Influence of vitamin D receptor signaling and vitamin D on colonic epithelial cell fate decisions in ulcerative colitis.

BACKGROUND AND AIMS: Epidemiological studies have shown that subnormal levels of vitamin D (25(OH)D) are associated with a more aggravated clinical course of ulcerative colitis (UC). Despite an increased focus on the therapeutic importance of vitamin D and vitamin D receptor (VDR) signaling, the mechanisms underlying the effects of the vitamin D-VDR axis on UC remain elusive. Therefore, we aimed to investigate whether exposure to active vitamin D (1,25(OH)2D3)/VDR signaling in human organoids could influence the maintenance of the colonic epithelium. METHODS: Intestinal VDR expression was studied by immunohistochemistry, RNA expression arrays, and single-cell RNA sequencing of colonic biopsy specimens obtained from patients with UC and healthy individuals. To characterize the functional and transcriptional effects of 1,25(OH)2D3, we used patient-derived colonic organoids. The dependency of VDR was assessed by knocking out the receptor with CRISPR/Cas9. RESULTS: Our results suggest that 1,25(OH)2D3/VDR stimulation supports differentiation of the colonic epithelium and that impaired 1,25(OH)2D3/VDR signaling thereby may compromise the structure of the intestinal epithelial barrier, leading to flares of UC. Furthermore, a transcriptional response to VDR activity was observed primarily in fully differentiated cells at the top of the colonic crypt, and this response was reduced during flares of UC. CONCLUSIONS: We identified an important role of vitamin D signaling in supporting differentiated cell states in the human colonic epithelium, and thereby maintenance of the intestinal barrier integrity. This makes the vitamin D-VDR signaling axis an interesting target for therapeutic efforts to achieve and maintain remission in patients with UC.

Lipid nanoparticle delivery limits antisense oligonucleotide activity and cellular distribution in the brain after intracerebroventricular injection.

Antisense oligonucleotide (ASO) therapeutics are being investigated for a broad range of neurological diseases. While ASOs have been effective in the clinic, improving productive ASO internalization into target cells remains a key area of focus in the field. Here, we investigated how the delivery of ASO-loaded lipid nanoparticles (LNPs) affects ASO activity, subcellular trafficking, and distribution in the brain. We show that ASO-LNPs increase ASO activity up to 100-fold in cultured primary brain cells as compared to non-encapsulated ASO. However, in contrast to the widespread ASO uptake and activity observed following free ASO delivery in vivo, LNP-delivered ASOs did not downregulate mRNA levels throughout the brain after intracerebroventricular injection. This lack of activity was likely due to ASO accumulation in cells lining the ventricles and blood vessels. Furthermore, we reveal a formulation-dependent activation of the immune system post dosing, suggesting that LNP encapsulation cannot mask cellular ASO backbone-mediated toxicities. Together, these data provide insights into how LNP encapsulation affects ASO distribution as well as activity in the brain, and a foundation that enables future optimization of brain-targeting ASO-LNPs.

COX-2-PGE2 Signaling Impairs Intestinal Epithelial Regeneration and Associates with TNF Inhibitor Responsiveness in Ulcerative Colitis.

BACKGROUND: Inhibition of tumor necrosis factor-α (TNF) signaling is beneficial in the management of ulcerative colitis (UC), but up to one-third of patients do not have a clinical response of relevance to TNF inhibitors during induction therapy (i.e. primary non-responders [PNRs]). Through production of prostaglandins (PGs) and thromboxanes, cyclooxygenase-2 (COX-2) affects inflammation and epithelial regeneration and may in this way be implicated in treatment resistance to TNF inhibitors. METHODS: In this study, COX-2 expression was analyzed in human intestinal biopsies and patient-derived monocytes, and the downstream consequences of COX-2 activity was evaluated by assessing the influence of the down-stream effector, PGE2, on intestinal epithelial stem cell self-renewal and differentiation using primary human intestinal organoids ("mini-guts"). FINDINGS: We found that TNF stimulation induced COX-2 expression in monocytes isolated from responders (Rs), whereas COX-2 expression was constitutively high and non-inducible in monocytes from PNRs. Additionally, PGE2 in combination with proliferative signals transformed human intestinal epithelial cells to a proinflammatory state akin to flaring UC, whereas PGE2 in combination with differentiation signals supported robust mucin induction. INTERPRETATION: Our work indicates that COX-2-PGE2 signaling could be a novel target for the management of PNRs to TNF inhibitors. We additionally demonstrate that COX-2-PGE2 signaling has dual functions during tissue repair and normal lineage differentiation, explaining in part the lack of response to TNF inhibitors among PNRs. FUND: This work was funded by grants from the Novo Nordisk Foundation, the Lundbeck Foundation, the Vanderbilt Digestive Disease Research Center, NIH Grants, Aase and Ejnar Danielsen's Foundation and the A.P. Møller Foundation.

Targeting JAK-STAT signal transduction in IBD.

An unmet medical need exists for novel targeted therapies for inflammatory bowel disease (IBD) as many patients experience inadequate responses to antibody-based biologics. An oral drug formulation with reduced production costs and redundancy for healthcare staff to administer therapy ideally should result in diminished healthcare expenses and improved patient compliance. A new drug class of small molecules, the Janus kinase (JAK) inhibitors (jakinibs), fulfills these criteria and has recently shown efficacy in IBD. Here we provide an overview of the mode of action of jakinibs and provide a comprehensive overview of existing clinical studies. Convincing clinical data show that a complex cytokine-driven inflammation can efficiently be modulated by therapeutic inhibition of the JAK proteins.

Putative biomarkers of vedolizumab resistance and underlying inflammatory pathways involved in IBD.

OBJECTIVES: Characterise the circulating inflammatory cytokine pattern among patients failing consecutive anti-tumour necrosis factor (anti-TNF) and anti-integrin treatments to identify predictors of response. METHODS: A retrospective single-centre cohort study of 28 patients with inflammatory bowel disease (IBD) receiving anti-integrin therapy (vedolizumab) subsequent to the failure of anti-TNF treatment was conducted. Blood samples were obtained immediately prior to initiation of vedolizumab therapy, and the response to treatment was evaluated after completion of the 14-week induction regimen. Multiplex ELISA was applied to quantify 47 preselected plasma proteins based on their putative involvement in the inflammatory process in IBD. RESULTS: Anti-TNF and vedolizumab non-responders (n=20) had significantly higher levels of circulating interleukin (IL)-6 than anti-TNF non-responders with subsequent response to vedolizumab (n=8): median 9.5 pg/mL versus 5.9 pg/mL, p<0.05. Following stratification by diagnosis, patients with Crohn's disease who failed vedolizumab therapy (n=7) had higher soluble CD40 ligand (sCD40L) than responders (n=4): 153.0 pg/mL versus 45.5 pg/mL, p<0.01; sensitivity 100% (95% CI 59% to 100%), specificity 100% (95% CI 40% to 100%). Osteocalcin was higher among patients with ulcerative colitis responding to vedolizumab (n=4) compared with those not responding (n=13): 4219 pg/mL versus 2823 pg/mL, p=0.01; sensitivity 85% (95% CI 55% to 98%), specificity 100% (95% CI 40% to 100%). CONCLUSIONS: Patients with IBD failing vedolizumab induction and anti-TNF therapy have persistent IL-6 pathway activity, which could be a potential alternative treatment target. sCD40L, osteocalcin and the IL-6 pathway activity might be predictors for response to vedolizumab.

Rational Management of Iron-Deficiency Anaemia in Inflammatory Bowel Disease.

Anaemia is the most frequent, though often neglected, comorbidity of inflammatory bowel disease (IBD). Here we want to briefly present (1) the burden of anaemia in IBD, (2) its pathophysiology, which mostly arises from bleeding-associated iron deficiency, followed by (3) diagnostic evaluation of anaemia, (4) a balanced overview of the different modes of iron replacement therapy, (5) evidence for their therapeutic efficacy and subsequently, (6) an updated recommendation for the practical management of anaemia in IBD. Following the introduction of various intravenous iron preparations over the last decade, questions persist about when to use these preparations as opposed to traditional and other novel oral iron therapeutic agents. At present, oral iron therapy is generally preferred for patients with quiescent IBD and mild iron-deficiency anaemia. However, in patients with flaring IBD that hampers intestinal iron absorption and in those with inadequate responses to or side effects with oral preparations, intravenous iron supplementation is the therapy of choice, although information on the efficacy of intravenous iron in patients with active IBD and anaemia is scare. Importantly, anaemia in IBD is often multifactorial and a careful diagnostic workup is mandatory for optimized treatment. Nevertheless, limited information is available on optimal therapeutic start and end points for treatment of anaemia. Of note, neither oral nor intravenous therapies seem to exacerbate the clinical course of IBD. However, additional prospective studies are still warranted to determine the optimal therapy in complex conditions such as IBD.

Intestinal barrier integrity and inflammatory bowel disease: Stem cell-based approaches to regenerate the barrier.

Disruption of normal barrier function is a fundamental factor in the pathogenesis of inflammatory bowel disease, which includes increased epithelial cell death, modified mucus configuration, altered expression and distribution of tight junction proteins, along with a decreased expression of antimicrobial peptides. Inflammatory bowel disease is associated with life-long morbidity for affected patients, and both the incidence and prevalence is increasing globally, resulting in substantial economic strain for society. Mucosal healing and re-establishment of barrier integrity are associated with clinical remission, as well as with an improved patient outcome. Hence, these factors are vital treatment goals, which conventionally are achieved by a range of medical treatments, although none are effective in all patients, resulting in several patients still requiring surgery at some point. Therefore, novel treatment strategies to accomplish mucosal healing and to re-establish normal barrier integrity in inflammatory bowel disease are warranted, and luminal stem cell-based approaches might have an intriguing potential. Transplantation of in vitro expanded intestinal epithelial stem cells derived either directly from mucosal biopsies or from directed differentiation of human pluripotent stem cells may constitute complementary treatment options for patients with mucosal damage, as intestinal epithelial stem cells are multipotent and may give rise to all epithelial cell types of the intestine. This review provides the reader with a comprehensive state-of-the-art overview of the intestinal barrier's role in healthy and diseased states, discussing the clinical application of stem cell-based approaches to accomplish mucosal healing in inflammatory bowel disease.

Characterization of Growth Hormone Resistance in Experimental and Ulcerative Colitis.

Growth hormone (GH) resistance may develop as a consequence of inflammation during conditions such as inflammatory bowel disease, encompassing ulcerative colitis (UC). However, the specific role of the GH-insulin growth factor (IGF)-1-axis and/or the functional consequences of GH resistance in this condition are unclear. In situ hybridization targeting the GH receptor (GHR) and relevant transcriptional analyses were performed in patients with UC and in IL-10 knock-out mice with piroxicam accelerated colitis (PAC). Using cultured primary epithelial cells, the effects of inflammation on the molecular mechanisms governing GH resistance was verified. Also, the therapeutic potential of GH on mucosal healing was tested in the PAC model. Inflammation induced intestinal GH resistance in UC and experimental colitis by down-regulating GHR expression and up-regulating suppressor of cytokine signalling (SOCS) proteins. These effects are driven by pro-inflammatory mediators (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6) as confirmed using primary epithelial cells. Treatment of experimental colitis with GH increased IGF-1 and body weight of the mice, but had no effects on colonic inflammation or mucosal healing. The high transcriptional similarity between UC and experimental colitis accentuates the formation of intestinal GH resistance during inflammation. Inflammation-induced GH resistance not only impairs general growth but induces a state of local resistance, which potentially impairs the actions of GH on mucosal healing during colitis when using long-acting GH therapy.

Growth Hormone Resistance-Special Focus on Inflammatory Bowel Disease.

Growth hormone (GH) plays major anabolic and catabolic roles in the body and is important for regulating several aspects of growth. During an inflammatory process, cells may develop a state of GH resistance during which their response to GH stimulation is limited. In this review, we will emphasize specific mechanisms governing the formation of GH resistance in the active phase of inflammatory bowel disease. The specific molecular effects mediated through individual inflammatory mediators and processes will be highlighted to provide an overview of the transcriptional, translational and post-translational inflammation-mediated impacts on the GH receptor (GHR) along with the impacts on GH-induced intracellular signaling. We also will review GH's effects on mucosal healing and immune cells in the context of experimental colitis, human inflammatory bowel disease and in patients with short bowel syndrome.

HAI-2 stabilizes, inhibits and regulates SEA-cleavage-dependent secretory transport of matriptase.

It has recently been shown that hepatocyte growth factor activator inhibitor-2 (HAI-2) is able to suppress carcinogenesis induced by overexpression of matriptase, as well as cause regression of individual established tumors in a mouse model system. However, the role of HAI-2 is poorly understood. In this study, we describe 3 mutations in the binding loop of the HAI-2 Kunitz domain 1 (K42N, C47F and R48L) that cause a delay in the SEA domain cleavage of matriptase, leading to accumulation of non-SEA domain cleaved matriptase in the endoplasmic reticulum (ER). We suggest that, like other known SEA domains, the matriptase SEA domain auto-cleaves and reflects that correct oligomerization, maturation, and/or folding has been obtained. Our results suggest that the HAI-2 Kunitz domain 1 mutants influence the flux of matriptase to the plasma membrane by affecting the oligomerization, maturation and/or folding of matriptase, and as a result the SEA domain cleavage of matriptase. Two of the HAI-2 Kunitz domain 1 mutants investigated (C47F, R48L and C47F/R48L) also displayed a reduced ability to proteolytically silence matriptase. Hence, HAI-2 separately stabilizes matriptase, regulates the secretory transport, possibly via maturation/oligomerization and inhibits the proteolytic activity of matriptase in the ER, and possible throughout the secretory pathway.

Regulation of Laminin γ2 Expression by CDX2 in Colonic Epithelial Cells Is Impaired During Active Inflammation.

The expression of Caudal-related homeobox transcription factor 2 (CDX2) is impaired by tumor necrosis factor-α (TNF-α)-mediated activation of nuclear factor-κB (NF-κB) in ulcerative colitis (UC). Laminin subunit γ2 (LAMC2) is an epithelial basement membrane protein implicated in cell migration, proliferation, differentiation, as well as tumor invasion and intestinal inflammation, and its expression is enhanced by TNF-α in a NF-κB-dependent regulation of the recently identified LAMC2 enhancer. The aim was to determine whether CDX2 is involved in the basal regulation of LAMC2 in epithelial cells and to assess the influence of inflammation. Transcriptional regulation of LAMC2 was examined by reporter gene assays, overexpression, and shRNA-mediated knock-down of CDX2. CDX2-DNA interactions were assessed by chromatin immunoprecipitation on Caco-2 cells without or with TNF-α, as well as in purified colonic human epithelial cells. Immunohistochemical staining and quantitative reverse-transcription polymerase chain reaction analyses were used to measure the expression of CDX2 and LAMC2 in colonic biopsies from healthy controls and patients with UC. These data indicate that CDX2 directly regulates LAMC2 gene expression through interaction with elements in the LAMC2 promoter region. We further revealed an inverse effect of inflammation on CDX2 and LAMC2. The data presented provide a novel insight into how CDX2 is implicated in the transcriptional regulation of LAMC2 in intestinal epithelial cells, a function that is impaired during mucosal inflammation where a high level of TNF-α is present. J. Cell. Biochem. 118: 298-307, 2017. © 2016 Wiley Periodicals, Inc.

Objective Quantification of Immune Cell Infiltrates and Epidermal Proliferation in Psoriatic Skin: A Comparison of Digital Image Analysis and Manual Counting.

Digital pathology and image analysis have developed extensively during the last couple of years. Especially the advance in whole-slide scanning, software, and computer processing makes it possible to apply these methods in tissue-based research. Today this task is dominated by tedious manual assessments by pathologists with the interobserver and intraobserver variation this includes. Automated quantitative assessment of immunohistochemical staining has the potential to objectively extract numerical measures from cell and tissue structures, and allows efficient high throughput analysis in clinical research. Published data of manual cell counts in psoriatic skin samples were in this study reevaluated using the digital image analysis (DIA) software. Whole slides immunohistochemically stained for CD3, CD4, CD8, CD45R0, and Ki-67 were scanned and quantitatively evaluated using simple threshold analysis. Regression analysis with R values in the range of 0.85 to 0.95 indicates a good correlation between the manual count of cell numbers and the staining density obtained by automated DIA. Moreover, we show that the automated image analysis is reliable over a broad range of thresholds and that it is robust to differences in staining intensities and hence useful for high throughput analysis. DIA is a viable technical approach for automated cell quantification. Its output highly correlates to the conventional manual cell counting and hence allows for increasing the throughput and reducing the analysis time significantly.

Systemic and intestinal levels of factor XIII-A: the impact of inflammation on expression in macrophage subtypes.

BACKGROUND: Subunit A of coagulation factor XIII (FXIII-A) is important for clot stability and acts in the subsequent wound healing process. Loss of plasma FXIII-A has been reported after surgery, sepsis, and inflammatory conditions. In the intestinal mucosa, FXIII-A is expressed by macrophages and cellular FXIII-A has been associated with phagocytosis and migration of macrophages. The objective was to evaluate the consequences of intestinal inflammation on resident mucosal macrophages, focusing on the level and distribution of FXIII-A. METHODS: Plasma and colonic biopsies were collected from 67 patients with ulcerative colitis and controls. Intestinal samples were stained using immunohistochemistry for FXIII-A and macrophages (CD68, CD163 and iNOS). In situ hybridization were used to assess the intestinal expression of FXIII-A. FXIII-A antigen and activity levels were measured in plasma. RESULTS: Increased infiltration of CD68 positive macrophages in the inflamed mucosa coincided with increased extracellular deposited FXIII-A and decreased expression and intracellular protein levels of FXIII-A. A decreased proportion of FXIII-A/CD68/CD163 triple-positive macrophages was observed in inflamed mucosa, indicating a reduction of the M2 phenotype with consequent loss of FXIII-A. No induction of iNOS positive macrophages was observed. Stimulation of naïve monocytes with physiological concentrations of pro-inflammatory mediators negatively affected the expression of FXIII-A. Measurements in plasma confirmed the loss of both FXIII antigen and activity during active disease. CONCLUSIONS: Intestinal inflammation in UC induces loss of M2 macrophages with subsequent loss of FXIII-A synthesis. The loss of cellular FXIII-A may impact migration and phagocytosis, and hence limit pathogen eradication in UC.

Alpha-1 antitrypsin and granulocyte colony-stimulating factor as serum biomarkers of disease severity in ulcerative colitis.

BACKGROUND: Initial assessment of patients with ulcerative colitis (UC) is challenging and relies on apparent clinical symptoms and measurements of surrogate markers (e.g., C-reactive protein [CRP] or similar acute phase proteins). As CRP only reliably identifies patients with severe disease, novel biomarkers are currently needed for identification of patients with mild or moderate disease activity. Using a commercially available platform, we aimed at identifying serum biomarkers that are able to grade the disease severity. METHODS: Serum samples from 65 patients with UC with varying disease activity (Mayo score) and from 40 healthy controls were analyzed by multiplex enzyme-linked immunosorbent assay for 78 potential disease biomarkers. Using the statistical software SIMCA-P+ and GraphPad Prism, multivariate statistical analyses were conducted to identify a limited number of biomarkers to assess disease severity. RESULTS: Alpha-1 antitrypsin (AAT) differentiated between mild and moderate UC (area under the curve [AUC] = 0.79) with a sensitivity of 0.90 and a specificity of 0.70, thereby exceeding the predictive ability of CRP (AUC = 0.52). Combining alpha-1 antitrypsin and granulocyte colony-stimulating factor produced a predictive model with an AUC of 0.72 when differentiating mild and moderate UC, and an AUC of 0.96 when differentiating moderate and severe UC, the latter being as reliable as CRP. CONCLUSIONS: Alpha-1 antitrypsin is identified as a potential serum biomarker of mild-to-moderate disease activity in UC. With the ability to differentiate between mild, moderate, and severe stages of UC using a simple serum biomarker that is already commercially available, clinicians can initiate individualized treatment regimens at an earlier stage before endoscopic examinations are available.

Collagenous sprue: a coeliac disease look-alike with different treatment strategy.

Collagenous sprue is a rare clinicopathological condition of the small bowel. It is characterised by abnormal subepithelial collagen deposition and is typically associated with malabsorption, diarrhoea and weight loss. The clinical features of collagenous sprue often resemble those of coeliac disease and together with frequent histological findings like mucosal thinning and intraepithelial lymphocytosis the diagnosis may be hard to reach without awareness of this condition. While coeliac disease is treated using gluten restriction, collagenous sprue is, however, not improved by this intervention. In cases of diet-refractory 'coeliac disease' it is therefore essential to consider collagenous sprue to initiate treatment at an early stage to prevent the fibrotic progression. Here, we report a case of a 78-year-old man with collagenous sprue and present the clinical and histological manifestations as well as the successful treatment course that he underwent.

Proximal collagenous gastroenteritides: clinical management. A systematic review.

AIM: While collagenous colitis represents the most common form of the collagenous gastroenteritides, the collagenous entities affecting the proximal part of the gastrointestinal tract are much less recognized and possibly overlooked. The aim was to summarize the latest information through a systematic review of collagenous gastritis, collagenous sprue, and a combination thereof. METHOD: The search yielded 117 studies which were suitable for inclusion in the systematic review. Excluding repeated cases, 89 case reports and 28 case series were reported, whereas no prospective studies with or without control groups were identified. Further, no randomized, controlled trials were identified. The total number of patients with proximal collagenous gastroenteritides reported was 330. RESULTS: An overview of clinical presentations, prognosis, pathophysiology and histopathology, as well as management of these disorders is presented. The prognosis of both collagenous gastritis and sprue seems not to be as dismal as considered previously. Data point to involvement of immune or autoimmune mechanisms potentially driven by luminal antigens initiating the fibroinflammatory condition. CONCLUSIONS: To reach the diagnosis it is recommended that biopsies are obtained during gastroduodenoscopies. Therapies with anti-secretory strategies, glucocorticoids, and in some cases iron supplementation are suggested, although rational treatment options from randomized, controlled trials do not exist for these rare or even overlooked disorders.

Inflammatory pathways of importance for management of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a group of chronic disorders of the gastrointestinal tract comprising Crohn's disease (CD) and ulcerative colitis (UC). Their etiologies are unknown, but they are characterised by an imbalanced production of pro-inflammatory mediators, e.g., tumor necrosis factor (TNF)-α, as well as increased recruitment of leukocytes to the site of inflammation. Advantages in understanding the role of the inflammatory pathways in IBD and an inadequate response to conventional therapy in a large portion of patients, has over the last two decades lead to new therapies which includes the TNF inhibitors (TNFi), designed to target and neutralise the effect of TNF-α. TNFi have shown to be efficient in treating moderate to severe CD and UC. However, convenient alternative therapeutics targeting other immune pathways are needed for patients with IBD refractory to conventional therapy including TNFi. Indeed, several therapeutics are currently under development, and have shown success in clinical trials. These include antibodies targeting and neutralising interleukin-12/23, small pharmacologic Janus kinase inhibitors designed to block intracellular signaling of several pro-inflammatory cytokines, antibodies targeting integrins, and small anti-adhesion molecules that block adhesion between leukocytes and the intestinal vascular endothelium, reducing their infiltration into the inflamed mucosa. In this review we have elucidated the major signaling pathways of clinical importance for IBD therapy and highlighted the new promising therapies available. As stated in this paper several new treatment options are under development for the treatment of CD and UC, however, no drug fits all patients. Hence, optimisations of treatment regimens are warranted for the benefit of the patients either through biomarker establishment or other rationales to maximise the effect of the broad range of mode-of-actions of the present and future drugs in IBD.

Detection of active matriptase using a biotinylated chloromethyl ketone peptide.

Matriptase is a member of the family of type II transmembrane serine proteases that is essential for development and maintenance of several epithelial tissues. Matriptase is synthesized as a single-chain zymogen precursor that is processed into a two-chain disulfide-linked form dependent on its own catalytic activity leading to the hypothesis that matriptase functions at the pinnacle of several protease induced signal cascades. Matriptase is usually found in either its zymogen form or in a complex with its cognate inhibitor hepatocyte growth factor activator inhibitor 1 (HAI-1), whereas the active non-inhibited form has been difficult to detect. In this study, we have developed an assay to detect enzymatically active non-inhibitor-complexed matriptase by using a biotinylated peptide substrate-based chloromethyl ketone (CMK) inhibitor. Covalently CMK peptide-bound matriptase is detected by streptavidin pull-down and subsequent analysis by Western blotting. This study presents a novel assay for detection of enzymatically active matriptase in living human and murine cells. The assay can be applied to a variety of cell systems and species.

Transplantation of expanded fetal intestinal progenitors contributes to colon regeneration after injury.

Regeneration and homeostasis in the adult intestinal epithelium is driven by proliferative resident stem cells, whose functional properties during organismal development are largely unknown. Here, we show that human and mouse fetal intestine contains proliferative, immature progenitors, which can be expanded in vitro as Fetal Enterospheres (FEnS). A highly similar progenitor population can be established during intestinal differentiation of human induced pluripotent stem cells. Established cultures of mouse fetal intestinal progenitors express lower levels of Lgr5 than mature progenitors and propagate in the presence of the Wnt antagonist Dkk1, and new cultures can be induced to form mature intestinal organoids by exposure to Wnt3a. Following transplantation in a colonic injury model, FEnS contribute to regeneration of colonic epithelium by forming epithelial crypt-like structures expressing region-specific differentiation markers. This work provides insight into mechanisms underlying development of the mammalian intestine and points to future opportunities for patient-specific regeneration of the digestive tract.

ERK controls epithelial cell death receptor signalling and cellular FLICE-like inhibitory protein (c-FLIP) in ulcerative colitis.

Intestinal epithelial cell (IEC) death signalling through the Fas receptor is impaired in active ulcerative colitis (UC). This is possibly due to the activation of cytoprotective pathways resulting in limitation of the tissue injury secondary to inflammation. We hypothesized that inflammatory signalling like the nuclear factor (NF)-κB or mitogen activated protein kinase (MAPK) pathways could be involved in (a) the modification of Fas mediated apoptosis responses and (b) the regulation of the Fas receptor inhibitor cellular FLICE-like inhibitory protein (c-FLIP). Phospho-ERK was upregulated in IECs in active UC as well as IECs exposed to pro-inflammatory cytokines in vitro. Similarly, the short form of c-FLIP (c-FLIPS) was found to be upregulated in IECs from patients with active UC. c-FLIPS was the main splice variant found in both HT-29 cells and primary human IECs. Both splice variants were induced by TNF-α, IL-1ß and IFN-γ, while IL-10 induced c-FLIPL expression; TNF-α also induced c-FLIPS in primary IECs. Inhibition of NF-κB, JNK and p38 pathways did not affect c-FLIP expression, whereas ERK inhibition by MEK1 RNA silencing and pharmacologic inhibitors decreased c-FLIPS expression. Similarly, ERK - but not NF-κB - inhibited Fas ligand and TNF-α-mediated apoptosis responses in both cell line experiments and primary IECs. The present study identifies the MEK-ERK pathway as a major regulator of apoptosis in IECs during flares of UC and an inducer of c-FLIPS. The results explain the resistance to receptor mediated epithelial apoptosis in active UC. Oncogenic c-FLIP could promote propagation of DNA-damaged IECs and contribute to cancer development in UC.