Background
The commensal intestinal microbiota helps to maintain stability and prevent overgrowth or infection with pathogenic bacteria. Gut microbiota dysbiosis has been associated with inflammatory bowel diseases (IBDs) [
1]. IBDs are chronic inflammatory diseases of the gastro-intestinal (GI) tract, and include Crohn’s disease (CD) and ulcerative colitis (UC) [
2]. Disruption of the gut microbiota as well as impairment of host immunity give rise to perturbations promoting the overgrowth of opportunistic yeast species such as
Candida albicans, leading to increased yeast translocation and susceptibility to systemic infection [
3‐
5]. Like
C. albicans,
C. glabrata can cause infections ranging from mucosal lesions to bloodstream or invasive infections in patients with altered immune or physiological responses [
6]. The cell wall of
C. glabrata plays a crucial role in the interaction of the yeast with its host. The yeast cell wall mainly contains of carbohydrate polymers linked with glycoproteins and phospholipids [
7]. The three major components are β-glucans (polymers of glucose), chitin (a polymer of
N-acetylglucosamine), and mannans [
7]. β-1,3-glucans provide the major structural scaffold of the yeast cell wall and have different amounts of β-1,6 branches.
Different studies have demonstrated the biological properties of β-glucans, particularly their anti-inflammatory, antioxidant, and anti-tumor effects [
8‐
10]. β-glucans can directly activate leukocytes, and further stimulate their phagocytic, cytotoxic, and antimicrobial activities. β-glucans are recognized by several receptors, in particular integrin α
Mβ
2, dectin-1 and TLR-4, thereby triggering an immune response [
11‐
13].
In the present study, we studied the effect of β-glucans on the biodiversity of the gastrointestinal tract microbiota in a dextran sulfate sodium (DSS)-induced colitis model. We also analyzed the effect of β-glucans on intestinal inflammation and elimination of C. glabrata from the gut.
Discussion
IBDs are a group of chronic multifactorial disorders. Although the etiology of IBDs remains unknown, several studies have reported that the immune system, genetic susceptibility, and the environmental factors have all been implicated in its pathogenesis [
1,
14,
15]. Involvement of the gut microbiota in the pathophysiology of IBDs has been highlighted [
16]. Several studies have shown reduced diversity of the gut microbiota in IBD patients [
4,
17]. Furthermore, patients with CD are highly colonized with
Candida spp., in particular,
C. albicans [
18]. Consistently, in a murine model, intestinal inflammation induced chemically by DSS promotes
Candida spp. colonization. In turn, fungal colonization exacerbates intestinal inflammation [
3,
19]. The present study focused on the most representative cultivable anaerobic and aerobic bacteria that can undergo changes during intestinal inflammation [
1,
20]. We focused in particular on cultivable bacteria belonging to the phyla Firmicutes, Bacteroidetes, Proteobacteria or Actinobacteria by using selective bacterial media since these bacteria are known for their possible involvement in Crohn’s disease, i.e.
E. coli and
E. faecalis [
21].
An increase in
E. coli and
E. faecalis populations in mice that developed colitis was observed. These data are consistent with clinical and experimental studies, which show an increase in
E. coli and
E. faecalis in CD patients [
4,
20]. In addition to an increase in the population of opportunistic
E. coli, the development of colitis is characterized by high levels of reactive oxygen species (ROS) and nitrogen species, and these are considered hazardous to bacteria. However, the
E. coli population is armed with multiple strategies to help their survival from ROS [
22]. Additionally, tissue damage during colitis could serve as potential nutrients, such as ethanolamine, to encourage
E. coli overgrowth [
23,
24].
In terms of anaerobic bacteria,
B. thetaiotaomicron and
L. johnsonii decreased in response to intestinal inflammation, while
C. glabrata colonization increased during the development of colitis. Interestingly, the reduction in the
L. johnsonii population was more pronounced in the
C. glabrata-DSS group, suggesting that
Lactobacillus growth can antagonize colonization by
Candida spp. [
25].
The first point of contact between
C. glabrata and its host is the fungal cell wall, whose major constituent is β-glucan. The biological activity of β-glucans is related to their molecular structure, including their solubility, their degree of branching, and their effect on the inhibition or activation of host pattern recognition receptors [
13]. This study used soluble short fractions of β-glucans, purified from
C. glabrata in a DSS-induced colitis model. Orally administered β-glucans decreased intestinal inflammation and
C. glabrata overgrowth. This observation is consistent with our previous study, which showed that oral administration of soluble β-glucans derived from
C. albicans reduced leukocyte infiltration into the gut mucosa and enhanced mouse survival in the DSS model [
9]. Additionally, oral administration of β-glucans increased
B. thetaiotaomicron and
L. johnsonii populations in
C. glabrata-DSS groups. The combination of oat β-glucans and
Lactobacillus spp. has consistently been shown to increase the viability of these microorganisms within food matrices [
26].
The anti-inflammatory activities of β-glucans from fungi have been emphasized in different studies, which have shown that soluble β-glucans can activate or block different receptors such as CD11b/CD18, dectin-1, or TLR-4 [
11,
27]. Recently, short fractions of β-glucans were shown to block the activation of platelets mediated by TLR-4 expression [
13]. β-glucans can initiate the innate immune response and then trigger effective immune responses including phagocytosis and cytokine production that lead to fungal elimination. It has been shown that soluble β-glucans were captured via macrophages and dendritic cells (DCs) present in Peyer’s patches and induced DC maturation via the dectin‐1 pathway [
28]. In line with this observation, α-glucan induced the maturation of DCs and was dependent on TLR2 and promoted anti-inflammatory regulatory T cell responses [
29].
In the present study,
C. glabrata overgrowth reduced IL-10 expression during the development of colitis, while β-glucan treatment increased IL-10 production via PPARγ sensing. These data are consistent with the previous study, which indicates that β-glucan treatment reduces pro-inflammatory cytokine expression [
9]. Mice deficient in IL-10 showed an increase in IL-1β levels that promoted uncontrolled inflammation in the intestine [
30]. Additionally, inhibition of IL-1β activity with a receptor antagonist has been shown to decrease intestinal inflammation in a murine model of colitis [
31].
Conclusions
The development of colitis promotes an increase in the aerobic bacteria population, in particular
E. coli and
E. faecalis, but a reduction in the anaerobic bacteria population, such as
L. johnsonii, and
B. thetaiotaomicron. In terms of fungal cell wall components, oral administration of β-glucans to mice decreased the overgrowth of aerobic bacteria and
C. glabrata as well as the production of inflammatory parameters. β-glucan treatment increased IL-10 production via PPARγ sensing, promoting the attenuation of colitis and
C. glabrata elimination. We are currently analyzing the metabolites released by anaerobic bacteria during the development of colitis to determine the molecules involved in the anti-inflammatory process. In a similar way, our previous work showed an interaction between
Candida spp.,
E. coli, and
Serratia marcescens to form robust biofilms where lipopolysaccharide produced by
S. marcescens and
E. coli significantly increased fungal biofilm maturation [
4].
Methods
C. glabrata strain and culture conditions
Candida glabrata wild-type strain (ATCC, Cg) is used. The yeast is grown in liquid YPD medium (yeast extract 1%, peptone 1%, dextrose 1%), on a rotary shaker for 18 h at 37 °C. The culture obtained is then centrifuged at 2500 rpm for 5 min and washed twice in phosphate buffer saline (PBS).
The extraction and assay of β-glucans are carried out as described previously [
9]. Briefly,
C. glabrata cell pellet was treated twice with 200 mL of 1 M NaOH at 70 °C for 30 min. The cell pellet was then washed twice with distilled water, the supernatant was discarded and the pellet was oxidized with 100 mM NaIO4 (Sigma-Aldrich, France) at room temperature for 3 days in the dark room. After completion of the reaction, excess periodate was destroyed by adding ethylene glycol. The reduction of the pellet was performed with 1 M NaBH4 (Sigma Aldrich, France) at room temperature. The reaction was ended by decreasing the pH to 5 by the addition of acetic acid. After different times of washing with distilled water, the insoluble fraction was incubated with zymolyase 20T (0.2 mg/mL, Immuno™; ICN Biomedicals Inc.) at 37 °C for 3 h. Zymolyase was inactivated at 80 °C for 10 min. After centrifugation, the supernatant was dialyzed against distilled water. The dialyzed solution was loaded onto a Sep-Pak C18 column (Alltech) equilibrated with 0.1% trifluoroacetic acid. The evaporation of the Eluate from Sep-Pak column was performed and the resulting oligoglucosaccharides were resuspended in distilled water. Further, these oligoglucosaccharides were loaded onto a carbograph column (Alltech carbograph SPE column). Eluate from the carbograph column was the soluble β-glucan fractions. The nature and purity of the soluble β-glucan fractions are then confirmed by mass spectrometry and NMR analyses. The concentration of β-glucan fractions is measured using sulfuric acid and phenol (5%) in 96-well plates [
9,
13].
Animals
Ten-to-12-week-old, female C57BL/6 mice are used (Charles River Laboratories, France). Mice are allocated to one of three experimental groups or three control groups. A group of healthy mice is used as controls (CTL, 5 mice). A second group of mice receive oral gavage with C. glabrata without any other treatment (Cg, 5 mice). A third group are treated with β-glucan fractions (β-glu, 5 mice). A fourth group are treated with DSS (D, 10 mice). A fifth group are treated with DSS and oral gavage with C. glabrata (DCg, 12 mice). A sixth group are given DSS and oral gavage with C. glabrata and treated with β-glucans (β-gluDCg, 6 mice).
All animal experiments were approved by the subcommittee for Research Animal Care, Regional Hospital Center, Lille, France (00550.05), and were in accordance with institutional (86/609/CEE) and European guidelines for the care and use of laboratory animals.
Inoculum preparation and induction of colitis
The mice are given 200 µL PBS containing 5 × 10
7 live
C. glabrata cells on day 1 by oral gavage. From day 1 to day 14, mice are also treated with 2% DSS (36–50 kDa; MP Biomedicals, LLC, Germany) in drinking water in order to induce colitis. For β-glucan treatment, mice are given β-glucans purified from
C. glabrata (3 mg per mouse) orally and daily for 5 days, starting on day 1. The presence of
C. glabrata in the gut is determined by measuring the number of colonies in feces (approximately 0.1 g/sample) collected from each animal at day 14. Fecal samples are suspended in 1 mL PBS, and samples are then cultured on Candi-Select medium (Bio-Rad Laboratories, France). The results are expressed as CFU/mg of feces [
32].
Isolation of bacteria
Serial dilutions of fecal samples collected from the mice are performed daily. The samples are cultured on non-selective bacterial media (AC agar) focusing on the most representative cultivable anaerobic and aerobic bacteria that can undergo changes during intestinal inflammation. Isolation of aerobic bacteria and anaerobic bacteria is carried out as described previously [
33]. Identification of bacteria isolated on selective media is performed by MALDI TOF mass spectrometry [
33].
Assessment of clinical and histologic scores
The body weight of the mice is recorded daily. Data are expressed as mean percent change from initial body weight. Clinical scores ranging from 0 to 12 and histologic scores ranging from 0 to 6 are assessed as described previously [
19,
33].
Real-time mRNA quantification of cytokines and receptor expression
Nucleospin RNA kit is used to extract total RNA from mouse colons (Macherey–Nagel). RNA is measured by spectrophotometry (Nanodrop; Nyxor Biotech, France). mRNA reverse transcription is performed from 1 µg total RNA using a high capacity cDNA-RT kit (Applied Biosystems) in a final volume of 20 µL [
34,
35]. cDNA synthesis is carried out by PCR in the one-step system (Applied Biosystems) using Fast SYBR green (Applied Biosystems). SYBR green dye intensity is assessed using one-step software [
35]. The reference gene used to normalize the results is POLR2A47 [
33].
Statistical analysis
All data are expressed as the mean ± standard deviation for each experimental group. Pairs of groups are compared using the Mann–Whitney U test. Data are considered to be statistically significant when the P value is as follows: P < 0.05; P < 0.01; P < 0.001. All statistical analyses are performed using GraphPad Prism 4.0 and XLSTAT.
Authors’ contributions
RC, CB, MB, and SJ performed the experiments. RC, CB, BS and SJ analyzed data. RC, CB, MB, BS and SJ interpreted results of experiments. SJ designed the experiments and drafted the manuscript. All authors read and approved the final manuscript.