Expression Profile of the REG Gene Family in Colorectal Carcinoma

Regenerating (REG) gene family belongs to the calcium-dependent lectin gene superfamily and encodes small multifunctional secretory proteins, which might be involved in cell proliferation, differentiation, and carcinogenesis. To clarify REG expression profile in colorectal carcinoma (CRC), the authors examined the expression of REG Iα, Iβ, III, HIP/PAP, and REG IV by immunohistochemistry on tissue microarray. The expression of REG Iα, III, and HIP/PAP was more frequently observed in the CRCs than adjacent non-neoplastic mucosa (p < 0.001), whereas it was the converse for REG Iβ and IV (p < 0.001). The expression of REG Iα, Iβ, III, and HIP/PAP was negatively correlated with the depth of invasion of CRCs (p < 0.05). The REG Iβ and HIP/PAP were less expressed in CRCs with than without venous invasion (p < 0.05). The positive rates of REG Iα and HIP/PAP were significantly higher in CRCs without than with lymph node metastasis (p < 0.05). Mucinous carcinoma more frequently expressed REG IV protein than well- and moderately differentiated ones (p < 0.05). There was a positive relationship between REG Iα, Iβ, III, and HIP/PAP expression (p < 0.05). Survival analysis indicated the REG Iβ or HIP/PAP expression was positively linked to favorable prognosis of carcinoma patients (p < 0.05). This study indicated that aberrant REG expression might be closely linked to the pathogenesis, invasion, or lymph node metastasis of CRCs. REG Iβ and HIP/PAP could be considered reliable markers of favorable prognosis of CRC patients.     

Whole Genome Gene Expression Meta-Analysis of Inflammatory Bowel Disease Colon Mucosa Demonstrates Lack of Major Differences between Crohn's Disease and Ulcerative Colitis

Background

In inflammatory bowel disease (IBD), genetic susceptibility together with environmental factors disturbs gut homeostasis producing chronic inflammation. The two main IBD subtypes are Ulcerative colitis (UC) and Crohn’s disease (CD). We present the to-date largest microarray gene expression study on IBD encompassing both inflamed and un-inflamed colonic tissue. A meta-analysis including all available, comparable data was used to explore important aspects of IBD inflammation, thereby validating consistent gene expression patterns.

Methods

Colon pinch biopsies from IBD patients were analysed using Illumina whole genome gene expression technology. Differential expression (DE) was identified using LIMMA linear model in the R statistical computing environment. Results were enriched for gene ontology (GO) categories. Sets of genes encoding antimicrobial proteins (AMP) and proteins involved in T helper (Th) cell differentiation were used in the interpretation of the results. All available data sets were analysed using the same methods, and results were compared on a global and focused level as t-scores.

Results

Gene expression in inflamed mucosa from UC and CD are remarkably similar. The meta-analysis confirmed this. The patterns of AMP and Th cell-related gene expression were also very similar, except for IL23A which was consistently higher expressed in UC than in CD. Un-inflamed tissue from patients demonstrated minimal differences from healthy controls.

Conclusions

There is no difference in the Th subgroup involvement between UC and CD. Th1/Th17 related expression, with little Th2 differentiation, dominated both diseases. The different IL23A expression between UC and CD suggests an IBD subtype specific role. AMPs, previously little studied, are strongly overexpressed in IBD. The presented meta-analysis provides a sound background for further research on IBD pathobiology.     

Epigenetic regulation of REG1A and chemosensitivity of cutaneous melanoma

Regenerating gene 1A (REG1A) plays an important role in tissue regeneration and in cell proliferation in epithelium origin tumors; however, its role in melanoma has not been explored in details. The objective of this study was to identify whether REG1A is expressed in cutaneous melanoma and if REG1A expression status can predict prognosis in cutaneous melanoma patients with metastasis. We also determined whether epigenetic regulation of the promoter region regulates REG1A expression. AJCC stage III cutaneous melanoma specimens with clinically well annotated stage III lymph node melanoma metastasis tissue microarray were assessed by IHC. MALDI-TOF-mass spectrometry and HM450K array were used to identify REG1A promoter region CpG site methylation. Chemotherapeutic agent response by melanoma cells as related to REG1A protein expression was assessed. Post-surgery melanoma patients followed by adjuvant chemotherapy with high REG1A expression had a significantly better prognosis (disease-specific survival) compared with patients with low REG1A expression (log rank test; p = 0.0013). The demethylating reagent 5-Aza-2′-deoxycytidine activated REG1A promoter region resulting in enhanced REG1A mRNA and protein expression in melanoma cell lines. Promoter region CpG methylation was shown to regulate REG1A expression in melanoma cells. Moreover, melanoma lines with high REG1A mRNA expression were more susceptible to Dacarbazine and Cisplatin, as compared with those with low REG1A mRNA expression. In conclusion, REG1A expression status may be useful as a biomarker in melanoma patients for sensitivity to these chemotherapeutic agents. The epigenetic regulation of the REG1A promoter region may offer a potential therapeutic approach to improve chemotherapy for metastatic melanoma patients.     

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.     

R-Spondins Are Expressed by the Intestinal Stroma and are Differentially Regulated during Citrobacter rodentium- and DSS-Induced Colitis in Mice

The R-spondin family of proteins has recently been described as secreted enhancers of β-catenin activation through the canonical Wnt signaling pathway. We previously reported that Rspo2 is a major determinant of susceptibility to Citrobacter rodentium-mediated colitis in mice and recent genome-wide association studies have revealed RSPO3 as a candidate Crohn’s disease-specific inflammatory bowel disease susceptibility gene in humans. However, there is little information on the endogenous expression and cellular source of R-spondins in the colon at steady state and during intestinal inflammation. RNA sequencing and qRT-PCR were used to assess the expression of R-spondins at steady state and in two mouse models of colonic inflammation. The cellular source of R-spondins was assessed in specific colonic cell populations isolated by cell sorting. Data mining from publicly available datasets was used to assess the expression of R-spondins in the human colon. At steady state, colonic expression of R-spondins was found to be exclusive to non-epithelial CD45^- lamina propria cells, and Rspo3/RSPO3 was the most highly expressed R-spondin in both mouse and human colon. R-spondin expression was found to be highly dynamic and differentially regulated during C. rodentium infection and dextran sodium sulfate (DSS) colitis, with notably high levels of Rspo3 expression during DSS colitis, and high levels of Rspo2 expression during C. rodentium infection, specifically in susceptible mice. Our data are consistent with the hypothesis that in the colon, R-spondins are expressed by subepithelial stromal cells, and that Rspo3/RSPO3 is the family member most implicated in colonic homeostasis. The differential regulation of the R-spondins in different models of intestinal inflammation indicate they respond to specific pathogenic and inflammatory signals that differ in the two models and provides further evidence that this family of proteins plays a key role in linking intestinal inflammation and homeostasis.     

The Goblet Cell Is the Cellular Source of the Anti-Microbial Angiogenin 4 in the Large Intestine Post Trichuris muris Infection

Background

Mouse angiogenin 4 (Ang4) has previously been described as a Paneth cell–derived antimicrobial peptide important in epithelial host defence in the small intestine. However, a source for Ang4 in the large intestine, which is devoid of Paneth cells, has not been defined.

Methodology/Principal Findings

Analysis was performed on Ang4 expression in colonic tissue by qPCR and immunohistochemistry following infection with the large intestine dwelling helminth parasite Trichuris muris. This demonstrated an increase in expression of the peptide following infection of resistant BALB/c mice. Further, histological analysis of colonic tissue revealed the cellular source of this Ang4 to be goblet cells. To elucidate the mechanism of Ang4 expression immunohistochemistry and qPCR for Ang4 was performed on colonic tissue from T. muris infected mouse mutants. Experiments comparing C3H/HeN and C3H/HeJ mice, which have a natural inactivating mutation of TLR4, revealed that Ang4 expression is TLR4 independent. Subsequent experiments with IL-13 and IL-4 receptor alpha deficient mice demonstrated that goblet cell expression of Ang4 is controlled either directly or indirectly by IL-13.

Conclusions

The cellular source of mouse Ang4 in the colon following T. muris infection is the goblet cell and expression is under the control of IL-13.     

Loss of Sirt1 Function Improves Intestinal Anti-Bacterial Defense and Protects from Colitis-Induced Colorectal Cancer

Dysfunction of Paneth and goblet cells in the intestine contributes to inflammatory bowel disease (IBD) and colitis-associated colorectal cancer (CAC). Here, we report a role for the NAD⁺-dependent histone deacetylase SIRT1 in the control of anti-bacterial defense. Mice with an intestinal specific Sirt1 deficiency (Sirt1^int−/−) have more Paneth and goblet cells with a consequent rearrangement of the gut microbiota. From a mechanistic point of view, the effects on mouse intestinal cell maturation are mediated by SIRT1-dependent changes in the acetylation status of SPDEF, a master regulator of Paneth and goblet cells. Our results suggest that targeting SIRT1 may be of interest in the management of IBD and CAC.     

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.     

CD4CD8αα Lymphocytes,A Novel Human Regulatory T Cell Subset Induced by Colonic Bacteria and Deficient in Patients with Inflammatory Bowel Disease

How the microbiota affects health and disease is a crucial question. In mice, gut Clostridium bacteria are potent inducers of colonic interleukin (IL)-10-producing Foxp3 regulatory T cells (Treg), which play key roles in the prevention of colitis and in systemic immunity. In humans, although gut microbiota dysbiosis is associated with immune disorders, the underlying mechanism remains unknown. In contrast with mice, the contribution of Foxp3 Treg in colitis prevention has been questioned, suggesting that other compensatory regulatory cells or mechanisms may exist. Here we addressed the regulatory role of the CD4CD8 T cells whose presence had been reported in the intestinal mucosa and blood. Using colonic lamina propria lymphocytes (LPL) and peripheral blood lymphocytes (PBL) from healthy individuals, and those with colon cancer and irritable bowel disease (IBD), we demonstrated that CD4CD8αα (DP8α) T lymphocytes expressed most of the regulatory markers and functions of Foxp3 Treg and secreted IL-10. Strikingly, DP8α LPL and PBL exhibited a highly skewed repertoire toward the recognition of Faecalibacterium prausnitzii, a major Clostridium species of the human gut microbiota, which is decreased in patients with IBD. Furthermore, the frequencies of DP8α PBL and colonic LPL were lower in patients with IBD than in healthy donors and in the healthy mucosa of patients with colon cancer, respectively. Moreover, PBL and LPL from most patients with active IBD failed to respond to F. prausnitzii in contrast to PBL and LPL from patients in remission and/or healthy donors. These data (i) uncover a Clostridium-specific IL-10-secreting Treg subset present in the human colonic LP and blood, (ii) identify F. prausnitzii as a major inducer of these Treg, (iii) argue that these cells contribute to the control or prevention of colitis, opening new diagnostic and therapeutic strategies for IBD, and (iv) provide new tools to address the systemic impact of both these Treg and the intestinal microbiota on the human immune homeostasis.     

Modifications of mesenteric adipose tissue during moderate experimental colitis in mice

Aims

Adipose tissue secretes various proteins referred to as adipokines, being involved in inflammation. It was recognized that mesenteric adipose tissue (MAT) is altered by inflammation, and pathologies such as inflammatory bowel disease (IBD). The aim of this study was to investigate the alterations of the mesenteric adipose tissue in two experimental colitis models in mice adapted to obtain moderate colonic inflammation.

Main methods

Colonic inflammation was obtained using two models, either DSS dissolved in drinking water or intra-colonic instillation of DNBS. The expression of adipokines (leptin and adiponectin) and inflammatory markers (IL-6, MCP-1, F4/80) was studied by qRT-PCR in the MAT of treated and control mice.

Key findings

Observations of the colon and IL-6 plasma level determination demonstrated that DNBS treatment led to stronger inflammation. Colitis induced a decrease of mRNA encoding to leptin and adiponectin in MAT. In contrast, colonic inflammation led to an increase of mRNA encoding to IL-6, MCP-1 and F4/80, a specific marker of macrophages.

Significance

The mesenteric adipose tissue, in two models of moderate colitis, shows a loss of adipose profile and a strong increase of inflammatory pattern, close to the observations made in MAT of IBD patients. These data suggest that these pro-inflammatory modifications of MAT have to be taken into account in the pathophysiology of IBD.     

Dietary vitamin B6 intake modulates colonic inflammation in the IL10−/− model of inflammatory bowel disease

Pyridoxal-5-phosphate, the biologically active form of vitamin B₆, is a cofactor for over 140 biochemical reactions. Although severe vitamin B₆ deficiency is rare, mild inadequacy [plasma pyridoxal 5’-phosphate (PLP) <20 nmol/L] is observed in 19–27% of the US population. Plasma PLP concentrations are inversely related to markers of inflammation such as C-reactive protein. Furthermore, plasma PLP is diminished in those with inflammatory conditions and, in the case of inflammatory bowel disease (IBD), more so in those with active versus quiescent disease. Restricting B₆ intake attenuates IBD pathology in mice; however, the effects of supplementation are unclear. We therefore sought to determine the effects of mild inadequacy and moderate supplementation of B6 on the severity of colonic inflammation. Weanling IL-10^?/? (positive for Helicobacter hepaticus) mice were fed diets containing 0.5 (deficient), 6.0 (replete) or 24 (supplemented) mg/kg pyridoxine HCl for 12 weeks and then assessed for histological and molecular markers of colonic inflammation. Both low and high plasma PLP were associated with a significant suppression of molecular (TNFα, IL-6, IFN-γ, COX-2 and iNOS expression) and histological markers of inflammation in the colon. PLP is required for the breakdown of sphingosine 1-phosphate (S1P), a chemotactic lipid, by S1P lyase. Colonic concentrations of S1P and PLP were significantly and inversely correlated. If confirmed, vitamin B6 supplementation may offer an additional tool for the management of IBD. Although B6 is required in dozens of reactions, its role in the breakdown of S1P may explain the biphasic relationship observed between PLP and inflammation.