Human Intestinal Inflammation Research Articles

Gut-on-chip devices as intestinal inflammation models and their future for studying multifactorial diseases

Inflammatory bowel disease (IBD), celiac disease, and other inflammatory conditions of the gastrointestinal tract are highly prevalent in society. Due to the complexity of factors involved, detailed pathogenesis is difficult to determine and thus remains elusive in many cases. The advent of gut-on-chip devices has permitted more accurate modeling of the gut microenvironment with the inclusion of fluid flow and mechanical cues that are true to intestinal architecture and physiology. This review outlines the evolution of gut-on-chip platforms towards greater physiological relevance and elucidates how these devices have been used to model and study intestinal inflammation in humans. In addition, we identify key elements from both technological and disease standpoints that are integral for accurate gut-on-chip models of IBD and celiac disease.

Bioaccessible Organosulfur Compounds in Broccoli Stalks Modulate the Inflammatory Mediators Involved in Inflammatory Bowel Disease.

Inflammatory diseases are strongly associated with global morbidity and mortality. Several mediators are involved in this process, including proinflammatory interleukins and cytokines produced by damaged tissues that, somehow, act as initiators of the autoreactive immune response. Bioactive compounds present in plant-based foods and byproducts have been largely considered active agents with the potential to treat or prevent inflammatory diseases, being a valuable alternative to traditional therapeutic agents used nowadays, which present several side effects. In this regard, the present research uncovers the anti-inflammatory activity of the bioaccessible fraction of broccoli stalks processed, by applying different conditions that render specific concentrations of bioactive sulforaphane (SFN). The raw materials' extracts exhibited significantly different contents of total glucosinolates (GSLs) that ranged between 3993.29 and 12,296.48 mg/kg dry weight (dw), with glucoraphanin as the most abundant one, followed by GI and GE. The indolic GSLs were represented by hydroxy-glucobrassicin, glucobrassicin, methoxy-glucobrassicin, and neo-glucobrassicin, with the two latter as the most abundant. Additionally, SFN and indole-3-carbinol were found in lower concentrations than the corresponding GSL precursors in the raw materials. When exploring the bioaccessibility of these organosulfur compounds, the GSL of all matrices remained at levels lower than the limit of detection, while SFN was the only breakdown product that remained stable and at quantifiable concentrations. The highest concentration of bioaccessible SFN was provided by the high-ITC materials (~4.00 mg/kg dw). The results retrieved on the cytotoxicity of the referred extracts evidenced that the range of supplementation of growth media tested (0.002-430.400 µg of organosulfur compounds/mL) did not display cytotoxic effects on Caco-2 cells. The obtained extracts were assessed based on their capacity to reduce the production of key proinflammatory cytokines (interleukin 6 (IL-6), IL-8, and TNF-α) by the intestinal epithelium. Most of the tested processing conditions provided plant material with significant anti-inflammatory activity and the absence of cytotoxic effects. These data confirm that SFN from broccoli stalks, processed to optimize the bioaccessible concentration of SFN, may be potential therapeutic leads to treat or prevent human intestinal inflammation.

Protective Application of Chinese Herbal Compounds and Formulae in Intestinal Inflammation in Humans and Animals.

Intestinal inflammation is a chronic gastrointestinal disorder with uncertain pathophysiology and causation that has significantly impacted both the physical and mental health of both people and animals. An increasing body of research has demonstrated the critical role of cellular signaling pathways in initiating and managing intestinal inflammation. This review focuses on the interactions of three cellular signaling pathways (TLR4/NF-κB, PI3K-AKT, MAPKs) with immunity and gut microbiota to explain the possible pathogenesis of intestinal inflammation. Traditional medicinal drugs frequently have drawbacks and negative side effects. This paper also summarizes the pharmacological mechanism and application of Chinese herbal compounds (Berberine, Sanguinarine, Astragalus polysaccharide, Curcumin, and Cannabinoids) and formulae (Wumei Wan, Gegen-Qinlian decoction, Banxia xiexin decoction) against intestinal inflammation. We show that the herbal compounds and formulae may influence the interactions among cell signaling pathways, immune function, and gut microbiota in humans and animals, exerting their immunomodulatory capacity and anti-inflammatory and antimicrobial effects. This demonstrates their strong potential to improve gut inflammation. We aim to promote herbal medicine and apply it to multispecies animals to achieve better health.

Recent Advances in Cell-Based In Vitro Models to Recreate Human Intestinal Inflammation.

Inflammatory bowel disease causes a major burden to patients and healthcare systems, raising the need to develop effective therapies. Technological advances in cell culture, allied with ethical issues, have propelled in vitro models as essential tools to study disease aetiology, its progression, and possible therapies. Several cell-based in vitro models of intestinal inflammation have been used, varying in their complexity and methodology to induce inflammation. Immortalized cell lines are extensively used due to their long-term survival, in contrast to primary cultures that are short-lived but patient-specific. Recently, organoids and organ-chips have demonstrated great potential by being physiologically more relevant. This review aims to shed light on the intricate nature of intestinal inflammation and cover recent works that report cell-based in vitro models of human intestinal inflammation, encompassing diverse approaches and outcomes.

Fecal Calprotectin Concentrations in Cats with Chronic Enteropathies.

Diagnosis of feline chronic inflammatory enteropathies (CIE) and the differentiation from small cell intestinal lymphoma (SCL) can be challenging. Intestinally expressed calprotectin (S100A8/A9 protein complex) appears to be part of the complex pathogenesis of feline chronic enteropathies (FCE). Fecal calprotectin is a non-invasive biomarker for intestinal inflammation in humans and dogs but has not yet been evaluated in cats. We hypothesized that fecal calprotectin (fCal) concentrations are increased in FCE, correlate with clinical and/or histologic disease severity, and distinguish cases of CIE from SCL. This case-control study included fecal samples and patient data from cats with CIE (n = 34), SCL (n = 17), other gastrointestinal (GI) diseases (n = 16), and cats with no clinical signs of GI disease (n = 32). fCal concentrations were measured using the immunoturbidimetric fCal turbo assay (Bühlmann Laboratories). Compared to healthy cats, fCal concentrations were significantly increased in CIE, SCL, and other diseases (all p < 0.0001), but were not different between these three groups (all p > 0.05), or between cats with extra-GI diseases and healthy controls. These findings suggest that fCal may have utility as a clinical biomarker for FCE but not for intestinal disease differentiation. It further supports the role of calprotectin in the pathogenesis of the spectrum of FCE, which includes CIE and SCL.

Berberine and rifaximin effects on small intestinal bacterial overgrowth: Study protocol for an investigator-initiated, double-arm, open-label, randomized clinical trial (BRIEF-SIBO study).

Introduction: Small intestinal bacterial overgrowth (SIBO) leads to non-specific abdominal discomfort and nutrient malabsorption. Currently, rifaximin is widely applied in SIBO based on its antibacterial and non-absorbable nature. Berberine is a natural component of many popular medicine plants that ameliorates intestinal inflammation in humans through its modification of the gut microbiota. Potential effect of berberine to the gut may provide therapeutic target for SIBO. We aimed to evaluate the effect of berberine compared with rifaximin on SIBO patients. Methods: This is an investigator-initiated, single-center, open-label, double-arm randomized controlled trial, termed BRIEF-SIBO (Berberine and rifaximin effects for small intestinal bacterial overgrowth). In total, 180 patients will be recruited and allocated to an intervention group (berberine) and a control group (rifaximin). Each participant will receive one 400mg drug twice a day (800mg daily) for 2weeks. The total follow-up period is 6weeks from the start of medication. The primary outcome is a negative breath test. The secondary outcomes include abdominal symptom relief and alteration in gut microbiota. Efficacy assessment will be performed every 2weeks, as well as safety assessment during the treatment. The primary hypothesis is that berberine is not inferior to rifaximin for SIBO. Discussion: The BRIEF-SIBO study is the first clinical trial assessing the eradication effects of 2weeks of berberine treatment in SIBO patients. The effect of berberine will be fully verified by using rifaximin as the positive control. The findings of this study may have implications for the management of SIBO, especially increasing the awareness of both physicians and patients who are suffering from long-term abdominal discomfort and avoiding excessive examination.

P009 IL-3 receptor signalling suppresses chronic intestinal inflammation by controlling mechanobiology and tissue egress of regulatory T cells

Abstract Background The pathogenesis of inflammatory bowel diseases (IBD) such as Crohn’s disease (CD) and ulcerative colitis (UC) is still incompletely understood. However, despite having been described back in the 1980s and reported to be involved in chronic inflammatory arthritis, the role of interleukin 3 (IL-3) signaling via the IL-3 receptor (IL-3R) in the development and perpetuation of chronic intestinal inflammation remains largely unexplored. In this study, we therefore aimed to explore the role of IL-3 signaling via IL-3R in experimental colitis as well as human IBD. Methods We analysed IL-3 expression in lamina propria mononuclear cells (LPMCs) from patients with IBD and from mice with experimental colitis. We compared the development of T cell transfer colitis in Rag1-/- mice after transfer of Il3r-/- and Il3r+/+ naïve T cells and investigated the underlying mechanisms by immunofluorescence and functional cell trafficking assays. The mechanical properties of Il3r-/- and Il3r+/+ T cells were determined by real-time deformability cytometry (RT-DC) and atomic force microscopy (AFM) and cytoskeleton structure was analysed by scanning electron microscopy (SEM) and fluorescence recovery after photobleaching (FRAP). Results The expression of IL-3 and IL3+ T cells were increased in the gut of patients with IBD and IL-3 expression was associated to shorter flare-free survival. In vivo, experimental chronic colitis upon T cell transfer was exacerbated in the absence of IL-3 or IL-3R signalling. This was attributable to IL-3-induced changes in kinase signalling and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of regulatory T (Treg) cells from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Treg cells. (A) IL3 mRNA expression in colon tissue from patients.(B) Flare-free survival of IBD patients.(C) Colitis in Rag1–/– mice after transfer of naïve CD4+ T cells from Il3r+/+ and Il3r-/- mice. Mini-endoscopy (left) and IVIS (right).(D) RT-DC plot (left) and quantification (right) of thymic lymphocytes from Il3r-/- and Il3r+/+ mice.(E) Lightsheet microscopy (left) and quantification of TReg index (right, TReg fraction in Ozanimod/DATK32 per fraction in placebo) in mLNs from Rag1-/- mice with transfer colitis induced by Il3r-/- or Il3r+/+ T cells. Conclusion We uncover a crucial role of IL-3R signalling in regulating TReg mechanobiology and tissue egress. Together, our data demonstrate a central – probably counter-regulatory – role for IL-3 receptor signalling in Treg cells in the pathogenesis of murine and human chronic intestinal inflammation and therefore suggest that IL-3 might be a novel target for future therapeutic approaches in IBD.

Minimal Influence of Cayenne Pepper on the Human Gastrointestinal Microbiota and Intestinal Inflammation in Healthy Adult Humans-A Pilot Study.

Diet impacts human gut microbial composition. Phytochemicals in cayenne pepper (CP), such as capsaicin, have anti-inflammatory properties and alter bacterial growth in vitro. However, the evidence that CP impacts the human microbiota and intestinal inflammation in free-living adults is lacking. Thus, the objective of this randomized cross-over study was to determine the influence of CP on human gut microbiota and intestinal inflammation in vivo. A total of 29 participants were randomly allocated to consume two 250 mL servings of tomato juice plus 1.8 g of CP each day or juice only for 5 days before crossing over to the other study arm. Fecal samples were analyzed. CP reduced Oscillibacter and Phascolarctobacterium but enriched Bifidobacterium and Gp6. When stratified by BMI (body mass index), only the increase in Gp6 was observed in all BMI groups during CP treatment. Stool concentrations of lipocalin-2 and calprotectin were similar regardless of CP treatment. However, lipocalin-2 and calprotectin levels were positively correlated in samples taken after CP consumption. Neither lipocalin-2 nor calprotectin levels were related to gut microbial composition. In conclusion, in healthy adult humans under typical living conditions, consumption of CP minimally influenced the gut microbiota and had little impact on intestinal inflammation.

Moderate intestinal immunopathology after acute oral infection with Toxoplasma gondii oocysts is associated with expressive levels of serotonin

BackgroundInvasion of the intestinal mucosa by T. gondii elicits a local immune response of variable intensity. These reactions can be lethal in C57BL/6 mice. The tissue damage caused by inflammation and the functional effects depend on the host immunity, strain, and developmental form of the parasite. We investigated the effects of acute oral infection with T. gondii on histoarchitecture, enteric nervous system (ENS), and inflammatory markers in the jejunum and ileum of mice. MethodsFemale C57BL/6 mice were divided into a control group and a group orally infected with 1000 sporulated T. gondii oocysts (ME-49 strain). After 5 days, jejunum and ileum were collected and processed for analyzes (e.g., histological and histopathological examinations, ENS, cytokine dosage, myeloperoxidase, nitric oxide activity). Main resultsIn infected mice, we observed a significant increase in serotonin-immunoreactive cells (5-HT IR) in the intestinal mucosa, as well as cellular infiltrates in the lamina propria, periganglionitis, and ganglionitis in the myenteric plexus. We also noted decreased neuron density in the jejunum, increased population of enteric glial cells in the ileum, histomorphometric changes in the intestinal wall, villi, and epithelial cells, remodeling of collagen fibers, and increased myeloperoxidase activity, cytokines, and nitric oxide in the intestine. Conclusions and inferencesAcute infection of female mice with T. gondii oocysts resulted in changes in ENS and a marked increase in 5-HT. These changes are consistent with its modulatory role in the development of moderate acute inflammation. The use of this experimental model may lend itself to studies aimed at understanding the pathophysiological mechanisms of intestinal inflammation in humans involving ENS.

P096 Store-Operated Calcium Entry Controls Innate and Adaptive Immune Cell Function in Inflammatory Bowel Disease

Abstract Background Inflammatory bowel disease (IBD) represents a major clinical challenge in need of new treatment modalities to improve patient care. Store-operated calcium entry (SOCE) is the predominant calcium influx pathway in T cells, regulating many of their functional properties. However, it is currently unknown whether the pharmacologic inhibition of SOCE is a suitable drug target in IBD. Therefore, we aimed to investigate the effects of SOCE inhibitors (SOCEi) on lymphocytes and epithelial cells isolated from IBD patients. Methods Peripheral blood mononuclear cells (PBMC), lamina propria lymphocytes (LPMCs) and epithelial cells were isolated from IBD patients undergoing colon resection. LPMCs or PBMCs were ex-vivo stimulated with Ionomycin/PMA ± SOCEi and subsequently stained with a panel of 37 immunological markers for mass cytometry acquisition. Ca2+ influx measurements were performed in order to assess the metabolic status of immune (PBMCs and LPMCs) and epithelial cells after SOCE blockade. Finally, murine models of colitis were used in order to investigate the effects of SOCE blockade on the induction of intestinal inflammation in-vivo. Results Data on B, T, NK, and myeloid cells revealed that treatment with SOCEi caused a dose-dependent inhibition of Ca2+ signaling in human gut-resident lymphocytes and PBMCs. The inhibition of SOCE attenuated the production of pathogenic cytokines including IL-2, IL-4, IL-6, IL-17, TNF-α and IFNγ by colonic T cells and ILCs, reduced the activation of B cells and the production of IL-6 by myeloid cells, without affecting the viability of primary human epithelial cells. Remarkably, mouse models of transfer colitis, in which SOCE signaling components have been blocked, revealed that SOCE is required for the induction of intestinal inflammation in-vivo. Conclusion Our data revealed for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis.

P044 Enteric dopaminergic pathways in mouse and human intestinal inflammation

Abstract Background Changes in dopamine beta-hydroxylase (DBH) and anomalies in dopaminergic machinery have been shown in inflammatory bowel disease (IBD) patients and related animal models. Thus, we aimed to evaluate the dopaminergic pathways in IBD patients as well as in a mouse model of dextran sodium sulphate (DSS)-induced ileitis. Methods Colon biopsies (CB) obtained from healthy volunteers (N=3) and matched-IBD patients (N=3), were used to evaluate DBH immunoreactivity by confocal microscopy. Male C57/Bl6 (8±2 weeks old; N=16 mice) received 1.5% DSS in drinking water for 5 days, then switched to regular drinking water for 3 days. Inflammatory cytokines (IL-1β, TNFα, IL-6) were measured to assess ileitis severity. Changes in ileal muscle tension were isometrically recorded following 30 μM dopamine or 30 μM SKF38393 (a dopamine receptor 1 (D1R) agonist) or 30 μM bromocriptine (a D2R agonist). Immunofluorescence distribution of Iba1 (a macrophage specific marker), D1R, DBH and dopamine transporter (DAT) were determined in longitudinal muscle-myenteric plexus whole-mount preparations (LMMPs) by confocal microscopy. D1R and D2R mRNA transcripts were evaluated by qRT-PCR. Results CB from IBD patients and LMMPs from DSS mice showed a significant increase of DBH immunoreactivity compared to healthy patients and sham mice (+25% [p&amp;lt;0.01], +20% [p&amp;lt;0.01], respectively). DSS treatment caused a significant increase of DAT and D1R immunoreactivity as well as D1R mRNA levels (+27% [p&amp;lt;0.05], +24% [p&amp;lt;0.05], +6-fold [p&amp;lt;0.05], respectively), accompanied by a significant reduction of dopamine-mediated relaxation (-27% [p&amp;lt;0.01]). SKF38393 determined a marked inhibitory response in ileal preparations from DSS mice compared to sham mice (+73% [p&amp;lt;0.01]), suggesting that dopamine responses are mainly mediated through D1R. A 2-fold increase of resident Iba1+ macrophages was observed in the myenteric plexus of DSS mice associated with a 2.9- and 1.5-fold enhancement of IL-6 and IL-1β mRNA levels, respectively. Conclusion Human colitis and mouse ileitis affect dopamine machinery in the enteric nervous system. Experimentally induced ileitis impairs dopaminergic neurotransmission altering D1R-mediated responses. These findings provide novel information on the involvement of dopaminergic pathways in IBD.

Hormesis Effect of Berberine against Klebsiella pneumoniae Is Mediated by Up-Regulation of the Efflux Pump KmrA.

Berberine (BBR) is an effective drug for human intestinal inflammation by preventing intestinal adhesion of bacterial pathogens, while its antibacterial activity is ineffective. Although the antimicrobial mechanisms of BBR are intensively studied at high concentrations, the response of pathogens to its low concentrations remains poorly understood. Here we demonstrated that low concentrations of BBR (3 and 6 μg/mL) conferred by hormesis accelerated cell growth of an important Gram-negative pathogen, Klebsiella pneumoniae, in vitro, while higher concentrations (25 and 50 μg/mL) resulted in the opposite. Transcriptome analysis of K. pneumoniae revealed the up-regulated expression of the KmrA efflux pump and further confirmed it was hypersensitive to BBR stress. Strikingly, when cultivated in tetracycline, the growth-promoting effect of BBR became more significant, while this effect was reversed in the presence of the efflux pump inhibitor cyanide-m-chlorophenylhydrazone. The hormesis was also found in Enterobacter cloacae and Acinetobacter baumannii. More importantly, the presence of BBR at low concentrations resulted in higher minimal inhibitory concentrations of efflux-related antibiotics such as rifampicin and azithromycin. Overall, our data demonstrated the hormesis of BBR and revealed the potential risk of its applications against Gram-negative pathogens at low concentrations.

P008 Store-operated calcium entry controls immune cell function and activation in Inflammatory Bowel Disease

Abstract Background Inflammatory bowel disease (IBD) comprises ulcerative colitis (UC) and Crohn’s disease (CD), both representing major clinical challenges in need of new treatment modalities to improve patient care. Store-operated calcium entry (SOCE) is the predominant calcium influx pathway in immune cells regulating many of their functional properties. Conditional knockout mice, in which SOCE signaling components are deleted in T cells, have revealed that SOCE is required for the induction of intestinal inflammation in mouse models of colitis. However, it is currently unknown whether the pharmacologic inhibition of SOCE is a suitable drug target in IBD and it remains elusive, which immune cell subsets are affected by the pharmaceutical blockade of SOCE. Therefore, we here aimed to investigate the effects of the SOCE inhibitor BTP-2 on the function and activation of lymphocytes isolated from IBD patients. Methods PBMC and/or lamina propria lymphocytes (LPMC) were isolated from UC and CD patients undergoing colon resection. Cells were ex -vivo stimulated with PMA/Ionomycin in the presence or absence of increasing concentrations of BTP-2, fixed and stored at -80°C until acquisition by mass cytometry. LPMCs were stained using 37 lineage and functional markers targeting B, T, NK or myeloid cells and the resulting flow cytometry standard (FCS) files were analyzed by using cytobank and the R/Bioconductor 9 packages. Additionally, Ca2+ influx measurements were performed in order to assess the impact of SOCE inhibition on the calcium homeostasis of various immune cell subsets. Results Data on B, T, NK, and myeloid cells revealed that treatment with BTP-2 causes a dose-dependent inhibition of calcium influx, demonstrating that BTP-2 is a potent inhibitor of SOCE in human LPMCs and PBMCs. By applying mass cytometry, we investigated how the gradual pharmacologic inhibition of SOCE affects the activation and pro-inflammatory functions of various immune cell subsets obtained from colon specimens of patients with UC and CD, which were characterized by the accumulation of CD4+ effector T cells, IFNγ-producing CD8+ T cells and IL-17-producing innate lymphoid cells (ILCs) compared to non-inflamed specimens. Remarkably, inhibition of SOCE attenuated the production of cytokines including IL-2, IL-4, IL-6, IL-17, TNF-α and IFNγ by T cells and ILCs, reduced the activation of B cells and decreased the production of IFNγ by myeloid cells without affecting the viability of primary human epithelial cells. Conclusion Our data reveal for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis.

Investigations of acute effects of polystyrene and polyvinyl chloride micro- and nanoplastics in an advanced in vitro triple culture model of the healthy and inflamed intestine

The continuous degradation of plastic waste in the environment leads to the generation of micro- and nanoplastic fragments and particles. Due to the ubiquitous presence of plastic particles in natural habitats as well as in food, beverages and tap water, oral exposure of the human population with plastic particles occurs worldwide. We investigated acute toxicological effects of polystyrene (PS) and polyvinyl chloride (PVC) micro- and nanoparticles in an advanced in vitro triple culture model (Caco-2/HT29-MTX-E12/THP-1) mimicking the healthy and inflamed human intestine to study the effect of inflammatory processes on plastic particle toxicity. We monitored barrier integrity, cytotoxicity, cell layer integrity, DNA damage, the release of pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α) and mucus distribution after 24 h of particle exposure. In addition, we investigated cytotoxicity, DNA damage and IL-1β release in monocultures of the three cell lines. Amine-modified polystyrene nanoparticles (PS–NH2) served as a positive control for particle-induced toxicity. No acute effects in the investigated endpoints were observed in the model of the healthy intestine after PS or PVC exposure. However, during active inflammatory processes, exposure to PVC particles was found to augment the release of IL-1β and to cause a loss of epithelial cells. Our results suggest that prevalent intestinal inflammation might be an important factor to consider when assessing the hazard of ingested micro- and nanoplastic particles.

Inhibitory Effect of Steamed Soybean Wastewater Against DSS-Induced Intestinal Inflammation in Mice.

This study was performed to examine the beneficial potential of steamed soybean wastewater (SSW), which is generated during the manufacture of fermented soybean products and usually discarded as a by-product. The SSW was found to contain considerable amounts of isoflavones and had concentration-dependent radical scavenging capabilities. Moreover, oral administration of SSW effectively prevented colonic damage induced by dextran sulfate sodium (DSS), based on improvement of morphological and histological features, reduction of oxidative stress indicators, suppression of proinflammatory cytokine production, downregulation of inflammatory marker expression in the colonic tissue, and inhibition of the inflammatory activation of macrophages. It suggests that SSW could prevent intestinal inflammation in humans, although its efficacy should be verified through careful study design in humans. These findings have implications for enhancement of the value-added of SSW and for reduction of wastewater treatment costs incurred by the food industry.

Lipopolysaccharide neutralizing protein in Miso, Japanese fermented soybean paste.

Miso, a fermented paste made from soybeans, is used traditionally for seasoning of food. It has been a protein and nitrogen source since ancient times in Japan because of its high nutritional value. Furthermore, it has important health functions such as the estrogen-like activity of isoflavones, anti-oxidation, and angiotensin-converting-enzyme inhibition activity. Moreover, it has activity for neutralization of lipopolysaccharide (LPS) from Escherichia coli. Nevertheless, the mechanisms of that activity remain unclear. For this study, we purified and identified the proteins responsible for LPS-neutralization. After proteins were isolated from a miso extract using Blue native polyacrylamide gel electrophoresis, a protein found at 10 to 30kDa on the polyacrylamide gel was identified using nano LC-MS/MS as 2S albumin in soybean (Glycine max). The protein had two LPS binding motifs: SKWQHQ (22 amino acid residues) and EKQKKKMEKE (131 amino acid residues). The protein in miso was found to have LPS neutralization activity, as assayed by prostaglandin D2 (PGD2 ) production from macrophage cells. The PGD2 production by macrophage cells was inhibited by LPS-neutralizing protein (LNP) from miso. Particularly, 50mg/mL of LNP solution and LPS (10µg/mL) inhibited production of PGD2 from the cells. The data were inferred as significantly different (P < 0.05) from statistical analyses by analysis of variance testing and Tukey tests. The 2S albumin in soybean is LNP, an LPS-neutralizing protein, produced in miso. PRACTICAL APPLICATION: A protein from miso fermented soy paste neutralizes an Escherichia coli intestinal bacterial product, lipopolysaccharide (LPS), which causes intestinal inflammation. Miso and its protein are anticipated for use as a probiotic agent to prevent intestinal inflammation in humans and domestic animals. Miso is useful not only as a seasoning for food, but also as a health-functional food because it is an LPS-neutralizing agent.

P107 The expression and regulation of Oncostatin M and its receptor in intestinal inflammation

Abstract Background Intestinal homeostasis depends on the interplay between the gut microbiota, epithelium and immune cells. A novel role of Oncostatin M (OSM), a pro-inflammatory cytokine has recently been identified in mouse and human intestinal inflammation. Previous studies have shown OSM as a key driver of chronic inflammation in anti-TNF-α-refractory colitis. A single-nucleotide polymorphism in the human OSM genetic locus is strongly associated with risk of developing inflammatory bowel disease (IBD), thus, biological therapies targeting OSM could have therapeutic potential. Our project aims to explore the impact of OSM on intestinal barrier function in health and disease. Methods To evaluate the role of OSM in intestinal inflammation, we utilized a combination of in vitro and in vivo techniques. This included the generation of 3D intestinal organoids from mice and patients. Organoids were stimulated with a repertoire of different cytokines to determine the responsiveness of OSM receptor (OSMR) to different cytokine signals using a quantitative-PCR-based approach. For in vivo modelling of disease, the Helicobacter hepaticus colitis model was used, as it combines both immune and dysbiosis-driven aspects of disease. This allowed us to measure OSM and OSMR expression in response to inflammation and within specific organs and cell subsets. Furthermore, RNAscope in situ hybridisation was used to determine the localisation of OSM- and OSMR-expressing cells in inflamed mucosal tissue from colitic mice and IBD patients. Results RNAscope in situ hybridisation as well as gene expression analysis have shown that the OSM and OSMR were highly expressed in C57BL/6 mice upon induction of colitis in the H. hepaticus model of disease and in mucosal tissues of IBD patients. In addition, a plethora of pro-inflammatory cytokines were upregulated during colitis, with colitic mice showing increased tissue pathology. Furthermore, FACS analysis shows excessive immune cell infiltration in the spleen, colon and mesenteric lymph nodes of colitic mice. Conclusion Our preliminary results have shown that different gut-resident hematopoietic and non-hematopoietic cell types express OSM and OSMR and this expression was modulated by pro-inflammatory cytokines. We therefore hypothesis that OSM might drive distinct transcriptional responses in various gut-resident cell populations. Thus, differential targeting of the OSM receptor might be a potential therapeutic approach in IBD.

P060 The pharmacologic inhibition of store-operated calcium entry pathway in inflammatory bowel disease

Abstract Background Store-operated calcium entry (SOCE) represents the major calcium influx pathway in T cells which not only controls the activation and function of lymphocytes, but which also has been implicated in the metabolic homeostasis and survival of murine CD4+ and CD8+ T cells. Conditional knockout mice, in which SOCE signalling components are deleted in T cells, revealed that SOCE is required for the induction of intestinal inflammation in mouse models of colitis. However, the effects of SOCE inhibition have not been studied in inflammatory bowel disease (IBD) and it remains elusive, which immune cell subset is affected by the pharmaceutical blockade of SOCE. We therefore aim to investigate the effects of SOCE inhibitor BTP-2 on functions and metabolic homeostasis of human lymphocytes isolated from IBD patients. Methods PBMC and/or lamina propria lymphocytes were isolated from colitis patients undergoing colon resection and mass cytometry served in order to evaluate the cytokine production and activation of B, T, NK as well as myeloid cells. Additionally, Ca2+ influx measurement and Seahorse analyses were performed in order to assess the metabolic status of immune cell subsets after SOCE inhibition. Results Data on B, T, NK, myeloid cells and neutrophils isolated from peripheral blood or colon lamina propria revealed that each immune cell subset harbours a distinctive SOCE-dependent Ca2+ influx rate, suggesting that SOCE might differentially regulate the activation and function of each cell subtype. In particular, CD4+ and CD8+ T cells, B and NK cells as well as monocytes were highly susceptible to extracellular Ca2+ influx, followed by granulocytes. Furthermore, inhibition of SOCE in lymphocytes resulted in impaired metabolic fitness, reduced glycolytic capacity and impaired fatty acid oxidation. Finally, BTP-2 was able to decrease the production of key pro-inflammatory cytokines involved in IBD, including TNFα and IL-17 in lamina propria resident T cells. Conclusion Our data revealed for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis. Moreover, we hope that a wide phenotypical characterisation of immune cells via mass cytometry will provide a better insight into positive as well as negative effects of SOCE inhibitors that might interfere with the clinical applicability of SOCE inhibitors for treating IBD.

NAD metabolism fuels human and mouse intestinal inflammation

ObjectiveNicotinamide phosphoribosyltransferase (NAMPT, also referred to as pre-B cell colony-enhancing factor or visfatin) is critically required for the maintenance of cellular nicotinamide adenine dinucleotide (NAD) supply catalysing the rate-limiting step...

Tu1780 - Regulated Expression of Phosphatidylserine-Specific Phospholipase A1 and Lysophosphatidylserine Receptors in Human Intestinal Inflammation

Tu1780 - Regulated Expression of Phosphatidylserine-Specific Phospholipase A1 and Lysophosphatidylserine Receptors in Human Intestinal Inflammation