a member of the gut mycobiota modulates host purine...

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

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1Division of Microbiology and Immunology, Department of Pathology, Universityof Utah School of Medicine, Salt Lake City, UT 84112, USA. 2ARUP Laboratories,500 Chipeta Way, Salt Lake City, UT 84108, USA. 3Metabolomics Core, University ofUtah Health Sciences Center, Salt Lake City, UT 84112, USA. 4Department of Bio-chemistry, University of Utah School of Medicine, Salt Lake City, UT 84112, USA.*Corresponding author. Email: [email protected]

Chiaro et al., Sci. Transl. Med. 9, eaaf9044 (2017) 8 March 2017

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A member of the gut mycobiota modulates host purinemetabolism exacerbating colitis in miceTyson R. Chiaro,1 Ray Soto,1 W. Zac Stephens,1 Jason L. Kubinak,1 Charisse Petersen,1

Lasha Gogokhia,1 Rickesha Bell,1 Julio C. Delgado,1,2 James Cox,3,4 Warren Voth,1 Jessica Brown,1

David J. Stillman,1 Ryan M. O’Connell,1 Anne E. Tebo,1,2 June L. Round1*

The commensal microbiota has an important impact on host health, which is only beginning to be elucidated.Despite the presence of fungal, archaeal, and viral members, most studies have focused solely on the bacterialmicrobiota. Antibodies against the yeast Saccharomyces cerevisiae are found in some patients with Crohn’s disease(CD), suggesting that the mycobiota may contribute to disease severity. We report that S. cerevisiae exacerbatedintestinal disease in a mouse model of colitis and increased gut barrier permeability. Transcriptome analysis of colontissue from germ-free mice inoculated with S. cerevisiae or another fungus, Rhodotorula aurantiaca, revealed thatS. cerevisiae colonization affected the intestinal barrier and host metabolism. A fecal metabolomics screen of germ-free animals demonstrated that S. cerevisiae colonization enhanced host purine metabolism, leading to an increasein uric acid production. Treatment with uric acid alone worsened disease and increased gut permeability. Allopuri-nol, a clinical drug used to reduce uric acid, ameliorated colitis induced by S. cerevisiae in mice. In addition, we founda positive correlation between elevated uric acid and anti-yeast antibodies in human sera. Thus, yeast in the gut maybe able to potentiate metabolite production that negatively affects the course of inflammatory bowel disease.

INTRODUCTIONInflammatory bowel disease (IBD) is a chronic inflammatory disorderof the gastrointestinal (GI) tract that includes Crohn’s disease (CD)and ulcerative colitis (UC). Unrestricted inflammation at intestinalsites leads to malabsorption of nutrients, severe abdominal pain,and increased chance of developing colorectal cancers (1). Althoughcurrent therapies such as immunosuppression, intestinal resections,and antibiotics ameliorate symptoms, there is no cure for this disease.Emerging evidence supports a role for the microbiota in the modula-tion of IBD. Multiple studies have demonstrated that both communitymembership and abundance within the microbiota in patients withIBD are different from those of healthy individuals (2, 3). Moreover,probiotic treatment during experimental colitis can alleviate disease(4–6). The human microbiota is a complex ecosystem composed ofbacteria, fungi, archaea, and viruses; however, most studies to datehave focused solely on bacterial members and their involvement inIBD (2, 3).

Fungi comprise a diverse kingdom of eukaryotic organisms com-posed of yeasts, molds, and mushrooms. Several yeast species havebeen identified in the GI tract, where they are estimated to makeup 0.1% of the intestinal microbiota (7). Common members includeCandida, Saccharomyces, Aspergillus, Cryptococcus, and Rhodotorula,which indicate the existence of a diverse resident fungal community(8–14). Fungi are large, complex organisms known to act opportunis-tically during immune-mediated and antibiotic therapy (15–17). Thereare several clinical and experimental indications that yeast might in-fluence intestinal inflammation. The first was the discovery of elevatedanti–Saccharomyces cerevisiae antibodies (ASCAs) in the serum of CDpatients (16, 18), which suggests that an aberrant immune response toyeast might be involved in IBD progression. Patients suffering from

IBD are often prescribed antibiotics to kill bacteria thought to drivethe chronic inflammatory response (19–22). Because yeasts are nottargets of commonly used antibiotics, a well-documented side effectof extended antibiotic use is the overgrowth of fungal species (23–25).Moreover, yeasts are a common component of many foods, whichmight provide daily exposure to these organisms. Polymorphisms ingenes such as CARD9 and CLEC7A (dectin-1) that function in fungalrecognition by the host have been described in patients who suffer fromincreased fungal infections (26–28). Deletion of these genes in miceleads to worsened intestinal disease (29, 30). Thus, there is evidencein both mouse models and humans that supports a role for fungi inintestinal disease, yet the mechanisms by which this occurs remainpoorly defined. Here, we investigate a role for S. cerevisiae in the patho-genesis of colitis.

The study of resident fungal communities is still in its infancy;however, there are a handful of papers that have surveyed fungalpopulations in patients with IBD (8, 12, 31). Several of these studieshave reported a loss of bacterial diversity with a concomitant increasein fungal variety and load during IBD (8, 32). Similarly, the most re-cent and largest patient survey of fungal communities identified ahigher fungal-to-bacterial ratio in patients with IBD (12), furthersupporting the hypothesis that increases in fungal load might be asso-ciated with disease. Several species of fungi have now been identifiedfrom both colitogenic and healthy individuals, including S. cerevisiae,Candida albicans, Penicillium italicum, Rhodotorula aurantiaca, andMalassezia sympodialis (8, 12, 14). These species belong to two fungalphyla, the Ascomycota and Basidomycota, which dominate the intes-tinal fungal community. To investigate how different yeast species iden-tified in the human GI tract influence intestinal disease, we chose oneorganism from each of these fungal phyla that are readily available,easily cultured, and present in individuals with IBD—S. cerevisiae,a member of the Ascomycota, and R. aurantiaca, a member of theBasidomycota. S. cerevisiae represents one of the most highly abundantand most commonly detected fungal members in the human GI tractand is also found in food and the environment (12, 14), making it highlyrelevant to human biology. Moreover, we studied a wild, prototrophic,

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diploid strain that represents one of the more common strains found inthe human gut. R. aurantiaca is commonly found within the environ-ment and is a member of a genus that is beginning to emerge as a newclass of opportunistic pathogens (33).

RESULTSS. cerevisiae, but not R. aurantiaca, exacerbatesexperimental colitisC57Bl/6 mice were orally gavaged with either S. cerevisiae orR. aurantiaca and subsequently challenged with two different re-agents, 2,4,6-trinitrobenzenesulfonic acid (TNBS) or dextran sulfatesodium (DSS), which yielded two chemical models of colitis (34). Be-cause animals are resistant to stable yeast colonization without priortreatment with antibiotics (35), a phenomenon termed colonizationresistance, animals were orally gavaged with 106 colony-forming units(CFU) every day for 7 days. This treatment mimics consumption offood enriched with yeast products. Animals treated with S. cerevisiaeconsistently developedworsened disease, as measured by hematoxylinand eosin (H&E) staining of paraffin-embedded sections of the colon.Animals treated with S. cerevisiae had enhanced epithelial damage,greater crypt loss, and increased cellular infiltration when comparedto either vehicle- or R. aurantiaca–treated animals (Fig. 1, A to D).This observation suggests that the presence of S. cerevisiae, but notR. aurantiaca, exacerbates intestinal disease. To determine whetherlive S. cerevisiaewas required to worsen colitis or whether a host reac-tion to fungal cell wall constituents was the cause, we fed either live orheat-killed S. cerevisiae to C57Bl/6mice and subsequently induced co-litis with DSS. Whereas live S. cerevisiae induced aggravated colitis,heat-killed S. cerevisiae failed to elicit heightened disease. Colonicshortening occurs during the course of disease and is used as anindicator of disease severity. Animals treated with live S. cerevisiaehad the shortest colons, whereas control and heat-killed groups hadsimilar colon lengths (Fig. 1E). Moreover, heat-killed S. cerevisiae re-sulted in histology scores similar to those of control animals (Fig. 1F).Therefore, worsened disease was not simply a reflection of a host re-sponse to fungal components; rather, it required ametabolically activeorganism.

Inflammatory responses are known to play a critical role duringcolitis. Therefore, we analyzed the mesenteric lymph nodes (MLNs)for inflammatory interferon-g (IFN-g)– and interleukin-17 (IL-17)–producing T helper 1 (TH1) and TH17 cells, respectively. We didnot detect significant differences in inflammatory T cell subsets inS. cerevisiae– or R. aurantiaca–treated animals compared to controls(fig. S1, A to C). In addition, we did not detect transcriptional changesin the colon of commonly elevated inflammatory cytokines such as IL-6or tumor necrosis factor–a (fig. S1, D and E). These data imply thatworsened disease in S. cerevisiae–treated mice was not the result of agreater inflammatory response and suggest that exposure to S. cerevisiae,but not R. aurantiaca, had the potential to exacerbate the course ofintestinal disease.

The prominent commensal fungus S. cerevisiae diminishesintestinal barrier functionWe next sought to better understand how commensal yeast couldhave such disparate outcomes during the course of intestinal inflam-mation using an unbiased approach. To this end, we performed tran-scriptome analysis on colonic RNA isolated from either S. cerevisiae orR. aurantiaca monoassociated germ-free animals. Whereas S. cerevisiae


stably colonized germ-free animals (fig. S2A), R. aurantiaca couldnot be detected in feces of germ-free animals after a single oral ga-vage of 106 CFU. Because these organisms are commonly found inthe environment and food and to ensure equal exposure of animalsto yeast, we placed S. cerevisiae and R. aurantiaca in the drinkingwater of germ-free animals. Yeast maintained equal viability indrinking water.

The density of yeast is highest in the colon; thus, we focused ourinitial analysis at this site (29). Colonization with yeast did not inducea host transcriptional response as large as that reported for some bac-terial strains (36, 37). Monoassociation with either yeast strains led tomaximum gene expression changes of just 10-fold. Although the hostgene expression profile induced by either yeast species was very simi-lar, several genes were differentially regulated between the two strains(fig. S2, B and C). KEGG (Kyoto Encyclopedia of Genes and Ge-nomes) pathway analysis of the microarray data revealed that yeastcolonization most significantly influenced host metabolic pathwaysand genes involved in maintaining the intestinal barrier (fig. S2, Bto C). With the exception of changes in antimicrobial peptides, therewere few changes in immune-mediated pathways identified by thisanalysis. A secondary analysis using Ingenuity software also confirmedthat intestinal barrier pathways were highly influenced in response tofungal colonization. Some of the most significant changes in gene ex-pression between the two species were for genes involved in control-ling bacterial populations and maintaining intestinal epithelial integrity(Fig. 2, A and B). Many antimicrobial peptides and genes involved incellular tight junction formation were more highly up-regulated by thepresence of R. aurantiaca compared to S. cerevisiae, suggesting that thepresence of S. cerevisiae supported epithelial barrier integrity lessstrongly (Fig. 2, A and B). Consistent with these observations, quanti-tative reverse transcription polymerase chain reaction (qRT-PCR) anal-ysis from colonic tissue of monoassociated animals revealed thatmultiple genes involved in forming epithelial junctions in the intestinewere more highly up-regulated by R. aurantiaca, including claudin-2,Zonula occludens (ZO)-2, and claudin-7 (Fig. 2, C to F, and fig. S2D).

Given these results, we sought to functionally test how fungalorganisms influenced intestinal barrier function. Animals received adaily oral gavage of S. cerevisiae or R. aurantiaca and were placedon 2.5% DSS in drinking water. Seven days after disease induction,animals were orally gavaged with fluorescein isothiocyanate (FITC)–dextran, and blood was collected 4 hours later to determine the level ofintestinal leakage of FITC-dextran in the systemic compartment.Animals treated with R. aurantiaca had similar levels of intestinal pen-etration when compared to control animals (Fig. 2G). However,animals treated with S. cerevisiae demonstrated a significant increasein the amount of FITC-dextran detected in their blood (Fig. 2G).These data indicate that the presence of S. cerevisiae led to greater in-testinal leakage.

To determine whether S. cerevisiae could directly influence perme-ability of the intestinal epithelium, we developed an in vitro intestinalpermeability assay (fig. S2E). Yeast strains were incubated with a con-fluent layer of MODE-K cells, a cell line derived from mouse smallintestine. Penetration of FITC-dextran into the bottom of a Transwellwas used as a measure of permeability. Intestinal epithelia coculturedwith S. cerevisiae showed significantly enhanced, albeit modest, per-meability when compared to cells incubated with medium alone orR. aurantiaca–treated cells (fig. S2F), suggesting that S. cerevisiaecan directly act on intestinal epithelia to influence permeability. Main-taining intestinal epithelial barrier integrity has been shown to be a

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critical component to prevent intestinal disease in mouse models andhumans (38, 39). Thus, our data suggest that a common member ofthe intestinal and environmental mycobiota, S. cerevisiae, can worsenintestinal disease by enhancing gut epithelial leakage.


S. cerevisiae enhances degradation of purine produced byintestinal epitheliaOther major gene expression pathways influenced by yeast coloniza-tion in germ-free mice were host metabolic pathways (fig. S2, B and

C). The microbiota contributes impor-tant functions to their mammalian hosts,including colonization resistance and in-duction of immune responses; however,one of the most well-known roles forthe microbiota is the breakdown of dietarycomponents for use by host metabolism(40). How commensal fungi influence me-tabolism is largely unknown. Therefore,we generated a fecal metabolomic profileusing gas chromatography–mass spec-trometry (GC-MS) from SPF, germ-free,and S. cerevisiae or R. aurantiaca monoas-sociated animals to determine how yeastcolonization might influence metabolism.SPF animals are markedly different fromthe other three groups, such that yeastmonoassociated animals cluster moreclosely with germ-free animals (Fig. 3A).Despite the similarity between the me-tabolomic profile of yeast monocolonizedand germ-free animals, there were 20metabolites that were influenced by yeastcolonization (Fig. 3B). Many of the metab-olites found to be elevated in germ-freemice were sugars such as ribitol andmannitol (fig. S3, A and B). These sugarswere depleted upon monoassociationwith either yeast species and likely indi-cated the use of these sugars by coloniz-ing yeast.

Of the few metabolites that differedbetween S. cerevisiae and R. aurantiaca,five were part of the purine degradationpathway (Fig. 3C and fig. S3C) and in-cluded adenosine, adenine, xanthine, hy-poxanthine, and uric acid. Uric acid is theoxidative product of hypoxanthine andxanthine through the action of the enzymexanthine oxidase (XO) (41). Most yeastspecies lack XO; rather, they convert hy-poxanthine to xanthine via xanthine di-oxygenase. However, S. cerevisiae lacks allof the enzymes necessary to catabolize pu-rines (42). These observations support theidea that increased levels of uric acid area host response to monoassociation withS. cerevisiae rather than themetabolic activ-ity of the yeast. Supporting this notion, uricacid cannot be detected in the growth me-dium of S. cerevisiae. To determine wheth-er S. cerevisiae can elevate serum uric acidin animals with an intact microbiota duringdisease, we treated mice with S. cerevisiaeor R. aurantiaca and subsequently induced

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Fig. 1. S. cerevisiae, but not R. aurantiaca, exacerbates intestinal disease in two mouse models of colitis.(A and B) C57Bl/6 specific pathogen–free (SPF) mice were orally gavaged daily for 7 days with 106 of S. cerevisiae (n =7) or R. aurantiaca (n = 7), and control mice were mock-gavaged with phosphate-buffered saline (PBS) (n = 6) eachday for 1 week. Colitis was induced via administration of TNBS. These data are representative of two independentexperiments with six to seven mice per group in each experiment. (C and D) C57Bl/6 SPF animals were treated as in(A) [S. cerevisiae (n = 7), R. aurantiaca (n = 7), and PBS (n = 6)], and colitis was induced by placing 2.5% DSS in drinkingwater. Animals were sacrificed 8 days after DSS induction, and colons were analyzed by histology for disease severity.These are representative of three independent experiments with six to seven mice per group in each experiment. (E andF) C57Bl/6 SPF animals were orally gavaged for 1 week with 106 live (n = 4) or heat-killed (n = 5) S. cerevisiae; controls (n = 5)were mock-gavaged with PBS each day for 1 week. Colitis was induced by placing 2.5% DSS in drinking water. Animals weresacrificed 8 days after DSS treatment, and colons were analyzed for disease severity. Colon length (E) and histology scores (F)are shown. These data are representative of two replicate experiments with four to six mice per group in each experiment.Each dot indicates an individual mouse in all panels. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001, t test. ns, not significant.

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DSS colitis. Treatment with S. cerevisiae, but not R. aurantiaca,elevated uric acid above controls (Fig. 3D). Moreover, treatment ofMODE-K small intestinal epithelial cells with S. cerevisiae in vitro di-rectly stimulated uric acid production, whereas heat-killed and chem-ically killed yeast failed to do so (Fig. 3E and fig. S3D), indicating thatlive yeast was necessary for induction of uric acid production by cells.Last, production of uric acid from intestinal epithelia was specific toS. cerevisiae because both R. aurantiaca and C. albicans did not inducethis response (Fig. 3E). Together, these data establish that S. cerevisiaecan elevate host uric acid production by the intestinal epithelia, evenin animals with a complete microbiota. Thus, exposure to S. cerevisiaein the gut led to enhanced purine metabolism and promoted increaseduric acid.

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Fig. 2. S. cerevisiae colonization disrupts intestinal barrier function. (A and B)C57Bl/6 germ-free animals were colonized with S. cerevisiae or R. aurantiaca byplacing 108 yeast in 200 ml of drinking water. RNA was collected from the colonsof the indicated animals 1 month after colonization and used for microarray anal-ysis. Genes shown were statistically significantly different (P < 0.05) with at least a1.5-fold change. GF, germ-free (n = 4); SC, S. cerevisiae monoassociated germ-freeanimals (n = 4); RA, R. aurantiaca monoassociated germ-free animals (n = 4). (C to F)Some of the genes involved in intestinal epithelial tight junction formation were ver-ified by qPCR; y axis values represent 106 gene transcripts per L32 copies. (G) C57Bl/6SPF animals were treated as described in Fig. 1 [S. cerevisiae (n = 6), R. aurantiaca (n =8), and DSS control (PBS, n = 7)]. Eight days after DSS treatment, animals were orallygavaged with FITC-dextran. Serum was collected and analyzed for the presence ofFITC-dextran. Two independent experiments were performed and are shown herewith three to four mice per group in each experiment. *P < 0.05; **P < 0.01, t test.

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Fig. 3. S. cerevisiae colonization induces elevated purine metabolism inmouse gut. (A and B) C57Bl/6 germ-free animals were monoassociated with eitherS. cerevisiae or R. aurantiaca as described in Fig. 2. Fecal metabolites were analyzedusing GC-MS (n = 4 to 6 in each group). Principal component analysis (PCA) of meta-bolic profiles between germ-free and yeast monoassociated animals (A). Volcano plotshowing individual metabolites between yeast monoassociated and germ-freeanimals (B). (C) Pairwise comparisons of significantly changed metabolites involvedin purine metabolism between the indicated groups. (D) Animals were treated asdescribed in Fig. 1, and colitis was subsequently induced with DSS. Eight days afterDSS induction, serum uric acid levels were measured in the indicated animals.(E) MODE-K cells were treated with the indicated fungal strains for 24 hours, anduric acid production in the medium was analyzed. *P < 0.05; **P < 0.01; ***P <0.005; ****P < 0.001, t test. MOI, multiplicity of infection.

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Host immune reactions to commensal yeast positivelycorrelate with elevated uric acid production in humansElevated titers of ASCAs are often used as a diagnostic tool to differ-entiate CD from UC (18, 43). About 60 to 70% of CD patients haveASCA immunoglobulin G (IgG) and 35% have ASCA IgA, but thepresence of both IgA and IgG at high levels is highly specific forCD (44). However, ASCAs are often found in healthy patients as wellas in people with other forms of intestinal inflammation such as celiacdisease. ASCAs react to components of fungal cell walls, specificallyphosphopeptidomannan structures, common in multiple types ofyeast (45). Although the role of ASCAs during intestinal inflammationremains unclear, elevated titers suggest that an immune responsetoward yeast or yeast products might influence the disease course.

To determine whether there is a connection between uric acid andimmune responses to yeast in humans, we obtained serum from 168individuals (84 men and 84 women) who did not report any symp-toms of disease and were not currently on any medications. Thesesamples were tested for ASCA IgG, IgA, and uric acid. We discovereda positive correlation between levels of ASCA IgG or IgA and uric acidin these serum samples (Fig. 4, A to C). To determine the specificity ofthe relationship between ASCAs and uric acid, we performed an analysisof anti-chromatin antibodies, which are used as a diagnostic marker foranother autoimmune disease, systemic lupus erythematosus. There wasno significant correlation between anti-chromatin antibodies and uricacid (fig. S4, A and B), ruling out the possibility that elevated uric acidis related to all autoimmune diseases. Furthermore, a comparison ofASCAs in individuals with normal or elevated uric acid revealed thatsubjects whose uric acid levels were above the range for average adultshad more ASCA IgA (Fig. 4, D to F). Our analysis suggests that indivi-duals with greater immune reaction to fungal species in the GI tract alsohave elevated uric acid. These data validate our findings in animal modelsthat members of the mycobiota have effects on human metabolism.

Worsened intestinal disease induced by S. cerevisiae ismediated by uric acidUric acid is most commonly recognized as the etiological agent of gout,a disorder of purine metabolism, resulting in the deposition of mono-sodium urate crystals in joints (46); it is also the cause of uric acid neph-rolithiasis (kidney stones). Patients with IBD, compared to healthyindividuals, are more prone to develop uric acid nephrolithiasis. More-over, uric acid is known to bind the NLRP3 (NALP3) inflammasomeand is thus associated with inflammatory disorders (46). These clinicalstudies suggest that elevated uric acid in the intestine during an inflam-matory response could exacerbate intestinal disease.

To test this hypothesis, we orally gavaged animals with uric acidand subsequently induced experimental colitis with DSS. Oral treat-ment with uric acid increased fecal uric acid by only 1.5-fold, elevatingit to the same levels as seen with S. cerevisiae colonization of germ-freeanimals (Fig. 5A). Uric acid supplementation during colitis inducedworsened disease, as evidenced by greater intestinal crypt loss and ep-ithelial damage (Fig. 5, B and C). In addition, increased uric acid alonealso caused greater intestinal permeability, as measured by leakage ofFITC-dextran into the blood (Fig. 5D). Consistent with this finding,treatment of small intestinal epithelial cell lines in vitro with uric acidalso resulted in increased permeability of FITC-dextran across the cellmonolayer (fig. S5A). Because uric acid is known to bind to the NLRP3inflammasome, we investigated a role for NLRP3 in eliciting exacerbatedcolitis during S. cerevisiae treatment. To this end, NLRP3−/− or wild-type(WT) mice were orally treated with S. cerevisiae, and DSS colitis was


induced. There were no differences in colon length between the twogroups, and NLRP3−/− animals actually had slightly elevated histologyscores when compared to WT controls (fig. S5, B to D), demonstratingthat heightened disease induced by S. cerevisiae is not dependent onNLRP3. Collectively, these data demonstrate that uric acid productioninduced by S. cerevisiae can enhance intestinal permeability and increasedisease.

To demonstrate whether aggravated colitis induced by S. cerevisiaewas dependent on the induction of purine metabolism, we sought toblock this pathway using a clinical inhibitor of XO, allopurinol (41).S. cerevisiae–colonized animals were orally gavaged with allopurinol(10 mg/kg) during DSS-induced colitis. Administration of allopurinolreduced uric acid levels (Fig. 5E) and decreased intestinal disease com-pared to those animals that received only S. cerevisiae or DSS (Fig. 5, F

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Fig. 4. A positive correlation between ASCAs and uric acid in human sera.(A to C) Human serum was obtained from healthy donors, and ASCA IgA (A), IgG (B),both ASCA IgG and IgA combined titers (C), and uric acid were determined. Spearmancorrelation analysis was performed on the data, and P values are provided. (D to F) Ser-um uric acid levels were divided into two groups. Each group was then evaluated forthe presence of ASCA IgA (D), IgG (E), or combined antibody titers (F). Normal range forfemale andmale uric acid is 2.5 to 6.0 mg/dl and 3.5 to 8.0 mg/dl, respectively (normal).Above these ranges, uric acid is considered elevated. *P < 0.05, **P < 0.01, t test.

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to H). Colons from S. cerevisiae–treated animals were shorter comparedto control groups, whereas colons from allopurinol-treated animals werelonger than those from animals that received S. cerevisiae alone (Fig.5F). Moreover, animals treated with allopurinol had less pathologywhen compared to S. cerevisiae–treated animals (Fig. 5, G and H).Together, these data demonstrate that certain members of the myco-biota can influence purine metabolism of the host and modulate dis-ease pathology.


DISCUSSIONDuring hominid evolution, the loss of urate oxidase (uricase) activityled to higher levels of uric acid found in the blood of humans com-pared to other vertebrate species (47). Although a certain level of uricacid is thought to be beneficial as an antioxidant, excessive uric acid isassociated with several disorders, including gout, vascular disease, andkidney stone formation. The increased incidence of developing uricacid nephrolithiasis during IBD is consistent with higher uric acid

levels in people with intestinal disorders.Allopurinol has been used in patientswith CD to increase the efficacy of theIBD medications 6-mercaptopurine andazathioprine (48), and many patientswho received adjunctive allopurinol ther-apy were reported to have major clinicalimprovement (48). Our results suggest thatsome of the improvement might comefrom preventing yeast-induced uric acidbuildup in the intestine. Thus, allopurinoltreatment in some IBD patients with ad-verse reactions to yeast and high uric acidmight be of therapeutic benefit and shouldbe explored.

The presence of ASCAs in CD pa-tients suggests fungal involvement, buthow these antibodies or the presence ofyeast functionally influence IBD remainsundefined. Using genetic and metabolicapproaches, we experimentally connectedthese two clinical phenotypes by showingthat a common intestinal commensal,S. cerevisiae, induced uric acid productionand caused worsened intestinal diseaseby increasing intestinal permeability. Astudy performed 45 years ago demon-strated that feeding yeast to healthy youngindividuals elevated serum uric acid, cor-roborating our metabolic profiling ex-periments in germ-free mice (49). Ourresults suggest that uric acid testing com-bined with ASCA testing in patients withCD would represent an easy clinical assayto better inform therapeutic interventionregimens.

The mechanism by which S. cerevisiaeincreases intestinal permeability and ex-acerbates disease remains unclear. Uricacid is a ligand of the NLRP3 inflamma-some that induces inflammatory mediatorssuch as IL-1b and IL-18 (46); therefore, wetested a role for this protein complex inincreased disease severity in responseto S. cerevisiae. However, we found thatS. cerevisiae induced similar colitis inNLRP3−/− animals, ruling out a role forNLRP3 in this setting. This finding is alsoconsistent with our data that enhancedimmune responses were not detected inanimals colonized with S. cerevisiae when

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Fig. 5. S. cerevisiae colonization causes worsened colitis by inducing production of uric acid. (A to D) C57Bl/6SPF mice were treated daily with uric acid 3 days before the induction of colitis with DSS. Eight days after colitisinduction, animals were analyzed for fecal uric acid levels (A), disease severity were analyzed by histology (B and C),and intestinal barrier penetration were analyzed by FITC-dextran (D). These data are representative of threeindependent experiments with five to seven animals in each group per experiment. (E to H) C57Bl/6 animals weretreated with S. cerevisiae (n = 4) as described in Fig. 1. Animals were also treated with allopurinol (n = 4) or vehiclecontrol (n = 2). Eight days after colitis, animals were analyzed for serum uric acid levels (E), colon length (F), andcolitis severity as determined by histology of H&E-stained colon sections (G and H). These data are representative of twoindependent experiments with five to seven mice per group in each experiment. *P < 0.05; **P < 0.01, ***P < 0.005, t test.

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compared to control animals. The observation that heat-killed S. cerevisiaedid not elicit worsened disease or elevate uric acid in mouse small in-testinal epithelial cell lines suggests that the yeast must be metabolical-ly active to induce this host response. Therefore, S. cerevisiae eitherproduces a metabolite or influences the expression or function of agene involved in purine metabolism. Future studies will be necessaryto identify this mechanism and can take advantage of all the genetictools available for this organism.

Patients with IBD are often treated with antibiotics to kill theresident bacteria that are eliciting chronic gut inflammation. Becausefungi are not affected by most antibiotics, a common side effect ofantibiotic therapy is an expansion of these organisms. Thus, the useof antibiotics in people with IBD might not only kill bacteria that arepromoting healthy immune responses but also create an environmentwith little competition and allow for overgrowth of fungal populations.Even after cessation of antibiotic therapy, environmental fungal spe-cies might continue to colonize the host in greater numbers and dis-rupt the normal microbiota from recolonizing to the same levels.Supporting this hypothesis, two studies have demonstrated that patientswith IBD have a greater diversity of fungi in their stool (8, 12). Al-though more studies are required, our findings suggest that anti-yeasttherapy or avoidance of foods with live yeast might be therapeuticallybeneficial for patients with IBD. Note that not all yeasts are capable ofexacerbating intestinal disease. Persistent exposure to R. aurantiaca, acommon environmental fungus, did not alter the course of IBD. In ad-dition, Saccharomycopsis fibuligera appeared to ameliorate intestinaldisease in a mouse model of IBD (29), and Saccharomyces boulardiiis often used as a probiotic, underscoring an important role for fungiin human health.

One limitation of our study is that numerous S. cerevisiae strainsfrom a multitude of environmental, industrial, and clinical sourcesare known to exist (50, 51). Although S. cerevisiae, like most yeastspecies, is generally considered nonpathogenic, it can cause diseasein immunocompromised individuals (52). In the current study, wechose to use an environmentally isolated diploid strain (RM11) torepresent how exogenous yeast, acquired or encountered throughdiet, can influence the course of IBD (53). Although vaginal S. cerevisiaehas been isolated and therefore could be passed vertically to offspring,S. cerevisiae has not been reported to date to be a common vaginalcommensal (54, 55). Therefore, it is likely that most of the S. cerevisiaefound in the GI tract is acquired initially from the environment,making use of an environmentally isolated strain appropriate. Furtherstudies will be needed to address how different strains of S. cerevisiaebehave in vivo. Investigating the transcriptional, proteomic, andmetabolomic profiles of S. cerevisiae as a member of a complex mi-crobiota, during both homeostatic and inflammatory states in theGI tract, will be instrumental in understanding its biology, its con-tribution to mammalian commensalism, and its ability to potenti-ate disease.

Because there are numerically more bacteria than fungi in the gut,it is likely that these organisms interact with one another. Consistentwith this, a recent study showed correlations between the abundanceof certain bacterial and fungal members of the human microbiota(12). Many bacterial and fungal species are able to degrade uric acid;thus, S. cerevisiae–induced uric acid production could influence thebiology of bacterial and other fungal members of the microbiota.However, little is known about these interkingdom interactions inthe gut, further highlighting the complexity of host-microbe symbiosisand its role in human health and disease.


MATERIALS AND METHODSStudy designThe aim of this study was to explore how commensal yeast speciesinfluence the course of murine experimental colitis. Preliminary exper-iments demonstrated that S. cerevisiae exacerbated colitis, as measuredby histology and colon length, in the absence of traditional inflamma-tory markers, that is, TH1 and TH17 cells and corresponding cytokines,as assayed by flow cytometry of MLNs and qRT-PCR from colonictissue. Microarray data of colonic RNA from germ-free mice monoas-sociated with S. cerevisiae or R. aurantiaca pointed to disrupted barrierintegrity and multiple host metabolic pathways being affected. This ledus to functionally investigate barrier disruption during experimental colitis,using FITC-dextran as a marker of intestinal permeability, as well as tocharacterize the metabolic profile of feces from animals that had beenmonoassociated with S. cerevisiae or R. aurantiaca, using GS-MS. Ourmetabolic profiling highlighted components of the purine degradationpathway, including uric acid. As ASCAs are used as a diagnostic markerof CD, we wanted to know whether there was a correlation betweenASCAs and uric acid in human patients. Human subjects were chosenat random, with male and female subjects represented equally. A blindedpathologist performed histology: Slides were assigned random numbersthat had no relevance to the experiment. After grading, histology scoreswere sent back to the investigator via email, and scores were decoded andassigned to their experimental group. Experimental replicates are cited inthe figure legends. A statistician was consulted before the study to deter-mine the minimum number of animals that would be required for a studybased on published literature using a power analysis. All experimentswere repeated two to four times as indicated in the figure legends.

AnimalsAll experiments were performed on age-matched, 8- to 10-week-oldmale mice on a C57Bl/6 background. C57Bl/6 (WT controls) andNLRP3−/− animals were purchased from the Jackson Laboratoryand housed under SPF conditions. C57Bl/6 germ-free mice were bredat the University of Utah, and sterility was checked by anaerobic andaerobic microbial plating and PCR. All animal use was in compliancewith federal regulations and guidelines set by the University of Utah’sInstitutional Animal Care and Use Committee (protocol #14-05009).

Yeast strainsS. cerevisiae used in all experiments was a prototrophic diploid strainon an RM11 background provided from the laboratory of D. Stillman.S. cerevisiae was grown in yeast peptone, adenine, and dextrose(YPAD) at 30°C. R. aurantiaca was obtained from the American TypeCulture Collection (ATCC 10655) and was grown in Sabouraud dextroseagar at room temperature. C. albicans was provided by the laboratory ofS. Nobel and was a 50:50 mixture ofWT:WOR1mutant grown in YPADat 30°C. Heat-killed S. cerevisiae (HKY) at a concentration of 107/mlwas incubated in a water bath (70°C) for 1 hour. HKY was struck outon YPAD agar plates and incubated for 48 hours at 30°C to verify nogrowth. HKY stocks were stored at −80°C.

MicroarraysRNA was collected from the indicated samples using a QiagenmiRNA RNA isolation kit. RNA was then provided to the Universityof Utah Microarray/High Throughput Genomics Core using the Agi-lent platform. RNA was analyzed by an Agilent Bioanalyzer, labeledwith modified nucleotide samples (Cy dyes or biotin), hybridized, andscanned using standard procedures.

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ASCA/uric acid quantification in human seraHuman serum samples were collected and processed at ARUP Labora-tories and approved under Institutional Review Board protocol #7740.Sera from deidentified females and males aged 20 to 66 years werecollected in plasma serum separator tubes. Patients were not currentlyon any therapies at the time of sample collection. Human serum wasanalyzed for the presence of ASCA IgG and IgA using a U.S. Food andDrug Administration–approved enzyme-linked immunosorbent assay(Inova Diagnostics). Uric acid from human serum was detected usingquantitative spectrometry.

Flow cytometryMLNs were gently pushed through a 40-mM filter to obtain lympho-cytes. Surface staining for lymphocytes was done in sterile 1× Hank’sbalanced salt solution (HBSS) (Corning) supplementedwith10mMHepes(Cellgro), 2mMEDTA(Cellgro), and0.5% (v/v) fetal bovine serum(FBS)(Gibco BRL) for 20 min at 4°C. Cells were then washed twice in supple-mented 1×HBSS and enumerated via flow cytometry. The following anti-bodies were used: anti-CD4 (FITC) and anti-CD3 (Pacific Blue).

For intracellular staining, cells were first stimulated with ionomycin(500 ng/ml), phorbol 12-myristate 13-acetate (5 ng/ml), and brefeldinA (5 mg/ml; BioLegend) for 4 hours at 37°C. Cells were surface-stained, washed, and then permeabilized and fixed in 100 ml of Perm/Fix buffer (eBioscience) overnight at 4°C. Cells were washed twice inPerm/Wash buffer (eBioscience) and then stained for intracellular cyto-kines with the following antibodies: anti–IFN-g (phycoerythrin) andanti–IL-17A (allophycocyanin). These data were collected with a BDLSRFortessa and analyzed with FlowJo software.

Quantitative PCRFor inflammatory cytokine and tight junction transcript analysis, a1-cm cut of colon tissue about 1 cm proximal to the cecum was washedin sterile PBS and placed in QIAzol (Qiagen) and stored at −20°C untilextraction. A standard phenol-chloroform extraction was performed toisolate RNA. RNA samples were deoxyribonuclease-treated (AMPD-1KT, Sigma-Aldrich) before reverse transcription (qScript, cDNASuperMix, Quanta Biosciences Inc.) following the manufacturer’sinstructions. qPCR was performed with a LightCycler LC480 instru-ment (Roche) under the following cycling parameters: for tight junc-tions, 45 cycles: denaturation for 15 s at 95°C, annealing for 1 min at60°C, and elongation for 1 min at 72°; for inflammatory cytokines,45 cycles: denaturation for 15 s at 95°C, annealing for 30 s at 60°C,and elongation for 30 s at 72°. Primer sets are shown in table S1.

TNBS colitisFor induction of TNBS colitis, C57Bl/6 mice were anesthetized withisoflurane, and 50:50 (v/v) ethanol (EtOH)/TNBS was administeredintrarectally. Mice were then held tail up for 30 s to allow the solutionto adsorb. Animals were weighed daily and sacrificed 5 days afterTNBS administration.

DSS colitisFor induction of DSS colitis, WT C57Bl/6 or NLRP3−/− mice wereprovided 2.5% DSS in drinking water for 8 days. Animals were sacri-ficed on day 8. DSS water was changed every other day.

Oral gavageBefore and during colitis, mice were supplemented with daily doses(~1.0 × 106 CFU/mouse per dose) of either S. cerevisiae or R. aurantiaca.


Uric acid [molecular weight (MW), 168.1, Sigma-Aldrich] was suspendedin sterile PBS at a concentration of 100 mg/ml. Before each mousewas orally gavaged, the suspension was shaken vigorously to obtain ahomogeneous mixture. One hundred microliters (10 mg) of the mix-ture was administered twice a day for 10 days. Allopurinol (10 mg/kg)(MW, 136.11; Sigma-Aldrich) was orally gavaged every other day dur-ing DSS colitis.

HistologyWhole colons were removed and washed in sterile PBS before beingfixed in 10% formalin. Colons were then paraffin-embedded, sec-tioned, and stained with H&E for pathology assessment. Colons werescored by a blinded pathologist using the following scoring system: 0,no crypt loss; 1 to 2, mild crypt loss; 3 to 4, medium crypt loss; and 5to 6, severe crypt loss. The amount of colon affected was also takeninto consideration: 1, 1 to 10%; 2, 10 to 20%; 3, 20 to 40%; 4, 40 to60%; and 5, >60%. The level of inflammation was also assessed: 1 to 2,mild; 3, medium; and 4, severe.

Metabolomic analysisMetabolite extraction from pellet.Mouse fecal pellets were extracted using a modified method derivedfrom Canelas et. al (56). To each cell pellet, we added 5 ml of boiling75% EtOH (aqueous), followed by vortex mixing, and then incubatedit at 90°C for 5 min. The EtOH solution contained a mixture of U-13C/U-15N stable isotope-labeled amino acids (CNLM-6696-1, CambridgeIsotope Laboratories) at 5 mg per sample, and d4-succinate was addedat 1 mg per sample. Cell debris was removed by centrifugation at 5000gfor 3 min. The supernatant was removed to new tubes and dried en vacuo.

GC-MS analysisAll GC-MS analysis was performed with a Waters GCT PremierMass Spectrometer fitted with an Agilent 6890 Gas Chromatographand a GERSTEL MPS2 Autosampler. Dried samples were suspendedin 100 ml of O-methoxylamine hydrochloride (40 mg/ml) in pyri-dine and incubated for 1 hour at 30°C. To the autosampler vials,we added 25 ml of this solution. Ten microliters of N-methyl-N-trimethylsilyltrifluoracetamide was added automatically via the auto-sampler and incubated for 60 min at 37°C with shaking. After incubation,3 ml of a fatty acid methyl ester standard solution was added via theautosampler, and 1 ml of the prepared sample was then injected to thegas chromatograph inlet in the split mode, with the inlet temperatureheld at 250°C. Two GC-MS runs were performed: one at a 10:1 splitratio to detect low-level metabolites and the second at a 100:1 splitratio to accurately measure high-concentration metabolites, which sat-urate the detector at the 10:1 split ratio. For the 10:1 split ratio anal-ysis, the gas chromatograph had an initial temperature of 95°C for1 min, followed by a 40°C/min ramp to 110°C, and a hold time of2 min. This was followed by a second 5°C/min ramp to 250°C, a thirdramp to 350°C, and then a final hold time of 3 min. For the 100:1 splitratio analysis, the gas chromatograph had an initial temperature of 95°C for 1 min, followed by a 40°C/min ramp to 110°C, and a hold timeof 2 min. This was followed by a second 25°C/min ramp to 330°C. A30-m Phenomenex ZB-5 MSi column with a 5-m-long guard columnwas used for chromatographic separation. Helium was used as thecarrier gas at 1 ml/min.

Below is a description of the two-step derivatization process usedto convert nonvolatile metabolites to a volatile form amenable to GC-MS. Pyruvic acid is used here as an example.

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O

OHO

MOX/pyridine, 30°C 1 hour

O

OHN

O

MSTFA, 37°C 1 hour

O

ON

O

Si


Analysis of GC-MS dataData were collected using MassLynx 4.1 software (Waters). A two-stepprocess was used for data analysis, targeted followed by nontargetedanalysis. For the targeted approach, known metabolites were identified,and their peak area was recorded using QuanLynx. Data were normal-ized to the internal standard d4-succinate, and the pellet was weighed.

For the nontargeted approach, peak picking and analysis were per-formed using MarkerLynx and the freely available online software www.metaboanalyst.ca. Chemical entities were identified using MarkerLynx,followed by the generation of an XML file. This file was formatted to anExcel format, and the peak areas were normalized to weight. These datawere analyzed in MetaboAnalyst version 2.0 (57). PCA and volcanoplots were generated.

Metabolite identity was established using a combination of an in-house metabolite library developed using pure purchased standardsand commercially available NIST library. When reporting each metab-olite, those with absolute identity are not qualified, whereas those thatare identified using the NIST library are noted using a percentage ofcertainty produced by the NIST software. Metabolites, which are com-pletely unknown, are labeled as unRTm/z, where un is the unknown,RT is the retention time, and m/z is the mass-to-charge ratio. An ex-ample of this is an unknown metabolite that elutes at 15.22 min andhas a characteristic mass of 247 would be written as un15.22_247. On-ly those unknowns that are statistically relevant were recorded.

FITC-dextran permeability assayFor assessment of intestinal permeability, animals were fasted for 4 hoursbefore receiving an oral gavage of 60 mg/100 g of FITC-dextran 4 (MW,3000 to 5000; Sigma-Aldrich). Animals were then fasted for another4 hours before sacrificing. Blood was taken via cardiac puncture andcollected in serum separator tubes. Blood was allowed to sit at roomtemperature for 30 min before being centrifuged at 1300g for 10 min.One hundred microliters of serum was then assayed for the presence ofFITC fluorophores using a microplate reader at 488-nm excitation and519-nm emission (Biotek Synergy H1).

Uric acid measurementSerum was collected as previously described. To remove interferingsubstances, we filtered serum in 10,000-MW filters and spun it at14,000g for 20 min. Filtered serum was then used in the QuantiChromUric Acid Assay Kit (DIUA-250, BioAssay Systems) or Amplex RedUric Acid/Uricase Assay Kit (Life Technologies) following the manu-facturer’s guidelines. For uric acid measurement in feces, mouse fecalpellets were weighed and then homogenized in 200 to 300 ml of sterilePBS. Feces were filtered as described above, and filtrates were used todetect uric acid levels using the same detection kit. Uric acid levelswere then normalized to fecal weight in grams.

In vitro experiments using mouse intestinal epithelial cells(MODE-K cells)Mouse intestinal epithelial cells were maintained in Dulbecco’s mod-ified Eagle’s medium (DMEM), with L-glutamine and sodium pyr-


uvate. DMEM was supplemented with 10% FBS, 1% (v/v) glutamine,penicillin-streptomycin, and 1% Hepes. For uric acid determination, aconfluent monolayer of cells was incubated with different MOIs for24 hours. Medium was then assayed for the presence of uric acid as pre-viously described. For Transwell experiments, FITC-dextran was addedto a confluent monolayer in the apical compartment for a final concen-tration of 25 mg/ml. After 24 hours, the medium in the basolateral com-partment was assayed for FITC fluorophores as previously described.

StatisticsStatistics were carried out using Prism 6.0 (GraphPad) and JMP9.0(SAS). Two-tailed unpaired Student’s t test was used for all pairwisestatistical comparisons unless otherwise noted. NonparametricMann-Whitney t test was used on human data. Error bars in all figuresrepresent means ± SEM. Before data analysis, outliers were identifiedand excluded using the ROUT method in Prism 6.0. Sample size, num-ber of replicates, and statistical test are reported in all figure captions.

SUPPLEMENTARY MATERIALSwww.sciencetranslationalmedicine.org/cgi/content/full/9/380/eaaf9044/DC1Fig. S1. S. cerevisiae does not induce greater inflammation.Fig. S2. S. cerevisiae leads to worsened intestinal barrier.Fig. S3. S. cerevisiae induces purine metabolism.Fig. S4. There is a positive correlation between elevated uric acid and ASCAs but not withanti-chromatin antibodies.Fig. S5. S. cerevisiae does not exacerbate disease through NLRP3.Table S1. Primer sets for qPCR.

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Acknowledgments: We thank members of the Round and O’Connell laboratories for theircritical review of the manuscript and ARUP Laboratories (specifically, the Autoimmune IIand Core laboratories) for the processing of clinical samples. Some of the germ-free mice usedin this publication were provided by the University of North Carolina’s Gnotobiotic Rodent


Core Facility, which is supported by grants 5-P39-DK034987 and 5-P40-OD010995.Funding: C.P. is supported by a T32 fellowship in microbial pathogenesis (AI-055434). R.S. issupported by a T32 genetics training grant (GM007464). R.M.O. is supported by the NIHNew Innovator Award (DP2GM111099-01), the National Heart, Lung, and Blood Institute(R00HL102228-05), an American Cancer Society Research Grant, and a Kimmel Scholars Award.This project was supported by the National Institute of Allergy and Infectious Disease(NIAID) (R21 AI109122), the Edward Mallinckrodt Jr. Foundation, Pew Scholars Program,NSF CAREER award (IOS-1253278), Packard Fellowships in Science and Engineering and NIAIDK22 (AI95375), and an NIH Innovator Award (DP2AT008746-01 to J.L.R.). Authorcontributions: J.L.R., C.P., and T.R.C. conceived the project. J.L.R., T.R.C., and R.M.O. designedthe experiments. T.R.C. performed most of the experiments. C.P. performed initial colitisexperiment. R.S. and J.C. performed the metabolomic analysis and analyzed the data with helpfrom J.L.R., W.Z.S., and J.L.K. W.Z.S. and J.L.K. helped with the statistical analysis. A.E.T. andJ.C.D. performed human serum studies. J.B. helped with yeast assays. D.J.S. and W.V. providedyeast strains and advice for yeast in vitro work. J.L.R. and T.R.C. analyzed all data and wrotethe manuscript. L.G. assisted with in vitro assays. R.B. maintained gnotobiotic facility andanimals for all germ-free experiments. All authors read and edited the manuscript. Competinginterests: The authors declare that they have no competing interests.

Submitted 2 September 2015Resubmitted 17 April 2016Accepted 10 February 2017Published 8 March 201710.1126/scitranslmed.aaf9044

Citation: T. R. Chiaro, R. Soto, W. Z. Stephens, J. L. Kubinak, C. Petersen, L. Gogokhia, R. Bell,J. C. Delgado, J. Cox, W. Voth, J. Brown, D. J. Stillman, R. M. O’Connell, A. E. Tebo, J. L. Round, Amember of the gut mycobiota modulates host purine metabolism exacerbating colitis in mice.Sci. Transl. Med. 9, eaaf9044 (2017).

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