Murine Colitis Model Research Articles

A Technique to Generate a Murine Model of Dextran Sulfate Sodium-Induced Colitis

A Technique to Generate a Murine Model of Dextran Sulfate Sodium-Induced Colitis

Nanoprobiotics for Remolding the Pro-inflammatory Microenvironment and Microbiome in the Treatment of Colitis.

Despite the great progress of current bacterially based biotherapeutics, their unsatisfying efficacy and underlying safety problems have limited their clinical application. Herein, inspired by probiotic Escherichia coli strain Nissle 1917, probiotic-derived outer membrane vesicles (OMVs) are found to serve as an effective therapeutic platform for the treatment of inflammatory bowel disease (IBD). To further enhance the therapeutic effect, the probiotic-derived OMV-encapsulating manganese dioxide nanozymes are constructed, named nanoprobiotics, which can adhere to inflamed colonic epithelium and eliminate intestinal excess reactive oxygen species in the murine IBD model. Moreover, combined with the anti-inflammatory medicine metformin, nanoprobiotics could further remold the pro-inflammatory microenvironment, improve the overall richness and diversity of the gut microbiota, and exhibit better therapeutic efficacy than commercial IBD chemotherapeutics. Importantly, insignificant overt systemic toxicity in this treatment was observed. By integrating cytokine storm calm with biotherapy, we develop a safe and effective bionanoplatform for the effective treatment of inflammation-mediated intestinal diseases.

Analysis of the interferon-γ-induced secretome of intestinal endothelial cells: putative impact on epithelial barrier dysfunction in IBD.

The development of inflammatory bowel diseases (IBD) involves the breakdown of two barriers: the epithelial barrier and the gut-vascular barrier (GVB). The destabilization of each barrier can promote initiation and progression of the disease. Interestingly, first evidence is available that both barriers are communicating through secreted factors that may accordingly serve as targets for therapeutic modulation of barrier functions. Interferon (IFN)-γ is among the major pathogenesis factors in IBD and can severely impair both barriers. In order to identify factors transmitting signals from the GVB to the epithelial cell barrier, we analyzed the secretome of IFN-γ-treated human intestinal endothelial cells (HIEC). To this goal, HIEC were isolated in high purity from normal colon tissues. HIEC were either untreated or stimulated with IFN-γ (10U/mL). After 48h, conditioned media (CM) were harvested and subjected to comparative hyper reaction monitoring mass spectrometry (HRM™ MS). In total, 1,084 human proteins were detected in the HIEC-CM. Among these, 43 proteins were present in significantly different concentrations between the CM of IFN-γ- and control-stimulated HIEC. Several of these proteins were also differentially expressed in various murine colitis models as compared to healthy animals supporting the relevance of these proteins secreted by inflammatory activated HIEC in the inter-barrier communication in IBD. The angiocrine pathogenic impact of these differentially secreted HIEC proteins on the epithelial cell barrier and their perspectives as targets to treat IBD by modulation of trans-barrier communication is discussed in detail.

Stimulation-responsive mucoadhesive probiotics for inflammatory bowel disease treatment by scavenging reactive oxygen species and regulating gut microbiota

Inflammatory bowel disease (IBD) is characterized by the high level of reactive oxygen species (ROS) and highly dysfunctional intestinal flora. Here, a stimulation-responsive mucoadhesive probiotic Lac@HDP was rationally constructed for achieving specific adhesion of colitis site and depleting high level of ROS in inflammatory site. Briefly, Lac is Lactobacillus acidophilus, HDP is obtained by hyaluronic acid grafted with dopamine protected by phenylboric acid. Specifically, by consuming a large amount of ROS, phenyl borate group of Lac@HDP is oxidized and fractured, thus exposing the catechol hydroxyl group and obtaining strong mucosal adhesion ability, thereby significantly prolong the retention time of Lac in the inflammatory site. In the murine model of acute and chronic colitis, the stimulation-responsive mucoadhesive probiotics were significantly more effective in alleviating colitis symptoms than antioxidants and probiotics alone. In addition, the abundance and diversity of intestinal flora were increased after treatment with Lac@HDP, which was helpful to alleviate IBD. Importantly, the stimulation-responsive mucoadhesive probiotics have good biological safety in vivo, which provides the prospect of clinical application in the future.

Myenteric Plexus Immune Cell Infiltrations and Neurotransmitter Expression in Crohn's Disease and Ulcerative Colitis.

Pain is a cardinal symptom in inflammatory bowel disease [IBD]. An important structure in the transduction of pain signalling is the myenteric plexus [MP]. Nevertheless, IBD-associated infiltration of the MP by immune cells lacks in-depth characterisation. Herein, we decipher intra- and periganglionic immune cell infiltrations in Crohn´s disease [CD] and ulcerative colitis [UC] and provide a comparison with murine models of colitis. Full wall specimens of surgical colon resections served to examine immune cell populations by either conventional immuno-histochemistry or immunofluorescence followed by either bright field or confocal microscopy. Results were compared with equivalent examinations in various murine models of intestinal inflammation. Whereas the MP morphology was not significantly altered in IBD, we identified intraganglionic IBD-specific B cell- and monocyte-dominant cell infiltrations in CD. In contrast, UC-MPs were infiltrated by CD8+ T cells and revealed a higher extent of ganglionic cell apoptosis. With regard to the murine models of intestinal inflammation, the chronic dextran sulphate sodium [DSS]-induced colitis model reflected CD [and to a lesser extent UC] best, as it also showed increased monocytic infiltration as well as a modest B cell and CD8+ T cell infiltration. In CD, MPs were infiltrated by B cells and monocytes. In UC, mostly CD8+ cytotoxic T cells were found. The chronic DSS-induced colitis in the mouse model reflected best the MP-immune cell infiltrations representative for IBD.

Reduced smooth muscle-fibroblasts transformation potentially decreases intestinal wound healing and colitis-associated cancer in ageing mice

Cancer and impaired tissue wound healing with ageing are closely related to the quality of life of the elderly population. Given the increased incidence of cancer and the population ageing trend globally, it is very important to explore how ageing impairs tissue wound healing and spontaneous cancer. In a murine model of DSS-induced acute colitis and AOM/DSS-induced colitis-associated cancer (CAC), we found ageing significantly decreases intestinal wound healing and simultaneous CAC initiation, although ageing does not affect the incidence of AOM-induced, sporadic non-inflammatory CRC. Mechanistically, reduced fibroblasts were observed in the colitis microenvironment of ageing mice. Through conditional lineage tracing, an important source of fibroblasts potentially derived from intestinal smooth muscle cells (ISMCs) was identified orchestrating intestinal wound healing and CAC initiation in young mice. However, the number of transformed fibroblasts from ISMCs significantly decreased in ageing mice, accompanied by decreased intestinal wound healing and decreased CAC initiation. ISMCs-fibroblasts transformation in young mice and reduction of this transformation in ageing mice were also confirmed by ex-vivo intestinal muscular layer culture experiments. We further found that activation of YAP/TAZ in ISMCs is required for the transformation of ISMCs into fibroblasts. Meanwhile, the reduction of YAP/TAZ activation in ISMCs during intestinal wound healing was observed in ageing mice. Conditional knockdown of YAP/TAZ in ISMCs of young mice results in reduced fibroblasts in the colitis microenvironment, decreased intestinal wound healing and decreased CAC initiation, similar to the phenotype of ageing mice. In addition, the data from intestine samples derived from inflammatory bowel disease (IBD) patients show that activation of YAP/TAZ also occurs in ISMCs from these patients. Collectively, our work reveals an important role of the ageing stromal microenvironment in intestinal wound healing and CAC initiation. Furthermore, our work also identified a potential source of fibroblasts involved in colitis and CAC.

Engineered calprotectin-sensing probiotics for IBD surveillance in humans

Inflammatory bowel disease (IBD) is a spectrum of autoimmune diseases affecting the gastrointestinal tract characterized by a relapsing and remitting course of gut mucosal inflammation. Disease flares can be difficult to predict, and the current practice of IBD disease activity surveillance through endoscopy is invasive and requires medical expertise. Recent advancements in synthetic biology raise the possibility that symbiotic microbes can be engineered to selectively detect disease biomarkers used in current clinical practice. Here, we introduce an engineered probiotic capable of detecting the clinical gold standard IBD biomarker, calprotectin, with sensitivity and specificity in IBD patients. Specifically, we identified a bacterial promoter in the probiotic strain Escherichia coli Nissle 1917 (EcN) which exhibits a specific expression increase in the presence of calprotectin. Using murine models of colitis, we show that the reporter signal is activated in vivo during transit of the GI tract following oral delivery. Furthermore, our engineered probiotic can successfully discriminate human patients with active IBD from those in remission and without IBD using patient stool samples, where the intensity of reporter signal quantitatively tracks with clinical laboratory-measured levels of calprotectin. Our pilot study sets the stage for probiotics that can be engineered to detect fecal calprotectin for precise noninvasive disease activity monitoring in IBD patients.

Qing Hua Chang Yin alleviates chronic colitis of mice by protecting intestinal barrier function and improving colonic microflora.

Background: Qing Hua Chang Yin (QHCY) is a famous formula of traditional Chinese medicine (TCM) and has been proven to have protective effect on ulcerative colitis. However, its protective effect and potential therapeutic mechanisms in chronic colitis remain unclear. The purpose of this study is to explore the effects and underlying mechanisms of QHCY on dextran sulfate sodium (DSS)-induced chronic colitis mice model. Methods: The chronic colitis model was established by administration of 2% DSS for three consecutive cycles of 7 days with two intervals of 14 days for recovery by drinking water. The experiment lasted 49 days. The DSS + QHCY group received QHCY administration by oral gavage at doses of 1.6g/kg/d, DSS + Mesalazine group was administrated Mesalazine by oral gavage at doses of 0.2g/kg/d. The control and DSS group were given equal volume of distilled water. The body weight, stool consistency and blood in stool were monitored every 2days. The disease activity index (DAI) was calculated. The colon length was measured after the mice were sacrificed. The histomorphology of colonic tissues was checked by the HE and PAS staining. Immunohistochemistry was performed to detect the expressions of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), tight junction proteins (ZO-1, occludin) and Mucin2 (MUC2). 16S rRNA sequencing analysis was conducted to study the diversity and abundance of gut microbiota changes. Results: QHCY treatment not only significantly attenuated DSS-induced the weight loss, DAI score increase, colon shortening and histological damage in mice, but also decreased the expression of pro-inflammatory cytokines in colonic tissues and increased the expression of ZO-1, occludin, and MUC2. Furthermore, QHCY enhanced the diversity of gut microbes and regulated the structure and composition of intestinal microflora in mice with chronic colitis. Conclusion: QHCY has a therapeutic effect on a murine model of chronic colitis. It can effectively reduce the clinical and pathological manifestations of colitis and prevent alterations in the gut microbiota.

ADP as a novel stimulus for NLRP3-inflammasome activation in mice fails to translate to humans

The NLRP3-inflammasome is a cytosolic multiprotein complex that triggers an inflammatory response to certain danger signals. Recently adenosine diphosphate (ADP) was found to activate the NLRP3-inflammasome in murine macrophages via the P2Y1 receptor. Blockade of this signaling pathway reduced disease severity in a murine colitis-model. However, the role of the ADP/P2Y1-axis has not yet been studied in humans. This present study confirmed ADP-dependent NLRP3-inflammasome activation in murine macrophages, but found no evidence for a role of ADP in inflammasome activation in humans. We investigated the THP1 cell line as well as primary monocytes and further looked at macrophages. Although all cells express the three human ADP-receptors P2Y1, P2Y12 and P2Y13, independent of priming, neither increased ASC-speck formation could be detected with flow cytometry nor additional IL-1β release be found in the culture supernatant of ADP stimulated cells. We now show for the first time that the responsiveness of monocytes and macrophages to ADP as well as the regulation of its purinergic receptors is very much dependent on the species. Therefore the signaling pathway found to contribute to colitis in mice is likely not applicable to humans.

Murine Models of Salmonella Infection.

The pathogen Salmonella enterica encompasses a range of bacterial serovars that cause intestinal inflammation and systemic infections in humans. Mice are a widely used infection model due to their relative simplicity and versatility. Here, we provide standardized protocols for culturing the prolific zoonotic pathogen S. enterica serovar Typhimurium for intragastric inoculation of mice to model colitis or systemic dissemination, along with techniques for direct extraintestinal infection. Furthermore, we present procedures for quantifying pathogen burden and for characterizing the immune response by analyzing tissue pathology, inflammatory markers, and immune cells from intestinal tissues. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Murine colitis model utilizing oral streptomycin pretreatment and oral S. Typhimurium administration Basic Protocol 2: Intraperitoneal injection of S. Typhimurium for modeling extraintestinal infection Support Protocol 1: Preparation of S. Typhimurium inoculum Support Protocol 2: Preparation of mixed S. Typhimurium inoculum for competitive infection Basic Protocol 3: Assessment of S. Typhimurium burden Support Protocol 3: Preservation and pathological assessment of S. Typhimurium-infected tissues Support Protocol 4: Measurement of inflammatory marker expression in intestinal tissues by qPCR Support Protocol 5: Preparation of intestinal content for inflammatory marker quantification by ELISA Support Protocol 6: Immune cell isolation from Salmonella-infected intestinal tissues.

Spermidine protects intestinal mucosal barrier function in mice colitis via the AhR/Nrf2 and AhR/STAT3 signaling pathways

Aryl hydrocarbon receptor (AhR) activation promotes intestinal barrier repair and enhances the gut mucosal barrier function in inflammatory bowel diseases (IBD). Spermidine is beneficial in several murine models of IBD and may affect AhR activity. However, the precise effects of spermidine on the intestinal barrier and AhR remain unclear. This study was designed to investigate whether spermidine affects AhR and gut barrier function in IBD models as well as, its underlying mechanism. We used dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mice, as well as, Caco2 cells incubated with TNF-α and IFN-γ to establish multiple IBD models, followed by spermidine intervention. Alcian blue/Periodic acid-Schiff (AB/PAS) staining, Fluorescein isothiocyanate (FITC)-dextran permeability assay, transepithelial electrical resistance (TER), tight junction protein (TJs) expression, and 16S rRNA scope in situ hybridization were performed to assess intestinal barrier function. AhR expression and the associated pathways were measured. AhR-targeted adeno-associated virus (AAV) and siRNA were used to explore the related molecular mechanisms. Spermidine significantly attenuated the increased intestinal permeability, decreased TER, abnormal distribution of TJs in colitis, and bacterial translocation from the gut tract. Additionally, it significantly increased AhR and Nrf2 expression and inhibited STAT3 phosphorylation. However, the protective effects of spermidine and the related alterations in pathway proteins were largely abolished by the specific inhibition of AhR. Our study demonstrated that spermidine rescues intestinal barrier defects in mice with colitis via the AhR-Nrf2 and AhR-STAT3 pathways, providing a potential therapeutic agent for IBD and other conditions associated with dysregulated gut barrier function.

Therapeutic Potential of a Small-Molecule STAT3 Inhibitor in a Mouse Model of Colitis.

Inflammatory bowel disease (IBD) predisposes to colorectal cancer (CRC). In the current studies, we used the dextran sodium sulfate (DSS) murine model of colitis, which is widely used in preclinical studies, to determine the contribution of STAT3 to IBD. STAT3 has two isoforms: (STAT3 α; which has pro-inflammatory and anti-apoptotic functions, and STAT3β; which attenuates the effects of STAT3α). In the current study, we determined the contribution of STAT3 to IBD across all tissues by examining DSS-induced colitis in mice that express only STAT3α and in mice treated with TTI-101, a direct small-molecule inhibitor of both isoforms of STAT3. We examined mortality, weight loss, rectal bleeding, diarrhea, colon shortening, apoptosis of colonic CD4+ T-cells, and colon infiltration with IL-17-producing cells following 7-day administration of DSS (5%) to transgenic STAT3α knock-in (STAT3β-deficient; ΔβΔβ) mice and wild-type (WT) littermate cage control mice. We also examined the effect of TTI-101 on these endpoints in DSS-induced colitis in WT mice. Each of the clinical manifestations of DSS-induced colitis examined was exacerbated in ΔβΔβ transgenic versus cage-control WT mice. Importantly, TTI-101 treatment of DSS-administered WT mice led to complete attenuation of each of the clinical manifestations and also led to increased apoptosis of colonic CD4+ T cells, reduced colon infiltration with IL-17-producing cells, and down-modulation of colon mRNA levels of STAT3-upregulated genes involved in inflammation, apoptosis resistance, and colorectal cancer metastases. Thus, small-molecule targeting of STAT3 may be of benefit in treating IBD and preventing IBD-associated colorectal cancer.

New Insights into the Mechanism of Ulva pertusa on Colitis in Mice: Modulation of the Pain and Immune System

Inflammatory bowel diseases (IBDs) involving Crohn's disease (CD) and ulcerative colitis (UC) are gastrointestinal (GI) disorders in which abdominal pain, discomfort, and diarrhea are the major symptoms. The immune system plays an important role in the pathogenesis of IBD and, as indicated by several clinical studies, both innate and adaptative immune response has the faculty to induce gut inflammation in UC patients. An inappropriate mucosal immune response to normal intestinal constituents is a main feature of UC, thus leading to an imbalance in local pro- and anti-inflammatory species. Ulva pertusa, a marine green alga, is known for its important biological properties, which could represent a source of beneficial effects in various human pathologies. We have already demonstrated the anti-inflammatory, antioxidant, and antiapoptotic effects of an Ulva pertusa extract in a murine model of colitis. In this study, we aimed to examine thoroughly Ulva pertusa immunomodulatory and pain-relieving properties. Colitis was induced by using the DNBS model (4 mg in 100 μL of 50% ethanol), whereas Ulva pertusa was administered daily at the dosage of 50 and 100 mg/kg by oral gavage. Ulva pertusa treatments have been shown to relieve abdominal pain while modulating innate and adaptative immune-inflammatory responses. This powerful immunomodulatory activity was specifically linked with TLR4 and NLRP3 inflammasome modulation. In conclusion, our data suggest Ulva pertusa as a valid approach to counteract immune dysregulation and abdominal discomfort in IBD.

Evaluation of microbial and vancomycin treatments in ulcerative colitis in murine models.

Despite the number of available therapies for ulcerative colitis (UC), severe side effects and high cost has limited their clinical application. Thus, finding new alternative strategies with minimal side effects is inevitable. Therefore, this study aimed to compare the effectiveness of different therapeutic approaches in DSS-induced colitis. Firstly, we designed oral bio-therapeutic products, Live Bacterial Products (LBP), which include a mixture of fecal bacteria strains isolated from healthy mice and prepared by microencapsulation and freeze-dried techniques. Then we investigated the efficiency of 7 days of freeze-dried FMT, LBP, and vancomycin treatments in DSS-induced colitis. Secondly, we compared the effect of 15 days of microbial therapies (freeze-dried powder of FMT and LBP microcapsules) and seven days of oral vancomycin on the severity of colitis in mice. Furthermore, the levels of IL-1β and TNF-α were measured in serum by ELISA, and the fecal microbiota diversity was analyzed by high-throughput sequencing for all mice groups. After seven days of treatments, our results indicated that oral vancomycin reduced the severity of DSS-induced colitis in mice, where weight gain and a decrease in IL-1 β and TNF-α levels were observed in the vancomycin group compared with other treatment groups. While after two weeks of treatment, the LBP microcapsules were able to reduce the severity of colitis. And at the end of the treatment period, weight gain and a decrease in the DAI scores and the levels of IL-1β and TNF-α were noted in the LBP treatment group compared to other treatment groups. By high-throughput sequencing of the 16S rRNA gene, our results showed that while the microcapsules LBP treatment increased the fecal microbial diversity, after vancomycin therapy, most of the fecal microbiota genera and operational taxonomic units (OTUs) were depleted. Our results concluded that treatment duration and preparation methods affect the microbial therapies' efficiency in UC. Furthermore, this study highlighted the negative consequences of oral vancomycin administration on gut health that should be known before using this medication.

Lafoensia pacari alleviates intestinal damage by modulating cyclooxygenase-2: In silico and in vivo evaluation in a colitis model.

Inflammatory bowel diseases (IBD) are a worldwide health problem and mainly affect young people, consequently affecting the workforce. Available treatments are often associated with side effects, and new therapeutic options are needed. For centuries, plants have represented important substrates in the field of drug development. Lafoensia pacari (L. pacari) is a plant whose pharmaceutical potential has been described, and may have biological activity relevant to the treatment of IBD symptoms. To investigate the activity of keto-alcoholic extracts of L. pacari with respect to ameliorating the inflammatory and nociceptive symptoms of acute experimental colitis in mice. Keto-alcoholic extracts of L. pacari leaves and bark were administered to male and female Swiss mice weighing 25 g to 30 g (n = 8 male mice and n = 8 female mice). The effect of these extracts was observed in an acetic acid-induced acute experimental model of colitis with regard to antinociception/analgesia and inflammatory tissue damage. Recorded macroscopic indices included the Wallace score and the colon weight obtained using a precision scale. Mechanical hyperalgesia was determined using an electronic analgesimeter. Behavior related to overt pain was determined by quantifying the number of writhing instances within 20 min of administration of acetic acid. Molecular docking was performed using human and murine cyclooxygenase-2 (COX-2) with 3 flavonoids (ellagic acid, kaempferol, and quercetin) on the AutoDock Vina software. Analysis of variance followed by Tukey's posttest was used with P < 0.05 indicating significance. In this murine model of colitis, administration of extracts from L. pacari ameliorated acetic acid-induced writhing and colitis-associated inflammatory pain. These improvements may be attributable to the reduction in edema, inflammation (e.g., ulcers, hyperemia, and bowel wall damage), and the intensity of abdominal hyperalgesia. The keto-alcoholic extracts of L. pacari leaves and bark administered at a dose of either 100 mg/kg or 300 mg/kg significantly reduced the number of writhing events when compared to the negative control (P < 0.05). Additionally, extracts of L. pacari bark also performed better than Dipyrone. Leaf extracts administered at 10 mg/kg, 30 mg/kg, and 100 mg/kg and bark extracts administered at 30 mg/kg significantly reduced or prevented the development of edema in the colon of treated mice, while mesalazine did not. Moreover, using molecular docking, we observed that the flavonoids present in L. pacari extracts bind to COX-2, an event not unique to ellagic acid. The results of this study demonstrate a potential novel application of L. pacari extracts for the reduction of inflammation and promotion of antinociception/analgesia as demonstrated by our findings in a murine model of colitis. These findings were also corroborated by in silico analyses, and suggest that L. pacari extracts may be a promising therapeutic agent in the treatment of IBD.

Regulation of CD4 T-cell immunity by eukaryotic Elongation Factor 2 Kinase

Abstract CD4 T cells are a major source of inflammatory cytokines to manage the immune responses, and there is evidence showing that IL-17-secreting helper CD4 T cells (Th17 cells), play a critical role in autoimmune diseases. Eukaryotic Elongation Factor 2 Kinase (eEF2K) is a protein kinase that negatively regulates the elongation step of protein synthesis. eEF2K regulates vascular inflammatory responses and hypertension development, yet a complete immunologic characterization of eEF2K, particularly in CD4 T cells and autoimmune diseases, remains obscure. Defining eEF2K-mediated CD4 T cell activities, including CD4 T cell inflammation and metabolism, will significantly enhance our knowledge toward the goal of improving health span. Here, we reported that eEF2K is essential for the survival and proliferative capacity of CD4 T cells. eEF2K deficient mice displayed a higher metabolic profile and increased production of inflammatory cytokines. By analyzing CD4 T cells from the wild type (WT) and eEF2K deficient mice, we revealed that eEF2K regulates mitochondrial stress capacity and the production of reactive oxygen species (ROS) through the master transcription factor, STAT3. Depletion of eEF2K agitated Th17-related autoimmune diseases, including arthritis and colitis. A STAT3 inhibitor, C188–9, attenuated Th17 cell-mediated autoimmune colitis by reducing inflammation in the murine colitis model that received eEF2K deficient CD4 T cells. Our findings provide insights into the regulation of CD4 T cells and autoimmunity by eEF2K, and further investigations would help design novel strategies by targeting eEF2K-based immunotherapy in autoimmune diseases.

Bacterial targeting of the P-glycoprotein to reduce intestinal inflammation in ulcerative colitis

Ulcerative colitis (UC) is a devastating chronic disease characterized by aberrant colonic inflammation and intestinal dysbiosis. The pathognomonic ulceration as well as intestinal epithelial damage is driven by excess luminal infiltration and activation of neutrophils. Recently our group discovered a system of lipid mediators regulating transmigration of neutrophils from the colonic lamina propria across the epithelial barrier. During homeostasis the multidrug resistance efflux pump p-glycoprotien (P-gp) maintains a gradient of endocannabinoids, which limits neutrophil transmigration. During inflammation however, P-gp is downregulated and Multidrug resistance-associated protein 2 (MRP2), which secretes the neutrophil chemoattractant hepoxilin A3 is upregulated, promoting luminal infiltration of neutrophils. In ulcerative colitis P-gp is significantly downregulated, resulting in the excess luminal accumulation of neutrophils found in ulcer bases. We discovered that the balance of these multidrug resistant efflux pumps is dependent on the microbiome and its secreted metabolites, which is significantly altered in UC. Consequently, we are designing a consortium of bacteria to optimize the expression of P-gp on the host epithelium and thus restrain neutrophil transmigration in UC.Using antibiotic stratification experiments and fecal xenograft models we have identified specific bacterial species that increase P-gp expression, and thus likely restrain neutrophil transmigration. Thus far we have validated the role of 15 candidate bacteria and 78 bacterial metabolites in regulating P-gp expression and function either directly or through interactions with other bacterial species. Leveraging this understanding of microbial regulation of host efflux pumps as well as in-vivo population dynamics data we are developing an algorithm to predict the P-gp induction ability of all possible combinations of candidate microbe. Using this algorithm we will design P-gp high, mid, and low inducing consortia, to study the role of these populations on intestinal inflammation using murine models of ulcerative colitis. The development of such consortia has wide ranging implications for research and clinical practice alike from facilitating further study of the underlying inter-microbial networks regulating key host defense systems in the intestine to the development of a probiotic with the potential to limit colonic neutrophil infiltration in patients with UC. This work is supported by R01DK125407, T32AI132152, and F30DK134110 by National Institutes of Health. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Loss of Claudin-3 Impairs Intestinal Barrier Function and Gut Microbiota to Promote Colitis

Objective: Inflammatory Bowel Disease (IBD) is multifactorial autoimmune disorder where dysregulation of the gut barrier and gut microbiota (Dysbiosis) play critical role in disease onset and progression. A leaky gut often coexists with the dysbiotic gut microbiota, however a pathobiological integration is not well understood. Intestinal claudin-3 expression is robust, and we have reported that its loss promotes gut permeability and colon cancer. Increased gut permeability and colon cancer risk characterize Inflammatory Bowel Disease (IBD). The current study was undertaken to delineate the status and role of claudin-3 expression in IBD. Methods: Multiple murine models of colitis and IBD patient biopsy were used. Claudin-3KO mice were used to determine causal role. Multiplex ELISA, RNAseq and 16S DNA sequencing helped determine inflammatory cytokines, transcriptomic changes, and gut microbiota diversity, respectively. Fecal microbiota transplantation (FMT) and antibiotic treatment studies were done to test the role of claudin-3/gut dysbiosis axis in promoting IBD. Results: A significant decrease in claudin-3 expression was observed in IBD patient samples or the colon of mice subjected to DSS- (2.5% w/v) or C. rodentium-colitis (5 x108 CFU/oral). colitis (p&lt;0.001). To test the causal role, claudin-3KO mice were subjected to DSS-colitis (acute and chronic), and infectious colitis. A significant change in the body weight, colon thickness, and mucosal injury score (p&lt;0.001) characterized the colitis challenged claudin-3KO versus WT mice. P-Stat3 expression was highly upregulated in colitis challenged claudin-3KO mice. Interestingly, administration of an antibiotic cocktail rescued the colitis severity in claudin-3KO mice. The 16s metagenomics further revealed that the gut microbiota of claudin-3KO mice is diverse and clustered distinctly from the WT mice. To determine if gut dysbiosis in claudin-3KO mice promotes susceptibility to colitis, we performed FMT from naïve Claudin-3KO mice to germ-free WT mice. When subjected to DSS-colitis, germ-free mice transplanted with Claudin3-KO gut microbiota showed significantly higher colitis severity versus WT (p&lt;0.05). Conclusion: We summarize that the loss of colonic Cldn3 deregulates mucosal barrier integrity to induce gut dysbiosis and susceptibility to IBD. We propose that claudin-3 can be a novel therapeutic target in strengthening the gut barrier integrity to diminish IBD susceptibility. National Institute of Health, Veterans Administration This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Probiotic potential of Saccharomyces cerevisiae GILA with alleviating intestinal inflammation in a dextran sulfate sodium induced colitis mouse model

Recently, several probiotic products have been developed; however, most probiotic applications focused on prokaryotic bacteria whereas eukaryotic probiotics have received little attention. Saccharomyces cerevisiae yeast strains are eukaryotes notable for their fermentation and functional food applications. The present study investigated the novel yeast strains isolated from Korean fermented beverages and examined their potential probiotic characteristics. We investigated seven strains among 100 isolates with probiotic characteristics further. The strains have capabilities such as auto-aggregation tendency, co-aggregation with a pathogen, hydrophobicity with n-hexadecane,1,1-diphenyl-2-picrylhydrazyl scavenging effect, survival in simulated gastrointestinal tract conditions and the adhesion ability of the strains to the Caco-2 cells. Furthermore, all the strains contained high cell wall glucan content, a polysaccharide with immunological effects. Internal transcribed spacer sequencing identified the Saccharomyces strains selected in the present study as probiotics. To examine the effects of alleviating inflammation in cells, nitric oxide generation in raw 264.7 cells with S. cerevisiae showed that S. cerevisiae GILA could be a potential probiotic strain able to alleviate inflammation. Three probiotics of S. cerevisiae GILA strains were chosen by in vivo screening with a dextran sulfate sodium-induced colitis murine model. In particular, GILA 118 down-regulates neutrophil–lymphocyte ratio and myeloperoxidase in mice treated with DSS. The expression levels of genes encoding tight junction proteins in the colon were upregulated, cytokine interleukin-10 was significantly increased, and tumor necrosis factor-α was reduced in the serum.

Does caffeine have a double-edged sword role in inflammation and carcinogenesis in the colon?

Caffeine (1,3,7-trimethylxanthine, also abbreviated to CAF) is a natural chemical with stimulant effects and is commonly included in many drinks and foods, including coffee, tea, cola, energy drinks, cocoa, chocolates, and so on. Our group previously reported that oral administration of CAF efficiently suppressed the development of intestinal inflammation in a dextran sulfate sodium (DSS)-induced murine acute colitis model by suppressing the expression of chitinase 3-like 1, one of the mammalian chitinases without enzymatic activity. Chitinases are hydrolytic enzymes that break down chitin, a polymer of N-acetylglucosamine, and chitinase-like proteins have no enzymatic activity with preserving chitin-binding ability. CAF binds a cleft of the chitinase active site and plays a role as a pan-chitinase inhibitor. Although CAF showed an anti-inflammatory effect in the above model, oral administration of low-dose CAF with 10% sucrose showed potentially neoplastic effects in colonic epithelial cells in a DSS-induced murine chronic colitis model. In this review, we would like to discuss the pros and cons of coffee/CAF in colonic inflammation and neoplasia with an example of pathological finding.