Influence of Smoking on Colonic Gene Expression Profile in Crohn's Disease

Background

The development and course of Crohn''s disease (CD) is related to both genetic and environmental factors. Smoking has been found to exacerbate the course of CD by increasing the risk of developing fistulas and strictures as well as the need for surgery, possibly because of an interaction between smoking or nicotine on macrophage function and the intestinal microvasculature. Several genes are involved in the pathogenesis of CD, and in this study the gene expression differences of the descending colonic mucosa were investigated in CD (smokers or never smokers) and controls (smokers or never smokers).

Aim

To identify any difference in gene expression of the descending colonic mucosa between smoking and never-smoking CD patients (and controls) by determining genetic expression profiles from microarray analysis.

Methods

Fifty-seven specimens were obtained by routine colonoscopy from the included material: CD smokers (n = 28) or never-smokers (n = 14) as compared to fifteen healthy controls (8 smokers and 7 never-smokers). RNA was isolated and gene expression assessed with Affymetrix GeneChip Human Genome U133 Plus 2.0. Data were analyzed by principal component analysis (PCA), Wilcoxon rank sum test and multiple linear regressions. Real-time (RT) PCR was subsequently applied to verify microarray results.

Results

The PCA analysis showed no intrinsic clustering of smokers versus never-smokers. However, when Wilcoxon rank sum test corrected with Q values were performed, six known genes were significantly expressed differently in the inflamed CD smokers as compared to the inflamed CD never-smokers: ring finger protein 138 (RNF138), metalothionein 2A (MT2A) and six transmembrane epithelial antigen of the prostate 3 (STEAP3), SA hypertension-associated homolog, PGM2L1 and KCNJ2. The subsequent RT-PCR-analyses verified, however, that only RNF138, MT2A and STEAP3 were significantly up-regulated in CD smokers in specimens with inflammatory activity of the descending colon.

Conclusions

The present study demonstrates that the genes, RNF138, MT2A, and STEAP3 are differently expressed in the inflamed descending colon of smoking versus never-smoking CD patients, which might be of relevance for the poorer clinical course among CD smokers. Many gastroenterologists are still not totally aware of the benefits of smoking cessation in relation to CD, and do not put much effort into getting the patients to quit, therefore more information on the negative effects of smoking, seems warranted.     

Spatial heterogeneity of TNF-alpha-induced T cell migration to colonic mucosa is mediated by MAdCAM-1 and VCAM-1

Relatively little is known about how recirculation of lymphocytes through the inflamed intestinal mucosa is regulated. The aim of this study was to investigate the dynamic process of T lymphocyte-endothelial cell adhesion in TNF-alpha-challenged murine colonic mucosa by intravital microscopy. T lymphocytes from spleen (SPL) and intestinal lamina propria (LPL) were fluorescence labeled, and their adhesion to microvessels in the colonic mucosa was observed. In TNF-alpha (25 microg/kg)-stimulated colonic venules, an enhanced adhesion of SPL and LPL was demonstrated, with dominant recruitment of LPLs. The magnitude of the increased LPL adhesion was more significant in the colon than in the small intestine. These T lymphocyte interactions in the colonic mucosa were significantly reduced by blocking MAbs against either mucosal addressin cell adhesion molecule-1 (MAdCAM-1), VCAM-1, alpha(4)-integrin, or beta(7)-integrin but not by anti-ICAM-1. Immunohistochemistry revealed significant MAdCAM-1 expression in the lamina propria and VCAM-1 expression in the submucosa of TNF-alpha-treated colon. Spatial heterogeneity of MAdCAM-1 and VCAM-1 activation following TNF-alpha challenge may promote specific T lymphocyte recruitment in the inflamed colonic mucosa.     

MAdCAM mediates lymphocyte-endothelial cell adhesion in a murine model of chronic colitis

Previous studies have revealed that the expression of several endothelial cell adhesion molecules [e.g., intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and mucosal addressin cell adhesion molecule 1 (MAdCAM-1)] is dramatically elevated in the chronically inflamed colonic vasculature of severe combined immunodeficient (SCID) mice reconstituted with congenic CD4+, CD45RB(high) T lymphocytes. The objective of this study was to define the contribution of different endothelial cell adhesion molecules to the lymphocyte-endothelial cell (L/E) adhesion observed in the colonic microvasculature in this experimental model of inflammatory bowel disease. Fluorescently labeled T lymphocytes, isolated from spleens of normal BALB/C mice, were injected intravenously into SCID mice that had been reconstituted with CD4+, CD45RB(high) T lymphocytes either before (3 wk after reconstitution) or after (7 wk postreconstitution) the onset of clinical signs of colitis (i.e., diarrhea, loss of body wt). Intravital fluorescence microscopy was used to quantify L/E adhesion in different-sized venules of the colonic submucosa during the development of colitis. L/E adhesion was noted in some segments of the vasculature in precolitic SCID mice (3 wk after reconstitution) but not in similar-sized vessels of control (wild type and SCID) mice. L/E adhesion was observed in a greater proportion of venules and occurred with greater intensity in the mucosa of colitic mice (7 wk postreconstitution). Pretreatment with a blocking monoclonal antibody against MAdCAM-1, but not ICAM-1 or VCAM-1, significantly and profoundly reduced L/E adhesion in colitic mice. Immunohistochemical staining also revealed the localization of T cells on colonic endothelial cells expressing MAdCAM-1. These findings indicate that MAdCAM-1 is largely responsible for recruiting T lymphocytes into inflamed colonic tissue.     

Identification of Restricted Subsets of Mature microRNA Abnormally Expressed in Inactive Colonic Mucosa of Patients with Inflammatory Bowel Disease

Background

Ulcerative Colitis (UC) and Crohn''s Disease (CD) are two chronic Inflammatory Bowel Diseases (IBD) affecting the intestinal mucosa. Current understanding of IBD pathogenesis points out the interplay of genetic events and environmental cues in the dysregulated immune response. We hypothesized that dysregulated microRNA (miRNA) expression may contribute to IBD pathogenesis. miRNAs are small, non-coding RNAs which prevent protein synthesis through translational suppression or mRNAs degradation, and regulate several physiological processes.

Methodology/Findings

Expression of mature miRNAs was studied by Q-PCR in inactive colonic mucosa of patients with UC (8), CD (8) and expressed relative to that observed in healthy controls (10). Only miRNAs with highly altered expression (>5 or <0.2 -fold relative to control) were considered when Q-PCR data were analyzed. Two subsets of 14 (UC) and 23 (CD) miRNAs with highly altered expression (5.2->100 -fold and 0.05–0.19 -fold for over- and under- expression, respectively; 0.001<p≤0.05) were identified in quiescent colonic mucosa, 8 being commonly dysregulated in non-inflamed UC and CD (mir-26a,-29a,-29b,-30c,-126*,-127-3p,-196a,-324-3p). Several miRNA genes with dysregulated expression co-localize with acknowledged IBD-susceptibility loci while others, (eg. clustered on 14q32.31), map on chromosomal regions not previously recognized as IBD-susceptibility loci. In addition, in silico clustering analysis identified 5 miRNAs (mir-26a,-29b,-126*,-127-3p,-324-3p) that share coordinated dysregulation of expression both in quiescent and in inflamed colonic mucosa of IBD patients. Six miRNAs displayed significantly distinct alteration of expression in non-inflamed colonic biopsies of UC and CD patients (mir-196b,-199a-3p,-199b-5p,-320a,-150,-223).

Conclusions/Significance

Our study supports miRNAs as crucial players in the onset and/or relapse of inflammation from quiescent mucosal tissues in IBD patients. It allows speculating a role for miRNAs as contributors to IBD susceptibility and suggests that some of the miRNA with altered expression in the quiescent mucosa of IBD patients may define miRNA signatures for UC and CD and help develop new diagnostic biomarkers.     

Differential protein expression profile in the intestinal epithelium from patients with inflammatory bowel disease

The loss of intestinal epithelial cell (IEC) function is a critical component in the initiation and perpetuation of chronic intestinal inflammation in the genetically susceptible host. We applied proteome analysis (PA) to characterize changes in the protein expression profile of primary IEC from patients with Crohn's disease (CD) and ulcerative colitis (UC). Surgical specimens from 18 patients with active CD (N = 6), UC (N = 6), and colonic cancer (N = 6) were used to purify primary IEC from ileal and colonic tissues. Changes in protein expression were identified using 2D-gel electrophoreses (2D SDS-PAGE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS) as well as Western blot analysis. PA of primary IEC from inflamed ileal tissue of CD patients and colonic tissue of UC patients identified 21 protein spots with at least 2-fold changes in steady-state expression levels compared to the noninflamed tissue of control patients. Statistical significance was achieved for 9 proteins including the Rho-GDP dissociation inhibitor alpha that was up-regulated in CD and UC patients. Additionally, 40 proteins with significantly altered expression levels were identified in IEC from inflamed compared to noninflamed tissue regions of single UC (N = 2) patients. The most significant change was detected for programmed cell death protein 8 (7.4-fold increase) and annexin 2A (7.7-fold increase). PA in primary IEC from IBD patients revealed significant expression changes of proteins that are associated with signal transduction, stress response as well as energy metabolism. The induction of Rho GDI alpha expression may be associated with the destruction of IEC homeostasis under condition of chronic intestinal inflammation.     

Inhibition of the intrinsic but not the extrinsic apoptosis pathway accelerates and drives MYC-driven tumorigenesis towards acute myeloid leukemia

Myc plays an important role in tumor development, including acute myeloid leukemia (AML). However, MYC is also a powerful inducer of apoptosis, which is one of the major failsafe programs to prevent cancer development. To clarify the relative importance of the extrinsic (death receptor-mediated) versus the intrinsic (mitochondrial) pathway of apoptosis in MYC-driven AML, we coexpressed MYC together with anti-apoptotic proteins of relevance for AML; BCL-X(L)/BCL-2 (inhibiting the intrinsic pathway) or FLIP(L) (inhibiting the extrinsic pathway), in hematopoietic stems cells (HSCs). Transplantation of HSCs expressing MYC into syngeneic recipient mice resulted in development of AML and T-cell lymphomas within 7-9 weeks as expected. Importantly, coexpression of MYC together with BCL-X(L)/BCL-2 resulted in strongly accelerated kinetics and favored tumor development towards aggressive AML. In contrast, coexpression of MYC and FLIP(L) did neither accelerate tumorigenesis nor change the ratio of AML versus T-cell lymphoma. However, a change in distribution of immature CD4(+)CD8(+) versus mature CD4(+) T-cell lymphoma was observed in MYC/FLIP(L) mice, possibly as a result of increased survival of the CD4+ population, but this did not significantly affect the outcome of the disease. In conclusion, our findings provide direct evidence that BCL-X(L) and BCL-2 but not FLIP(L) acts in synergy with MYC to drive AML development.     

IL-4 protects tumor cells from anti-CD95 and chemotherapeutic agents via up-regulation of antiapoptotic proteins

We recently proposed that Th1 and Th2 cytokines exert opposite effects on the pathogenesis and clinical outcome of organ-specific autoimmunity by altering the expression of genes involved in target cell survival. Because a Th2 response against tumors is associated with poor prognosis, we investigated the ability of IL-4 to protect tumor cells from death receptor- and chemotherapy-induced apoptosis. We found that IL-4 treatment significantly reduced CD95 (Fas/APO-1)- and chemotherapeutic drug-induced apoptosis in prostate, breast, and bladder tumor cell lines. Analysis of antiapoptotic protein expression revealed that IL-4 stimulation resulted in up-regulation of cellular (c) FLIP/FLAME-1 and Bcl-x(L). Exogenous expression of cFLIP/FLAME-1 inhibited apoptosis induced by CD95 and to a lesser extent by chemotherapy, while tumor cells transduced with Bcl-x(L) were substantially protected both from CD95 and chemotherapeutic drug stimulation. Moreover, consistent IL-4 production and high expression of both cFLIP/FLAME-1 and Bcl-x(L) were observed in primary prostate, breast, and bladder cancer in vivo. Finally, primary breast cancer cells acquired sensitivity to apoptosis in vitro only in the absence of IL-4. Thus, IL-4 protects tumor cells from CD95- and chemotherapy-induced apoptosis through the up-regulation of antiapoptotic proteins such as cFLIP/FLAME-1 and Bcl-x(L). These findings may provide useful information for the development of therapeutic strategies aimed at restoring the functionality of apoptotic pathways in tumor cells.     

Prevention of experimental colitis in SCID mice reconstituted with CD45RBhigh CD4+ T cells by blocking the CD40-CD154 interactions

Increased expression of CD40 and CD40 ligand (CD40L or CD154) has been found in inflamed mucosa of human inflammatory bowel disease (IBD), and interactions between these molecules seem to be involved in local cytokine production by macrophages. However, the precise role of CD40 signaling in the pathogenesis of IBD is still poorly understood. The aim of the present study was to investigate the in vivo relevance of CD40 signaling in experimental colitis in SCID mice reconstituted with syngeneic CD45RBhighCD4+ T cells. The results demonstrated that CD40+ and CD40L+ cells as well as their mRNA levels were significantly increased in inflamed mucosa. Administration of anti-CD40L neutralizing mAb over an 8-wk period starting immediately after CD45RBhighCD4+ T cell reconstitution completely prevented symptoms of wasting disease. Intestinal mucosal inflammation was effectively prevented, as revealed by abrogated leukocyte infiltration and decreased CD54 expression and strongly diminished mRNA levels of the proinflammatory cytokines IFN-gamma, TNF, and IL-12. When colitic SCID mice were treated with anti-CD40L starting at 5 wk after T cell transfer up to 8 wk, this delayed treatment still led to significant clinical and histological improvement and down-regulated proinflammatory cytokine secretion. These data suggest that the CD40-CD40L interactions are essential for the Th1 inflammatory responses in the bowel in this experimental model of colitis. Blockade of CD40 signaling may be beneficial to human IBD.     

MicroRNA-193a-3p Reduces Intestinal Inflammation in Response to Microbiota via Down-regulation of Colonic PepT1

Intestinal inflammation is characterized by epithelial disruption, leading to the loss of barrier function, recruitment of immune cells, and host immune responses to gut microbiota. PepT1, a di/tripeptide transporter that uptakes bacterial products, is up-regulated in inflamed colon tissue, which implies its role in bacterium-associated intestinal inflammation. Although microRNA (miRNA)-mediated gene regulation has been found to be involved in various processes of inflammatory bowel disease (IBD), the biological function of miRNAs in the pathogenesis of IBD remains to be explored. In this study we detected miRNA expression patterns in colon tissues during colitis and investigated the mechanism underlying the regulation of colonic PepT1 by miRNAs. We observed an inverse correlation between PepT1 and miR-193a-3p in inflamed colon tissues with active ulcerative colitis, and we further demonstrated that miR-193a-3p reduced PepT1 expression and activity as a target gene and subsequently suppressed the NF-κB pathway. Intracolonic delivery of miR-193a-3p significantly ameliorated dextran sodium sulfate-induced colitis, whereas the overexpression of colonic PepT1 via PepT1 3′-untranslated region mutant lentivirus vector abolished the anti-inflammatory effect of miR-193a-3p. Furthermore, antibiotic treatment eliminated the difference in the dextran sodium sulfate-induced inflammation between the presence and absence of miR-193a-3p. These findings suggest that miR-193a-3p regulation of PepT1 mediates the uptake of bacterial products and is a potent mechanism during the colonic inflammation process. Overall, we believe miR-193a-3p may be a potent regulator of colonic PepT1 for maintaining intestinal homeostasis.     

Heat shock protein 72 associated with CD44v6 in human colonic adenocarcinoma

CD44v6 is a splice variant of CD44 (CD44v), probably promoting cancer cell adherence to vascular endothelium and base membranes and enhancing the invasion and metastasis of colonic carcinomas. Heat shock protein 72 (HSP72) as a molecular chaperone has been confirmed to be overexpressed in epithelial carcinoma cells. There may be a possible association between the expression of HSP72 and CD44v6 during the growth and progression of colonic carcinoma cells. The aim of the study was to investigate the interaction between heat shock protein 72 and CD44v6 in human colonic carcinomas. The localization of HSP72 and CD44v6 in human colonic carcinomas was determined by immunohistochemistry and confocal laser microscopy. The interaction between HSP72 and CD44v6 in colonic carcinoma cells was analyzed by immunoprecipitation and Western immunoblots. Our results revealed that colonic carcinoma synchronously co-expressed higher levels of HSP72 and CD44v6 than that in adjacent normal colonic tissues. HSP72 and CD44v6 were mainly immunolocalized in the cytoplasm, and also immunolabelled on the cell membrane. Based on immunoprecipitation and Western immunoblots, we found that HSP72 was associated with CD44v6 precursor fragments in human colonic carcinoma cells. The interaction between HSP72 and CD44v6 in human colonic carcinoma cells may contribute to study the pathogenesis and immunotherapy of colonic carcinoma.     

Serpin B1 protects colonic epithelial cell via blockage of neutrophil elastase activity and its expression is enhanced in patients with ulcerative colitis

Serpin B1 is a monocyte neutrophil elastase (NE) inhibitor and is one of the most efficient inhibitors of NE. In the present study, we investigated the role of serpin B1 in the pathogenesis of ulcerative colitis by using clinical samples and an experimental model. The colonic expression of serpin B1 was determined by real-time polymerase chain reaction (PCR), Western blot analysis, and immunohistological studies in both normal and inflamed mucosa from patients with ulcerative colitis. Serpin B1 mRNA expression was determined by real-time PCR in the mouse dextran sodium sulfate (DSS)-induced colitis model. Young adult mouse colonic epithelial (YAMC) cells were used to determine the role of serpin B1. Serpin B1 gene transfected YAMC cells were treated with H(2)O(2) to measure cell viability. The expression of NE was determined in YAMC cells treated with H(2)O(2). NE-silenced YAMC cells were also treated with H(2)O(2) and then measured for viability. Upregulated expression of serpin B1 in colonic mucosa was confirmed from patients with active ulcerative colitis. Immunohistochemical studies showed that serpin B1 expression was localized not only in inflammatory infiltration cells but also in epithelial cells. Serpin B1 mRNA expression was also increased in colonic mucosa of mouse DSS-induced colitis. Serpin B1-transfected YAMC cells were resistant against the treatment of H(2)O(2). H(2)O(2) treatment significantly induced NE in YAMC cells, and NE-silenced YAMC cells were also resistant against the treatment of H(2)O(2). These results suggest that serpin B1 may be a novel marker of active ulcerative colitis and may play an important role in the pathogenesis of inflammatory bowel disease.     

IL-7 exacerbates chronic colitis with expansion of memory IL-7Rhigh CD4+ mucosal T cells in mice

We have previously demonstrated that mucosal CD4(+) T cells expressing high levels of IL-7 receptor (IL-7R(high)) are pathogenic cells responsible for chronic colitis. Here we investigate whether IL-7 is directly involved in the expansion of IL-7R(high) memory CD4(+) mucosal T cells and the exacerbation of colitis. We first showed that CD4(+) lamina propria lymphocytes (LPLs) from wild-type, T cell receptor-alpha-deficient (TCR-alpha(-/-)), and recombinase-activating gene (RAG)-2(-/-)-transferred mice with or without colitis showed phenotypes of memory cells, but only CD4(+) LPLs from colitic mice showed IL-7R(high). In vitro stimulation by IL-7, but not by IL-15 and thymic stromal lymphopoietin, enhanced significant proliferative responses and survival of colitic CD4(+), but not normal CD4(+) LPLs. Importantly, in vivo administration of IL-7 mice accelerated the expansion of IL-7R(high) memory CD4(+) LPLs and thereby exacerbated chronic colitis in RAG-2(-/-) mice transferred with CD4(+) LPLs from colitic TCR-alpha(-/-) mice. Conversely, the administration of anti-IL-7R monoclonal antibody significantly inhibited the development of TCR-alpha(-/-) colitis with decreased expansion of CD4(+) LPLs. Collectively, the present data indicate that IL-7 is essential for the expansion of pathogenic memory CD4(+) T cells under pathological conditions. Therefore, therapeutic approaches targeting the IL-7R pathway may be feasible in the treatment of human inflammatory bowel disease.     

Involvement of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease: A Different Implication for Colonic and Ileal Disease?

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.     

Expression of heat shock protein 32 (hemoxygenase-1) in the normal and inflamed human stomach and colon: an immunohistochemical study

Heat shock protein 32 (Hsp32, hemoxygenase-1) is induced by reactive oxygen metabolites (ROM) and degrades heme leading to the formation of antioxidant bilirubin. Increased mucosal generation of ROM occurs in gastritis and inflammatory bowel disease. We aimed to assess mucosal expression of Hsp32 in normal stomach and colon and to test the hypothesis that disease-related differential expression occurs in inflamed tissue. Gastric body and antral mucosal biopsies were obtained from 33 patients comprising Helicobacter pylori-negative normal controls (n = 8), H pylori-negative gastritis patients (n = 11), and H pylori-positive gastritis patients (n = 14). Forty-seven archival colonic mucosal biopsies selected comprised normal histology (n = 10), active ulcerative colitis (UC) (n = 9), inactive UC (n = 8), active Crohn's disease (CD) (n = 8), inactive CD (n = 6), and other colitides (n = 6). Hsp32 expression in formalin-fixed sections was assessed by avidin-biotin peroxidase immunohistochemistry using a polyclonal rabbit anti-Hsp32 as the primary antibody. Immunohistochemical staining identified Hsp32 in all groups. Diffuse cytoplasmic staining was seen in gastric and colonic epithelial and lamina proprial inflammatory cells. Staining scores for Hsp32 were higher in antral H pylori-positive (P = 0.002) and H pylori-negative (P = 0.02) gastritis than in controls and in body H pylori-positive gastritis than in the other 2 groups (P < 0.01). Expression of Hsp32 was increased in active UC compared with inactive disease (P = 0.03) and normal controls (P = 0.02). In conclusion, Hsp32 is expressed constitutively in normal gastric and colonic mucosa, and differential expression occurs in these tissues when they are inflamed. Upregulation of Hsp32 may be an adaptive response to protect mucosa from oxidative injury in patients with gastritis and inflammatory bowel disease.