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Gene Expression Signature for Biliary Atresia and a Role for Interleukin-8

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Gene Expression Signature for Biliary Atresia and a Role for Interleukin-8 SUPPLEMENTARY “PATIENTS AND METHODS” “Gene expression signature for biliary atresia and a role for Interleukin-8 in pathogenesis of experimental disease” Bessho K, et al. PATIENTS AND METHODS Patients. Liver biopsies, serum samples and clinical data were obtained from infants with cholestasis enrolled into a prospective study (ClinicalTrials.gov Identifier: NCT00061828) of the NIDDK-funded Childhood Liver Disease Research and Education Network (www.childrennetwork.org) or from infants evaluated at Cincinnati Children’s Hospital Medical Center. For subjects with biliary atresia (BA), liver biopsies were obtained from 64 infants during the preoperative workup or at the time of intraoperative cholangiogram, with ages ranging from 22-169 days after birth (Supplementary Table 7). For subjects with intrahepatic cholestasis (serving as diseased controls, and referred to as non- BA), liver biopsy samples were obtained percutaneously or intraoperatively from 14 infants at the time of diagnostic evaluation, with ages ranging from 19-189 days. Their diagnosis were Alagille syndrome (N=1), multidrug resistance protein-3 deficiency (N=2), alpha-1-antitrypsin deficiency (N=2) and cholestasis with unknown etiology (N=9) (Supplementary Table 7). Representative liver biopsy photomicrographs are shown in Supplementary Figure 6A-D. A third group of normal controls (NC) consisted of liver biopsy samples obtained from 7 deceased-donor children aged 22-42 months as described previously (1). This group serves as a reference cohort, with the median levels of gene expression used to normalize gene expression across all patients in the BA and non-BA groups. This greatly facilitates the visual identification of key differences in gene expression levels between BA and non-BA groups. Further, the ability to compare these experimental groups with normal controls separately enables the discoveries of profiles that are unique for BA and non-BA groups individually. We also obtained a liver biopsy from an infant at 75 days of age being evaluated for hypotonia. The infant had normal bilirubin levels and normal synthetic function, with normal liver histology and electron microscopy. Serum samples were obtained from 81 subjects at the time of diagnosis of biliary atresia (ages: 10-116 days), and 66 patients with other causes of intrahepatic cholestasis (ages: 2-118 days). Serum samples were also obtained from 5 healthy age-matched controls. All samples were obtained after informed consent from patients' parents/legal guardians. The study protocols were approved by the human research review boards of all participating institutions. Mouse model of experimental biliary atresia. BALB/c mice carrying the genetic inactivation of Cxcr2 (C.129S2[B6]-Cxcr2tm1Mwm/J; Cxcr2-/- mice) were purchased from Jackson Laboratory (Bar Harbor, ME). Newborn mice were injected with 1.5x106 fluorescent-forming units of rhesus rotavirus (RRV) or 0.9% NaCl (saline) intraperitoneally within 24 hours of birth, and were examined daily for the development of cholestasis as described previously (2, 3). The extrahepatic bile ducts and gallbladder were microdissected en bloc (here collectively referred to as extrahepatic bile ducts) at 3, 7 and 14 days after RRV or saline injection, and snap frozen in liquid nitrogen or embedded in paraffin. Livers were harvested using snap frozen in liquid nitrogen, embedded in paraffin or subjected to protocols to purify mononuclear cells for flow cytometric analysis, as described previously (2, 3). All mice were maintained under specific pathogen–free conditions and diet that contained trimethoprim-sulfamethoxazole. The Institutional Animal Care and Use Committee of Cincinnati Children’s Hospital Medical Center approved the experimental protocols. To generate genome-wide expression datasets for extrahepatic bile ducts of mice, 3 sets of total RNA samples were generated for each time point and experimental and control groups, with each set containing 2-6 extrahepatic bile ducts depending on their sizes to make sure enough RNA was available for processing and hybridization to GeneChip® Mouse Gene ST 1.0 Arrays (Affymetrix) following the same experimental protocols as described above for human samples (data also deposited in GEO: GSE46995. Quantitative PCR and detection of IL8 protein. Quantitative PCR was performed as reported previously (2), with all primers are listed in the Supplementary Table 8. Sections of frozen liver biopsies fixed in paraformaldehyde were dual stained with anti-cytokeratin antibody (polyclonal rabbit anti-cytokeratin, DAKO, Carpinteria, CA; to stain cholangiocytes) simultaneously with anti-human IL8 antibody (Human CXCL8/IL-8 polyclonal antibody, goat IgG, R&D Systems, Minneapolis, MN), followed by incubation with fluorescence-labeled species-specific secondary antibodies, and DAPI counterstaining, as described previously (4). Serum IL8 concentrations were measured using the Luminex Technology Platform according to the instruction supplied by the manufacturer (Millipore Co., Bedford, MA). Cell isolation and flow cytometric analysis. Single-cell suspensions from neonatal livers and cellular phenotyping by flow activated cell sorting (FACS) were performed using protocols and antibodies as described previously (1, 2, 4); isotype-matched controls were used for all FACS acquisitions and analyses. REFERENCES 1. Li J, Bessho K, Shivakumar P, Mourya R, Mohanty SK, Dos Santos JL, Miura IK, et al. Th2 signals induce epithelial injury in mice and are compatible with the biliary atresia phenotype. J Clin Invest 2011;121:4244-4256. 2. Shivakumar P, Campbell KM, Sabla GE, Miethke A, Tiao G, McNeal MM, Ward RL, et al. Obstruction of extrahepatic bile ducts by lymphocytes is regulated by IFN-gamma in experimental biliary atresia. J Clin Invest 2004;114:322-329. 3. Carvalho E, Liu C, Shivakumar P, Sabla G, Aronow B, Bezerra JA. Analysis of the biliary transcriptome in experimental biliary atresia. Gastroenterology 2005;129:713-717. 4. Shivakumar P, Sabla GE, Whitington P, Chougnet CA, Bezerra JA. Neonatal NK cells target the mouse duct epithelium via Nkg2d and drive tissue- specific injury in experimental biliary atresia. J Clin Invest 2009;119:2281-2290. Supplementary Table 1 List of regulated probe sets differing by more than 2-fold change among the 3 conditions (BA, DC and NC). The probe sets in red font constitute the molecular signature of biliary atresia. Corrected P value equals to P value with Benjamini-Hochberg multiple testing correction (false discovery rate [FDR] 0.05). 574 probe sets regulated by more than at least 2-fold between biliary atresia (BA) and normal control (NC) Transcripts Fold change Regulation Corrected Gene Symbol Gene Description Entrezgene ID Cluster ID BA vs NC in BA P value 8175234 GPC3 glypican 3 2719 54.44 up 1.37E-18 8098439 20.67 up 1.26E-09 8131844 GPNMB glycoprotein (transmembrane) nmb 10457 10.12 up 5.49E-07 secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T- 8096301 SPP1 6696 9.56 up 8.01E-09 lymphocyte activation 1) 8016646 COL1A1 collagen, type I, alpha 1 1277 8.92 up 1.39E-06 8041853 TACSTD1 tumor-associated calcium signal transducer 1 4072 8.79 up 4.08E-15 7934979 ANKRD1 ankyrin repeat domain 1 (cardiac muscle) 27063 8.61 up 3.58E-16 8134263 COL1A2 collagen, type I, alpha 2 1278 8.31 up 2.15E-06 8104663 CDH6 cadherin 6, type 2, K-cadherin (fetal kidney) 1004 7.97 up 3.58E-16 7951217 MMP7 matrix metallopeptidase 7 (matrilysin, uterine) 4316 7.70 up 2.16E-12 8124196 DCDC2 doublecortin domain containing 2 51473 7.37 up 6.55E-13 8124650 UBD|GABBR1 ubiquitin D | gamma-aminobutyric acid (GABA) B receptor, 1 10537 | 2550 7.10 up 2.07E-04 8178295 UBD|GABBR1 ubiquitin D | gamma-aminobutyric acid (GABA) B receptor, 1 10537 | 2550 6.70 up 2.39E-04 7965403 LUM lumican 4060 6.49 up 2.76E-08 8174201 BEX1 brain expressed, X-linked 1 55859 6.33 up 4.64E-11 solute carrier family 12 (sodium/potassium/chloride transporters), 8107769 SLC12A2 6558 6.23 up 5.08E-14 member 2 7927681 BICC1 bicaudal C homolog 1 (Drosophila) 80114 6.18 up 4.36E-15 8101126 CXCL10 chemokine (C-X-C motif) ligand 10 3627 6.09 up 2.11E-04 7906919 RGS4 regulator of G-protein signaling 4 5999 5.81 up 1.64E-08 phospholipase A2, group VII (platelet-activating factor 8126784 PLA2G7 7941 5.76 up 1.40E-05 acetylhydrolase, plasma) collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, 8046922 COL3A1 1281 5.72 up 1.42E-05 autosomal dominant) 8029693 FOSB FBJ murine osteosarcoma viral oncogene homolog B 2354 5.62 up 4.84E-12 8104079 FAT 2195 5.54 up 1.53E-06 7954090 EMP1 epithelial membrane protein 1 2012 5.28 up 5.72E-08 8130867 THBS2 thrombospondin 2 7058 5.20 up 1.63E-06 8177222 CD24 934 5.16 up 1.77E-06 7976812 SNORD113-4 small nucleolar RNA, C/D box 113-4 767564 5.03 up 3.95E-07 cystic fibrosis transmembrane conductance regulator (ATP-binding 8135661 CFTR 1080 5.02 up 2.81E-12 cassette sub-family C, member 7) 7935553 LOXL4 lysyl oxidase-like 4 84171 5.02 up 2.78E-12 8152617 HAS2 hyaluronan synthase 2 3037 4.85 up 2.70E-06 7938366 WEE1 7465 4.83 up 6.35E-05 8136336 AKR1B10 aldo-keto reductase family 1, member B10 (aldose reductase) 57016 4.79 up 0.045463 7976814 SNORD114-2 small nucleolar RNA, C/D box 114-2 767578 4.77 up 2.96E-09 8049425 SPP2 secreted phosphoprotein 2, 24kDa 6694 4.71 up 7.52E-06 7946048 HBG1|HBG2 hemoglobin, gamma A | hemoglobin, gamma G 3047 | 3048 4.64 up 9.38E-09 7946054 HBG1|HBG2 hemoglobin, gamma A | hemoglobin, gamma G 3047 | 3048 4.63 up 9.38E-09 8059905 COL6A3 collagen, type VI, alpha 3 1293 4.63 up 1.54E-07
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