Anti-inflammatory Effects of Fungal Metabolites in Mouse Intestine as Revealed by In vitro Models.
ABSTRACT: Inflammatory bowel diseases (IBD), which include Crohn's disease and ulcerative colitis, are chronic inflammatory disorders that can affect the whole gastrointestinal tract or the colonic mucosal layer. Current therapies aiming to suppress the exaggerated immune response in IBD largely rely on compounds with non-satisfying effects or side-effects. Therefore, new therapeutical options are needed. In the present study, we investigated the anti-inflammatory effects of the fungal metabolites, galiellalactone, and dehydrocurvularin in both an in vitro intestinal inflammation model, as well as in isolated myenteric plexus and enterocyte cells. Administration of a pro-inflammatory cytokine mix through the mesenteric artery of intestinal segments caused an up-regulation of inflammatory marker genes. Treatment of the murine intestinal segments with galiellalactone or dehydrocurvularin by application through the mesenteric artery significantly prevented the expression of pro-inflammatory marker genes on the mRNA and the protein level. Comparable to the results in the perfused intestine model, treatment of primary enteric nervous system (ENS) cells from the murine intestine with the fungal compounds reduced expression of cytokines such as IL-6, TNF-?, IL-1?, and inflammatory enzymes such as COX-2 and iNOS on mRNA and protein levels. Similar anti-inflammatory effects of the fungal metabolites were observed in the human colorectal adenocarcinoma cell line DLD-1 after stimulation with IFN-? (10 ng/ml), TNF-? (10 ng/ml), and IL-1? (5 ng/ml). Our results show that the mesenterially perfused intestine model provides a reliable tool for the screening of new therapeutics with limited amounts of test compounds. Furthermore, we could characterize the anti-inflammatory effects of two novel active compounds, galiellalactone, and dehydrocurvularin which are interesting candidates for studies with chronic animal models of IBD.
Project description:BACKGROUND:Antimicrobial peptide expression is associated with disease activity in inflammatory bowel disease (IBD) patients. IBD patients have abnormal expression of elafin, a human elastase-specific protease inhibitor and antimicrobial peptide. We determined elafin expression in blood, intestine, and mesenteric fat of IBD and non-IBD patients. METHODS:Serum samples from normal and IBD patients were collected from two UCLA cohorts. Surgical resection samples of human colonic and mesenteric fat tissues from IBD and non-IBD (colon cancer) patients were collected from Cedars-Sinai Medical Center. RESULTS:High serum elafin levels were associated with a significantly elevated risk of intestinal stricture in Crohn's disease (CD) patients. Microsoft Azure Machine learning algorithm using serum elafin levels and clinical data identified stricturing CD patients with high accuracy. Serum elafin levels had weak positive correlations with clinical disease activity (Partial Mayo Score and Harvey Bradshaw Index), but not endoscopic disease activity (Mayo Endoscopic Subscore and Simple Endoscopic Index for CD) in IBD patients. Ulcerative colitis (UC) patients had high serum elafin levels. Colonic elafin mRNA and protein expression were not associated with clinical disease activity and histological injury in IBD patients, but stricturing CD patients had lower colonic elafin expression than non-stricturing CD patients. Mesenteric fat in stricturing CD patients had significantly increased elafin mRNA and protein expression, which may contribute to high circulating elafin levels. Human mesenteric fat adipocytes secrete elafin protein. CONCLUSIONS:High circulating elafin levels are associated with the presence of stricture in CD patients. Serum elafin levels may help identify intestinal strictures in CD patients.
Project description:CCL25/CCR9 is a non-promiscuous chemokine/receptor pair and a key regulator of leukocyte migration to the small intestine. We investigated here whether CCL25/CCR9 interactions also play a role in the regulation of inflammatory responses in the large intestine.Acute inflammation and recovery in wild-type (WT) and CCR9(-/-) mice was studied in a model of dextran sulfate sodium (DSS)-induced colitis. Distribution studies and phenotypic characterization of dendritic cell subsets and macrophage were performed by flow cytometry. Inflammatory bowel disease (IBD) scores were assessed and expression of inflammatory cytokines was studied at the mRNA and the protein level.CCL25 and CCR9 are both expressed in the large intestine and are upregulated during DSS colitis. CCR9(-/-) mice are more susceptible to DSS colitis than WT littermate controls as shown by higher mortality, increased IBD score and delayed recovery. During recovery, the CCR9(-/-) colonic mucosa is characterized by the accumulation of activated macrophages and elevated levels of Th1/Th17 inflammatory cytokines. Activated plasmacytoid dendritic cells (DCs) accumulate in mesenteric lymph nodes (MLNs) of CCR9(-/-) animals, altering the local ratio of DC subsets. Upon re-stimulation, T cells isolated from these MLNs secrete significantly higher levels of TNF?, IFN?, IL2, IL-6 and IL-17A while down modulating IL-10 production.Our results demonstrate that CCL25/CCR9 interactions regulate inflammatory immune responses in the large intestinal mucosa by balancing different subsets of dendritic cells. These findings have important implications for the use of CCR9-inhibitors in therapy of human IBD as they indicate a potential risk for patients with large intestinal inflammation.
Project description:Patients suffering from chronic inflammatory diseases have an increased mortality risk resulting from cardiovascular disorders due to enhanced atherosclerotic and thrombotic events. Until now, it is not completely understood in which way an abnormal expression of pro-inflammatory mediators contributes to this elevated cardiovascular risk, but there is a need for new drugs that on the one hand suppress the expression of pro-inflammatory mediators and on the other hand inhibit arterial platelet adhesion. Thus, we analyzed the anti-inflammatory and anti-thrombotic capacity of the fungal metabolite Galiellalactone in atherosclerosis-prone apolipoprotein E-deficient mice. Treatment of the mice with Galiellalactone lowered the inflammatory expression profile and improved blood clotting times, as well as platelet adhesion to the injured common carotid artery. The results indicate that administration of Galiellalactone is able to reduce the extent of inflammation and arterial platelet adhesion in this mouse model.
Project description:Intestinal disorders such as inflammatory bowel disease (IBD) result in chronic illness requiring lifelong therapy. Our aim was to evaluate the efficacy of recombinant adeno-associated virus (AAV) vector-mediated gene delivery to intestinal epithelial cells in vitro and in vivo. Human colon epithelial cell lines and colon biopsies were transduced using AAV pseudotypes 2/1, 2/2, and 2/5 encoding green fluorescence protein (GFP). Mice were administered the same vectors through oral, enema, intraperitoneal (IP) injection and superior mesenteric artery (SMA) injection routes. Tropism and efficiency were determined by microscopy, flow cytometry, immunohistochemistry and PCR. Caco2 cells were more permissive to AAV transduction. Human colon epithelial cells in organ culture were more effectively transduced by AAV2/2. SMA injection provided the most effective means of vector gene transfer to small intestine and colonic epithelial cells in vivo. Transgene detection 80 days post AAV treatment suggests transduction of crypt progenitor cells. This study shows the feasibility of AAV-mediated intestinal gene delivery, applicable for the investigation of IBD pathogenesis and novel therapeutic options, but also revealed the need for further studies to identify more efficient pseudotypes.
Project description:<b>Background:</b> Gut microbiota, which plays a crucial role in inflammatory bowel diseases (IBD), might have therapeutic benefits for ulcerative colitis or Crohn's disease. Targeting gut microbiota represents a new treatment strategy for IBD patients. Rhein is one of the main components of rhubarb and exhibits poor oral bioavailability but still exerts anti-inflammatory effects in some diseases. Therefore, we investigated the effect of rhein on colitis and studied its possible mechanisms. <b>Methods:</b> The chronic mouse colitis model was induced by four rounds of 2% dextran sulfate sodium (DSS) treatment. The mice were treated with 50 mg/kg and 100 mg/kg rhein daily, body weight, colon length, histological score, inflammatory cytokines in serum or intestine, and fecal lipocalin 2 concentration were determined. Th17 cell, Th1 cell and Th2 cell infiltration in the mesenteric lymph node were analyzed by flow cytometry. Metabolic profiles were collected by non-targeted metabolomics and key metabolic pathways were identified using MetaboAnalyst 4.0. We also assessed intestinal barrier permeability and performed 16s rDNA sequencing. <i>Lactobacillus sp.</i> was cultured, and fecal microbiota transplantation (FMT) was employed to evaluate the contribution of gut microbiota. <b>Results:</b> Rhein could significantly alleviate DSS-induced chronic colitis. Uric acid was identified as a crucial modulator of colitis and rhein treatment led to decreased uric acid levels. We determined that rhein changed purine metabolism indirectly, while the probiotic <i>Lactobacillus</i> was involved in the regulation of host metabolism. Uric acid resulted in a worsened intestinal barrier, which could be rescued by rhein. We further confirmed that rhein-treated gut microbiota was sufficient to relieve DSS-induced colitis by FMT. <b>Conclusion:</b> We showed that rhein could modulate gut microbiota, which indirectly changed purine metabolism in the intestine and subsequently alleviated colitis. Our study has identified a new approach to the clinical treatment of colitis.
Project description:Mast cells have been shown to regulate intestinal ion transport in animal models and normal human colon but their physiological role in human intestinal inflammatory disorders is unknown.To examine mast cell regulation of ion transport in inflammatory bowel disease (IBD).Small and large intestine was obtained from patients with and without IBD undergoing surgical resection. Short circuit current (Isc) responses to rabbit antihuman IgE, histamine, and electrical stimulation were measured in Ussing chambers. Specimens were also examined for mast cell numbers and degree of inflammation.Isc responses to anti-IgE and histamine were smaller in magnitude in IBD compared with non-IBD tissues. In all tissues, anti-IgE Isc responses were reduced by about 80% in chloride free buffer. The histamine H1 receptor antagonist, pyrilamine, decreased anti-IgE responses in non-IBD tissues. Greater inhibition with pyrilamine was seen in IBD small intestine but its effect was less in IBD colon. Histamine pretreatment of non-IBD control tissues reduced anti-IgE responses to levels seen in IBD colon but had no effect in small intestine. Mast cell numbers were greater in IBD compared with non-IBD small intestine while no differences were observed between the colonic groups. Isc responses to anti-IgE were not correlated with the degree of mucosal inflammation.This study provides further evidence that mast cells are capable of mediating alterations of ion transport in human gut but that this regulatory role may be altered in IBD. The data suggest that prior activation of mast cells with release of histamine may account for the reduced secretory response to anti-IgE observed in IBD colonic tissues.
Project description:The dysregulation of intestinal microbial communities is associated with inflammatory bowel diseases (IBD). Studies aimed at understanding the contribution of the microbiota to inflammatory diseases have primarily focused on bacteria, yet the intestine harbours a viral component dominated by prokaryotic viruses known as bacteriophages (phages). Phage numbers are elevated at the intestinal mucosal surface and phages increase in abundance during IBD, suggesting that phages play an unidentified role in IBD. We used a sequence-independent approach for the selection of viral contigs and then applied quantitative metagenomics to study intestinal phages in a mouse model of colitis. We discovered that during colitis the intestinal phage population is altered and transitions from an ordered state to a stochastic dysbiosis. We identified phages specific to pathobiotic hosts associated with intestinal disease, whose abundances are altered during colitis. Additionally, phage populations in healthy and diseased mice overlapped with phages from healthy humans and humans with IBD. Our findings indicate that intestinal phage communities are altered during inflammatory disease, establishing a platform for investigating phage involvement in IBD.
Project description:The receptor for advanced glycation endproducts (RAGE) can engage a diverse class of ligands and contribute to the immune and inflammatory response to infection and injury. It is known to be a pathogenic receptor in many inflammatory diseases, including ischemia/reperfusion (IR) injuries in several tissues; however, its role has not been investigated in IR injuries of the intestine to date. Mesenteric (or intestinal) IR leads to recruitment of inflammatory cells into intestinal interstitial spaces, which markedly disrupts intestinal mucosa. IR-induced mucosal injury is accompanied by the development of a local and systemic inflammatory response and remote organ injury, and results in high mortality in the clinic. We hypothesized that elimination of RAGE signaling using RAGE(-/-) mice would result in decreased local and remote organ injury and reduced inflammation in a mesenteric IR model, and thus be a target for therapeutic intervention. We found that RAGE ligands including HMGB-1 and C3a were elevated after mesenteric IR indicating the potential for enhanced RAGE activation in this model. However despite this, wild-type and RAGE(-/-) mice both displayed similar degrees of mesenteric injury, neutrophil infiltration, intestinal edema, cytokine generation, neutrophil mobilization, and remote organ injury after mesenteric IR. We, therefore, conclude that despite its role in other organ IR injuries, and the robust production of RAGE ligands after intestinal ischemia, RAGE itself does not directly influence tissue injury and the inflammatory response in mesenteric IR.
Project description:Inflammatory bowel disease (IBD) and chronic obstructive pulmonary disease (COPD) are chronic inflammatory diseases of the gastrointestinal and respiratory tracts, respectively. These mucosal tissues bear commonalities in embryology, structure and physiology. Inherent similarities in immune responses at the two sites, as well as overlapping environmental risk factors, help to explain the increase in prevalence of IBD amongst COPD patients. Over the past decade, a tremendous amount of research has been conducted to define the microbiological makeup of the intestine, known as the intestinal microbiota, and determine its contribution to health and disease. Intestinal microbial dysbiosis is now known to be associated with IBD where it impacts upon intestinal epithelial barrier integrity and leads to augmented immune responses and the perpetuation of chronic inflammation. While much less is known about the lung microbiota, like the intestine, it has its own distinct, diverse microflora, with dysbiosis being reported in respiratory disease settings such as COPD. Recent research has begun to delineate the interaction or crosstalk between the lung and the intestine and how this may influence, or be influenced by, the microbiota. It is now known that microbial products and metabolites can be transferred from the intestine to the lung via the bloodstream, providing a mechanism for communication. While recent studies indicate that intestinal microbiota can influence respiratory health, intestinal dysbiosis in COPD has not yet been described although it is anticipated since factors that lead to dysbiosis are similarly associated with COPD. This review will focus on the gut-lung axis in the context of IBD and COPD, highlighting the role of environmental and genetic factors and the impact of microbial dysbiosis on chronic inflammation in the intestinal tract and lung.
Project description:Inflammatory bowel disease (IBD) is characterized by alterations in the intestinal microbiota and altered immune responses to gut microbiota. Evidence is accumulating that IBD is influenced by not only commensal bacteria but also commensal fungi. We characterized fungi directly associated with the intestinal mucosa in healthy people and Crohn's disease patients and identified fungi specifically abundant in patients. One of these, the common skin resident fungus Malassezia restricta, is also linked to the presence of an IBD-associated polymorphism in the gene for CARD9, a signaling adaptor important for anti-fungal defense. M. restricta elicits innate inflammatory responses largely through CARD9 and is recognized by Crohn's disease patient anti-fungal antibodies. This yeast elicits strong inflammatory cytokine production from innate cells harboring the IBD-linked polymorphism in CARD9 and exacerbates colitis via CARD9 in mouse models of disease. Collectively, these results suggest that targeting specific commensal fungi may be a therapeutic strategy for IBD.