Project description:Inflammatory bowel diseases (IBD) in humans are characterized by chronic inflammation and gastrointestinal tissue damage, caused by a combination of genetic and environmental factors. It has been largely documented that IBD frequently lead to colorectal cancers (CRC). The identification of causative factors of IBD is thus essential to understand CRC progression and develop therapeutical approaches. Models have been described in which molecular alterations are combined with inflammatory treatments in order to recapitulate IBD-associated CRC. Here, we describe a mouse line, Î±6fl/fl Villin-Cre, in which inactivation of the gene encoding the integrin alpha-6 subunit (ITGA6) specifically in the intestinal mucosa results into chronic inflammation and intestinal carcinogenesis. In these mice, the loss of integrin alpha-6 beta-4, a receptor mediating the attachment of epithelial cells to laminins, leads to epithelial detachment, hyperplasia, chronic inflammation, rectal prolapses, and ultimately adenocarcinomas. Alterations of differentiation affecting mucus secreting (goblet) cells as well as changes in expression of essential intestinal transcription factors were detected. Thus alpha-6 beta-4 integrin is a key factor for the maintenance of intestinal integrity and its loss may represent a risk factor for tumor progression associated with IBD. Transcriptome analysis of RNA from normal versus inflamed and carcinomatous rectal mucosa of Î±6 integrin deficient intestinal epithelium mice was performed. RNAs were prepared from 5 adenocarcinomas, which were macrodissected from the rectal prolapses and 4 flanking inflamed rectal mucosa of 53-80 week-old Î±6fl/fl Villin-Cre mice. RNAs from normal rectal mucosa were obtained by rectum scraping from 4 matched control animals. The transcriptome analysis was performed using the Affymetrix Mouse Gene 1.0 ST arrays. Images were processed using affymetrix GeneChipÂ® Command ConsoleÂ® Software (AGCC) (version 2.0) and numerical value were generated using Affymetrix Expression Consoleâ¢ Software (version 1.1).

Project description:Inflammatory bowel disease (IBD) is a complex and relapsing inflammatory disease, and patients with IBD exhibit a higher risk of developing colorectal cancer (CRC). Epithelial barrier disruption is one of the major causes of inflammatory bowel disease (IBD) in which epigenetic modulations are pivotal elements. However, the epigenetic mechanisms underlying the epithelial barrier integrity regulation remain largely unexplored. Here, we investigated how SETD2, a histone H3K36 trimethyltransferase, maintains intestinal epithelial homeostasis under inflammatory conditions. GEO public database and IBD tissues were used to investigate the clinical relevance of SetD2 in IBD. To define the role of SetD2 in the colitis, we generated mice with epithelial-specific deletion of Setd2 (Setd2Vil-KO mice). Acute colitis was induced by 2% dextran sodium sulfate (DSS), and colitis-associated CRC was induced by injecting azoxymethane (AOM), followed by three cycles of 2% DSS treatments. Colon tissues were collected from mice and analyzed by histology, immunohistochemistry and immunoblots. Organoids were generated from Setd2Vil-KO and control mice, and were stained with 7-AAD to detect apoptosis. We isolated intestinal epithelial cells (IECs), performed RNA-seq and H3K36me3 ChIP-seq analysis to uncover the mechanism. Results were validated in functional rescue experiments by N-acetyl-l-cysteine (NAC) treatment and transgenes expression in IECs. SETD2 expression was decreased in IBD patients and DSS-treated colitis mice. Setd2Vil-KO mice had abnormal loss of mucosa-producing goblet cells and antimicrobial peptide (AMP)-producing Paneth cells, and promoted early intestinal inflammation development. Consistent with the reduced SETD2 expression in IBD patients, Setd2Vil-KO mice exhibited increased susceptibility to DSS-induced colitis, accompanied by more severe epithelial barrier disruption. Intestinal permeability was markedly increased in Setd2Vil-KO mice. Setd2 ablation drived inﬂammation-associated CRC. Deletion of Setd2 resulted in excess reactive oxygen species (ROS), which led to cellular apoptosis and the defects in barrier integrity. N-acetyl-l-cysteine (NAC) treatment in Setd2Vil-KO mice rescued epithelial barrier injury and apoptosis. Moreover, overexpression of antioxidase PRDX6 in Setd2Vil-KO IECs largely alleviated the overproductions of ROS and improved the cellular survival. Deficiency of Setd2 specifically in the intestine aggravates epithelial barrier disruption and inflammatory response in colitis via a mechanism dependent on oxidative stress. More importantly, we show that Setd2 depletion results in excess ROS by directly down-regulating PRDX6, an antioxidant protein that inhibit excess ROS. Thus, our results highlight an epigenetic mechanism by which Setd2 mediates oxidative stress to modulate intestinal epithelial homeostasis.

Project description:Background; MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. MUC2 homo-oligomerizes intracellularly into large secreted polymers which give mucus its viscous properties. The inflammatory bowel disease (IBD) ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, whereas goblet cells and the mucus layer are increased in the other major inflammatory bowel disease, Crohnâs disease. Methods and Findings; By murine N-ethyl-N-nitrosourea-mutagenesis we identified two distinct non-complementing missense mutations in Muc2 exons encoding N- and C-terminal homo-oligomerization domains causing an ulcerative colitis-like phenotype. Both strains developed mild spontaneous distal intestinal inflammation, chronic diarrhea, rectal bleeding and prolapse, increased susceptibility to acute and chronic colitis induced by a luminal toxin, aberrant Muc2 biosynthesis, smaller goblet cell thecae (less stored mucin) and a diminished mucus barrier. Enhanced local production of IL-1beta, TNF-alpha and IFN-gamma was seen in the distal colon. The number of leukocytes within mesenteric lymph nodes was increased five-fold and leukocytes cultured in vitro produced both Th1 and Th2 cytokines (IFN-gamma, TNF-alpha and IL-13). Intestinal permeability was increased and the luminal bacterial flora were more heavily coated with immunoglobulin as occurs in IBD. This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis and wound repair. Expression of mutated Muc2 oligomerization domains in vitro demonstrated that aberrant Muc2 oligomerization underlies the ER stress. These models show that mutations in Muc2 oligomerization domains can lead to aberrant assembly of the Muc2 complex leading to ER stress, a depleted mucus barrier and intestinal inflammation. In ulcerative colitis we demonstrate similar accumulation of non-glycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in non-inflamed intestinal tissue. Conclusions; The observations that mucin misfolding and ER stress lead directly to intestinal inflammation and that ER stress and goblet cell pathology occur in ulcerative colitis suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of colitis. Experiment Overall Design: 3 individual mice from the Eeyore, Winnie or Wild-type strains were compared as groups. An Affymetrix ID was compared between groups if the ID was Present within two of the three mice within each grouping. IDs were compared by calculating the log2 of Group One average signal divided by Group 2 average signal.

			Action	DRS
	20230220_ZKhan_sample_AP.mgf	Mgf
	20230220_ZKhan_sample_AP.raw.raw	Raw
	20230220_ZKhan_sample_BP.mgf	Mgf
	20230220_ZKhan_sample_BP.raw.raw	Raw
	20230220_ZKhan_sample_CP.mgf	Mgf

Dataset Information

Label free-DDA nano-LC/MS/MS analysis of Human Mesenteric lymph collected from Inflammatory Bowel Disease (IBD) subjects.

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Role of the afferent lymph as an immunological conduit to analyze tissue antigenic and inflammatory load.

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