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Despite the longstanding awareness of the presence of mesenteric alterations in Crohn's disease, the functional and clinical consequences of these alterations remain a topic of debate. Guidelines advise a limited resection without resection of the adjacent mesentery to prevent short bowel syndrome and postoperative complications. However, recently mesenteric resection has been proposed as an alternative to reduce recurrence rates in Crohn's disease patients. Here, we evaluate the data available on this topic in terminal ileitis, both from a fundamental research point of view and clinical perspective.

Background and AimsHistone deacetylase inhibitors [HDACi] exert potent anti-inflammatory effects. Because of the ubiquitous expression of HDACs, clinical utility of HDACi is limited by off-target effects. Esterase-sensitive motif [ESM] technology aims to deliver ESM-conjugated compounds to human mononuclear myeloid cells, based on their expression of carboxylesterase 1 [CES1]. This study aims to investigate utility of an ESM-tagged HDACi in inflammatory bowel disease [IBD].MethodsCES1 expression was assessed in human blood, in vitro differentiated macrophage and dendritic cells, and Crohn’s disease [CD] colon mucosa, by mass cytometry, quantitative polymerase chain reaction [PCR], and immunofluorescence staining, respectively. ESM-HDAC528 intracellular retention was evaluated by mass spectrometry. Clinical efficacy of ESM-HDAC528 was tested in dextran sulphate sodium [DSS]-induced colitis and T cell transfer colitis models using transgenic mice expressing human CES1 under the CD68 promoter.ResultsCES1 mRNA was highly expressed in human blood CD14+ monocytes, in vitro differentiated and lipopolysaccharide [LPS]-stimulated macrophages, and dendritic cells. Specific hydrolysis and intracellular retention of ESM-HDAC528 in CES1+ cells was demonstrated. ESM-HDAC528 inhibited LPS-stimulated IL-6 and TNF-α production 1000 times more potently than its control, HDAC800, in CES1high monocytes. In healthy donor peripheral blood, CES1 expression was significantly higher in CD14++CD16- monocytes compared with CD14+CD16++ monocytes. In CD-inflamed colon, a higher number of mucosal CD68+ macrophages expressed CES1 compared with non-inflamed mucosa. In vivo, ESM-HDAC528 reduced monocyte differentiation in the colon and significantly improved colitis in a T cell transfer model, while having limited potential in ameliorating DSS-induced colitis.ConclusionsWe demonstrate that monocytes and inflammatory macrophages specifically express CES1, and can be preferentially targeted by ESM-HDAC528 to achieve therapeutic benefit in IBD.

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Deletion of endoplasmic reticulum resident chaperone Grp78 results in activation of the unfolded protein response and causes rapid depletion of the entire intestinal epithelium. Whether modest reduction of Grp78 may affect stem cell fate without compromising intestinal integrity remains unknown. Here, we employ a model of epithelial-specific, heterozygous Grp78 deletion by use of VillinCreERT2-Rosa26ZsGreen/LacZ-Grp78+/fl mice and organoids. We examine models of irradiation and tumorigenesis, both in vitro and in vivo. Although we observed no phenotypic changes in Grp78 heterozygous mice, Grp78 heterozygous organoid growth was markedly reduced. Irradiation of Grp78 heterozygous mice resulted in less frequent regeneration of crypts compared with nonrecombined (wild-type) mice, exposing reduced capacity for self-renewal upon genotoxic insult. We crossed mice to Apc-mutant animals for adenoma studies and found that adenomagenesis in Apc heterozygous-Grp78 heterozygous mice was reduced compared with Apc heterozygous controls (1.43 vs. 3.33; P < 0.01). In conclusion, epithelium-specific Grp78 heterozygosity compromises epithelial fitness under conditions requiring expansive growth such as adenomagenesis or regeneration after γ-irradiation. These results suggest that Grp78 may be a therapeutic target in prevention of intestinal neoplasms without affecting normal tissue.Significance: Heterozygous disruption of chaperone protein Grp78 reduces tissue regeneration and expansive growth and protects from tumor formation without affecting intestinal homeostasis. Cancer Res; 78(21); 6098–106. ©2018 AACR.

Background Previous studies have indicated that the appendix may be a priming site of ulcerative colitis (UC). Appendectomy is inversely associated with the development of UC, and is suggested to have a beneficial effect on the disease course in patients with refractory disease. Objective The aim of the current study was to assess histological features of appendices from patients with UC and their clinical relevance. Methods Patients with UC in remission and active UC (therapy refractory) that underwent appendectomy between 2012 and 2019 were included. Histological features of UC appendices were compared to those of patients with acute appendicitis and colon carcinoma. The Robarts Histopathology Index (RHI) was used to assess appendiceal inflammation. In patients with active UC, histological and clinical characteristics were compared between patients with and without endoscopic response following appendectomy. Results In total, 140 appendix specimens were assessed (n = 35 UC remission, n = 35 active UC, n = 35 acute appendicitis, n = 35 colon carcinoma). Histological features of appendices from UC patients looked like UC rather than acute appendicitis. The presence of active appendiceal inflammation was comparable between patients in remission versus active disease (53.7% vs. 46.3%, p = 0.45) and limited versus extensive disease (58.5% vs. 41.5%, p = 0.50). Endoscopic response (Mayo 0-1) following appendectomy, assessed in 28 therapy refractory patients, was more frequently seen in patients with higher RHI scores (RHI > 6: 81.8% vs. RHI ≤ 6: 9.1%, p = 0.03) and limited disease (proctitis/left sided 63.6% vs. pancolitis 36.4%, p = 0.02). Conclusion The presence of active appendiceal inflammation is common in UC and does not correlate with colonic disease activity. More than 50% of UC patients in remission showed active histological disease in the appendix. Favorable response to appendectomy for refractory UC was seen in cases with ulcerative appendicitis. These findings might support the role of the appendix as a pivotal organ in UC.

Background Epidemiological studies and meta-analyses show an association between statin use and a reduced incidence of colorectal cancer (CRC). We have shown that statins act on CRC through bone morphogenetic protein (BMP) signalling, but the exact cellular targets and underlying mechanism of statin action remain elusive. In this study, we set out to assess the influence of statins on global cancer cell signalling by performing an array-based kinase assay using immobilised kinase substrates spanning the entire human kinome. Methods CRC cells with or without Lovastatin treatment were used for kinome analysis. Findings on kinome arrays were further confirmed by immunoblotting with activity-specific antibodies. Experiments in different CRC cell lines using immunoblotting, siRNA-mediated knockdown and treatment with specific BMP inhibitor Noggin were performed. The relevance of in vitro findings was confirmed in xenografts and in CRC patients treated with Simvastatin. Results Kinome analysis can distinguish between non-specific, toxic effects caused by 10 mu M of Lovastatin and specific effects on cell signalling caused by 2 mu M Lovastatin. Statins induce upregulation of PTEN activity leading to downregulation of the PI3K/Akt/mTOR signalling. Treatment of cells with the specific BMP inhibitor Noggin as well as PTEN knockdown and transfection of cells with a constitutively active form of AKT abolishes the effect of Lovastatin on mTOR phosphorylation. Experiments in xenografts and in patients treated with Simvastatin confirm statin-mediated BMP pathway activation, activation of PTEN and downregulation of mTOR signalling. Conclusions Statins induce BMP-specific activation of PTEN and inhibition of PI3K/Akt/mTOR signalling in CRC.

Indian Hedgehog (Ihh) is a morphogen expressed by epithelial cells in the small intestine and colon that signals in a paracrine manner to gp38+ stromal cells. The loss of Ihh signaling results in increased epithelial proliferation, lengthening and multiplication of intestinal crypts and the activation of a stromal cell immune response. How Ihh controls epithelial proliferation through the stroma and how it affects colorectal cancer development remains poorly defined. To study the influence of Ihh signaling on the earliest stage of colorectal carcinogenesis, we used a well characterized mouse model in which both alleles of the Adenoma Polyposis Coli (Apc) gene could be inducibly deleted, leading to instant transformation of the colonic epithelium to an adenomatous phenotype. Concurrent deletion of Ihh from the adenomatous colonic epithelium of Apc inducible double mutant mice resulted in a remarkable increase in the hyperproliferative epithelial phenotype and increased accumulation of Lgr5+ stem cells. Transcriptional profiling of sorted colonic gp38+ fibroblasts showed upregulation of three ErbB pathway ligands (EREG, BTC, and NRG1) in Apc−/−Ihh−/− double mutant mice. We found that recombinant EREG, BTC, and NRG1 but not Lgr5 ligand R-Spondin promoted growth and proliferation of Apc double mutant colonic organoids. Thus, the loss of Ihh enhances Apc-driven colonic adenomagenesis via upregulation of ErbB pathway family members in colonic stromal cells. Our findings highlight the critical role of epithelium-derived Indian Hedgehog as a stromal tumor suppressor in the intestine.

Background & Aims The use of antibiotics (ABs) is a common practice during the first months of life. ABs can perturb the intestinal microbiota, indirectly influencing the intestinal epithelial cells (IECs), but can also directly affect IECs independent of the microbiota. Previous studies have focused mostly on the impact of AB treatment during adulthood. However, the difference between the adult and neonatal intestine warrants careful investigation of AB effects in early life. Methods Neonatal mice were treated with a combination of amoxicillin, vancomycin, and metronidazole from postnatal day 10 to 20. Intestinal permeability and whole-intestine gene and protein expression were analyzed. IECs were sorted by a fluorescence-activated cell sorter and their genome-wide gene expression was analyzed. Mouse fetal intestinal organoids were treated with the same AB combination and their gene and protein expression and metabolic capacity were determined. Results We found that in vivo treatment of neonatal mice led to decreased intestinal permeability and a reduced number of specialized vacuolated cells, characteristic of the neonatal period and necessary for absorption of milk macromolecules. In addition, the expression of genes typically present in the neonatal intestinal epithelium was lower, whereas the adult gene expression signature was higher. Moreover, we found altered epithelial defense and transepithelial-sensing capacity. In vitro treatment of intestinal fetal organoids with AB showed that part of the consequences observed in vivo is a result of the direct action of the ABs on IECs. Lastly, ABs reduced the metabolic capacity of intestinal fetal organoids. Conclusions Our results show that early life AB treatment induces direct and indirect effects on IECs, influencing their maturation and functioning., Graphical abstract

Background and Aims Tyrosine kinase 2 [TYK2] is required for the signalling of key cytokines in the pathogenesis of inflammatory bowel disease [IBD]. We assessed the efficacy of a novel selective TYK2 inhibitor [TYK2i] in experimental colitis, using pharmacological and genetic tools. Methods At onset of T cell transfer colitis, RAG1-/- mice received vehicle or TYK2i daily by oral gavage. T cells lacking TYK2 kinase activity [TYK2KE] were used to confirm selectivity of the inhibitor. To this end, RAG1-/- or RAG1-/-TYK2KE animals were transferred with either wild type [WT] or TYK2KE-CD45RBhigh colitogenic T cells. Loss of body weight, endoscopic disease, the disease activity index [DAI], and histopathology scores were recorded. Tissues were analysed ex vivo for lymphocyte populations by flow cytometry. The impact of TYK2 inhibition on human DC-T cell interactions were studied using autologous Revaxis specific T cell assays. Results TYK2i [70 mg/kg] prevented weight loss and limited endoscopic activity during T cell transfer colitis. TYK2i [70 mg/kg] decreased DAI. Whereas transfer of WT T cells into RAG-/-TYK2KE hosts induced colitis, TYK2KE T cells transferred into RAG1-/-TYK2KErecipients failed to do so. Ex vivo analysis showed a decrease in colon tissue Th1 cells and an increase in Th17 cells upon transfer of TYK2KE-CD45RBhigh cells. In human antigen-triggered T cells, TYK2i displayed reduced Th1 differentiation, similar to murine Th1 cells. Conclusions Oral administration of TYK2i, as well as transfer of T cells lacking TYK2 activity, reduced human Th1 differentiation and ameliorated the course of murine T cell transfer colitis. We conclude that TYK2 is a promising drug target for the treatment of IBD.

Background & Aims Activation factor-1 transcription factor family members activating transcription factors 2 and 7 (ATF2 and ATF7) have highly redundant functions owing to highly homologous DNA binding sites. Their role in intestinal epithelial homeostasis and repair is unknown. Here, we assessed the role of these proteins in these conditions in an intestine-specific mouse model. Methods We performed in vivo and ex vivo experiments using Villin-CreERT2Atf2fl/flAtf7ko/ko mice. We investigated the effects of intestinal epithelium-specific deletion of the Atf2 DNA binding region in Atf7-/- mice on cellular proliferation, differentiation, apoptosis, and epithelial barrier function under homeostatic conditions. Subsequently, we exposed mice to 2% dextran sulfate sodium (DSS) for 7 days and 12 Gy whole-body irradiation and assessed the response to epithelial damage. Results Activating phosphorylation of ATF2 and ATF7 was detected mainly in the crypts of the small intestine and the lower crypt region of the colonic epithelium. Under homeostatic conditions, no major phenotypic changes were detectable in the intestine of ATF mutant mice. However, on DSS exposure or whole-body irradiation, the intestinal epithelium showed a clearly impaired regenerative response. Mutant mice developed severe ulceration and inflammation associated with increased epithelial apoptosis on DSS exposure and were less able to regenerate colonic crypts on irradiation. In vitro, organoids derived from double-mutant epithelium had a growth disadvantage compared with wild-type organoids, impaired wound healing capacity in scratch assay, and increased sensitivity to tumor necrosis factor-α–induced damage. Conclusions ATF2 and ATF7 are dispensable for epithelial homeostasis, but are required to maintain epithelial regenerative capacity and protect against cell death during intestinal epithelial damage and repair., Graphical abstract

Background & Aims Recent evidence has suggested that the intact intestinal epithelial barrier protects our body from a range of immune-mediated diseases. The epithelial layer has an impressive ability to reconstitute and repair upon damage and this process of repair increasingly is seen as a therapeutic target. In vitro models to study this process in primary intestinal cells are lacking. Methods We established and characterized an in vitro model of intestinal damage and repair by applying γ-radiation on small-intestinal organoids. We then used this model to identify novel regulators of intestinal regeneration. Results We identified hepatocyte nuclear factor 4α (HNF4α) as a pivotal upstream regulator of the intestinal regenerative response. Organoids lacking Hnf4a were not able to propagate in vitro. Importantly, intestinal Hnf4a knock-out mice showed impaired regeneration after whole-body irradiation, confirming intestinal organoids as a valuable alternative to in vivo studies. Conclusions In conclusion, we established and validated an in vitro damage–repair model and identified HNF4α as a crucial regulator of intestinal regeneration. Transcript profiling: GSE141515 and GSE141518., Graphical abstract

The ATG16L1 T300A single-nucleotide polymorphism (SNP) is associated with Crohn's disease and causes an autophagy impairment. We have previously shown that this SNP is involved in the migration and hyperactivation of Rac1 in dendritic cells. Mucosal healing, currently the main target for inflammatory bowel disease treatment, depends on restoration of the epithelial barrier and requires appropriate migration of epithelial cells towards and over mucosal lesions. Therefore, we here further investigated the impact of autophagy on epithelial migration. ATG16L1 knockdown was established in the HT29 human colonic epithelial cell line using lentiviral transduction. Migratory capacity was evaluated using scratch assays and RhoAGTP was measured using G-LISA. Immunofluorescent ARHGAP18 and sequestome 1 (SQSTM1; also known as p62) staining was performed on HT29 cells and primary colonic tissue of Crohn's disease patients. We observed that ATG16L1 knockdown cells exhibited decreased autophagy and decreased migration capacity. Furthermore, activity of RhoA was decreased. These characteristics were phenocopied using ATG5 knockdown and pharmacological inhibition of autophagy. The migration defect was dependent on accumulation of SQSTM1 and was alleviated upon SQSTM1 knockdown. Strikingly, thiopurines also mitigated the effects of impaired autophagy. RhoA dysregulation appeared mediated through accumulation of the upstream regulator ARHGAP18, which was observed in cell lines, human foetal organoids and primary colonic tissue. Our results indicate that the ATG16L1 T300A Crohn's disease-associated SNP causes a decrease in migration capacity in epithelial cells, mediated by an increase in SQSTM1 and ARHGAP18 protein and subsequent reduced RhoA activation., Summary: Analyses of HT29 cells after ATG16L1 knockdown reveal a novel biological mechanism for the association between the autophagy pathway and Crohn's disease, and could aid personalized medicine strategies in Crohn's disease therapy.

Primary sclerosing cholangitis (PSC) is an inflammatory disease of the biliary tree, characterised by stricturing bile duct disease and progression to liver fibrosis. The pathophysiology of PSC is still unknown. The concurrence with inflammatory bowel disease (IBD) in about 70% of cases has led to the hypothesis that gut-homing lymphocytes aberrantly traffic to the liver, contributing to disease pathogenesis in patients with both PSC and IBD (PSC-IBD). The discovery of mutual trafficking pathways of lymphocytes to target tissues, and expression of gut-specific adhesion molecules and chemokines in the liver has pointed in this direction. There is now increasing interest in using drugs that intervene with these trafficking pathways (e.g. vedolizumab, etrolizumab) for the treatment of PSC-IBD. In this review we discuss what is currently known about the immunological interactions between the gut and the liver in concomitant PSC and IBD, as well as potential therapeutic options for intervening in these mechanisms.

Background and aims Rectal resection in inflammatory bowel disease [IBD] is frequently complicated by disturbed perineal wound healing. Close rectal dissection, where the mesorectum remains in situ, is hypothesized to reduce complications by minimizing dead space, compared to total mesorectal excision. The aim of this study was to analyse post-operative outcomes of both techniques. In addition, immune activity in mesorectal tissue was assessed. Methods Perineal complications and healing were retrospectively assessed in a series of 74 IBD patients undergoing proctectomy using close rectal dissection or total mesorectal excision. The mesorectums of 15 patients were analysed by fluorescence-activated cell sorting, immunofluorescence and in situ hybridization. Based on the clinical and in vitro findings, a novel surgical approach for Crohn's disease patients with disturbed perineal healing after proctectomy was developed. Results In Crohn's disease, perineal complications were more frequent after close rectal dissection than after total mesorectal excision [59.5% vs 17.6%; p = 0.007] with lower healing rates [51.4% vs 88.2%; p = 0.014]. No differences were observed in ulcerative colitis. The mesorectal tissue in Crohn's disease contained enhanced numbers of tumour necrosis factor α-producing CD14+ macrophages, with less expression of the wound-healing marker CD206. Based on these findings, mesorectal excision with omentoplasty was performed in eight patients with perineal complications after close rectal dissection, resulting in complete perineal wound closure in six. Pro-inflammatory characteristics remained present in the mesorectum after close rectal dissection in these patients. Conclusions In Crohn's disease, close rectal dissection resulted in more perineal complications, associated with a pro-inflammatory immune status of the mesorectal tissue. Excision of this pro-inflammatory mesenteric tissue resulted in improved perineal healing rates.

Clinical trials suggest that vagus nerve stimulation presents an alternative approach to classical immune suppression in Crohn's disease. T cells capable of producing acetylcholine (ChAT+ T cells) in the spleen are essential mediators of the anti-inflammatory effect of vagus nerve stimulation. Besides the spleen, ChAT+ T cells are found abundantly in Peyer’s patches of the small intestine. However, the role of ChAT+ T cells in colitis pathogenesis is unknown. Here, we made use of CD4creChATfl/fl mice (CD4ChAT−/− mice) lacking ChAT expression specifically in CD4+ T cells. Littermates (ChATfl/fl mice) served as controls. In acute dextran sulfate sodium (DSS)-induced colitis (7 days of 2% DSS in drinking water), CD4ChAT−/− mice showed attenuated colitis and lower intestinal inflammatory cytokine levels compared with ChATfl/fl mice. In contrast, in a resolution model of DSS-induced colitis (5 days of 2% DSS followed by 7 days without DSS), CD4ChAT−/− mice demonstrated a worsened colitis recovery and augmented colonic histological inflammation scores and inflammatory cytokine levels as compared with ChATfl/fl mice. In a transfer colitis model using CD4+CD45RBhigh T cells, T cells from CD4ChAT−/− mice induced a similar level of colitis compared with ChATfl/fl T cells. Together, our results indicate that ChAT+ T cells aggravate the acute innate immune response upon mucosal barrier disruption in an acute DSS-induced colitis model, whereas they are supporting the later resolution process of this innate immune-driven colitis. Surprisingly, ChAT expression in T cells seems redundant in the context of T cell-driven colitis. NEW & NOTEWORTHY By using different mouse models of experimental colitis, we provide evidence that in dextran sulfate sodium-induced colitis, ChAT+ T cells capable of producing acetylcholine worsen the acute immune response, whereas they support the later healing phase of this innate immune-driven colitis.

Background Inflammatory bowel disease (IBD) is characterised by substantial heterogeneity in treatment response. With an expanding number of therapeutic agents, identifying optimal treatment at the patient level remains a major challenge. Aim To systematically review the available literature on predictive biomarkers of therapeutic response in IBD. Methods An electronic literature search was performed on 30 January 2018 using MEDLINE, EMBASE and the Cochrane Library. Retrospective, prospective, uncontrolled and controlled studies reporting on biomarkers predicting therapeutic response in paediatric and adult IBD populations were eligible for inclusion. The methodological quality of the included studies was assessed using the QUIPS tool. Due to anticipated heterogeneity and limited data, a qualitative, rather than quantitative, assessment was planned. Results Of the 10 638 citations identified, 92 articles met the inclusion criteria. Several potential DNA, mRNA and protein markers were evaluated as predictive biomarkers. Most studies focused on predicting response to anti-TNF agents. Substantial between-study heterogeneity was identified with respect to both the biomarkers studied and the definition of response. None of the included studies received a low risk of bias rating for all six domains. Currently, none of the biomarkers is sufficiently predictive for clinical use. Conclusions The search for predictive biomarkers is still in its infancy and current evidence is limited. Future research efforts should take into account the high patient heterogeneity within prospective trials with objective response assessment. Predictive models will most likely comprise a combination of several molecular markers from integrated omics-levels and clinical characteristics.

Background and aims Immortalized cell lines have been long used as substitute for ex vivo murine and human material, but exhibit features that are not found in healthy tissue. True human dendritic cells (DC) cannot be cultured or passaged as opposed to immortalized cell lines. Research in the fields of immunogenic responses and immunotolerance in DCs has increased over the last decade. Autophagy has gained interest in these fields as well, and has been researched extensively in many other cell types as well. Here we have studied the applicability of cell line-derived dendritic cell-like cells of six myeloid cell lines aimed at research focussed on autophagy. Methods Six myeloid leukaemia cell lines were differentiated towards cell line-derived dendritic cell-like cells (cd-DC) using GM-CSF, IL-4, Ionomycine and PMA: HL60, KG1, MM6, MV-4-11, THP1 and U937. Autophagy was modulated using Rapamycin, Bafilomycin A1 and 3MA. Cell lines were genotyped for autophagy-related SNPs using RFLP. Marker expression was determined with FACS analysis and cytokine profiles were determined using Human Cytometric Bead Assay. Antigen uptake was assessed using Fluoresbrite microspheres. Results and discussion All researched cell lines harboured SNPs in the autophagy pathways. MM6 and THP1 derived cd-DCs resembled monocyte-derived DCs (moDC) most closely in marker expression, cytokine profiles and autophagy response. The HL60 and U937 cell lines proved least suitable for autophagy-related dendritic cell research. Conclusion The genetic background of cell lines should be taken into account upon studying (the effects of) autophagy in any cell line. Although none of the studied cell lines recapitulate the full spectrum of DC characteristics, MM6 and THP1 derived cd-DCs are most suitable for autophagy-related research in dendritic cells.

Background A fistula is an abnormal tract connecting two epithelialized surfaces, for example the intestine and the skin. Perianal fistula are a common complication of patients suffering Crohn’s Disease (CD), but also occur in non-IBD patients in the form of cryptoglandular fistula. Around one third of all CD patients develop fistula at some point during their disease course. Fistula are often refractory to therapy, due to poor wound healing responses. In contrast, cryptoglandular fistula often respond to standard therapy. The biological background of this difference is unknown, and comparative studies between the two groups are lacking. The aim of this study was to characterize the cellular composition in fistula tracts of CD and cryptoglandular patients. Methods Curettage material of perianal fistula tracts was obtained during surgical intervention from patients with CD (n=15) and cryptoglandular fistulas (n=5). Single-cell suspensions were stained with a 35-antibody panel, focusing on myeloid and T-cell markers and were analyzed using mass cytometry (CyTOF). To visualize macrophages in the fistula tract we performed in situ hybridization with CD68 and TNF-α. Results The main cellular component of both fistula tracts consisted of CD66a+ granulocytes (64 +/- 24%). However, the remaining mononuclear compartment differed significantly between Crohn and cryptoglandular fistula. In CD, the majority was of lymphoid nature (CD3+ T cells 57 +/-21%, CD19+ B cells 14 +/-15%), while in cryptoglandular tracts, the majority consisted of myeloid origin (61+/- 15%). Within the T cell compartment, the majority of cells was CD45RO+, indicating activation. Presence of a seton increased the proportion of CD45RO+ T cells, in particular in CD4+ cells. In the myeloid compartment, CD14high/HLA-int monocytes, CD14int/HLA-high inflammatory macrophages and CD14high/CD163+ resident macrophages were identified. Interestingly, CD patient samples contained less monocyte-like cells, and substantially more resident macrophages compared to cryptoglandular samples. This feature tended to be even more enhanced in the presence of a seton, although this did not reach statistical significance. In situ hybridization showed a high production of TNF-α in epithelial-like cells in fistula tract of Crohn’s disease patients, but not in macrophages. Conclusion Despite granulocytes being the main contributor to the cellular composition of fistula tracts, striking differences were found between Crohns and cryptoglandular fistula, both in lymphoid/myeloid balance, and in the presence of resident macrophages. We also showed that epithelial-like cells in Crohns’s disease fistula tracts produce high amounts of TNF-α. These differences may contribute to the lack of response to therapy in CD.

Background The aim of the current study was to assess histological features of appendices from patients with UC and its clinical relevance. Methods Patients with UC in remission and active UC (therapy refractory) that underwent an appendectomy in the frame of clinical trials between 2012–2019 were included. Histological features of UC appendices were compared to those of patients with acute appendicitis and colon carcinoma. The Robarts Histopathology Index score (RHI) was used to assess appendiceal inflammation. In patients with active UC, clinical and histological characteristics were compared between patients with and without endoscopic response after appendectomy. Results In total, 140 appendix specimens were assessed (n=35 UC remission, n=35 active UC, n=35 acute appendicitis, n=35 colon carcinoma). Histological features of appendices from UC patients looked like UC rather than acute appendicitis. The incidence of active appendiceal inflammation was not different between UC patients in remission versus active disease (53.7% versus 46.3%, p=0.45) and limited versus extensive disease (58.5% versus 41.5%, p=0.50). Endoscopic response, assessed in 28 therapy refractory patients, was more frequently seen in patients with higher RHI scores (RHI>9 81.8% versus RHI≤9 9.1%, p=0.004) and limited disease (proctitis/left sided 63.6% versus pancolitis 36.4%, p=0.02). Conclusion The presence of active appendiceal inflammation is common in UC and not related to disease activity in the colon. More than 50% of UC patients in remission show active histological disease in the appendix. Favorable response to appendectomy for refractory UC was seen in cases with ulcerative appendicitis. These findings might support the role of the appendix as a driving force in UC.

Background. The human digestive tract is structurally mature at birth, yet maturation of gut functions such as digestion and mucosal barrier continues for the next 1&ndash, 2 years. Human milk and infant milk formulas (IMF) seem to impact maturation of these gut functions differently, which is at least partially related to high temperature processing of IMF causing loss of bioactive proteins and formation of advanced glycation end products (AGEs). Both loss of protein bioactivity and formation of AGEs depend on heating temperature and time. The aim of this study was to investigate the impact of mildly pasteurized whey protein concentrate (MP-WPC) compared to extensively heated WPC (EH-WPC) on gut maturation in a piglet model hypersensitive to enteral nutrition. Methods. WPC was obtained by cold filtration and mildly pasteurized (73 &deg, C, 30 s) or extensively heat treated (73 &deg, C, 30 s + 80 &deg, C, 6 min). Preterm (~90% gestation) and near-term piglets (~96% gestation) received enteral nutrition based on MP-WPC or EH-WPC for five days. Macroscopic and histologic lesions in the gastro-intestinal tract were evaluated and intestinal responses were further assessed by RT-qPCR, immunohistochemistry and enzyme activity analysis. Results. A diet based on MP-WPC limited epithelial intestinal damage and improved colonic integrity compared to EH-WPC. MP-WPC dampened colonic IL1-&beta, IL-8 and TNF-&alpha, expression and lowered T-cell influx in both preterm and near-term piglets. Anti-microbial defense as measured by neutrophil influx in the colon was only observed in near-term piglets, correlated with histological damage and was reduced by MP-WPC. Moreover, MP-WPC stimulated iALP activity in the colonic epithelium and increased differentiation into enteroendocrine cells compared to EH-WPC. Conclusions. Compared to extensively heated WPC, a formula based on mildly pasteurized WPC limits gut inflammation and stimulates gut maturation in preterm and near-term piglets and might therefore also be beneficial for preterm and (near) term infants.

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

Background and Aims Although several endoscopic and histopathologic indices are available for evaluating the severity of inflammation in mouse models of colitis, the reliability of these scoring instruments is unknown. Our aim was to evaluate the reliability of the individual items in the existing indices and develop new scoring systems by selection of the most reliable index items. Methods Two observers scored the histological slides [n = 224] and endoscopy videos [n = 201] from treated and untreated Interleukin[IL]-10 knock-out and T-cell transferred SCID mice. Intra-rater and inter-rater reliability for endoscopy and histology scores, and each individual item, were measured using intraclass correlation coefficients [ICCs]. The Mouse Colitis Histology Index [MCHI] and Mouse Colitis Endoscopy Index [MCEI] were developed using the most reliable items. Both were correlated to the colon density and to each other and were evaluated for their ability to detect changes in pathobiology. Results The intraclass correlation coefficients (ICCs) for inter-rater agreement (95% CIs) for the total histology and endoscopy scores were 0.90 [0.87–0.92] and 0.80 [0.76–0.84], respectively. The MCHI and MCEI were highly correlated with colon density, with a Spearman Rho = 0.81[0.75–0.85] and 0.73 [0.66–0.79], respectively, and with each other, Spearman Rho = 0.71 [0.63–0.77]. The MCHI and MCEI were able to distinguish between the experimental groups within the models, with pairwise differences between the treated and untreated groups being statistically significant [p < 0.001]. Conclusions These histological and endoscopic indices are valid and reliable measures of intestinal inflammation in mice, and they are responsive to treatment effects in pre-clinical studies.

Background Flow cytometric (FC) analysis of intestinal tissue biopsies requires prompt cell isolation and processing to prevent cell death and generate valid data. We examined the effect of storage conditions prior to cell isolation and FC on viable cell yield and the proportions of immune cell phenotypes from intestinal biopsies. Methods Biopsies (N = 224) from inflamed or non-inflamed ileal and/or colonic tissue from three patients with Crohn's disease were processed and analyzed immediately in duplicate, or stored under different conditions. Cells were isolated and stained for specific markers, followed by FC. Results Decreased mean live CD45+ cell counts were observed after storage of biopsies at −80 °C dimethyl sulfoxide (DMSO)/citrate buffer compared with immediate processing (1794.3 vs. 19,672.7; p = 0.006]). A non-significant decrease in CD45+ live cell count occurred after storage at −20 °C in DMSO/citrate buffer and cell yield was adequate for subsequent analysis. CD3+ cell proportions were significantly lower after storage at 4 °C in complete medium for 48 h compared with immediate analysis. Mean CD14+ cell proportions were significantly higher after storage of biopsies at −80 °C in DMSO/citrate buffer compared with immediate analysis (2.61% vs. 1.31%, p = 0.007). CD4+, CD8+ and CD4+/CD8+ cell proportions were unaffected by storage condition. Conclusion Storage of intestinal tissue biopsies at −20 °C in DMSO/citrate buffer for up to 48 h resulted in sufficient viable cell yield for FC analysis without affecting subsequent marker-positive cell proportions. These findings support the potential shipping and storage of intestinal biopsies for centralized FC analysis in multicenter clinical trials.

The Na+ -taurocholate cotransporting polypeptide (NTCP/SLC10A1) is believed to be pivotal for hepatic uptake of conjugated bile acids. However, plasma bile acid levels are normal in a subset of NTCP knockout mice and in mice treated with myrcludex B, a specific NTCP inhibitor. Here, we elucidated which transport proteins mediate the hepatic uptake of conjugated bile acids and demonstrated intestinal sensing of elevated bile acid levels in plasma in mice. Mice or healthy volunteers were treated with myrcludex B. Hepatic bile acid uptake kinetics were determined in wild-type (WT), organic anion transporting polypeptide (OATP) knockout mice (lacking Slco1a/1b isoforms), and human OATP1B1-transgenic mice. Effects of fibroblast growth factor 19 (FGF19) on hepatic transporter mRNA levels were assessed in rat hepatoma cells and in mice by peptide injection or adeno-associated virus-mediated overexpression. NTCP inhibition using myrcludex B had only moderate effects on bile acid kinetics in WT mice, but completely inhibited active transport of conjugated bile acid species in OATP knockout mice. Cholesterol 7α-hydroxylase Cyp7a1 expression was strongly down-regulated upon prolonged inhibition of hepatic uptake of conjugated bile acids. Fgf15 (mouse counterpart of FGF19) expression was induced in hypercholanemic OATP and NTCP knockout mice, as well as in myrcludex B-treated cholestatic mice, whereas plasma FGF19 was not induced in humans treated with myrcludex B. Fgf15/FGF19 expression was induced in polarized human enterocyte-models and mouse organoids by basolateral incubation with a high concentration (1 mM) of conjugated bile acids. CONCLUSION NTCP and OATPs contribute to hepatic uptake of conjugated bile acids in mice, whereas the predominant uptake in humans is NTCP mediated. Enterocytes sense highly elevated levels of (conjugated) bile acids in the systemic circulation to induce FGF15/19, which modulates hepatic bile acid synthesis and uptake. (Hepatology 2017;66:1631-1643).

Inflammatory bowel diseases, such as ulcerative colitis and Crohn’s disease, are disabling conditions characterised by chronic, relapsing inflammation of the gastrointestinal tract. Current treatments are not universally effective or, in the case of therapeutic antibodies, are hampered by immune responses. Janus kinase inhibitors are orally delivered small molecules that target cytokine signalling by preventing phosphorylation of Janus kinases associated with the cytokine receptor. Subsequently, phosphorylation of signal transducers and activators of transcription that relay Janus kinase signalling and transcription of cytokines in the nucleus will be diminished. Key cytokines in the pathogenesis of inflammatory bowel diseases are targeted by Janus kinase inhibitors. Several Janus kinase inhibitors are in development for the treatment of inflammatory bowel diseases. Tofacitinib, inhibiting signalling via all Janus kinase family members, was effective in phase 2 and 3 trials in moderate-severe ulcerative colitis. GSK2586184, a Janus kinase 1 selective inhibitor, induced clinical and endoscopic response in ulcerative colitis; however, the study was discontinued at an early stage due to liver toxicity observed in systemic lupus patients receiving the drug. Filgotinib, a Janus kinase 1 selective inhibitor investigated in treatment of Crohn’s disease, was superior to placebo. As adverse events associated with the broad immunological effect of these agents have been reported, the future application of these drugs is potentially limited. We will discuss the treatment efficacy of Janus kinase inhibition in inflammatory bowel diseases, how current Janus kinase inhibitors available target immune responses relevant in inflammatory bowel disease, and whether more specific kinase inhibition could be effective.

Background Alterations in the mesentery of Crohn’s disease (CD) patients have long been described, although the functional importance of this tissue is less clear. Some studies hypothesise an anti-inflammatory role for enhanced mesentery, whereas others consider a more pathologic function, given its close proximity to the inflamed intestine. Better phenotypic and functional evaluation of the cells present in this tissue is warranted to improve our understanding of its role in disease. We have previously shown a disproportionate presence of macrophages in CD mesorectum. In this study, we aimed to better characterise the phenotype and function of the macrophages in the mesentery in CD. Methods We collected mesenteric specimens from Crohn’s disease, ulcerative colitis (UC) and non-inflammatory bowel disease (IBD) patients, undergoing surgical bowel resections. In IBD patients, mesentery was collected contiguous to the inflamed region of the intestine, in non-IBD patients, mesentery adjacent to the resection margin. Macrophages were sorted by flow cytometry as CD45+ CD66b-CD14+CD11b+ cells. Sorted cells were analysed by expression profiling and functional analysis. Results Macrophages were present in all analysed tissues. Within the population, a discriminating expression pattern for CD11b was present, dividing CD14+ macrophages in a CD11bhigh and a CD11bdim subset. Transcriptional profiling and gene set enrichment showed the CD11bhigh population to be consistent with IFN-induced pro-inflammatory macrophages, while the CD11bdim population was most consistent with IL4-induced regulatory macrophages. Among the top upregulated genes in CD11bhigh macrophages were S100A8 and S100A9, the two heterodimeric subunits for calprotectin. Flow cytometry confirmed higher expression of calprotectin in the CD11bhigh population also on the protein level. Functionally, CD11bdim macrophages showed higher phagocytic capacity, again consistent a role in tissue repair. Strikingly, the CD11bdim subset was diminished significantly in the mesentery of CD patients, with a near absence in some patients, while the profile in UC was comparable to non-IBD patients. Conclusion Mesenteric macrophages contain two populations, CD11bhigh with a pro-inflammatory profile and CD11bdim with a regulatory profile. In the mesentery of CD patients, the CD11bdim population was strongly decreased, consistent with a pro-inflammatory role for this tissue.

Background To facilitate personalised treatment of inflammatory bowel disease (IBD), biomarkers for disease entity and disease severity in easily accessible samples are required. Therefore, we aimed to (i) identify proteins that discriminate Crohn’s disease (CD) from ulcerative colitis (UC), (ii) assess the association of proteomic profiles with disease severity and (iii) assess the potential to use serum samples as a proxy for intestinal biopsies. Methods In this prospective cross-sectional study, IBD patients with active endoscopic disease (presence of ≥1 ulcer (CD) or Mayo score ≥1 (UC)) and age matched non IBD controls (normal endoscopy) were included. In IBD, biopsies were taken from inflamed tissue; in CD from the edge of an ulcer and in UC and controls between 20 and 25 cm from the anal verge. Disease severity was dichotomised in mild vs. severe defined as SES CD Results Forty-one CD, 39 UC and 10 controls were included. Median SES-CD score [IQR] was 9 [7–16] in CD. In UC, 20 (51.3%), 15 (38.5%) and 4 (10.3%) patients had an endoscopic Mayo score of 3, 2 and 1, respectively. Three proteins differed significantly between UC and CD in tissue (IL-17A, IL13 and CCL4). However, this was not reflected in serum levels of these molecules. For prediction of disease severity in CD, elastic net regression identified a set of 11 serum proteins that discriminated mild from severe disease (accuracy 0.667). Top five proteins were IFNγ (OR 3.03), CCL23 (OR 1.28), Cystatin D (OR 0.61), TNF (OR 1.28) and HGF (OR 1.17) (Figure 1). In tissue, no discriminatory profile was identified. Conversely, in serum of UC, no proteins were selected, while a set of three tissue proteins was associated with disease severity (accuracy 0.725). These proteins were osteoprotegerin (OPG) (OR 1.66), IL8 (OR 1.09) and CCL25 (OR 0.97). The discrepancy between serum and tissue was further supported by correlation analyses. Of all 92 proteins, only three showed significant correlation between tissue and serum: IL17A (r = 0.44), TGF-α (r = 0.42) and IL6 (r = 0.39). Conclusion Proteomic profiles between tissue and serum in active IBD correlated poorly. Only in CD, serum markers differentiated mild from severe disease. In UC, no proteins in serum were selected whereas in tissue, expression of OPG, IL-8 and CCL25 was associated with disease severity. These data again emphasise the difference between CD and UC on a molecular level.

Indian Hedgehog (Ihh) is a morphogen expressed by epithelial cells in the small intestine and colon that signals in a paracrine manner to gp38+ stromal cells. The loss of Ihh signaling results in increased epithelial proliferation, lengthening and multiplication of intestinal crypts and the activation of a stromal cell immune response. How Ihh controls epithelial proliferation through the stroma and how it affects colorectal cancer development remains poorly defined. To study the influence of Ihh signaling on the earliest stage of colorectal carcinogenesis, we used a well characterized mouse model in which both alleles of the Adenoma Polyposis Coli (Apc) gene could be inducibly deleted, leading to instant transformation of the colonic epithelium to an adenomatous phenotype. Concurrent deletion of Ihh from the adenomatous colonic epithelium of Apc inducible double mutant mice resulted in a remarkable increase in the hyperproliferative epithelial phenotype and increased accumulation of Lgr5+ stem cells. Transcriptional profiling of sorted colonic gp38+ fibroblasts showed upregulation of three ErbB pathway ligands (EREG, BTC, and NRG1) in Apc

During the suckling‐to‐weaning transition, the intestinal epithelium matures, allowing digestion of solid food. Transplantation experiments with rodent fetal epithelium into subcutaneous tissue of adult animals suggest that this transition is intrinsically programmed and occurs in the absence of dietary or hormonal signals. Here, we show that organoids derived from mouse primary fetal intestinal epithelial cells express markers of late fetal and neonatal development. In a stable culture medium, these fetal epithelium‐derived organoids lose all markers of neonatal epithelium and start expressing hallmarks of adult epithelium in a time frame that mirrors epithelial maturation in vivo . In vitro postnatal development of the fetal‐derived organoids accelerates by dexamethasone, a drug used to accelerate intestinal maturation in vivo . Together, our data show that organoids derived from fetal epithelium undergo suckling‐to‐weaning transition, that the speed of maturation can be modulated, and that fetal organoids can be used to model the molecular mechanisms of postnatal epithelial maturation.

Background and Aims The objective of this study was to examine the modulating effect of an appendectomy on the disease course of therapy-refractory ulcerative colitis [UC] patients, and to analyse appendiceal pathological characteristics predictive of pathological response. Methods Patients with therapy-refractory UC, and referred for proctocolectomy, were invited to undergo laparoscopic appendectomy first. The primary end points were clinical response after 3 and 12 months. Secondary end points were endoscopic remission, failure, and pathologic response. Appendiceal specimens, and pre- and post-operative biopsies were histologically graded according to the validated Geboes score. Results Thirty patients [53% male] with a median age of 40 (interquartile range [IQR], 33–47) underwent appendectomy, with a median preoperative total Mayo score of 9 [IQR, 8–11]. After 12 months, 9 patients [30%] had lasting clinical response, of whom 5 [17%] were in endoscopic remission. Pathological evaluation was possible in 28 patients. After a median of 13.0 weeks [range 7–51], pathological response was seen in 13 patients [46%], with a median decrease of 2 points [range 1–3]. Appendiceal inflammation was highly predictive of pathological response when compared with no inflammation or extensive ulcerations [85% vs 20%, p = 0.001]. Conclusions Appendectomy was effective in one-third of therapy-refractory UC patients, with a substantial proportion of patients demonstrating complete endoscopic remission after 1 year. Pathological response was seen in almost 50% of patients and was related to active inflammation in the appendix, limited disease, and shorter disease duration. These early results suggest that there is a UC patient group that may benefit from appendectomy.