Dietary and Microbial Oxazoles Induce Intestinal Inflammation by Modulating Aryl Hydrocarbon Receptor Responses

Article

Dietary and Microbial Oxazoles Induce Intestinal

Inﬂammation by Modulating Aryl Hydrocarbon

Receptor Responses

Graphical Abstract

Authors

Shankar S. Iyer, Thomas Gensollen,

Amit Gandhi, ..., Anthony Maxwell,

Amadeu Llebaria, Richard S. Blumberg

Oxazoles

(environment, diet, microbes)

INTESTINAL EPITHELIAL CELL

Correspondence

APICAL

rblumberg@bwh.harvard.edu

In Brief

Tryptophan

A class of microbial and environmental

compounds triggers inﬂammation in gut

epithelial cells through the action of

natural killer T cells and aryl hydrocarbon

receptor signaling.

IDO1

AHR

AHR ligands

ER

BASOLATERAL

MUCOSAL BARRIER IL-10

PROTECTION

IL-13/IFN-

NKT

PATHOGENESIS

Highlights

d

Oxazoles derived from diet, industrial sources, and microbes

activate IDO1

Environmental oxazoles induce tryptophan-derived

metabolites to activate AhR in IECs

AhR activation in IECs limits CD1d-restricted production of

IL-10

Oxazole activation of AhR in IECs results in iNKT-mediated

intestinal inﬂammation

Iyer et al., 2018, Cell 173, 1123–1134

May 17, 2018 Crown Copyright ª 2018 Published by Elsevier Inc.

https://doi.org/10.1016/j.cell.2018.04.037

Article

Dietary and Microbial Oxazoles Induce

Intestinal Inﬂammation by Modulating

Aryl Hydrocarbon Receptor Responses

Shankar S. Iyer,¹Thomas Gensollen,¹Amit Gandhi,¹Sungwhan F. Oh,¹Joana F. Neves,¹Frederic Collin,²Richard Lavin,³

Carme Serra,⁴Jonathan Glickman,⁵Punyanganie S.A. de Silva,¹R. Balfour Sartor,⁶Gurdyal Besra,⁷Russell Hauser,⁸

Anthony Maxwell,⁹Amadeu Llebaria,⁴and Richard S. Blumberg^1,10,

*

¹Division of Gastroenterology, Hepatology, and Endoscopy, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical

School, Boston, MA, USA

²Inspiralis, Innovation Centre, Norwich Research Park, Colney Lane, Norwich NR4 7GJ, UK

³Center for Clinical and Translational Metagenomics, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School,

Boston, MA, USA

⁴Laboratory of Medicinal Chemistry, Department of Biomedicinal Chemistry, Institute of Advanced Chemistry of Catalonia (IQAC–CSIC),

Barcelona, Spain

⁵Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA, USA

⁶Departments of Medicine, and Microbiology and Immunology, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill,

NC, USA

⁷School of Biosciences, University of Birmingham, Birmingham, UK

⁸Department of Environmental Health, Harvard School of Public Health, Boston, MA, USA

⁹Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK

¹⁰Lead Contact

*Correspondence: rblumberg@bwh.harvard.edu

https://doi.org/10.1016/j.cell.2018.04.037

SUMMARY

INTRODUCTION

Genome-wide association studies have identiﬁed Inﬂammatory bowel disease (IBD) is a complex disorder that

evolves from the interactions between poorly understood envi-

ronmental factors and a host’s genetic framework that together

risk loci associated with the development of in-

ﬂammatory bowel disease, while epidemiological

studies have emphasized that pathogenesis likely

involves host interactions with environmental ele-

ments whose source and structure need to be

deﬁned. Here, we identify a class of compounds

derived from dietary, microbial, and industrial sour-

deﬁne susceptibility to and severity of disease. Pathology is

inﬂuenced by speciﬁc host elements that include the autochtho-

nous commensal microbiota, which is acquired at birth, the in-

testinal epithelial cell (IEC) barrier, and subjacent immune cells

within the intestinal mucosa (Kaser et al., 2010). One of the great

challenges of understanding IBD pathogenesis stems from ef-

ces that are characterized by the presence of a

ﬁve-membered oxazole ring and induce CD1d-

dependent intestinal inﬂammation. We observe

forts to elucidate the molecular details surrounding the environ-

mental basis for these disorders. This is increasingly important,

since epidemiologic studies have revealed a rapid global expan-

that minimal oxazole structures modulate natural sion of these diseases that includes geographic regions, which

have heretofore been unaffected (Molodecky and Kaplan 2010).

A potential opportunity to investigate this question has

killer T cell-dependent inﬂammation by regulating

lipid antigen presentation by CD1d on intestinal

epithelial cells (IECs). CD1d-restricted produc-

tion of interleukin 10 by IECs is limited through

activity of the aryl hydrocarbon receptor (AhR)

pathway in response to oxazole induction of

tryptophan metabolites. As such, the depletion of

emerged from recent studies on the role of CD1d and natural killer

T (NKT) cells (NKT) in mucosal biology. CD1d is a major histocom-

patibility complex (MHC) class I-related molecule that presents

cell-associated and microbial lipid antigens to NKT cells (Brennan

et al., 2013). In the intestines, CD1d is expressed by IECs and he-

matopoietic cells and presents endogenous (self) or exogenous

the AhR in the intestinal epithelium abrogates oxa-

zole-induced inﬂammation. In summary, we iden-

tify environmentally derived oxazoles as triggers

lipid antigens to NKT cells expressing an invariant T cell receptor

(TCR) a chain (iNKT cells) or a semi-diverse (d)set ofTCR-a chains

(dNKT), which are present in human and mouse intestines (Ross-

of CD1d-dependent intestinal inﬂammatory res- john et al., 2012).

Experimental models using chemical induction of colitis using

a classic hapten, oxazolone (Wirtz et al., 2007), implicate a role

ponses that occur via activation of the AhR in the

intestinal epithelium.

Cell 173, 1123–1134, May 17, 2018 Crown Copyright ª 2018 Published by Elsevier Inc. 1123

for NKT cells in the pathogenesis of IBD and have been corrobo- much larger collection of environmental chemical moieties

rated in some patient studies (Fuss et al., 2004). Boirevant and capable of triggering CD1d-restricted iNKT cell responses.

colleagues ﬁrst demonstrated that administration of oxazolone Consistent with this, we have identiﬁed environmental mimetics

directly to the colon in ethanol of SJL/J mice, or in later studies of oxazolone that are derived from dietary, industrial, or microbial

indirectly after skin painting and sensitization, suggesting a sources that are characterized by the presence of a ﬁve-

model of haptenization, resulted in a severe acute, superﬁcial membered oxazole ring and capable of driving CD1d-dependent

inﬂammation due to the production of interleukin (IL)-4 and IL- inﬂammation through activation of the aryl hydrocarbon receptor

13 that was counterbalanced by tumor growth factor b, which de- (AhR) pathway within IECs of the colon. As efforts to deﬁne and

limited the inﬂammation to the distal colon (Boirivant et al., 1998). evaluate natural and synthetic chemicals involved in homeosta-

This restriction to type 2 cytokines was eventually recognized to sis or disease have generally been elusive, these results have

be genetically based as similar studies in C57BL/6 mice revealed broad implications for understanding the environmental basis

inﬂammation in association with oxazolone that was due to type 1 of mucosal diseases, such as IBD.

cytokines (namely interferon g, IFN-g) derived from hematopoiet-

ic cells (Iijima et al., 2004). In later groundbreaking studies by RESULTS

Heller and colleagues, it was recognized that oxazolone-induced

colitis was dependent upon CD1d and iNKT cells, as an inﬂam- Deﬁning a Structural Moiety that Modulates Epithelial-

matory response to oxazolone was abrogated by the deletion Derived CD1d-Dependent Inﬂammatory Responses

of Cd1d or Ja18, encoding the invariant TCR-a chain (Heller 4-ethoxymethylene-2-phenyl-2-oxazol-5-one (referred to as

et al., 2002) primarily derived from the activity of professional an- oxazolone; Figure 1A) has been widely utilized in models of con-

tigen-presenting cells (APCs) in the lamina propria using bone tact hypersensitivity and, when applied to the mucosa, has been

marrow chimeras (Olszak et al., 2014). In contrast, CD1d-ex- proposed to cause colitis through its properties as a hapten

pressing IECs were shown to secrete anti-inﬂammatory IL-10 in (Gorbachev and Fairchild 2001). However, to date, there is

response to iNKT cells in a CD1d-dependent manner, which limited evidence for hapten-speciﬁc antibody production or

serves to restrain oxazolone-induced inﬂammation derived responses following topical oxazolone sensitization or hapten-

from the hematopoietic system (Colgan et al., 1999; Olszak modiﬁed autologous proteins or luminal antigens identiﬁed at

et al., 2014). Thus, when CD1d-restricted pathways in the IEC mucosal sites (Singleton et al., 2016; Wirtz et al., 2007). Interest-

are speciﬁcally eliminated by conditional genetic deletion of ingly, upon analysis of previously published microarray studies

Cd1d, microsomal triglyceride transfer protein (Mttp), an endo- derived from epithelial-enriched colon fractions following intra-

plasmic reticulum resident protein that serves to regulate CD1d rectal oxazolone challenge (Olszak et al., 2014), we observed

lipidation and its ability to function, or Il10, oxazolone-induced decreased expression of Mttp/MTP and increased expression

colitis is unrestrained and severe (Dougan et al., 2005, 2007; for elements associated with AhR signaling, including P450

Sagiv et al., 2007; Olszak et al., 2014; Brozovic et al., 2004). Hu- enzymes (cyp1a1) and indoleamine 2,3-dioxygenase (Ido1). We

man studies have further identiﬁed a potential role for dNKT cells therefore asked whether oxazolone could affect the expression

in IBD by showing elevated IL-13 production by lamina propria of these transcripts in the intestinal epithelium in a cell-intrinsic

mononuclear cells in response to sulfatide, a CD1d-restricted manner by directly stimulating an immortalized IECs derived

self-antigen that can activate iNKT and dNKT cells (Fuss et al., from mouse small intestine, MODE-K, with oxazolone. We

2004, 2014). Further, TCR transgenic mice expressing a non- observed downregulation of Mttp and induction of metabolic

invariant CD1d-restricted TCR develop colitis (Wang et al., genes cyp1a1 and Ido1 (Figures 1B and S1A).

2018), though their exact role during the pathogenesis of IBD re-

mains unknown (Grose et al., 2007; van der Vliet et al., 2001).

MTP is a critical regulator of mucosal homeostasis that pro-

motes epithelial barrier activity by controlling CD1d-restricted

Importantly, host inﬂammatory responses to oxazolone occur IL-10 production by epithelial cells (Colgan et al., 1999; Olszak

even in animals raised under germ-free (GF) conditions, indi- et al., 2014). Thus, IEC-speciﬁc deletion of Mttp results in

cating that NKT-cell-mediated response to self (rather than decreased CD1d-stimulated IL-10 production by the IEC, lead-

microbial)-lipid antigens presented by CD1d on professional ing to the exaggerated inﬂammatory activity of hematopoietic

and/or non-professional APCs is sufﬁcient for pathogenesis cells in response to oxazolone challenge, resulting in increased

(Olszak et al., 2012). Instead, commensal bacteria themselves colitis (Olszak et al., 2014). We modeled interactions between

can modulate the magnitude of NKT cell responses either by NKT cells and IECs using MODE-K cells and CD1d-restricted

directly altering the CD1d-restricted lipid antigen reservoir (An T cell hybridomas in a previously established CD1d-dependent

et al., 2014, Wieland Brown et al., 2013) or indirectly by regu- co-culture system (van de Wal et al., 2003). These studies have

lating the quantity of iNKT cells in the colon (Olszak et al., shown that loading of CD1d with a-galactosyl ceramide (GC), a

2012). Conversely, CD1d itself plays a critical role in regulating cognate lipid antigen, in this co-culture system leads to MTP-

the composition and extent of colonization by both commensal dependent, CD1d-restricted IL-10 production that is primarily

and pathogenic microbial species potentially through disruption derived from the IEC (Colgan et al., 1999; Olszak et al., 2014).

of the antimicrobial activity of Paneth cells (Nieuwenhuis et al., Here, we found that MODE-K cells pre-conditioned with oxazo-

2002, 2009).

lone exhibited attenuated IL-10 production in response to a-GC

The similarities between these biological observations in when placed in co-culture with an iNKT cell hybridoma (24.7),

mouse models and current hypotheses for IBD pathogenesis which speciﬁcally reacts to a-GC presented on CD1d (Figure 1C).

lead us to hypothesize that oxazolone may be an example of a Interestingly, epithelial-derived IL-10 was also suppressed when

1124 Cell 173, 1123–1134, May 17, 2018

A

B

C

D

E

G

H

F

Figure 1. A Minimal Oxazole Structure Modulates Expression of Speciﬁc Gene Targets and Attenuates CD1d Restricted in Intestinal

Epithelial Cells, Leading to Colonic Inﬂammation

(A) Panel of oxazole-containing and control heterocylic natural and synthetic compounds.

(B) Relative transcript abundance in MODE-K cells stimulated with indicated compounds, normalized to b-actin. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s

t test).

(C) IL-10 production in MODE-K cells conditioned with the indicated compounds, loaded with a-GC, followed by 24.7 iNKT hybridoma co-culture. ***p < 0.001

(Student’s t test).

(D and E) Representative traces and quantiﬁcation of surface CD1d (D) loaded with a-GC (E) in MODE-K cells conditioned with indicated compounds.

(F) Weight change after intra-rectal administration of 1% TMO or EtOH (50% v/v) vehicle in WT or CD1d-deﬁcient (KO) animals (n = 16–20). *p < 0.05 (Mann-

Whitney U test)

(G) Quantitative scoring for colitis after intra-rectal administration of 1% TMO or ethanol (EtOH; 50% v/v) vehicle in WT or CD1d-deﬁcient (KO) animals (n = 8).

(H) Quantiﬁcation (ELISA) of Il-13 and IFN-g production from colon explants 2 days after intra-rectal administration of 1% TMO, TMC, or EtOH (50% v/v) vehicle

(n = 3). ***p < 0.001 (Student’s t test).

See also Figures S1 and S2 and Table S1.

IECs stimulated with oxazolone were co-cultured with an autor- shown), which was not affected by oxazolone or any of the oxa-

eactive iNKT cell hybridoma (24.8) or an autoreactive non- zolone-related compounds described below.

invariant dNKT cell hybridoma (14S.6) in the absence of exoge-

In light of the decreased MTP expression observed in oxazo-

nously administered a-GC (Figures S1B and S1C). These effects lone-treated IECs (Figure 1B) and given that MTP loss alters

were speciﬁc to the IEC, as co-culture of bone-marrow-derived the ability of CD1d to acquire exogenous antigens, such as

dendritic cells with the 24.7 iNKT cell hybridoma and a-GC lead a-GC, and CD1d-elicited IL-10 responses by epithelial cells

to production of IFN-g (Figure S1D), but not IL-10 (data not (Dougan et al., 2005, 2007; Sagiv et al., 2007; Brozovic et al.,

Cell 173, 1123–1134, May 17, 2018 1125

2004; Olszak et al., 2014), we investigated CD1d expression and induced cyp1a1 and Ido1 transcripts in IECs (Figures 1B and

lipid loading on oxazolone-conditioned IECs. IECs stimulated S1A). In contrast, two structurally related non-OxC heterocyclic

with oxazolone did not display altered CD1d protein expression aromatic compounds—1,2,4-trimethylcyclopentane (TMC;

or distribution between intracellular and cell surface pools (Fig- MCS = 0.5) and 2-methyl-1-pyrroline (1-MP; MCS = 0.83)—that

ures S2A and S2B) but instead exhibited decreased loading substitute carbons at the 1’-oxygen and/or 3⁰-nitrogen did not

of a-GC on CD1d at the cell surface (Figures 1D and 1E). These elicit any transcriptional changes in the target genes assessed

results were consistent with the decreased CD1d-restricted (Figure 1B). Moreover, IECs conditioned with oxazolone or

IL-10 protein production we observed in MODE-K:iNKT cell TMO, but not with TMC or 1-MP, lead to decreased loading of

co-cultures (Figures 1C, S1B, and S1C) and suggest that oxazo- a-GC on CD1d at the cell surface, thereby limiting CD1d-

lone-induced alterations in MTP expression were associated restricted IL-10 production in co-cultures with an iNKT

with decreased CD1d function, affecting IL-10 production in hybridoma (24.7) (Figures 1C–1E). Overall, ﬁve of seven commer-

response to iNKT cells.

cially available naturally occurring dietary OxCs, as well as vinclo-

Oxazolone is administered in experimental models of colitis zolin (Figure 1A and Table S1), an OxC fungicide used in agricul-

and contact sensitivity but is also widely used in industry and ture, phenocopied the transcriptional and CD1d-restricted

pharmaceuticals as a pharmacophore for the generation of syn- epithelial responses observed after oxazolone stimulation.

thetic compounds, suggesting it may act as a potential environ-

mental trigger of inﬂammation in sensitive hosts (Turchi, 2008). A minimal Oxazole Structure Induces Colonic

We therefore sought to identify a library of structural mimetics Inﬂammation in a CD1d-Dependent Manner

of oxazolone and test whether these molecules behaved in a We next asked if a dietary-sourced minimal oxazole structure

similar manner. We performed a two-part in silico screen against was sufﬁcient to induce intestinal inﬂammation in vivo. Indeed,

three chemical databases of naturally occurring compounds (the intra-rectal administration of TMO was observed to lead to

Human Metabolome Database, the Super Natural II database, increased weight loss and pathology characterized by superﬁcial

Dictionary of Natural Products) (Banerjee et al., 2015; Wishart inﬂammation of the gut wall, neutrophil accumulation, and ulcer-

et al., 2013), thereby limiting our search to compounds within a ation of the epithelial layer of the colon that was similar to the

deﬁned human enviroment. Natural compounds were queried inﬂammation associated with oxazolone (Figures 1F, 1G, and

in the standard simpliﬁed molecular input line entry system S3A). Response to TMO, but not to TMC or vehicle, was charac-

(SMILES) format, and Tanimoto similarities between oxazolone terized by production of Th2 and Th1 cytokines, including IL-13

and test compounds were computed with the ChemMine Simi- and IFN-g from colon explants (Figure 1H). Importantly, intestinal

larity Workbench, which calculated maximum common sub- inﬂammation after TMO challenge was dependent on the pres-

structure (MCS) similarities with the Tanimoto coefﬁcient. ence of iNKT cells, as CD1d-deﬁcient animals displayed no evi-

Further, Euclidean distance scores were calculated using a dence of clinical symptoms or pathology (Figures 1F and 1G), as

non-continuous atom-matching structural similarity function previously shown for oxazolone (Heller et al., 2002). In addition,

(NAMS) (Cao et al., 2008; Teixeira and Falcao, 2013). An initial host in vivo IEC responses to TMO, but not to TMC or vehicle,

screen identiﬁed 33 compounds with MCS > 0.5 and Euclidean recapitulated the transcriptional changes observed in microarray

distance scores cutoff of 0.9 (Table S1). Oxazolone consists of and in vitro analyses, including downregulation of Mttp and in-

four major functional moieties: a ﬁve-membered oxazole ring, a duction of cyp1a1 (Figures S3B and S3C). Importantly, these

2⁰phenylgroup, a 4’ ethoxymethylene substituent, and the studies were performed by direct administration of TMO to the

5⁰-carbonyl lactone. Our computational screen revealed the ox- colonic epithelium by rectal challenge without preceding skin

azole ring to be a distinctive and shared chemical substructure sensitization, further suggesting that the OxC-induced effects

between the 33 compounds identiﬁed, all of which derived observed were direct and did not require prior immune activa-

from dietary, microbial, and/or industrial sources. We then tion, as would be expected from a hapten-induced model. To

employed a second screen using the same parameters but conﬁrm this, we compared the ability of oxazolone skin pre-

instead substituting a minimal oxazole ring as our template sensitization to affect the responses to rectal challenge with

structure. The analysis led to the identiﬁcation of 63 naturally TMO and vice versa (Figure S3D). These studies showed that

occurring oxazole-containing compounds (OxCs; including the pre-sensitization with oxazolone or TMO did not affect rectal

33 described above). Of particular interest, we noticed that responses to TMO or oxazolone, respectively; corroborating

many of these compounds derived from two major sources: evidence that oxazolone or TMO was having direct effects on

diet and microbes. This led us to ask whether oxazolone is a mo- the colonic mucosa.

lecular prototype for a broader class of environmental stimuli that

feature an oxazole substructure capable of modulating epithelial A Microbial-Derived Oxazole Structure Modulates

CD1d-restricted responses in a cell-intrinsic manner.

Epithelial Responses and Leads to Intestinal

To test this, we devised a systematic in vitro system to assess Inﬂammation

transcriptional and CD1d-restricted responses in IECs in Our in silico analysis also identiﬁed a class of bacterial-derived

response to stimulation with dietary OxC structures, including toxins, featuring oxazole moieties with a high degree of similarity

2,4,5-trimethyl-2,5-dihydro-1,3-oxazole (referred to as TMO; Fig- to a minimal oxazole template. These are termed thiazole-/oxa-

ure 1A)—found in coffee, peanuts, and meats—that lacks the zole-modiﬁed microcins (TOMMs) and make up a family of

2-phenyl ring, 5-ketone, and 4-ethoxymethylene present in oxa- antimicrobial peptides featuring the presence of thiazol(in)e

zolone (Maga, 1978). Like oxazolone, TMO suppressed Mttp and and oxazol(in)e heterocycles derived from cysteine, serine, and

1126 Cell 173, 1123–1134, May 17, 2018

Figure 2. Microbial-Derived Oxazoles Modulate CD1d-Restricted Responses and Regulate Transcriptional Targets in Intestinal Epithe-

lial Cells

(A and B) IL-10 response in MODE-K cells pre-conditioned with vehicle (DMSO) or indicated compounds—(A) lysates from bacterial transformants (empty/

pUC19, B17) grown in Luria broth or M63 supplement and (B) puriﬁed MccB17 (B17) microcin and/or B17 microcin proteolytic digest (subtilisin, trypsin)—and

subsequently loaded with a-GC followed by 24.7 iNKT hybridoma co-culture. *p < 0.05, **p < 0.01 (Student’s t test).

(C) Relative transcript abundance of target genes, Mttp and Ido1, in MODE-K cells to b-actin in MODE-K pre-conditioned as in (B). **p < 0.01, ***p < 0.001

(Student’s t test).

(D) Panel of synthetic analogs of B17 microcin proteolysis products with indicated anti-microbial activity.

(E) IL-10 response in MODE-K cells pre-conditioned with vehicle, oxazolone, or synthetic MccB17 microcin analogs and subsequently loaded with a-GC followed

by 24.7 iNKT hybridoma co-culture. n.s., not signiﬁcant; ***p < 0.001 (Student’s t test).

(F) Relative transcript abundance of target genes in MODE-K cells to b-actin in MODE-K pre-conditioned as in (E). n.s., not signiﬁcant; ***p < 0.001 (Student’s

t test).

threonine residues on a ribosomally produced precursor peptide from strains of E. coli from the intestinal tract of newborns whose

that confer a range of antimicrobial activities, including DNA gyr- expression is linked to a plasmid (pMccB17) that carries the mcb

ase inhibitors, translation inhibitors, and hemolytic toxins (Table operon, encoding the MccB17 toxin that acts as a DNA gyrase

S1) (Melby et al., 2011; Collin et al., 2013; Lee et al., 2008). inhibitor (Table S1) (Li et al., 1996). The mcb operon was cloned

Though chemically and functionally diverse, as a rule, TOMM into a pUC19 vector and transformed into a competent E. coli

biosynthetic gene clusters (BGCs) encode a leader peptide, BSL1 human commensal strain (MG1655) (Collin et al., 2013).

enzymatic machinery including a cyclodehydratase and cyclo- Bacteria transformed with mcb17 or empty plasmid was grown

dehydrogenase, that mediate post-translational installation of in Luria broth or M63 supplemented media to induce MccB17

oxazole/thiazole moieties required for biological activity and, synthesis (Collin et al., 2013), and bacterial lysates were incu-

in some cases, an immunity gene that protects the host bated with IECs, after which we measured CD1d-restricted

strain from the antimicrobial activities of the TOMM itself IL-10 production. We observed that lysates from bacterial

(Melby et al., 2011). Importantly, many TOMM BGCs have isolates transformed with mcb and grown under MccB17

been described in a variety of human commensal communities permissive conditions attenuated CD1d-restricted IL-10 produc-

as a means of providing host strains with ecological ﬁtness ad- tion in MODE-K:iNKT cell co-cultures, whereas lysates isolated

vantages (Lee et al., 2008). Within the commensal populations, from bacteria grown under non-permissive conditions or trans-

these BGC-encoded small-molecule bacterial signaling factors formed with empty plasmid had no effect (Figure 2A). To further

collectively have an enormous potential to mediate microbe- examine whether suppression of CD1d-restricted responses

microbe interactions (Lee et al., 2008; Mazmanian et al., 2008; was due to a MccB17 product, we puriﬁed full-length mature

Sassone-Corsi et al., 2016). Therefore, we considered if micro- MccB17 by high-performance liquid chromatography (HPLC).

cins also regulated microbe-host interactions by modulating To our surprise, puriﬁed MccB17 exerted no effects on

epithelial-derived CD1d immune responses in the host, similar CD1d-dependent IL-10 production (Figure 2B). Interestingly,

to other OxCs. To address this question, we focused our atten- although the full-length MccB17 microcin consists of

tion on microcin B17 (MccB17, MCS = 0.75), originally isolated 3,093-Da peptide, several smaller heterocyclic mcb17-derived

a

Cell 173, 1123–1134, May 17, 2018 1127

heterocyclic species, some of which retain DNA gyrase inhibitory 2013), to formally distinguish whether the intestinal inﬂammation

activity, have been identiﬁed by matrix-assisted laser desorp- observed was due to its antimicrobial properties or its direct ef-

tion/ionization time of ﬂight (MALDI-ToF) mass spectrometry fects on the host, we challenged GF and speciﬁc-pathogen-

(MS) analysis. We hypothesized that the immunomodulatory ef- free (SPF) mice with frag-oz or vehicle. Consistent with the

fects of the MccB17-positive lysate might be a result of proteo- former, GF animals exhibited increased weight loss compared

lytic and/or degradative products rather than the mature full- to animals raised under SPF conditions, together with severe

length MccB17 itself (Collin et al., 2013). B17 microcin is sensi- inﬂammation as deﬁned by quantitative histopathology (Figures

tive to cleavage by subtilisin, but not by trypsin (Asensio and 3C and 3F). These studies thus identify an oxazole-containing mi-

´

Perez-Dı

´az,

1976). Therefore, we digested puriﬁed full-length crobial mimic that promotes CD1d-dependent intestinal inﬂam-

B17 with each protease and measured CD1d-restricted IL-10 mation independent of its classical antimicrobial activity.

responses in the IEC:iNKT cell co-culture assay to these proteo-

lytic products (Figure 2B). In this case, cleavage products from Activation of Epithelial AhR Pathways Exacerbates

subtilisin, but not from trypsin, were able to attenuate IL-10 pro- Response to Dietary and Microbial Oxazole Compounds

duction in IEC:iNKT co-cultures with a-GC. Similarly, we by Limiting IL-10 Responses

observed decreased Mttp and increased Cyp1a1 transcripts in The data presented support a model in which exposure to a

MODE-K cells incubated with MccB17 subtilisin proteolytic broad class of oxazole (or thiazole) ring-containing compounds

products, but not with full-length B17 or MccB17 digested with can direct transcriptional changes in responsive tissue compart-

trypsin (Figure 2C).

ments, such as the intestinal epithelium, that inﬂuence CD1d-

Taken together, these results suggested that the ability of restricted antigen presentation pathways, thereby augmenting

MccB17-derived toxins to affect epithelial transcriptional and iNKT (or non-invariant NKT) cell inﬂammatory responses, leading

CD1d-restricted responses is limited by the size of the product. to colitis. These data also suggest the presence of cellular sen-

To clarify this size restriction, we synthesized and screened a sor(s) that are responsible for recognizing and transducing oxa-

library of eight MccB17-derivative products with molecular zole-dependent signals. Review of our previous microarray ana-

weights (MWs) ranging from 183.17–857.82 Da whose anti- lyses identiﬁed two signature AhR targets, cyp1a1 and Ido1,

DNA gyrase activities have been previously described (Figure 2D) involved in P450 and tryptophan metabolism, respectively, as

(Collin et al., 2013). Consistent with our hypothesis, four frag- inducible gene targets in the colonic epithelium (Olszak et al.,

ments, containing permutations of one or two oxazole/thiazole 2014). Here, we demonstrate that oxazolone, vinclozolin, an in-

moieties, with MW < 270 Da were able to modulate CD1d- dustrial agent, and the dietary and microbial oxazole ligands,

dependent IL-10 production and regulate Mttp and Cyp1a1, TMO and frag-oz, induced expression of these genes in vitro

whereas synthetic products > 500Da were not (Figures 2E and and in vivo. Interestingly, vinclozolin, an antifungal agent to which

2F and data not shown). Importantly, synthetic analogs capable humans are exposed through agriculture and which affects

of exerting immunomodulatory effects were within the relative CD1d-restricted pathways as an OxC as shown here, has been

size range of both oxazolone (217.22 Da) and 2,4,5-trimethyl- described as an AhR activator (Wambaugh et al., 2014).

2,5,-dihydro-1,3-oxazole (113.16 Da), but surprisingly, even syn-

We therefore transfected IECs with an AhR ﬁreﬂy luciferase

thetic products that did not confer anti-DNA gyrase activity were reporter and observed that oxazolone, TMO, and ﬁve additional

able to generate epithelial responses (Figures 2D–2F). Further- dietary oxazoles, as well as four synthetic MccB17 derivatives,

more, both oxazole- and thiazole-containing compounds gener- induced robust AhR transcriptional activity compared to vehicle

ated similar effects, consistent with their shared physico-chem- or a non-oxazole heterocyclic control, TMC (Figure 4A). Consis-

ical properties (Figure 2D–2F).

tent with this, small interfering RNA (siRNA)-mediated knock-

down of AhR in MODE-K cells abrogated induction of cyp1a1

and IDO1 and partially restored Mttp expression (Figures S4A–

S4C). Given that AhR regulates MTP expression, we asked

whether this pathway could modulate epithelial-derived CD1d-

A Synthetic B17 Microcin Derivative Induces Colonic

Inﬂammation in a CD1d-Dependent Manner

Independent of Antimicrobial Activity

We next asked whether B17 microcin derivatives could induce in- restricted responses. Indeed, AhR deﬁciency caused by

testinal inﬂammation in vivo. We therefore synthesized adequate silencing AhR expression in epithelial cells reversed oxazo-

quantities of a minimal B17 derivative (termed frag-oz; Figure 2D) lone-, TMO-, or frag-oz-mediated inhibition of CD1d-restricted

to perform in vivo experiments and challenged animals via intra- IL-10 production in IEC:iNKT cell co-cultures via reduction of

rectal administration. Like with oxazolone and TMO, we cell surface CD1d lipid antigen loading (Figures 4B and 4C).

observed CD1d-dependent pathology, as well as induction of This was conﬁrmed in hepatocytes obtained from Ahr^ꢀ/ꢀmice,

both Th1 and Th2 cytokines from colon explants, suggesting which also exhibited resistance to the effects of oxazolone-,

that this molecule induces inﬂammation in an iNKT-dependent TMO-, frag-oz-, or vinclozolin-induced inhibition of IL-10 pro-

manner by comparing response in wild-type (WT) versus duction in response to iNKT cells in the presence of a-GC (Fig-

CD1d-deﬁcient animals (CD1d knockout, KO) (Figures 3A–3C ure S5). Interestingly, these effects were not attributed to gen-

and S3A). We also observed decreased Mttp levels and induction eral AhR function but instead reﬂected a speciﬁc response to

of Cyp1a1 in IEC-enriched colonic fractions, suggesting that oxazole-containing molecules, as a human cognate AhR ligand,

frag-oz exerts immunomodulatory effects via the epithelial 2-(1H-Indol-3-carbonyl)-4-thiazolecarboxylic acid methyl ester

compartment in vivo (Figures 3D and 3E). Though in vitro studies (ITE), did not modulate Mttp expression or CD1d-restricted re-

showed that frag-oz confers no anti-gyrase activity (Collin et al., sponses (Figures 4B and 4C).

1128 Cell 173, 1123–1134, May 17, 2018

A

B

C

F

D

E

Figure 3. NKT Cells Are Required for Colonic Inﬂammation Induced by Frag-oz

(A) Quantitative scoring for colitis after intra-rectal administration of 1% TMO or EtOH (50% v/v) vehicle in WT or CD1d-deﬁcient (KO) littermates (n = 8). **p < 0.01

(Student’s t test).

(B) Quantiﬁcation of IL-13 and IFN-g production in overnatant of colon explants 2 days after intra-rectal administration of 1% TMO or EtOH (50% v/v) vehicle

(n = 6). **p < 0.01 (Student’s t test).

(C) Weight change after intra-rectal administration of 1% Frag-oz or EtOH (50% v/v) vehicle in GF or SPF animals (n = 8). **p < 0.01, ***p < 0.001 (Mann-Whitney

U test).

(D and E) Quantiﬁcation of (D) Mttp and (E) Cyp1a1 transcripts (normalized to b-actin) from mucosal scraping 2 days after intra-rectal administration of 1% Frag-oz

or EtOH (50% v/v) vehicle (n = 4). **p < 0.01, (Student’s t test)

(F) Quantitative scoring for colitis after intra-rectal administration of 1% TMO or EtOH (50% v/v) vehicle in GF or SPF animals (n = 8–9). **p < 0.01, p < 0.001

(Student’s t test).

See also Figures S4 and S5.

kynurenic acid and 3-hydroxy kynurenic acid (referred to as xan- had no effect on CD1d-restricted IL-10 production (Figures 5B

thurenic acid) only upon stimulation with oxazolone or TMO and and 5D). In contrast, IECs cultured under tryptophan-deﬁcient

frag-oz, respectively, within 6 hr of exposure to oxazoles (Fig- conditions and stimulated with oxazolone, TMO, or frag-oz

ure 5C). To further investigate whether tryptophan-derived me- when supplemented with kynurenic acid led to decreased

tabolites attenuate CD1d-restricted responses, we cultured IL-10 production that was reduced when AhR was silenced (Fig-

IECs in tryptophan-deﬁcient media supplemented with L-Trp, ure 5E). Taken together, these latter studies are consistent with a

kynurenic acid, xanthurenic acid, or an upstream tryptophan model in which exposure of IECs to oxazole-containing com-

product, kynurenine, in the presence or absence of oxazolone. pounds leads to the activation of IDO1 and generation of trypto-

Supplementation with exogenous tryptophan or its downstream phan-derived metabolites that induce the AhR pathway to

products of catabolism restored IEC responses to oxazolone, as downregulate CD1d-restricted responses associated with the

shown by reduced CD1d-restricted IL-10 production (Figure 5D). production of IL-10 (Figure 6).

In contrast, supplementation with DL-methyl tryptophan (DL-

Mtrp), a tryptophan competitive inhibitor of IDO1, did not restore DISCUSSION

suppression of CD1d-restricted IL-10 production in IECs

cultured in tryptophan-deﬁcient conditions and stimulated with In the studies described herein, we have used structural ana-

oxazolone (Figure 5D). Finally, to ascertain whether these lyses of oxazolone, a compound previously shown to be capable

same metabolites identiﬁed in our LC-MS analyses functioned of inducing inﬂammation of the colon (Boirivant et al., 1998, Hell-

through the AhR pathway, we performed siRNA-mediated er et al., 2002), to identify a new and potentially broad class of

knockdown of AhR in IECs cultured under tryptophan-deﬁcient environmental factors—derived from diet, microbes, and other

conditions in the presence of exogenously administered kynur- sources—that we name OxCs. OxCs are capable of direct induc-

enic acid and measured CD1d-restricted IL-10 production after tion of intestinal inﬂammation through crosstalk of CD1d-

stimulation with OxCs (Figure 5E). Incubation of IECs with L-Trp restricted and AhR pathways in IECs and, together, suggest

or kynurenic acid alone, or in conjunction with TMC stimulation, that these environmental agents are capable of activating

1130 Cell 173, 1123–1134, May 17, 2018

A

B

C

D

E

Figure 5. Oxazoles Induce the Generation of Tryptophan-Derived Metabolites that Attenuate Epithelial CD1d-Restricted Responses in an

AhR-Dependent Manner

(A) Recombinant IDO1 was incubated with L-tryptophan in the presence of oxazole (oxazolone, TMO, Frag-oz), control (TMC) compounds, or vehicle and formyl-

kynurenine production measured via calorimetric assay. ***p < 0.001 (Student’s t test)

(B) MODE-K cells cultured with L-Trp-deﬁcient media or media supplemented with exogenous L-Trp and CD1d-restricted IL-10 responses measured in MODE-K

cells pre-conditioned with vehicle or indicated compounds. n.s., not signiﬁcant; ***p < 0.001 (Student’s t test).

(C) HPLC-MS quantiﬁcation of label-retaining metabolites in MODE-K cells grown under tryptophan-deﬁcient conditions and supplemented with deuterated

tryptophan and conditioned with the indicated compounds. ***p < 0.001 (Student’s t test)

(D) CD1d-restricted IL-10 responses in MODE-K cells pre-conditioned with vehicle or oxazolone in tryptophan-deﬁcient media with supplementation with L-Trp,

DL-Mtrp, kynurenine, kynurenic acid, or xanthurenic acid and then loaded with a-GC followed by 24.7 iNKT hybridoma co-culture. ***p < 0.001 (Student’s t test)

(E) CD1d-restricted IL-10 production by MODE-K cells transfected with scrambled (solid ﬁll) or siRNA targeted against AhR (clear ﬁll) in tryptophan-deﬁcient

media. CD1d-restricted IL-10 responses were measured following pre-conditioning with vehicle, TMC, oxazolone, TMO, or frag-oz with or without supple-

mentation of L-Trp or kynurenic acid. ***p < 0.001 (Student’s t test).

CD1d-restricted pathways by phenocopying host responses to mally processed foods, pesticides, and other environmental

microbial factors.

sources (Maga, 1978). In addition, microbes are another abun-

Environmental and lifestyle factors have been identiﬁed as crit- dant source of oxazoles as part of a structurally functionally

ical-risk elements for development of disorders of chronic diverse class of ribosomally derived peptides, dubbed TOMMs,

inﬂammation, such as IBD (Molodecky and Kaplan, 2010). that are widely disseminated across the phylogenetic spectra of

Consistent with this, chemical-induced models of intestinal bacterial secretion systems, including potential pathobionts

inﬂammation have been crucial experimental tools for investi- associated with IBD pathogenesis or skin inﬂammation (Mazma-

gating the pathophysiology and the testing of therapeutic strate- nian et al., 2008; Melby et al., 2011). Together, this supports the

gies for IBD (Wirtz et al., 2007; Kaser et al., 2010). Here, we have notion that dietary and microbial-derived compounds with a

isolated the colitogenic activity within oxazolone to a ﬁve- common substructure, like oxazoles, can modulate host

membered heterocyclic oxazole moiety. Based on these obser- response by converging on conserved host immune cellular

vations, we inquired whether oxazolone is representative of a pathways (Figure 6).

broader class of environmental factors that may be involved in

We observed that exposure to select oxazole species modu-

the pathogenesis of IBD and possess a conserved or shared lates CD1d-restricted responses in epithelial cells in vitro that

substructure. Oxazolone, ﬁrst synthesized in 1883 by Erlen- are characterized by downregulation of Mttp and reduced

¨

meyer and Plochl (Plochl, 1884; Erlenmeyer, 1893), has been IL-10 production after CD1d crosslinking with NKT cell-associ-

widely utilized as a synthetic building block for compounds ated receptors. This mechanism has been previously shown

with a range of pharmacological properties (Turchi, 2008) and to reduce epithelial barrier activity in response to similar concen-

is also naturally abundant in diet as a component of many ther- trations of oxazolone in the colon, leading to exacerbated

Cell 173, 1123–1134, May 17, 2018 1131

supports a role for the AhR as a potential cytosolic sensor or

signal transducer of oxazole-induced products in the intestinal

epithelium (Lanis et al., 2017). Indeed, speciﬁc depletion of the

AhR in the epithelium was shown to alleviate the oxazole-

induced suppression of CD1d-restricted anti-inﬂammatory re-

sponses, including production of IL-10 (Figure 4B). MS analyses

identiﬁed at least two products of tryptophan catabolism, kynur-

enic acid and xanthurenic acid, that are speciﬁcally generated in

IECs exposed to oxazolone or dietary and/or microbial oxazoles

and their capacity to activate the AhR pathway in a manner that

results in attenuation of CD1d-restricted IL-10 production. This

observation is interesting, given that levels of tryptophan and

its byproducts in the serum have provided a robust marker for

gastrointestinal pathology in clinical settings (Ciorba, 2013).

In addition, we have shown that oxazoles also downregulate

MTP, which is normally responsible for the loading of phospho-

lipid antigens onto CD1d (Dougan et al., 2005, 2007). Consistent

with our observations, altering MTP expression or activity has

been previously shown to prevent transfer of phospholipids to

CD1d or the ability of CD1d to acquire exogenous lipid antigens,

such as a-GC (Dougan et al., 2005). Further, loss of MTP in IECs

has been shown to disable the ability of iNKT cells to induce the

barrier-protective cytokine, IL-10, by this cell type, as we have

shown previously and demonstrated here (Olszak et al., 2014).

Together, our data suggest that oxazoles alter presentation of

lipid autoantigens (Figures S1B and S1C) and exogenous lipid

antigens as shown with a-GC (Figures 1D, 1E, and 4C) to

CD1d-restricted invariant and non-invariant NKT cells in a

pathway that is dependent on AhR through the generation of

tryptophan-derived metabolites. As both oxazolone and frag-

oz induce inﬂammation in animals under GF conditions (Olszak

et al., 2012) (Figures 3D and 3E), NKT-mediated responses to

host-derived lipid antigens may be particularly important to oxa-

zole-induced inﬂammation and further suggest that oxazoles

may thus modulate autoimmune responses to self-lipid antigens.

Conversely, it is also interesting to consider whether microbes

Figure 6. Exposure to Environmental Oxazoles (from Diet or

Microbes) Inﬂuences CD1d-Restricted Responses in Intestinal

Epithelial Cells through Production of Tryptophan Derivatives that

Activate the Aryl Hydrocarbon Receptor Pathway

IECs promote mucosal barrier protection through microsomal triglyceride

protein (MTP)-mediated CD1d-restricted production of IL-10 (blue circles).

Upon exposure to oxazole compounds (derived from diet, microbes, or other

environmental sources), these CD1d-restricted responses are attenuated

through a mechanism involving the production of tryptophan-derived metab-

olites (kynurenic acid, xanthurenic acid; orange squares) through the activity of

IDO1 that trigger the AhR. Decreased, CD1d-restricted IL-10 production

results in unrestrained inﬂammatory responses (IL-13/IFNg, red dots) and

resulting pathology. ER, endoplasmic reticulum.

inﬂammatory responses (Olszak et al., 2014). To what degree ox-

azole products accumulate within the intestinal tract and the use oxazole products to target CD1d in order to establish

physiological concentrations and circumstances required for their niche during primary colonization, potentially resulting in

triggering inﬂammatory responses are unknown. However, at mucosal inﬂammation and pathology. We have previously

least two of the structures we identiﬁed, TMO and vinclozolin, demonstrated an important physiological role for CD1d during

have been detected in human urine through studies associated initial colonization of commensal and pathogenic bacteria (Nieu-

with deﬁning the human exposome (Table S1) (Wambaugh wenhuis et al., 2009). Consequently, generation of products that

et al., 2014). Although not directly addressed in this study, it is modulate CD1d activity, such as oxazoles, which are capable

interesting to consider whether accumulated exposure to of speciﬁcally inhibiting IEC function, may affect the ability

oxazoles derived from multifactorial sources, including diet, of microbes to colonize the intestine and potentially cause inﬂam-

microbes, industry, and/or agriculture, acts in an additive or mation. While full-length MccB17 itself shared relatively low

combinatorial manner that includes other potential non-oxazole structural similarity with oxazolone, derivative products below

agents in modulating host responses that are associated with 270 Da generated either by alkaline hydrolysis or proteolytic

disease pathogenesis.

cleavage showed relatively high similarity with both oxazolone

Our gene expression proﬁling of IECs exposed to oxazole and TMO (Table S1). Importantly, we have observed that both

compounds has identiﬁed a several genes (Figures 1B, 2C, 2F, the natural products and their synthetic analogs (Figure S6)

3E, S1A, and S4) involved in xenobiotic metabolism and trypto- display similar ability to attenuate CD1d-lipid antigen presenta-

phan catabolism, including IDO1, a marker highly expressed in tion and subsequent CD1d-restricted IL-10 production, as well

patients suffering from IBD, as well as in many animal models as activate AhR responses speciﬁcally in IECs, resulting in inﬂam-

of colitis (Olszak et al., 2014; Ciorba, 2013; Wolf et al., 2004). mation associated with decreased epithelial-derived IL-10. That

Data presented here suggest that oxazole compounds directly inﬂammation to MccB17 derivatives occurs in GF animals con-

enhance the ability of IDO1, the rate-limiting enzyme in trypto- ﬁrms that the effects of these compounds are directly impacting

phan catabolism, to metabolize tryptophan (Figure 5A). This the host and not a result of their antimicrobial capacity. It is

1132 Cell 173, 1123–1134, May 17, 2018

B Luciferase promoter-reporter assays

B In-silica similarity screening

therefore intriguing to consider then that the accumulation of ox-

azole compounds derived from MccB17-producing and other

TOMM-producing bacterial strains during a period of microbial

competition may have a bystander effect on host immune re-

sponses, resulting in intestinal inﬂammation or other effects

that beneﬁt the organism at the expense of the host.

SUPPLEMENTAL INFORMATION

Supplemental Information includes six ﬁgures and one table and can be found

with this article online at https://doi.org/10.1016/j.cell.2018.04.037.

In conclusion, our studies illustrate a strategic approach for

identifying potential triggers of inﬂammation by interrogating

and evaluating structural mimics of lead compounds with estab-

lished host responses and pathology. In this manner, by

leveraging the knowledge that oxazolone acts as a colitogenic

agent via modulation of CD1d-restricted pathways, we have

identiﬁed OxCs as a new class of potential environmental agents

with inﬂammation-inducing potential that are contained within

a wide range of environmental sources, including diet and

microbes. With respect to the latter, our studies further show

that bacterial microcins, which are well known to play a role in

microbe-microbe interactions, also possess important and

previously unappreciated immune-regulating properties via their

effects on CD1d-restricted pathways. Further, in both the case of

environmental and microbial OxCs, we have found that they

involve epithelial sensing by the AhR, which in turn adversely

affects CD1d-restricted presentation of lipid antigens that are

associated with barrier-protective production of IL-10. As a

result, we also identiﬁed a novel pro-inﬂammatory role of AhR

sensing that is associated with the intestinal epithelium and

have brought forth the concept that environmental triggers of

inﬂammation that are potentially associated with IBD may func-

tion in this manner by phenocopying the activity of microbial

products with similar activity.

ACKNOWLEDGMENTS

R.S.B. is supported by NIH grants DK044319, DK053056, DK051362,

DK088199, and 5P01AI073748 and the Harvard Digestive Diseases Center

(P30DK034854). S.S.I. and T.G. are supported by the Crohn’s and Colitis

Foundation of America Research Fellow Awards (383527 and 418509).

S.F.O is supported by NIH grant DK102771. We thank Drs. Roberto Kolter

and Dennis Kasper (Harvard Medical School) for reagents and discussion of

data, Drs. Keith Houck and Jill Franzosa (Environmental Protection Agency)

for helpful discussions and Toxcast21 data, and Vladimir Yeliseyev and Marla

Marquez for technical assistance. We thank Amanjot Riar and Blumberg labo-

ratory members for assistance in manuscript preparation.

AUTHOR CONTRIBUTIONS

Conceptualization, S.S.I., T.G., and R.S.B.; Methodology, S.S.I., T.G., A.G.,

S.F.O., F.C., C.S., A.L., A.M., J.F.N., R.L., and R.S.B.; Analysis, S.S.I., T.G.,

A.G., C.S., F.C., A.L., A.M., J.G., and R.S.B.; Writing and Editing S.S.I., T.G.,

A.G., P.S.A.d.S., R.B.S., G.B., R.H., A.L., A.M., J.F.N., R.B.S., and R.S.B.

DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: May 30, 2017

Revised: December 7, 2017

Accepted: April 25, 2018

Published: May 17, 2018

STAR+METHODS

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1134 Cell 173, 1123–1134, May 17, 2018

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

anti-mouse CD1d (1B1) FITC

anti-mouse a-GalCer:CD1d complex PE

anti-mouse aryl hydrocarbon receptor PE

BD Biosciences

Biolegend

Cat# 553845

Cat# 140505

Cat# 5657711

BD Biosciences

Chemicals, Peptides, and Recombinant Proteins

Dialyzed FBS

GIBCO

custom for R.S.B. Laboratory

Dialyzed RPMI

Critical Commercial Assays

Mouse IL-13 DuoSet ELISA

Mouse IFN-g ELISA MAX

Mouse IL-10 ELISA Ready-Set-Go

IDO1 Inhibitor Screening Assay Kit

Luciferase Assay System

R&D Systems

Biolegend

Cat# DY413

Cat# 430801

Cat# 501125188

Cat# 72021

eBioscience

BPS Biosciences

Promega

Cat# E4550

Lipofectamine 2000

Thermo Fisher Scientiﬁc

eBioscience

QIAGEN

Cat# 11668019

Cat# 88882400

Cat# 205111

Cat# 3003230001

Intracellular Fixation and Permeablization Buffer Set

Omniscript RT Kit

SYBR Green I

Roche

Oligonucleotides

5⁰-GATGCTCCCCGGGCTGTATT-3⁰

5⁰-GGGGTA CTTCAGGGTCAGGA-3⁰

5⁰-GAGAGCTGCAGGGCCCTTTGC-3⁰

5⁰-CTCCCTGGTTTCTCTTCCCAAGACC-3⁰

5⁰-GCAGCCAGTACGCTCTTTTC-3⁰

5⁰-ACAGCTTGTTTCTGGC AGGT-3⁰

⁰-GGACTTTTTGGATTTCAAAAGTGAC-3⁰

5⁰-GGAGAAACGGTCATAATTGTG-3⁰

5⁰-TGGCACTCAGTAAAATATCTCCT-3⁰

5⁰CAGGCAGATTTCTAGCCACA-3⁰

5⁰-GGCCACTTTGACCCTTACAA-3⁰

5⁰-CAGGTAACGGAGGACAGGAA-3⁰

mouse AhR siRNA

Mouse Bactin qPCR Forward Primer

Mouse Bactin qPCR Reverse Primer

Mouse Interleukin 10 qPCR Forward Primer

Mouse Interleukin 10 qPCR Reverse Primer

Mouse CD1d qPCR Forward Primer

Mouse CD1d qPCR Reverse Primer

Mouse Mttp qPCR Forward Primer

Mouse Mttp qPCR Reverse Primer

Mouse IDO1 qPCR Forward Primer

Mouse IDO1 qPCR Reverse Primer

Mouse Cyp1a1 qPCR Forward Primer

Mouse Cyp1a1 qPCR Reverse Primer

Thermo Fisher Scientiﬁc

R.S.B. Laboratory

Cat# 100360

control siRNA

Thermo Fisher Scientiﬁc

Cat# AM4611

Experimental Models: Cell Lines

MODE-K small intestinal epithelial cell line

24.7 invariant Natural Killer T cell hybridoma

24.8 invariant Natural Killer T cell hybridoma

14S.6 non-invariant Naturall Killer T cell hybridoma

Vidal et al., 1993

R.S.B. Laboratory

N/A

Experimental Models: Organisms/Strains

C57BL/6J Speciﬁc Pathogen Free

CD1d ꢀ/ꢀ

The Jackson Laboratory

Cat# 000664

N/A

R.S.B. Laboratory

Cat# 006203

Ahr tm3.1Bra/J

The Jackson Laboratory

N/A

Villin-Cre

R.S.B. Laboratory

(Continued on next page)

C57BL/6 Germ Free

Harvard Digestive Disease Center

Cell 173, 1123–1134.e1–e5, May 17, 2018 e1

Continued

REAGENT or RESOURCE

SOURCE

Taconic

IDENTIFIER

Cat# 9166

Cat# B6

AhR ꢀ/ꢀ

C57BL/6

Recombinant DNA

pGL4.43 Luc2pXRE

pGL4.75

Promega

Cat# E4121

Cat# E6931

Cat# E2261

Renilla luciferase

Software and Algorithms

Graphpad Prism 7.0

FloJo

GraphPad Sofware

https://www.graphpad.com/

https://www.ﬂowjo.com/

FloJo, LLC and Illumina, Inc

Other

Nikon Eclipse Ti Microscope

BD FACS Aria II

Nikon

N/A

BD Biosciences

Miltenyi Biotec

Thermo Fisher Scientiﬁc

Bio-Rad

N/A

MACSQuant Analyzer

LTQ-XL Linear Ion Trap Mass Spectrometer

CFX96 Real-Time System

4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one

2,4,5-trimethyl-2,5-dihydro-1,3-oxazole

2-methyl-1-pyrroline

Frag-oz

N/A

Sigma

Cat# 862207

Cat# CDS001470

381055

Sigma

Collin et al., 2013

Sigma

A.L Laboratory

A.L. Laboratory

A.M. Laboratory

Cat# 45705

Cat# T0254

Cat# 860646

Cat# sc-391262A

Cat# 1803

Frag-tz

frag-oztz

frag-tzoz

Microcin B17

Vinclozolin

L-Tryptophan

Sigma

DL-methyl tryptophan

L-trytophan-D5

Sigma

ITE

Tocris

Subtilisin

Sigma

Cat# P5380

Cat# D-4391

Cat# K8625

Cat# K3375

Cat# D120804

Cat# 867000

Cat# 860547P

Cat# R289728

Kynurenic-3,5,6,7,8-d5

L-Kynurenine

CDN Isotopes

Sigma

Kynurenic Acid

Sigma

4,8-dihydroxyquinoline-2-carboxylic acid

KRN7000

Sigma

Avanti Polar Lipids, Inc

Sigma

beta-glucosyl ceramide

1,2,4-trimethyl cyclopentane

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulﬁlled by the Lead Contact,

Dr. Richard S. Blumberg (blumberg@bwh.harvard.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animals

Mice (C57BL/6J) were housed in a speciﬁc pathogen-free (SPF) barrier facility at Harvard Medical School: Wild-type, Ahr^ﬂ/ﬂ, Villin-

Cre, mice were purchased from The Jackson Laboratory. Ahr^ꢀ/ꢀmice were purchased from Taconic USA and maintained as hetero-

zygotes. Cd1d1^ꢀ/ꢀCd1d2^ꢀ/ꢀhave been described. Animal studies were conducted in a gender and age matched (8-10 week)

manner using littermates for each experiment, except for wild-type versus Cd1d^ꢀ/ꢀcomparisons. SPF and Germ-free animals

e2 Cell 173, 1123–1134.e1–e5, May 17, 2018

were bred and maintained in vinyl isolators in the Harvard Digestive Disease Center Gnotobiotic and Microbiology Core. For colitis

experiments, mice were transferred to Optimice cages (Animal Care Systems) for daily monitoring under sterile conditions. All

procedures were approved by the Harvard Medical Area Standing Committee on Animals.

Primary Cultures

Bone marrow derived cells (BMDC) were puriﬁed from mouse femurs and cultured for 7 days in RPMI (supplemented with 10% fetal

bovine serum, 1% antibiotic-antimycotic, GIBCO and 20 ng/mL granulocyte-macrophage colon-stimulating factor, Biosource). For

primary hepatocyte isolation, mice were anesthetized with intraperitoneal injection of ketamine (87 mg/kg body weight) plus xylazine

(13 mg/kg body weight), after which the inferior vena cava was exposed, cannulated and perfused for 5 min with liver perfusion media

(Invitrogen), followed by a 10 min perfusion with liver digestion media (Invitrogen). The digested liver was minced in hepatocyte was

media (Invitrogen) and cells were pelleted and resuspended in Williams E medium (10% FBS, 10^ꢀ7M dexamethasone, 10 mg/ml

insulin and 5 mg/ml transferrin.

Bacterial Stocks and Puriﬁcation

MccB17 was generated and puriﬁed as described (Collin et al., 2013; Li et al., 1996). Plasmid pUC19, wild-type or carrying the com-

plete biosynthetic operon (mcbABCDEFG), was transformed into competent DH5a or MG1655 E. coli.

Synthesis of Microcin B17 and Derivatives

Overnight colonies were grown in LB media and used to inoculate M63 media (3 g/l KH₂PO₄, 7 g/l K₂HPO₄, 2 g/l (NH₄)SO₄, 1 mM

MgSO₄, 1 mg/ml thiamine, 0.2% glucose, 0.1 mg/ml ampicillin) or LB Media (0.1mg/mL ampicillin) and incubated for 48 h. Cells

were pelleted and lysed in a boiling solution of 1 mM EDTA and 100 mM acetic acid. Cellular debris was removed by centrifugation,

protein was quantiﬁed by BCA Assay kit (ThermoFisher Scientiﬁc) according to manufacturer’s instructions and lysates were serially

titrated (1 mg-10 mg/mL) incubated with MODE-K cells for co-culture assays as described. Digestion of full length MccB17 ( Collin

et al., 2013) by subtilisin (Sigma) (0.1 mM MccB17, 10% DMSO, 10 mM NaAcO, 5 mM Ca(AcO)₂) or sequence grade trypsin (Prom-

ega) (0.5 mM 50 mg/mL trypsin, 1%DMSO in buffer (Promega) for 24 h to completion (Collin et al., 2013). Entire digested products (or

equivalent buffer with enzyme) was then incubated with MODE-K cells for co-culture and qPCR analyses.

Synthesis of Frag-oz

Frag-oz, frag-tz, frag-oztz, and frag-tzoz originally were kindly provided by R. Payne, School of Chemistry University of Sydney or

later synthesized by A.L. The compounds were synthesized according to established procedures for microcin B17-related oxazole

and thiazole synthesis. Refer to Figure S6A. Step 1. Synthesis of 2-(aminomethyl)oxazole-4-carboxamide (Figure S6A) Tert-butyl

((4-carbamoyloxazol-2-yl)methyl)carbamate 1¹(0.895 g, 3.71 mmol) was stirred in TFA/CH₂Cl₂(1:1 v/v, 40 mL) at room temperature

for 1 h. The solvent was removed under reduced pressure affording the title compound as a brown oil (0.367 g, 70%). ¹H NMR

(400 MHz, Methanol-d₄) d 8.42 (s, 1H), 4.28 (s, 2H). Step 2. 2-(acetamidomethyl)oxazole-4-carboxamide (Frag-oz): To a solution

of 2-(aminomethyl)oxazole-4-carboxamide 2 (630mg, 4.46 mmol) in DMF (2.2 mL) at 0^ꢁC TEA (0.7 mL, 4.9 mmol) was added followed

by Acetyl Chloride (0.35 mL, 4.9 mmol). The solution was stirred for 5 h at 0^ꢁC. The DMF was removed under reduced pressure and

the crude product was puriﬁed by column chromatography on silica [DCM-Methanol, 9:1 (v/v)] affording the title compound as a white

solid (400mg, 49%). ¹H NMR (400 MHz, Methanol-d₄) d 8.30 (s, 1H), 4.48 (s, 2H), 1.99 (s, 3H). ¹³C NMR (101 MHz, Methanol-d₄)

d 172.12, 163.71, 161.52, 142.01, 135.76, 35.87, 20.92.

Tert-butyl ((4-carbamoylthiazol-2-yl)methyl)carbamate (4)

Aqueous ammonia (28% w/w, 3,1 mL, 40 mmol) was added to a solution of ethyl 2-(((tert-butoxycarbonyl)amino)methyl)thiazole-

4-carboxylate²3 (381 mg, 1.33 mmol) in methanol (1,3 mL). After 16 h at 45^ꢁC, the reaction mixture was concentrated affording

the title compound as a brown oil (315mg, 92%) which was used directn˜ y in the next step. ¹H NMR (400 MHz, Methanol-d₄)

d 8.10 (s, 1H), 4.49 (s, 2H), 1.45 (s, 9H).

2-(aminomethyl)thiazole-4-carboxamide (5)

Tert-butyl ((4-carbamoylthiazol-2-yl)methyl)carbamate 4 (0.342 g, 1.331 mmol) was stirred in TFA/CH₂Cl₂(1:1 v/v, 13 mL) at room

temperature for 1.5 h. The solvent was removed under reduced pressure affording the title compound as a green oil (0.150, 72%)

which was used directly in the next step. ¹H NMR (400 MHz, Methanol-d₄) d 8.25 (s, 1H), 4.41 (s, 2H).

Synthesis of Frag-tz 2-(acetamidomethyl)thiazole-4-carboxamide

Refer to Figure S6B. To a solution of 2-(aminomethyl)thiazole-4-carboxamide 5 (204mg, 1.30 mmol) in DMF (6.5mL) at 0^ꢁC, Triethyl-

amine (TEA, 0.2 mL, 1.43 mmol) was added followed by Acetyl Chloride (0.1 mL, 1.43 mM). The solution was stirred for 5 h at 0^ꢁC. The

solvent was removed under reduced pressure and the crude product was puriﬁed by column chromatography on silica [CH₂Cl₂/

Methanol, 9:1 (v/v)] affording the title compound as a white solid (80mg, 31%). ¹H NMR (400 MHz, Methanol-d₄) d 8.11 (s, 1H),

4.63 (s, 2H), 2.01 (s, 3H). ¹³C NMR (101 MHz, Methanol-d₄) d 172.13, 169.43, 164.11, 149.10, 124.41, 40.47, 21.01.

Cell 173, 1123–1134.e1–e5, May 17, 2018 e3

METHOD DETAILS

Experimental colitis model

For reciprocal sensitization studies (Figure S3), mice were pre-sensitized by epicutaneous application of 3% w/v oxazolone

(4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, Sigma-Aldrich) or 2,4,5-trimethyl-2,5-dihydro-1,3-oxazole (Sigma-Aldrich) in

100% ethanol (200 mL volume). Five days later, animals were re-challenged intra-rectally (through a 3.5F catheter) with 1% vehicle,

oxazolone or 2,4,5-trimethyl-2,5-dihydro-1,3-oxazole in 50% ethanol (5 mL/g of body weight) as indicated. For all other studies,

vehicle, oxazolone, 2,4,5-trimethyl-2,5-dihydro-1,3-oxazole, 1,2,4-trimethylcyclopentane (Sigma-Aldrich) were administered intra-

rectally (through a 3.5F catheter) as a 1% solution in 50% ethanol (5 ml/g of body weight). Body weight, rectal bleeding, and stool

consistency were analyzed 1-2 times daily. Tissues obtained at the indicated time points were embedded in parafﬁn, stained with

hematoxylin and eosin, and examined by a pathologist (Dr. Jon Glickman) in a blinded fashion for evidence of colitis according to

ﬁve established criteria: mononuclear inﬂammation, crypt hyperplasia, epithelial injury, neutrophilic inﬂammation, and hypervascu-

larization grading on 4 point scale (0 = absent, 1 = mild, 2 = moderate, 3 = severe).

Colon organ culture

Standardized segments (1 cm 3 1 cm) of the transverse colon were washed in cold PBS supplemented with penicillin and strepto-

mycin (GIBCO) and cultured in 24-well ﬂat-bottom culture plates (Falcon) in RPMI 1640 media (GIBCO) for 24 h at 37^ꢁC. Supernatants

were analyzed for cytokines IL-10, IL-13 and IL-1b by ELISA (all BD).

Cells and Antigen presentation assays

1 3 10⁵BMDCs, primary hepatocytes or 0.5 3 10⁵MODE-K cells (Vidal et al., 1993) were incubated in 96 well plates (Corning)

maintained in RPMI (supplemented with 2% fetal bovine serum, 1% antibiotic-antimycotic, GIBCO) were pulsed with the indicated

compounds dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM for 36-48h. Cells were washed three times with ster-

ile phosphate buffered saline (PBS, GIBCO) after which cells were incubated with 100 ng ml^-1alpha-galactosyl ceramide/KRN7000 or

beta-glucosyl ceramide (Avanti Polar Lipids) for 3h, washed three times with sterile PBS, before addition of 2 3 10⁴NKT hybridoma

cells including 24.7, 24.8, and 14S6 overnight co-culture after which supernatant was collected and clariﬁed by centrifugation. Mouse

IL-10, IFNg production was assessed by ELISA (OptEIA, BD Biosciences). For aryl hydrocarbon receptor siRNA mediated knock-

down, MODE-K cells were transfected with AM100441, AM 100360, or Silencer negative control siRNAs (ThermoFisher Scientiﬁc)

using Lipofectamine 2000 (Life Technologies) according to manufacturer’s instructions. AhR silencing was veriﬁed 48h after trans-

fection by quantitative polymerase chain reaction.

Identiﬁcation of Tryptophan Derived Metabolites

MODE-K cells were cultured in dialyzed FBS and RPMI (GIBCO #26400044, RPMI 1640 cGMP #100001229, all amino acids < 1nM,)

and supplemented with essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, and valine

at 1 mM) and supplemented with deuterated tryptophan (1 mM D5-Trp, Santa Cruz sc-391262, see Key Resources Table). Lysates

were extracted from cell pellets in 80% Methanol and analyzed with reverse phase HPLC-MS (Agilent C18 column connected

with Thermo Scientiﬁc LTQ-XL, see Key Resources Table).

IDO1 enzymatic assay

Recombinant His-tagged IDO1 was incubated with 10nM concentrations of TMC, oxazolone, TMO, or frag-oz and assay was per-

formed according to the manufacturer’s instruction (BPS Bioscience, San Diego, CA, see Key Resources Table)

CD1d and CD1d-lipid complex measurements by ﬂow cytometry

MODE-K cells were pulsed with 10 mM indicated compounds dissolved in DMSO for 36-48h. Cells were washed three times with

sterile PBS after which cells were incubated with 100 ng ml^-1alpha-galactosyl ceramide/KRN7000 or the non-agonistic compound,

beta-glucosyl ceramide (data not shown), for 3 h, washed three times with sterile PBS. Cells were lifted in 10 mM EDTA/PBS and

stained with the following antibodies: mouse L363 (eBiosciences, see Key Resources Table), mouse CD1d (BD Bioscience, see

Key Resources Table). For intracellular staining, cells were ﬁxed using the Intracellular Fixation and Permeabilization Buffer Set

(eBioscience, see Key Resources Table) according to the manufacturer’s instructions. Flow cytometry was performed on

MACSQuant Analyzer (Miltenyi Biotec) and analyzed using FlowJo software.

Quantitative polymerase chain reaction assays

RNA samples were prepared using an RNeasy Mini Kit and cDNAs were synthesized using the Omniscript RT Kit (QIAGEN, see Key

Resources Table). Real-time RT–PCR was performed using SYBR Green I Master Mix (Roche) and a CFX96 Real-Time System

(Bio-Rad, see Key Resources Table). Values were normalized to b-actin (see Key Resources Table).

e4 Cell 173, 1123–1134.e1–e5, May 17, 2018

Luciferase promoter-reporter assays

For aryl hydrocarbon receptor luciferase assays, MODE-K cells were cultured on 6-well plates (Corning). pGL4.43 Luc2pXRE

(Promega) or empty pGL4.43 (Promega) and 100 ng Renilla luciferase (Promega) was transfected into MODE-K cells using Lipofect-

amine 2000. Twenty four hours later, MODE-K cells were conditioned with indicated compounds for 48 h. Cells were lysed and ﬁreﬂy

luciferase (normalized to renilla luciferase) was measured using Dual Luciferase Reporter Assay System (Promega)

In-silica similarity screening

We used two-step in-silico screening process. First, broad similarity screening was performed to identify oxazole related natural

compounds from various databases including Pubchem, Super Natural II a database of natural products, Dictionary of natural prod-

ucts Chemnet database, and The Human Metabolome Database (Wishart et al., 2013) (Table S1). Second, focused screen search

was performed based on the results of ﬁrst screening to identify natural compounds related to a 5-membered oxazole ring of

oxazolone with emphasis to ﬁnd minimal structural features of oxazolone for its inﬂammatory activity. This focused screen selection

was based on identiﬁcation of natural compounds with Tanimoto similarity of more than 0.5 and/or Euclidean distance scores cutoff

of 0.9 (Table S1). Natural compounds were queried in the standard SMILES format. Tanimoto similarities between compounds were

computed with the Chemmine Similarity Workbench, which calculated maximum common substructure (MCS) similarities with the

Tanimoto coefﬁcient (Cao et al., 2008). Euclidean distance scores were calculated using Noncontiguous atom matching structural

similarity function (NAMS) (Teixeira and Falcao, 2013). The following formulas were used to calculate MCS similarity and TS score

of natural compounds with Oxazolone and Oxazole ring substituted with a methyl group at the 2, 4 and 5 position. MCS similarity

score: Atom number (AN) in MCS/AN in smaller molecule, TS score: AN in MCS/ (AN in smaller molecule + AN in larger molecule

–AN in MCS). Based on the results of the screening process, TMO with Tanimoto/Euclidean similarity score of 0.78/0.97 was selected

as one of the natural compound for in vivo experiments. To further investigate the role of nitrogen and oxygen atoms of the oxazole

ring and correlate them with colitogenic activity, Trimethylcyclopentane (TMC) and 2-Methyl-1-pyrroline (1MP) were selected as

additional controls. In addition, Vinclozolin was selected based on Euclidean distance score of 0.94 and widespread fungicide usage.

Cell 173, 1123–1134.e1–e5, May 17, 2018 e5

Supplemental Figures

Figure S1. A Minimal Oxazole Structure Modulates Intestinal Epithelial Transcriptional and Antigen Presentation Capacity to Invariant and

Non-variant NKT Cells, Related to Figure 1

(A) Relative transcript abundance in MODE-K cells conditioned with the indicated compounds, normalized to b-actin. **p < 0.01 (Student’s t test).

(B and C) CD1d-restricted Interleukin 10 production in the absence of exogenous lipid supplementation in MODE-K cells conditioned with the indicated com-

pounds followed by co-culture with (B) invariant NKT (24.8) or (C) non-invariant NKT (14S.6) hybridomas. ***p < 0.001 (Student’s t test).

(D) Interferon g production in bone marrow derived dendritic cells conditioned with the indicated compounds followed by loading with a-galactosyl ceramide and

co-cultured with 24.7 iNKT hybridoma.

Figure S2. A minimal Oxazole Structure Does Not Alter CD1d Protein Expression or Cellular Distribution, Related to Figure 1

(A and B) Quantiﬁcation of surface and intra-cellular CD1d protein expression in MODE-K cells conditioned with the indicated compounds for 48hr, as measured

by (A) ﬂow cytometry and (B) relative proportions of intracellular and cell surface CD1d protein abundance.

Mttp

n.s.

A

B

EtOH

TMC

TMO

*

1.5

1.0

0.5

0.0

Frag-oz

Oxazolone

C

Cyp1a1

Colitis Score

D

***

500

400

300

200

100

0

***

6

4

n.s.

2

Sensitization

Intrarectal

TMO

Ox

TMO

Ox

TMO

TMC

TMO

EtOH

EtOH EtOH

TMO TMO

Figure S3. Intestinal Inﬂammation, Epithelial Response, and Pathology to Oxazole-Containing Compounds Does Not Require Apriori Host

Sensitization, Related to Figure 3

(A) Representative sections of wild-type animals administered 1% of the indicated compounds (50% EtOH v/v) by intra-rectal challenge and analyzed by H&E

stain after 3 days. 10X magniﬁcation, scale bar (40 mM).

(B and C) Quantiﬁcation of B. Mttp and C. Cyp1a1 transcripts (normalized to b-actin) from mucosal scrapings 2 days after intra-rectal challenge with 1% TMO, 1%

TMC or EtOH (50%v/v) vehicle (n = 3). n.s. not signiﬁcant, *p < 0.05, ***p < 0.001 (Student’s t test).

(D) Animals were sensitized by topical application of the indicated compounds followed by intra-rectal administration of 1% TMO, 1% oxazolone, or EtOH (50%

v/v) vehicle in wild-type C57BL/6 animals. Quantitative colitis scoring was assessed on colons harvested 3 days after intra-rectal challenge. ***p < 0.001

(Student’s t test).

Figure S4. Related to Figure 4

(A–C) Aryl hydrocarbon receptor mediates expression of a subset of gene targets in response to oxazole containing compounds: MODE-K cells transfected with

control or AhR speciﬁc siRNA, conditioned with the indicated compounds and relative transcript abundance was measured normalized to b-actin. (A) cyp1a1, (B)

Ido1, (C) Mttp n.s. not signiﬁcant, ***p < 0.001 (Student’s t test).

Figure S5. Aryl Hydrocarbon Receptor Attenuates CD1d-Restricted Responses in Primary Hepatocytes, Related to Figure 4

Interleukin 10 production from primary hepatocytes derived from WT or AhR-deﬁcient (KO) animals were conditioned with the indicated compounds, loaded with

a-galactosyl ceramide followed by co-culture with 24.7 iNKT hybridoma. ***p < 0.001 (Student’s t test).

A

B

Figure S6. Synthesis of Frag-oz and Frag-tz, Related to STAR Methods

(A and B) A schematic outline for synthesis of MccB17 derived products (A) Frag-oz. (B) Frag-tz.

Article Doi

Dietary and Microbial Oxazoles Induce Intestinal Inflammation by Modulating Aryl Hydrocarbon Receptor Responses

DOI: 10.1016/j.cell.2018.04.037

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Article abstract of DOI:10.1016/j.cell.2018.04.037

Full text of DOI:10.1016/j.cell.2018.04.037

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