GSE107933
Transcriptome analysis of TNFR1-knockout mouse colon
We have compared mRNA expression in full-thickness mouse colon between wildtype mice and mice with a genetic deletion in tumor necrosis factor receptor 1 (TNFR1, encoded by the Tnfrsf1a gene). This experiment was motivated by our observation that Il10-/- Tnfr1-/- double-knockout mice develop very-early-onset colitis at the time of weaning, significantly earlier than disease onset in Il10-/- single-knockout mice. This suggests that TNFR1 mediates protection from colitis. To understand these protective mechanisms at the transcript level in a non-inflammatory context, we performed transcriptome profiling (mRNA-Seq) on colons from 12-week-old Tnfr1-/- mice, which do not develop colitis, and wildtype littermates. These experiments revealed that an estimated 510 transcripts were upregulated and 377 downregulated in colonic tissue of Tnfr1-/- mice. We aggregated the transcript information to calculate gene expression and performed gene set enrichment analysis to identify signaling pathways altered in Tnfr1-/- mice. When queried against a high-confidence set of 50 signaling pathways, the Tnfr1-/- gene expression profile was associated with reduced mitosis and increased glycolysis, transforming growth factor beta signaling, DNA repair, and reactive oxygen species signaling. Gene ontological analysis classified some of the differentially expressed genes into cytokines, junctional proteins, and transcription factors, but within these groups there was no consensus on the direction of regulation. We also examined how differentially expressed transcripts (called the TNFR1-regulated transcriptome) compared to differentially expressed colonic transcripts from previously collected Tnfr2-/- animals (or the TNFR2-regulated transcriptome, available at the NCBI Gene Expression Omnibus under the accession GSE65408). The raw data were re-analyzed with the current pipeline to enable comparison. There was almost no correlation between TNFR1- and TNFR2-regulated transcripts. Only 30 (~3%) TNFR1-regulated transcripts were also TNFR2-regulated transcripts, and ~4% of the TNFR2-regulated genes were also TNFR1-regulated genes. Of the few co-regulated transcripts, there was a significant negative correlation in their direction of regulation in Tnfr1-/- versus Tnfr2-/- colons. Collectively these results indicate that TNFR1 and TNFR2 have mostly different and opposing effects on gene expression in the mouse colon. MANUSCRIPT ABSTRACT:  BACKGROUND & AIMS: Very-early-onset inflammatory bowel disease (VEO-IBD) is a devastating disease beginning in early childhood, refractory to anti-tumor necrosis factor (anti-TNF) therapies, and associated with mutations in the interleukin 10 (IL-10) pathway. Contrary to mainstream clinical practice, cellular and animal studies have shown beneficial roles of TNF signaling in colitis, but how these roles are encoded through TNF's receptors remains unknown. Here we examined the role of TNF receptor 1 (TNFR1, or p55) in modulating the onset and severity of colitis in the interleukin 10 (IL-10)-deficient mouse model.  METHODS: Colitis severity was compared between Il10-/- Tnfr1-/- and Il10-/-- littermate mice at 2-12 weeks of age. To assess dysbiosis as a potential pathogenic mechanism, Il10-/- Tnfr1-/- mice were treated with neomycin and metronidazole and their cecal contents analyzed by 16S rRNA sequencing. Gene expression, barrier function, and epithelial proliferation and apoptosis were examined in adult colitis-free Tnfr1-/- wildtype littermates.  RESULTS: In contrast to relatively healthy Il10-/- mice, Il10-/- Tnfr1-/- mice developed severe, antibiotic-treatable colitis shortly after weaning. Microbiotal composition was similar between Il10-/- Tnfr1-/- mice and Il10-/- littermate controls. Tnfr1-/- mice expressed transcripts associated with reactive oxygen species and DNA repair pathways. Tnfr1-/- mice exhibited focal areas of crypt branching, higher colonic epithelial permeability, and hallmarks of DNA damage.  CONCLUSIONS: TNFR1 promotes epithelial homeostasis and regulates commensal exposure. The lack of TNFR1 accelerates the onset and severity of IBD in the absence of tolerogenic signaling (i.e., lack of IL-10). Il10-/- Tnfr1-/- mice may serve as a valuable model of very-early-onset IBD (VEO-IBD). Overall design: Total RNA from whole colons of 12 week-old male wildtype C57Bl/6 and TNFR1-/- mice (n=3 each) was isolated using the PureLink RNA kit (Ambion, Life Technologies). RNA samples were submitted to the Genomic Services Lab at the HudsonAlpha Institute for Biotechnology (Huntsville, AL) for multiplex library preparation, mRNA enrichment, and sequencing. Sequencing was performed to an average depth of 30M paired-end 50bp reads per sample (HiSeq, Illumina, San Diego, CA). Data files containing raw reads were aligned to the mouse genome using kallisto, and statistical tests for differential expression were performed with sleuth.