ABSTRACT
Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.
Subject(s)
Information Services , Polymerase Chain Reaction/methods , Data CollectionABSTRACT
BACKGROUND: Endoplasmic reticulum (ER) stress has been suggested to play a role in inflammatory bowel disease (IBD). The three branches (ATF6, IRE1 and PERK) of the unfolded protein response (UPR) have different roles and are not necessarily activated simultaneously. METHODOLOGY/PRINCIPAL FINDINGS: Expression of UPR-related genes was investigated in colonic and ileal biopsies of 23 controls, 15 ulcerative colitis (UC) and 54 Crohn's disease (CD) patients. This expression was confirmed at protein level in colonic and ileal samples of five controls, UC and CD patients. HSPA5, PDIA4 and XBP1s were significantly increased in colonic IBD at mRNA and/or protein levels, indicating activation of the ATF6 and IRE1 branch. Colonic IBD was associated with increased phosphorylation of EIF2A suggesting the activation of the PERK branch, but subsequent induction of GADD34 was not observed. In ileal CD, no differential expression of the UPR-related genes was observed, but our data suggested a higher basal activation of the UPR in the ileal mucosa of controls. This was confirmed by the increased expression of 16 UPR-related genes as 12 of them were significantly more expressed in ileal controls compared to colonic controls. Tunicamycin stimulation of colonic and ileal samples of healthy individuals revealed that although the ileal mucosa is exhibiting this higher basal UPR activation, it is still responsive to ER stress, even more than colonic mucosa. CONCLUSIONS/SIGNIFICANCE: Activation of the three UPR-related arms is seen in colonic IBD-associated inflammation. However, despite EIF2A activation, inflamed colonic tissue did not increase GADD34 expression, which is usually involved in re-establishment of ER homeostasis. This study also implies the presence of a constitutive UPR activation in healthy ileal mucosa, with no further activation during inflammation. Therefore, engagement of the UPR differs between colon and ileum and this could be a factor in the development of ileal or colonic disease.
Subject(s)
Colitis, Ulcerative/pathology , Colon/pathology , Crohn Disease/pathology , Endoplasmic Reticulum Stress/genetics , Ileum/pathology , Transcriptome , Activating Transcription Factor 6/genetics , Activating Transcription Factor 6/metabolism , Adolescent , Adult , Aged , Case-Control Studies , Child , Colitis, Ulcerative/diagnosis , Colitis, Ulcerative/genetics , Colitis, Ulcerative/metabolism , Colon/metabolism , Crohn Disease/diagnosis , Crohn Disease/genetics , Crohn Disease/metabolism , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/drug effects , Endoribonucleases/genetics , Endoribonucleases/metabolism , Endoscopy, Gastrointestinal , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Ileum/metabolism , Interleukin-8/genetics , Interleukin-8/metabolism , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Middle Aged , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tunicamycin/pharmacology , Unfolded Protein Response/drug effects , Unfolded Protein Response/genetics , Young AdultABSTRACT
BACKGROUND: Immunological and genetic findings implicate Th17 effector cytokines in the pathogenesis of inflammatory bowel disease (IBD). Expression of Th17 pathway-associated genes is mainly studied in colonic disease. The present study assessed the mRNA expression levels of Th17 effector cytokines (IL17A, IL17F, IL21, IL22 and IL26) and genes involved in differentiation (IL6, IL1B, TGFB1, IL23A and STAT3) and recruitment of Th17 cells (CCR6 and CCL20) by quantitative real-time PCR analysis of colonic and ileal biopsies from 22 healthy control subjects, 26 patients with Crohn's disease (CD) and 12 patients with ulcerative colitis (UC). Inflammation was quantified by measuring expression of the inflammatory mediators IL8 and TNF. RESULTS: Evaluation of mRNA expression levels in colonic and ileal control samples revealed that TNF, TGFB1, STAT3 and CCR6 were expressed at higher levels in the ileum than in the colon. Expression of all the Th17 pathway-associated genes was increased in inflamed colonic samples. The increased expression of these genes was predominantly observed in samples from UC patients and was associated with more intense inflammation. Although increased expression of IL17A, IL17F, IL21 and IL26 was detected in inflamed ileal samples, expression of the indispensable Th17 cell differentiation factors TGFB1 and IL23A, the signaling molecule STAT3 and the Th17 recruitment factors CCR6 and CCL20 were unchanged. CONCLUSIONS: Our findings suggest that immune regulation is different in colonic and ileal disease, which might have important consequences for therapeutic intervention.
Subject(s)
Colon/metabolism , Ileum/metabolism , Inflammation Mediators/metabolism , Inflammatory Bowel Diseases/immunology , Th17 Cells/metabolism , Adolescent , Adult , Aged , Colon/immunology , Colon/pathology , Female , Gene Expression Profiling , Humans , Ileum/immunology , Ileum/pathology , Immunity, Mucosal/genetics , Inflammatory Bowel Diseases/genetics , Male , Middle Aged , RNA, Messenger/analysis , Th17 Cells/immunology , Th17 Cells/pathologyABSTRACT
Hydroxylase inhibitors stabilize hypoxia-inducible factor-1 (HIF-1), which has barrier-protective activity in the gut. Because the inflammatory cytokine TNF-α contributes to inflammatory bowel disease in part by compromising intestinal epithelial barrier integrity, hydroxylase inhibition may have beneficial effects in TNF-α-induced intestinal epithelial damage. The hydroxylase inhibitor dimethyloxalylglycin (DMOG) was tested in a murine model of TNF-α-driven chronic terminal ileitis. DMOG-treated mice experienced clinical benefit and showed clear attenuation of chronic intestinal inflammation compared with that of vehicle-treated littermates. Additional in vivo and in vitro experiments revealed that DMOG rapidly restored terminal ileal barrier function, at least in part through prevention of TNF-α-induced intestinal epithelial cell apoptosis. Subsequent transcriptional studies indicated that DMOG repressed Fas-associated death domain protein (FADD), a critical adaptor molecule in TNFR-1-mediated apoptosis, in an HIF-1α-dependent manner. Loss of this FADD repression by HIF-1α-targeting small interfering RNA significantly diminished the antiapoptotic action of DMOG. Additional molecular studies led to the discovery of a previously unappreciated HIF-1 binding site in the FADD promoter, which controls repression of FADD during hypoxia. As such, the results reported in this study allowed the identification of an innate mechanism that protects intestinal epithelial cells during (inflammatory) hypoxia, by direct modulation of death receptor signaling. Hydroxylase inhibition could represent a promising alternative treatment strategy for hypoxic inflammatory diseases, including inflammatory bowel disease.
Subject(s)
Enzyme Inhibitors/pharmacology , Fas-Associated Death Domain Protein/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Intestinal Mucosa/metabolism , Mixed Function Oxygenases/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , Amino Acids, Dicarboxylic/pharmacology , Animals , Blotting, Western , Cell Hypoxia/genetics , Cell Hypoxia/immunology , Chromatin Immunoprecipitation , Enzyme-Linked Immunosorbent Assay , Fas-Associated Death Domain Protein/genetics , Gene Expression Regulation/genetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Ileitis/genetics , Ileitis/metabolism , Immunity, Mucosal/genetics , Immunity, Mucosal/immunology , Immunohistochemistry , Intestinal Mucosa/pathology , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Promoter Regions, Genetic/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The role of the vagus nerve in controlling and modulating inflammatory responses under physiological conditions has been investigated. The purpose of this study is to assess changes in the immunological state evoked by vagus nerve stimulation in humans, by measuring cytokines produced by peripheral blood mononuclear cells (PBMC). We compared induction of IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha by lipopolysaccharide (LPS)-stimulated PBMC which were isolated from patients treated with vagus nerve stimulation for refractory epilepsy. We observed a significant decrease in IL-8 induction by LPS-stimulated PBMC after 6 months of vagus nerve stimulation in comparison to the pre-stimulation state. No significant changes were seen in the induction of IL-1beta, TNF-alpha, IL-6 or IL-10. The present study shows that cytokine induction by PBMC isolated from patients with refractory epilepsy is altered by long-term vagus nerve stimulation.
