Role of fungal polysaccharides and gut microbiota in the digestive clearance of Candida glabrata

La maladie de Crohn (MC) est une maladie inflammatoire chronique de l'intestin qui peut s'expliquer par une dysbiose et une dérégulation de la réponse immune chez des individus génétiquement susceptibles. Des observations expérimentales et cliniques suggèrent le rôle prépondérant de certains microorganismes du microbiote intestinal comme agents pouvant contribuer au déclenchement ou au maintien de l'inflammation associée à la MC. Parmi ces derniers, nous retrouvons les levures commensales du genre Candida dont la colonisation digestive est accrue chez les patients atteints de MC. Dans ce contexte, notre équipe étudie le rôle des composants pariétaux fongiques dans la régulation de l'inflammation intestinale. Dans le cas de Candida glabrata (C. glabrata), ces études montrent que la chitine fongique est capable d'atténuer l'inflammation tout en réduisant la prolifération fongique dans un modèle murin de colite chimio-induite. Dans ce même modèle, nous avons montré que la biodiversité bactérienne intestinale se réduit au fur et à mesure du développement de la colite et que cette diminution est corrélée à une augmentation de la prolifération de C. glabrata. De plus, l'ajout de la chitine a pour conséquence de modifier le microbiote intestinal en faveur des bactéries anaérobies : Bacteroides thetaiotaomicron et Lactobacillus johnsonii. Par la suite et dans ce même modèle, l'administration orale de ces deux bactéries anaérobies ont permis de révéler que celles-ci participent au même titre que la chitine à l'atténuation de l'inflammation intestinale et à la réduction des populations pathobiontes.Toutefois, les mécanismes qui régissent l'interaction Candida-bactéries anaérobies ainsi que les déterminants moléculaires mis en jeu restent à caractériser. La première partie de ce projet a été d'étudier les métabolites impliqués dans l'interaction bactéries anaérobies- cellules épithéliales intestinales coliques de souris. Nos études montrent que l'acide oléique (OA), seul ou combiné avec l'acide palmitique (PA), présente des propriétés anti-inflammatoires notables en réduisant l'expression de plusieurs marqueurs inflammatoires exprimés par les cellules Caco-2 exposées au DSS (Dextran sulfate de sodium). Nos investigations ont également démontré que l'activité de l'OA sur les macrophages induirait une augmentation de l'expression de l'IL-10 et une diminution de divers marqueurs pro-inflammatoires, probablement en lien avec la reconnaissance de l'OA par les récepteurs FFARs et AhR. Ces AG présentent aussi des propriétés inhibitrices sur la formation du biofilm, l'adhérence et la viabilité fongique. L'ensemble de ces propriétés a été confirmé dans un modèle murin de colite DSS-induite où l'administration orale de ces deux AG atténue les paramètres inflammatoires tout en réduisant la prolifération de C. glabrata et des populations bactériennes pathobiontes.Dans la seconde partie de ce projet, nos investigations confirment les propriétés anti-inflammatoires et immunomodulatrices de la chitine administrée de manière curative (par voie intra-péritonéale) à des souris avec colite DSS-induite. Ce traitement induit une diminution des paramètres inflammatoires (scores clinique et histologique), de l'expression des cytokines et médiateurs pro-inflammatoires conduisant à une diminution des charges fongique et bactérienne aérobie fécales. Les souris, prétraitées avec de la chitine (administrée par voie sous-cutanée) préalablement à leur exposition au DSS, sont protégées contre la colonisation digestive par C. glabrata. Bien qu'il s'agisse d'un résultat déterminant, les mécanismes qui régissent la clairance fongique associés à ce traitement restent encore à explorer. Nos données montrent aussi que ce traitement préventif permet d'induire des anticorps dirigés contre la chitine. Cependant et contrairement au traitement curatif, ce traitement ne permet pas de réduire l'inflammation intestinale.Crohn's disease (CD) is a chronic inflammatory disease of the intestine caused by dysbiosis and dysregulation of the immune response in genetically-susceptible individuals. Experimental and clinical studies suggest that certain microorganisms in the intestinal microbiota play a major role in the triggering or maintenance of inflammation associated with CD. These microorganisms include commensal yeasts of the genus Candida, which increase significantly in the digestive tract of patients with CD. In this context, our group studied the role of fungal cell wall components in the regulation of intestinal inflammation. In the case of Candida glabrata, these studies showed that fungal chitin is capable of attenuating inflammation while reducing fungal proliferation in a murine model of chemically-induced colitis. In this same model, we also showed that the intestinal bacterial biodiversity decreases gradually as colitis develops and that this decrease is correlated with an increase in proliferation of C. glabrata. In addition, the addition of chitin modifies the intestinal microbiota in favour of the anaerobic bacteria Bacteroides thetaiotaomicron and Lactobacillus johnsonii. As a result, and in this same model, oral administration of these two anaerobic bacteria revealed that they participate in the same way as chitin in the attenuation of intestinal inflammation and the reduction of disease-causing populations.However, the mechanisms that regulate the interaction between Candida and anaerobic bacteria, as well as the molecular determinants brought into play, remain to be characterised. The first part of our work studied the metabolites involved in the interaction between anaerobic bacteria and colonic epithelial cells of mice. These studies showed that oleic acid (OA), alone or combined with palmitic acid (PA), had notable anti-inflammatory properties by reducing the expression of several inflammatory markers expressed by Caco-2 cells exposed to DSS (dextran-sulphate sodium). Our investigations also demonstrated that the action of OA on macrophages induced an increase in expression of IL-10 and a decrease in diverse pro-inflammatory markers, probably linked to the recognition of OA by the receptors FFARs and AhR. These fatty acids also had inhibitory properties on biofilm formation, adherence and fungal viability. All of these properties were confirmed in a murine model of DSS-induced colitis where the oral administration of these two fatty acids attenuated inflammation by reducing the proliferation of C. glabrata and disease-causing bacterial populations.In the second part of this project, our investigations confirmed the anti-inflammatory and immunomodulating properties of chitin administered curatively (by intra-peritoneal administration) to mice with DSS-induced colitis. This treatment induced a decrease in inflammatory parameters (clinical and histological scores) and the expression of cytokines and pro-inflammatory mediators, leading to a decrease in the fungal and aerobic bacterial load in the faeces. Mice, pretreated with chitin (administered subcutaneously) prior to their exposure to DSS, were protected against digestive colonisation by C. glabrata. Although this is a significant result, the mechanisms that lead to fungal clearance associated with this treatment are unknown. Our data show that this preventive treatment induces antibodies directed against chitin. However, and contrary to curative treatment, this treatment does not reduce intestinal inflammation

H89 Treatment Reduces Intestinal Inflammation and Candida albicans Overgrowth in Mice

International audienceDeregulation of the dynamic crosstalk between the gut microbiota, intestinal epithelial cells, and immune cells is critically involved in the development of inflammatory bowel disease and the overgrowth of opportunistic pathogens, including the human opportunistic fungus Candida albicans. In the present study, we assessed the effect of N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), a protein kinase A inhibitor, on the migration of macrophages to C. albicans through dextran sulphate sodium (DSS)-challenged Caco-2 cells. We also investigated the impact of H89 on intestinal inflammation and C. albicans clearance from the gut, and determined the diversity of the gut microbiota in a murine model of DSS-induced colitis. H89 reduced the migration of macrophages to C. albicans through DSS-challenged Caco-2 cells. In addition, H89 decreased C. albicans viability and diminished the expression of pro-inflammatory cytokines and innate immune receptors in macrophages and colonic epithelial Caco-2 cells. In mice with DSS-induced colitis, H89 attenuated the clinical and histological scores of inflammation and promoted the elimination of C. albicans from the gut. H89 administration to mice decreased the overgrowth of Escherichia coli and Enterococcus faecalis populations while Lactobacillus johnsonii populations increased significantly. Overall, H89 reduced intestinal inflammation and promoted the elimination of C. albicans from the gut

A Small Aromatic Compound Has Antifungal Properties and Potential Anti-Inflammatory Effects against Intestinal Inflammation

Resistance of the opportunistic pathogen Candida albicans to antifungal drugs has increased significantly in recent years. After screening 55 potential antifungal compounds from a chemical library, 2,3-dihydroxy-4-methoxybenzaldehyde (DHMB) was identified as having potential antifungal activity. The properties of DHMB were then assessed in vitro and in vivo against C. albicans overgrowth and intestinal inflammation. Substitution on the aromatic ring of DHMB led to a strong decrease in its biological activity against C. albicans. The MIC of DHMB was highly effective at eliminating C. albicans when compared to that of caspofungin or fluconazole. Additionally, DHMB was also effective against clinically isolated fluconazole- or caspofungin-resistant C. albicans strains. DHMB was administered to animals at high doses. This compound was not cytotoxic and was well-tolerated. In experimental dextran sodium sulphate (DSS)-induced colitis in mice, DHMB reduced the clinical and histological score of inflammation and promoted the elimination of C. albicans from the gut. This finding was supported by a decrease in aerobic bacteria while anaerobic bacteria populations were re-established in mice treated with DHMB. DHMB is a small organic molecule with antifungal properties and anti-inflammatory activity by exerting protective effects on intestinal epithelial cells

Oleic Acid and Palmitic Acid from <i>Bacteroides thetaiotaomicron</i> and <i>Lactobacillus johnsonii</i> Exhibit Anti-Inflammatory and Antifungal Properties

A decrease in populations of Bacteroides thetaiotaomicron and Lactobacillus johnsonii is observed during the development of colitis and fungal overgrowth, while restoration of these populations reduces inflammatory parameters and fungal overgrowth in mice. This study investigated the effect of two fatty acids from B. thetaiotaomicron and L. johnsonii on macrophages and Caco-2 cells, as well as their impact on the inflammatory immune response and on Candida glabrata overgrowth in a murine model of dextran sulfate sodium (DSS)-induced colitis. Oleic acid (OA) and palmitic acid (PA) from L. johnsonii and B. thetaiotaomicron were detected during their interaction with epithelial cells from colon samples. OA alone or OA combined with PA (FAs) reduced the expression of proinflammatory mediators in intestinal epithelial Caco-2 cells challenged with DSS. OA alone or FAs increased FFAR1, FFAR2, AMPK, and IL-10 expression in macrophages. Additionally, OA alone or FAs decreased COX-2, TNFα, IL-6, and IL-12 expression in LPS-stimulated macrophages. In the DSS murine model, oral administration of FAs reduced inflammatory parameters, decreased Escherichia coli and Enterococcus faecalis populations, and eliminated C. glabrata from the gut. Overall, these findings provide evidence that OA combined with PA exhibits anti-inflammatory and antifungal properties

Intravenous Immunoglobulin Therapy Eliminates <i>Candida</i> <i>albicans</i> and Maintains Intestinal Homeostasis in a Murine Model of Dextran Sulfate Sodium-Induced Colitis

Intravenous immunoglobulin (IVIg) therapy has diverse anti-inflammatory and immunomodulatory effects and has been employed successfully in autoimmune and inflammatory diseases. The role of IVIg therapy in the modulation of intestinal inflammation and fungal elimination has not been yet investigated. We studied IVIg therapy in a murine model of dextran sulfate sodium (DSS)-induced colitis. Mice received a single oral inoculum of Candida albicans and were exposed to DSS treatment for 2 weeks to induce colitis. All mice received daily IVIg therapy starting on day 1 for 7 days. IVIg therapy not only prevented a loss of body weight caused by the development of colitis but also reduced the severity of intestinal inflammation, as determined by clinical and histological scores. IVIg treatment significantly reduced the Escherichia coli, Enterococcus faecalis, and C. albicans populations in mice. The beneficial effects of IVIg were associated with the suppression of inflammatory cytokine interleukin (IL)-6 and enhancement of IL-10 in the gut. IVIg therapy also led to an increased expression of peroxisome proliferator-activated receptor gamma (PPAR&#947;), while toll-like receptor 4 (TLR-4) expression was reduced. IVIg treatment reduces intestinal inflammation in mice and eliminates C. albicans overgrowth from the gut in association with down-regulation of pro-inflammatory mediators combined with up-regulation of anti-inflammatory cytokines

New Efficient Eco‐Friendly Supported Catalysts for the Synthesis of Amides with Antioxidant and Anti‐Inflammatory Properties

International audienceA new environmentally friendly approach for the synthesis of idrocilamide (1), a marketed myorelaxant and anti-inflammatory agent, is reported herein. The synthetic strategy involves a solvent-free aminolysis reaction catalyzed by zinc-containing species (ZnCl2 , montmorillonite K10 (MK10) impregnated with ZnCl2 or eco-catalysts). The latter have been prepared from the aerial parts of Lolium perenne L. plants grown on contaminated soils from northern France without and with thermal activation at 120 °C and supported on MK10 (Ecocat1 and Ecocat2, respectively). The best aminolysis catalysts in the current study (ZnCl2 and Ecocat2) were selected for additional aminolyses. Compared to ZnCl2 , Ecocat2 had the advantage of being reusable over five test runs and constituted a sustainable catalyst allowing a green route to idrocilamide. Synthesized derivatives 1-4, 6 and 9 were first evaluated for their effect on reactive oxygen species (ROS) generation from macrophages and displayed antioxidant properties by preventing ROS production. Next, the analysis of the effect of molecules 1-4, 6 and 9 on macrophage migration between epithelial cells to human opportunistic fungus Candida albicans indicated that molecules 2-4, 6 and 9 exert anti-inflammatory properties via reducing macrophage migration while the parent idrocilamide (1) did not show any significant effect. This work opens the way for the discovery of new analogues of idrocilamide with improved properties

Two New Compounds Containing Pyridinone or Triazine Heterocycles Have Antifungal Properties against Candida albicans

Candidiasis, caused by the opportunistic yeast Candida albicans, is the most common fungal infection today. Resistance of C. albicans to current antifungal drugs has emerged over the past decade leading to the need for novel antifungal agents. Our aim was to select new antifungal compounds by library-screening methods and to assess their antifungal effects against C. albicans. After screening 90 potential antifungal compounds from JUNIA, a chemical library, two compounds, 1-(4-chlorophenyl)-4-((4-chlorophenyl)amino)-3,6-dimethylpyridin-2(1H)-one (PYR) and (Z)-N-(2-(4,6-dimethoxy-1,3,5-triazin-2-yl)vinyl)-4-methoxyaniline (TRI), were identified as having potential antifungal activity. Treatment with PYR and TRI resulted in a significant reduction of C. albicans bioluminescence as well as the number of fungal colonies, indicating rapid fungicidal activity. These two compounds were also effective against clinically isolated fluconazole- or caspofungin-resistant C. albicans strains. PYR and TRI had an inhibitory effect on Candida biofilm formation and reduced the thickness of the mannan cell wall. In a Caenorhabditis elegans infection model, PYR and TRI decreased the mortality of nematodes infected with C. albicans and enhanced the expression of antimicrobial genes that promote C. albicans elimination. Overall, PYR and TRI showed antifungal properties against C. albicans by exerting fungicidal activities and enhancing the antimicrobial gene expression of Caenorhabditis elegans

Adherent invasive escherichia coli (aiec) strain lf82, but not candida albicans, plays a profibrogenic role in the intestine

International audienceBACKGROUND: Intestinal fibrosis is a frequent complication of Crohn's disease. However, the factors that cause chronicity and promote fibrogenesis are not yet understood.OBJECTIVE: In the present study, we evaluated the profibrotic effects of adherent-invasive Escherichia coli (AIEC) LF82 strain and Candida albicans in the gut.METHODS: Colonic fibrosis was induced in C57BL/6 mice by administration of three cycles of 2.5% (w/v) dextran sulfate sodium (DSS) for 5 weeks. LF82 and C. albicans were administered orally once at the start of each week or each cycle, respectively. Expression of markers of myofibroblast activation was determined in TGF-β1-stimulated human intestinal epithelial cells (IECs).RESULTS: LF82 administration exacerbated fibrosis in DSS-treated mice, revealed by increased colonic collagen deposition and expression of the profibrotic genes Col1a1, Col3a1, Fn1 and Vim. This was accompanied by enhanced gene expression of proinflammatory cytokines and chemokines, as well as more recruited inflammatory cells into the intestine. LF82 also potentiated TGF-β1-stimulated epithelial-mesenchymal transition and myofibroblast activation in IECs, by further inducing gene expression of the main mesenchymal cell markers FN1 and VIM and downregulating the IEC marker OCLN. Proinflammatory cytokines were overexpressed with LF82 in TGF-β1-stimulated IECs. Conversely, C. albicans did not affect intestinal fibrosis progression in DSS-treated mice or myofibroblast activation in TGF-β1-stimulated IECs.CONCLUSIONS: These results demonstrate that AIEC strain LF82, but not C. albicans, may play a major profibrogenic role in the gut

Pyroglutamide-Based P2X7 Receptor Antagonists Targeting Inflammatory Bowel Disease

International audienceThis report deals with the design, the synthesis, and the pharmacological evaluation of pyroglutamide-based P2X7 antagonists. A dozen were shown to possess improved properties, among which inhibition of YO-PRO-1/TO-PRO-3 uptake and IL1β release upon BzATP activation of the receptor and dampening signs of DSS-induced colitis on mice, in comparison with reference antagonist GSK1370319A. Docking study and biological evaluation of synthesized compounds has highlighted new SAR, and low toxicity profiles of pyroglutamides herein described are clues for the finding of a usable h-P2X7 antagonist drug. Such a drug would raise the hope for a cure to many P2X7-dependent pathologies, including inflammatory, neurological, and immune diseases