Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse IL10-/- knocked out colitis model

ABSTRACT: Objetives: study and characterization of the IL10-/- knocked out colitis model in mice at genomic level and the study of the influence of bacteria in the development of the disease. Experiment Overall Design: We used three groups of animasl: 1)IL10-/- mice grown under conventional condition, 2)IL10-/- mice grown under specific pathogen free (SPF) conditions, 3) Wild type mice grown under conventional conditions (CONTROL group). For each group, we recovered samples at 7, 9 and 12 weeks after birth According to colon length and colonic damage, three replicates were selected for each time point and each group (27 samples), for genomic analysis. Experiment Overall Design: RNA was extracted from homogenized full-thickness colonic tissues in TrizolÂ® reagent (Invitrogen) and purified with RNeasy affinity columns (Qiagen), according to manufacturerÂ´s protocol. The microarray analysis was performed by Progenika Biopharma (Bilbao, Spain) on GeneChipÂ® Rat Genome 230 2.0 Array (Affymetrix). All sample labeling (biotin), hybridization, staining and scanning procedures were carried out using Affimetrix, standard protocols (www.affymetrix.com). Normalization was carried out using Bioconductor sofware (affyPLM package).

ORGANISM(S): Mus musculus

SUBMITTER: Antonio Suarez

PROVIDER: E-GEOD-9297 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Inflammatory Bowel Disease (IBD) is a term describing a collection of conditions characterised by chronic inflammatory disorder of the gastrointestinal tract involving an inappropriate immune response to commensal microorganisms in a genetically susceptible host. Four kiwifruit extracts, aqueous and ethyl acetate extracts of gold kiwifruit (Actinidia chinensis) or green kiwifruit (A. deliciosa), have previously demonstrated anti-inflammatory activity using in vitro models of IBD. This study examined whether these kiwifruit extracts had immune modulating effects in vivo against inflammatory processes known to be increased in patients with IBD. KFEs were used as a dietary intervention in Il10-/- mice (an in vivo model of IBD) and the C57BL/6J background strain in a 3 x 2 factorial design. While all Il10-/- mice developed significant colonic inflammation compared to the C57BL/6J mice, this was not affected by the inclusion of KFE in the diet. Whole genome gene and protein expression level profiling indicated that KFEs influenced immune signalling pathways and metabolic processes within the colonic tissue; however, the effects were subtle. In particular, adaptive immune pathways were reduced by three out of four kiwifruit extracts, with greater reduction seen in the C57BL/6J mice. This suggests that while immune-modulating activity was present in vivo, KFEs did not reduce inflammatory processes relevant to IBD. Experimental design. Two experiments were conducted, one using extracts from gold kiwifruit and one using extracts from green kiwifruit. Within each experiment, both Il10-/- and C57 mice were randomly divided into three diet treatment groups, to ive the following 12 treatments: 1) Gold Kiwifruit Experiment, C57BL/6J mice, Control diet (AIN-76A), 2) Gold Kiwifruit Experiment, C57BL/6J mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 3) Gold Kiwifruit Experiment, C57BL/6J mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) 4) Gold Kiwifruit Experiment, Il10-/- mice, Control diet (AIN-76A), 5) Gold Kiwifruit Experiment, Il10-/- mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 6) Gold Kiwifruit Experiment, Il10-/- mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) 7) Green Kiwifruit Experiment, C57BL/6J mice, Control diet (AIN-76A), 8) Green Kiwifruit Experiment, C57BL/6J mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 9) Green Kiwifruit Experiment, C57BL/6J mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) 10) Green Kiwifruit Experiment, Il10-/- mice, Control diet (AIN-76A), 11) Green Kiwifruit Experiment, Il10-/- mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 12) Green Kiwifruit Experiment, Il10-/- mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) Six biological replicates were analysed from each treatment group, except for groups 3 and 7 where five replicates were analysed due to array quality issues with the sixth replicate. Each replicate contained mRNA from one mouse. A reference design was used, where each slide was hybridised with mRNA from one sample (green channel) and mRNA from a common reference pool (red channel).

Project description:BACKGROUND: Appendicitis followed by appendectomy (AA) at a young age protects against later inflammatory bowel disease (IBD). Using a novel murine appendicitis model we earlier demonstrated that AA proffered significant protection against subsequent experimental colitis. AIM: To delineate genes and biological pathways involved in the protective effect of AA against subsequent colitis using gene set enrichment analysis (GSEA) of DNA microarray data. METHODS: Appendicitis and appendicectomy was done (5 week old male BALB/c mice) near the most proximal colon (caecal lymphoid follicles) and colonic samples were harvested from the most distal colon. Two consecutive laparotomies were done in control Sham-Sham (SS) mice. RNA was extracted (TRIzol®) from 4 individual colonic samples per group (AA group vs. SS group) with each sample taken independently through Affymetrix® microarray hybridization. For GSEA, data for more than 23,000 genes were exported from Partek and analyzed with GSEA software (with 2852 gene sets encoded) to establish correlates with phenotypes of the gene sets. RESULTS: Distal colonic expression of 636 gene-sets were significantly upregulated in AA group samples (False Discovery Rates (FDR) values < 1 % and p value < 0.001; stringent statistical selection). These were validated by quantitative PCR of 14 selected genes across the immunological spectrum and over time-intervals of 3 days, 14 days and 28 days. CONCLUSIONS: Many key immunological, apoptosis-related and cellular function-associated gene-sets involved in the protective effect of AA in experimental colitis were identified. Further analysis of these profiles and biological pathways will assist utilizing these gene products and manipulating various aspects of these pathways to develop better therapeutic strategies in the management of intractable IBD. Appendicitis and appendicectomy was done (5 week old male BALB/c mice) near the most proximal colon (caecal lymphoid follicles) and colonic samples were harvested from the most distal colon. Two consecutive laparotomies were done in control Sham-Sham (SS) mice. RNA was extracted (TRIzol®) from 4 individual colonic samples per group (AA group vs. SS group) with each sample taken independently through Affymetrix® microarray hybridization. variable_protocol: appendicitis and appendectomy: AA1, AA2, AA3, AA4 variable_protocol: sham/sham surgery: SS1, SS2, SS3, SS4 repeat_biological replicate: AA1, AA2, AA3, AA4 repeat_biological replicate: SS1, SS2, SS3, SS4 Upregulated gene-set linked as supplementary file.

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Transcription profiling of mouse IL10-/- knocked out colitis model

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