Differential expression of prolyl hydroxylase 1 in patients with ulcerative colitis versus patients with Crohn's disease/infectious colitis and healthy controls

Background: Inhibition of prolyl hydroxylases (PHDs) leads to the induction of a transcriptional program that, in the gut, promotes intestinal epithelial cell survival. PHD inhibitors have recently been suggested as a promising alternative treatment for inflammatory bowel disease (IBD). In this study, we explored the colonic mucosal expression of the different PHD-isoforms (PHD1, 2 and 3) in order to identify the key isoform(s) involved in the pathogenesis of IBD. Methods: The mRNA expression of inflammatory cytokines (IL-8 and TNF-alpha), an apoptosis marker (caspase 3) and PHD1, 2 and 3 was analysed in biopsies of IBD patients (UC and CD), patients with infectious colitis and healthy controls using qRT-PCR. PHD protein levels were evaluated using western blot. Cellular localization of PHD 1, 2 and 3 was determined by immunohistochemistry. Results: PHD1 was significantly up-regulated in IBD patients, both at the mRNA (UC: p < 0.0001 and CD: p < 0.05) and at the protein level (UC: p < 0.05 and CD: p < 0.05), and showed a very good correlation with the expression of the inflammatory cytokines IL-8 and TNF-alpha and the apoptosis marker caspase 3. Colonic mucosal PHD2 mRNA and protein expressions were not altered in IBD. PHD3 expression was increased in inflamed biopsies from UC patients (p < 0.0001), but only at the mRNA level. PHD1 and PHD2 expression was found both in the colonic lamina propria and the epithelium while PHD3 was mainly located in the endothelium of blood vessels. Conclusions: In this exploratory expression analysis, PHD1 comes forward as the primary therapeutic target for UC and, to a lesser extent, for (colonic) CD

Ly49E Expression on CD8αα-expressing intestinal intraepithelial lymphocytes plays no detectable role in the development and progression of experimentally induced inflammatory bowel diseases

The Ly49E NK receptor is a unique inhibitory receptor, presenting with a high degree of conservation among mouse strains and expression on both NK cells and intraepithelial-localised T cells. Amongst intraepithelial-localised T cells, the Ly49E receptor is abundantly expressed on CD8 alpha alpha-expressing innate-like intestinal intraepithelial lymphocytes (iIELs), which contribute to front-line defense at the mucosal barrier. Inflammatory bowel diseases (IBDs), encompassing Crohn's disease and ulcerative colitis, have previously been suggested to have an autoreactive origin and to evolve from a dysbalance between regulatory and effector functions in the intestinal immune system. Here, we made use of Ly49E-deficient mice to characterize the role of Ly49E receptor expression on CD8 alpha alpha-expressing iIELs in the development and progression of IBD. For this purpose we used the dextran sodium sulphate (DSS)- and trinitrobenzenesulfonic-acid (TNBS)-induced colitis models, and the TNF Delta ARE ileitis model. We show that Ly49E is expressed on a high proportion of CD8 alpha alpha-positive iIELs, with higher expression in the colon as compared to the small intestine. However, Ly49E expression on small intestinal and colonic iIELs does not influence the development or progression of inflammatory bowel diseases

Open Access

Differential expression of prolyl hydroxylase 1 in patients with ulcerative colitis versus patients with Crohn’s disease/infectious colitis and healthy control

Long-term environmental hypoxia exposure and haematopoietic prolyl hydroxylase-1 deletion do not impact experimental Crohn's like ileitis

Simple Summary Hypoxia-induced signalling represents an important contributor to inflammatory bowel disease (IBD) pathophysiology. However, available data solely focus on colonic inflammation while the primary disease location in Crohn's disease patients is the terminal ileum. Therefore, we explored the effects of environmental hypoxia and immune cell-specific deletion of oxygen sensor prolyl hydroxylase (PHD) 1 in a Crohn's like ileitis mouse model. Five-week-old TNF (increment ARE/+) mice and wildtype (WT) littermates were housed in normoxia (21% O-2) or hypoxia (8% O-2) for 10 weeks. Although environmental hypoxia increased both systemic as ileal markers of hypoxia, the body weight evolution in both WT and TNF (increment ARE/+) mice was not affected. Interestingly, hypoxia did increase circulatory monocytes, ileal mononuclear phagocytes and proinflammatory cytokine expression in WT mice. However, no histological or inflammatory gene expression differences in the ileum could be identified between TNF (increment ARE/+) mice housed in hypoxia versus normoxia nor between TNF (increment ARE/+) and WT mice with additional loss of immune cell-specific Phd1 expression. This is the first study showing that long-term environmental hypoxia or haematopoietic Phd1-deletion does not impact experimental ileitis. Therefore, it strongly questions whether targeting hypoxia-induced signalling via currently available PHD inhibitors would exert an immune suppressive effect in IBD patients with ileal inflammation. Environmental hypoxia and hypoxia-induced signalling in the gut influence inflammatory bowel disease pathogenesis, however data is limited to colitis. Hence, we investigated the effect of environmental hypoxia and immune cell-specific deletion of oxygen sensor prolyl hydroxylase (PHD) 1 in a Crohn's like ileitis mouse model. Therefore, 5-week-old C57/BL6 TNF (increment ARE/+) mice and wildtype (WT) littermates were housed in normoxia (21% O-2) or hypoxia (8% O-2) for 10 weeks. Systemic inflammation was assessed by haematology. Distal ileal hypoxia was evaluated by pimonidazole staining. The ileitis degree was scored on histology, characterized via qPCR and validated in haematopoietic Phd1-deficient TNF (increment ARE/+) mice. Our results demonstrated that hypoxia did not impact body weight evolution in WT and TNF (increment ARE/+) mice. Hypoxia increased red blood cell count, haemoglobin, haematocrit and increased pimonidazole intensity in the ileum. Interestingly, hypoxia evoked an increase in circulatory monocytes, ileal mononuclear phagocytes and proinflammatory cytokine expression in WT mice. Despite these alterations, no histological or ileal gene expression differences could be identified between TNF (increment ARE/+) mice housed in hypoxia versus normoxia nor between haematopoietic Phd1-deficient TNF (increment ARE/+) and their WT counterparts. Therefore, we demonstrated for the first time that long-term environmental hypoxia or haematopoietic Phd1-deletion does not impact experimental ileitis development

Can serum metabolomics discriminate the Crohn’s disease fibrostenotic phenotype?

Background: Crohn’s disease (CD) patients are highly prone to develop fibrotic strictures, for which surgery is the only treatment option. Therefore, an unmet need exists for convenient diagnostic tools and clinical markers to assess onset and progression of this life-threatening complication. Aim: This study aimed to identify discriminating metabolic markers in the serum of CD patients with and without fibrostenosis. Methods: Samples of 66 CD patients with (n=28) and without (n=38) ileal fibrotic strictures were selected from a local biobank (UZ Gent, BB190100). Fibrostenosis was defined as a narrowing of lumen and prestenotic dilation on CT/MRI. Metabolomics analysis was performed applying UHPLC-Q-Orbitrap-HRMS. The in-house method for metabolite extraction and mass spectrometry analysis was validated for serum compatibility. Statistical analysis of the untargeted MS data was performed using SIMCA 15.0 and MetaboAnalyst 4.0, allowing multivariate statistical modelling through Principal Component Analysis and sparse Partial Least-Squares Discriminant Analysis (sPLS-DA). Results: Validation of serum metabolomics analysis, including instrumental precision, intra-assay, and inter-day analyses, showed excellent coverage of the measured metabolites. After analysis, CD samples yielded 5,959 features in total. Using sPLS-DA models, 1,000 features were retained to build an Orthogonal PLS-DA model which had R2Y of 0.99 and Q2 of 0.83, suggesting excellent predictivity and fitting of the existent data. After further filtering, 47 most differentiating features were determined. Conclusion: Our comprehensive metabolomics approach unveiled a discriminative metabolic fingerprint in the serum of fibrostenotic CD patients which has clinical application potential as novel diagnostic tool. Identification and validation of these metabolites is ongoing

T84 monolayers are superior to Caco-2 as a model system of colonocytes

Colonic adenocarcinoma-derived Caco-2 and T84 epithelial cell lines are frequently used as in vitro model systems of functional epithelial barriers. Both are utilised interchangeably despite evidence that differentiated Caco-2 cells are more reminiscent of small intestinal enterocytes than of colonocytes, whereas differentiated T84 cells are less well characterised. The aim of this study was, therefore, to further characterise and compare differentiated Caco-2 and T84 cells. The objectives were to (1) compare the brush border morphology, (2) measure the expression of enterocyte- and colonocyte-specific genes and (3) compare their response to butyrate, which is dependent on the monocarboxylate transporter 1 (MCT1), an apical protein expressed primarily in colonocytes. T84 microvilli were significantly shorter than those of Caco-2 cells, which is a characteristic difference between small intestinal enterocytes and colonocytes. Also, enterocyte-associated brush border enzymes expressed in differentiated Caco-2 cells were not increased during T84 maturation, whereas colonic markers such as MCT1 were more abundant in differentiated T84 cells compared to differentiated Caco-2 cells. Consequently, T84 cells displayed a dose-responsive improvement of barrier function towards butyrate, which was absent in Caco-2 cells. On the other hand, differences in epithelial toll-like receptor expression between Caco-2 and T84 monolayers did not result in a corresponding differential functional response. We conclude that differentiated Caco-2 and T84 cells have distinct morphological, biochemical and functional characteristics, suggesting that T84 cells do not acquire the biochemical signature of mature small intestinal enterocytes like Caco-2 cells, but retain much of their original colonic characteristics throughout differentiation. These findings can help investigators select the appropriate intestinal epithelial cell line for specific in vitro research purposes

Tauroursodeoxycholic acid protects bile acid homeostasis under inflammatory conditions and dampens Crohn’s disease-like ileitis

Bile acids regulate the expression of intestinal bile acid transporters and are natural ligands for nuclear receptors controlling inflammation. Accumulating evidence suggests that signaling through these receptors is impaired in inflammatory bowel disease. We investigated whether tauroursodeoxycholic acid (TUDCA), a secondary bile acid with cytoprotective properties, regulates ileal nuclear receptor and bile acid transporter expression and assessed its therapeutic potential in an experimental model of Crohn's disease (CD). Gene expression of the nuclear receptors farnesoid X receptor, pregnane X receptor and vitamin D receptor and the bile acid transporters apical sodium-dependent bile acid transporter and organic solute transporter a and (3 was analyzed in Caco-2 cell monolayers exposed to tumor necrosis factor (TNF)alpha, in ileal tissue of TNF Delta ARE/WT mice and in inflamed ilea) biopsies from CD patients by quantitative real-time polymerase chain reaction. TNF Delta ARE/WT mice and wild-type littermates were treated with TUDCA or placebo for 11 weeks and ileal histopathology and expression of the aforementioned genes were determined. Exposing Caco-2 cell monolayers to TNFa impaired the mRNA expression of nuclear receptors and bile acid transporters, whereas co-incubation with TUDCA antagonized their downregulation. TNF Delta ARE/WT mice displayed altered ileal bile acid homeostasis that mimicked the situation in human CD ileitis. Administration of TUDCA attenuated ileitis and alleviated the downregulation of nuclear receptors and bile acid transporters in these mice. These results show that TUDCA protects bile acid homeostasis under inflammatory conditions and suppresses CD-like ileitis. Together with previous observations showing similar efficacy in experimental colitis, we conclude that TUDCA could be a promising therapeutic agent for inflammatory bowel disease, warranting a clinical trial