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1、ORIGINAL ARTICLEFungal microbiota dysbiosis in IBDHarry Sokol,1,2,3,4Valentin Leducq,1,4Hugues Aschard,5Hang-Phuong Pham,6Sarah Jegou,1,4Cecilia Landman,3,4David Cohen,1,2Giuseppina Liguori,7Anne Bourrier,3,4Isabelle Nion-Larmurier,3,4Jacques Cosnes,3,4Philippe Seksik,3,4Philippe Langella,2,4David S
2、kurnik,8,9Mathias L Richard,2,4Laurent Beaugerie3,4ABSTRACTObjective The bacterial intestinal microbiota playsmajor roles in human physiology and IBDs.Althoughsome data suggest a role of the fungal microbiota in IBDpathogenesis,the available data are scarce.The aim ofour study was to characterise th
3、e faecal fungalmicrobiota in patients with IBD.Design Bacterial and fungal composition of the faecalmicrobiota of 235 patients with IBD and 38 healthysubjects(HS)was determined using 16S and ITS2sequencing,respectively.The obtained sequences wereanalysed using the Qiime pipeline to assess compositio
4、nand diversity.Bacterial and fungal taxa associated withclinical parameters were identified using multivariateassociation with linear models.Correlation betweenbacterial and fungal microbiota was investigated usingSpearmans test and distance correlation.Results We observed that fungal microbiota is
5、skewedin IBD,with an increased Basidiomycota/Ascomycotaratio,a decreased proportion of Saccharomycescerevisiae and an increased proportion of Candidaalbicans compared with HS.We also identified disease-specific alterations in diversity,indicating that a Crohnsdisease-specific gut environment may fav
6、our fungi at theexpense of bacteria.The concomitant analysis ofbacterial and fungal microbiota showed a dense andhomogenous correlation network in HS but adramatically unbalanced network in IBD,suggesting theexistence of disease-specific inter-kingdom alterations.Conclusions Besides bacterial dysbio
7、sis,our studyidentifies a distinct fungal microbiota dysbiosis in IBDcharacterised by alterations in biodiversity andcomposition.Moreover,we unravel here disease-specificinter-kingdom network alterations in IBD,suggestingthat,beyond bacteria,fungi might also play a role in IBDpathogenesis.INTRODUCTI
8、ONCrohns disease(CD)and UC,the two primarytypes of IBD,are lifelong conditions that usuallyaffect young subjects and substantially alter theirquality of life.The exact pathogenesis of IBDremains unknown;however,studies over the lastdecade have demonstrated that IBD involves analtered immune response
9、 towards gut microbiota ingenetically predisposed subjects and under theinfluence of environmental factors.The bacterialmicrobiota in IBD has been thoroughly investi-gated,and several groups worldwide observed abacterial dysbiosis(an imbalance in composition)that is characterised by a reduced biodiv
10、ersity,adecreaseinsomebacteriabelongingtotheFirmicutes phylum(such as Faecalibacterium praus-nitzii)and an increase in bacteria belonging to theProteobacteria phylum such as Escherichia coli.15However,other microorganism types colonisingthe human gut have not been thoroughly investi-gated.With the e
11、xception of a recent study high-lighting the possible role of the enteric virome inIBD,6the data are scarce,particularly regardingfungal microbiota.Fungi have long been suspectedin IBD pathogenesis.Many years ago,antibodiesdirected against Saccharomyces cerevisiae mannan(Anti Saccharomyces cerevisia
12、e antibody(ASCA)were shown to be associated with CD.Moreover,several IBD-associated genes,such as Card9,areinvolved in immune responses to fungi.7In mice,gut inflammation promotes fungi proliferation;8conversely,some fungi can modulate susceptibilitySignificance of this studyWhat is already known on
13、 this subject?The bacterial intestinal microbiota isunbalanced in IBDs and plays a role in itspathogenesis.The fungal microbiota has been poorly studieddespite several clues of its role in IBDpathogenesis.Card9 and Dectin1,two key molecules involvedin the innate immunity against fungi,stronglyinflue
14、nce mice susceptibility to intestinalinflammation and the fungal microbiota.What are the new findings?The faecal fungal microbiota is imbalanced inpatients with IBD.The concomitant analysis of the bacterialmicrobiota in the same subjects showed manycorrelations between bacterial and fungalcomponents
15、 with differences between IBD andhealthy subjects,suggesting the existence ofdisease-specific inter-kingdom alterations.How might it impact on clinical practice inthe foreseeable future?These results support the role of fungalmicrobiota in IBD pathogenesis and indicate anew potential therapeutic tar
16、get.1039Sokol H,etal.Gut 2017;66:10391048.doi:10.1136/gutjnl-2015-310746To cite:SokolH,LeducqV,AschardH,etal.Gut 2017;66:10391048.Additional material is published online only.To view please visit the journal online(http:/dx.doi.org/10.1136/gutjnl-2015-310746).For numbered affiliations see end of art
17、icle.Correspondence toDr Harry Sokol,Service de Gastroentrologie et Nutrition,Hpital Saint-Antoine,184 rue du faubourg St Antoine,Paris 75571,cedex 12,France;harry.sokolaphp.frReceived 17 September 2015Revised 12 January 2016Accepted 14 January 2016Published Online First 3February2016Inflammatory bo
18、wel diseaseto inflammation in a negative(Candida albicans)or positive(Saccharomyces boulardii)manner.811Finally,mice lackingmajor genes involved in fungi sensing,such as Dectin-1 orCard9,have an increased fungal microbiota load and are moresusceptible to colitis.12 13These data suggest a link betwee
19、nfungal microbiota and IBD pathogenesis.Here,we characterised the fungal microbiota in both healthysubjects(HS)and patients with well-phenotyped IBD usinghigh-throughput sequencing technology.In the correspondingpatients,we also determined the bacterial microbiota compos-ition and the sequence of 22
20、 single-nucleotide polymorphisms(SNPs)in genes known to be involved in fungal susceptibility.We observed a clear fungal dysbiosis in patients with IBD.Moreover,a correlation analysis suggested altered inter-kingdomrelations in IBD.Finally,while somewhat lacking in power,ourgenotypefungal microbiota
21、analysis suggested that genes maybe a driving factor of the fungal microbiota dysbiosis in IBD.Overall,the data presented in this study represent the mostcomprehensive analysis of fungal microbiota in patients withIBD to date and provide a rationale to support the role offungal microbiota in IBD pat
22、hogenesis.These data thus pave theway for intervention studies targeting fungal microbiota.RESULTSBacterial dysbiosis in IBDOur study population was composed of 235 patients with well-phenotyped IBD and 38 HS(see online supplementary 1).Wefirst analysed the bacterial fraction of the microbiota using
23、 16Ssequencing.A beta diversity analysis showed a clustering ofsamples according to disease phenotypes(figure 1A,B,onlinesupplementary figure S1).Compared with HS samples,thealpha diversity(assessed using four different indexes)was sig-nificantly decreased in UC and CD and particularly in samplesfro
24、m patients in flare(figure 1C,D,online supplementary figureS2).In all phenotypes,the bacterial microbiota was dominatedby bacteria from Firmicutes,Bacteroidetes and Proteobacteriaphyla(figure1E,F,online supplementary figure S3).These dataare in accordance with the published literature and validate t
25、hequality of our cohort and the methods used.Altered fungal microbiota diversity in IBDUsing ITS2 sequencing,we then assessed the composition of thefungal microbiota in our population.The clustering between thesamples according to disease phenotype was weaker than withbacterial microbiota(figure 2A,
26、B,online supplementary figureS4A).Notably,no statistically significant difference was observedbetween samples from patients with CD and UC or betweensamples from patients with IBD in remission and HS.However,a difference was observed between samples from patients withIBD in flare and HS(p=0.0008)or
27、patients with IBD in remis-sion(p=0.0007).This fungal microbiota-specific signal in flarewas observed with a higher intensity in UC(p=0.0002)than inCD(p=0.006;see online supplementary figure S4B-C).Similarto the results found with the bacterial microbiota,the alphadiversity of the fungal microbiota
28、was decreased in patients withIBD(figure 2C,online supplementary figure S5AD).Thisfeature was primarily found in samples from patients with UC,whereas fungi diversity was largely similar among the HS andpatients with CD.To explore the equilibrium between bacteriaand fungi diversity in the gut,we the
29、n determined thefungi-to-bacteria diversity ratio.This ratio was increased in IBDsamples and particularly in CD and flares(figure 2D,online sup-plementary figure S5EG).The highest ratio was found amongpatients with CD and demonstrated ileal involvement.In bothHS and patients with IBD,the fungal micr
30、obiota was dominatedby fungi from the Ascomycota and Basidiomycota phyla withsome variations according to disease phenotype(figure 2E,F,online supplementary figure S6).Among the most dominantgenerawereSaccharomyces,Debaryomyces,Penicillium,KluyveromycesandCandida.Interestingly,Saccharomyces,Debaryom
31、yces and Kluyveromyces are found in food(cheese,bread,beer notably);they might be routinely ingested in thisFrench cohort,suggesting a possible influence of the diet habits,although a specific study is needed to explore this hypothesis.These data show specific alterations in fungal microbiotadiversi
32、ty in parallel with modifications of the bacterial micro-biota.Taken together,this suggests that the environmentalchanges during inflammation might affect differently fungi andbacteria and induce an altered fungalbacterial inter-kingdomrelationship in IBD.Distortion in bacterial and fungal microbiot
33、a composition inIBDWe identified the microbial features associated with diseasephenotype and used a multivariate association test to control forthe effects of potential confounding factors such as age,gender,smoking and treatment(multivariate association with linearmodels(MaAsLin);see Materials and
34、methods for details).Regarding bacterial microbiota,we observed an alteration in theabundance of several taxa in IBD compared with HS,in flarecompared with remission and in IBD with ileal involvementcompared with IBD without ileal involvement(figure 3A;seeonline supplementary figures S7A and 8,table
35、 S1 for fullMaAsLin output).Many of these taxa have been reported in pre-vious studies,1 5including Ruminococcaceae,Lachnospiraceae,Enterobacteriaceae,Pasteurellaceae,RikenellaceaeandGemellaceae.Notably,Ruminococcus,Coprococcus,Blautia,Eubacterium and Dorea abundance were decreased in IBD,Roseburia,
36、Faecalibacterium,Dorea and Blautia abundance weredecreased in IBD flare and Ruminococcus gnavus was increasedin ileal CD.In addition to confirming these already demon-strated associations,we also found new associations such as adecrease in Anaerostipes in IBD and particularly in flare and inileal CD
37、.We also found an increase of Streptococcus anginosusinIBDandanincreaseofAggregatibactersegnisandActinobacillus(two members of the Pasteurellaceae family)inIBD flare compared with remission.When analysing fungal microbiota,we identified a lowernumber of associations with the disease phenotype(figure
38、 3B;see online supplementary figure S7B,table S2 for full MaAsLinoutput).One of the most striking features was the increasedabundance of Basidiomycota in IBD and particularly in flare,which was balanced by an equivalent decrease in Ascomycota.Among the decreased Ascomycota in IBD and in flare,Malass
39、eziasympodialis was identified.However,the Saccharomyces genusand particularly S.cerevisiae species exhibited the strongestsignals.We thus performed real-time qPCR targeting S.cerevisiaeon the same samples and confirmed a clear decrease in S.cerevi-siae both in the absolute number and regarding the
40、proportion inIBD and particularly in flare(figure 3C,online supplementaryfigure S9A-B).Although C.albicans abundance is increased inCD,14Candida was not identified to be associated with diseasephenotype in the multivariate analysis.We thus performed real-time qPCR targeting C.albicans on the same sa
41、mples to specific-ally examine this microorganism.The C.albicans proportion andabsolute number were significantly increased in IBD flare com-pared with IBD in remission(figure 3D,E,online supplementaryfigure S9C).We investigated correlations between their respective1040 Sokol H,etal.Gut 2017;66:1039
42、1048.doi:10.1136/gutjnl-2015-310746Inflammatory bowel diseaseabundance.BasidiomycotaandAscomycotaabundancesexhibitedastrongnegativecorrelationwitheachother(seeonlinesupplementaryfigureS9D).Moreover,theBasidiomycota-to-Ascomycota abundance ratio was dramaticallydifferent according to the disease phen
43、otype with higher valuesinIBDflarecomparedwithIBDinremissionandHS(figure 3F-G),suggesting that this ratio could represent a fungaldysbiosis index.S.cerevisiae induces a regulatory response of dendritic cellsDue to the sequencing and qPCR results,we hypothesised thatS.cerevisiae and C.albicans could
44、respectively exert a protectiveand worsening role in the inflammatory process.As a proof ofprinciple,we assessed the interleukin(IL)6 and IL10 productionof murine bone marrow-derived dendritic cells(BMDCs)afterstimulation with the two heat-killed yeast strains.The IL6 pro-duction was similar among S
45、.cerevisiae and C.albicans;however,the production of the anti-inflammatory cytokine IL10was significantly higher following stimulation with S.cerevisiae,suggesting an anti-inflammatory effect of S.cerevisiae comparedwith C.albicans(figure 4).Interestingly,the observed effectswere Card9-dependent bec
46、ause cytokine production was nearlyabolished in DC from Card9 KO mice.This confirmed thecentral role of this gene in hostfungi interactions.IBD microbiota show-specific bacteriafungi associationsWe next assessed whether the fungi microbiota composition wascorrelated with the bacterial composition.To
47、 address this,weFigure 1Altered bacterial microbiota biodiversity and composition in IBD.(A and B)Beta diversity.Principal coordinate analysis of BrayCurtisdistance with each sample coloured according to the disease phenotype.PC1,PC2 and PC3 represent the top three principal coordinates thatcaptured
48、 most of the diversity.The fraction of diversity captured by the coordinate is given as a percentage.Groups were compared usingPermanova method.(C and D)Observed species number describing the alpha diversity of the bacterial microbiota in the various groups studied(KruskalWallis test with Dunns mult
49、iple comparison test).(E and F)Global composition of bacterial microbiota at the phyla and family levels.Healthy subjects(HS)and patient subgroups are labelled on the x-axis and expressed as the relative operational taxonomic unit(OTUs)abundancefor each group.In all panels:*p0.05;*p0.01;*p0.001.CD,C
50、rohns disease.1041Sokol H,etal.Gut 2017;66:10391048.doi:10.1136/gutjnl-2015-310746Inflammatory bowel diseaseinvestigated a correlation at the genus level according to diseasephenotype.We observed a disease-specific pattern with a highernumber of significant correlations in UC compared with HS(figure