Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

Intelectin-1 binds and alters the localization of the mucus barrier-modifying bacterium Akkermansia muciniphila.

Intelectin-1 (ITLN1) is a lectin secreted by intestinal epithelial cells (IECs) and upregulated in human ulcerative colitis (UC). We investigated how ITLN1 production is regulated in IECs and the biological effects of ITLN1 at the host-microbiota interface using mouse models. Our data show that ITLN1 upregulation in IECs from UC patients is a consequence of activating the unfolded protein response. Analysis of microbes coated by ITLN1 in vivo revealed a restricted subset of microorganisms, including the mucolytic bacterium Akkermansia muciniphila. Mice overexpressing intestinal ITLN1 exhibited decreased inner colonic mucus layer thickness and closer apposition of A. muciniphila to the epithelial cell surface, similar to alterations reported in UC. The changes in the inner mucus layer were microbiota and A. muciniphila dependent and associated with enhanced sensitivity to chemically induced and T cell-mediated colitis. We conclude that by determining the localization of a select group of bacteria to the mucus layer, ITLN1 modifies this critical barrier. Together, these findings may explain the impact of ITLN1 dysregulation on UC pathogenesis.

Real-world experience of switching from intravenous to subcutaneous vedolizumab maintenance treatment for inflammatory bowel diseases.

BACKGROUND: Subcutaneous (SC) vedolizumab is effective in inflammatory bowel diseases (IBD) when administered after induction with two infusions. AIM: To assess the effectiveness, safety and pharmacokinetics of a switch from intravenous (IV) to SC maintenance vedolizumab in patients with IBD METHODS: In this prospective cohort study, patients with IBD who had ≥4 months IV vedolizumab were switched to SC vedolizumab. We studied the time to discontinuation of SC vedolizumab, adverse events (AEs), changes in clinical and biochemical outcomes and vedolizumab concentrations at baseline, and weeks 12 and 24. RESULTS: We included 82 patients with Crohn's disease (CD) and 53 with ulcerative colitis (UC). Eleven (13.4%) patients with CD and five (9.4%) with UC discontinued SC vedolizumab after a median of 18 (IQR 8-22) and 6 weeks (IQR 5-10), respectively. Four patients with CD switched to a different drug due to loss of response, nine switched back to IV vedolizumab due to adverse events, and three due to needle fear. Common AEs were injection site reactions (n = 15) and headache (n = 6). Median clinical and biochemical disease activity remained stable after the switch. Median serum vedolizumab concentrations increased from 19 µg/ml at the time of the switch to 31 µg/ml 12 weeks after the switch (p < 0.005). CONCLUSIONS: Switching from IV to SC vedolizumab maintenance treatment is effective in patients with CD or UC. However, 9% of patients were switched back to IV vedolizumab due to adverse events or fear of needles.

Emerging therapies for ulcerative colitis.

INTRODUCTION: Despite advances in the medical management of ulcerative colitis (UC), a subgroup of patients does not respond to currently available therapies. A number of novel drugs are in late stages of clinical development or have recently received regulatory approval for UC. AREAS COVERED: This review focuses on three drug classes that have recently been approved or are awaiting approval for UC: antibodies against interleukin (IL)-23, sphingosine-1-phosphate receptor (S1PR) modulators, and selective inhibitors of Janus kinases (JAK). We provide an overview of their mechanism of action and summarize available evidence for their efficacy and safety. Finally, we discuss expected future challenges in UC management. EXPERT OPINION: The evaluated drugs have demonstrated efficacy with an acceptable safety profile. IL-23 antagonists appear to be safest with very few (serious) adverse events, while the use of S1PR modulators or JAK inhibitors has been associated with infectious and cardiovascular/thromboembolic events, albeit in low numbers. Although advances in drug development are promising, there is an urgent need for (validated) biomarkers to guide rational treatment selection. The scarcity of head-to-head trials also complicates comparisons between available drugs. Breaking the therapeutic ceiling of efficacy in UC will require marked advances in management extending well beyond drug development.

The Role of the Lymphatic System in the Pathogenesis and Treatment of Inflammatory Bowel Disease.

Although the number of therapeutic options for the treatment of inflammatory bowel disease (IBD) has increased in recent years, patients suffer from decreased quality of life due to non-response or loss of response to the currently available treatments. An increased understanding of the disease's etiology could provide novel insights for treatment strategies in IBD. Lymphatic system components are generally linked to immune responses and presumably related to inflammatory diseases pathophysiology. This review aims to summarize findings on immune-mediated mechanisms in lymphoid tissues linked with IBD pathogenesis and (potential) novel treatments. Enhanced innate and adaptive immune responses were observed in mesenteric lymph nodes (MLNs) and other lymphoid structures, such as Peyer's patches, in patients with IBD and in animal models. Furthermore, the phenomenon of lymphatic obstruction in the form of granulomas in MLNs and lymphatic vessels correlates with disease activity. There is also evidence that abnormalities in the lymphatic stromal components and lymph node microbiome are common in IBD and could be exploited therapeutically. Finally, novel agents targeting lymphocyte trafficking have been added to the treatment armamentarium in the field of IBD. Overall, gut-associated lymphoid tissue plays a key role in IBD immunopathogenesis, which could offer novel therapeutic targets.

Temporal Transcript Profiling Identifies a Role for Unfolded Protein Stress in Human Gut Ischemia-Reperfusion Injury.

BACKGROUND & AIMS: Intestinal ischemia-reperfusion injury is a serious and life-threatening condition. A better understanding of molecular mechanisms related to intestinal ischemia-reperfusion injury in human beings is imperative to find therapeutic targets and improve patient outcome. METHODS: First, the in vivo dynamic modulation of mucosal gene expression of the ischemia-reperfusion-injured human small intestine was studied. Based on functional enrichment analysis of the changing transcriptome, one of the predominantly regulated pathways was selected for further investigation in an in vitro human intestinal organoid model. RESULTS: Ischemia-reperfusion massively changed the transcriptional landscape of the human small intestine. Functional enrichment analysis based on gene ontology and pathways pointed to the response to unfolded protein as a predominantly regulated process. In addition, regulatory network analysis identified hypoxia-inducing factor 1A as one of the key mediators of ischemia-reperfusion-induced changes, including the unfolded protein response (UPR). Differential expression of genes involved in the UPR was confirmed using quantitative polymerase chain reaction analysis. Electron microscopy showed signs of endoplasmic reticulum stress. Collectively, these findings point to a critical role for unfolded protein stress in intestinal ischemia-reperfusion injury in human beings. In a human intestinal organoid model exposed to hypoxia-reoxygenation, attenuation of UPR activation with integrated stress response inhibitor strongly reduced pro-apoptotic activating transcription factor 4 (ATF4)-CCAAT/enhancer-binding protein homologous protein (CHOP) signaling. CONCLUSIONS: Transcriptome analysis showed a crucial role for unfolded protein stress in the response to ischemia-reperfusion in human small intestine. UPR inhibition during hypoxia-reoxygenation in an intestinal organoid model suggests that downstream protein kinase R-like ER kinase (PERK) signaling may be a promising target to reduce intestinal ischemia-reperfusion injury. Microarray data are available in GEO (https://www.ncbi.nlm.nih.gov/gds, accession number GSE37013).

The Role of the Immune System in IBD-Associated Colorectal Cancer: From Pro to Anti-Tumorigenic Mechanisms.

Patients with inflammatory bowel disease (IBD) have increased incidence of colorectal cancer (CRC). IBD-associated cancer follows a well-characterized sequence of intestinal epithelial changes, in which genetic mutations and molecular aberrations play a key role. IBD-associated cancer develops against a background of chronic inflammation and pro-inflammatory immune cells, and their products contribute to cancer development and progression. In recent years, the effect of the immunosuppressive microenvironment in cancer development and progression has gained more attention, mainly because of the unprecedented anti-tumor effects of immune checkpoint inhibitors in selected groups of patients. Even though IBD-associated cancer develops in the background of chronic inflammation which is associated with activation of endogenous anti-inflammatory or suppressive mechanisms, the potential role of an immunosuppressive microenvironment in these cancers is largely unknown. In this review, we outline the role of the immune system in promoting cancer development in chronic inflammatory diseases such as IBD, with a specific focus on the anti-inflammatory mechanisms and suppressive immune cells that may play a role in IBD-associated tumorigenesis.

Overexpression of Transmembrane TNF Drives Development of Ectopic Lymphoid Structures in the Bone Marrow and B Cell Lineage Alterations in Experimental Spondyloarthritis.

TNF is important in immune-mediated inflammatory diseases, including spondyloarthritis (SpA). Transgenic (tg) mice overexpressing transmembrane TNF (tmTNF) develop features resembling human SpA. Furthermore, both tmTNF tg mice and SpA patients develop ectopic lymphoid aggregates, but it is unclear whether these contribute to pathology. Therefore, we characterized the lymphoid aggregates in detail and studied potential alterations in the B and T cell lineage in tmTNF tg mice. Lymphoid aggregates developed in bone marrow (BM) of vertebrae and near the ankle joints prior to the first SpA features and displayed characteristics of ectopic lymphoid structures (ELS) including presence of B cells, T cells, germinal centers, and high endothelial venules. Detailed flow cytometric analyses demonstrated more germinal center B cells with increased CD80 and CD86 expression, along with significantly more T follicular helper, T follicular regulatory, and T regulatory cells in tmTNF tg BM compared with non-tg controls. Furthermore, tmTNF tg mice exhibited increased IgA serum levels and significantly more IgA+ plasma cells in the BM, whereas IgA+ plasma cells in the gut were not significantly increased. In tmTNF tg × TNF-RI-/- mice, ELS were absent, consistent with reduced disease symptoms, whereas in tmTNF tg × TNF-RII-/- mice, ELS and clinical symptoms were still present. Collectively, these data show that tmTNF overexpression in mice results in osteitis and ELS formation in BM, which may account for the increased serum IgA levels that are also observed in human SpA. These effects are mainly dependent on TNF-RI signaling and may underlie important aspects of SpA pathology.

Evaluating the safety of two human experimental intestinal ischemia reperfusion models: A retrospective observational study.

BACKGROUND: We developed a jejunal and colonic experimental human ischemia-reperfusion (IR) model to study pathophysiological intestinal IR mechanisms and potential new intestinal ischemia biomarkers. Our objective was to evaluate the safety of these IR models by comparing patients undergoing surgery with and without in vivo intestinal IR. METHODS: A retrospective study was performed comparing complication rates and severity, based on the Clavien-Dindo classification system, in patients undergoing pancreatoduodenectomy with (n = 10) and without (n = 20 matched controls) jejunal IR or colorectal surgery with (n = 10) and without (n = 20 matched controls) colon IR. Secondary outcome parameters were operative time, blood loss, 90-day mortality and length of hospital stay. RESULTS: Following pancreatic surgery, 63% of the patients experienced one or more postoperative complications. There was no significant difference in incidence or severity of complications between patients undergoing pancreatic surgery with (70%) or without (60%, P = 0.7) jejunal IR. Following colorectal surgery, 60% of the patients experienced one or more postoperative complication. Complication rate and severity were similar in patients with (50%) and without (65%, P = 0.46) colonic IR. Operative time, amount of blood loss, postoperative C-reactive protein, length of hospital stay or mortality were equal in both intervention and control groups for jejunal and colon IR. CONCLUSION: This study showed that human experimental intestinal IR models are safe in patients undergoing pancreatic or colorectal surgery.

Females Are More Resistant to Ischemia-Reperfusion-induced Intestinal Injury Than Males: A Human Study.

BACKGROUND AND OBJECTIVE: Sex differences in responses to intestinal ischemia-reperfusion (IR) have been recognized in animal studies. We aimed to investigate sexual dimorphism in human small intestinal mucosal responses to IR. METHODS: In 16 patients (8 men and 8 women) undergoing pancreaticoduodenectomy, an isolated part of jejunum was subjected to IR. In each patient, intestinal tissue and blood was collected directly after 45 minutes of ischemia without reperfusion (45I-0R), after 30 minutes of reperfusion (45I-30R), and after 120 minutes of reperfusion (45I-120R), as well as a control sample not exposed to IR, to assess epithelial damage, unfolded protein response (UPR) activation, and inflammation. RESULTS: More extensive intestinal epithelial damage was observed in males compared to females. Intestinal fatty acid binding protein (I-FABP) arteriovenous (V-A) concentrations differences were significantly higher in males compared to females at 45I-0R (159.0 [41.0-570.5] ng/mL vs 46.9 [0.3-149.9] ng/mL). Male intestine showed significantly higher levels of UPR activation than female intestine, as well as higher number of apoptotic Paneth cells per crypt at 45I-30R (16.4% [7.1-32.1] vs 10.6% [0.0-25.4]). The inflammatory response in male intestine was significantly higher compared to females, with a higher influx of neutrophils per villus at 45I-30R (4.9 [3.1-12.0] vs 3.3 [0.2-4.5]) and a higher gene expression of TNF-α and IL-10 at 45I-120R. CONCLUSION: The human female small intestine seems less susceptible to IR-induced tissue injury than the male small intestine. Recognition of such differences could lead to the development of novel therapeutic strategies to reduce IR-associated morbidity and mortality.

Epithelial endoplasmic reticulum stress orchestrates a protective IgA response.

Immunoglobulin A (IgA) is the major secretory immunoglobulin isotype found at mucosal surfaces, where it regulates microbial commensalism and excludes luminal factors from contacting intestinal epithelial cells (IECs). IgA is induced by both T cell-dependent and -independent (TI) pathways. However, little is known about TI regulation. We report that IEC endoplasmic reticulum (ER) stress induces a polyreactive IgA response, which is protective against enteric inflammation. IEC ER stress causes TI and microbiota-independent expansion and activation of peritoneal B1b cells, which culminates in increased lamina propria and luminal IgA. Increased numbers of IgA-producing plasma cells were observed in healthy humans with defective autophagy, who are known to exhibit IEC ER stress. Upon ER stress, IECs communicate signals to the peritoneum that induce a barrier-protective TI IgA response.

Histopathology of human small intestinal and colonic ischemia-reperfusion: Experiences from human IR-models.

Intestinal ischemia-reperfusion (IR) injury is a frequent, but potentially life-threatening condition. Although much has been learned about its pathophysiology from animal IR models, the translation to the human setting is imperative for better understanding of its etiology. This could provide us with new insight into development of early detection and potential new therapeutic strategies. Over the past decade, we have studied the pathophysiology of human small intestinal and colonic ischemia-reperfusion (IR) in newly developed human in vivo IR models. In this review, we give an overview of new insights on the sequelae of human intestinal IR, with particular attention for the differences in histopathology between small intestinal and colonic IR.

New Insights Into the Regulation of Natural-Killer Group 2 Member D (NKG2D) and NKG2D-Ligands: Endoplasmic Reticulum Stress and CEA-Related Cell Adhesion Molecule 1.

Natural-killer group 2 member D (NKG2D) is a well-characterized activating receptor expressed by natural killer (NK) cells, NKT cells, activated CD8+ T cells, subsets of γδ+ T cells, and innate-like T cells. NKG2D recognizes multiple ligands (NKG2D-ligands) to mount an innate immune response against stressed, transformed, or infected cells. NKG2D-ligand surface expression is tightly restricted on healthy cells through transcriptional and post-transcriptional mechanisms, while transformed or infected cells express the ligands as a danger signal. Recent studies have revealed that unfolded protein response pathways during endoplasmic reticulum (ER) stress result in upregulation of ULBP-related protein via the protein kinase RNA-like ER kinase-activating factor 4-C/EBP homologous protein (PERK-ATF4-CHOP) pathway, which can be linked to the pathogenesis of autoimmune diseases. Transformed cells, however, possess mechanisms to escape NKG2D-mediated immune surveillance, such as upregulation of carcinoembryonic antigen (CEA)-related cell adhesion molecule 1 (CEACAM1), a negative regulator of NKG2D-ligands. In this review, we discuss mechanisms of NKG2D-ligand regulation, with a focus on newly discovered mechanisms that promote NKG2D-ligand expression on epithelial cells, including ER stress, and mechanisms that suppress NKG2D-ligand-mediated killing of cancer cells, namely by co-expression of CEACAM1.

Intestinal epithelial cell endoplasmic reticulum stress promotes MULT1 up-regulation and NKG2D-mediated inflammation.

Endoplasmic reticulum (ER) stress is commonly observed in intestinal epithelial cells (IECs) and can, if excessive, cause spontaneous intestinal inflammation as shown by mice with IEC-specific deletion of X-box-binding protein 1 (Xbp1), an unfolded protein response-related transcription factor. In this study, Xbp1 deletion in the epithelium (Xbp1ΔIEC ) is shown to cause increased expression of natural killer group 2 member D (NKG2D) ligand (NKG2DL) mouse UL16-binding protein (ULBP)-like transcript 1 and its human orthologue cytomegalovirus ULBP via ER stress-related transcription factor C/EBP homology protein. Increased NKG2DL expression on mouse IECs is associated with increased numbers of intraepithelial NKG2D-expressing group 1 innate lymphoid cells (ILCs; NK cells or ILC1). Blockade of NKG2D suppresses cytolysis against ER-stressed epithelial cells in vitro and spontaneous enteritis in vivo. Pharmacological depletion of NK1.1+ cells also significantly improved enteritis, whereas enteritis was not ameliorated in Recombinase activating gene 1-/-;Xbp1ΔIEC mice. These experiments reveal innate immune sensing of ER stress in IECs as an important mechanism of intestinal inflammation.

Human small intestine is capable of restoring barrier function after short ischemic periods.

AIM: To assess intestinal barrier function during human intestinal ischemia and reperfusion (IR). METHODS: In a human experimental model, 6 cm of jejunum was selectively exposed to 30 min of ischemia (I) followed by 30 and 120 min of reperfusion (R). A sham procedure was also performed. Blood and tissue was sampled at all-time points. Functional barrier function was assessed using dual-sugar absorption tests with lactulose (L) and rhamnose (R). Plasma concentrations of citrulline, an amino acid described as marker for enterocyte function were measured as marker of metabolic enterocytes restoration. Damage to the epithelial lining was assessed by immunohistochemistry for tight junctions (TJs), by plasma marker for enterocytes damage (I-FABP) and analyzed by electron microscopy (EM) using lanthanum nitrate as an electrondense marker. RESULTS: Plasma L/R ratio's were significantly increased after 30 min of ischemia (30I) followed by 30 min of reperfusion (30R) compared to control (0.75 ± 0.10 vs 0.20 ± 0.09, P < 0.05). At 120 min of reperfusion (120R), ratio's normalized (0.17 ± 0.06) and were not significantly different from control. Plasma levels of I-FABP correlated with plasma L/R ratios measured at the same time points (correlation: 0.467, P < 0.01). TJs staining shows distortion of staining at 30I. An intact lining of TJs was again observed at 30I120R. Electron microscopy analysis revealed disrupted TJs after 30I with paracellular leakage of lanthanum nitrate, which restored after 30I120R. Furthermore, citrulline concentrations closely paralleled the histological perturbations during intestinal IR. CONCLUSION: This study directly correlates histological data with intestinal permeability tests, revealing that the human gut has the ability of to withstand short episodes of ischemia, with morphological and functional recovery of the intestinal barrier within 120 min of reperfusion.

Cyclooxygenase-2 Is Essential for Colorectal Anastomotic Healing.

OBJECTIVE: To study the effects of COX-2 on colonic surgical wound healing. BACKGROUND: Cyclooxygenase-2 (COX-2) is a key enzyme in gastrointestinal homeostasis. COX-2 inhibitors have been associated with colonic anastomotic leakage. METHODS: Wildtype, COX-2 knockout and COX-2 heterozygous mice were subjected to a model of colonic anastomotic leakage, and were treated with vehicle, diclofenac, or prostaglandin E2 (PGE2), the most important COX-2 product in the intestine. We assessed anastomotic leakage, mortality, angiogenesis, and inflammation. Furthermore, we investigated the association between anastomotic leakage and a human polymorphism of the COX-2 gene resulting in low COX-2 levels. RESULTS: Diclofenac, a nonsteroidal anti-inflammatory drug inhibiting COX-2, increased anastomotic leakage compared to vehicle-treated mice (100% vs 25%, respectively). Similarly, 92% of COX-2-deficient mice developed anastomotic leakage (P = 0.003) compared to WT. PGE2 partly rescued this severe phenotype because only 46% of PGE2-administered COX-2 knockout mice developed anastomotic leakage (P = 0.02). This may be related to decreased neovascularization, because decreased CD31 staining, indicating less blood vessels, was observed in COX-2 mice (2 vessels/mm vs 6 vessels/mm in controls (P = 0.03)). This effect could partly be reversed by administration of PGE2 to COX-2 mice. No significant differences in inflammation were found. PTGS2-765G>C polymorphism in humans, associated with reduced COX-2 expression, was associated with higher anastomotic leakage rates. CONCLUSIONS: COX-2-induced PGE2 production is essential for intestinal wound healing after colonic surgery, possibly via its effects on angiogenesis. These data emphasize that COX-2 inhibitors should be avoided after colonic surgery, and administration of PGE2 might be favorable for a selection of patients.

The unfolded protein response in immunity and inflammation.

The unfolded protein response (UPR) is a highly conserved pathway that allows the cell to manage endoplasmic reticulum (ER) stress that is imposed by the secretory demands associated with environmental forces. In this role, the UPR has increasingly been shown to have crucial functions in immunity and inflammation. In this Review, we discuss the importance of the UPR in the development, differentiation, function and survival of immune cells in meeting the needs of an immune response. In addition, we review current insights into how the UPR is involved in complex chronic inflammatory diseases and, through its role in immune regulation, antitumour responses.

Life and death at the mucosal-luminal interface: New perspectives on human intestinal ischemia-reperfusion.

Intestinal ischemia is a frequently observed phenomenon. Morbidity and mortality rates are extraordinarily high and did not improve over the past decades. This is in part attributable to limited knowledge on the pathophysiology of intestinal ischemia-reperfusion (IR) in man, the paucity in preventive and/or therapeutic options and the lack of early diagnostic markers for intestinal ischemia. To improve our knowledge and solve clinically important questions regarding intestinal IR, we developed a human experimental intestinal IR model. With this model, we were able to gain insight into the mechanisms that allow the human gut to withstand short periods of IR without the development of severe inflammatory responses. The purpose of this review is to overview the most relevant recent advances in our understanding of the pathophysiology of human intestinal IR, as well as the (potential) future clinical implications.