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Table S1. and assays selected for reverse transcription quantitative PCR, including ratio between mean relative expression of Marsh 3 biopsies

(n=10) and reference population (n=4), and results from Mann-whitney comparing the same two groups.

Official symbol name Gene Expression assay** Relative expression‡ P-value§ Reference genes HPRT1 hypoxanthine phosphoribosyltransferase 1 Hs99999909_m1 PGK1 phosphoglycerate kinase 1 Hs99999906_m1 Villi genes ACSL5 (1) acyl-CoA synthetase long-chain family member 5 Hs00212106_m1 0.77 0.054 ADA* (2) adenosine deaminase Hs01110949_m1 0.52 0.008 ALPI* (3) alkaline phosphatase, intestinal Hs00357578_m1 0.46 0.014 APOA1* (4, 5) apolipoprotein A-I Hs00985000_g1 0.05 0.002 APOA4* (5) apolipoprotein A-IV Hs01573751_g1 0.16 0.002 APOB* (5) apolipoprotein B Hs01071209_m1 0.13 0.002 APOC3* (1) apolipoprotein C-III Hs00163644_m1 0.11 0.002 CYP3A4* (6) cytochrome P450, family 3, subfamily A, polypeptide 4 Hs00430021_m1 0.04 0.002 DPP4* (1) dipeptidyl-peptidase 4 Hs00175210_m1 0.33 0.004 ENPP3* (1) ectonucleotide pyrophosphatase/phosphodiesterase 3 Hs01038411_m1 0.11 0.002 FABP1 (7) fatty acid binding 1, liver Hs00155026_m1 0.85 0.454 FABP2* (7) fatty acid binding protein 2, intestinal Hs01573162_m1 0.53 0.008 LCT* (2) lactase Hs00158722_m1 0.09 0.002 VIL1 (2) villin 1 Hs00200229_m1 0.74 0.106 Crypt genes ABCC1 (8) ATP-binding cassette, sub-family C (CFTR/MRP), member 1 Hs00219905_m1 1.19 0.188 APOA2 (2) apolipoprotein A-II Hs00155788_m1 0.78 0.076 BTC (2) betacellulin Hs01101204_m1 0.90 0.454 CDK1* (1) cyclin-dependent kinase 1 Hs00364293_m1 1.58 0.004 CDK2* (9) cyclin-dependent kinase 2 Hs01548894_m1 1.45 0.024 CDK4 (9) cyclin-dependent kinase 4 Hs00175935_m1 1.23 0.188 CLU (2) clusterin Hs00156548_m1 0.93 0.454 DMBT1 (1) deleted in malignant brain tumors 1 Hs00244838_m1 0.69 0.054 GLUL (2) glutamate-ammonia ligase (glutamine synthetase) Hs00365928_g1 1.15 0.374 HES1 (10) Hairy and enhancer of split 1 Hs00172878_m1 0.92 0.839

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KRT19 (2) keratin 19 Hs01051611_gH 1.49 0.454 MAD2L1* (1) MAD2 mitotic arrest deficient-like 1 (yeast) Hs01554513_g1 1.53 0.004 MKI67* (1) antigen identified by monoclonal antibody Ki-67 Hs00606991_m1 1.93 0.002 MYC (9) v-myc myelocytomatosis viral oncogene homolog (avian) Hs99999003_m1 0.65 0.188 OLFM4 (1) olfactomedin 4 Hs00910490_m1 0.65 0.054 PCNA* (9) proliferating cell nuclear antigen Hs00952870_g1 1.56 0.004 REG3A (11) regenerating islet-derived 3 alpha Hs00170171_m1 0.77 0.240 RRM2* (1) ribonucleotide reductase M2 Hs01072069_g1 2.23 0.004 SEMA3F* (11) sema domain, (Ig), short basic domain, secreted, (semaphorin) 3F Hs01030912_m1 0.55 0.004 TOP2A* (2) topoisomerase (DNA) II alpha 170kDa Hs00172214_m1 1.51 0.004 Epithelial permeability genes CDH1* (12) cadherin 1, type 1, E-cadherin (epithelial) Hs00170423_m1 0.75 0.024 CLDN1 (12) claudin 1 Hs00221623_m1 1.67 0.076 CLDN2 (12) claudin 2 Hs00252666_s1 1.54 0.374 CLDN3 (12) claudin 3 Hs00265816_s1 0.95 0.454 CLDN4 (12) claudin 4 Hs00976831_s1 0.86 0.304 CLDN5 (12) claudin 5 Hs00533949_s1 1.23 0.454 CLDN8 (12) claudin 8 Hs00273282_s1 n.d.¶ CLDN15* (13) claudin 15 Hs00204982_m1 0.43 0.004 CTNNB1 (12) catenin (cadherin-associated protein), beta 1, 88kDa Hs00170025_m1 0.93 0.106 F11R* (12) Hs00375889_m1 0.75 0.014 JAM2 (12) junctional adhesion molecule 2 Hs01022007_m1 0.88 0.142 JAM3* (12) junctional adhesion molecule 3 Hs01113227_m1 0.79 0.036 MAGI2 (14) membrane associated guanylate kinase, WW and PDZ domain containing 2 Hs00202321_m1 1.02 1.000 OCLN* (12) Hs00170162_m1 0.51 0.002 PARD3 (14) par-3 partitioning defective 3 homolog (C. elegans) Hs00969067_m1 0.68 0.014 TJP1* (12) (zona occludens 1) Hs00268480_m1 0.59 0.002 TJP2* (12) (zona occludens 2) Hs00910543_m1 0.79 0.036 TJP3* (12) tight junction protein 3 (zona occludens 3) Hs00274276_m1 0.70 0.004 Immune response related genes CCR9 (15) chemokine (C-C motif) receptor 9 Hs01890924_s1 0.98 0.945 CD28 (16) CD28 molecule Hs01007419_m1 1.12 0.839 CSF2 (17) colony stimulating factor 2 (granulocyte-macrophage) Hs00171266_m1 1.16 0.454

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CSF3 (17) colony stimulating factor 3 (granulocyte) Hs99999083_m1 10.60 0.733 CTLA4* (18) cytotoxic T-lymphocyte-associated protein 4 (soluble isoform) Hs03044419_m1 2.46 0.002 CTLA4* (18) cytotoxic T-lymphocyte-associated protein 4 (membrane bound isoform) Hs01011591_m1 2.41 0.002 CXCL11* (17) chemokine (C-X-C motif) ligand 11 Hs00171138_m1 12.55 0.002 FGF7* (15) fibroblast growth factor 7 (keratinocyte growth factor) Hs00940253_m1 2.13 0.024 GZMA (19) granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine esterase 3) Hs00196206_m1 1.04 0.839 GZMB* (20) granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1) Hs00188051_m1 6.38 0.002 ICOS (17) inducible T-cell co-stimulator Hs00359999_m1 1.39 0.304 IFNA1 (21) interferon, alpha 1 Hs03044218_g1 0.31 0.240 IFNG*† interferon, gamma Hs00174143_m1 21.72 0.002 IL1A† interleukin 1, alpha Hs00174092_m1 2.46 0.304 IL1B† interleukin 1, beta Hs00174097_m1 4.23 0.106 IL2*† interleukin 2 Hs00174114_m1 0.31 0.014 IL4† Interleukin 4 Hs99999030_m1 1.30 0.635 IL6* (17) interleukin 6 (interferon, beta 2) Hs00985639_m1 2.25 0.036 IL8*† interleukin 8 Hs00174103_m1 7.63 0.002 IL10*† interleukin 10 Hs00174086_m1 2.23 0.008 IL11 (22) interleukin 11 Hs00174148_m1 3.44 0.106 IL12A*† interleukin 12A (natural killer cell stimulatory factor 1, cytotoxic lymphocyte maturation factor 1, p35) Hs00168405_m1 2.61 0.002 IL12B† interleukin 12B (natural killer cell stimulatory factor 2, cytotoxic lymphocyte maturation factor 2, p40) Hs01011516_g1 1.26 0.839 IL13 (23) interleukin 13 Hs00174379_m1 n.d.¶ IL15† interleukin 15 Hs01003716_m1 0.90 0.454 IL17A* (24) interleukin 17A Hs00174383_m1 14.44 0.002 IL17B (25) interleukin 17B Hs00975262_m1 0.55 0.188 IL18* (17) interleukin 18 (interferon-gamma-inducing factor) Hs00155517_m1 0.49 0.036 IL21 (26) interleukin 21 Hs00222327_m1 2.47 0.142 ITGAX (27) integrin, alpha X (complement component 3 receptor 4 subunit) Hs01015064_m1 1.66 0.054 KLRK1 (28) killer cell lectin-like receptor subfamily K, member 1 Hs00183683_m1 1.05 0.839 LTA (17) lymphotoxin alpha (TNF superfamily, member 1) Hs00236874_m1 1.35 0.142 MICA (28) MHC class I polypeptide-related sequence A Hs00792195_m1 1.38 0.240 MICB (21) MHC class I polypeptide-related sequence B Hs00792952_m1 1.31 0.054 RGS1 (29) regulator of G-protein signaling 1 Hs00175260_m1 1.67 0.539 TBX21 (17) T-box 21 Hs00203436_m1 1.46 0.240

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TGFB1 (30) transforming growth factor, beta 1 Hs00171257_m1 1.20 0.374 TLR3* (31) toll-like receptor 3 Hs01551078_m1 0.38 0.004 TNF† tumor necrosis factor (TNF superfamily, member 2) Hs00174128_m1 0.92 0.839 TNFRSF4 (27) tumor necrosis factor receptor superfamily, member 4 Hs00533968_m1 1.79 0.142 TNFRSF9* (27) tumor necrosis factor receptor superfamily, member 9 Hs00155512_m1 4.31 0.002 TNFRSF18 (17) tumor necrosis factor receptor superfamily, member 18 Hs02572926_s1 1.46 0.635 TYROBP (32) TYRO protein tyrosine kinase binding protein Hs00182426_m1 0.72 0.106 Other potential marker genes ARG1 (33) arginase, liver Hs00968979_m1 1.91 0.539 ARG2 (33) arginase, type II Hs00982833_m1 0.94 0.539 CYP4F2* (17) cytochrome P450, family 4, subfamily F, polypeptide 2 Hs02557757_s1 0.16 0.002 CYP4F3 (17) cytochrome P450, family 4, subfamily F, polypeptide 3 Hs00168521_m1 1.12 0.945 E2F4 (34) E2F transcription factor 4, p107/p130-binding Hs01053570_m1 0.84 0.142 ECE1* (17) endothelin converting enzyme 1 Hs01043741_m1 0.59 0.008 EDN1* (17) endothelin 1 Hs01116576_g1 0.15 0.002 MYO9B (35) myosin IXB Hs00994622_m1 1.11 0.635 NOS2* (17, 33) nitric oxide synthase 2, inducible Hs00167257_m1 3.06 0.008 SELE* (17) E Hs00174057_m1 10.24 0.036 SMAD3* (17) SMAD family member 3 Hs00232219_m1 0.48 0.004 SMAD7* (17) SMAD family member 7 Hs00178696_m1 0.44 0.002 TAGAP* (29) T-cell activation RhoGTPase activating protein Hs01097871_m1 1.60 0.036 TM4SF4* (36) transmembrane 4 L six family member 4 Hs00270335_m1 0.08 0.002

* Selected for further analyses. † Gene present on Taqman cytokine gene expression plate I (Applied Biosystems). ** Final concentrations of primers and probe 900 nM and 250 nM, respectively. Minor groove binding Taqman probes, fluorescently labelled in the 5’ end with FAM and in the 3’ end with a non- fluorescent quencher. ‡ Ratio between mean relative expression of Marsh 3 biopsies (n=10) and the reference population (n=4). § P-value based on expression in Marsh 3 biopsies (n=10) compared to the reference population (n=4) (Mann-Whitney). ¶ Not detected.

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Table S1: References 1. Gassler N, Newrzella D, Bohm C, Lyer S, Li L, Sorgenfrei O, van Laer L, Sido B, Mollenhauer J, Poustka A, Schirmacher P, Gretz N 2006 Molecular characterisation of non-absorptive and absorptive enterocytes in human small intestine. Gut 55:1084-1089. 2. Olsen L, Hansen M, Ekstrom CT, Troelsen JT, Olsen J 2004 CVD: the intestinal crypt/villus in situ hybridization database. Bioinformatics 20:1327-1328. 3. Prasad KK, Thapa BR, Nain CK, Sharma AK, Singh K 2008 Brush border enzyme activities in relation to histological lesion in pediatric celiac disease. J Gastroenterol Hepatol 23:e348-352. 4. Floren CH, Alm P 1988 Defective synthesis of apolipoprotein A-I in jejunal mucosa in coeliac disease. Scand J Gastroenterol 23:856-860. 5. Green PH, Lefkowitch JH, Glickman RM, Riley JW, Quinet E, Blum CB 1982 Apolipoprotein localization and quantitation in the human intestine. Gastroenterology 83:1223-1230. 6. Johnson TN, Tanner MS, Taylor CJ, Tucker GT 2001 Enterocytic CYP3A4 in a paediatric population: developmental changes and the effect of coeliac disease and cystic fibrosis. Br J Clin Pharmacol 51:451-460. 7. Derikx JP, Vreugdenhil AC, Van den Neucker AM, Grootjans J, van Bijnen AA, Damoiseaux JG, van Heurn LW, Heineman E, Buurman WA 2009 A pilot study on the noninvasive evaluation of intestinal damage in celiac disease using I-FABP and L-FABP. J Clin Gastroenterol 43:727-733. 8. Anderle P, Sengstag T, Mutch DM, Rumbo M, Praz V, Mansourian R, Delorenzi M, Williamson G, Roberts MA 2005 Changes in the transcriptional profile of transporters in the intestine along the anterior-posterior and crypt-villus axes. BMC Genomics 6:69. 9. Mariadason JM, Nicholas C, L'Italien KE, Zhuang M, Smartt HJ, Heerdt BG, Yang W, Corner GA, Wilson AJ, Klampfer L, Arango D, Augenlicht LH 2005 Gene expression profiling of intestinal epithelial cell maturation along the crypt-villus axis. Gastroenterology 128:1081-1088. 10. Kayahara T, Sawada M, Takaishi S, Fukui H, Seno H, Fukuzawa H, Suzuki K, Hiai H, Kageyama R, Okano H, Chiba T 2003 Candidate markers for stem and early progenitor cells, Musashi-1 and Hes1, are expressed in crypt base columnar cells of mouse small intestine. FEBS Lett 535:131-135. 11. George MD, Wehkamp J, Kays RJ, Leutenegger CM, Sabir S, Grishina I, Dandekar S, Bevins CL 2008 In vivo gene expression profiling of human intestinal epithelial cells: analysis by laser microdissection of formalin fixed tissues. BMC Genomics 9:209. 12. Niessen CM 2007 Tight junctions/adherens junctions: basic structure and function. J Invest Dermatol 127:2525-2532. 13. Tamura A, Kitano Y, Hata M, Katsuno T, Moriwaki K, Sasaki H, Hayashi H, Suzuki Y, Noda T, Furuse M, Tsukita S 2008 Megaintestine in claudin-15-deficient mice. Gastroenterology 134:523-534. 14. Wapenaar MC, Monsuur A, van Bodegraven A, Weersma RK, Bevova M, Linskens R, Howdle P, Holmes G, Mulder CJ, Dijkstra G, van Heel DA, Wijmenga C 2007 Associations With Tight Junction Genes Pard3 And Magi2 In Dutch Patients Point To A Common Barrier Defect For Celiac Disease And Ulcerative Colitis. Gut. 15. Kabelitz D, Wesch D 2003 Features and functions of gamma delta T lymphocytes: focus on chemokines and their receptors. Crit Rev Immunol 23:339-370. 16. Wolters VM, Wijmenga C 2008 Genetic background of celiac disease and its clinical implications. Am J Gastroenterol 103:190-195. 17. Diosdado B, van Bakel H, Strengman E, Franke L, van Oort E, Mulder CJ, Wijmenga C, Wapenaar MC 2007 Neutrophil recruitment and barrier impairment in celiac disease: a genomic study. Clin Gastroenterol Hepatol 5:574-581.

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18. Hunt KA, McGovern DP, Kumar PJ, Ghosh S, Travis SP, Walters JR, Jewell DP, Playford RJ, van Heel DA 2005 A common CTLA4 haplotype associated with coeliac disease. Eur J Hum Genet 13:440-444. 19. Augustin MT, Kokkonen J, Karttunen R, Karttunen TJ 2005 Serum granzymes and CD30 are increased in children's milk protein sensitive enteropathy and celiac disease. J Allergy Clin Immunol 115:157-162. 20. Oberhuber G, Vogelsang H, Stolte M, Muthenthaler S, Kummer AJ, Radaszkiewicz T 1996 Evidence that intestinal intraepithelial lymphocytes are activated cytotoxic T cells in celiac disease but not in giardiasis. Am J Pathol 148:1351-1357. 21. Jabri B, Kasarda DD, Green PH 2005 Innate and adaptive immunity: the yin and yang of celiac disease. Immunol Rev 206:219-231. 22. Garrote JA, Gomez-Gonzalez E, Bernardo D, Arranz E, Chirdo F 2008 Celiac disease pathogenesis: the proinflammatory cytokine network. J Pediatr Gastroenterol Nutr 47 Suppl 1:S27-32. 23. Turner JR 2009 Intestinal mucosal barrier function in health and disease. Nat Rev Immunol 9:799-809. 24. Castellanos-Rubio A, Santin I, Irastorza I, Castano L, Carlos Vitoria J, Ramon Bilbao J 2009 TH17 (and TH1) signatures of intestinal biopsies of CD patients in response to gliadin. Autoimmunity 42:69-73. 25. Shires J, Theodoridis E, Hayday AC 2001 Biological insights into TCRgammadelta+ and TCRalphabeta+ intraepithelial lymphocytes provided by serial analysis of gene expression (SAGE). Immunity 15:419-434. 26. van Heel DA, Franke L, Hunt KA, Gwilliam R, Zhernakova A, Inouye M, Wapenaar MC, Barnardo MC, Bethel G, Holmes GK, Feighery C, Jewell D, Kelleher D, Kumar P, Travis S, Walters JR, Sanders DS, Howdle P, Swift J, Playford RJ, McLaren WM, Mearin ML, Mulder CJ, McManus R, McGinnis R, Cardon LR, Deloukas P, Wijmenga C 2007 A genome-wide association study for celiac disease identifies risk variants in the region harboring IL2 and IL21. Nat Genet 39:827-829. 27. Montufar-Solis D, Garza T, Klein JR 2007 T-cell activation in the intestinal mucosa. Immunol Rev 215:189-201. 28. Hue S, Mention JJ, Monteiro RC, Zhang S, Cellier C, Schmitz J, Verkarre V, Fodil N, Bahram S, Cerf-Bensussan N, Caillat-Zucman S 2004 A direct role for NKG2D/MICA interaction in villous atrophy during celiac disease. Immunity 21:367-377. 29. Hunt KA, Zhernakova A, Turner G, Heap GA, Franke L, Bruinenberg M, Romanos J, Dinesen LC, Ryan AW, Panesar D, Gwilliam R, Takeuchi F, McLaren WM, Holmes GK, Howdle PD, Walters JR, Sanders DS, Playford RJ, Trynka G, Mulder CJ, Mearin ML, Verbeek WH, Trimble V, Stevens FM, O'Morain C, Kennedy NP, Kelleher D, Pennington DJ, Strachan DP, McArdle WL, Mein CA, Wapenaar MC, Deloukas P, McGinnis R, McManus R, Wijmenga C, van Heel DA 2008 Newly identified genetic risk variants for celiac disease related to the immune response. Nat Genet. 30. Benahmed M, Meresse B, Arnulf B, Barbe U, Mention JJ, Verkarre V, Allez M, Cellier C, Hermine O, Cerf-Bensussan N 2007 Inhibition of TGF-beta signaling by IL-15: a new role for IL-15 in the loss of immune homeostasis in celiac disease. Gastroenterology 132:994-1008. 31. Zhou R, Wei H, Sun R, Zhang J, Tian Z 2007 NKG2D recognition mediates Toll-like receptor 3 signaling-induced breakdown of epithelial homeostasis in the small intestines of mice. Proc Natl Acad Sci U S A 104:7512-7515. 32. Jabri B, Ebert E 2007 Human CD8+ intraepithelial lymphocytes: a unique model to study the regulation of effector cytotoxic T lymphocytes in tissue. Immunol Rev 215:202-214. 33. Mori M, Gotoh T 2000 Regulation of nitric oxide production by arginine metabolic enzymes. Biochem Biophys Res Commun 275:715-719.

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34. Sancho E, Batlle E, Clevers H 2003 Live and let die in the intestinal epithelium. Curr Opin Cell Biol 15:763-770. 35. Monsuur AJ, de Bakker PI, Alizadeh BZ, Zhernakova A, Bevova MR, Strengman E, Franke L, van't Slot R, van Belzen MJ, Lavrijsen IC, Diosdado B, Daly MJ, Mulder CJ, Mearin ML, Meijer JW, Meijer GA, van Oort E, Wapenaar MC, Koeleman BP, Wijmenga C 2005 Myosin IXB variant increases the risk of celiac disease and points toward a primary intestinal barrier defect. Nat Genet 37:1341-1344. 36. Diosdado B, Wapenaar MC, Franke L, Duran KJ, Goerres MJ, Hadithi M, Crusius JB, Meijer JW, Duggan DJ, Mulder CJ, Holstege FC, Wijmenga C 2004 A microarray screen for novel candidate genes in coeliac disease pathogenesis. Gut 53:944-951.

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Table S2. Results from gene expression analysis of eleven potential reference genes

(Human endogenous control plate (Applied Biosystems)) using cDNA obtained from reference population biopsies (n = 2) and cases with varying degrees of mucosal

damage in the small intestine (n = 4).

Gene SD* (Normfinder (1)) M-value** (geNorm (2)) HPRT1 0,13 0,19 PGK1 0,20 0,19 TBP 0,29 0,34 GUSB 0,34 0,30 PPIA 0,34 0,40 ACTB 0,35 0,24 GAPDH 0,35 0,27 RPLP0 0,39 0,43 B2M 0,42 0,32 18S rRNA 0,55 0,47 TFRC 1,0 0,56

* Standard devation.

** Average pairwise variation of a particular gene with all other control genes (2). Genes with the lowest M-value have the most stable expression.

Table S2: References 1. Andersen CL, Jensen JL, Orntoft TF 2004 Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245-5250 2. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F 2002 Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034.1-0034.11

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