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Project description:Background & Aims: Gut microbiota dysbiosis is associated with the pathogenesis of Crohn's disease (CD), but the underlying mechanisms by which colon bacteria contribute to the development of CD remain largely unclear. Here, we investigated whether colon bacteria, particularly Bacteroides acidifaciens (B. acidifaciens), promote CD pathogenesis through their small RNAs (sRNAs). Methods: The composition of gut bacteria were analyzed in the fecal samples of IL-10 knockout (KO) mice and CD45RBhigh mice that spontaneously develop colitis via 16S amplification sequencing, and the expression pattern of B. acidifaciens in both experimental colitis mice and CD patients was confirmed by quantitative RT-PCR (qRT-PCR). The effects of B. acidifaciens and their total and small RNA contents on colitis development were evaluated in IL-10 KO mice. Furthermore, the expression profile of B. acidifaciens-derived sRNA (BA-sRNA) in IL-10 KO mice was established by RNA sequencing, and BA-sRNA-172 levels in experimental colitis mice and CD patients were determined. Lentivirus carrying BA-sRNA-172 and BA-sRNA-172-deficient B. acidifaciens were then constructed and used to investigate the role of BA-sRNA-172 in the development of colitis. The activities of BA-sRNA-172 in regulating mucin2 (MUC2) expression and gut mucosal barrier function was systematically studied by in situ hybridization, immunofluorescence staining, qRT-PCR, western blot, RNA pull-down and luciferase assays in vitro. Finally, the effects of anti-BA-sRNA-172 and anti-MUC2 shRNA on intestinal barrier function and colitis development were evaluated. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
2023-08-13 | GSE155040 | GEO
Project description:Background & Aims: Gut microbiota dysbiosis is associated with the pathogenesis of Crohn's disease (CD), but the underlying mechanisms by which colon bacteria contribute to the development of CD remain largely unclear. Here, we investigated whether colon bacteria, particularly Bacteroides acidifaciens (B. acidifaciens), promote CD pathogenesis through their small RNAs (sRNAs). Methods: The composition of gut bacteria were analyzed in the fecal samples of IL-10 knockout (KO) mice and CD45RBhigh mice that spontaneously develop colitis via 16S amplification sequencing, and the expression pattern of B. acidifaciens in both experimental colitis mice and CD patients was confirmed by quantitative RT-PCR (qRT-PCR). The effects of B. acidifaciens and their total and small RNA contents on colitis development were evaluated in IL-10 KO mice. Furthermore, the expression profile of B. acidifaciens-derived sRNA (BA-sRNA) in IL-10 KO mice was established by RNA sequencing, and BA-sRNA-172 levels in experimental colitis mice and CD patients were determined. Lentivirus carrying BA-sRNA-172 and BA-sRNA-172-deficient B. acidifaciens were then constructed and used to investigate the role of BA-sRNA-172 in the development of colitis. The activities of BA-sRNA-172 in regulating mucin2 (MUC2) expression and gut mucosal barrier function was systematically studied by in situ hybridization, immunofluorescence staining, qRT-PCR, western blot, RNA pull-down and luciferase assays in vitro. Finally, the effects of anti-BA-sRNA-172 and anti-MUC2 shRNA on intestinal barrier function and colitis development were evaluated. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
2023-08-13 | GSE155039 | GEO
Project description:Background & Aims: Gut microbiota dysbiosis is associated with the pathogenesis of Crohn's disease (CD), but the underlying mechanisms by which colon bacteria contribute to the development of CD remain largely unclear. Here, we investigated whether colon bacteria, particularly Bacteroides acidifaciens (B. acidifaciens), promote CD pathogenesis through their small RNAs (sRNAs). Methods: The composition of gut bacteria were analyzed in the fecal samples of IL-10 knockout (KO) mice and CD45RBhigh mice that spontaneously develop colitis via 16S amplification sequencing, and the expression pattern of B. acidifaciens in both experimental colitis mice and CD patients was confirmed by quantitative RT-PCR (qRT-PCR). The effects of B. acidifaciens and their total and small RNA contents on colitis development were evaluated in IL-10 KO mice. Furthermore, the expression profile of B. acidifaciens-derived sRNA (BA-sRNA) in IL-10 KO mice was established by RNA sequencing, and BA-sRNA-172 levels in experimental colitis mice and CD patients were determined. Lentivirus carrying BA-sRNA-172 and BA-sRNA-172-deficient B. acidifaciens were then constructed and used to investigate the role of BA-sRNA-172 in the development of colitis. The activities of BA-sRNA-172 in regulating mucin2 (MUC2) expression and gut mucosal barrier function was systematically studied by in situ hybridization, immunofluorescence staining, qRT-PCR, western blot, RNA pull-down and luciferase assays in vitro. Finally, the effects of anti-BA-sRNA-172 and anti-MUC2 shRNA on intestinal barrier function and colitis development were evaluated. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
2023-08-13 | GSE155038 | GEO
Project description:Background & Aims: Gut microbiota dysbiosis is associated with the pathogenesis of Crohn's disease (CD), but the underlying mechanisms by which colon bacteria contribute to the development of CD remain largely unclear. Here, we investigated whether colon bacteria, particularly Bacteroides acidifaciens (B. acidifaciens), promote CD pathogenesis through their small RNAs (sRNAs). Methods: The composition of gut bacteria were analyzed in the fecal samples of IL-10 knockout (KO) mice and CD45RBhigh mice that spontaneously develop colitis via 16S amplification sequencing, and the expression pattern of B. acidifaciens in both experimental colitis mice and CD patients was confirmed by quantitative RT-PCR (qRT-PCR). The effects of B. acidifaciens and their total and small RNA contents on colitis development were evaluated in IL-10 KO mice. Furthermore, the expression profile of B. acidifaciens-derived sRNA (BA-sRNA) in IL-10 KO mice was established by RNA sequencing, and BA-sRNA-172 levels in experimental colitis mice and CD patients were determined. Lentivirus carrying BA-sRNA-172 and BA-sRNA-172-deficient B. acidifaciens were then constructed and used to investigate the role of BA-sRNA-172 in the development of colitis. The activities of BA-sRNA-172 in regulating mucin2 (MUC2) expression and gut mucosal barrier function was systematically studied by in situ hybridization, immunofluorescence staining, qRT-PCR, western blot, RNA pull-down and luciferase assays in vitro. Finally, the effects of anti-BA-sRNA-172 and anti-MUC2 shRNA on intestinal barrier function and colitis development were evaluated. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.
2023-08-13 | GSE155035 | GEO
Project description:Sites of inflammation are associated with profound changes in tissue metabolism. Studies in vitro and in vivo have shown that the activation of the hypoxia-inducible factor (HIF) serves as an adaptive pathway for the resolution of inflammation associated with various murine disease models. The resolution of disease occurs, at least in part, through transcriptional regulation of non-classical epithelial barrier genes. There is significant recent interest in harnessing hypoxia-inducible pathways, including targeting the HIF and the proyl-hydroxylase (PHD) enzymes that stabilize HIF, to promote mucosal healing. Here, we review the signaling pathways involved and define how hypoxia-associated signaling provides mechanistic insight into augmenting barrier function in mucosal inflammatory disease.
| S-EPMC5560425 | biostudies-other
Project description:Aberrant neutrophil extracellular trap (NET) formation and the loss of barrier integrity in inflamed intestinal tissues have long been associated with inflammatory bowel disease (IBD). However, whether NETs alter intestinal epithelium permeability during colitis remains elusive. Here, we demonstrated that NETs promote the breakdown in intestinal barrier function for the pathogenesis of intestinal inflammation in mouse models of colitis. NETs were abundant in the colon of mice with colitis experimentally induced by dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS). Analysis of the intestinal barrier integrity revealed that NETs impaired gut permeability, enabling the initiation of luminal bacterial translocation and inflammation. Furthermore, NETs induced the apoptosis of epithelial cells and disrupted the integrity of tight junctions and adherens junctions. Intravenous administration of DNase I, an enzyme that dissolves the web-like DNA filaments of NETs, during colitis restored the mucosal barrier integrity which reduced the dissemination of luminal bacteria and attenuated intestinal inflammation in both DSS and TNBS models. We conclude that NETs serve a detrimental factor in the gut epithelial barrier function leading to the pathogenesis of mucosal inflammation during acute colitis.
| S-EPMC7459452 | biostudies-literature
Project description:In colitis associated cancer (CAC), chronic inflammation exposes the epithelial mucosal defensive lining to inflammatory mediators such as cytokines and anti-microbial peptides (AMPs) causing the dysbiosis of microbiota population and the dysregulation of immune response. Matrix Metalloproteinases (MMPs) are zinc dependent endopeptidases which mediate inflammation, tissue remodeling, and carcinogenesis. MMP9 is undetectable in healthy tissue, although highly upregulated during inflammation and cancer. We have previously shown that MMP9 plays a protective role in CAC opposite to its conventional role of acute inflammation and cancer mediator. In this study, we investigated the mechanistic role of MMP9 in preserving the epithelial mucosal integrity to suppress the progression of tumor microenvironment in CAC. We used transgenic mice constitutively expressing MMP9 in colonic epithelium (TgM9) as an in vivo model and intestinal cell line CaCo2BBE as an in vitro model. We induced CAC with three cycles of dextran sodium sulfate (DSS). We observed that MMP9 expression in colonic epithelium maintains the microbiota. We also observed that MMP9 mediates pro-inflammatory cytokine levels and AMPs but suppresses IL-22 resulting in lower levels of REG3-g and S100A8 AMPs. We also found that MMP9 maintains an efficient barrier function and the integrity of tight junctions. We also observed increased levels of mucin and intestinal trefoil factor among TgM9 mice in CAC. We also found that MMP9 expressing CaCo2BBE cells had increased expressions of EGFR and nuclear transcription factor- specificity protein 1 (Sp1). These data imply that MMP9 acts as a tumor suppressor in CAC by sustaining the epithelial mucosal integrity due to the activation of EGFR-Sp1 signaling pathway.
| S-EPMC5706902 | biostudies-other
Project description:ObjectiveThe pathogenesis of UC relates to gut microbiota dysbiosis. We postulate that alterations in the viral community populating the intestinal mucosa play an important role in UC pathogenesis. This study aims to characterise the mucosal virome and their functions in health and UC.DesignDeep metagenomics sequencing of virus-like particle preparations and bacterial 16S rRNA sequencing were performed on the rectal mucosa of 167 subjects from three different geographical regions in China (UC=91; healthy controls=76). Virome and bacteriome alterations in UC mucosa were assessed and correlated with patient metadata. We applied partition around medoids clustering algorithm and classified mucosa viral communities into two clusters, referred to as mucosal virome metacommunities 1 and 2.ResultsIn UC, there was an expansion of mucosa viruses, particularly Caudovirales bacteriophages, and a decrease in mucosa Caudovirales diversity, richness and evenness compared with healthy controls. Altered mucosal virome correlated with intestinal inflammation. Interindividual dissimilarity between mucosal viromes was higher in UC than controls. Escherichia phage and Enterobacteria phage were more abundant in the mucosa of UC than controls. Compared with metacommunity 1, metacommunity 2 was predominated by UC subjects and displayed a significant loss of various viral species. Patients with UC showed substantial abrogation of diverse viral functions, whereas multiple viral functions, particularly functions of bacteriophages associated with host bacteria fitness and pathogenicity, were markedly enriched in UC mucosa. Intensive transkingdom correlations between mucosa viruses and bacteria were significantly depleted in UC.ConclusionWe demonstrated for the first time that UC is characterised by substantial alterations of the mucosa virobiota with functional distortion. Enrichment of Caudovirales bacteriophages, increased phage/bacteria virulence functions and loss of viral-bacterial correlations in the UC mucosa highlight that mucosal virome may play an important role in UC pathogenesis.
| S-EPMC6582748 | biostudies-literature
Project description:The intestinal epithelial barrier is critical to limit potential harmful consequences from exposure to deleterious luminal contents on the organism. Although this barrier is functionally important along the entire gut, specific regional regulatory mechanisms involved in the maintenance of this barrier are poorly defined. Herein, we identified Gata4 as a crucial regulator of barrier integrity in the mouse proximal intestinal epithelium. Conditional deletion of Gata4 in the intestine led to a drastic increase in claudin-2 expression that was associated with an important increase of gut barrier permeability without causing overt spontaneous inflammation. Administration of indomethacin, a non-steroidal anti-inflammatory drug (NSAID) that causes enteritis, led to rapid and restricted proximal small intestinal injuries in Gata4 mutant mice as opposed to control mice. Comparative analysis of gene transcript profiles from indomethacin-challenged control and Gata4 mutant mice identified defects in epithelial cell survival, inflammatory cell recruitment and tissue repair mechanisms. Altogether, these observations identify Gata4 as a novel crucial regulator of the intestinal epithelial barrier and as a critical epithelial transcription factor implicated in the maintenance of proximal intestinal mucosal integrity after injury.
| S-EPMC5101531 | biostudies-literature
Project description:Psychological disorders are associated with increased risk of severe inflammatory bowel disease (IBD) by causing gut microbiota dysbiosis and colonic mucosal barrier damage. However, the interaction between chronic restraint stress (CRS), gut microbiota composition, and colonic mucus remains unclear. We demonstrated that mice under CRS conditions exhibited alterations in microbiota composition, disruption of colonic mucus, and aggravation of colitis. In addition, the abundance of Akkermansia muciniphila was significantly decreased in mice under CRS and UC patients with depression, and positively associated with the expression of MUC2. After antibiotic treatment, the recipient mice colonized with CRS microbiota showed barrier defects and severe colitis. Administration of Akkermansia muciniphila was found to restore colonic mucus and modify the gut microbiota. We confirm that CRS-mediated gut microbiota dysbiosis results in colonic mucosal barrier damage and aggravation of colitis. Our results suggest that A. muciniphila is expected to be a potential probiotic to protect and treat colonic mucus that is involved in IBD with psychological disorders.
| S-EPMC8551916 | biostudies-literature