Intestinal Inflammatory Conditions Research Articles

Bowel function and inflammation: Is motility the other side of the coin?

Digestion and intestinal absorption allow the body to sustain itself and are the emblematic functions of the bowel. On the flip side, functions also arise from its role as an interface with the environment. Indeed, the gut houses microorganisms, collectively known as the gut microbiota, which interact with the host, and is the site of complex immune activities. Its role in human pathology is complex and scientific evidence is progressively elucidating the functions of the gut, especially regarding the pathogenesis of chronic intestinal diseases and inflammatory conditions affecting various organs and systems. This editorial aims to highlight and relate the factors involved in the pathogenesis of intestinal and systemic inflammation.

Polyphenolic Characterization and Anti-Inflammatory Effect of In Vitro Digested Extracts of Echinacea purpurea L. Plant Parts in an Inflammatory Model of Human Colon Cells.

Echinacea purpurea L. (EP) preparations are globally popular herbal supplements known for their medicinal benefits, including anti-inflammatory activities, partly related to their phenolic composition. However, regarding their use for the management of inflammation-related intestinal diseases, the knowledge about the fate of orally ingested constituents throughout the human gastrointestinal tract and the exposition of in vitro digested extracts in relevant inflammatory models are unknown. This study investigated for the first time the impact of in vitro gastrointestinal digestion (INFOGEST) on the phenolic composition and anti-inflammatory properties of EP extracts from flowers (EF), leaves (EL), and roots (ER) on IL-1β-treated human colon-derived CCD-18Co cells. Among the seven hydroxycinnamic acids identified using HPLC-UV-MS/MS, chicoric and caftaric acids showed the highest concentrations in EL, followed by EF and ER, and all extracts exerted significant reductions in IL-6, IL-8, and PGE2 levels. After digestion, despite reducing the bioaccessibility of their phenolics, the anti-inflammatory effects were preserved for digested EL and, to a lesser extent, for EF, but not for digested ER. The lower phenolic content in digested EF and ER could explain these findings. Overall, this study emphasizes the potential of EP in alleviating intestinal inflammatory conditions and related disorders.

M6A Methylated Long Noncoding RNA LOC339803 Regulates Intestinal Inflammatory Response.

Cytokine mediated sustained inflammation increases the risk to develop different complex chronic inflammatory diseases, but the implicated mechanisms remain unclear. Increasing evidence shows that long noncoding RNAs (lncRNAs) play key roles in the pathogenesis of inflammatory disorders, while inflammation associated variants are described to affect their function or essential RNA modifications as N6-methyladenosine (m6A) methylation, increasing predisposition to inflammatory diseases. Here, the functional implication of the intestinal inflammation associated lncRNA LOC339803 in the production of cytokines by intestinal epithelial cells is described. Allele-specific m6A methylation is found to affect YTHDC1 mediated protein binding affinity. LOC339803-YTHDC1 interaction dictates chromatin localization of LOC339803 ultimately inducing the expression of NFκB mediated proinflammatory cytokines and contributing to the development of intestinal inflammation. These findings are confirmed using human intestinal biopsy samples from different intestinal inflammatory conditions and controls. Additionally, it is demonstrated that LOC339803 targeting can be a useful strategy for the amelioration of intestinal inflammation in vitro and ex vivo. Overall, the results support the importance of the methylated LOC339803 lncRNA as a mediator of intestinal inflammation, explaining genetic susceptibility and presenting this lncRNA as a potential novel therapeutic target for the treatment of inflammatory intestinal disorders.

Sleep Deprivation Impairs Intestinal Mucosal Barrier by Activating Endoplasmic Reticulum Stress in Goblet Cells

Sleep deficiency is associated with intestinal inflammatory conditions and is increasingly recognized as a public health concern worldwide. However, the effects of sleep deficiency on intestinal goblet cells (GCs), which play a major role in intestinal barrier formation, remain elusive. Herein, the effects of sleep deprivation on intestinal GCs were determined using a sleep-deprivation mouse model. Sleep deprivation impaired the intestinal mucosal barrier and decreased the expression of tight junction proteins. According to single-cell RNA sequencing and histologic assessments, sleep deprivation significantly reduced GC numbers and mucin protein levels in intestinal tissues. Furthermore, sleep deprivation initiated endoplasmic reticulum stress by activating transcription factor 6 and binding Ig protein. Treatment with melatonin, an endoplasmic reticulum stress regulator, significantly alleviated endoplasmic reticulum stress responses in intestinal GCs. In addition, melatonin increased the villus length, reduced the crypt depth, and restored intestinal barrier function in mice with sleep deprivation. Overall, the findings revealed that sleep deprivation could impair intestinal mucosal barrier integrity and GC function. Targeting endoplasmic reticulum stress could represent an ideal strategy for treating sleep deficiency-induced gastrointestinal disorders.

A33 EFFECTS OF THE NEXT GENERATION PROBIOTIC, AKKERMANSIA MUCINIPHILA, ON INTESTINAL INFLAMMATION AND BARRIER FUNCTION

Abstract Background Inflammatory bowel disease (IBD), characterised by chronic intestinal inflammation, is hypothesised to arise from the interplay between susceptibility genes, the immune system, environmental factors, and gut microbiota. Akkermansia muciniphila is a symbiotic bacterium that accounts for 1-5% of the human fecal microbiota. This microbe has been hailed as a next-generation probiotic, principally with regard to its plethora of beneficial host interactions, including the ability to influence mucin secretion and strengthen the intestinal barrier. Purpose Though a clear-cut role and mechanism by which A. muciniphila influences inflammatory conditions is unknown, evidence indicates this microbe is depleted in IBD, suggesting it may have protective effects that are lost in these conditions. Here, we investigate the role and mechanism of A. muciniphila in intestinal inflammation and its influence on intestinal barrier function by utilizing barrier-disrupting models of colitis. Method Across several experimental models of intestinal inflammation including the chemically-induced dextran sulphate sodium (DSS) model, the parasitic-based model of Trichuris muris infection, and the spontaneous Muc2-/- model, A.muciniphila was administered by oral gavage. Disease activity index, macroscopic scoring and histological scoring were all performed to assess the severity of intestinal inflammation. Various pro- and anti-inflammatory cytokines were assessed within colonic tissue using commercially available ELISA kits.To investigate the effects that A. muciniphila has on barrier function in the context of colitis, reverse transcriptase qPCR was used to explore several factors, including several TJPs, AMPs, and mucins. To analyse the composition of the microbiota and changes in diversity with A. muciniphila supplementation, 16S rRNA sequencing of fecal samples was performed. Result(s) Though only minor benefits were derived from this microbe in germ-free mice, in specific pathogen-free (SPF) mice, administration of pasteurized A. muciniphila in a DSS recovery model ameliorated inflammation severity and promoted recovery compared to controls. When gavaged prior to DSS administration, both live and pasteurized A. muciniphila failed to diminish inflammatory markers indicating minimal preventative effects. T. muris-infected SPF mice treated with live A. muciniphila showed increased levels of Th2 and anti-inflammatory cytokines, decreased worm burden, and enhanced levels of the mucin, Muc5ac, compared with those receiving control broth or pasteurized bacteria. Further, both live and pasteurized A. muciniphila ameliorated the severity of inflammation in a mucin 2 deficient (Muc2-/-) mouse model of spontaneous colitis, indicating that these protective effects are Muc2-independent. Conclusion(s) These observations provide us not only with an enhanced understanding of the role A. muciniphila plays in the pathogenesis of intestinal inflammatory conditions but also may fuel novel avenues of treatment for those with IBD. Please acknowledge all funding agencies by checking the applicable boxes below CIHR Disclosure of Interest None Declared

Ozoile Reduces the LPS-Induced Inflammatory Response in Colonic Epithelial Cells and THP-1 Monocytes

Inappropriate activation of immune functions in intestinal epithelial cells can lead to inflammation that is characterized also by infiltration into intestinal tissue of monocytes/macrophages. Current therapies for intestinal inflammation include anti-inflammatory, immunosuppressive and biological drugs. Ozoile (stable ozonides) has been reported to exert anti-inflammatory effects. However, ozonated oil has been used mainly for topical applications and no data are available about its effects on intestinal cells or immune cells. In this study, we evaluated Ozoile effects on human HT-29 colonic cells and THP-1 monocytic cells stimulated with LPS to induce inflammation. HT-29 and THP-1 cells were treated with LPS in the presence/absence of Ozoile for 4 h. Biomarkers of inflammation, some members of tight junctions and the adhesion molecule ICAM were assessed by qRT-PCR. Protein expression was analyzed by Western blotting. The release of TNF-α and IL-1β was measured by ELISA. In HT-29, Ozoile inhibited LPS-induced expression of TNF-α, IL-1β, ZO-1, CLDN1, NOS2 and MMP-2 and increased the expression of Nrf2 and SOD2 antioxidant proteins. In THP-1 cells, the LPS induction of TNF-α, IL-1β and ICAM was counteracted by Ozoile treatment. Our in vitro results demonstrate the effectiveness of Ozoile in reducing the inflammatory response in intestinal and monocytic cells. Further in vivo studies are necessary to confirm its possible use for intestinal inflammatory conditions.

Anti-inflammatory activity of a fixed combination of probiotics and herbal extract in an in-vitro model of intestinal inflammation by stimulating Caco-2 cells with LPS-conditioned THP-1 cells medium.

Inflammatory bowel disease (IBD) is an inflammatory condition of the gastrointestinal tract, characterized by chronic and relapsing immune system activation, often diagnosed in adolescence, with a rising incidence in pediatric populations. IBD results from altered interactions between gut microbes and the intestinal immune system which induce an aberrant immune response, thus anti-inflammatory or immunosuppressive therapies are generally used. Recent interest has been given to the identification of integrative and complementary approaches that could be able to restore and preserve the intestinal barrier function. In this work, we tested the effect of a fixed combination of probiotics and herbal extract (Colikind Gocce® [CKG], Schwabe Pharma, Egna-Neumarkt, Bolzano, Italy) in an in-vitro model of intestinal inflammation. Caco-2 cells stimulated with LPS-conditioned monocytes culture medium was used as a paradigm of intestinal inflammation. The possible effect of CKG in maintaining the homeostasis of the intestinal epithelial barrier was investigated by measurement of the trans-epithelial electrical resistance, the paracellular permeability, and the release of inflammatory cytokines (TNF-α, IL-8, and IL-10). Results obtained in this work demonstrated that CKG is able to prevent the impairment of intestinal barrier function induced by inflammation, ameliorating the transepithelial electrical resistance and the paracellular permeability of the Caco-2 monolayer; moreover, CKG is able to counteract the increased release of TNF-a and IL-8 induced by inflammatory stimulus, thus reducing the intestinal inflammation. This work underlines the protective effect of CKG on intestinal barrier, reducing the damages induced by inflammatory stimulus. This suggests CKG as an interesting product in the management of intestinal inflammatory conditions.

Tissue Niches Formed by Intestinal Mesenchymal Stromal Cells in Mucosal Homeostasis and Immunity.

The gastrointestinal tract is the largest mucosal surface in our body and accommodates the majority of the total lymphocyte population. Being continuously exposed to both harmless antigens and potentially threatening pathogens, the intestinal mucosa requires the integration of multiple signals for balancing immune responses. This integration is certainly supported by tissue-resident intestinal mesenchymal cells (IMCs), yet the molecular mechanisms whereby IMCs contribute to these events remain largely undefined. Recent studies using single-cell profiling technologies indicated a previously unappreciated heterogeneity of IMCs and provided further knowledge which will help to understand dynamic interactions between IMCs and hematopoietic cells of the intestinal mucosa. In this review, we focus on recent findings on the immunological functions of IMCs: On one hand, we discuss the steady-state interactions of IMCs with epithelial cells and hematopoietic cells. On the other hand, we summarize our current knowledge about the contribution of IMCs to the development of intestinal inflammatory conditions, such as infections, inflammatory bowel disease, and fibrosis. By providing a comprehensive list of cytokines and chemokines produced by IMCs under homeostatic and inflammatory conditions, we highlight the significant immunomodulatory and tissue niche forming capacities of IMCs.

Yersiniosis: a forgotten mimicker and confounder of Crohn’s disease

Abstract Yersiniosis is a zoonosis caused by the Yersinia bacterium. The route of infection is most commonly oral and is caused by consumption of Yersinia-contaminated food. The clinical presentations of chronic yersiniosis are abdominal pain, diarrhea, relapsing arthritis, and skin lesions, that is, nodular erythema. The diagnosis is based on culture-dependent identification of Yersinia in stool, positive serologic test results, or molecular techniques. The treatment of choice is combination antibiotic therapy. Mild forms of the disease do not usually require treatment. Yersiniosis frequently mimics or confounds other chronic intestinal and extraintestinal inflammatory conditions, particularly Crohn’s disease. Therefore, diagnosis of yersiniosis may be a challenge for medical practitioners. Not including Yersinia infection in the differential diagnosis of abdominal symptoms can lead to an incorrect diagnosis and inappropriate treatment. This review summarises the current knowledge of Yersinia enterocolitica and pseudotuberculosis infection, with special focus on differential diagnosis between this infection and Crohn’s disease.

Melatonin prevents the dysbiosis of intestinal microbiota in sleep-restricted mice by improving oxidative stress and inhibiting inflammation.

Background:Intestinal inflammation caused by sleep restriction (SR) threatens human health. However, radical cure of intestinal inflammatory conditions is considerably difficult. This study focuses on the effect of melatonin on SR-induced intestinal inflammation and microbiota imbalance in mice.Methods:We successfully established a water platform to induce long-term SR in mice for 28 days with or without melatonin supplementation. The SR-induced oxidative stress and inflammatory changes were evaluated in plasma and jejunum tissue samples using in vitro assays. Additionally, changes in the intestinal microbiota were explored using high-throughput sequencing of the 16S rRNA gene.Results:After 20 h of chronic sleep restriction for 28 consecutive days, plasma melatonin was significantly reduced by 48.91% (P < 0.05), while GLU, NE, and CORT were significantly increased (34.32%–90.28%, P < 0.05). The activities of antioxidant enzymes (SOD, GSH-Px, and CAT) and T-AOC in intestinal tissues of SR mice were decreased (17.02%–40.92%, P < 0.05), while the content of MDA was increased (15.12%, P = 0.0089). The levels of pro-inflammatory cytokines (IL-6 and TNF-α) ware increased (65.27%–123.26%, P < 0.05), while the levels of anti-inflammatory cytokines (IL-10 and IFN-γ) were decreased (26.53%–60.41%, P < 0.05). High-throughput pyrosequencing of 16S rRNA from jejunum samples demonstrated an overall increase in the number of OTUs (30.68%, P = 0.015). The α-diversity (Shannon, ACE and Chao1) of jejunum was increased (28.18%–48.95%, P < 0.05), and the β-diversity (PCoA and NMDS) was significantly different from that of the control group (P = 0.001). Furthermore, the prevalences of Helicobacter and Clostridium were higher, whereas that of Bacteroidetes and Lactobacillus were lower in SR mice than in controls (P < 0.05). However, melatonin supplementation reversed the SR-induced changes and improved oxidative stress, inflammatory response, and microbiota dysbiosis in the jejunum, and there was not significant difference compared with the control group (P > 0.05).Conclusions:Melatonin prevents the dysbiosis of intestinal microbiota in SR mice by improving oxidative stress and inhibiting inflammation. Our results may provide a theoretical basis for conducting clinical research on insufficient sleep leading to intestinal health in humans and hence facilitate a better understanding of the role of melatonin.

The Mechanism of Secretion and Metabolism of Gut-Derived 5-Hydroxytryptamine.

Serotonin, also known as 5-hydroxytryptamine (5-HT), is a metabolite of tryptophan and is reported to modulate the development and neurogenesis of the enteric nervous system, gut motility, secretion, inflammation, sensation, and epithelial development. Approximately 95% of 5-HT in the body is synthesized and secreted by enterochromaffin (EC) cells, the most common type of neuroendocrine cells in the gastrointestinal (GI) tract, through sensing signals from the intestinal lumen and the circulatory system. Gut microbiota, nutrients, and hormones are the main factors that play a vital role in regulating 5-HT secretion by EC cells. Apart from being an important neurotransmitter and a paracrine signaling molecule in the gut, gut-derived 5-HT was also shown to exert other biological functions (in autism and depression) far beyond the gut. Moreover, studies conducted on the regulation of 5-HT in the immune system demonstrated that 5-HT exerts anti-inflammatory and proinflammatory effects on the gut by binding to different receptors under intestinal inflammatory conditions. Understanding the regulatory mechanisms through which 5-HT participates in cell metabolism and physiology can provide potential therapeutic strategies for treating intestinal diseases. Herein, we review recent evidence to recapitulate the mechanisms of synthesis, secretion, regulation, and biofunction of 5-HT to improve the nutrition and health of humans.

A grass carp model with an antibiotic-disrupted intestinal microbiota

Intestinal microbiota has increasingly been recognized as a major determinant of host health due to its critical role in the maintenance of immune homeostasis. An experimental fish model with disrupted intestinal microbiota allows us to identify potential strategies for restoring or modulating microbiota composition. The aim of this study was to develop a reliable grass carp model with disrupted intestinal microbiota by treatment with an antibiotic cocktail consisting of vancomycin, enrofloxacin, florfenicol, and metronidazole at different concentrations. To identify the appropriate concentration and exposure time of antibiotic treatment required to disrupt intestinal microbiota, five antibiotic regimens were compared in terms of their effects on intestinal microbiota composition, growth and health status, and histological changes in the intestine, hepatopancreas, and spleen. Antibiotic-induced changes in the expression of inflammation- and apoptosis-related genes in grass carp intestines were also examined. The results revealed that the highest antibiotic concentration over a 28 day period was required to disrupt both luminal and mucosal microbiota through significant expansion of Proteobacteria and suppression of Fusobacteria. Treatment with this regimen induced significant down-regulation of caspase-8, MLCK, ZO-1, IL-17N, and IL-23R, and up-regulation of IL-1β and IL-6 in the intestine, suggesting possible effects on intestinal epithelial tight junction and inflammatory conditions. This treatment did not produce substantial histopathological changes, and was not directly detrimental to growth and health. Therefore, this antibiotic regimen may provide a suitable option to disrupt intestinal microbiota in grass carp. In addition, our study also implies that fish may serve as a promising model for investigating growth-promoting effects of antibiotics, when treated with antibiotics in low doses.

DSS-induced colitis is associated with adipose tissue dysfunction and disrupted hepatic lipid metabolism leading to hepatosteatosis and dyslipidemia in mice

Considering high prevalence of non-alcoholic fatty liver diseases (NAFLD) in patients with inflammatory bowel disease (IBD), this study aimed to elucidate molecular mechanisms for how intestinal inflammatory conditions are causally linked to hepatic steatosis and dyslipidemia. Both younger and older mice treated with acute or chronic dextran sodium sulfate (DSS) developed colitis, which was evidenced by weight loss, colon length shortening, and elevated disease activity index and inflammation score. They also showed decreased expression of intestinal barrier function-related proteins and elevated plasma lipopolysaccharide level, indicating DSS-induced barrier dysfunction and thereby increased permeability. Interestingly, they displayed phenotypes of hepatic fat accumulation and abnormal blood lipid profiles. This DSS-induced colitis-associated lipid metabolic dysfunction was due to overall disruption of metabolic processes including fatty acid oxidation, lipogenesis, lipolysis, reverse cholesterol transport, bile acid synthesis, and white adipose tissue browning and brown adipose tissue thermogenesis, most of which are mediated by key regulators of energy homeostasis such as FGF21, adiponectin, and irisin, via SIRT1/PGC-1α- and LXRα-dependent pathways. Our study suggests a potential molecular mechanism underlying the comorbidity of NAFLD and IBD, which could provide a key to understanding how the two diseases are pathogenically linked and discovering critical therapeutic targets for their treatment.

Machine Learning Based Risk Assessment and Early Diagnostic Model of Inflammatory Bowel Disease Associated Arthropathy

Inflammatory bowel disease (IBD) is a persistent idiopathic disorder responsible for intestinal inflammatory conditions. IBD exhibits several extraintestinal manifestations, the most common being arthropathy, that are important triggers and risk factors for adverse progression of the disease. Due to a lack of definite diagnostic criteria and treatment regime, a need arises for early diagnosis and management of IBD associated arthropathy to reduce its morbidity. The present study utilizes a machine learning approach for development and validation of a risk prediction and early diagnostic disease model for IBD associated arthropathy. A publically available IBD cases vs control dataset from University of Massachusetts Medical School’s institutional repository was taken and the data was filtered into 54 IBD individuals along with their demographic and clinical characteristics. The arthropathy characteristics were incorporated from literature in the IBD dataset. Data was randomly split into training (50%; n=27) and testing (50%; n=27) for development and validation of model using logistic regression. Out of 54 IBD cases, 21 had a higher risk of developing arthropathy. The area under the receiver operator curve for the validated model was 0.90 (95% CI 0.80–0.99; accuracy 96%). This disease model can aid in identification of high-risk individuals and for early diagnosis of arthropathy in IBD cases before reaching the imaging and invasive diagnostic stage. This model warrants for prospective case-control trials validation.

Vedolizumab for the Treatment of Noninflammatory Bowel Disease Related Enteropathy

Vedolizumab has become an integral part of the therapeutic armamentarium for patients with inflammatory bowel disease (IBD) who have failed conventional medical therapy. Vedolizumab is an α4β7 integrin antagonist that inhibits intestinal T-cell translocation by blocking integrin interactions with mucosal vascular addressin cellular adhesion molecule 1, reducing lymphocyte-mediated inflammation.1 Its gut selective mode of action and favorable safety profile have led to reports of off-label use for non-IBD-related inflammatory intestinal disorders, such as microscopic colitis,2 and small intestinal inflammatory conditions including autoimmune enteropathy3 and common variable immune deficiency-related enteritis.4 Treatment of non-IBD-related enteropathies is challenging with no approved therapies or established treatment algorithms. We conducted a literature review to assess clinical, endoscopic, and histologic improvement in patients treated with vedolizumab for non-IBD enteropathies refractory to conventional therapy.

KCNN4 Expression Is Elevated in Inflammatory Bowel Disease: This Might Be a Novel Marker and Therapeutic Option Targeting Potassium Channels.

The K + channel KCNN4 is involved in many inflammatory diseases. Previous work has shown that this channel is involved in epithelial ion transport and intestinal restitution. In inflammatory bowel diseases (IBD) a defective epithelial barrier can lead to typical symptoms like secretory diarrhea and the formation of intestinal ulcers. We compared surgical samples from patients with IBD, diverticulitis and controls without inflammation to determine the potential role of KCNN4 as a diagnostic marker and/or therapeutic target. mRNA-levels of KCNN4 and a control K + channel were determined in intestinal epithelial cells (IEC) from patients with IBD, diverticulitis and controls. In addition, we performed a Western blot analysis of KCNN4 and a respective control K + channel in IEC from patients with IBD. Furthermore, we determined epithelial barrier integrity by measuring the flux of fluorescent-labeled dextran beads across a cell monolayer upon incubation with interferon-γ. KCNN4 mRNA and protein levels were elevated in IEC from patients with Crohn`s disease (CD) and ulcerative colitis (UC). Of note, KCNN4 was not elevated in non-IBD intestinal inflammatory conditions e.g. diverticulitis. Of clinical relevance, pharmacological KCNN4 channel openers stabilized epithelial barrier function in vitro. Thus, KCNN4 may have a protective role in IBD and constitute a therapeutic target. Our data demonstrate elevated KCNN4 both at mRNA and protein level in IEC specifically from patients with IBD. Therefore, we conclude that KCNN4 could be used as a novel marker for IBD, especially for the establishment of initial diagnosis. Of therapeutic consequence, we show that pharmacological KCNN4 openers stabilize the epithelial barrier. Thus, KCNN4 might be a novel target to diagnose and treat IBD.

Salicylates Ameliorate Intestinal Inflammation by Activating Macrophage AMPK.

Inflammatory bowel diseases are the most common chronic intestinal inflammatory conditions, and their incidence has shown a dramatic increase in recent decades. Limited efficacy and questionable safety profiles with existing therapies suggest the need for better targeting of therapeutic strategies. Adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of cellular metabolism and has been implicated in intestinal inflammation. Macrophages execute an important role in the generation of intestinal inflammation. Impaired AMPK in macrophages has been shown to be associated with higher production of proinflammatory cytokines; however, the role of macrophage AMPK in intestinal inflammation and the mechanism by which it regulates inflammation remain to be determined. In this study, we investigated the role of AMPK with a specific focus on macrophages in the pathogenesis of intestinal inflammation. A dextran sodium sulfate-induced colitis model was used to assess the disease activity index, histological scores, macroscopic scores, and myeloperoxidase level. Proinflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-1β were measured by enzyme-linked immunosorbent assay. Transient transfection of AMPKβ1 and LC3-II siRNA in RAW 264.7 cells was performed to elucidate the regulation of autophagy by AMPK. The expression of p-AMPK, AMPK, and autophagy markers (eg, LC3-II, p62, Beclin-1, and Atg-12) was analyzed by Western blot. Genetic deletion of AMPKβ1 in macrophages upregulated the production of proinflammatory cytokines, aggravated the severity of dextran sodium sulfate-induced colitis in mice, which was associated with an increased nuclear translocation of nuclear factor-κB, and impaired autophagy both in vitro and in vivo. Notably, the commonly used anti-inflammatory 5-aminosalicylic acid (ie, mesalazine) and sodium salicylate ameliorated dextran sodium sulfate-induced colitis through the activation of macrophage AMPK targeting the β1 subunit. Together, these data suggest that the development of therapeutic agents targeting AMPKβ1 may be effective in the treatment of intestinal inflammatory conditions including inflammatory bowel disease.

A209 ROLE OF SEROTONIN-AUTOPHAGY AXIS IN REGULATION OF EPITHELIAL CELL FUNCTION AND MICROBIOTA COMPOSITION IN GUT

Abstract Background Serotonin (5-hydroxytryptamine; 5-HT), an enteric signalling molecule mainly produced by the enterochromaffin (EC) cells of the intestinal epithelium regulates various processes of the gut. Tryptophan hydroxylase 1 (Tph1) is the rate-limiting enzyme of 5-HT biosynthesis in EC cells. In inflammatory bowel disease (IBD) and experimental colitis, there are alterations in 5-HT content and microbiota composition in the gut. Previously we reported, Tph1-deficient (Tph1-/-) mice with reduced 5-HT in the gut exhibit reduced susceptibility to colitis. The mechanism by which 5-HT regulates colitis is unknown. Autophagy, a catabolic process regulates the function of intestinal epithelial cells (IECs), gut microbiota, and protects against intestinal inflammation. Both aberrant 5-HT signalling and autophagy is implicated in colitis. It is unclear whether they interact in regulation of production of pro-inflammatory cytokines from IECs and gut microbiota composition in relation to colitis. Our hypothesis is, an increase in 5-HT signalling inhibits autophagy in the IECs, which results in up-regulation of colitis by increasing the production of pro-inflammatory cytokines, and by selection for a more colitogenic microbiota. Aims To define the role of 5-HT-autophagy axis in the production of pro-inflammatory cytokines from IECs and gut microbiota composition in intestinal inflammation. Methods We investigated level of autophagy with or without 5% dextran sodium sulphate (DSS) in colons, mucosal scraping and IECs of Tph1-/- and their wild-type (WT) littermates. In addition, autophagy and proinflammatory cytokine production were investigated in human colonic epithelial cells (HT-29) following stimulation by 5-HT. We evaluated colitis and gut microbiota composition in WT, Tph1-/-, epithelial-specific autophagy gene Atg7 deficient (Atg7ΔIEC), and Atg7ΔIECTph1-/- (double knock out; DKO) mice. Results Tph1 -/- mice, with less 5-HT in the gut than WT mice following DSS administration exhibited an up-regulation of autophagy markers in the colon, mucosal scraping and IECs along with reduction of colitis severity. 5-HT treatment of HT-29 cells resulted in down-regulation of autophagy and upregulation of pro-inflammatory cytokine, IL-8. DKO mice exhibited increased severity of DSS-colitis, and altered microbiota composition compared to Tph1-/- mice. Conclusions These findings suggest, an increase in 5-HT in colitis inhibits autophagy in the IECs that contribute to alteration of the gut microbiota and disease severity. Blocking 5-HT signalling may promote autophagy in the IECs and alleviate the severity of colitis. Understanding the contribution of 5-HT in autophagy may identify new therapeutic target in IBD and other intestinal inflammatory conditions that exhibit dysregulated autophagy. Funding Agencies CAG, CIHR

Detrimental Role of Nerve Injury-Induced Protein 1 in Myeloid Cells under Intestinal Inflammatory Conditions.

Nerve injury-induced protein 1 (Ninjurin1, Ninj1) is a cell-surface adhesion molecule that regulates cell migration and attachment. This study demonstrates the increase in Ninj1 protein expression during development of intestinal inflammation. Ninj1-deficient mice exhibited significantly attenuated bodyweight loss, shortening of colon length, intestinal inflammation, and lesser pathological lesions than wild-type mice. Although more severe inflammation and serious lesions are observed in wild-type mice than Ninj1-deficient mice, there were no changes in the numbers of infiltrating macrophages in the inflamed tissues obtained from WT and Ninj1-deficient mice. Ninj1 expression results in activation of macrophages, and these activated macrophages secrete more cytokines and chemokines than Ninj1-deficient macrophages. Moreover, mice with conditional deletion of Ninj1 in myeloid cells (Ninj1fl/fl; Lyz-Cre+) alleviated experimental colitis compared with wild-type mice. In summary, we propose that the Ninj1 in myeloid cells play a pivotal function in intestinal inflammatory conditions.

Modulatory Mechanisms of the NLRP3 Inflammasomes in Diabetes.

The inflammasome is a multiprotein complex that acts to enhance inflammatory responses by promoting the production and secretion of key cytokines. The best-known inflammasome is the NLRP3 (nucleotide-binding oligomerization domain-like receptor [NLR] family pyrin domain-containing 3) inflammasome. The evidence has shown that the NLRP3 inflammasome, IL-1β, thioredoxin-interacting protein (TXNIP), and pyroptosis play vital roles in the development of diabetes. This review summarizes the regulation of type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) by NLRP3 via modulation of glucose tolerance, insulin resistance, inflammation, and apoptosis mediated by endoplasmic reticulum stress in adipose tissue. Moreover, NLRP3 participates in intestinal homeostasis and inflammatory conditions, and NLRP3-deficient mice experience intestinal lesions. The diversity of an individual’s gut microbiome and the resultant microbial metabolites determines the extent of their involvement in the physiological and pathological mechanisms within the gut. As such, further study of the interaction between the NLRP3 inflammasome and the complex intestinal environment in disease development is warranted to discover novel therapies for the treatment of diabetes.