Gregory Da Costa

Drinking habits as cofactors of risk for alcohol induced liver damage

Background—The Dionysos Study is a cohort study of the prevalence of chronic liver disease in the general population of two northern Italian communities. It included 6917 subjects, aged 12–65 (69% of the total population). Aims—The aim of this part of the study was to examine the relationship of daily alcohol intake, type of alcoholic beverage consumed, and drinking patterns to the presence of alcohol induced liver damage in an open population. Patients and methods—6534 subjects, free of virus related chronic liver disease and participating in the first cross-sectional part of the study, were fully examined. Each subject underwent: (a) medical history and physical examination, (b) evaluation of alcohol intake using an illustrated dietary questionnaire, and (c) routine blood tests. More invasive diagnostic procedures were performed when indicated. Results—Multivariate analysis showed that the risk threshold for developing either cirrhosis or non-cirrhotic liver damage (NCLD) was ingestion of more than 30 g alcohol per day in both sexes. Using this definition, 1349 individuals (21% of the population studied) were at risk. Of these, only 74 (5.5% of the individuals at risk) showed signs of liver damage. The prevalence of “pure” alcoholic cirrhosis was 0.43% (30 of 6917), representing 2.2% of the individuals at risk, with a ratio of men to women of 9:1, while 44 (3.3% of the individuals at risk) showed persistent signs of NCLD. After 50 years of age, the cumulative risk of developing both NCLD and cirrhosis was significantly higher (p<0.0001) for those individuals who regularly drank alcohol both with and without food than for those who drank only at mealtimes. Conclusions—Our data show that in an open population the risk threshold for developing cirrhosis and NCLD is 30 g ethanol/day, and this risk increases with increasing daily intake. Drinking alcohol outside mealtimes and drinking multiple different alcoholic beverages both increase the risk of developing alcohol induced liver damage.

CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands

Complex interactions between the host and the gut microbiota govern intestinal homeostasis but remain poorly understood. Here we reveal a relationship between gut microbiota and caspase recruitment domain family member 9 (CARD9), a susceptibility gene for inflammatory bowel disease (IBD) that functions in the immune response against microorganisms. CARD9 promotes recovery from colitis by promoting interleukin (IL)-22 production, and Card9−/− mice are more susceptible to colitis. The microbiota is altered in Card9−/− mice, and transfer of the microbiota from Card9−/− to wild-type, germ-free recipients increases their susceptibility to colitis. The microbiota from Card9−/− mice fails to metabolize tryptophan into metabolites that act as aryl hydrocarbon receptor (AHR) ligands. Intestinal inflammation is attenuated after inoculation of mice with three Lactobacillus strains capable of metabolizing tryptophan or by treatment with an AHR agonist. Reduced production of AHR ligands is also observed in the microbiota from individuals with IBD, particularly in those with CARD9 risk alleles associated with IBD. Our findings reveal that host genes affect the composition and function of the gut microbiota, altering the production of microbial metabolites and intestinal inflammation.

Fungal dysbiosis in mucosa-associated microbiota of Crohn’s disease patients

BACKGROUND AND AIMS Gut microbiota is involved in many physiological functions and its imbalance is associated with several diseases, particularly with inflammatory bowel diseases. Mucosa-associated microbiota could have a key role in induction of host immunity and in inflammatory process. Although the role of fungi has been suggested in inflammatory disease pathogenesis, the fungal microbiota has not yet been deeply explored. Here we analysed the bacterial and fungal composition of the mucosa-associated microbiota of Crohns disease patients and healthy subjects. METHODS Our prospective, observational study evaluated bacterial and fungal composition of mucosa-associated microbiota of 23 Crohns disease patients [16 in flare, 7 in remission] and 10 healthy subjects, using 16S [MiSeq] and ITS2 [pyrosequencing] sequencing, respectively. Global fungal load was assessed by real time quantitative polymerase chain reaction. RESULTS Bacterial microbiota in Crohns disease patients was characterised by a restriction in biodiversity. with an increase of Proteobacteria and Fusobacteria. Global fungus load was significantly increased in Crohns disease flare compared with healthy subjects [p < 0.05]. In both groups, the colonic mucosa-associated fungal microbiota was dominated by Basidiomycota and Ascomycota phyla. Cystofilobasidiaceae family and Candida glabrata species were overrepresented in Crohns disease. Saccharomyces cerevisiae and Filobasidium uniguttulatum species were associated with non-inflamed mucosa, whereas Xylariales order was associated with inflamed mucosa. CONCLUSIONS Our study confirms the alteration of the bacterial microbiota and is the first demonstration of the existence of an altered fungal microbiota in Crohns disease patients, suggesting that fungi may play a role in pathogenesis.

Unexpected Role for a Serine/Threonine-Rich Domain in the Candida albicans Iff Protein Family

ABSTRACT Glycosylphosphatidylinositol (GPI)-anchored proteins are an important class of cell wall proteins in Candida albicans because of their localization and their function, even if more than half of them have no characterized homolog in the databases. In this study, we focused on the IFF protein family, investigating their exposure on the cell surface and the sequences that determine their subcellular localization. Protein localization and surface exposure were monitored by the addition of a V5 tag on all members of the family. The data obtained using the complete proteins showed for Iff3 (or -9), Iff5, Iff6, and Iff8 a covalent linkage to the β-1,6-glucan network but, remarkably, showed that Iff2/Hyr3 was linked through disulfide bridges or NaOH-labile bonds. However, since some proteins of the Iff family were undetectable, we designed chimeric constructions using the last 60 amino acids of these proteins to test the localization signal. These constructions showed a β-1,6-glucan linkage for Iff1/Rbr3, Iff2/Hyr3, Iff4 and Iff7/Hyr4 C-terminal–Iff5 fusion proteins, and a membrane localization for the Iff10/Flo9 C terminus-Iff5 fusion protein. Immunofluorescence analyses coupled to these cell fraction data confirmed the importance of the length of the central serine/threonine-rich region for cell surface exposure. Further analysis of the Iff2/Hyr3 linkage to the cell surface showed for the first time that a serine/threonine central region of a GPI-anchored protein may be responsible for the disulfide and the NaOH bonds to the glucan and glycoproteins network and may also override the signal of the proximal ω site region.

Rbt1 protein domains analysis in Candida albicans brings insights into hyphal surface modifications and Rbt1 potential role during adhesion and biofilm formation.

Cell wall proteins are central to the virulence of Candida albicans. Hwp1, Hwp2 and Rbt1 form a family of hypha-associated cell surface proteins. Hwp1 and Hwp2 have been involved in adhesion and other virulence traits but Rbt1 is still poorly characterized. To assess the role of Rbt1 in the interaction of C. albicans with biotic and abiotic surfaces independently of its morphological state, heterologous expression and promoter swap strategies were applied. The N-terminal domain with features typical of the Flo11 superfamily was found to be essential for adhesiveness to polystyrene through an increase in cell surface hydrophobicity. A 42 amino acid-long domain localized in the central part of the protein was shown to enhance the aggregation function. We demonstrated that a VTTGVVVVT motif within the 42 amino acid domain displayed a high β-aggregation potential and was responsible for cell-to-cell interactions by promoting the aggregation of hyphae. Finally, we showed through constitutive expression that while Rbt1 was directly accessible to antibodies in hyphae, it was not so in yeast. Similar results were obtained for another cell wall protein, namely Iff8, and suggested that modification of the cell wall structure between yeast and hyphae can regulate the extracellular accessibility of cell wall proteins independently of gene regulation.

Card9 mediates susceptibility to intestinal pathogens through microbiota modulation and control of bacterial virulence

Objective In association with innate and adaptive immunity, the microbiota controls the colonisation resistance against intestinal pathogens. Caspase recruitment domain 9 (CARD9), a key innate immunity gene, is required to shape a normal gut microbiota. Card9 –/– mice are more susceptible to the enteric mouse pathogen Citrobacter rodentium that mimics human infections with enteropathogenic and enterohaemorrhagic Escherichia coli. Here, we examined how CARD9 controls C. rodentium infection susceptibility through microbiota-dependent and microbiota-independent mechanisms. Design C. rodentium infection was assessed in conventional and germ-free (GF) wild-type (WT) and Card9 –/– mice. To explore the impact of Card9 –/–microbiota in infection susceptibility, GF WT mice were colonised with WT (WT→GF) or Card9 –/– (Card9–/– →GF) microbiota before C. rodentium infection. Microbiota composition was determined by 16S rDNA gene sequencing. Inflammation severity was determined by histology score and lipocalin level. Microbiota–host immune system interactions were assessed by quantitative PCR analysis. Results CARD9 controls pathogen virulence in a microbiota-independent manner by supporting a specific humoral response. Higher susceptibility to C. rodentium-induced colitis was observed in Card9–/– →GF mice. The microbiota of Card9 –/– mice failed to outcompete the monosaccharide-consuming C. rodentium, worsening the infection severity. A polysaccharide-enriched diet counteracted the ecological advantage of C. rodentium and the defective pathogen-specific antibody response in Card9 –/– mice. Conclusions CARD9 modulates the susceptibility to intestinal infection by controlling the pathogen virulence in a microbiota-dependent and microbiota-independent manner. Genetic susceptibility to intestinal pathogens can be overridden by diet intervention that restores humoural immunity and a competing microbiota.

Mucosa-associated microbiota dysbiosis in colitis associated cancer.

ABSTRACT Gut microbiota dysbiosis has been associated with inflammatory bowel diseases (IBD). In colorectal cancer, the gut microbiota has also been recognized as potentially involved in aggravating or favoring the tumor development. However, very little is known on the structure and role of the microbiota in colitis associated cancer (CAC), an important complication of IBD in human. Here we analyzed the bacterial and fungal composition of the mucosa associated microbiota of patients suffering CAC, sporadic cancer (SC) and of healthy subjects (HS) by barcode sequences analysis on the following cohort: 7 CAC patients, 10 SC patients and 10 HS using 16S (MiSeq) and ITS2 (pyrosequencing) sequencing, for bacteria and fungi respectively. Mucosa-associated bacterial microbiota in CAC was significantly different from the ones in SC or in HS, while the fungal showed no differences. Comparison between mucosa-associated microbiota on the tumor site or in normal mucosa near the tumor showed very similar patterns. The global mucosa-associated bacterial microbiota in cancer patients was characterized by a restriction in biodiversity but no change for the fungal community. Compared to SC, CAC was characterized by an increase of Enterobacteriacae family and Sphingomonas genus and a decrease of Fusobacterium and Ruminococcus genus. Our study confirms the alteration of the mucosa-associated bacterial microbiota in IBD and SC. Although the cohort is limited in number, this is the first evidence of the existence of an altered bacterial microbiota in CAC clearly different from the one in SC patients.

Enterobacteriaceae are essential for the modulation of colitis severity by fungi

BackgroundHost-microbe balance maintains intestinal homeostasis and strongly influences inflammatory conditions such as inflammatory bowel diseases (IBD). Here we focused on bacteria-fungi interactions and their implications on intestinal inflammation, a poorly understood area.MethodsDextran sodium sulfate (DSS)-induced colitis was assessed in mice treated with vancomycin (targeting gram-positive bacteria) or colistin (targeting Enterobacteriaceae) and supplemented with either Saccharomyces boulardii CNCM I-745 or Candida albicans. Inflammation severity as well as bacterial and fungal microbiota compositions was monitored.ResultsWhile S. boulardii improved DSS-induced colitis and C. albicans worsened it in untreated settings, antibiotic treatment strongly modified DSS susceptibility and effects of fungi on colitis. Vancomycin-treated mice were fully protected from colitis, while colistin-treated mice retained colitis phenotype but were not affected anymore by administration of fungi. Antibacterial treatments not only influenced bacterial populations but also had indirect effects on fungal microbiota. Correlations between bacterial and fungal relative abundance were dramatically decreased in colistin-treated mice compared to vancomycin-treated and control mice, suggesting that colistin-sensitive bacteria are involved in interactions with fungi. Restoration of the Enterobacteriaceae population by administrating colistin-resistant Escherichia coli reestablished both beneficial effects of S. boulardii and pathogenic effects of C. albicans on colitis severity. This effect was at least partly mediated by an improved gut colonization by fungi.ConclusionsFungal colonization of the gut is affected by the Enterobacteriaceae population, indirectly modifying effects of mycobiome on the host. This finding provides new insights into the role of inter-kingdom functional interactions in intestinal physiopathology and potentially in IBD.

Bilophila wadsworthia aggravates high fat diet induced metabolic dysfunctions in mice

Dietary lipids favor the growth of the pathobiont Bilophila wadsworthia, but the relevance of this expansion in metabolic syndrome pathogenesis is poorly understood. Here, we showed that B. wadsworthia synergizes with high fat diet (HFD) to promote higher inflammation, intestinal barrier dysfunction and bile acid dysmetabolism, leading to higher glucose dysmetabolism and hepatic steatosis. Host-microbiota transcriptomics analysis reveal pathways, particularly butanoate metabolism, which may underlie the metabolic effects mediated by B. wadsworthia. Pharmacological suppression of B. wadsworthia-associated inflammation demonstrate the bacterium’s intrinsic capacity to induce a negative impact on glycemic control and hepatic function. Administration of the probiotic Lactobacillus rhamnosus CNCM I-3690 limits B. wadsworthia-induced immune and metabolic impairment by limiting its expansion, reducing inflammation and reinforcing intestinal barrier. Our results suggest a new avenue for interventions against western diet-driven inflammatory and metabolic diseases.Lipid intake is known to promote Bilophila wadsworthia growth. Here the authors show that B. wadsworthia aggravates high fat diet induced metabolic dysfunctions and its suppression, both pharmacologically or mediated by Lactobacillus rhamnosus, limits the severity of metabolic impairment.