Sarah Devriese

Butyrate-producing bacteria supplemented in vitro to Crohn’s disease patient microbiota increased butyrate production and enhanced intestinal epithelial barrier integrity

The management of the dysbiosed gut microbiota in inflammatory bowel diseases (IBD) is gaining more attention as a novel target to control this disease. Probiotic treatment with butyrate-producing bacteria has therapeutic potential since these bacteria are depleted in IBD patients and butyrate has beneficial effects on epithelial barrier function and overall gut health. However, studies assessing the effect of probiotic supplementation on microbe-microbe and host-microbe interactions are rare. In this study, butyrate-producing bacteria (three mono-species and one multispecies mix) were supplemented to the fecal microbial communities of ten Crohn’s disease (CD) patients in an in vitro system simulating the mucus- and lumen-associated microbiota. Effects of supplementation in short-chain fatty acid levels, bacterial colonization of mucus environment and intestinal epithelial barrier function were evaluated. Treatment with F. prausnitzii and the mix of six butyrate-producers significantly increased the butyrate production by 5–11 mol%, and colonization capacity in mucus- and lumen-associated CD microbiota. Treatments with B. pullicaecorum 25-3T and the mix of six butyrate-producers improved epithelial barrier integrity in vitro. This study provides proof-of-concept data for the therapeutic potential of butyrate-producing bacteria in CD and supports the future preclinical development of a probiotic product containing butyrate-producing species.

Reduced Mucosa-associated Butyricicoccus Activity in Patients with Ulcerative Colitis Correlates with Aberrant Claudin-1 Expression.

Background and Aims: Butyricicoccus is a butyrate-producing clostridial cluster IV genus whose numbers are reduced in the stool of ulcerative colitis [UC] patients. Conditioned medium of Butyricicoccus [B.] pullicaecorum prevents tumour necrosis factor alpha [TNF&agr;]-induced increase in epithelial permeability in vitro. Since butyrate influences intestinal barrier integrity, we further investigated the relationship between the abundance of mucosa-associated Butyricicoccus and the expression of butyrate-regulated tight junction [TJ] genes. Methods: Tight junction protein 1 [TJP1], occludin [OCLN], claudin-1 [CLDN1], and Butyricicoccus 16S rRNA expression was analysed in a collection of colonic biopsies of healthy controls and UC patients with active disease. The effect of butyrate and B. pullicaecorum conditioned medium on TJ gene expression was investigated in TNF&agr;-stimulated Caco-2 monolayers and inflamed mucosal biopsies of UC patients. Results: TJP1 expression was significantly decreased in inflamed UC mucosa, whereas CLDN1 mRNA levels were increased. OCLN did not differ significantly between the groups. Mucosa-associated Butyricicoccus 16S rRNA transcripts were reduced in active UC patients compared with healthy controls. Interestingly, Butyricicoccus activity negatively correlated with CLDN1 expression. Butyrate reversed the inflammation-induced increase of CLDN1 protein levels, and stimulation of inflamed UC biopsies with B. pullicaecorum conditioned medium normalized CLDN1 mRNA levels. Conclusions: Butyricicoccus is a mucosa-associated bacterial genus under-represented in colonic mucosa of patients with active UC, whose activity inversely correlates with CLDN1 expression. Butyrate and B. pullicaecorum conditioned medium reduce CLDN1 expression, supporting its use as a pharmabiotic preserving epithelial TJ integrity.

Haematopoietic prolyl hydroxylase-1 deficiency promotes M2 macrophage polarization and is both necessary and sufficient to protect against experimental colitis

Prolyl hydroxylase domain‐containing proteins (PHDs) regulate the adaptation of cells to hypoxia. Pan‐hydroxylase inhibition is protective in experimental colitis, in which PHD1 plays a prominent role. However, it is currently unknown how PHD1 targeting regulates this protection and which cell type(s) are involved. Here, we demonstrated that Phd1 deletion in endothelial and haematopoietic cells (Phd1f/fTie2:cre) protected mice from dextran sulphate sodium (DSS)‐induced colitis, with reduced epithelial erosions, immune cell infiltration, and colonic microvascular dysfunction, whereas the response of Phd2f/+Tie2:cre and Phd3f/fTie2:cre mice to DSS was similar to that of their littermate controls. Using bone marrow chimeras and cell‐specific cre mice, we demonstrated that ablation of Phd1 in haematopoietic cells but not in endothelial cells was both necessary and sufficient to inhibit experimental colitis. This effect relied, at least in part, on skewing of Phd1‐deficient bone marrow‐derived macrophages towards an anti‐inflammatory M2 phenotype. These cells showed an attenuated nuclear factor‐κB‐dependent response to lipopolysaccharide (LPS), which in turn diminished endothelial chemokine expression. In addition, Phd1 deficiency in dendritic cells significantly reduced interleukin‐1β production in response to LPS. Taken together, our results further support the development of selective PHD1 inhibitors for ulcerative colitis, and identify haematopoietic cells as their primary target. Copyright

Ursodeoxycholic acid and its taurine/glycine conjugated species reduce colitogenic dysbiosis and equally suppress experimental colitis in mice

ABSTRACT The promising results seen in studies of secondary bile acids in experimental colitis suggest that they may represent an attractive and safe class of drugs for the treatment of inflammatory bowel diseases (IBD). However, the exact mechanism by which bile acid therapy confers protection from colitogenesis is currently unknown. Since the gut microbiota plays a crucial role in the pathogenesis of IBD, and exogenous bile acid administration may affect the community structure of the microbiota, we examined the impact of the secondary bile acid ursodeoxycholic acid (UDCA) and its taurine or glycine conjugates on the fecal microbial community structure during experimental colitis. Daily oral administration of UDCA, tauroursodeoxycholic acid (TUDCA), or glycoursodeoxycholic acid (GUDCA) equally lowered the severity of dextran sodium sulfate-induced colitis in mice, as evidenced by reduced body weight loss, colonic shortening, and expression of inflammatory cytokines. Illumina sequencing demonstrated that bile acid therapy during colitis did not restore fecal bacterial richness and diversity. However, bile acid therapy normalized the colitis-associated increased ratio of Firmicutes to Bacteroidetes. Interestingly, administration of bile acids prevented the loss of Clostridium cluster XIVa and increased the abundance of Akkermansia muciniphila, bacterial species known to be particularly decreased in IBD patients. We conclude that UDCA, which is an FDA-approved drug for cholestatic liver disorders, could be an attractive treatment option to reduce dysbiosis and ameliorate inflammation in human IBD. IMPORTANCE Secondary bile acids are emerging as attractive candidates for the treatment of inflammatory bowel disease. Although bile acids may affect the intestinal microbial community structure, which significantly contributes to the course of these inflammatory disorders, the impact of bile acid therapy on the fecal microbiota during colitis has not yet been considered. Here, we studied the alterations in the fecal microbial abundance in colitic mice following the administration of secondary bile acids. Our results show that secondary bile acids reduce the severity of colitis and ameliorate colitis-associated fecal dysbiosis at the phylum level. This study indicates that secondary bile acids might act as a safe and effective drug for inflammatory bowel disease.

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 α and β was analyzed in Caco-2 cell monolayers exposed to tumor necrosis factor (TNF)α, in ileal tissue of TNFΔARE/WT mice and in inflamed ileal biopsies from CD patients by quantitative real-time polymerase chain reaction. TNFΔARE/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 TNFα impaired the mRNA expression of nuclear receptors and bile acid transporters, whereas co-incubation with TUDCA antagonized their downregulation. TNFΔARE/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.

P025 Reduced Butyricicoccus pullicaecorum levels in mucosa of UC patients correlate with aberrant CLDN1 expression

Background: Intestinal fibrosis (IF) is one of the major complications in inflammatory bowel disease patients. IF can cause narrowing of the intestinal lumen, which may lead to stricture formation. The mechanism of IF is still unknown and adequate models are lacking. By using precision-cut intestinal slice (PCIS) from different regions of the bowel, we studied the early onset of fibrosis in mouse jejunum, ileum and colon PCIS, in the presence of transforming growth factor (TGF)-beta1 and platelet-derived growth factor (PDGF). Methods: Mouse jejunum, ileum and colon were excised and prepared as a segment embedded in agarose. PCIS (estimated 300 400mm) was prepared and incubated up to 48 hr with or without the presence of TGF-beta1 and PDGF. ATP content of the PCIS was used to assess the general viability. The gene expression of different fibrosis markers including Pro-Collagen 1 A1 (COL1A1), heat shock protein 47 (HSP47), alpha-smooth muscle actin (SMA), connective tissue growth factor (CTGF), synaptophysin (SYN) and fibronectin (FN2) were determined. Results: Mouse PCIS from different segments were viable up to 48 hr. After 48 hr of incubation, HSP47 and FN2 gene expression were significantly up-regulated, compared to PCIS directly after slicing, in jejunum (3.6 and 4.9 fold, respectively) and in ileum (4.9 and 5.5 fold, respectively). When incubated with 5 ng/mL TGF-beta1, in jejunum PCIS, COL1A1, HSP47, CTGF and FN2 were significantly up-regulated compared to 48 hr control. In ileum PCIS the gene expression of HSP47 (1.9 fold) and FN2 (3.9 fold) were also significantly increased. In the presence of 50 ng/mL PDGF, only in ileum PCIS, CTGF (1.4 fold) and SYN (1.9 fold) were significantly increased compared to 48 hr control. Interestingly, in PCIS from the colon, 5 ng/ml TGF-beta1 did not affect the gene expression of the fibrosis markers. However, HSP47 (1.4 fold) and FN2 (1.7 fold) were significantly increased when colon PCIS were incubated with 50 ng/mL PDGF. Conclusions: TGF-beta1, but not PDGF, was able to induce HSP47 and FN2 in mouse jejunum and ileum PCIS. This is in contrast to the result in colon PCIS, where only PDGF was able to induce these fibrosis markers. Moreover, PDGF increased CTGF and SYN only in ileum PCIS. These results indicate differences in the effect of TGF-beta1 and PDGF on the early onset of fibrosis in different regions of the intestine.