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.

Polyp size measurement during colonoscopy using a virtual scale: variability and systematic differences.

BACKGROUND: Accurate polyp size measurement is important for polyp risk stratification and decision-making regarding polypectomy and surveillance. Recently, a virtual scale (VS) function has been developed that allows polyp size measurement through projection of an adaptive VS onto colorectal polyps during real-time endoscopy. We aimed to evaluate the VS in terms of variability and systematic differences. METHODS: We conducted a video-based study with 120 colorectal polyps, measured by eight dedicated colorectal gastroenterologists (experts) and nine gastroenterology residents following endoscopy training (trainees). Three endoscopic measurement methods were compared: (1) visual, (2) snare and (3) VS measurement. We evaluated the method-specific variance (as measure of variability) in polyp size measurements and systematic differences between these methods. RESULTS: Variance in polyp size measurements was significantly lower for VS measurements compared to visual and snare measurements for both experts (0.52 vs. 1.59 and 1.96, p<0.001) and trainees (0.59 vs. 2.21 and 2.53, p<0.001). VS measurement resulted in a higher percentage of polyps assigned to the same size category by all endoscopists compared to visual and snare measurements (experts: 69% vs. 55% and 59%; trainees: 67% vs. 51% and 47%) and reduced the maximum difference between individual endoscopists regarding the percentage of polyps assigned to the >10 mm size category (experts: 1.7% vs. 10.0% and 5.0%; trainees: 2.5% vs. 6.7% and 11.7%). Systematic differences between methods were <0.5 mm. Conclusions Use of the VS leads to lower polyp size measurement variability and more uniform polyp sizing by individual endoscopists compared to visual and snare measurements.

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.

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.

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.

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.

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 Human Colon Is More Resistant to Ischemia-reperfusion-induced Tissue Damage Than the Small Intestine: An Observational Study.

OBJECTIVE: Aim of this study was to draw comparisons between human colonic and jejunal ischemia-reperfusion sequelae in a human in vivo experimental model. BACKGROUND: In patients, colonic ischemia-reperfusion generally has a milder course than small intestinal ischemia-reperfusion. It is unclear which pathophysiologic processes are responsible for this difference. METHODS: In 10 patients undergoing colonic surgery and 10 patients undergoing pancreaticoduodenectomy, 6 cm colon or jejunum was isolated and exposed to 60 minutes ischemia followed by various reperfusion periods. Morphology (hematoxylin and eosin), apoptosis (M30), tight junctions (zonula occludens 1), and neutrophil influx (myeloperoxidase) were assessed using immunohistochemistry. Quantitative polymerase chain reaction and enzyme-linked immunosorbent assay were performed for interleukin-6 and tumor necrosis factor-α. RESULTS: Hematoxylin and eosin staining revealed intact colonic epithelial lining, but extensive damage in jejunal villus tips after 60 minutes ischemia. After reperfusion, the colonic epithelial lining was not affected, whereas the jejunal epithelium was seriously damaged. Colonic apoptosis was limited to scattered cells in surface epithelium, whereas apoptosis was clearly observed in jejunal villi and crypts, (42 times more M30 positivity compared with colon, P < 0.01). Neutrophil influx and increased tumor necrosis factor-α mRNA expression were observed in jejunum after 30 and 120 minutes of reperfusion (P < 0.05). Interleukin-6 mRNA expression was increased in jejunum after 120 minutes of reperfusion (3.6-fold increase, P < 0.05), whereas interleukin-6 protein expression was increased in both colon (1.5-fold increase, P < 0.05) and small intestine (1.5-fold increase, P < 0.05) after 30 and 120 minutes of reperfusion. CONCLUSIONS: Human colon is less susceptible to IR-induced tissue injury than small intestine.

Plasma intestinal fatty acid-binding protein levels correlate with morphologic epithelial intestinal damage in a human translational ischemia-reperfusion model.

BACKGROUND AND AIM: Intestinal fatty acid-binding protein (I-FABP) is a useful marker in the detection of intestinal ischemia. However, more insight into the test characteristics of I-FABP release is needed. This study aimed to investigate the relationship between plasma I-FABP levels and the severity of ischemic mucosal injury, and define the clinical usefulness of systemic I-FABP following ischemia. METHODS: In a human experimental model, 6 cm of the jejunum, to be removed for surgical reasons, was selectively exposed to either 15, 30, or 60 minutes of ischemia (I) followed by 30 and 120 minutes of reperfusion (R). Blood and tissue was sampled at all time points. Arteriovenous (V-A) concentration differences of I-FABP were measured. Tissue sections were stained with hematoxylin/eosin, and villus height was measured to score epithelial damage. RESULTS: Histologic analysis showed only minor reversible intestinal damage following 15 I and 30 I; however, severe irreversible epithelial damage was observed in the jejunum exposed to 60 I. I-FABP V-A differences paralleled the degree of tissue damage over time [7.79 (± 1.8) ng/mL, 128.6 (± 44.2) ng/mL, 463.3 (± 139.8) ng/mL for 15 I, 30 I and 60 I, respectively]. A good correlation was found between histologic epithelial damage and V-A I-FABP (r=-0.82, P<0.001). Interestingly, systemic I-FABP levels were significantly increased after 60 I of this short small intestinal segment. CONCLUSIONS: This study demonstrates the relationship between the duration of ischemia and the extent of tissue damage, which is reflected by I-FABP V-A plasma levels. In addition, systemic I-FABP levels appear valuable in detecting irreversible intestinal ischemia-reperfusion damage.

Ischaemia-induced mucus barrier loss and bacterial penetration are rapidly counteracted by increased goblet cell secretory activity in human and rat colon.

OBJECTIVE: Colonic ischaemia is frequently observed in clinical practice. This study provides a novel insight into the pathophysiology of colon ischaemia/reperfusion (IR) using a newly developed human and rat experimental model. DESIGN: In 10 patients a small part of colon that had to be removed for surgical reasons was isolated and exposed to 60 min of ischaemia (60I) with/without different periods of reperfusion (30R and 60R). Tissue not exposed to IR served as control. In rats, colon was exposed to 60I, 60I/30R, 60I/120R or 60I/240R (n=7 per group). The tissue was snap-frozen or fixed in glutaraldehyde, formalin or methacarn fixative. Mucins were stained with Periodic Acid Schiff/Alcian Blue (PAS/AB) and MUC2/Dolichos biflorus agglutinin (DBA). Bacteria were studied using electron microscopy (EM) and fluorescent in situ hybridisation (FISH). Neutrophils were studied using myeloperoxidase staining. qPCR was performed for MUC2, interleukin (IL)-6, IL-1ß and tumour necrosis factor α. RESULTS: In rats, PAS/AB and MUC2/DBA staining revealed mucus layer detachment at ischaemia which was accompanied by bacterial penetration (in EM and FISH). Human and rat studies showed that, simultaneously, goblet cell secretory activity increased. This was associated with expulsion of bacteria from the crypts and restoration of the mucus layer at 240 min of reperfusion. Inflammation was limited to minor influx of neutrophils and increased expression of proinflammatory cytokines during reperfusion. CONCLUSIONS: Colonic ischaemia leads to disruption of the mucus layer facilitating bacterial penetration. This is rapidly counteracted by increased secretory activity of goblet cells, leading to expulsion of bacteria from the crypts as well as restoration of the mucus barrier.

Mapping the complexity and diversity of tertiary lymphoid structures in primary and peritoneal metastatic gastric cancer.

BACKGROUND: Tertiary lymphoid structures (TLSs) are thought to stimulate antitumor immunity and positively impact prognosis and response to immune checkpoint blockade. In gastric cancers (GCs), however, TLSs are predominantly found in GC with poor prognosis and limited treatment response. We, therefore, hypothesize that immune cell composition and function of TLS depends on tumor location and the tumor immune environment. METHODS: Spatial transcriptomics and immunohistochemistry were used to characterize the phenotype of CD45+ immune cells inside and outside of TLS using archival resection specimens from GC primary tumors and peritoneal metastases. RESULTS: We identified significant intrapatient and interpatient diversity of the cellular composition and maturation status of TLS in GC. Tumor location (primary vs metastatic site) accounted for the majority of differences in TLS maturity, as TLS in peritoneal metastases were predominantly immature. This was associated with higher levels of tumor-infiltrating macrophages and Tregs and less plasma cells compared with tumors with mature TLS. Furthermore, mature TLSs were characterized by overexpression of antitumor immune pathways such as B cell-related pathways, MHC class II antigen presentation while immature TLS were associated with protumor pathways, including T cell exhaustion and enhancement of DNA repair pathways in the corresponding cancer. CONCLUSION: The observation that GC-derived peritoneal metastases often contain immature TLS which are associated with immune suppressive regulatory tumor-infiltrating leucocytes, is in keeping with the lack of response to immune checkpoint blockade and the poor prognostic features of peritoneal metastatic GC, which needs to be taken into account when optimizing immunomodulatory strategies for metastatic GC.

T-cell responses in colorectal peritoneal metastases are recapitulated in a humanized immune system mouse model.

Background: The occurrence of peritoneal metastasis (PM) in patients with colorectal cancer (CRC) has a dismal prognosis. There is often limited response to systemic- and immunotherapy, even in microsatellite unstable (MSI) CRC. To overcome therapy resistance, it is critical to understand local immune environment in the peritoneal cavity, and to develop models to study anti-tumor immune responses. Here, we defined the peritoneal immune system (PerIS) in PM-CRC patients and evaluate the pre-clinical potential of a humanized immune system (HIS) mouse model for PM-CRC. Methods: We studied the human PerIS in PM-CRC patients (n=20; MSS 19/20; 95%) and in healthy controls (n=3). HIS mice (NODscid gamma background; n=18) were generated, followed by intraperitoneal injection of either saline (HIS control; n=3) or human MSS/MSI CRC cell lines HUTU80, MDST8 and HCT116 (HIS-PM, n=15). Immune cells in peritoneal fluid and peritoneal tumors were analyzed using cytometry by time of flight (CyTOF). Results: The human and HIS mouse homeostatic PerIS was equally populated by NK cells and CD4+- and CD8+ T cells, however differences were observed in macrophage and B cell abundance. In HIS mice, successful peritoneal engraftment of both MSI and MSS tumors was observed (15/15; 100%). Both in human PM-CRC and in the HIS mouse PM-CRC model, we observed that MSS PM-CRC triggered a CD4+ Treg response in the PerIS, while MSI PM-CRC drives CD8+ TEMs responses. Conclusion: In conclusion, T cell responses in PM-CRC in HIS mice mirror those in human PM-CRC, making this model suitable to study antitumor T cell responses in PM-CRC.

New insights in intestinal ischemia-reperfusion injury: implications for intestinal transplantation.

PURPOSE OF REVIEW: Ischemia-reperfusion injury is inevitable during intestinal transplantation and can negatively affect the transplant outcome. Here, an overview is provided of the recent advances in the pathophysiological mechanisms of intestinal ischemia-reperfusion injury and how this may impact graft survival. RECENT FINDINGS: The intestine hosts a wide range of microorganisms and its mucosa is heavily populated by immune cells. Intestinal ischemia-reperfusion results in the disruption of the epithelial lining, affecting also protective Paneth cells (antimicrobials) and goblet cells (mucus), and creates a more hostile intraluminal microenvironment. Consequently, both damage-associated molecular patterns as well as pathogen-associated molecular patterns are released from injured tissue and exogenous microorganisms, respectively. These 'danger' signals may synergistically activate the innate immune system. Exaggerated innate immune responses, involving neutrophils, mast cells, platelets, dendritic cells, as well as Toll-like receptors and complement proteins, may shape the adaptive T-cell response, thereby triggering the destructive alloimmune response toward the graft and resulting in transplant rejection. SUMMARY: Innate immune activation as a consequence of ischemia-reperfusion injury may compromise engraftment of the intestine. More dedicated research is required to further establish this concept in man and to design more effective therapeutic strategies to better tolerize intestinal grafts.

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.

Physiology and pathophysiology of splanchnic hypoperfusion and intestinal injury during exercise: strategies for evaluation and prevention.

Physical exercise places high demands on the adaptive capacity of the human body. Strenuous physical performance increases the blood supply to active muscles, cardiopulmonary system, and skin to meet the altered demands for oxygen and nutrients. The redistribution of blood flow, necessary for such an increased blood supply to the periphery, significantly reduces blood flow to the gut, leading to hypoperfusion and gastrointestinal (GI) compromise. A compromised GI system can have a negative impact on exercise performance and subsequent postexercise recovery due to abdominal distress and impairments in the uptake of fluid, electrolytes, and nutrients. In addition, strenuous physical exercise leads to loss of epithelial integrity, which may give rise to increased intestinal permeability with bacterial translocation and inflammation. Ultimately, these effects can deteriorate postexercise recovery and disrupt exercise training routine. This review provides an overview on the recent advances in our understanding of GI physiology and pathophysiology in relation to strenuous exercise. Various approaches to determine the impact of exercise on the individual athlete's GI tract are discussed. In addition, we elaborate on several promising components that could be exploited for preventive interventions.

Level of activation of the unfolded protein response correlates with Paneth cell apoptosis in human small intestine exposed to ischemia/reperfusion.

BACKGROUND & AIMS: In the intestine, Paneth cells participate in the innate immune response. Their highly secretory function makes them susceptible to environmental conditions that cause endoplasmic reticulum (ER) stress. We investigated whether intestinal ischemia/reperfusion (I/R) induces ER stress, thereby activating the unfolded protein response (UPR), and whether excessive UPR activation affects Paneth cells. In addition, we investigated the consequences of Paneth cell compromise during physical barrier damage. METHODS: Jejunal I/R was studied using a human experimental model (n = 30 patients). Activation of the UPR was assessed using immunofluorescence for binding protein and quantitative polymerase chain reaction analyses for C/EBP homologous protein (CHOP), growth arrest and DNA-damage inducible protein 34 (GADD34), and X-box binding protein 1 (XBP1) splicing. Paneth cell apoptosis was assessed by double staining for lysozyme and M30. Male Sprague-Dawley rats underwent either intestinal I/R to investigate UPR activation and Paneth cell apoptosis, or hemorrhagic shock with or without intraperitoneal administration of dithizone, to study consequences of Paneth cell compromise during physical intestinal damage. In these animals, bacterial translocation and circulating tumor necrosis factor-α and interleukin-6 levels were assessed. RESULTS: In jejunum samples from humans and rats, I/R activated the UPR and resulted in Paneth cell apoptosis. Apoptotic Paneth cells showed signs of ER stress, and Paneth cell apoptosis correlated with the extent of ER stress. Apoptotic Paneth cells were shed into the crypt lumen, significantly lowering their numbers. In rats, Paneth cell compromise increased bacterial translocation and inflammation during physical intestinal damage. CONCLUSIONS: ER stress-induced Paneth cell apoptosis contributes to intestinal I/R-induced bacterial translocation and systemic inflammation.

Starvation compromises Paneth cells.

Lack of enteral feeding, with or without parenteral nutritional support, is associated with increased intestinal permeability and translocation of bacteria. Such translocation is thought to be important in the high morbidity and mortality rates of patients who receive nothing by mouth. Recently, Paneth cells, important constituents of innate intestinal immunity, were found to be crucial in host protection against invasion of both commensal and pathogenic bacteria. This study investigates the influence of food deprivation on Paneth cell function in a mouse starvation model. Quantitative PCR showed significant decreases in mRNA expression of typical Paneth cell antimicrobials, lysozyme, cryptdin, and RegIIIγ, in ileal tissue after 48 hours of food deprivation. Protein expression levels of lysozyme and RegIIIγ precursor were also significantly diminished, as shown by Western blot analysis and IHC. Late degenerative autophagolysosomes and aberrant Paneth cell granules in starved mice were evident by electron microscopy, Western blot analysis, and quantitative PCR. Furthermore, increased bacterial translocation to mesenteric lymph nodes coincided with Paneth cell abnormalities. The current study demonstrates the occurrence of Paneth cell abnormalities during enteral starvation. Such changes may contribute to loss of epithelial barrier function, causing the apparent bacterial translocation in enteral starvation.

Rapid lamina propria retraction and zipper-like constriction of the epithelium preserves the epithelial lining in human small intestine exposed to ischaemia-reperfusion.

To ensure a sufficient barrier between a host and noxious luminal content, the intestinal epithelium must be equipped with efficient mechanisms to limit damage to the epithelial lining. Using a human model, we were able to investigate these mechanisms in the human gut exposed to ischaemia-reperfusion (IR) over the time course of 150 min. In 10 patients a part of jejunum, to be removed for surgical reasons, was selectively exposed to IR. Control tissue was collected, as well as tissue exposed to 30 min of ischaemia with 0, 30 or 120 min of reperfusion. Haematoxylin/eosin staining demonstrated the appearance of subepithelial spaces following 30 min of ischaemia, while the epithelial lining remained intact at this stage. Western blot for myosin light chain kinase (MLCK) revealed a significant increase in protein levels after ischaemia (p < 0.01), and selective staining of MLCK and phosphorylated MLC (pMLC) in lamina propria muscle fibres indicated that appearance of subepithelial spaces was a consequence of active villus contraction. Early during reperfusion, accumulation of pMLC was observed exclusively at the basal side of enterocytes that had lost contact with the collagen-IV-positive basement membrane. These epithelial sheets were pulled together like a zipper, even before these cells were shed. This constriction, verified by increased F-actin and pMLC double staining, accounted for a 45% reduction in virtual wound surface (p < 0.001) at 30 min of reperfusion. In addition, these mechanisms were involved in resealing remaining small epithelial defects, resulting in a fully restored epithelial lining within 120 min of reperfusion. In conclusion, we show in a human in vivo model that the human jejunum has the ability to preserve the epithelial lining during intestinal IR by rapid lamina propria contraction and zipper-like constriction of epithelial cells that are to be shed into the lumen. These newly described phenomena limit exposure to noxious luminal content.

Reduced Paneth cell antimicrobial protein levels correlate with activation of the unfolded protein response in the gut of obese individuals.

The intestinal microbiota is increasingly acknowledged to play a crucial role in the development of obesity. A shift in intestinal microbiota composition favouring the presence of Firmicutes over Bacteroidetes has been observed in obese subjects. A similar shift has been reported in mice with deficiency of active Paneth cell α-defensins. We aimed at investigating changes in Paneth cell antimicrobial levels in the gut of obese subjects. Next, we studied activation of the unfolded protein response (UPR) as a possible mechanism involved in altered Paneth cell function. Paneth cell numbers were counted in jejunal sections of 15 severely obese (BMI > 35) and 15 normal weight subjects. Expression of Paneth cell antimicrobials human α-defensin 5 (HD5) and lysozyme were investigated using immunohistochemistry, qPCR, and western blot. Activation of the UPR was assessed with western blot. Severely obese subjects showed decreased protein levels of both HD5 and lysozyme, while Paneth cell numbers were unchanged. Lysozyme protein levels correlated inversely with BMI. Increased expression of HD5 (DEFA5) and lysozyme (LYZ) transcripts in the intestine of obese subjects prompted us to investigate a possible translational block caused by UPR activation. Binding protein (BiP) and activating transcription factor 4 (ATF4) levels were increased, confirming activation of the UPR in the gut of obese subjects. Furthermore, levels of both proteins correlated with BMI. Involvement of the UPR in the lowered antimicrobial protein levels in obese subjects was strongly suggested by a negative correlation between BiP levels and lysozyme levels. Additionally, indications of ER stress were apparent in Paneth cells of obese subjects. Our findings provide the first evidence for altered Paneth cell function in obesity, which may have important implications for the obesity-associated shift in microbiota composition. In addition, we show activation of the UPR in the intestine of obese subjects, which may underlie the observed Paneth cell compromise.