Non-inflamed Intestinal Mucosa Research Articles

Reduced diversity of intestinal T-cell receptor repertoire in patients with Crohn's disease.

BackgroundThe intestinal microenvironment directly determines the human T-cell receptor (TCR) repertoire. Despite its extreme diversity, TCR repertoire analysis may provide a better understanding of the immune system in patients with inflammatory bowel disease.MethodsTo investigate TCR repertoires in the intestinal mucosa, RNA sequencing was performed for inflamed and non-inflamed intestinal mucosa samples obtained from 13 patients with Crohn’s disease (CD) and healthy mucosa from nine non-IBD controls.ResultsThe gene expression frequency of the TCR repertoire showed a clear separation between inflamed mucosa of patients with CD and healthy mucosa of non-IBD controls in the hierarchical clustering heatmap. The richness of TCR repertoires measured by the Chao1 index did not show a significant difference among groups, whereas diversity measured by the D50 diversity index was decreased in the inflamed mucosa of CD patients. Rare/small TCR clonotypes occupied a large proportion of TCR repertoires in healthy mucosa of controls, whereas expanded clonotypes were common in inflamed mucosa of patients with CD. Segment usages of TRAV2, TRAV22, TRAV40, TRJ14, TRAJ51, TRBV1, TRBV21.1, and TRBJ1.5 were significantly decreased in CD patients. KEGG enrichment analysis identified the enrichment of several KEGG pathways, including inflammatory bowel disease (p = 0.0012), Th1 and Th2 cell differentiation (p = 0.0011), and intestinal immune network for IgA production (p = 0.0468).ConclusionsThe diversity of the TCR repertoire is reduced in inflamed mucosa of CD patients, which might contribute to intestinal inflammation.

Identification of Diagnostic Biomarkers by Bioinformatics Analysis in the Inflamed and Non-inflamed Intestinal Mucosa of Patients With Crohn’s Disease

Background: Crohn’s Disease (CD) is a type of inflammatory bowel disease that, despite its unknown etiology, is generally associated with genetics, immune system, and environmental factors. In this study, we uncover transcriptional signatures in patients with CD and subsequently explain the putative molecular pathways in the inflamed and non-inflamed intestinal mucosa. Materials and Methods: We obtain GSE83448 gene expression profiles from the Omnibus gene expression database. Also, for the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of Differentially Expressed Gene (DEG) pathways, we used DAVID software. DEGs were detected in the inflamed and non-inflamed intestinal mucosa of CD patients compared to the control group using the GEO2R instrument. Significant modules and hub genes were identified after producing protein-protein interaction (PPIs) networks of DEGs using Cytoscape software. Results: The 10 specific hub genes of CD, including Matrix Metallopeptidase 2 (MMP2), Cadherin 1 (CDH1), Periostin (POSTN), Collagen type I alpha 2 chain (COL1A2), C-X-C motif chemokine ligand 8 (CXCL8), Collagen type III alpha 1 chain (COL3A1), JUN, Serine Protease Inhibitor clade E member 1 (SERPINE1), Integrin alpha M (ITGAM), and Connective Tissue Growth Factor (CTGF), were used as biomarkers to discriminate between inflamed and non-inflamed intestinal mucosa groups in patients. Conclusion: These findings could lead to new molecular targets and diagnostic biomarkers for both inflamed and non-inflamed intestinal mucosa in CD patients.

Fungi in Gastrointestinal Tracts of Human and Mice: from Community to Functions.

Fungi are often ignored in studies on gut microbes because of their low level of presence (making up only 0.1% of the total microorganisms) in the gastrointestinal tract (GIT) of monogastric animals. Recent studies using novel technologies such as next generation sequencing have expanded our understanding on the importance of intestinal fungi in humans and animals. Here, we provide a comprehensive review on the fungal community, the so-called mycobiome, and their functions from recent studies in humans and mice. In the GIT of humans, fungi belonging to the phyla Ascomycota, Basidiomycota and Chytridiomycota are predominant. The murine intestines harbor a more diverse assemblage of fungi. Diet is one of the major factors influencing colonization of fungi in the GIT. Presence of the genus Candida is positively associated with dietary carbohydrates, but are negatively correlated with dietary amino acids, proteins, and fatty acids. However, the relationship between diet and the fungal community (and functions), as well as the underlying mechanisms remains unclear. Dysbiosis of intestinal fungi can cause invasive infections and inflammatory bowel diseases (IBD). However, it is not clear whether dysbiosis of the mycobiome is a cause, or a result of IBD. Compared to non-inflamed intestinal mucosa, the abundance and diversity of fungi is significantly increased in the inflamed mucosa. The commonly observed commensal fungal species Candida albicans might contribute to occurrence and development of IBD. Limited studies show that Candida albicans might interact with immune cells of the host intestines through the pathways associated with Dectin-1, Toll-like receptor 2 (TLR2), and TLR4. This review is expected to provide new thoughts for future studies on intestinal fungi and for new therapies to fungal infections in the GIT of human and animals.

RNA-seq Reveals Transcriptomic Differences in Inflamed and Noninflamed Intestinal Mucosa of Crohn's Disease Patients Compared with Normal Mucosa of Healthy Controls.

Aberrant gene expression in the gut mucosa might contribute to the initiation and progression of Crohn's disease (CD). RNA sequencing (RNA-seq) provides precise measurements of expression levels of transcripts and their isoforms. The aim of this study was to use RNA-seq to investigate transcriptomic differences and identify significantly differentially expressed transcripts in inflamed and noninflamed intestinal mucosa of CD patients. RNA-seq was performed on 13 pairs of inflamed and noninflamed intestinal mucosa from 13 CD patients and on sex-matched normal mucosa of 13 healthy controls. Significantly differentially expressed transcripts were validated by immunohistochemistry, quantitative reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay. RNA-seq revealed genome-wide transcriptomic differences between normal mucosa, noninflamed, and inflamed CD mucosa. Among 950 differentially expressed genes, 19 were up- or downregulated (upregulation: ANGPT2, CHN1, CPXM1, CPZ, CXCL1, FCN3, GJC1, HSD11B1, LZTS1, MEOX1, MMP12, PLA1A, SERPINE1, SGIP1, and TRPC4; downregulation: FAM189A1, PDE6A, SLC38A4, and HMGCS2) with statistical significance (p < 0.01 and q < 0.05). Among them, CXCL1 exhibited the highest fold change between groups. Immunohistochemistry for CXCL1 revealed no expression in normal mucosa, slightly increased expression in noninflamed CD mucosa, and highly increased expression in inflamed CD mucosa. Quantitative reverse transcriptase polymerase chain reaction showed that CXCL1 expression was significantly associated with epithelial damage, increased infiltration of polymorphonuclear leukocytes, and submucosal fibrosis. Serum CXCL1 concentration measured by enzyme-linked immunosorbent assay was better correlated with CD activity index (r = 0.660) than with C-reactive protein (r = 0.204). RNA-seq revealed transcriptomic differences between normal mucosa, noninflamed CD mucosa, and inflamed CD mucosa. Intestinal and serum CXCL1 was substantially increased with CD activity and can be used as a potential biomarker of CD.

Up-regulation and Pre-activation of TRAF3 and TRAF5 in Inflammatory Bowel Disease

Objective: TRAF3 and TRAF5 share a common ancestral gene, and interact as essential components of signaling pathways in immunity. TRAF3 and TRAF5 are overexpressed in the colon of rat/mouse models with colitis. However, the expressions of TRAF3 and TRAF5 in patients with inflammatory bowel disease have not been elucidated. The aim of the present study is to explore the potential roles of TRAF3 and TRAF5 in patients with inflammatory bowel disease.Methods: Plasma levels of TRAF3 and TRAF5 proteins were detected by Enzyme-linked Immunosorbent Assay (ELISA). Colonic expression of TRAF3 and TRAF5 proteins was detected by western blot analysis. Quantitative Real-time PCR (qRT-PCR) was applied for gene expression. Inflamed intestinal mucosa and non-inflamed intestinal mucosa in patients with inflammatory bowel disease and normal mucosa was analyzed from healthy controls.Results: The plasma levels of TRAF3 and TRAF5 were significantly higher both in patients with Crohn's disease and ulcerative colitis than in healthy controls. Only soluble TRAF5 showed a weak correlation with endoscopic disease activity index (Baron score) in patients with ulcerative colitis (spearman's r=0.358, P=0.022). Gene expressions of TRAF3 and TRAF5 in peripheral blood mononuclear cells were significantly higher both in patients with Crohn's disease and ulcerative colitis than in healthy controls (all P<0.0001). Gene and protein expressions of TRAF3 and TRAF5 were significantly higher in inflamed colonic mucosa of patients with Crohn's disease and ulcerative colitis than in non-inflamed colonic mucosa and normal mucosa of healthy controls (all P<0.0001). Furthermore, gene and protein expressions of TRAF3 and TRAF5 were also significantly higher in non-inflamed colonic mucosa of patients with Crohn's disease and ulcerative colitis than in normal mucosa of healthy controls.Conclusions: TRAF3 and TRAF5 are overexpressed in inflammatory bowel disease. Although the endoscopic appearance can be normal, TRAF3 and TRAF5 pre-activation can be detected in non-inflamed colonic segments.

Monocyte chemoattractant protein-1 (MCP-1) inhibits the intestinal-like differentiation of monocytes

Monocytes (MO) migrating into normal, non-inflamed intestinal mucosa undergo a specific differentiation resulting in a non-reactive, tolerogenic intestinal macrophage (IMAC). Recently we demonstrated the differentiation of MO into an intestinal-like macrophage (MAC) phenotype in vitro in a three-dimensional cell culture model (multi-cellular spheroid or MCS model). In the mucosa of patients with inflammatory bowel disease (IBD) in addition to normal IMAC, a reactive MAC population as well as increased levels of monocyte chemoattractant protein 1 (MCP-1) is found. The aim of this study was to investigate the influence of MCP-1 on the differentiation of MO into IMAC. MCS were generated from adenovirally transfected HT-29 cells overexpressing MCP-1, macrophage inflammatory protein 3 alpha (MIP-3alpha) or non-transfected controls and co-cultured with freshly elutriated blood MO. After 7 days of co-culture MCS were harvested, and expression of the surface antigens CD33 and CD14 as well as the intracellular MAC marker CD68 was determined by flow-cytometry or immunohistochemistry. MCP-1 and MIP-3alpha expression by HT-29 cells in the MCS was increased by transfection at the time of MCS formation. In contrast to MIP-3alpha, MCP-1 overexpression induced a massive migration of MO into the three-dimensional aggregates. Differentiation of IMAC was disturbed in MCP-1-transfected MCS compared to experiments with non-transfected control aggregates, or the MIP-3alpha-transfected MCS, as indicated by high CD14 expression of MO/IMAC cultured inside the MCP-1-transfected MCS, as shown by immunohistochemistry and FACS analysis. Neutralization of MCP-1 was followed by an almost complete absence of monocyte migration into the MCS. MCP-1 induced migration of MO into three-dimensional spheroids generated from HT-29 cells and inhibited intestinal-like differentiation of blood MO into IMAC. It may be speculated that MCP-1 could play a role in the disturbed IMAC differentiation in IBD mucosa.

Intestinal macrophages: unique effector cells of the innate immune system

The gastrointestinal mucosa is the largest reservoir of macrophages in the body. These important effector cells are derived from blood monocytes that are recruited to the lamina propria by endogenous chemoattractants in the non-inflamed mucosa and by inflammatory chemokines and bacterial products during inflammation. In the non-inflamed mucosa, newly recruited pro-inflammatory monocytes are exposed to lamina propria stromal (extracellular matrix) factors that induce phenotypic and functional differentiation into non-inflammatory macrophages. As a consequence of this differentiation, resident lamina propria macrophages are strikingly downregulated for the expression of innate response receptors, such as the receptors for lipopolysaccharide, immunoglobulin G (IgG), and IgA, and the production of pro-inflammatory cytokines, including interleukin-1 (IL-1), IL-6, IL-8, and tumor necrosis factor-alpha. Despite downregulated pro-inflammatory function, strong phagocytic and bactericidal activities remain intact. Thus, in the non-inflamed intestinal mucosa, lamina propria macrophages are non-inflammatory but retain avid scavenger and host defense functions, a unique but ideal phenotype and functional profile for effector cells in close proximity to immunostimulatory microorganisms and products.

Biochemical and immunohistochemical analysis of glycosaminoglycans in inflamed and non-inflamed intestinal mucosa of patients with Crohn’s disease

Changes in extracellular matrix glycosaminoglycans (GAGs) of the intestinal mucosa have been associated with inflammatory bowel disease. The aim of the present study was to follow the changes in GAGs metabolism during the progression from non-inflamed to inflamed intestinal colon of patients with Crohn's disease (CD), using direct biochemical analysis and specific immunohistochemistry against chondroitin/dermatan sulfate and heparan sulfate. The content of GAGs from inflamed and non-inflamed colon of eight patients with active CD was estimated by uronic acid per dry weight of tissue and analyzed by agarose gel electrophoresis and ion-exchange chromatography. Intestinal sections were stained using antibodies against dermatan sulfate/chondroitin 4-sulfate (DS/CS), heparan sulfate (HS), and ICAM-1 (CD54), and analyzed by confocal microscopy. There was a reduction in the amount of GAGs in the non-inflamed colon of patients with CD. In the inflamed colon, HS, CS and DS showed increased concentrations compared with the non-inflamed colon. GAGs showed a diffuse distribution in the lamina propria and in the basement membrane of both inflamed and non-inflamed mucosa of patients with CD. We observed a marked reduction in GAGs with altered patterns of distribution in the non-inflamed colon of patients with CD. The increase in the synthesis of GAGs observed in the inflamed colon may be a compensatory mechanism for the restoration of the integrity of the intestinal mucosa.

Apoptosis in the intestinal mucosa of patients with inflammatory bowel disease: evidence of altered expression of FasL and perforin cytotoxic pathways

Abnormal apoptosis may result in the persistence of activated intestinal T-cells in inflammatory bowel disease (IBD). We investigated apoptosis in distinct mucosal compartments, and the expression of Fas/Fas ligand and perforin in the inflamed and non-inflamed intestinal mucosa of patients with IBD. Colon specimens from 15 patients with ulcerative colitis (UC) and inflamed and non-inflamed mucosa from 15 patients with Crohn's disease (CD) were analysed for densities and distribution of apoptotic cells determined by the terminal deoxynucleotidyltransferase-mediated dUDP-biotin nick-end labelling (TUNEL) method. Fas, FasL, and perforin-expressing cells were assessed by immunoperoxidase, and with anti-CD3, anti-CD20 and anti-CD68, by double immunofluorescence with confocal microscopy. Quantitative analysis was performed using a computer-assisted image analyser. Colonic lamina propria (LP) and epithelium from patients with UC showed higher rates of apoptosis than controls, but no difference was shown regarding patients with CD. In LP, co-expression of Fas was reduced with T-cells in inflamed CD mucosa, and with macrophages in all patients with IBD. No difference was found in the expression of Fas on B-cells. Rates of FasL-expressing cells in LP were higher in IBD than in controls, with no correlation with the rates of apoptosis. Rates of perforin-expressing cells in LP were greater in UC than in controls, and correlated to the rates of apoptosis. No difference was shown regarding the inflamed and non-inflamed CD mucosa. Rates of FasL and perforin-expressing intra-epithelial lymphocytes showed no difference among groups. Increased expression of FasL in IBD colonic LP not parallelled by Fas on T-cells and macrophages may indicate a reduced susceptibility to the Fas/FasL-mediated apoptosis of lymphoid cells. Expression of perforin is correlated to the tissue damage, and may represent the enhancement of a distinct cytotoxic pathway in UC.

Ca2+ Signaling in Identified T-lymphocytes from Human Intestinal Mucosa: RELATION TO HYPOREACTIVITY, PROLIFERATION, AND INFLAMMATORY BOWEL DISEASE

Ca2+ entry across the plasma membrane is necessary for the activation and proliferation of T-lymphocytes. Human intestinal lamina propria lymphocytes physiologically exhibit minimal proliferation in response to antigen receptor stimulation when compared with peripheral blood T-lymphocytes. This hyporeactivity is partially abolished in inflammatory bowel disease. We hypothesized that differences in Ca2+ signaling could be related to the disease. To test this possibility, we measured Ca2+ signals in identified lymphocytes from human blood and human intestinal mucosa. Ca2+ signals in lamina propria T-lymphocytes from non-inflamed tissue were drastically reduced when compared with Ca2+ signals of blood T-lymphocytes from the same persons. However, Ca2+ signals in T-lymphocytes from inflamed intestinal mucosa were much higher than the ones from non-inflamed mucosa and almost reached levels of Ca2+ signals in peripheral blood T-cells. Furthermore, Ca2+ influx was closely linked to cell proliferation in both peripheral blood T-lymphocytes and lamina propria lymphocytes cells. We conclude that differences in Ca2+ signaling can explain the differences of T-lymphocyte reactivity in blood versus lamina propria and, importantly, also between T-lymphocytes from inflamed and non-inflamed intestinal mucosa. Ca2+ channels in the plasma membrane of T-lymphocytes might thus prove an excellent target to screen for immunosuppressiva to potentially treat the symptoms of inflammatory bowel disease.

Application of electroblotting technique to studies of the intestinal antibody response to extractable fecal antigens.

Water-soluble and sodium dodecyl sulfate (SDS)-extractable fractions were prepared from feces of four patients with inflammatory and six with non-inflammatory bowel disease. Both types of fractions were run on polyacrylamide gel electrophoresis, followed by electroblotting on nitrocellulose sheets of the separated components. The antibody reactivities in serum and in the intestinal wall against the extracted and separated fecal components were investigated. A striking lack of reactivity was observed when serum was used as antibody source against the fecal extracts both in patients with inflamed and in those with non-inflamed intestinal mucosa. The locally produced gut-associated IgG reacted more intensely with SDS-extractable fecal components than with water-soluble components. A strong reaction between intestinal-wall IgG extracts and a water-soluble antigen of 46-48 kD in the feces of two colitis patients was, however, observed. No clear correlation between the single bands and the occurrence of inflammatory bowel disease could be established because of the small number of patients.