Human Inflammatory Bowel Disease Research Articles

Evidence for a Causal Role for Escherichia coli Strains Identified as Adherent-Invasive (AIEC) in Intestinal Inflammation.

Enrichment of adherent-invasive Escherichia coli (AIEC) has been consistently detected in subsets of inflammatory bowel disease (IBD) patients. Although some AIEC strains cause colitis in animal models, these studies did not systematically compare AIEC with non-AIEC strains, and causal links between AIEC and disease are still disputed. Specifically, it remains unclear whether AIEC shows enhanced pathogenicity compared to that of commensal E. coli found in the same ecological microhabitat and if the in vitro phenotypes used to classify strains as AIEC are pathologically relevant. Here, we utilized in vitro phenotyping and a murine model of intestinal inflammation to systematically compare strains identified as AIEC with those identified as non-AIEC and relate AIEC phenotypes to pathogenicity. Strains identified as AIEC caused, on average, more severe intestinal inflammation. Intracellular survival/replication phenotypes routinely used to classify AIEC positively correlated with disease, while adherence to epithelial cells and tumor necrosis factor alpha production by macrophages did not. This knowledge was then applied to design and test a strategy to prevent inflammation by selecting E. coli strains that adhered to epithelial cells but poorly survived/replicated intracellularly. Two E. coli strains that ameliorated AIEC-mediated disease were subsequently identified. In summary, our results show a relationship between intracellular survival/replication in E. coli and pathology in murine colitis, suggesting that strains possessing these phenotypes might not only become enriched in human IBD but also contribute to disease. We provide new evidence that specific AIEC phenotypes are pathologically relevant and proof of principle that such mechanistic information can be therapeutically exploited to alleviate intestinal inflammation. IMPORTANCE Inflammatory bowel disease (IBD) is associated with an altered gut microbiota composition, including expansion of Proteobacteria. Many species in this phylum are thought to contribute to disease under certain conditions, including adherent-invasive Escherichia coli (AIEC) strains, which are enriched in some patients. However, whether this bloom contributes to disease or is just a response to IBD-associated physiological changes is unknown. Although assigning causality is challenging, appropriate animal models can test the hypothesis that AIEC strains have an enhanced ability to cause colitis in comparison to other gut commensal E. coli strains and to identify bacterial traits contributing to virulence. We observed that AIEC strains are generally more pathogenic than commensal E. coli and that bacterial intracellular survival/replication phenotypes contributed to disease. We also found that E. coli strains lacking primary virulence traits can prevent inflammation. Our findings provide critical information on E. coli pathogenicity that may inform development of IBD diagnostic tools and therapies.

Tofacitinib fails to prevent T cell transfer colitis in mice but ameliorates disease activity

Tofactinib is a JAK inhibitor approved for ulcerative colitis in humans. Despite of its’ proven effectiveness in humans, mechanistic data are scarce on the effectiveness of Tofactinib in experimental colitis in mice. We induced experimental colitis by transfer of CD4+CD25− isolated T cells into RAG2−/− (T and B cell deficient) mice and treated these mice with tofacitinib for 5–6 weeks either with a dosage of 10 or 40 mg/kg body weight immediately after CD4+ transfer or started treatment after first symptoms of disease for several weeks. While treatment with tofacitinib immediately after transfer resulted in an enhanced expansion of CD4+ T cells and did not prevent occurrence of colitis, treatment after start of symptoms of colitis ameliorated disease activity on a clinical basis and in histological analyses. Tofacitinib is effective in the treatment of murine experimental T cell transfer colitis, however does not prevent occurrence of disease.

Stiffness Restricts the Stemness of the Intestinal Stem Cells and Skews Their Differentiation Toward Goblet Cells

Fibrosis and tissue stiffening are hallmarks of inflammatory bowel disease (IBD). We have hypothesized that the increased stiffness directly contributes to the dysregulation of the epithelial cell homeostasis in IBD. Here, we aim to determine the impact of tissue stiffening on the fate and function of the intestinal stem cells (ISCs). We developed a long-term culture system consisting of 2.5-dimensional intestinal organoids grown on a hydrogel matrix with tunable stiffness. Single-cell RNA sequencing provided stiffness-regulated transcriptional signatures of the ISCs and their differentiated progeny. YAP-knockout and YAP-overexpression mice were used to manipulate YAP expression. In addition, we analyzed colon samples from murine colitis models and human IBD samples to assess the impact of stiffness on ISCs invivo. We demonstrated that increasing the stiffness potently reduced the population of LGR5+ ISCs and KI-67+-proliferating cells. Conversely, cells expressing the stem cell marker, olfactomedin-4, became dominant in the crypt-like compartments and pervaded the villus-like regions. Concomitantly, stiffening prompted the ISCs to preferentially differentiate toward goblet cells. Mechanistically, stiffening increased the expression of cytosolic YAP, driving the extension of olfactomedin-4+ cells into the villus-like regions, while it induced the nuclear translocation of YAP, leading to preferential differentiation of ISCs toward goblet cells. Furthermore, analysis of colon samples from murine colitis models and patients with IBD demonstrated cellular and molecular remodeling reminiscent of those observed invitro. Collectively, our findings highlight that matrix stiffness potently regulates the stemness of ISCs and their differentiation trajectory, supporting the hypothesis that fibrosis-induced gut stiffening plays a direct role in epithelial remodeling in IBD.

Clinical and Morphological Analysis of Efficacy of Intraperitoneal Ozone Application in Experimental Colitis: Preclinical Randomized Experimental Study

Background. Inflammatory bowel diseases — Crohn’s disease and ulcerative colitis — are chronic gastrointestinal diseases affecting young people of working age. An alternative to basic therapy (5-aminosalicylic acid) for inflammatory bowel disease is the use of ozone, which has anti-inflammatory, immunomodulatory, antibacterial properties and no side effects in therapeutic concentrations. Objective. To perform clinical and morphological analysis of efficacy of intraperitoneal ozone application in experimental colitis.Methods. The study was conducted on 73 male Wistar rats weighing 200-250 g. The animals were divided into four groups by simple randomization. Check studies were performed on the second, fourth and sixth days. Oxazolone-induced colitis was simulated in two stages using a 3%-alcohol oxazolone solution. Ozone-acid mixture was obtained on “UOTA-60-01” unit (“Medozone”, Russia). Rectal suppositories with 5-aminosalicylic acid were prepared on the basis of rectal suppositories “Salofalk”. Clinical status was assessed daily according to the disease activity index (DAI) scale. Morphological evaluation of colon lesion tissue fragments was carried out using a PrimoStar microscope (CarlZeiss, Germany). Colon tissue damage was assessed using tissue damage index (TDI). Statistical analysis was conducted with SPSS Statistics 19 (IBM, USA).Results. Clinical and morphological picture of the large intestine lesion in oxazolone-induced colitis on days 2, 4 and 6 is consistent with the changes typical of inflammatory bowel disease in humans. Daily intraperitoneal insufflation of ozone at a dose of 0.05 mg/kg in oxazolone-induced colitis leads to partial restoration of DAI, reduction in neutrophils, eosinophils, histiocytes, and fibroblasts in the lesion, as well as to a decrease in ulcerous defect diameter and TDI. The effects of intraperitoneal insufflations of ozone in oxazolone-induced colitis as compared to rectal suppositories with 50 mg of 5-aminosalicylic acid every 12 hours were less marked for the DAI index on day 4; for the number of eosinophils, plasma cells, histiocytes — on day 2, 4 and 6; for lymphocytes — on day 6.Conclusion. Clinical and morphological picture of the large intestine lesion in ozone-induced colitis correlates with the changes typical of inflammatory bowel disease in humans. The positive effect of ozone in ozone-induced colitis was driven by its anti-inflammatory properties through the activation of Nrf2 and by its antioxidant properties through the inhibition of Keap1.

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.

P054 Secreted inflammatory protein profiles following treatment of ex-vivo human inflammatory bowel disease explants with licenced biologic therapies

Abstract Background Patient-derived inflammatory bowel disease (IBD) ex-plants have potential for biomarker and therapy discovery. Infliximab (IFX), ustekinumab (USTK) and vedolizumab (VDZ) are biologic therapies licenced for the induction and maintenance of remission in ulcerative colitis (UC) and Crohn’s disease (CD). All are efficacious in IBD despite differing mechanisms of action. Treatment of IBD explants ex-vivo with licensed biologic therapies may result in further insights into their mechanism of action. We aimed to evaluate the effect of IFX, USTK and VDZ on inflammatory protein secretion profiles in ex-vivo human IBD ex-plants. Methods Patients with IBD, undergoing endoscopy, were prospectively recruited. Biopsies were collected from the sigmoid and IBD ex-plants generated as per previously described methods. IBD explants were then co-cultured for 24 hours with an IgG control vehicle, IFX, USTK and VDZ. After 24 hours, tissue conditioned media (TCM) from IBD explants was collected. TCM secreted inflammatory protein profiles were quantified using 54 V-plex ELISA (Meso Scale Diagnostics, USA). Secreted inflammatory protein profiles were compared between IgG vehicle (control) and USTK, IFX, VDZ treated ex-plants. All continuous variables are presented as median [interquartile range (IQR)]. P values < 0.05 were considered significant in analyses. Results 37 patients with IBD were included (51% CD, 49% UC); age (median, [IQR]) 41[33-53] years, 49% male; disease duration (median, [IQR]) 9 [5-14] years; 51% of patients were anti-TNF naïve. TNF-a secretion was significantly lower in IBD explants treated with IFX compared with IgG control, p=0.006. IL-23 secretion was significantly lower in IBD explants treated with USTK compared with IgG control, p=0.043. Biologic therapy ex-vivo resulted in significant differences in angiogenesis and vascular injury panel biomarkers compared to control; CRP, VEGF-A and SAA. Pro-inflammatory cytokines and chemokines were also significantly decreased with ex-vivo biologic therapy compared to control; IL-2, IL-10, IL-6, MCP-1, IL-1a, TNF-a and MDC. Three Th17 panel proteins were significantly decreased with ex-vivo biologic therapy compared to control; IL-22, IL-17A, IL17A Gen B. Conclusion The statistically significant decrease in both TNF-a and IL-23 secretion with ex vivo IFX and USTK treatment respectively demonstrates that the IBD explant model recapitulates expected IBD disease biology and therapy responses. These data demonstrate that treatment with licenced biologic therapies ex-vivo reduces multiple pro-inflammatory cytokines, chemokines and secreted proteins in IBD explants. Further study is required to completely understand the effects of licenced biologic therapies on the IBD tissue microenvironment.

P914 An adult zebrafish model for adherent-invasive Escherichia coli indicates protection from infection by treatment with probiotic E. coli Nissle

Abstract Background Adherent-invasive Escherichia coli (AIEC) is an opportunistic pathogen associated with major inflammatory bowel disease (IBD), Crohn’s disease (CD) and ulcerative colitis (UC). AIEC is a human commensal but during unfavorable gastric conditions it exhibits pathogenic properties and induces inflammation in the gut, sometimes progressing to inflammation-induced colon cancer. The exact mechanisms for AIEC pathogenesis are still being discovered. As a human opportunistic pathogen, AIEC has few animal models. In recent years, zebrafish have emerged as a useful model for studies of various human pathogens. Here, a zebrafish model to study AIEC infection was developed. Methods Colonization of AIEC from overnight culture was determined with different infectious doses (103, 104, 105 and 106 CFU/ml of infection water) in zebrafish, incubating fish with AIEC in water for 6 h and then in fresh water for 18 h at 28o C. The invasive property of AIEC in zebrafish gut was determine by gentamycin protection assay. Persistent colonization of AIEC in zebrafish gut was also assessed for 10 days. Induction of pro-inflammatory markers such as IL-1β, TNFα, IFNγ and S100A-10b (akin to human calprotectin) in zebrafish intestinal cells in response to AIEC infection was determined by qRT PCR and ELISA. A probiotic strain, E. coli Nissle 1917 (EcN) was used as a therapeutic and prophylactic against AIEC infection. Propionic acid was used to augment the AIEC infection and the efficacy of ECN was tested against the hyper-infectious condition of AIEC in zebrafish. Results Bath inoculation with AIEC resulted in colonization, invasion and tissue disruption in the zebrafish intestine. The AIEC infection induced neutrophil recruitment and showed significant pro-inflammatory responses in zebrafish gut. S100A-10b protein of zebrafish, equivalent of a human IBD marker, calprotectin, was significantly increased in the zebrafish gut during AIEC infection. The probiotic E. coli Nissle 1917 (EcN) reduced AIEC colonization, tissue damage and pro-inflammatory responses in zebrafish in both prophylactic and therapeutic ways. Furthermore, EcN diminished the propionic acid-augmented hyper infection of AIEC in the zebrafish model. Conclusion In this study a new zebrafish model was developed to study the infection and inflammation induced by AIEC. Probiotic strain, EcN was used successfully against AIEC to reduce the inflammation in AIEC-zebrafish model.

ESTABLISHMENT AND TREATMENT OF MURINE COLONOIDS TO STUDY THE EPITHELIAL EFFECTS OF INTESTINAL INFLAMMATION IN VITRO

Abstract BACKGROUND Disruption of the intestinal epithelial barrier is a hallmark of inflammatory bowel disease (IBD). The 3-dimensional colonoid culture system is a useful in vitro model to study intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice undergoing acute inflammation using a DSS model and primarily reflect stem cell physiology rather than that of terminally differentiated cells. We developed experimental methods to address this limitation. We terminally differentiate colonoids or create 2-D monolayers, and challenge them with a cocktail of inflammatory mediators that are typically elevated during IBD. METHODS Murine colonoids are established by isolating colonic crypts and plating them in basement membrane matrix. Over 3 to 5 days, crypt structures develop into spheroid colonoids 250 μm in diameter, forming a prominent lumen surrounded by a thin layer of epithelial stem cells. They are passaged a minimum of two times and then are either 1) terminally differentiated by incubating the colonoids with Differentiating Media or 2) used to establish intestinal epithelial monolayers. Inflammation is then induced by incubating cells with a cocktail of inflammatory mediators (TNFa, IL-1b, IFNg, H2O2). Protein is collected after 72 hours, and expression of proteins involved in mitochondrial biogenesis were assessed (n=3). RESULTS We found that changes in mitochondrial dynamics, reflective of key metabolic changes in the epithelium that are observed in colonic scrapings of DSS-treated mice are not recapitulated in traditional colonoids derived from mice on the same DSS regimen. We found that changes in expression and protein levels of peroxisome proliferator-activator receptor-gamma coactivator-1a (PGC1a), the master regulator protein of mitochondrial biogenesis, were not maintained in colonoids. After terminally differentiating colonoids and subsequently inducing inflammation, we were able to recapitulate a failure of mitochondrial biogenesis failure that is a hallmark of both human IBD and murine colitis. Protein expression of PGC1a as well as downstream transcription factor A, mitochondria (TFAM) were both decreased when assessed via western blot. DISCUSSION Colonoids are a useful tool for studying colonic physiology in vitro, however are limited in their ability to evaluate the effects of in vivo inflammation on the mitochondrial function and cellular metabolism. When removed from the intestinal milieu, crypt cells demonstrate a recovery of mitochondrial health and reflect stem primarily stem cell physiology. Our strategies provide opportunities for organoid manipulation which, when employed properly, can shed light on key aspects of intestinal epithelial mitochondrial function during inflammation.

Molecular Mechanisms of Shigella Pathogenesis; Recent Advances.

Shigella species are the main cause of bacillary diarrhoea or shigellosis in humans. These organisms are the inhabitants of the human intestinal tract; however, they are one of the main concerns in public health in both developed and developing countries. In this study, we reviewed and summarised the previous studies and recent advances in molecular mechanisms of pathogenesis of Shigella Dysenteriae and non-Dysenteriae species. Regarding the molecular mechanisms of pathogenesis and the presence of virulence factor encoding genes in Shigella strains, species of this bacteria are categorised into Dysenteriae and non-Dysenteriae clinical groups. Shigella species uses attachment, invasion, intracellular motility, toxin secretion and host cell interruption mechanisms, causing mild diarrhoea, haemorrhagic colitis and haemolytic uremic syndrome diseases in humans through the expression of effector delivery systems, protein effectors, toxins, host cell immune system evasion and iron uptake genes. The investigation of these genes and molecular mechanisms can help us to develop and design new methods to detect and differentiate these organisms in food and clinical samples and determine appropriate strategies to prevent and treat the intestinal and extraintestinal infections caused by these enteric pathogens.

Secreted glucose regulated protein78 ameliorates DSS-induced mouse colitis.

The secreted form of 78-kDa glucose-regulated protein (sGRP78) has been widely reported for its property in aiding resolution of inflammatory. However, little is known on its potential in the treatment of colitis. To investigate the expression pattern and functional outcome of GRP78 in ulcerative colitis, its expression was measured in human and murine colitis samples. It was found that GRP78 was spontaneously secreted to a high level in gut, which is a physiological site of immune tolerance. During the active phase of DSS-induced colitis, the sGRP78 level was significantly reduced but rebounded quickly during resolving phase, making it a potential candidate for the treatment of colitis. In the following experiments, the administration of sGRP78 was proved to decrease susceptibility to experimental colitis, as indicated by an overall improvement of intestinal symptoms, restoration of TJ integrity, decreased infiltration of immune cells and impaired production of inflammatory cytokines. And specific cleavage of endogenous sGRP78 could aggravate DSS colitis. Adoptive transfer of sGRP78-conditioned BMDMs reduced inflammation in the gut. We linked sGRP78 treatment with altered macrophage biology and skewed macrophage polarization by inhibiting the TLR4-dependent MAP-kinases and NF-κB pathways. Based on these studies, as a naturally occurring immunomodulatory molecule, sGRP78 might be an attractive novel therapeutic agent for acute intestinal inflammation.

BTNL2 promotes colitis-associated tumorigenesis in mice by regulating IL-22 production.

Interleukin 22 (IL-22) has an important role in colorectal tumorigenesis and many colorectal diseases such as inflammatory bowel disease and certain infections. However, the regulation of IL-22 production in the intestinal system is still unclear. Here, we present evidence that butyrophilin-like protein 2 (BTNL2) is required for colorectal IL-22 production, and BTNL2 knockout mice show decreased colonic tumorigenesis and more severe colitis phenotypes than control mice due to defective production of IL-22. Mechanistically, BTNL2 acts on group 3 innate lymphoid cells (ILC3s), CD4+ T cells, and γδ T cells to promote the production of IL-22. Importantly, we find that a monoclonal antibody against BTNL2 attenuates colorectal tumorigenesis in mice and that the mBTNL2-Fc recombinant protein has a therapeutic effect in a dextran sulfate sodium (DSS)-induced colitis model. This study not only identifies a regulatory mechanism of IL-22 production in the colorectal system but also provides a potential therapeutic target for the treatment of human colorectal cancer and inflammatory bowel diseases.

T-cell activation Rho GTPase-activating protein maintains intestinal homeostasis by regulating intestinal T helper cells differentiation through the gut microbiota.

Common variants of the T-cell activation Rho GTPase-activating protein (TAGAP) are associated with the susceptibility to human inflammatory bowel diseases (IBDs); however, the underlying mechanisms are still unknown. Here, we show that TAGAP deficiency or TAGAP expression downregulation caused by TAGAP gene polymorphism leads to decreased production of antimicrobial peptides (AMPs), such as reg3g, which subsequently causes dysregulation of the gut microbiota, which includes Akkermansia muciniphila and Bacteroides acidifaciens strains. These two strains can polarize T helper cell differentiation in the gut, and aggravate systemic disease associated with the dextran sodium sulfate-induced (DSS) disease's phenotype in mice. More importantly, we demonstrated that recombinant reg3g protein or anti-p40 monoclonal antibody exerted therapeutic effects for the treatment of DSS-induced colitis in wild-type and TAGAP-deficient mice, suggesting that they are potential medicines for human IBD treatment, and they may also have a therapeutic effect for the patients who carry the common variant of TAGAP rs212388.

Phytochemicals and Regulation of NF-kB in Inflammatory Bowel Diseases: An Overview of In Vitro and In Vivo Effects.

Inflammatory bowel diseases (IBD) are chronic relapsing idiopathic inflammatory conditions affecting the gastrointestinal tract. They are mainly represented by two forms, ulcerative colitis (UC) and Crohn's disease (CD). IBD can be associated with the activation of nuclear factors, such as nuclear factor-kB (NF-kB), leading to increased transcription of pro-inflammatory mediators that result in diarrhea, abdominal pain, bleeding, and many extra-intestinal manifestations. Phytochemicals can interfere with many inflammation targets, including NF-kB pathways. Thus, this review aimed to investigate the effects of different phytochemicals in the NF-kB pathways in vitro and in vivo models of IBD. Fifty-six phytochemicals were included in this study, such as curcumin, resveratrol, kaempferol, sesamol, pinocembrin, astragalin, oxyberberine, berberine hydrochloride, botulin, taxifolin, naringin, thymol, isobavachalcone, lancemaside A, aesculin, tetrandrine, Ginsenoside Rk3, mangiferin, diosgenin, theanine, tryptanthrin, lycopene, gyngerol, alantolactone, mangostin, ophiopogonin D, fisetin, sinomenine, piperine, oxymatrine, euphol, artesunate, galangin, and nobiletin. The main observed effects related to NF-kB pathways were reductions in tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, interferon-gamma (IFN-γ), and cyclooxygenase-2 (COX-2), and augmented occludin, claudin-1, zonula occludens-1, and IL-10 expression levels. Moreover, phytochemicals can improve weight loss, stool consistency, and rectal bleeding in IBD. Therefore, phytochemicals can constitute a powerful treatment option for IBD in humans.

Regulation of type I IFN responses by deubiquitinating enzyme A in inflammatory bowel diseases.

The development of Inflammatory bowel disease (IBD) is driven by excessive production of pro-inflammatory cytokines including TNF-α, IL-12, and IL-23. This notion is supported by the remarkable clinical success of biologics targeting these cytokines. Recognition of cell wall components derived from intestinal bacteria by Toll-like receptors (TLRs) induces the production of these pro-inflammatory cytokines by macrophages and dendritic cells in human IBD and experimental colitis model. Although sensing of bacterial nucleic acids by endosomal TLRs, specifically TLR3, TLR7, and TLR9 leads to robust production of type I IFNs, it remains debatable whether TLR-mediated type I IFN responses are pathogenic or protective in IBD patients. Additionally, recent studies identified deubiquitinating enzyme A (DUBA) as a novel negative regulator of TLR-mediated type I IFN responses. In light of these observations and their potential applications, in this review, we summarize recent findings on the roles of type I IFN responses and DUBA-mediated negative regulation of these responses in human IBD and experimental colitis model.

Modulation of Indoleamine 2,3-Dioxygenase 1 During Inflammatory Bowel Disease Activity in Humans and Mice.

Indoleamine 2,3 dioxygenase-1 (IDO1), a key enzyme in tryptophan metabolism, is strongly up-regulated both in human inflammatory bowel disease (IBD) and animal models of colitis, however its role in the pathogenesis is still controversial. In this study, we investigated IDO1 expression and activity in a mouse model of DSS-induced chronic colitis as well as in colon biopsies and sera from IBD patients. Chronic colitis was induced in mice through the oral administration of dextran sodium sulfate (DSS), and IDO1 activity was induced by i.p. treatment with N-acetyl serotonin (NAS). IDO1 expression and catalytic activity (measured as Kyn/Trp ratio) was evaluated in sera and tissue samples collected from mice and 93 IBD patients under immunotherapy with Vedolizumab (VDZ) or Ustekinumab (UST). Strong up-regulation of IDO1 was found in colons of mice with acute colitis, which follows disease activity. Enhanced IDO1 activity by NAS treatment protects the intestinal mucosa during the recovery phase of chronic colitis. In IBD patients, IDO1 expression and activity correlate with the severity of mucosal inflammation with inflamed regions showing higher IDO1 expression compared to non-inflamed regions within the same patient. Endoscopic response to VDZ/UST treatment is associated with decreased expression of IDO1. This is the first study demonstrating immunomodulatory activity of IDO1 in a chronic mouse model of DSS-induced colitis. As its expression and catalytic activity correlate with the grade of mucosal inflammation and treatment response, IDO1 could represent a promising biomarker for disease severity and treatment monitoring in IBD.

Disruption to tRNA Modification by Queuine Contributes to Inflammatory Bowel Disease

Transfer RNA (tRNA) is the most extensively modified RNA in cells. Queuosine modification is a fundamental process for ensuring the fidelity and efficiency of translation from RNA to protein. In eukaryotes, Queuosine tRNA (Q-tRNA) modification relies on the intestinal microbial product queuine. However, the roles and potential mechanisms of Q-containing tRNA (Q-tRNA) modifications in inflammatory bowel disease (IBD) are unknown. We explored the Q-tRNA modifications and expression of QTRT1 (queuine tRNA-ribosyltransferase 1) in patients with IBD by investigating human biopsies and reanalyzing datasets. We used colitis models, QTRT1 knockout mice, organoids, and cultured cells to investigate the molecular mechanisms of Q-tRNA modifications in intestinal inflammation. QTRT1 expression was significantly downregulated in ulcerative colitis and Crohn's disease patients. The 4 Q-tRNA-related tRNA synthetases (asparaginyl-, aspartyl-, histidyl-, and tyrosyl-tRNA synthetase) were decreased in IBD patients. This reduction was further confirmed in a dextran sulfate sodium-induced colitis model and interleukin-10-deficient mice. Reduced QTRT1 was significantly correlated with cell proliferation and intestinal junctions, including downregulation of β-catenin and claudin-5 and the upregulation of claudin-2. These alterations were confirmed invitro by deleting the QTRT1 gene from cells and invivo using QTRT1 knockout mice. Queuine treatment significantly enhanced cell proliferation and junction activity in cell lines and organoids. Queuine treatment also reduced inflammation in epithelial cells. Moreover, altered QTRT1-related metabolites were found in human IBD. tRNA modifications play an unexplored novel role in the pathogenesis of intestinal inflammation by altering epithelial proliferation and junction formation. Further investigation of the role of tRNA modifications will uncover novel molecular mechanisms for the prevention and treatment of IBD.

Extract of Laurus nobilis attenuates inflammation and epithelial ulcerations in an experimental model of inflammatory bowel disease

Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are classified as chronic inflammatory disorders and typically require anti-inflammatory drug therapies, such as glucocorticoid regimens, non-steroidal anti-inflammatory drugs, and biologics, aimed at reducing inflammation in the bowel wall. However, each of these therapies is accompanied by a list of possible serious side effects. Because of this, there remains an urgent need to identify new pharmacologic options to reduce or prevent the pro-inflammatory events of IBD while minimizing adverse side effects, and to make available more cost-effective treatment modalities. We have previously identified several herbal extracts that demonstrate potent bio-inhibitory activity of the innate immune response. In particular, Laurus nobilis (LN), or more commonly called bay laurel, demonstrated significant anti-inflammatory function by inhibiting nuclear factor-κB activation. Based upon our original in vitro findings, we have now examined the effects of this herbal extract on a murine dextran sodium sulfate (DSS) model of IBD. Hematoxylin and eosin-stained paraffin sections prepared from DSS treated animals show clear epithelial damage, including ulcerations, extensive neutrophil infiltration into the mucosal layer, and granuloma formation. Tissue from DSS treated animals that also received LN extract showed improved tissue morphology more closely resembling that from control animals. In addition, DSS treated mice with co-administration of LN extract showed a significant reduction in CD4+ antibody staining within the mucosal layer in colonic sections indicating reduced lymphocyte infiltration. Based on these findings, we believe that administration of LN extracts may be effective in reducing the intestinal epithelial damage seen in human IBD and warrants further investigation through clinical trials. Lay Summary: Inflammatory bowel diseases (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC), manifest as chronic inflammation and ulceration of tissues lining the digestive tract. CD involves inflammation of the deeper layers of the digestive tract, including both the small and large intestines, and less commonly, the upper digestive tract. UC involves inflammation along the lining of the colon and rectum. Steroid or biologic treatments for IBD are common, however, are limited due to significant side effects and/or prohibitive cost. In the present study, we provide evidence for use of the natural product, Laurus nobilis (bay leaf), as a safe and effective anti-inflammatory therapy for IBD.

Extract of Laurus nobilis attenuates inflammation and epithelial ulcerations in an experimental model of inflammatory bowel disease

Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are classified as chronic inflammatory disorders and typically require anti-inflammatory drug therapies, such as glucocorticoid regimens, non-steroidal anti-inflammatory drugs, and biologics, aimed at reducing inflammation in the bowel wall. However, each of these therapies is accompanied by a list of possible serious side effects. Because of this, there remains an urgent need to identify new pharmacologic options to reduce or prevent the pro-inflammatory events of IBD while minimizing adverse side effects and making available more cost-effective treatment modalities. We have previously identified several herbal extracts that demonstrate the potent bio-inhibitory activity of the innate immune response. In particular, Laurus nobilis (LN), or more commonly called bay laurel, demonstrated significant anti-inflammatory function by inhibiting nuclear factor-κB activation. Based upon our original in vitro findings, we have now examined the effects of this herbal extract on a murine dextran sodium sulfate (DSS) model of IBD. Hematoxylin and eosin-stained paraffin sections prepared from DSS-treated animals show clear epithelial damage, including ulcerations, extensive neutrophil infiltration into the mucosal layer, and granuloma formation. Tissue from DSS-treated animals that also received LN extract showed improved tissue morphology more closely resembling that from control animals. In addition, DSS-treated mice with co-administration of LN extract showed a significant reduction in CD4+ antibody staining within the mucosal layer in colonic sections indicating reduced lymphocyte infiltration. Based on these findings, we believe that the administration of LN extracts may be effective in reducing the intestinal epithelial damage seen in human IBD and warrants further investigation through clinical trials. Lay Summary: Inflammatory bowel diseases (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC), manifest as chronic inflammation and ulceration of tissues lining the digestive tract. CD involves inflammation of the deeper layers of the digestive tract, including both the small and large intestines, and less commonly, the upper digestive tract. UC involves inflammation along the lining of the colon and rectum. Steroid or biological treatments for IBD are common, however, they are limited due to significant side effects and/or prohibitive costs. In the present study, we provide evidence for use of the natural product, Laurus nobilis (bay leaf), as a safe and effective anti-inflammatory therapy for IBD.

Oral Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) Spore Powder Ameliorates Murine Colitis by Inhibiting Key Kinases Phosphorylation in MAPK Pathway.

The compound ganoderma lucidum spore powder (GLSP) has emerged as an anti-inflammatory and anti-oxidative regulator. In this study, we explored the roles of GLSP against dextran sulfate sodium (DSS)-induced mouse colitis that can mimic human inflammatory bowel disease (IBD). GLSP was administered by oral gavage at a dosage of 150 mg/kg/day to the acute colitis mice induced by DSS. The DSS-induced mouse weight loss, colonic shortening, diarrhea and bloody stool were observably alleviated after GLSP treatment. The lesion of macroscopic and microscopic signs of the disease was reduced significantly and DSS-induced gut barrier dysfunction was restored via increasing the level of claudin-1, ZO1, Occu, and ZO2 with GLSP. Meanwhile, the levels of IL-6, TNF-α, IL-1β, and IL-18 in the colon were reduced in the GLSP-treated groups. In addition, phosphorylation of the MAPKs ERK1/2, p38, and AKT was suppressed after GLSP treatment. All these results demonstrated that GLSP owned a protective effect on DSS-induced colitis by inhibition of MAPK pathway, which provides a promising therapeutic approach for the treatment of colitis.

Neutrophil-dependent Mitochondrial DNA Release Associated With Extracellular Trap Formation in Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is associated with increased circulating damage-associated molecular patterns, in particular, the highly pro-inflammatory mitochondrial DNA (mtDNA). Here, we study the importance of blood neutrophils in mtDNA release via neutrophil extracellular trap (NET) formation and mitochondrial NETosis, where neutrophils specifically expulse mtDNA as potential targetable biological pathways. We investigated the roles of A23187 (a known NET stimulant), granulocyte macrophage stimulating factor, lipopolysaccharide (LPS), and human IBD plasma in their ability to induce NET formation, mitochondrial NETosis, mtDNA, and total DNA release from human blood neutrophils; and the evidence for increased NET formation in IBD. We demonstrated that NET formation resulted in significant DNA (P < .0001) and mtDNA release (P < .0001) with long DNA fragments (>1000 base pairs) with NETs containing high levels of mtDNA. Using previously described invitro conditions for mitochondrial NETosis, granulocyte macrophage stimulating factor+ LPS triggered neutrophil mtDNA release at lower levels but not NETosis. LPS alone can trigger neutrophilic DNA release without NET formation. Heterologous coculture with plasma from patients with active IBD (vs remission [n= 6/group]) were not associated with significantly higher levels of NETs and mtDNA release. During coculture with active IBD plasma (vs remission), citrullinated histone 3 (CitH3) (a NETs biomarker) levels were significantly lower (P < .001). Similarly, CitH3 levels were lower in stool supernatants of patients with active IBD vs remission (n= 19/12, P= .0001). Stool CitH3 negatively correlates with stool calprotectin, a biomarker for gut inflammation (r=-0.47, P= .03). Hence, although blood neutrophils remain an important source of circulating mtDNA with defined mechanisms for release via NET formation and during neutrophil activation, our data do not support excessive systemic NET formation as a dominant underpinning pathobiological process in IBD.