Oncostatin M drives intestinal inflammation and predicts ... · [email protected] Nature Medicine, ... (Mitutoyo, UK), ... Cells were centrifuged for 15 minutes at 10°C

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Supplementary Methods, Figures 1–11, and Tables 1–4

Oncostatin M drives intestinal inflammation and predicts response

to tumor necrosis factor-neutralizing therapy in patients with

inflammatory bowel disease

Nathaniel R. West1,2,†, Ahmed N. Hegazy1,2,†, et al

Author Affiliations

1Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK. 2Translational Gastroenterology Unit,

Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK. †Co-first authors

Address for correspondence

Prof Fiona Powrie

Kennedy Institute of Rheumatology, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7FY, UK. [email protected] Nature Medicine, 2017

Nature Medicine: doi:10.1038/nm.4307

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Supplementary Methods ClueGO and CIBERSORT in silico analysis. ClueGo analysis1 for resolving gene ontology networks was

performed using right-sided hypergeometric tests, Bonferroni step-down correction, and a kappa score threshold

of 0.5 (Supplementary Fig. 4). To calculate enrichment of cell populations using CIBERSORT (Supplementary

Fig. 5e)2, we analyzed the GSE16879 dataset on default settings and processed the results as follows: for each

sample, the relative expression of PTPRC (CD45, representing relative leukocyte content in the tissue) was

calculated separately using the median-normalized microarray data. This value was then multiplied by

CIBERSORT cell type scores (e.g. proportion of memory CD4+ T cells in the total leukocyte fraction) to

estimate cell type enrichment levels for each tissue sample. Finally, for cell types of interest, fold differences in

enrichment scores between IBD and control specimens were calculated to estimate the relative abundance of

distinct cell types in active IBD lesions versus healthy control tissue.

Culture of epithelial organoids from mouse colon. Organoids were cultured as previously described3. Colonic

crypts were isolated from C57BL/6 mice, seeded in matrigel (Corning), and cultured in the presence of N2 and

B27 supplements (Invitrogen), mouse EGF (Invitrogen, 50ng/ml), Y-27632 (Sigma, 10uM), N-Acetylcysteine

(Sigma, 1mM) and conditioned medium containing Wnt3a (20%), Noggin (20%) and R-Spondin (10%).

Conditioned media was generated from Rspol-Fc Hek 293T cells (kind gift from Dr. Calvin Kuo, Stanford),

Nog-Fc Hek 293T cells (kind gift from Dr. Gjis van den Brink, Amsterdam), and Wnt3A L cells (ATCC® CRL-

2647™). After expansion by several passaging steps, organoids were seeded in 24 well plates for stimulation with

OSM (20ng/ml), TNF (20ng/ml) or IL-22 (20ng/ml).

Protein expression and purification. Both OR-Fc and mouse IgG2A-Fc were produced in Expi293™ cells

according to the manufacturer’s protocol (Invitrogen). Plasmid DNA for transient transfection was purified using

an EndoFree Plasmid Kit (Qiagen, UK). Proteins were purified from culture supernatants by a combination of

HiTrap rProtein A FF and size exclusion chromatography using an automated protocol implemented on an

ÄKTAxpress (GE Healthcare)4. Endotoxin testing of the purified proteins was conducted by Lonza (Belgium)

and levels were found to be below acceptable limits.

Imiquimod model of skin inflammation. Aldara cream (3M Pharmaceuticals), containing 5% imiquimod, was

applied to the ventral and dorsal sides of each ear of C57BL/6.Osm−/− mice or wild type littermates. Sufficient

cream was applied to completely cover each ear (approximately 20 mg per ear). Treatment was repeated daily

for 6 days and mice were sacrificed on day 7. Ear thickness was measured daily using a dial thickness gauge

(Mitutoyo, UK), and the mean of each ear was calculated. To isolate leukocytes from ear tissue, ears were


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removed at the base, split into two halves using forceps, and minced using surgical scissors. Tissue was then

digested using 3.5ml RPMI media with 0.1% BSA, 3 mg/ml collagenase D (Roche), and 400 ug/ml liberase

(Roche) at 37°C in a shaking incubator for 80 minutes. The cell digest was then homogenized using a syringe

and 18G mixing needle, neutralized with PBS containing 5 mM EDTA, and filtered through nylon mesh with

50um pores. Cells were centrifuged, resuspended in 6 ml RPMI (with 0.1% BSA and 5 mM EDTA) and layered

onto 4 ml of lymphoprep in 15 ml tubes. Cells were centrifuged for 15 minutes at 10°C at 900 rcf without brake.

Cells at the interface were collected for further analysis.

References

1. Bindea, G. et al. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and

pathway annotation networks. Bioinformatics 25, 1091–1093 (2009).

2. Newman, A. M. et al. Robust enumeration of cell subsets from tissue expression profiles. Nat Meth 12,

453–457 (2015).

3. Heijmans, J. et al. ER stress causes rapid loss of intestinal epithelial stemness through activation of the

unfolded protein response. Cell Rep 3, 1128–1139 (2013).

4. Nettleship, J. E., Rahman-Huq, N. & Owens, R. J. The production of glycoproteins by transient

expression in Mammalian cells. Methods Mol. Biol. 498, 245–263 (2009).


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Supplementary Figures

Supplementary Figure 1. Association of OSM and OSMR with clinical features of IBD. Q-PCR analysis of OSM and OSMR expression in intestinal biopsies from Oxford IBD patients. Panels depict patients categorized by (a) sex, (b) age at diagnosis, (c) disease duration from time of diagnosis to time of sample collection, (d) serum c-reactive protein, (e) peripheral blood leukocyte count, and (f) current treatment. Statistics: panels a and f, t-tests; panels b–e, one-way ANOVA with Tukey’s post-tests. *p=0.01–0.05, **p=0.001–0.01, ***p=0.0001–0.001, ****p<0.0001.


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a

Supplementary Figure 2. Hierarchical clustering of OSM with cytokine, chemokine, and stromal genes in human IBD. Continued on next page


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b

Supplementary Figure 2 continued. Hierarchical clustering of OSM with cytokine, chemokine, and stromal genes in human IBD. (a, previous page) Microarray gene expression profiling of colon and ileal mucosal biopsies from healthy controls (white, ileum; gray, colon) and IBD patients with active disease (violet, ileal CD; green, colonic CD; black, UC). All biopsies were collected from sites of active inflammation. Data are derived from GEO # GSE16879, are log2 transformed, and median-centered. The magnified region displays genes closely related to OSM. (b) RNA sequencing data from human intestinal mucosa were analyzed by unsupervised hierarchical clustering. All data are median-normalized and log2 transformed. Data are derived from ileal mucosal biopsies (regardless of diagnosis or site of inflammation) of newly diagnosed and untreated pediatric IBD patients or age-matched non-IBD controls (GEO # GSE57945). The color bar above the heatmap indicates the clinical phenotype of individual samples (white, non-IBD controls; gray, UC; green, colonic CD; violet, ileal CD without deep ulceration; black, ileal CD with deep ulceration). The magnified section displays a gene cluster with which OSM correlates closely. This cluster is also enriched in patients with ulcerated ileal CD. Genes associated with OSM in both the GSE57945 and GSE16879 datasets are shown in Fig. 2a (“OSM-associated module”) of the main manuscript.


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Supplementary Figure 3. Association of OSM, OSMR, and OSM-associated factors with non-responsiveness to anti-TNF therapy. Continued on next page.


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Supplementary Figure 3 continued. Association of OSM, OSMR, and OSM-associated factors with non-responsiveness to anti-TNF therapy. (a) Analytical approach for assessing the OSM-associated inflammatory module as a predictor of anti-TNF responsiveness. This also applies to data shown in Fig. 2 of the main manuscript. (b) Whole transcriptome microarray data from colon and ileal mucosal biopsies of IBD patients refractory to corticosteroids and/or immunosuppression prior to infliximab therapy. Data derived from GEO # GSE16879 (independent validation cohort). Data are median-normalized, log2 transformed, and analyzed by unsupervised hierarchical clustering. 12 healthy controls are included, indicated by white blocks in the grayscale bar above the heatmap. Infliximab responsive and refractory patients are identified by gray and black bars, respectively. The colored dendrograms identify patient clusters with differing response rates to infliximab, summarized in the accompanying table (which excludes healthy controls). (c) Pre-treatment expression of OSM and OSMR in infliximab responders and non-responders from the GSE16879 cohort. Examined using Mann-Whitney test. (d) OSM and OSMR expression in colon biopsies from a third cohort of UC patients refractory to corticosteroids or immune suppression (GSE23597). Patients were treated with infliximab and assessed for clinical response after 8 and 30 weeks. Asterisks; one-way ANOVA within a single timepoint; daggers, one-way ANOVA from samples within a single response group across all three timepoints. (e–i) In an additional validation cohort, patients refractory to immune suppression or dependent on corticosteroids were treated with subcutaneous golimumab as part of the PURSUIT phase 3 clinical trial (Sandborn et al, 2014). Clinical response to induction therapy was assessed after 6 weeks. (e) Mayo scores at baseline and after golimumab therapy in patients who achieved clinical remission (post-treatment Mayo score of 0–2), partial clinical response (decrease from baseline Mayo score by ≥30% and ≥3


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points, but post-treatment score ≥3), or no clinical response (decrease from baseline Mayo score of <30% and/or <3 points). See legend to the right of panel b. Statistics: one-way ANOVA with Tukey’s post-tests. (f) OSM expression in colon mucosal biopsies at baseline and after therapy. Statistics: one-way ANOVA with Tukey’s post-tests. (g) Association of baseline clinical features with treatment response. CRP, c-reactive protein; FCALP, fecal calprotectin; FLACT, fecal lactoferrin. Statistics: one-way ANOVA with Tukey’s post-tests. (h) Pearson correlation of baseline biopsy OSM expression and clinical features. (i) Pearson correlation of OSM expression and clinical features after golimumab therapy. In all panels, *p=0.01–0.05, **p=0.001–0.01, ***p=0.0001–0.001, ****p<0.0001. Absence of asterisks indicates a statistically non-significant difference.


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Supplementary Figure 4. ClueGo analysis of intestinal mucosa from human IBD patients. Whole transcriptome data from intestinal biopsies of UC (n=16) and CD (n=24) patients (GEO # GSE16879), were ranked in separate analyses by OSM, OSMR, or TNF expression. High and low expressing tissues in each analysis were compared using ClueGo to identify highly enriched gene ontology (GO) clusters (see Methods). The final GO clusters enriched in each analysis are shown here, with the most statistically significant single term for each cluster indicated in the tables. Comparison with TNF-enriched tissue suggests that high OSM and/or OSMR expression is associated with gene ontologies related to extracellular matrix regulation and mesenchymal development. Circles represent distinct GO terms, linked by lines to indicate similarity to GO terms with overlapping gene members. Circle size is proportional to statistical significance.


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Supplementary Figure 5. Analysis of OSM producer and responder cells in the human intestine. Continued on next page.


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Supplementary Figure 5. Analysis of OSM producer and responder cells in the human intestine. Continued on next page.


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q

Supplementary Figure 5 continued. Analysis of OSM producer and responder cells in the human intestine. (a) OSM expression in FACS-purified populations from healthy donor colon tissue (n=3). Statistics: one-way ANOVA with Dunnett’s post-tests. (b–f) Lamina propria leukocytes from digested human intestinal mucosa were cultured for 4 hours in the presence of brefeldin A (BFA) or BFA plus PMA (phorbol myristate acetate) and ionomycin. Intracellular OSM was then detected by flow cytometry. (b) OSM and TNF production by memory (CD45RA−) CD4+ T cells from a representative donor. (c, d) OSM production by CD4+ T cells (c) and antigen presenting cells (d) in a representative healthy donor, UC patient, and CD patient. (e) Relative enrichment of OSM+ CD4+ T cells and OSM+ antigen presenting cells, calculated based on flow cytometry observations and CIBERSORT analysis (see supplementary methods). Statistics: Mann-Whitney tests. (f) Frequencies of OSM expression in human intestinal CD45RA− CD4+ T cells according to their expression of TNF, IFN-γ, or IL-17A (n=27). (g) Q-PCR analysis of IL6R and OSMR expression in unstimulated primary ex vivo cultured intestinal stroma from n=9 donors (left) and CCD18Co cells (right). Statistics: Mann-Whitney test. (h) Frequency of endothelial and stromal cells in the CD45-negative fraction of human colon mucosa from n=11 donors, analyzed by flow cytometry. Statistics: Mann-Whitney tests. (i) In humans, OSM can transduce signals through either OSMR/gp130 or LIFR/gp130 heterodimers, while LIF signals only via LIFR/gp130 complexes. To demonstrate that OSMR is required for OSM signal transduction, we knocked down OSMR expression in CCD18Co cells using siRNA and stimulated with OSM (10 ng/ml) for 2 hours. Q-PCR analysis revealed nearly complete abrogation of OSM sensitivity upon OSMR knockdown. Experiment was performed in duplicate and repeated twice. (j) To determine if human intestinal stromal cells are responsive to LIFR activation, we treated CCD18Co cells with recombinant human LIF (10 ng/ml) for 2 hours and assessed responses using Q-PCR analysis of STAT3-driven genes (SOCS3 and CCL2). As a positive control, we stimulated HEK293 cells, which express LIFR and


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are known to be LIF-responsive. The results indicate that CCD18Co cells do not express sufficient LIFR to mediate signaling by LIF. Experiment was performed in triplicate and repeated twice. (k) Strategy for FACS-sorting OSMRlow and OSMRhigh stroma from normal colon mucosa. (l) Expression of CD45, EpCAM, CD31, and stromal markers in different cell types purified using the strategy illustrated in panel k. (m) Expression of selected genes by CCD18Co cells following 2 hours of stimulation with OSM, TNF, IL-6, or combinations of the three. Experiment performed in triplicate and repeated twice. Statistics: one-way ANOVA with 6 degrees of freedom. F=33.83 (CXCL10), 28.27 (CXCL11), and 347 (ICAM1). (n) Expression of selected genes by primary human intestinal stromal cells cultured ex vivo, following 2 hours of stimulation with OSM, TNF, IL-6, or combinations of the three. n=12 cultures from independent donors. For each donor, data are represented as fold changes relative to untreated conditions. Statistics: Wilcoxon signed rank test, versus a theoretical median of 1. (o) Q-PCR analysis of OSMR expression in resting HUVEC and CCD18Co cells. Each cell type was examined in triplicate. (p) CCD18Co cells and HUVEC were stimulated with OSM (10 ng/ml) for 2 hours with or without addition of TNF (5 ng/ml) and analyzed by Q-PCR. Endothelial cells were found to be weakly responsive to OSM, but strongly responsive to TNF. Assays were performed in triplicate and repeated twice. In all panels, *p=0.01–0.05, **p=0.001–0.01, ***p=0.0001–0.001, ****p<0.0001. (q) Full-length un-cropped western blots associated with Figure 3g in the main manuscript. Lanes 1–5 refer to stimulation with nothing, IL-6, TNF, OSM, or IL-1β.


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e

Supplementary Figure 6. Characterization of the Hh+αIL-10R colitis model. (a–c) Mean colon histopathology scores of mice subjected to Hh+αIL-10R colitis. Each data point represents a single animal. Statistics: Mann-Whitney tests. (a) Wild type mice treated with isotype control antibody or anti-TNF antibody beginning at day 7. (b) Wild type mice treated with isotype control antibody or anti-IL6R antibody starting at day 0. (c) Wild type mice versus IL-1R1 (IL-1 receptor)-knockout littermates. IL-1R1 deletion abrogates responsiveness to both IL-1α and IL-1β. (d) Colon mRNA expression kinetics following Hh+αIL-10R treatment (n≥5 mice per group). (e) Expression of key lineage-specific genes was assessed by Q-PCR to confirm the purity of FACS-sorted cell types isolated from mouse colon tissue (associated with Fig. 5e of the main manuscript). nd, not detected.


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a

Supplementary Figure 7. Characterization of the murine OSM-OSMR axis. Continued on next page.


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Supplementary Figure 7 continued. Characterization of the murine OSM-OSMR axis. (a) Osmr and Ppib (positive control) mRNA was detected using RNAscope in situ hybridization (punctate red signal). Tissues were counterstained with hematoxylin. Examples of Osmr-expressing cells in the lamina propria and lymphoid clusters are indicated by arrows, while submucosal endothelial cells positive for Osmr are indicated by arrowheads. (b) C57BL/6 mouse colon stroma cultured ex vivo were stimulated for 20 minutes with 10 ng/ml of recombinant mouse OSM before lysis and preparation for western blot analysis. CD45+ leukocytes isolated from healthy mouse colon or spleen were rested for 3 hours in RPMI media and similarly stimulated with OSM. Equal amounts of protein for each cell type were used. Data are representative of 2 independent experiments. (c) Organoids were generated ex vivo from healthy mouse colon epithelial cells and stimulated for various time-points with 20 ng/ml mouse OSM or IL-22 before harvesting for western blot analysis. Data are representative of 2 independent experiments. (d) Ex vivo cultured colon stroma from healthy C57BL/6 mice (n=4) were stimulated with 10 ng/ml OSM and analyzed by Q-PCR. Statistics: Student’s t-tests. Data are representative of 2 independent experiments. (e) Effect of additional TNF stimulation (5 ng/ml) on Il6 induction. Statistics: one-way ANOVA with Tukey’s post-tests. Data are representative of 2 independent experiments. In all panels, *p=0.01–0.05, **p=0.001–0.01, ***p=0.0001–0.001, ****p<0.0001.


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Supplementary Figure 8. Comparative histology of Osm−/− mice and heterozygous littermates. Continued on next page.


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Supplementary Figure 8 continued. Comparative histology of Osm"/" mice and heterozygous littermates. (a) Selected organs were harvested from healthy Osm!/! mice and co-housed heterozygous littermates. Tissues were formalin-fixed and paraffin embedded and stained with haematoxylin / eosin. Scale bar = 100µm. (b) B cells and T cells were detected in formalin-fixed lymph nodes and spleens using immunohistochemical dual staining of B220 (brown) and CD3 (pink). Reproducible differences between steady state Osm!/! mice and heterozygous littermates were observed only in the spleen, which displayed a slightly higher proportion of red pulp in OSM-deficient animals. (c) Immunohistochemical detection of splenic macrophages expressing F4/80. (d) Immunohistochemical staining of B220 (brown) and CD3 (pink) in small intestinal peyer’s patches and cecal patches.


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Supplementary Figure 9. Steady state immune phenotype of Osm−/− mice. Continued on next page.


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Supplementary Figure 9 continued. Steady state immune phenotype of Osm−/− mice. (a–d) Frequencies of major leukocyte populations in the colon lamina propria (a), mesenteric lymph nodes (mLN; (b)), spleen (c), and thymus (d). For colon, mLN, and spleen, n=7 for both wild type and Osm−/− littermates, pooled from 2 independent experiments. For thymus, n=3 for both wild type and Osm−/− littermates (representative of 2 independent experiments).


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(e) Frequencies of the indicated non-hematopoietic cell populations (among CD45-negative cells) in the colon lamina propria. Data derived from n=3 mice, representative of 3 independent experiments. All comparisons were made using t-tests. DC, dendritic cells. Tregs, regulatory T cells. All data were generated using flow cytometry. (f–h) Absolute numbers of major leukocyte populations in the colon lamina propria (f), mesenteric lymph nodes (mLN; (g)), and spleen (h). For colon, mLN, and spleen, n=7 for both wild type and Osm−/− littermates, pooled from 2 independent experiments. (i, j) Frequencies and absolute numbers of colonic CD4+ T cells expressing the indicated cytokines after stimulation with PMA/ionomycin. Data derived from n=3 mice, representative of 3 independent experiments. All comparisons were made using t-tests. DC, dendritic cells. Tregs, regulatory T cells. All data were generated using flow cytometry. *p=0.01–0.05, **p=0.001–0.01. (k) H. hepaticus genomic DNA was quantified by Q-PCR detection of the cdtb gene in total DNA extracted from the cecal contents of mice 2 weeks following infection. Data are pooled from two independent experiments and compared using Mann-Whitney tests.


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Supplementary Figure 10. Molecular and cellular kinetics of OSM-knockout mice during colon and skin inflammation. Continued on next page.


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Supplementary Figure 10 continued. Molecular and cellular kinetics of OSM-knockout mice during colon and skin inflammation. (a–f) Osm–/– or wild type littermates were induced with Hh+αIL-10R colitis and sacrificed at days 4, 9, and 14 (n=4–6 per group). Statistics: two-way ANOVA. *p=0.01–0.05, **p=0.001–0.01, ***p=0.0001–0.001, ****p<0.0001. (a) Expression of cytokine and chemokine genes in whole colon tissue (determined by Q-PCR), averaged for mice within each genotype and timepoint group, and converted to z-scores. (b) Histopathological colitis scores over the experimental time-course. (c) Expression of selected genes over the experimental timecourse, showing similar kinetics until day 9, and OSM-dependent divergence thereafter.


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(d) Total numbers of individual leukocyte populations in the colon lamina propria. (e) Percentage of Ki67+ cells among CD4+ T cells in the colon. (f) Total cell numbers in mesenteric lymph nodes. (g–l) Mice were treated daily with aldara cream (containing imiquimod) topically on each ear to stimulate TLR7 activation and acute inflammation. (g) Ear widths were measured daily using calipers. (h) Spleen weights on day 6. (i–l) Flow cytometry analysis of cervical lymph nodes and ear tissue (digested with collagenase, dispase and DNAse) on day 6. (i) Total live CD45+ cells in the ears and (j) total live CD45+ cells in the cervical lymph nodes. (k) Frequencies among CD45+ cells (top row) and absolute numbers (bottom row) of the indicated leukocyte lineages in ear skin at steady state and after 6 days of imiquimod treatment. (l) Frequencies among CD45+ cells (top row) and absolute numbers (bottom row) of the indicated leukocyte lineages in cervical lymph nodes at steady state and after 6 days of imiquimod treatment. No statistically significant differences were detected between imiquimod-treated Osm–/– and wild type mice.


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Supplementary Figure 11. Attenuation of OSM during colitis and a model of its effects in the intestine. (a) Whole-colon gene expression in mice after 14 days of Hh+αIL-10R colitis. Data represent z-scores of averaged expression of the OSM-associated inflammatory module (see Fig. 2 of main manuscript). (b) Comparison of the Fc-tagged mouse OSMR-gp130 fusion protein (OR-Fc) with a commercially available goat anti-mouse OSM polyclonal antibody (pAb). OSM neutralization capacity was determined by treating primary mouse intestinal fibroblasts with 10 ng/ml OSM and increasing concentrations of the pAb or OR-Fc. At equal molarities, OR-Fc was found to have a greater OSM neutralization capacity than the pAb, based on measurement of Socs3 expression (a STAT3 response gene). Thin dotted lines represent the 95% confidence intervals of the titration curve.


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(c) Whole colon gene expression (Q-PCR analysis) of mice after 7 days of Hh+αIL-10R colitis, compared using Mann-Whitney tests. (d) Histopathology sub-scores of OR-Fc or Fc-treated mice (associated with Fig. 6h in the main manuscript). Analyzed using two-way ANOVA. In all panels, *p=0.01–0.05, **p=0.001–0.01, ***p=0.0001–0.001, ****p<0.0001. (e) Intestinal leukocytes (such as antigen presenting cells and T cells) produce several key cytokines (including TNF and IL-23) following exposure to inflammatory insults that initiate pathogenic colitis if left unchecked. Further activation of gut resident and newly recruited leukocytes leads to accumulation of OSM, which drives diverse inflammatory activities by OSMR-expressing intestinal stromal cells. This includes chemokine, cytokine, and adhesion factor production, which further enhances leukocyte recruitment, activation, and retention in the tissue. This process may be further amplified by OSM-TNF synergy. Furthermore, stroma-derived cytokines like IL-6 may contribute to epithelial defects and tissue remodeling. As intestinal stromal cells accumulate with disease progression, this system strengthens, driving disease severity, chronicity, and therapeutic resistance.


Supplementary Table 1. Cytokines differentially expressed in CD1 or UC2 intestinal mucosaversus healthy control tissue.*

Cytokines significantly different in CD and UC versus healthy controlsCD UC

Gene Mean difference (log2) p-value Mean difference (log2) p-valueOSM 4.130 6.64E-22 1.432 3.10E-04IL1B 3.468 6.06E-18 3.619 3.24E-12IL1A 2.392 1.96E-08 2.309 2.82E-08IL6 2.266 1.68E-07 1.938 3.90E-04IFNG 1.901 6.93E-09 0.906 1.48E-04IL26 1.427 6.92E-11 0.242 1.06E-03TNFSF13 0.985 2.40E-18 0.349 1.90E-11IL17A 0.984 5.16E-05 0.555 4.12E-05TNF 0.962 1.13E-05 0.735 2.23E-05TGFB2 0.601 2.09E-03 0.975 7.32E-06IL33 0.571 1.62E-03 1.699 1.67E-09EBI3 0.541 1.58E-04 0.460 5.04E-04TGFB3 0.518 3.38E-03 0.826 4.31E-04TGFB1 0.363 8.17E-04 1.207 4.60E-09LIF 0.348 1.03E-04 0.634 6.84E-05CSF1 0.318 2.43E-05 0.483 8.52E-05

Cytokines significantly different in CD only versus healthy controlsCD UC

Gene Mean difference (log2) p-value Mean difference (log2) p-valueCSF3 3.795 9.70E-13 0.655 6.21E-03IL27 2.040 3.95E-15 -0.146 1.49E-02IL11 1.688 5.82E-06 0.751 4.03E-03CSF2 1.571 7.48E-06 0.423 8.85E-03IL22 1.076 1.03E-04 0.519 1.29E-02IL12A 0.960 6.38E-08 0.085 9.93E-02IL17F 0.844 1.78E-05 0.009 9.02E-01IL15 0.299 1.02E-05 0.183 1.21E-02IL32 -0.462 2.51E-03 -0.260 3.50E-02IL21 -0.654 4.72E-05 0.129 1.69E-01TSLP -0.674 1.86E-05 -0.021 7.64E-01

Cytokines significantly different in UC only versus healthy controlsCD UC

Gene Mean difference (log2) p-value Mean difference (log2) p-valueTL1A -0.039 7.32E-01 0.637 2.77E-06IL23A 0.274 2.17E-01 0.514 4.51E-06IL16 -0.195 1.36E-01 0.485 9.78E-05IL34 0.038 6.92E-01 0.424 1.84E-05IL29 -0.053 5.54E-01 -0.208 3.56E-03IL2 -0.136 3.56E-01 -0.221 3.93E-08IL37 -0.119 2.70E-01 -0.467 2.49E-09


Supplementary Table 1 Continued

Cytokines not significantly different in CD or UC versus healthy controlsCD UC

Gene Mean difference (log2) p-value Mean difference (log2) p-valueIFNA1 -0.056 1.45E-01 -0.055 3.02E-01IFNB1 0.051 2.24E-01 -0.140 1.05E-02IFNE 0.222 2.94E-01 0.137 5.76E-02IFNK -0.146 1.15E-01 -0.055 2.34E-01IFNW1 -0.031 3.05E-01 -0.186 4.84E-03IL10 0.046 7.34E-01 0.364 4.19E-03IL12B 0.599 8.98E-03 0.043 5.79E-01IL13 0.304 2.71E-02 -0.094 1.59E-01IL17B 0.196 2.66E-01 -0.132 3.91E-02IL17C 0.439 6.05E-03 0.021 7.79E-01IL17D -0.147 2.91E-01 -0.035 6.12E-01IL18 0.112 1.74E-01 0.309 2.33E-01IL19 0.376 7.55E-03 0.351 4.53E-03IL20 0.030 5.30E-01 -0.068 1.70E-01IL24 0.563 1.09E-01 1.275 4.08E-03IL25 -0.049 2.72E-01 -0.060 3.68E-01IL28A 0.013 4.58E-01 0.201 1.58E-02IL28B 0.004 7.88E-01 0.110 1.79E-01IL3 0.023 7.05E-01 -0.008 8.85E-01IL31 -0.003 4.93E-02 -0.047 4.40E-01IL36A -0.059 1.54E-01 -0.134 3.07E-02IL36B -0.153 2.87E-02 -0.047 5.44E-01IL36G 0.182 1.19E-01 0.015 8.22E-01IL4 -0.021 7.73E-01 -0.112 6.15E-03IL5 0.035 5.20E-01 -0.009 8.96E-01IL7 0.156 1.95E-01 0.126 2.63E-01IL9 0.023 4.84E-01 -0.077 1.83E-01LIGHT 0.119 4.00E-01 0.121 9.49E-02LTA 0.010 9.62E-01 0.175 7.54E-02LTB -0.202 3.13E-01 0.262 6.65E-03

* Significance determined by multiple t-tests with false discovery rate (1%) correction. P-values shown are uncorrected.

1. GEO dataset GSE57945. Haberman et al. J Clin Invest, 2014.2. GEO dataset GSE59071. Vanhove et al. Inflammatory Bowel Diseases, 2015.


Supplementary Table 2. Clinical characteristics of Oxford cohort patients assessed in this study.

Characteristic Control (n=13) UC (n=31) CD (n=27)Male/female (%) 3/9 (25/75) 13/17 (43/57) 11/16 (41/59)

Median (IQR) age at sampling (years) 58 (41–65) 48 (35–62) 37 (24–61)

Median (IQR) age at diagnosis (years) n/a 31 (24–39) 25 (18–46)

Median (IQR) disease duration (years) n/a 9 (3–23) 9 (6–13)

Median (IQR) C-reactive protein (mg/l) n/a 6.0 (1.1–14.9) 11.1 (3.8–46.3)

Median (IQR) peripheral blood leukocytes (10^9/l) 8.8 (8.3–9.0) 8.3 (7.0–9.7) 8.0 (6.5–10.7)

Current medication at sampling (%)

5-Aminosalicylates 21 (68) 1 (4)

Corticosteroids 8 (26) 1 (4)

Purine pathway inhibitors (e.g. azathioprine) 7 (23) 10 (37)

Infliximab or adalimumab 1 (3) 9 (33)

Methotrexate 0 (0) 1 (4)

NSAIDs 3 (10) 1 (4)

Monotherapy only (one of the above) 13 (42) 12 (44)

Combination of two or more of the above 11 (35) 5 (19)

No treatment 3 (10) 3 (11)

Unknown 4 (13) 7 (26)

IQR, interquartile range; n/a, not applicable


Supplementary Table 3. Mean fold changes in OSM and OSMR expression in active IBD intestinal mucosa versushealthy controls in independent cohorts.

OSM OSMRCountry Data type* Disease Control n IBD n Mean Δ (95% CI) p-value Mean Δ (95% CI) p-valueEnglanda RT-qPCR Ulcerative colitis 13 24 71.2 (19.0−266) 2.1 x 10−8 7.99 (3.33−19.1) 2.3 x 10−9

Englanda RT-qPCR Crohn's 13 19 37.7 (8.29−171) 2.9 x 10−7 4.37 (1.70−11.2) 3.0 x 10−5

North Americab RNA-Seq Crohn's (ileal) 42 162 17.5 (10.4−29.5) 6.6 x 10−22 1.61 (1.31−1.98) 1.0 x 10−5

Belgiumc Microarray‡ Ulcerative colitis 11 74 2.02 (1.51−3.36) <0.0001 7.31 (5.61−10.8) <0.0001Belgiumd Microarray Ulcerative colitis 6 24 9.89 (3.68−26.6) 5.6 x 10−5 6.15 (3.85−9.82) 1.1 x 10−8

Belgiumd Microarray Crohn's (colonic) 6 19 3.05 (0.656−14.2) 1.5 x 10−1 3.75 (2.09−6.75) 1.0 x 10−4

Belgiumd Microarray Crohn's (ileal) 6 18 14.6 (3.68−57.9) 6.0 x 10−4 1.91 (1.19−3.07) 9.8 x 10−3

Hungarye Microarray Mixed IBD 8 15 6.13 (2.82−13.3) 8.6 x 10−5 2.43 (1.77−3.33) 8.8 x 10−6

Spainf Microarray‡ Ulcerative colitis 13 15 1.38 (1.03−1.68) 0.0002 8.44 (5.49−16.1) <0.0001Total n 99 370

*All data derived from intestinal mucosa‡Data not normally distributed. Results depict differences in medians and significance values derived from non-parametric Mann-Whitney U-testsaUniversity of Oxford / John Radcliffe Hospital Translational Gastroenterology Unit IBD cohortbHaberman et al, 2014 (Gene Expression Omnibus dataset GSE57945)cVanhove et al, 2015 (Gene Expression Omnibus dataset GSE59071)dArijs et al, 2009 (Gene Expression Omnibus dataset GSE16879)eGalamb et al, 2010 (Gene Expression Omnibus dataset GSE4183)fPlanell et al, 2013 (Gene Expression Omnibus dataset GSE38713)


Supplementary Table 4. Top 50 differentially expressed genes (top 20 shaded in pink) in pre-‐treatmentintestinal biopsies of infliximab-‐refractory versus infliximab-‐responsive UC and CD patients.a

Ulcerative colitis (GEO #GSE12251)b Crohn's disease (GEO #GSE16879)c

Gene Probe IDMean fold difference P-‐valued Gene Probe ID

Mean fold difference P-‐valued

CXCR1 207094_at 17.21 0.0200 S100A8 214370_at 18.77 0.0021IL11 206924_at 15.10 0.0200 PI15 229947_at 16.12 0.0034HCAR3 205220_at 11.70 0.0071 IL11 206924_at 15.67 0.0142PROK2 232629_at 10.69 0.0200 PROK2 232629_at 14.80 0.0024PI15 229947_at 10.18 0.0215 TNFAIP6 206026_s_at 14.70 0.0010AQP9 205568_at 9.42 0.0294 CSF3 207442_at 14.67 0.0024KCNJ15 210119_at 8.80 0.0196 TNFAIP6 206025_s_at 14.01 0.0003TREM1 219434_at 8.71 0.0131 HCAR3 205220_at 13.71 0.0024TNFRSF10C 234644_x_at 8.68 0.0300 OSM 230170_at 12.59 0.0219S100A12 205863_at 8.68 0.0382 CXCL8 211506_s_at 12.35 0.0027CXCL8 211506_s_at 8.37 0.0200 TREM1 219434_at 12.26 0.0205LILRA3 206881_s_at 8.35 0.0115 PTGS2 1554997_a_at 10.94 0.0124FPR2 210773_s_at 7.96 0.0131 VMO1 235751_s_at 10.47 0.0053SOD2 215078_at 7.90 0.0200 S100A12 205863_at 10.37 0.0006G0S2 213524_s_at 6.99 0.0049 LILRB1 207104_x_at 9.92 0.0034PTGS2 1554997_a_at 6.84 0.0131 MME 203435_s_at 9.90 0.0021OSM 230170_at 6.74 0.0173 S100A8 202917_s_at 9.57 0.0021MGAM 206522_at 6.73 0.0319 AQP9 205568_at 9.22 0.0024CXCR2 207008_at 6.66 0.0409 MGAM 206522_at 8.98 0.0114INHBA 210511_s_at 6.58 0.0431 CXCL6 206336_at 8.85 0.0029TNFAIP6 206025_s_at 6.47 0.0131 CALCRL 210815_s_at 8.80 0.0193TNFAIP6 206026_s_at 6.20 0.0168 KCNJ15 210119_at 8.58 0.0166PTGS2 204748_at 6.05 0.0131 PTGS2 204748_at 8.48 0.0009FPR1 205119_s_at 5.76 0.0183 PTX3 206157_at 8.33 0.0124CLEC4E 222934_s_at 5.62 0.0438 BCL2A1 205681_at 8.19 0.0024OSM 214637_at 5.59 0.0238 INHBA 210511_s_at 7.95 0.0103APOBEC3A 210873_x_at 5.57 0.0131 SELE 206211_at 7.88 0.0067GPR84 223767_at 5.53 0.0455 INHBA 227140_at 7.75 0.0024SELE 206211_at 5.50 0.0468 IL24 206569_at 7.68 0.0221CEMIP 212942_s_at 5.44 0.0168 FCGR3B 204007_at 7.59 0.0024NKAPL 230804_at 5.29 0.0195 SERPINE1 202628_s_at 7.50 0.0267LOC100288985 230746_s_at 5.20 0.0049 LILRB2 207697_x_at 7.44 0.0080FPR2 210772_at 5.17 0.0229 FCGR2C 210992_x_at 7.42 0.0193BCL2A1 205681_at 5.15 0.0280 FCGR3B/A 204006_s_at 7.26 0.0048CSF3R 203591_s_at 5.13 0.0173 FPR1 205119_s_at 7.16 0.0006FFAR2 221345_at 5.07 0.0354 CXCL8 202859_x_at 7.14 0.0024FCGR3B 204007_at 5.06 0.0294 CSF2 210229_s_at 7.14 0.0081


FCGR3B/A 204006_s_at 5.00 0.0200 FPR2 210773_s_at 7.04 0.0124CMTM2 229967_at 4.99 0.0173 CCL3 205114_s_at 7.03 0.0062CREB5 232555_at 4.93 0.0131 TLR4 1552798_a_at 6.80 0.0100IL1RN 212657_s_at 4.87 0.0131 MMP1 204475_at 6.76 0.0035ARL11 1552693_at 4.80 0.0200 BLVRA 203771_s_at 6.75 0.0201MNDA 204959_at 4.72 0.0278 S100A9 203535_at 6.72 0.0024CLEC4D 1552772_at 4.64 0.0468 LILRB2 210146_x_at 6.66 0.0064PHACTR1 213638_at 4.60 0.0468 CYTL1 219837_s_at 6.53 0.0121FCGR2C 210992_x_at 4.58 0.0441 MCEMP1 235568_at 6.49 0.0053IL1B 205067_at 4.51 0.0134 LCP2 244556_at 6.39 0.0034BRE-‐AS1 1568768_s_at 4.49 0.0466 TNC 201645_at 6.22 0.0054SLC1A3 1569054_at 4.45 0.0456 PLEK 203470_s_at 6.13 0.0024SRGN 1554676_at 4.45 0.0200 MMP3 205828_at 6.02 0.0011

a All patients had active disease and were refractory to immunosuppression and/or corticosteroids at time of infliximab therapyb n=11 refractory patients versus n=12 responsive patientsc n=7 refractory patients versus n=12 responsive patientsd After Benjamini & Hochberg false discovery rate correction*Differences calcuated using GEO2R (NCBI)


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