Scholarly article on topic 'Interleukin 6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients With Chronic Intestinal Inflammation'

Interleukin 6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients With Chronic Intestinal Inflammation Academic research paper on "Biological sciences"

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Abstract of research paper on Biological sciences, author of scientific article — Nick Powell, Jonathan W. Lo, Paolo Biancheri, Anna Vossenkämper, Eirini Pantazi, et al.

Background & Aims Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation. Methods ILCs were isolated from colons of Tbx21 -/- × Rag2 -/- mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota. Results IL17A- and IL22-producing, natural cytotoxicity receptor–negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor–positive cells, in a dose-dependent manner. Conclusions IL6 contributes to activation of colonic natural cytotoxicity receptor–negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor–positive cells.

Academic research paper on topic "Interleukin 6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients With Chronic Intestinal Inflammation"

Accepted Manuscript

Interleukin-6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients with Chronic Intestinal Inflammation

Nick Powell, Jonathan W. Lo, Paolo Biancheri, Anna Vossenkamper, Eirini Pantazi, Alan W. Walker, Emilie Stolarczyk, Francesca Ammoscato, Rimma Goldberg, Paul Scott, James B. Canavan, Esperanza Perucha, Natividad Garrido-Mesa, Peter M. Irving, Jeremy D. Sanderson, Bu Hayee, Jane K. Howard, Julian Parkhill, Thomas T. MacDonald, Graham M. Lord

PII: S0016-5085(15)00573-9

DOI: 10.1053/j.gastro.2015.04.017

Reference: YGAST 59726

To appear in: Gastroenterology Accepted Date: 21 April 2015

Please cite this article as: Powell N, Lo JW, Biancheri P, Vossenkamper A, Pantazi E, Walker AW, Stolarczyk E, Ammoscato F, Goldberg R, Scott P, Canavan JB, Perucha E, Garrido-Mesa N, Irving PM, Sanderson JD, Hayee B, Howard JK, Parkhill J, MacDonald TT, Lord GM, Interleukin-6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients with Chronic Intestinal Inflammation, Gastroenterology (2015), doi: 10.1053/j.gastro.2015.04.017.

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Interleukin-6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients with Chronic Intestinal Inflammation

Nick Powell1'2'3, Jonathan W. Lo1'3, Paolo Biancheri4, Anna Vossenkämper4, Eirini Pantazi1'3, Alan W. Walker5'6, Emilie Stolarczyk1,3,7, Francesca Ammoscato4, Rimma Goldberg1,2, Paul Scott5, James B. Canavan1,3,

1 o o OR

Esperanza Perucha ' , Natividad Garrido-Mesa ' , , Peter M. Irving , Jeremy D. Sanderson , Bu Hayee , Jane K. Howard1'3'7, Julian Parkhill5, Thomas T. MacDonald4, Graham M. Lord1'3

department of Experimental Immunobiology, Division of Transplantation Immunology and Mucosal Biology, King's College London, United Kingdom, SE1 9RT

Gastroenterology Department, Guy's and St Thomas' NHS Foundation Trust, London, United Kingdom

3NIHR Biomedical Research Centre at Guy's & St Thomas' NHS Foundation Trust and King's College London, United Kingdom

4Centre for Immunology and Infectious Disease, Blizard Institute, Barts and the London School of Medicine and Dentistry, United Kingdom

5Pathogen Genomics Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridgeshire, United Kingdom, CB10 1SA

6Microbiology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, United Kingdom, AB21 9SB

7Division of Diabetes and Nutritional Sciences, King's College London, United Kingdom Gastroenterology Department, Kings College Hospital, London, United Kingdom

Tel: +44 207 188 3053, Fax: +44 207 188 3638 Corresponding Author: graham.lord@kcl.ac.uk

Microbiota sequence data has been deposited in the European Nucleotide Archive under Study Accession Number ERP005850 and Sample Accession numbers ERS459682 - ERS459702.

Author contributions

Study concept and design (NP, GL), acquisition of data (NP, JL, PB, AV, EP, ES, AW, PS, JC, EP, NG, FA, PI, JS, BH, GL), microbiota studies (AW, PS, JW), human cytokine production experiments and analyses (AV, PB, EP), data analysis and interpretation (NP, JL, PB, AV, EP, ES, AW, PS, EP, NG, GL), statistical analysis (NP, JL, AW), technical support (JL, EP), obtained funding (NP, GL, TM, AW, JP), drafting of manuscript (NP), study supervision (NP, TM, GL), critical revision of the manuscript (GL, TM, JH, AW, PI, JS).

Disclosures

NP has received honoraria for acting in an advisory capacity or speaking on behalf of Actavis UK Ferring and AstraZeneca. PMI has received honoraria for acting in an advisory capacity or speaking on behalf of Abbvie, MSD, Actavis UK, Shire, Ferring, Falk, Genentech, Tillotts, Takeda, Vifor Pharma, Pharmacosmos, Symprove. TTM receives support from Glaxo Smith Kline, Janssen Pharmaceuticals, Grunenthal, VH2 and Topivert. BH has received honoraria for acting in an advisory capacity or speaking on behalf of AbbVie, Takeda, Actavis UK. JWL, PB, AV, EP, AWW, ES, FA, RG, PS, JBC, EP, NG, JDS, JKH, JP and GML have no conflicts of interest to declare.

Acknowledgements

This study was supported by grants awarded by the Wellcome Trust (NP grant number WT 101159AIA, NP, GML, TTM, grant number WT088747MA, GML, grant number 091009, AWW, PS, JP, grant number 098051) and the Medical Research Council (GML, TTM, grant number G0802068, GML, JKH, grant number MR/K002996/1). AWW is funded by the Scottish Government Rural and Environmental Science and Analysis Service (RESAS). We are grateful to the Wellcome Trust Sanger Institute's core sequencing team for carrying out 16S rRNA gene sequencing, and to C. Evagora and support staff at the Pathology core at Queen Mary University of London. Research was also supported by the National Institute for Health Research (NIHR) Biomedical Research Centre at Guy's and St Thomas and King's

College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health.

Abstract:

Background & Aims: Innate lymphoid cells (ILC) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and humans with chronic intestinal inflammation.

Methods: ILCs were isolated from colons of Tbx2r-~ x Rag2~'~ mice (TRUC), which develop colitis, patients with inflammatory bowel disease (IBD), and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by ELISA and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota.

Results: IL17A- and IL22-producing, natural cytotoxicity receptor (NCR)-negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and ILla induced production of cytokines by these cells, which increased further following administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-g by human intestinal CD3-negative, IL7 receptor-positive cells, in a dose-dependent manner.

Conclusions: IL6 contributes to activation of colonic NCR-negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and ILla-induced production of IL17A and IL22. This pathway might be targeted to treat patients with

IBD, because IL6, which is highly produced in colonic tissue by some IBD patients, also increased production of IL17A, IL22, and interferon-g by cultured human colon CD3-negative, IL7 receptor-positive cells.

KEYWORDS: UC, CD, innate immunity, immune regulation

Introduction

Inflammatory bowel disease (IBD), comprising Crohn's disease (CD) and ulcerative colitis

(UC), is an increasingly common immune-mediated disease of the gut of unknown cause ' . The genetic architecture of IBD is complex, with over 130 significantly associated susceptibility loci identified to date3, indicating that multiple mechanisms of disease may exist. Nevertheless, prominent roles for innate immunity and particular immune response pathways, including the interleukin (IL) 23/IL17 axis are strongly implicated.

Innate lymphoid cells (ILCs) are emerging as important players in mucosal immunity. Although recognized to perform protective roles against mucosal pathogens4, 5, they also contribute to chronic intestinal inflammation, which is particularly apparent in mice lacking conventional T and B-cells6, 7 This is in part dependent on their capacity to produce

inflammatory cytokines, including interferon-g,

IL17A and IL224-8. ILCs can be subdivided into discrete populations, which accumulate in mucosal tissues in different pathological settings9. At least 3 subsets exist, including ILC1s which produce interferon-g ILC2s which produce IL5/IL13 and ILC3 which can be further subdivided based on differential expression of natural cytotoxicity receptors (NCRs), CD4 and production of IL17 and/or IL229.

Tbx21~ ~ Rag2~ ~ ulcerative colitis (TRUC)

mice spontaneously develop severe colitis with striking similarities to some aspects of human UC10. Colon lesions histologically resemble UC with goblet cell depletion, crypt abscess formation, epithelial hyperplasia and infiltration

of colonic lamina propria with neutrophils and mononuclear cells , . TRUC mice develop inflammation associated epithelial dysplasia, which frequently progresses to frank adenocarcinoma11, one of the most severe complications in human forms of IBD. TRUC disease is dependent on interactions between intestinal CD11c+ mononuclear phagocytes and

CD90 IL7R receptor positive (IL7R ) ILCs . Depletion of ILCs or genetic deficiency of the

common g-chain cytokine receptor, which is necessary for ILC survival, prevents disease . Similarly, blockade of IL23 or IL17A significantly attenuates disease . ILCs accumulate in gut lesions from IBD patients12-14 and it has been speculated that targeting these cells might represent a viable therapeutic approach in

IBD15. IL236, 7 and IL1p16 contribute to ILC activation, although curiously TRUC mice additionally deficient for either IL23R or IL1R are

not fully protected from colitis , consistent with a possible role for alternative ILC activation pathways contributing to disease. The purpose of this study was to investigate the proximal signals responsible for driving intestinal ILC activation and to determine whether similar pathways might exist in human disease.

Materials and Methods Mice

Balb/C Rag2 and wild type (WT) mice were sourced commercially (Jackson Labs). TRUC mice were a gift from Laurie Glimcher. Animal experiments were performed in accredited facilities in accordance with the UK Animals (Scientific Procedures) Act 1986 (Home Office Licence Number PPL: 70/6792 & PPL: 70/7869 from November 2013).

Human studies

Studies in human tissues received ethical approval from the City and Hackney Local

Research Ethics Committee (REC reference: 10/H0704/74 and 10/H0804/65). Colonic lamina

propria mononuclear cells (cLPMCs) were isolated as described previously from colectomy specimens and endoscopically acquired biopsies. Normal colonic mucosal samples were collected from macroscopically unaffected areas of patients undergoing intestinal resection for colon cancer or polyps. Informed written consent was obtained in all cases.

Flow Cytometry and cell sorting

Intracellular cytokine expression was measured as described previously7. Cells were stimulated with IL23 (10-20 ng/mL), IL6 (10-100 ng/mL) or PMA (50ng/mL) and ionomycin (1^M) for 4-6 hours at 37°C with monensin (3p,M) added for the last 2 hours. In human work antibodies used to stain cell surface antigens were incubated with unstimulated cells for 25 minutes and then fixed in 2% paraformaldehyde pending analysis. For FACS purification of murine

ILCs CD45+ cells

were first immunomagnetically sorted from unfractionated cLPMCs using anti-CD45 beads (Miltenyi) and LS columns. CD45+ cells were stained with CD90, NKp46 and IL7R. Antibodies used in flow cytometry experiments are listed in Supplemental table 1.

Ex vivo organ culture

Colon explants cultures from murine experiments were performed as described previously7. 3 biopsy punches from the distal colon were cultured in 500 pl of complete medium for 24

hours at 37°C. In human studies explant cultures were set up as described

previously .Cytokine production in culture supernatants were measured by ELISA. Cell culture

Unfractionated murine splenocytes (2 x 10 /mL) and mesenteric lymph node (mLN) cells

66 (1x10 /mL) or cLPMCs (1x10 /mL) were cultured in complete medium for 24 hours at 37°C

as described previously . cLPMCs from IBD and non-inflammatory control patients were

cultured with recombinant human IL6 (R&D systems) (0-100 ng/mL) overnight at 37°C, 5%

CO2 and then re-stimulated with PMA (50ng/mL) and ionomycin (1pM). In some

experiments cLPMCs were cultured with IL6 (100ng/mL) for 6 hours in the presence of

monensin (3pM). FACs purified NCR- ILC3s (CD45+ CD90+ IL7R+ NKp46-) from TRUC

mice were cultured at 5x104/mL for 24 hours. Cytokine concentrations in culture supernatants

were measured by ELISA (R&D Systems and eBioscience).

Histology

Colon histology was processed, stained (H&E), and colitis scores calculated as described previously7. Proximal and distal colitis scores from individual mice were averaged, unless otherwise stated.

ELISA and cytokine bead arrays

Cytokine concentrations were measured in culture supernatants by ELISA or Th1, Th2, Th17 CBA (BD Biosciences).

Microarray and real time PCR

RNA was extracted from 3x Rag2'-' and 3x TRUC mice aged 10 weeks old using Trizol reagent (Invitrogen). Transcript expression was analyzed with Mouse Genome 430 2.0

Affymatrix Expression Array. For real time PCR experiments cells were lyzed in Trizol reagent (Invitrogen) and RNA extracted. cDNA was generated with the cDNA synthesis kit (Bioline). Quantitative PCR was used to quantify mRNA transcripts using TaqMan gene expression assays (Applied Biosystems). Gene expression was normalized to the expression

of ß-actin to generate ACT values and relative abundance quantified using the 2" method. Human RORC (Hs01076112_m1) and ß-actin (4326315E) Taqman qPCR primer sets were used.

In vivo antibody treatment

Intraperitoneal injections of anti-CD4 (1 mg, GK1.5), anti-CD90 (1 mg, 30H12,), anti-IL6 (750 p,g, MP5-20F3) or isotype matched control antibodies (LTF-2 or HRPN) (Bio X Cell) were administered via to age matched TRUC mice on days 0, 7, 14, 21, and 28 (anti-CD4, anti-CD90 or days 0, 4, 9, 14, 18, 23 and 27 (anti-IL6).

Microbiota analysis

See Supplemental methods

Results

NCR- CD4' ILC3 cells are the predominant colonic ILC subset in chronic intestinal inflammation in TRUC mice

We validated the phenotype of ILCs in TRUC mice, confirming excessive accumulation of IL17A and IL22 producing CD90+ IL7R+ NCR" ILC3 in diseased colons (Figure 1A and 1B, Supplemental Figure 1A-D). CD4 expressing NCR- ILC3s resembling lymphoid tissue inducer (LTi) cells participate in mucosal immune responses in the gut5, therefore, we considered the possibility that CD4+ ILCs might be the NCR- ILC3 subset responsible for mediating chronic inflammation in TRUC mice. CD4+ cells were present in mesenteric lymph nodes (mLNs) of TRUC mice (many of which co-expressed CD90); however, very few CD4+ cells were present in the colon (Figure 1C). Given the low frequency of intestinal CD4+ ILCs in TRUC mice we considered it unlikely that these cells would play a major role in disease. To test this assumption we depleted CD4 expressing cells in vivo. Administration of anti-CD4 antibodies successfully depleted CD4 expressing cells in mLNs and colon of TRUC mice (Figure 1C). However, many CD90+ cells still remained in the colon and there was no reduction in the number of IL17A or IL22 producing cells (Figure 1D). Depleting anti-CD4 treatment did not significantly alter the severity of TRUC disease (Figure 1E). In contrast, anti-CD90 treatment depleted both CD90 and CD4 expressing ILCs, reduced the number of ILl7 and IL22 producing cells in the colon and significantly attenuated disease (Figure 1C-E). Taken together these data indicate IL17A/IL22 producing CD90+ IL7R+ NCR-CD4- ILC3 are the key ILC population in the colon responsible for causing disease in TRUC mice.

IL6 is highly expressed and augments pathogenic cytokine production in TRUC mice

We sought to define the proximal immune signals responsible for triggering effector function of colonic NCR- ILC3 in TRUC mice. ILip and IL6 were among the most highly expressed

(>2-fold induction) cytokine transcripts in the colon of TRUC mice in comparison with Rag2-- mice (Figure 2A). The other IL1 family member Il1a and the IL23 subunit transcripts (Il23a and Il12b) were also increased. Proximal cytokines responsible for driving ILC1 (IL12, IL15, IL18) or ILC2 (IL25, IL33) responses were not upregulated, and indeed in most instances were down-regulated in the colon of TRUC mice in comparison with Ragl'~ controls.

Although IL23 and IL1 family cytokines have been reported to stimulate ILCs, to our knowledge there are no data regarding a potential role for IL6 in the regulation of intestinal ILCs in intestinal inflammation. In addition to increased Il6 transcripts in the colon, there were very high concentrations of IL6 in serum and significantly increased production of IL6 in colon explant cultures from TRUC mice (Supplemental Figure 2A). Transcripts of genes known to be regulated by IL619 were upregulated in the colon of TRUC mice in comparison with Rag2/ controls (Supplemental Figure 2B), including well-recognized immune genes (Socs1, Socs3, Icam1), IL6 signalling components (Stat1 and Stat3) and anti-apoptotic genes (Bcl3, Bcl6, Bcl-x1). The most highly expressed IL6 regulated gene in the colon of TRUC

mice (12 fold enrichment) was Pou2af1, which encodes a transcriptional co-activator

responsible for IL6 mediated regulation of IL17 responses in T-cells .

To determine whether IL6 might trigger ILC derived cytokines we stimulated unfractionated cLPMCs and mLN cells from TRUC mice with recombinant IL6. Strikingly, IL6 triggered IL17A production by both cLPMCs and mLN cells (Figure 2B). We also performed flow cytometry with intracellular cytokine staining following IL6 stimulation of unfractionated mLN cells. Although less potent than IL23, IL6 induced expression of IL17A in NCR- ILC3s (Figure 2C). To determine whether this was a cell intrinsic phenomenon we purified CD90+ IL7R+ NCR- ILC3s from TRUC colons by fluorescence activated cell sorting (Supplemental Figure 3A). To our surprise neither IL6, IL23, nor IL1a, by themselves induced significant cytokine production by purified colonic NCR- ILC3s (Figure 2D). However, the combination

of IL23 and IL1a was a potent trigger for ILC production of IL17A and IL22. The addition of IL6 together with IL23 and IL1a was the most potent trigger of all. Purified intestinal NCR-ILCs from TRUC mice produced little TNFa or interferon-y under these conditions (Supplemental Figure 3B). IL23 and IL1a were weak inducers of IL6 by colonic NCR- ILCs (Supplemental Figure 3B). Taken together these data demonstrated that IL6 augments IL23/IL1a induced pathogenic cytokine production by intestinal ILCs in TRUC mice in a cell intrinsic manner.

IL6 signals through a heterodimeric receptor comprising ubiquitously expressed gp130 and selectively expressed IL6Ra. However, IL6Ra also exists as a soluble form which can complex with IL6 in solution and then bind to cells expressing gp130, enabling cells which do not usually express IL6Ra to respond to IL6 stimulation. Therefore, we investigated IL6Ra and soluble IL6Ra (sIL6Ra) expression in TRUC mice. IL6Ra expression by ILCs was highly variable in the colon of TRUC mice, but was typically less than 10% (Supplemental Figure 4A, and data not shown). However, sIL6Ra was abundant in the serum of TRUC mice and was detected in supernatants from cultured colon explants and unfractionated splenocytes (Supplemental Figure 4B). Therefore, it is likely that ILCs respond to IL6 stimulation directly, but also potentially through trans-signalling given the abundance of sIL6R in TRUC mice.

IL6 blockade attenuated TRUC disease independently of changes to intestinal microbiota community profiles

To determine whether IL6 mediated activation of innate immunity was functionally important in TRUC disease mice were treated with monoclonal antibodies that neutralize the biological activity of IL6. Treatment with anti-IL6 resulted in loss of IL6 bioavailability (Supplemental Figure 5A). IL17A production by unfractionated cLPMCs and splenocytes was significantly reduced in anti-IL6 treated TRUC mice, although was not completely abolished (Figure 3A).

IL6 neutralization significantly attenuated TRUC disease; including reduced colitis scores and reduced splenomegaly (Figure 3B-C).

Similar to the situation in human IBD, TRUC disease is associated with perturbation of

intestinal microbial communities. Since IL6 directly influences the success of mucosal

colonization by some intestinal bacteria , we considered the possibility that attenuation of chronic TRUC disease following IL6 blockade might have occurred secondarily to changes in key components in the composition of the intestinal microbiota. To address this question we sequenced 16S ribosomal RNA genes PCR amplified from faecal samples from anti-IL6 or control antibody treated TRUC mice. Overall we identified 2,642 different Operational Taxonomic Units (OTUs). Treatment with anti-IL6 antibody appeared to have a relatively minor impact on the microbiota (Figure 4A). At the phylum level, Firmicutes were slightly reduced in proportional abundance in anti-IL6 treated mice (P=0.035) (Figure 4A) but, at finer taxonomic levels, anti-IL6 treatment did not significantly impact the proportional abundance of the most common 150 OTUs, which cumulatively accounted for >96% of the total amount of sequence data generated (Supplemental Table 2). Cluster analysis, using the Bray Curtis calculator, confirmed that there was no signature microbiota profile associated with anti-IL6 treatment (Supplemental Figure 5B). H. typhlonius was ubiquitously present in anti-IL6 or isotype control treated mice, however, the proportional abundance did not differ significantly between the two groups either before or after treatment (Figure 4B). There was a tendency for increased bacterial diversity in the gut of anti-IL6 treated mice in comparison with control antibody treated mice although this did not achieve statistical significance (P<0.095, Supplemental Figure 5C).

IL6 augments pathogenic cytokine production by colonic CD3' IL7R+ cells from IBD patients

Our preclinical data support the possibility that targeting IL6 may be therapeutically tractable in chronic gut inflammation. Therefore, we aimed to verify whether this pathway was relevant in human disease. As expected, stimulation of unfractionated intestinal immune cells with PMA and ionomycin resulted in production of pathogenic cytokines, including IL17A, IL22 and interferon-g by CD3+ T-cells (Figure 5A and Supplemental Figure 6A). However, we also observed production of these cytokines in the non-T-cell (CD3-) fraction, particularly in IBD patients. Within the non-T-cell fraction (CD3-) we could identify a population of IL7R expressing cells in the colon of patients with CD, UC as well as non-inflammatory control patients (Figure 5B). Although the frequency of these cells was variable, their proportional abundance within the lymphocyte population was increased in IBD patients in comparison with non-inflammatory controls (Figure 5B). Consistent with ILC3s being present among the CD3- IL7R+ population, there was enriched expression of RORgt and c-kit (CD117) (Figure 5C). RORC transcripts were also enriched in FACS purified CD3- IL7R+ cells analysed by real time PCR (Supplemental Figure 6B), corroborating the likelihood of ILCs being present in the CD3- IL7R+ population. c-kit expression was also variable among CD3- IL7R+ cells, although the median proportion of cells expressing c-kit was >60% in CD, UC and non-inflammatory control patients express, consistent with the majority of these cells being ILCs (Supplemental Figure S6C). Analysis of the CD3- IL7R+ population according to NCR expression revealed the presence of 3 discrete populations, comprising NKp46+ NKp44-cells, NKp44+ NKp46- cells and NCR- (NKp44- NKp46-) cells (Figure 5D and Supplemental Figure 6C), indicating that NCR+ and NCR- ILCs are present within this CD3- IL7R population. In most patients, including IBD and non-inflammatory control patients, CD3-IL7R+ NKp44+ NKp46- cells were the predominant subset present (Figure 5D and Supplemental Figure 6C).

To determine whether CD3- IL7R+ cells present in diseased mucosa of IBD patients were responsive to IL6, cLPMCs were incubated overnight with recombinant human IL6 before being restimulated with PMA and ionomycin. Production of IL17A, IL22 and interferon-g by CD3" IL7R+ cells was significantly increased when cLPMCs were cultured in the presence of IL6 (Figure 6A-B). Additionally, some samples were stimulated with IL6 directly without mitogen, which demonstrated induction of IL17A by CD3- IL7R+ cells in a dose dependent manner (Figure 6C).

Finally, we analyzed IL6 production by diseased mucosa from IBD patients to see whether blocking IL6 might be a reasonable therapeutic strategy in some or all IBD patients. IL6 was produced by colon explant cultures in CD, UC and non-inflammatory control patients (Supplemental Figure 6D). However, IL6 production was variable, especially in IBD patients ranging from 72.2pg/mg colonic tissue to 8426.4pg/mg colonic tissue. IBD patients could be stratified according to mucosal production of IL6, with half of IBD patients producing relatively low levels comparable to non-inflammatory control patients and the other half producing high amounts (>1000pg/mg tissue). Taken together these data indicate that IL6, which is produced in very high quantities in approximately 50% of CD and UC patients drives pathologically relevant immune pathways in chronic intestinal inflammation.

Discussion

CD4" NCR- ILC3s are the predominant CD90+ IL7R+ ILC population in the colon of TRUC mice responsible for causing disease. Purified intestinal NCR- ILC3 from TRUC mice produced IL17A and IL22 but were poor producers of interferon-y and TNFa. They were also a modest source of IL6. Few NCR+ ILCs were present in the colon of TRUC mice, consistent

with data from other groups reporting a requirement for T-bet in NKp46+ ILC development

and differentiation . Intestinal CD4 ILCs are important in host resistance to intestinal pathogens, such as Citrobacter rodentium5. Here, we show CD4+ ILCs, which are abundant in mLN, but infrequent in the colon, do not play a major role in TRUC disease. Depletion of CD4+ ILCs had no impact on pathogenic cytokine production or disease outcome.

In TRUC mice highly purified colonic NCR- ILC3 did not respond to IL23 or IL1a in isolation. Instead combinations of IL23 together with IL1a were required for production of effector cytokines by ILC3. Further, additional exposure to IL6 was required for optimal IL17A and IL22 production, revealing a novel role for IL6 in the innate immune system in chronic intestinal inflammation.

IL17A, IL22 and interferon-y producing CD3- IL7R+ cells were also identified in the colonic lamina propria of patients with IBD. Although this population is heterogeneous, there was enrichment of RORgt and c-kit, confirming the likelihood that ILC3 were present within this compartment. Most CD3- IL7R+ cells were NKp44+, although NKp46+ and NCR- (NKp44-NKp46-) cells were also present. These data are broadly consistent with previous reports of ILC populations in human gut12-14. Crucially, IL6 increased pathogenic cytokine production by CD3- IL7R+ cLPMCs from IBD patients in a dose dependent manner, consistent with our preclinical data showing IL6 responsive colonic ILC3s.

IL6 is a pleiotropic cytokine that may be important in IBD. Peripheral blood and cLPMCs

23 24 25

produce excess IL6 in IBD 24, often at levels correlating with disease activity . Genetic variation at the IL6 locus is linked with early-onset IBD26 and polymorphisms at loci encoding IL6 receptor (IL6R) signalling components are associated with increased IBD risk . IL6 blockade is therapeutic in some preclinical models of IBD, although it has been assumed that the therapeutic mechanism was likely attributable to limitation of T-cell mediated

27-30 31

pathology - , since IL6 contributes to intestinal Th17 differentiation . We now show a novel role of IL6 in innate immune mediated chronic intestinal pathology. It is interesting that cytokines contributing to CD4+ Th17 differentiation, including IL1, IL23 and IL6 have conserved roles promoting innate IL17 production. Our data build on other work implicating ILCs as potentially important mediators in

IBD12-14, and confirm NCR- ILC3 as a source of pathogenic cytokines in IBD. Polymorphisms at multiple susceptibility loci in IBD that were previously considered to impact adaptive immunity, could similarly impact ILC phenotype, including RORC, IL23R, IL12RB2, IL12B, IL22, IFNG, STAT1, STAT3, STAT4, CCR6, IL1R1, IL15RA, and IL6Sf.

Accordingly, it is possible that genetic variation at these loci in IBD could impact disease susceptibility by altering the activation and effector function of mucosal ILCs. However, the relative contribution of ILC to the initiation and propagation of chronic intestinal inflammation in IBD remains to be determined. Polyclonal stimulus of unfractionated cLPMCs from IBD patients demonstrated that most cytokine expressing cells reside within the CD3+ cell fraction. It should be remembered that cytokine responses induced by polyclonal stimuli may overestimate T-cell contribution, since under physiological conditions few of these tissue trafficking T-cells would be encountering their relevant antigen, so would unlikely be triggered to produce cytokine. By contrast, despite their numerical inferiority to T-cells, mucosal ILC are likely to be activated directly by cytokine signals abundant in chronically inflamed tissue, such as IL6. IL6 induced

stimulation of ILC effector function may prove to be especially pertinent in UC since IL23, the canonical ILC activating cytokine, is produced at low levels in UC in comparison with CD32.

In this study IL6 neutralization reduced innate production of IL17A in TRUC mice and significantly attenuated disease severity, although the magnitude of impact was less than seen with ILC depletion or

IL23 blockade7. This is in keeping with our observation that although IL6 is required for optimal activation of ILC effector function, other proximal cytokine signals, including IL23 and/or IL1 stimulation are additionally required. IL6 blockade had minimal impact on the intestinal microbiota, other than a minor shift in the proportional abundance of Firmicutes and a tendency for increased intestinal bacterial diversity. It is possible that this latter change occurred secondary to reduced intestinal inflammation in anti-IL6 treated mice. Indeed, IBD activity/severity is recognized to inversely correlate with

bacterial diversity in the gut33 34.

Our data support extending biological therapies targeting IL6 in IBD. The IL6R blocking antibody tocilizumab is efficacious in other inflammatory diseases, including arthritis35, 36 and

lupus , and a pilot study in CD showed promising initial results . In this study mucosal IL6 production was highly variable, yet only half of IBD patients produced more IL6 than non-IBD control patients. Similarly, the frequency of CD3- IL7R+ cells, and the magnitude of IL6 induced cytokine responses by these cells was also markedly variable. With the promise of

personalised medicine on the horizon,39 it is tempting to speculate that treatment strategies targeting IL6 might be favoured in patient subsets defined by high mucosal expression of IL6 and/or high frequencies of IL6 responsive effector cells in diseased tissue. In summary, we have shown that IL6 augments pathogenic cytokine production by intestinal ILCs in chronic intestinal inflammation and that this pathway may be operational in human

IBD. Novel therapeutic strategies targeting ILC or their proximal cytokine signals may offer a new treatment paradigm in IBD.

Figure Legends

Figure 1: IL17A/IL22 producing CD4- NCR- ILC3 mediate colitis in TRUC mice

(A) Flow cytometry dot plots of live, CD45+ cells according to expression of CD90 and IL7R in the colon of Rag2-/- and TRUC mice, and (B) absolute numbers of live, CD45+ CD90+ IL7R+ ILCs in the colon of TRUC and Rag2'- mice. Each dot/square represents an individual mouse. Line depicts median. (C) Representative flow cytometry dot plots (left side of panel) and statistical analysis (right side of panel) of the proportion of CD4+ and CD90+ cells (gated on live, CD45+ cells) in mLN and colon of TRUC mice treated with isotype matched control antibodies (n=5), anti-CD4 (n=4) or anti-CD90 (n=4) antibodies. Statistical analyses were performed on colonic cells and show the proportion of colonic ILCs (cILCs) after treatment. *P<0.019, **P<0.04. (D) Representative flow cytometry dot plots (left side of panel) and statistical analysis (right side of panel) of the proportion of IL17A+ and IL22+ cells (gated on live, CD45+ cells) in the colon of TRUC mice treated with isotype matched control antibodies (n=5), anti-CD4 (n=4) or anti-CD90 (n=4) antibodies. Cells were stimulated with PMA and ionomycin prior to intracellular cytokine staining *P<0.01. (E) Representative colon micrographs (haematoxylin and eosin stained, left side of panel) and statistical analysis of colitis scores (right side of panel) of TRUC mice treated with isotype matched control antibodies, anti-CD4 or anti-CD90 antibodies. *P<0.03 (for both anti-CD90 vs control antibody and anti-CD90 vs anti-CD4). Each dot/square represents an individual mouse. Lines depict median.

Figure 2: IL6 promotes cytokine production by NCR- ILC3s in a cell intrinsic manner

(A) Microarray analysis showing abundance of cytokine transcripts in the colon of TRUC mice relative to Rag2'-' mice. Blue dotted line depicts 2-fold induction. (B) IL17A production by unfractionated cLPMCs and mLN cells isolated from TRUC mice in medium alone (-) or following supplementation with recombinant IL6 or IL23. Columns represent mean cytokine

and error bars depict SEM. Analysis of cLPMCs comprised 4 biological replicates. Analysis of mLN included 9 biological replicates for the unstimulated condition and 7 biological replicates for each of the stimulated conditions. *P<0.02. **P<0.003. (C) Flow cytometry plots of intracellular IL17A expression by CD90+ NKp46- cells following stimulation of unfractionated mLN cells with IL6, IL23 or unstimulated cells (-), which were incubated with monensin alone. Data are representative of 3 separate experiments. (D) Cytokine production by FACS sorted CD45+ CD90+ IL7R+ NKp46- ILCs purified from the colon of TRUC mice (Gating strategy for cell sorting is illustrated in Supplemental Figure 3A). Purified NCR-ILCs were stimulated with combinations of IL1a, IL6 and IL23 as depicted. After 24 hours cytokine concentrations were measured in culture supernatant by ELISA or CBA. Data are representative of 2 individual experiments with ILCs pooled from 10-15 colons. Bars show mean cytokine production and error bars depict SEM. See also Supplemental Figure 3B.

Figure 3: IL6 blockade reduces IL17A production and attenuates TRUC disease

(A) IL17A concentration in culture supernatants of unfractionated cLPMCs and splenocytes from TRUC mice treated with anti-IL6 (n=8) or isotype matched control antibodies (n=8).

(B) Representative colon micrographs (haematoxylin and eosin stained, left panel) and statistical analysis (right panel) of colitis scores of distal colon of TRUC mice treated with anti-IL6 or isotype matched control antibodies. (C) Spleen and colon mass of TRUC mice treated with anti-IL6 or isotype matched control antibodies. Each dot/square represents an individual mouse. Lines represent medians. Results from 2 separate antibody blockade experiments conducted under the same experimental conditions were pooled.

Figure 4: IL6 blockade does not significantly impact on the composition of the intestinal microbiota in TRUC mice

(A) Mean percentage of sequences of particular phyla present in the intestinal microbiota of TRUC mice before (top panel) and after (bottom panel) treatment with anti-IL6 (red bars) or isotype matched control antibodies (white bars). (B) Mean proportional abundance of Helicobacter typhlonius in the intestinal microbiota of TRUC mice before and after treatment with anti-IL6 (red bars) or isotype matched control antibodies (white bars). Error bars depict SEM.

Figure 5: CD3- IL7R+ cells are expanded in IBD patients

(A) Flow cytometry plots showing CD3 and intracellular IL17A expression in unfractionated cLPMCs following stimulation with PMA and ionomycin. Additional representative flow cytometry plots are illustrated in Supplemental Figure 6A. (B) Representative flow cytometry plots (left panel) and statistical analysis (right panel) of CD3 and IL7R staining by LPMCs cells in the colon of non-inflammatory control and IBD patients. Individual dots represent individual patients. * P<0.04. (C) Flow cytometric analysis of the phenotype of colonic CD3-IL7R+ cells. Grey histograms depict isotype control antibody staining. White histograms show staining with specific antibody. Data are representative of >3 independent experiments in IBD patients. (D) Flow cytometry dot plots showing expression of NKp46 and NKp44 by colonic CD3" IL7R+ cells in non-inflammatory control and IBD patients. Further analyses of additional patient replicates are shown in Supplemental Figures 6C-D.

Figure 6: IL6 responsive CD3- IL7R+ cells are present in the colon of IBD patients.

(A) Flow cytometry histograms and (B) statistical analyses of intracellular cytokine expression by CD3- IL7R+ cells following overnight culture in the presence or absence of IL6 (100 ng/mL). Cells were restimulated with PMA and ionomycin prior to staining. In (B) each

connected pair of dots represents an individual patient. (C) Flow cytometry histograms showing number of CD3- IL7R+ IL17A+ cells following culture with increasing doses of IL6.

References

1. Abraham C, Cho JH. Inflammatory bowel disease. N Engl J Med 2009;361:2066-78.

2. Molodecky NA, Soon IS, Rabi DM, et al. Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology 2012;142:46-54 e42; quiz e30.

3. Jostins L, Ripke S, Weersma RK, et al. Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature 2012;491:119-24.

4. Satoh-Takayama N, Vosshenrich CA, Lesjean-Pottier S, et al. Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense. Immunity 2008;29:958-70.

5. Sonnenberg GF, Monticelli LA, Elloso MM, et al. CD4(+) lymphoid tissue-inducer cells promote innate immunity in the gut. Immunity 2011;34:122-34.

6. Buonocore S, Ahern PP, Uhlig HH, et al. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology. Nature 2010;464:1371-5.

7. Powell N, Walker AW, Stolarczyk E, et al. The transcription factor T-bet regulates intestinal inflammation mediated by interleukin-7 receptor+ innate lymphoid cells. Immunity 2012;37:674-84.

8. Takatori H, Kanno Y, Watford WT, et al. Lymphoid tissue inducer-like cells are an innate source of IL-17 and IL-22. J Exp Med 2009;206:35-41.

9. Spits H, Artis D, Colonna M, et al. Innate lymphoid cells--a proposal for uniform nomenclature. Nat Rev Immunol 2013;13:145-9.

10. Garrett WS, Lord GM, Punit S, et al. Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system. Cell 2007;131:33-45.

11. Garrett WS, Punit S, Gallini CA, et al. Colitis-associated colorectal cancer driven by T-bet deficiency in dendritic cells. Cancer Cell 2009;16:208-19.

12. Geremia A, Arancibia-Carcamo CV, Fleming MP, et al. IL-23-responsive innate lymphoid cells are increased in inflammatory bowel disease. J Exp Med 2011;208:1127-33.

13. Bernink JH, Peters CP, Munneke M, et al. Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues. Nat Immunol 2013;14:221-9.

14. Fuchs A, Vermi W, Lee JS, et al. Intraepithelial type 1 innate lymphoid cells are a unique subset of IL-12- and IL-15-responsive IFN-gamma-producing cells. Immunity 2013;38:769-81.

15. Goldberg R, Prescott N, Lord GM, et al. The unusual suspects - innate lymphoid cells: novel therapeutic targets in IBD. Nature Reviews in Gastroenterology and Hepatology 2015;(In press).

16. Coccia M, Harrison OJ, Schiering C, et al. IL-1beta mediates chronic intestinal inflammation by promoting the accumulation of IL-17A secreting innate lymphoid cells and CD4(+) Th17 cells. J Exp Med 2012;209:1595-609.

17. Ermann J, Staton T, Glickman JN, et al. Nod/Ripk2 signaling in dendritic cells activates IL-17A-secreting innate lymphoid cells and drives colitis in T-bet-/-.Rag2-/-(TRUC) mice. Proc Natl Acad Sci U S A 2014;111:E2559-66.

18. Rovedatti L, Kudo T, Biancheri P, et al. Differential regulation of interleukin 17 and interferon gamma production in inflammatory bowel disease. Gut 2009;58:1629-36.

19. Brocke-Heidrich K, Kretzschmar AK, Pfeifer G, et al. Interleukin-6-dependent gene expression profiles in multiple myeloma INA-6 cells reveal a Bcl-2 family-independent survival pathway closely associated with Stat3 activation. Blood 2004;103:242-51.

20. Yosef N, Shalek AK, Gaublomme JT, et al. Dynamic regulatory network controlling TH17 cell differentiation. Nature 2013;496:461-8.

21. Dann SM, Spehlmann ME, Hammond DC, et al. IL-6-dependent mucosal protection prevents establishment of a microbial niche for attaching/effacing lesion-forming enteric bacterial pathogens. J Immunol 2008;180:6816-26.

22. Klose CS, Kiss EA, Schwierzeck V, et al. A T-bet gradient controls the fate and function of CCR6-RORgammat+ innate lymphoid cells. Nature 2013;494:261-5.

23. Reinecker HC, Steffen M, Witthoeft T, et al. Enhanced secretion of tumour necrosis factor-alpha, IL-6, and IL-1 beta by isolated lamina propria mononuclear cells from patients with ulcerative colitis and Crohn's disease. Clin Exp Immunol 1993;94:174-81.

24. Suzuki Y, Saito H, Kasanuki J, et al. Significant increase of interleukin 6 production in blood mononuclear leukocytes obtained from patients with active inflammatory bowel disease. Life Sci 1990;47:2193-7.

25. Hyams JS, Fitzgerald JE, Treem WR, et al. Relationship of functional and antigenic interleukin 6 to disease activity in inflammatory bowel disease. Gastroenterology 1993;104:1285-92.

26. Sagiv-Friedgut K, Karban A, Weiss B, et al. Early-onset Crohn disease is associated with male sex and a polymorphism in the IL-6 promoter. J Pediatr Gastroenterol Nutr;50:22-6.

27. Kitamura K, Nakamoto Y, Kaneko S, et al. Pivotal roles of interleukin-6 in transmural inflammation in murine T cell transfer colitis. J Leukoc Biol 2004;76:1111-7.

28. Naito Y, Takagi T, Uchiyama K, et al. Reduced intestinal inflammation induced by dextran sodium sulfate in interleukin-6-deficient mice. Int J Mol Med 2004;14:191-6.

29. Mitsuyama K, Matsumoto S, Rose-John S, et al. STAT3 activation via interleukin 6 trans-signalling contributes to ileitis in SAMP1/Yit mice. Gut 2006;55:1263-9.

30. Atreya R, Mudter J, Finotto S, et al. Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in crohn disease and experimental colitis in vivo. Nat Med 2000;6:583-8.

31. Hu W, Troutman TD, Edukulla R, et al. Priming microenvironments dictate cytokine requirements for T helper 17 cell lineage commitment. Immunity 2011;35:1010-22.

32. Kamada N, Hisamatsu T, Okamoto S, et al. Unique CD14 intestinal macrophages contribute to the pathogenesis of Crohn disease via IL-23/IFN-gamma axis. J Clin Invest 2008;118:2269-80.

33. Manichanh C, Rigottier-Gois L, Bonnaud E, et al. Reduced diversity of faecal microbiota in Crohn's disease revealed by a metagenomic approach. Gut 2006;55:205-11.

34. Ott SJ, Musfeldt M, Wenderoth DF, et al. Reduction in diversity of the colonic mucosa associated bacterial microflora in patients with active inflammatory bowel disease. Gut 2004;53:685-93.

35. Smolen JS, Beaulieu A, Rubbert-Roth A, et al. Effect of interleukin-6 receptor inhibition with tocilizumab in patients with rheumatoid arthritis (OPTION study): a double-blind, placebo-controlled, randomised trial. Lancet 2008;371:987-97.

36. Yokota S, Imagawa T, Mori M, et al. Efficacy and safety of tocilizumab in patients with systemic-onset juvenile idiopathic arthritis: a randomised, double-blind, placebo-controlled, withdrawal phase III trial. Lancet 2008;371:998-1006.

37. Illei GG, Shirota Y, Yarboro CH, et al. Tocilizumab in systemic lupus erythematosus: data on safety, preliminary efficacy, and impact on circulating plasma cells from an open-label phase I dosage-escalation study. Arthritis Rheum 2010;62:542-52.

38. Ito H, Takazoe M, Fukuda Y, et al. A pilot randomized trial of a human anti-interleukin-6 receptor monoclonal antibody in active Crohn's disease. Gastroenterology 2004;126:989-96; discussion 947.

39. Biancheri P, Powell N, Monteleone G, et al. The challenges of stratifying patients for trials in inflammatory bowel disease. Trends Immunol 2013;34:564-71.

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Supplemental methods Microbiota analysis

DNA was extracted from mouse faecal samples using the FastDNA® SPIN Kit for Soil and a FastPrep 24 machine (MP Biomedicals) according to the protocol provided by the manufacturer. Bacterial 16S rRNA genes were PCR amplified using barcoded primers MiSeq 27F(5'AATGATACGGCGACCACCGAGATCTACACTATGGTAATTCCAGMGTTYGA TYMTGGCTCAG-3') and MiSeq-338R (5' -CAAGCAGAAGACGGCATACGAGAT-barcode-AGTCAGTCAGAAGCTGCCTCCCGTAGGAGT-3'), which target variable regions V1-V2 of the 16S rRNA gene. Q5™ Taq polymerase (New England Biolabs) was used for the PCR step, and four PCR reactions were carried out per sample. Cycling conditions were: 98°C for 2 mins, followed by 20 cycles of 98°C for 30 secs, 50°C for 30 secs, 72°C for 1 min 30 secs and then a final extension step at 72°C for 5 mins. The four PCR reactions from each DNA extraction were then pooled and concentrated down to 25 |il volumes per sample. PCR amplicons were then quantified using a Qubit 2.0 Fluorometer (Life Technologies Ltd) and equimolar concentrations of each were added to a final mastermix for sequencing, using an Illumina MiSeq machine with 2 x 250 bp read length. Sequence data has been deposited in the European Nucleotide Archive under Study Accession Number ERP005850 and Sample Accession numbers ERS459682 - ERS459702. The sequence data was processed by following the MiSeq SOP of the mothur software package(Kozich et al., 2013). Paired-read contigs were created from the forward and reverse read sequence data, and preliminary quality processing was carried out by removing all contigs that were shorter than 260 bp, longer than 450 bp or those that contained any ambiguous bases or homopolymeric stretches longer than 7 bases. Perseus(Quince et al., 2011), as implemented in mothur, was used to remove putative chimeras, and any reads mapping to chloroplasts, mitochondria, eukarya, or archaea were also removed. After these

steps over 486,000 sequences were left in the final dataset (range of 447 to 51,571 sequences per sample). Next, following a pre-clustering step (diffs=3), 97% similarity OTUs were generated. Taxonomic classifications for each of these OTUs were created using the RDP taxonomy provided at the mothur web page. Metastats(White et al., 2009), as implemented in mothur, was used to test for significant differences in the proportional abundance of each of the phyla present, and the 150 most abundant OTUs between anti-IL6 and control mice. Bacterial diversity was measured by generating Shannon and inverse Simpson indices in mothur. Prior to these calculations each sample was randomly subsampled down to 447 reads to ensure equal sequencing depth for each. Kruskall-Wallis and Mann Whitney U tests, implemented in Minitab v16, were used to test for significant differences in diversity measures between the anti-IL6 and control mouse groups. A cluster dendrogram, using the Bray Curtis calculator, was also generated in mothur from the subsampled dataset, and subsequently visualised using the iTOL online software resource (Letunic and Bork, 2011).

Supplemental methods references

Kozich, J.J., Westcott, S.L., Baxter, N.T., Highlander, S.K., and Schloss, P.D. (2013). Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Applied and environmental microbiology 79, 5112-5120. Letunic, I., and Bork, P. (2011). Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucleic acids research 39, W475-478. Quince, C., Lanzen, A., Davenport, R.J., and Turnbaugh, P.J. (2011). Removing noise from pyrosequenced amplicons. BMC bioinformatics 12, 38.

White, J.R., Nagarajan, N., and Pop, M. (2009). Statistical methods for detecting differentially abundant features in clinical metagenomic samples. PLoS computational biology 5, e1000352.

Supplemental Table 1: Flow cytometry antibodies used

Anti-mouse antibodies

Antigen Clone Supplier

CD45 30-F11 eBioscience

CD90.2 53-2.1 eBioscience

CD127 A7R34 eBioscience

NKp46 29A1.4 eBioscience

CD126 D7715A7 Biolegend

CCR6 140706 R&D Systems

ICOS 7E.17G9 eBioscience

CD4 RM4.5 eBiosciences

Hematopoietic lineage cocktail (CD3, CD45R, B220, CD11b, TER-119, Gr-1) clones: 17A2, RA3-6B2, M1/70, TER-119) eBioscience

CD62L MEL-14 eBiosciences

IL17RB 752101 R&D Systems

CD69 H1.2F3 eBioscience

RORyt AFKJS-9 eBioscience

IL17A eBio17B7 eBioscience

IL22 1H8PWSR eBioscience

Interferon-y XMG1.2 eBioscience

IL4 11B11 eBioscience

Anti-human antibodies

Antigen Clone Supplier

CD3 UCHT-1 Biolegend

CD127 A019D5 (eBioRDR5) Biolegend (eBioscience)

CD117 104D2 eBioscience

NKp46 9E2 eBioscience

NKp44 44.189 eBioscience

IL17RB I170220 R&D Systems

RORyt AFKJS-9 eBioscience

IL17A eBio64DEC17 eBioscience

IL22 22URTI eBioscience

Interferon-y 4S.B3 eBioscience

Supplemental Table - Metastats comparison between the proportional abundance of top 150 most abundant OTUs in the microbiota dataset between anti-IL-6 and control mice groups.

RDP Taxonomic Classifications

OTU No. Phylum Class Order Family Genus NCBI MegaBLAST ID p-value q-value

Otu0001 Firmicutes(100) Bacilli(100) Lactobacillales( 100) Lactobacillaceae( 100) Lactobacillus(100) Lactobacillus animalis/murinus 0.287595 1

Otu0002 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) Butyrivibrio sp. P79 (86% similarity) 0.022376 1

Otu0003 Firmicutes(100) Bacilli(100) Lactobacillales( 100) Lactobacillaceae( 100) Lactobacillus(100) Lactobacillus taiwanensis/johnsonii/acidophilus 0.947484 1

Otu0004 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Clostridium_XlVb(100) Clostridium lactatifermentans (90% similarity) 0.15343 1

Otu0005 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) Eubacterium plexicaudatum (92% similarity) 0.82004 1

Otu0006 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) unclassified(100) unclassified(100) Butyricimonas sp. JCM 18677 (80% similarity) 0.012182 1

Otu0007 Proteobacteria(100) Epsilonproteobacteria(100) Campylobacterales(100) Helicobacteraceae(100) Helicobacter(100) Helicobacter typhlonius 0.173889 1

Otu0008 Deferribacteres(100) Deferribacteres(100) Deferribacterales(100) Deferribacteraceae(100) - Mucispirillum( 100) Mucispirillum colimuris 0.063365 1

Otu0009 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Rikenellaceae(100) Alistipes(100) Alistipes onderdonkii (90% similarity) 0.874014 1

Otu0010 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Rikenellaceae(100) Alistipes(100) Alistipes senegalensis (91% similarity) 0.800781 1

Otu0011 Firmicutes(100) Clostridia(100) Clostridiales(100) unclassified(100) unclassified(100) Clostridium scindens (88% similarity) 0.766219 1

Otu0012 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) Coprococcus catus (88% similarity) 0.571687 1

Otu0013 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Oscillibacter(100) Oscillibacter valericigenes (91% similarity) 0.03155 1

Otu0014 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Bacteroidaceae(100) Bacteroides(100) Bacteroides acidofaciens/uniformis 0.082054 1

Otu0015 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Anaerotruncus(100) Anaerotruncus colihominis (92% similarity) 0.489023 1

Otu0016 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) Clostridium hathewayi (92% similarity) 0.861923 1

Otu0017 Firmicutes(100) Bacilli(100) Lactobacillales( 100) Lactobacillaceae( 100) Lactobacillus(100) Lactobacillus reuteri 0.644746 1

Otu0018 Proteobacteria(100) Deltaproteobacteria( 100) unclassified(100) unclassified(100) unclassified(100) Desulfocurvus vexinensis (87% similarity) 0.44467 1

Otu0019 Deferribacteres(100) Deferribacteres(100) Deferribacterales(100) Deferribacteraceae(100) Mucispirillum( 100) Mucispirillum colimuris (97% similarity) 0.039672 1

Otu0020 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) unclassified(99) unclassified(99) Tannerella forsythensis (82% similarity) 0.262652 1

Otu0021 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Bacteroidaceae(100) Bacteroides(100) Bacteroides acidifaciens 0.733786 1

Otu0022 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Prevotellaceae(100) Paraprevotella(100) Prevotella sp. (87% similarity) 0.137208 1

Otu0023 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae(52) unclassified(52) Coprococcus catus (84% similarity) 0.501202 1

Otu0024 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) Pseudobutyrivibrio ruminis (88% similarity) 0.510185 1

Otu0025 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Porphyromonadaceae( 100) Paludibacter(77) Prevotella dentalis (81% similarity) 0.162484 1

Otu0026 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Bacteroidaceae(100) Bacteroides(100) Bacteroides uniformis (96% similarity) 0.195279 1

Otu0027 Firmicutes(100) Erysipelotrichia(99) Erysipelotrichales(99) Erysipelotrichaceae(99) Erysipelotrichaceae incertae sedis(99) Eubacterium cylindroides (88% similarity) 0.605205 1

Otu0028 Firmicutes(100) Clostridia(100) Clostridiales(100) unclassified(100) unclassified(100) Clostridium aminophilum (86% similarity) 0.246769 1

Otu0029 TM7(100) TM7_class_incertae_sedis(100) TM7_order_incertae_sedis(100) TM7 family incertae sedis(100) TM7_genus_incertae_sedis(100) TM7 phylum sp. (93% similarity) 0.222701 1

Otu0030 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) Clostridium phytofermentans (90% similarity) 0.574396 1

Otu0031 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) Clostridium celerecrescens (93% similarity) 0.664006 1

Otu0032 Firmicutes(100) Clostridia(100) Clostridiales(100) unclassified(100) unclassified(100) Clostridia scindens (84% similarity) 0.737231 1

Otu0033 Proteobacteria(100) Gammaproteobacteria( 100) Enterobacteriales( 100) Enterobacteriaceae(100) unclassified Escherichia/Enterobacter/Citrobacter/Shigella spp. 0.497501 1

Otu0034 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Rikenellaceae(100) Alistipes(100) Alistipes senegalensis (92% similarity) 0.127302 1

Otu0035 Firmicutes(100) unclassified(100) unclassified(100) unclassified(100) unclassified(100) Segmented filamentous bacterium 0.798342 1

Otu0036 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) Eubacterium oxidoreducens (87%) 0.334068 1

Otu0037 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Oscillibacter(100) 0.263709 1

Otu0038 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) unclassified(100) unclassified(100) 0.951701 1

Otu0039 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Bacteroidaceae(100) Bacteroides(100) 0.178105 1

Otu0040 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Lachnospiracea incertae sedis(79) 0.987598 1

Otu0041 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Oscillibacter(100) 0.948299 1

Otu0042 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Bacteroidaceae(100) Bacteroides(100) 0.334148 1

Otu0043 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Dorea(93) 0.850598 1

Otu0044 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Anaerotruncus(100) 0.622208 1

Otu0045 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.948849 1

Otu0046 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Rikenellaceae(100) Alistipes(100) 0.211071 1

Otu0047 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Clostridium_XlVb(100) 0.449205 1

Otu0048 Firmicutes(100) Clostridia(100) Clostridiales(100) unclassified(100) unclassified(100) 0.101219 1

Otu0049 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Butyricicoccus( 100) 0.597761 1

Otu0050 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Porphyromonadaceae(100) Odoribacter(100) 0.824846 1

Otu0051 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(72) 0.889006 1

Otu0052 Bacteroidetes(100) unclassified(99) unclassified(99) unclassified(99) unclassified(99) 0.352835 1

Otu0053 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.420749 1

Otu0054 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Oscillibacter(100) 0.807154 1

Otu0055 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Roseburia(100) 0.549208 1

Otu0056 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.795695 1

Otu0057 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Acetanaerobacterium( 100) 0.795544 1

Otu0058 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.594923 1

Otu0059 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.319484 1

Otu0060 Firmicutes(100) unclassified(100) unclassified(100) unclassified(100) unclassified(100) 0.815476 1

Otu0061 Firmicutes(100) Clostridia(100) Clostridiales(100) unclassified(100) unclassified(100) 0.245678 1

Otu0062 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Roseburia(100) 0.642752 1

Otu0063 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.866248 1

Otu0064 Firmicutes(100) Clostridia(100) Clostridiales(100) unclassified(100) unclassified(100) 0.684305 1

Otu0065 Actinobacteria(100) Actinobacteria(100) Coriobacteriales(100) Coriobacteriaceae(100) Enterorhabdus( 100) 0.470026 1

Otu0066 Firmicutes(100) unclassified(100) unclassified(100) unclassified(100) unclassified(100) 0.781442 1

Otu0067 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.075425 1

Otu0068 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.23604 1

Otu0069 Proteobacteria(100) Deltaproteobacteria( 100) Desulfovibrionales(100) Desulfovibrionaceae(100) Desulfovibrio(93) 0.122282 1

Otu0070 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Dorea(100) 0.335755 1

Otu0071 Firmicutes(100) unclassified(100) unclassified(100) unclassified(100) unclassified(100) 0.275564 1

Otu0072 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Porphyromonadaceae( 100) unclassified(100) 0.6759 1

Otu0073 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(98) unclassified(98) 0.739009 1

Otu0074 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Oscillibacter(100) 0.099638 1

Otu0075 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Porphyromonadaceae( 100) unclassified(100) 0.142259 1

Otu0076 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.446379 1

Otu0077 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Butyrivibrio(91) 0.048125 1

Otu0078 Actinobacteria(100) Actinobacteria(100) Coriobacteriales(100) Coriobacteriaceae(100) unclassified(100) 0.020051 1

Otu0079 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.995336 1

Otu0080 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Dorea(100) 0.580909 1

Otu0081 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Butyrivibrio(96) 0.231131 1

Otu0082 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.144752 1

Otu0083 Actinobacteria(100) Actinobacteria(100) Coriobacteriales(100) Coriobacteriaceae(100) Asaccharobacter( 100) 0.683732 1

Otu0084 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.059598 1

Otu0085 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Anaerotruncus(100) 0.557724 1

Otu0086 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Anaerotruncus(100) 0.829296 1

Otu0087 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) unclassified(100) 0.147604 1

Otu0088 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.325188 1

Otu0089 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.270353 1

Otu0090 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Clostridium_XlVa(98) 0.893607 1

Otu0091 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) unclassified(97) unclassified(97) 0.064746 1

Otu0092 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Syntrophococcus( 100) 0.465783 1

Otu0093 Proteobacteria(100) Deltaproteobacteria( 100) Desulfovibrionales(100) Desulfovibrionaceae(100) Desulfocurvus(73) 0.719197 1

Otu0094 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Pseudoflavoniiractor( 100) 0.223923 1

Otu0095 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Pseudoflavoniiractor(65) 0.323615 1

Otu0096 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.220402 1

Otu0097 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.755524 1

Otu0098 Firmicutes(100) Erysipelotrichia(100) Erysipelotrichales(100) Erysipelotrichaceae(100) Clostridium_XVIII(100) 0.167966 1

Otu0099 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.658952 1

Otu0100 Firmicutes(100) Bacilli(100) Lactobacillales( 100) Streptococcaceae( 100) Streptococcus( 100) 0.271556 1

Otu0101 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Oscillibacter(100) 0.529912 1

Otu0102 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) unclassified(98) 0.239692 1

Otu0103 Actinobacteria(100) Actinobacteria(100) Coriobacteriales(100) Coriobacteriaceae(100) unclassified(100) 0.438692 1

Otu0104 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Flavonifractor(97) 0.884299 1

Otu0105 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Porphyromonadaceae( 100) Parabacteroides(100) 0.604151 1

Otu0106 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.052835 1

Otu0107 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Lactonifactor(100) 0.688385 1

Otu0108 Firmicutes(100) unclassified(100) unclassified(100) unclassified(100) unclassified(100) 0.808444 1

Otu0109 unclassified(100) unclassified(100) unclassified(100) unclassified(100) unclassified(100) 0.580963 1

Otu0110 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Rikenellaceae(100) Alistipes(100) 0.590556 1

Qtu0111 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.70702 1

Otu0112 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Anaerotruncus(100) 0.629385 1

Otu0113 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Clostridium_XlVa(97) 0.690915 1

Otu0114 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Oscillibacter(100) 0.659852 1

Otu0115 Firmicutes(100) Clostridia(100) Clostridiales(100) unclassified(100) unclassified(100) 0.052071 1

Otu0116 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Clostridium_IV(100) 0.496749 1

Otu0117 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Lachnospiracea incertae sedis(64) 0.783083 1

Otu0118 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.450217 1

Otu0119 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Anaerotruncus(100) 0.474766 1

Otu0120 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Roseburia(100) 0.677578 1

Otu0121 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Pseudoflavonifractor(100) 0.764818 1

Otu0122 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Roseburia(71) 0.221575 1

Otu0123 Proteobacteria(100) Deltaproteobacteria(100) Desulfovibrionales(100) Desulfovibrionaceae(100) Bilophila(100) 0.759601 1

Otu0124 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Hydrogenoanaerobacterium(100) 0.641479 1

Otu0125 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Pseudoflavonifractor(100) 0.015658 1

Otu0126 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Flavonifractor( 100) 0.017547 1

Otu0127 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Butyrivibrio(100) 0.106234 1

Otu0128 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Oscillibacter(100) 0.090769 1

Otu0129 Firmicutes(100) Erysipelotrichia(100) Erysipelotrichales(100) Erysipelotrichaceae(100) Erysipelotrichaceae incertae sedis(100) 0.304972 1

Otu0130 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Syntrophococcus(100) 0.351336 1

Otu0131 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Flavonifractor( 100) 0.733134 1

Otu0132 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Pseudoflavonifractor( 100) 0.651459 1

Otu0133 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Oscillibacter(100) 0.336219 1

Otu0134 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Rikenellaceae(100) Alistipes(100) 0.364453 1

Otu0135 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.297057 1

Otu0136 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Anaerotruncus(100) 0.255234 1

Otu0137 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(66) 0.06594 1

Otu0138 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Clostridium_XlVa( 100) 0.934883 1

Otu0139 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Porphyromonadaceae( 100) unclassified(100) 0.364453 1

Otu0140 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.019222 1

Otu0141 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.486003 1

Otu0142 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.714399 1

Otu0143 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.641689 1

Otu0144 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Porphyromonadaceae(69) unclassified(69) 0.511826 1

Otu0145 Bacteroidetes(100) Flavobacteria(97) Flavobacteriales(97) unclassified(97) unclassified(97) 0.718342 1

Otu0146 Bacteroidetes(100) Bacteroidia(100) Bacteroidales( 100) Rikenellaceae(100) Alistipes(100) 0.977493 1

Otu0147 Firmicutes(100) Clostridia(100) Clostridiales(100) Ruminococcaceae(100) Oscillibacter(100) 0.345778 1

Otu0148 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.08833 1

Otu0149 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) Clostridium_XlVb(100) 0.548781 1

Otu0150 Firmicutes(100) Clostridia(100) Clostridiales(100) Lachnospiraceae( 100) unclassified(100) 0.633538 1

Supplemental Figure Legends Supplemental Figure 1

(S1A) Representative flow cytometry plot and statistical analysis (S1B) showing differential expression of NKp46 and CCR6 in live, CD45+ CD90+ IL7R+ cells in the colon of Rag!1' (n=6, white bar) and TRUC (n=5, red bar) mice. Error bars depict SEM. *P<0.01, **P<0.005. (S1C) Flow cytometric analysis of the phenotype of CD90+ IL7R+ ILCs in the colon of TRUC mice. Grey histograms depict staining with isotype matched control antibody in comparison with staining with specific antibody (white histograms with black lines). Dta are representative of >3 individual experiments. (S1D) Proportion of cytokine expressing CD45+ CD90+ IL7R+ ILCs in the colon of Rag2'-' and TRUC mice following stimulation of unfractionated cLPMCs with PMA and ionomycin. Each dot/square represents an individual mouse.

Supplemental Figure 2

(S2A) IL6 concentration in serum (left panel) and colon explant culture (right panel) of Rag2'1' and TRUC mice measured by ELISA. Dots/squares represent individual mice. Line depicts median. (S2B) Microarray analysis showing abundance of transcripts encoded by genes known to be regulated by IL6 in the colon of TRUC mice relative to Rag2'-' mice. Blue bars represent transcripts upregulated in the colon of TRUC mice and red bars represent down-regulated genes (in comparison with Rag2'-' mice).

Supplemental Figure 3

(S3A) FACs gating strategy used to purify ILCs from the colon of TRUC mice. CD45+ cells were first enriched from cLPMCs using anti-CD45 immunomagnetic beads. ILCs were FACs

purified >98%. (S3B) Cytokine production by FACs purified CD90+ IL7R+ NKp46" colonic ILCs from TRUC mice. Cells were cultured in the presence of combinations of IL1a, IL23, IL6 (as depicted) or medium alone (unstimulated). IL6 was measured in culture supernatant by CBA. Bars show mean cytokine production and error bars depict SEM. Data are from 2 independent experiments.

Supplemental Figure 4

(S4A) Flow cytometry plots showing IL6R expression by CD4+ T-cells, ILCs (lineage- IL7R+) in the colon of wild type (WT) and TRUC mice. Data are representative of 3 independent experiments. (S4B) Concentration of soluble IL6R (sIL6R) in serum (n=6) and supernatants of cultured colon explants (n=5) and unfractionated splenocytes (n=3) measured by ELISA. Bars represent mean sIL6R concentration and error mars depict SEM.

Supplemental Figure 5.

(S5A) Concentration of IL6 in serum of TRUC mice treated with anti-IL6 or control isotype antibody. Dots/squares represent individual mice. Line depicts median. (S5B) Bray Curtis cluster dendrogram showing that anti-IL6 treatment is not associated with a distinct microbiota profile. "Pre" indicates samples before anti-IL6 treatment and "Post" indicates those after treatment. Bacterial families coloured in shades of green belong to the Firmicutes phylum, blue to the Bacteroidetes phylum, yellow to the Deferribacteres phylum (Mucispirillum genus) and maroon/red to the Proteobacteria phylum. (S5C) Box and whisker plots of Simpson diversity index of the intestinal microbiota from TRUC mice treated with anti-IL6 or isotype matched

control antibodies. Diversity indices are highly sensitive to differential sequencing depth. Therefore, analyses were confined to 477 reads per sample.

Supplemental Figure 6

(S6A) Representative flow cytometry plots of intracellular cytokine and surface CD3 expression in non-inflammatory control, CD and UC patients. Cells were stimulated with PMA and ionomycin (S6B) Real time PCR analysis of RORC expression in sorted colonic CD3- IL7R+ cells in comparison with CD14+ monocytes (immunomagnetically selected from peripheral blood monocytes). Histogram shows mean expression of RORC in purified colonic CD3- IL7R+ cells (n=2 IBD patients) relative to monocytes. (S6C) Proportion of colonic CD3- IL7R+ cells expressing c-kit (CD117) in non-inflammatory control, CD and UC patients. (S6D) Proportion of colonic CD3- IL7R+ cells expressing NKp46, NKp44 or double negative cells (NKp46- NKp44-) in non-inflammatory control, CD and UC patients. (S6E) IL6 production in colon explant cultures from IBD patients. In graphs each dot represents an individual patient and line depicts median.

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