RNA-seq for inflamed and non-inflamed mucosa in patients with Crohn's disease
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ABSTRACT: To investigate the gene expression profile of inflamed and non-inflamed mucosa in patients with Crohn's disease. To investigate TCR repertoires in the intestinal mucosa, the expression profile of the TCR repertoire gene was analyzed.
Project description:Histology in the mesentery pointed to altered blood vessels. This experiment was designed to define the differences in gene expression in vessels from Crohn's disease versus controls. Crohn's disease was separately evaluated in inflamed (central disease) areas and in adjacent noninflamed areas. Laser capture microdissection was carried out on Carnoy's fixed mesenteric samples, comparing normal arteries or veins with Crohn's inflamed or nonifnlamed arteries or veins.
Project description:In Crohn's disease, creeping fat is the characteristic expansion of mesenteric adipose tissue wrapping around the inflamed intestine. Through a comparative transcriptomic analysis of creeping fat and normal-looking mesenteric adipose tissues from patients with Crohn's disease and non-Crohn's disease, we found that a dynamic transcriptional and cell compositional change occurs during the progression from non-Crohn's disease to Crohn's disease, and finally to creeping fat.
Project description:Rapid neutrophil (PMN) mobilization to sites of insult is critical for host defense and requires crossing of the vascular wall. PMN transendothelial migration (TEM) involves several well-studied sequential adhesive interactions with vascular ECs, however what initiates or terminates this process is not well-understood. Our findings identified a new mechanism where mucosal interstitial macrophages are recruited to interact with the vascular wall and locally prime endothelial cell (EC) responses to accommodate PMN TEM. Using real-time intravital microscopy (IVM) on lipopolysaccharides (LPS)-inflamed intestines in CX3CR1-EGFP macrophage-reporter mice, complemented by whole-mount tissue imaging and flow cytometry, we demonstrate that macrophage presence was critical for the initiation of PMN-EC adhesive interactions and subsequent PMN TEM and accumulation in the intestinal mucosa. Anti CSFR-1 antibody-mediated macrophage depletion in the lamina propria and at the vessel wall significantly reduced PMN adhesion and TEM in inflamed intestines. Vessel associated macrophages (VAMs) were found to trigger localized upregulation of EC ICAM-1 expression and their removal resulted in elimination of the ICAM-1 “hot spots”, impeding PMN-EC interactions. Further mechanistic studies using human clinical specimens, TNFa knockout macrophage chimeras, TNFa/TNF receptor (TNFR) neutralization and multi-cellular macrophage-EC-PMN cocultures revealed a new role for macrophage TNFa and EC TNFRII axis in regulating EC adhesion molecule expression and PMN TEM. As such, our findings identify new, clinically relevant mechanism by which macrophage regulate PMN trafficking in inflamed mucosa.
Project description:Rapid neutrophil (PMN) mobilization to sites of insult is critical for host defense and requires crossing of the vascular wall. PMN transendothelial migration (TEM) involves several well-studied sequential adhesive interactions with vascular ECs, however what initiates or terminates this process is not well-understood. Our findings identified a new mechanism where mucosal interstitial macrophages are recruited to interact with the vascular wall and locally prime endothelial cell (EC) responses to accommodate PMN TEM. Using real-time intravital microscopy (IVM) on lipopolysaccharides (LPS)-inflamed intestines in CX3CR1-EGFP macrophage-reporter mice, complemented by whole-mount tissue imaging and flow cytometry, we demonstrate that macrophage presence was critical for the initiation of PMN-EC adhesive interactions and subsequent PMN TEM and accumulation in the intestinal mucosa. Anti CSFR-1 antibody-mediated macrophage depletion in the lamina propria and at the vessel wall significantly reduced PMN adhesion and TEM in inflamed intestines. Vessel associated macrophages (VAMs) were found to trigger localized upregulation of EC ICAM-1 expression and their removal resulted in elimination of the ICAM-1 “hot spots”, impeding PMN-EC interactions. Further mechanistic studies using human clinical specimens, TNFa knockout macrophage chimeras, TNFa/TNF receptor (TNFR) neutralization and multi-cellular macrophage-EC-PMN cocultures revealed a new role for macrophage TNFa and EC TNFRII axis in regulating EC adhesion molecule expression and PMN TEM. As such, our findings identify new, clinically relevant mechanism by which macrophage regulate PMN trafficking in inflamed mucosa.
Project description:Histology in the mesentery pointed to altered blood vessels. This experiment was designed to define the differences in gene expression in vessels from Crohn's disease versus controls. Crohn's disease was separately evaluated in inflamed (central disease) areas and in adjacent noninflamed areas.
Project description:Inflammatory transcriptomic signatures and cell type compositions in inflamed and non-inflamed colonic mucosa of ulcerative colitis
Project description:The Illumina Infinium EPIC Human DNA methylation Beadchip was used to obtain DNA methylation profiles across approximately 867,000 CpGs in ileal fibroblast samples. Samples included 5 control samples and 13 Crohn’s disease samples. The Crohn’s disease samples can be further subdivided into inflamed (2), non-inflamed (7) and stenotic (4) samples. This experiment together was performed in conjunction with a gene expression experiment (GSE99816). Use Supplemental Table 2 of the manuscript to quickly find the samples that were present in both experiments.
Project description:The Illumina NextSeq500 was used to sequence the gene expression profiles of 21 ileal fibroblast samples. Samples included 6 control samples and 15 Crohn’s disease samples. The Crohn’s disease samples can be further subdivided into inflamed (4), non-inflamed (6) and stenotic (5) samples. This experiment together was performed in conjunction with a DNA methylation experiment (GSE99788). Use Supplemental Table 2 of the manuscript to quickly find the samples that were present in both experiments.