cerevisiae mannan were detected using ELISA tests [17, 18], which were initially designed to detect human antibodies. Antigens consisted of mannan extract from C. albicans VW32 yeasts for detection of anti–C. albicans mannan antibodies or S. cerevisiae SU1 for detection of ASCA. In brief, plates were coated with 100 μL of mannan at a concentration of 1 μg/mL of sugars in sodium carbonate buffer (60 mmol/L; pH 9.6). After incubation and washing in Tris-NaCl-Tween (TNT) (Tris-HCl, 50 mmol/L; NaCl, 150 mmol/L; Tween, 0.05%; pH 7.5), 100 μL of 1:100 diluted serum was added to the coated wells. Peroxidase-labeled anti-mouse immunoglobulins (G, A, M) (Zymed Laboratories) were diluted 1:1000 in TNT. Absorbance was read at 450 nm (reference filter, 620 nm) in a microplate reader (Bio-Rad Laboratories) after addition of tetramethylbenzydine. Results were expressed as optical density (OD)

MicroscopyRings of the transverse part of the colon were fixed overnight in 4% paraformaldehyde-acid and embedded in paraffin for histologic analysis. Cross-sections (4 μm thick) were stained with May-Grünwald-Giemsa stain (Merck). Histologic scores were evaluated by 2 independent, blinded investigators who observed 2 sections per mouse at magnifications of ×10 and ×100. The scores were determined in accordance with Siegmund et al. [19], and the sections were evaluated for the following 2 subscores: (1) a score for the presence and confluence of inflammatory cells, including neutrophils, in the lamina propria, and submucosal or transmural extension; and (2) a score for epithelial damage, focal lymphoepithelial lesions, mucosal erosion and/or ulceration, and extension to the bowel wall. The 2 subscores were added together, and the combined histologic score ranged from 0 (no changes) to 6 (extensive cell infiltration and tissue damage)

Fluorescence staining ofC. albicansin situParaffin-embedded sections were dewaxed and rehydrated with PBS. The tissue sections were then blocked in 1% bovine serum albumin (BSA) diluted in PBS for 30 min at room temperature. Slides were washed in PBS and sections were incubated for 1 h at room temperature with monoclonal antibody 5B2, which reacts with β-linked oligomannose epitopes of the C. albicans cell wall, diluted 1:100 in PBS [20]. After 3 washes with PBS, sections were incubated with fluorescein isothiocyanate–conjugated goat anti-mouse IgM (Zymed Laboratories) for 60 min at 37°C. The sections were then washed with PBS and counterstained with PBS containing 0.02% Evans blue. The sections were examined by immunofluorescence microscopy (Leica Microsystems AG)

Determination of tissue myeloperoxidase (MPO) activityColon tissue samples included tissue from the mid- to distal colon (adjacent to the tissue used for histology). Samples were rinsed with cold PBS, blotted dry, and immediately frozen in liquid nitrogen. The samples were stored at −80°C until MPO activity was estimated by the O-dianisidine method [21, 22]. In brief, tissue samples were weighed, suspended (10%, wt/vol) in 50 mmol/L potassium phosphate buffer (pH, 6.0) containing 0.5% hexadecyltrimethylammonium bromide (0.1 g/20 mL potassium phosphate), and homogenized. A 1-mL sample of the homogenate was sonicated for 30 s. The sample was then centrifuged at 14,000 g for 15 min at 4°C. The MPO level in the supernatant was determined by adding O-dianisidine dihydrochloride and H2O2 solution (O-dianisidine, 0.167 mg/mL; potassium phosphate buffer, 50 mmol/L, H2O20.0005%). The absorbance was read at 450 nm using a 96-well microplate reader. One unit of MPO activity was defined as the quantity able to convert 1 μmol of H2O2 to water in 1 minute at 20°C and was expressed as units/mg protein. Total protein concentrations were quantified using a Bio-Rad DC Protein assay kit

Real-time mRNA quantificationTotal RNA was isolated from colon samples using a NucleoSpin RNA II kit (Macherey-Nagel) following the manufacturer’s instructions, with 20–50 units of DNase I (RNase-free) at 37°C for 30 min to avoid contamination with genomic DNA. RNA quantification was performed by spectrophotometry (Nanodrop; Nyxor Biotech). Reverse transcription of mRNA was carried out in a final volume of 26 μL from 1 μg total RNA using 300 U Moloney murine leukemia virus reverse transcriptase (Invitrogen) according to the manufacturer’s instructions with 500 ng oligo (dT) 12–18 and 50 U ribonuclease inhibitor (RNase-Out; Promega). PCR was performed using an ABI 7000 prism sequence detection system (Applied Biosystems) with SYBR green (Applied Biosystems). Amplification was carried out in a total volume of 25 μL containing 0.5 μL of each primer (table 1) and 1 μL of cDNA prepared as described above. SYBR green dye intensity was analyzed using Abiprism 7000 SDS software. All results were normalized to the housekeeping gene β-actin

StatisticsData were expressed as the mean ± SE for each group. All comparisons were analyzed by the Mann-Whitney U test. Statistical analyses were performed using StatView statistical software (version 4.5; SAS Institute). Differences were considered significant when P<.05

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Results

DSS administration andC. albicanscolonization in WT andGal3−/− miceThe persistence of C. albicans in the intestinal tract of mice with or without DSS administration was evaluated on day 14 by counting colony-forming units of yeast in feces. In WT and Gal3−/− mice inoculated with C. albicans very few colony-forming units were recovered from stools in the absence of DSS treatment. In contrast, in DSS-treated mice, significantly higher numbers of colony-forming units were recovered from stool samples (P<.05) (figure 1). No significant difference was observed between WT and Gal3−/− mice

Figure 1
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Figure 1

Candida albicans cfu recovered from stools of wild-type mice (WT; black bars) and Gal3−/− mice (white bars) Each data set represents the mean value of C. albicans counts for CaCTL mice (control group of mice inoculated with C. albicans by oral gavage) and CaDSS mice (mice inoculated with C. albicans by oral gavage and treated with DSS for 7 days). * and **, significant differences (P<.05) between corresponding groups

The presence of yeast in the gut was examined by immunofluorescence staining of yeast with an anti–C. albicans β-mannose monoclonal antibody in colon sections from sacrificed mice. The results obtained showed that the presence of yeast in the stools was indeed associated with the presence of large quantities of C. albicans blastoconidia, either in the lumen of the gut (figure 2a) or adhering to the epithelium (figure 2b). We did not find any hyphae or pseudohyphae, nor did we observe epithelial invasion in any of the large number of sections examined

Figure 2
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Figure 2

Immunofluorescence staining of colon sections with an anti–β-1,2–linked oligomannose monoclonal antibody specific for Candida albicans Representative sections of colon from wild-type mice (WT; panel a) and Gal3−/− mice (panel b) inoculated with C. albicans alone. No yeast can be observed. Panel c and e WT mice (c) and Gal3−/− mice (e) treated with DSS and inoculated orally with C. albicans. C. albicans blastoconidia can be observed in the colon lumen and adhering to epithelial cells. Boxed regions in panel c and e are shown at a higher magnification than in panel d and f The scale bars represent 50 μm (panels a, b, c and e) and 10 μm (panels d and f)

Detection of anti-yeast mannan antibodies in serum from miceWe investigated the effect of DSS administration on the serologic response to yeast mannans. In both WT and Gal3−/− mice, oral administration of C. albicans to mice receiving DSS resulted in a significant production of anti–C. albicans mannan antibodies (figure 3A). Because C. albicans infection can induce an ASCA response, we also investigated the effect of C. albicans colonization and DSS treatment on the production of ASCA. As shown in figure 3B, mice that received DSS and C. albicans produced ASCA. With almost similar backgrounds for both tests, even higher increases in absorbance were detected in the ASCA ELISA as opposed to the C. albicans–specific mannan ELISA. The receipt of C. albicans alone did not lead to the generation of these antibodies (data not shown)

Figure 3
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Figure 3

Detection of anti-yeast mannan antibodies in serum from DSS-treated mice with or without Candida albicans administration. Results are expressed as the mean of optical densities (± SE) observed in serum from each group of mice: wild type (WT; black bars) and Gal3−/− (white bars), compared with C. albicans mannan (A) and anti–Saccharomyces cerevisiae mannan antibodies (ASCA) (B). * and **, significant differences (P<.05) between the corresponding groups

Determination of histologic scores and MPO activityHistologic analysis showed a significant difference in the number of colon lesions between DSS-treated WT mice and DSS-treated WT mice that received C. albicans by gavage (P<.05; figure 4A and figure 4B). By contrast, in Gal3−/− mice, DSS-induced histologic scores did not significantly increase after C. albicans gavage (figure 4A and figure 4B). As one of the hallmarks of induced colitis is marked infiltration of neutrophils into the mucosa, this was estimated by measuring mucosal MPO activity. DSS administration increased MPO levels in the colon of WT mice compared to Gal3−/− mice (P<.05). Colonization with C. albicans still enhanced MPO activity in the colon of WT mice with DSS-induced colitis, an augmentation which was less marked in Gal3−/− mice treated with DSS and Candida (P<.05; figure 4C). Thus, MPO activity paralleled colonic damage

Figure 4
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Figure 4

Histology and assessment of infiltrating neutrophil activity. A Histologic characteristics of colon sections from wild-type (WT) and Gal3−/− mice. Sections (4 μm thick) stained with May-Grünwald-Giemsa stain. At low magnification (panels a–f), living cells (e.g., panel a and b) display a typical purple color, whereas the gray color (e.g., panel c and f) indicates direct cell damage and death. Panel aand b control colon sections from WT (a) and Gal3−/−(b) mice. Panel c and d colon sections from WT (c) and Gal3−/−(d) mice with dextran sulfate sodium (DSS)-induced colitis. Panel e and f colon sections from mice with DSS-induced colitis inoculated with C. albicans by oral gavage; WT (e) and Gal3−/−(f) mice. Portions of boxed regions in panel e and f are shown at a higher magnification than in panels g and k Compared with colon sections from control animals (aand b), the colon sections from DSS-treated mice (c and d) showed severe inflammation with large numbers of infiltrating cells (asterisk), tissue destruction with almost complete loss of crypts, loss of epithelial integrity (arrowheads), and edema (arrows) In the presence of DSS, Candida albicans gavage generated the same morphologic damage in both WT and Gal3−/− mice with an extensive cellular infiltrate especially neutrophil cells (asterisk panels g and k), submucosal edema (arrows panels g and k), and epithelial destruction (arrowhead panel g), but cellular inflammation was more pronounced in WT mice (e). The scale bars represent 250 μm (panels a, b, c, d, e and f) and 25 μm (panel g and k). B Histologic scoring performed by 2 independent, blinded examiners (196 examinations). Data are expressed as mean ± SE for each group. WT and Gal3−/− mice (black bars and white bars respectively); CaCTL mice (control group of mice inoculated with C. albicans by oral gavage); and CaDSS mice (mice inoculated with C. albicans by oral gavage and treated with DSS for 7 days). *, **, and † , significant differences (P<.05y) between the corresponding groups. C Myeloperoxidase (MPO) activity. Data are expressed as mean ± SE for each group. †, P<.05 for CaDSS WT vs. DSS WT mice; ‡, P<.05 for CaDSS Gal3−/− vs. DSS Gal3−/− mice; *, P<.05 for CaDSS WT vs. CaDSS Gal3−/− mice; and **, P<.05 for DSS WT vs. DSS Gal3−/−mice

Inflammatory cytokine and TLR mRNA expression inGal3−/−mice and WT miceTo assess the specific contribution of proinflammatory cytokines and pathogen recognition receptors to the observed tissue damage, reverse transcriptase polymerase chain reaction (RT-PCR) amplification of mRNA isolated from colonic tissue was carried out for NOD-2, TLR-4, TLR-2, TNF-α and IL-1β (figure 5). DSS treatment and Candida colonization, or both, had little effect on NOD-2 and TLR-4 expression. Expression of these pathogen recognition receptors was not increased by the presence of C. albicans in DSS-treated mice. DSS alone induced TLR-2 mRNA expression that was less marked in Gal3−/− mice, but TLR-2 mRNA levels were clearly enhanced by C. albicans colonization and treatment with DSS alone in WT mice. TNF-α expression paralleled TLR-2 mRNA production. It was increased in WT colonized mice, whereas no change was observed in Gal3−/− mice. In contrast, for IL-1β levels, which responded significantly to DSS in WT and Gal3−/− mice (although at a lower level for Gal3−/− mice), C. albicans colonization enhanced the response in both types of mice

Figure 5
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Figure 5

Relative expression levels of NOD-2 (A) TLR-4 (B) TLR-2 (C) TNF-α (D) and IL-1β (E) mRNA in mouse colons. Data are expressed as the means ± SE for each group. Wild-type (WT) and Gal3−/− mice (black bars and white bars respectively); CaCTL mice (control group of mice inoculated with Candida albicans by oral gavage); CaDSS mice (group of mice inoculated with C. albicans by oral gavage and treated with DSS for 7 days). †, ‡, *, and **, significant (P<.05) differences between the corresponding groups

Table 1
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Table 1

Mouse oligonucleotide sequences

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Discussion

Several models have been developed to render the mouse, recognized as the most convenient laboratory animal, susceptible to colonization by C. albicans Numerous papers have been published with results based on the use of infant mice [7] or mice treated with antibiotics [8–10]. In this study, we have established a new and simple model of murine colonization by C. albicans that involves DSS-induced colitis. DSS is thought to induce mucosal injury and inflammation, initially through a direct toxic effect on epithelial cells, with subsequent recruitment and activation of inflammatory cells and upregulation of inflammatory mediators, leading to the development of severe colitis [23]. This model was chosen over a variety of other chemicals used to induce colonic inflammation, such as 2,4,6-trinitrobenzenesulfonic acid [24, 25], acetic acid, phorbol ester, sulfated polysaccharides [26], and formalin [27], which are limited by the lack of chronicity and rapid colonic healing. This DSS treatment, which is usually proposed for mimicking several pathologic conditions of humans, resulted in a significant increase in the number of C. albicans colonies recovered from murine stool samples. Examination by immunofluorescence suggested that isolation of colonies reflected the presence of C. albicans in the gut, because large numbers of yeasts were found, either in the lumen of the colon or adhering to epithelial cells

Gal-3 is an endogenous lectin, involved in a variety of normal and pathologic processes [28, 29]. Gal-3 also has an important role in inflammation [30]. In inflammatory bowel diseases, Gal-3 has been shown to be an autoantigenic target following cleavage after damage of epithelial cells [31] or impairment of its proapoptotic role [32]. Regarding C. albicans Gal-3 acts as a receptor for β-mannose adhesins expressed at the cell wall surface [12, 13]. In the present study, we used Gal3−/− mice to investigate the effect of Gal-3 on C. albicans colonization and its relationship with inflammation

After administration of DSS, Gal3−/− mice displayed lower histologic scores for inflammation. This was confirmed by determination of MPO activity, as a parameter of neutrophil accumulation, which was higher in the colon of WT mice with DSS-induced colitis, compared with Gal3−/− mice. Similarly, for pathogen recognition receptors and cytokine genes whose transcription was stimulated by DSS (i.e., TLR-2, IL1-β, and TNF-α), their induction was reduced significantly in Gal3−/− mice. All of these results strongly support a role for Gal-3 in inflammation

When considering C. albicans–host interactions against this DSS background, the first observation was that the absence of Gal3 did not significantly reduce the level of colonization by C. albicans compared with WT mice. This was a surprise in view of the strong expression of β-mannose epitopes at the C. albicans cell-wall surface (figure 2), which are ligands for Gal-3. However, this reflects the multiplicity of C. albicans adhesins in epithelial cells [4, 33]

Among the different possible interactions involved in C. albicans epithelial cell adherance, most molecular information has been gained over the past few years on glycan–pathogen recognition receptor interactions. The pathogen recognition receptors involved in C. albicans recognition comprise TLR -2, TLR-4 and TLR-6—among which TLR-2 and TLR-4 have prominent roles [34]—and endogenous lectins, including mannose-mannan–binding and glucan-binding lectins. Mannose-mannan–binding lectins are distributed among C-lectins specific for α-linked mannose residues, such as mannose-binding lectin [35], the mannose receptor present on macrophages [36], DC-SIGN (dendritic cell-specific intracellular adhesion molecule [ICAM]-grabbing nonintegrin) [37], Dectin-2 [38], and an S-lectin, galectin-3, that binds β-linked mannoses specific for C. albicans (the topic of the present study) [12, 39]. Finally, Dectin-1 recognizes C. albicans β-glucans [40]

The absence of C. albicans colonization in Gal3−/− mice is in agreement with previous in vitro observations on the role of this lectin in the binding and endocytosis capabilities of macrophages from Gal3−/− mice and leads to the conclusion that Gal-3 is not critical for binding/engulfment of C. albicans [14]. Numerous studies in humans have emphasized the role of C. albicans colonization in the generation of circulating anti-mannan antibodies [41, 42]. Treatment with DSS alone did not lead to the generation of such antibodies in mice. However, in this model, we confirmed the generation of anti–C. albicans mannan antibodies when C. albicans colonization was established. In this set of experiments, Gal-3 status had no influence on anti–C. albicans antibody levels. Interestingly, when these mouse serum samples were allowed to react with a S. cerevisiae mannan antigen, a significant antibody response was also observed. Anti–S. cerevisiae mannan antibodies are known as ASCA. ASCA are used extensively as serologic markers of Crohn disease, a chronic inflammatory bowel disease the etiology of which is still unknown but is considered to be a genetically based lack of tolerance to microbial and/or luminal antigens. It was shown that the major oligomannose epitopes of S. cerevisiae mannans supporting the ASCA response were expressed by the pathogenic phase of C. albicans This study suggested that C. albicans could be an immunogen for ASCA [3]. The data obtained with the present model show that DSS damage alone did not permit ASCA generation by endogenous fungal microbiota. This result is in agreement with previous observations showing that the administration of S. cerevisiae extracts to DSS-treated mice failed to generate ASCA [43]. Morever, our results confirm that when C. albicans encounters an inflamed bowel microenvironment, it triggers ASCA production

An unexpected observation was that C. albicans augmented the inflammation induced by DSS. This was clearly demonstrated in WT mice by histologic scores and MPO activity, as well as IL1-β and TNF-α expression. RT-PCR results for differential expression of pathogen recognition receptors also showed that TLR-2 expression was increased by C. albicans In contrast, TLR-4 mRNA expression was not induced by C. albicans stimulation. We also investigated NOD-2 because this sensor of endogenous microbes participates in genetic susceptibility to inflammatory bowel diseases [44]. Our data ruled out a role for NOD-2 in sensing C. albicans This is in accordance with a recently published study that demonstrated that NOD-2 was not involved in the recognition of C. albicans [45]

The last objective of this study was to define a role for Gal-3 in the sensing of C. albicans by comparing augmentation of the inflammation induced by C. albicans in WT and Gal3−/− mice treated with DSS. First, the absence of Gal3 reduced histologic scores. Qualitative analysis revealed that, for MPO activity and IL-1β levels, although the baseline values were lower than those observed for WT mice, increases resulted from C. albicans stimulation. In contrast, RT-PCR analysis revealed that TLR-2 and TNF-α expression, which were amplified by C. albicans stimulation in WT mice, became completely unresponsive to this stimulus in Gal3−/− mice

It is now established that the control of pathogens is based on proinflammatory and anti-inflammatory responses, an excess of which could be deleterious to the host by means of immunopathogenic mechanisms or by direct microbial damage, respectively [46]. This applies to C. albicans which is tolerated in the gut [47], can invade it [48, 49], and can cause inflammation [50, 51]

This experimental study has identified a role for C. albicans in the augmentation of intestinal inflammation for the first time, to our knowledge. It also showed that C. albicans induced the formation of ASCA in the intestinal niche [3]. These results support previous data concerning the link between Gal-3 and intestinal inflammation, as well as the growing body of evidence concerning the involvement of Gal-3 in C. albicans–host interactions [13]. A major role for TLR-2 in the recognition of C. albicans at the intestinal level was highlighted, in agreement with the results of a previous in vitro study that demonstrated that TLR-2 and Gal-3 associate to stimulate TNF-α synthesis [14]. This in vivo model shows that in the absence of Gal-3, neither TLR-2 nor TNF-α synthesis were stimulated by C. albicans Beside these specific conclusions, this new and easy model of mouse colonization may be valuable for further studies on molecular mechanisms controlling host–C. albicans interactions in the intestinal niche

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Acknowledgments

We thank Emilie Gantier and Edmone Erdual for their excellent technical assistance and Val Hopwood for editing the manuscript

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Footnotes

  • Presented in part: 16th congress of the International Society for Human and Animal Mycology (ISHAM), 25–29 June 2006, Paris, France (abstract 0-0004)

    Potential conflicts of interest: none reported

    Financial support: Institut National de la Santé et de la Recherche Médicale (Inserm), Région Nord Pas de Calais— FEDER (R06042EE), Région Nord Pas de Calais, France (03530111 to S.J.)

  • Received May 4, 2007.
  • Accepted October 4, 2007.
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