DSS Mice Research Articles

Targeted nuclear degranulation of neutrophils promotes the progression of pneumonia in ulcerative colitis.

Both intestinal and pulmonary systems are parts of the mucosal immune system, comprising ∼80% of all immune cells. These immune cells migrate or are transported between various mucosal tissues to maintain tissue homeostasis. In this study, we isolated neutrophils from the peripheral blood of patients and utilized immunofluorescence, flow cytometry, and Western blotting to confirm the incidence of "nucleus-directed degranulation" in vitro. Subsequently, we conducted a precise analysis using arivis software. Furthermore, using the DSS mouse model of colitis and tissue clearing technologies, we validated the "targeted nuclear degranulation" of neutrophils and their migration to the lungs in an inflammatory intestinal environment. In this study, we found that among patients with ulcerative colitis, the migration of neutrophils with "targeted nuclear degranulation" from the intestinal mucosa to the lungs significantly exacerbates lung inflammation during pulmonary infections. Notably, patients with ulcerative colitis exhibited a higher abundance of neutrophils with targeted nuclear degranulation. Using DSS mice, we observed that neutrophils with targeted nuclear degranulation from the intestinal mucosa migrated to the lung and underwent activation during pulmonary infections. These neutrophils rapidly released a high amount of neutrophil extracellular traps to mediate the progression of lung inflammation. Alterations in the neutrophil cytoskeleton and its interaction with the nuclear membrane represent the primary mechanisms underlying targeted nuclear degranulation. This study revealed that neutrophils accelerate lung inflammation progression in colitis, offering new insights and potential treatment targets for lung infections for patients with colitis.

Puerarin ameliorates colitis by direct suppression of macrophage M1 polarization in DSS mice

BackgroundUlcerative colitis (UC) is a type of inflammatory bowel disease primarily affecting the colon and rectum. The clinical symptoms of UC include persistent diarrhea, abdominal pain, and rectal bleeding, with chronic inflammation often limited to the mucosal layer of the colon. Macrophages play a significant role in the pathogenesis of UC in response to the presence of gut microbiota. Puerarin is an active compound derived from the root of pueraria lobata, a traditional Chinese herbal medicine, and exhibits potent anti-inflammatory properties in various diseases and disease models including UC-like colitis in mice. However, how the molecule achieves its therapeutic effect in colitis by re-polarizing macrophages remains poorly understood. PurposeUtilizing in vivo and in vitro experimental methods along with multi-omics analysis, we aimed to elucidate the potential mechanism by which puerarin targets macrophages to treat colitis. MethodsThe inflammation induced by DSS was assessed both locally in the gut and systemically, and the anti-inflammatory effect of puerarin was evaluated using molecular and histological assays such as H&E staining, qPCR, ELISA, Western blot, and immunofluorescence. Intestinal permeability parameters were measured by in vivo imaging, immunofluorescence, Western blot, qPCR, and PAS staining. The central role of macrophages in colitis was investigated through macrophage depletion/infusion using cytological methods. The direct effects of puerarin on the macrophages were examined by CCK8, flow cytometry, and qPCR in vitro. Additionally, 16S rRNA sequencing and metabolomics analysis of gut contents were conducted. Identification of key pathogenic flora was facilitated by a trans-omic approach and validated both in vitro and in vivo. ResultsPuerarin exerted a direct and robust suppression of M1-like polarization of macrophages in vitro, which was sufficient to confer therapeutic benefits in terms of colonic lesions and systemic inflammation in DSS mice. Puerarin also reduced the abundance of Akkermansia muciniphila in the gut, which was significantly upregulated in DSS mice in our experimental context. Further study demonstrated that puerarin effectively suppressed M1-like macrophage activation induced by Akkermansia muciniphila secreted protein Amuc_2172, thereby altering the pathology in the DSS model. ConclusionOur data suggest that the pathogenesis of DSS colitis is mediated by host cellular responses to toxic foreign molecules and the gut microbiota, and targeting specific cell populations, such as macrophages, with puerarin holds potential therapeutic value.

Synbiotics containing sea buckthorn polysaccharides ameliorate DSS-induced colitis in mice via regulating Th17/Treg homeostasis through intestinal microbiota and their production of BA metabolites and SCFAs

Inflammatory Bowel Disease (IBD) is a chronic condition whose incidence has been rising globally. Synbiotic (SYN) is an effective means of preventing IBD. This study investigated the preventive effects and potential biological mechanisms of SYN (Bifidobacterium longum, Lactobacillus acidophilus, and sea buckthorn polysaccharides) on DSS-induced colitis in mice. The results indicated that dietary supplementation with SYN has a significant improvement effect on DSS mice. SYN ameliorated disease activity index (DAI), colon length, and intestinal barrier permeability in mice. In addition, RT-qPCR results indicated that after SYN intervention, the expression levels of pro-inflammatory factors (IL-6, IL-1β, TNF-α, and IL-17F) and transcription factor RORγt secreted by Th17 cells were significantly reduced, and the expression levels of anti-inflammatory factors (IL-10 and TGF-β) and transcription factor Foxp3 secreted by Treg cells were robustly increased. 16S rDNA sequencing analysis revealed that key intestinal microbiota related to Th17/Treg balance (Ligilactobacillus, Lactobacillus, Bacteroides, and Akkermansia) was significantly enriched. At the same time, a significant increase in microbial metabolites SCFAs and BAs was observed. We speculate that SYN may regulate the Th17/Treg balance by restructuring the structure and composition of the intestinal microbiota, thereby mitigating DSS-induced colitis.

P1204 Oral Streptococcus mutans isolates aggravated colitis in mice

Abstract Background The incidence of inflammatory bowel disease (IBD) is increasing year by year and the mechanism is unclear. A great number of studies have shown that the gut microbiota composition of IBD patients has changed and the diversity of gut microbiota decreases. The abundance of firmicutes decreases, and the abundance of Proteobacteria increases, which eventually leads to the immune disorders and intestinal inflammation. The mouth is the "gateway" of the digestive tract, which has various microbiota. The entry of extraneous microbiota from the mouth into the intestines play an important role in influencing the composition of the gut microbiota. The continuous disorder of the oral microenvironment (such as periodontitis and dental caries) are also closely related to intestinal inflammation. Methods A series of in vivo and in vitro experiments were conducted to investigate the effect and mechanism of S. mutans on colitis mice. Results S.mutans strain UA159 which is closely related to dental caries significantly aggravated colitis in DSS induced mice, as shown by body weight loss, shorter colon length, and increased inflammatory histological scores (Fig1A). UA159 up-regulated intestinal pro-inflammatory cytokines and inhibited the expression of barrier molecules in DSS mice (Fig1B). The proportion of adaptive immune cells (Th1, Th17) was up-regulated in intestinal lamina propria of UA159-administered mice (Fig1C-D).The fecal microbiota composition of UA159 mice was different from control group. The pro-inflammatory effects of UA159 on DSS mice disappeared when antibiotics were used to eliminate the intestinal microbiota. It is suggested that S.mutans may play a proinflammatory role by influencing the intestinal microbiota of mice (Fig1E). We followed 55 IBD patients for more than 1 year and found that IBD patients with poor control had a higher proportion of S.mutans in saliva (Fig1F). S.mutans strains were isolated from IBD saliva samples and were found to cluster in different branches in the phylogenetic tree (Fig1G). S.mutans isolates have different pro-inflammatory effects on colitis mice and intestinal epithelial cells (Fig1H-I). Subsequent experiments are under way to further explore the mechanism leading to the differences of the pro-inflammatory effects. Conclusion Our study were the first to find that oral S.mutans has aggravated intestinal inflammation. S.mutans UA159 aggravated colitis in mice through damaging intestinal mucosal barrier function, enhancing immune response and disrupting intestinal microbiota (Fig1J). The difference in pro-inflammatory effect of S.mutans derived from different IBD patients saliva may be related to the individual clinical outcome.

DOP43 Histologic improvement, attenuation of inflammation and microbiome modulation by engineered high acetate producing Saccharomyces boulardii in DSS-induced colitis

Abstract Background The probiotic Saccharomyces cerevisiae var. boulardii (Sb) may be optimized for therapeutic use in ulcerative colitis by enhancing its acetate production, as acetate is a key factor for its probiotic efficacy and was shown to have anti-inflammatory and barrier-protective effects in vitro (Deleu, 2023). Therefore, higher acetate producing Sb strains have been engineered which were preclinically evaluated in an acute DSS mice model of colitis. Methods Nine-week-old female C57/Bl6 mice (N=120) were allocated to 12 treatment groups receiving drinking water or 2.75% DSS in combination with PBS (control), Baker’s yeast (non-probiotic control), SDH1 (no-acetate Sb), ENT (transient acetate strain-probiotic Enterol control), SbP (high acetate Sb) and ENT3 (extra high acetate Sb). Disease activity including weight loss, diarrhoea and the presence of occult blood were scored daily. On day 7, the DSS groups were transferred to regular drinking water and on day 14 mice were sacrificed. Colonic tissue and faeces (0, 9, 14d) were collected for resp. histology, RNA, and 16S rRNA sequencing. Results Disease activity in DSS subgroups (Fig.A), determined by the area under the curve, was lower for SbP compared to PBS and Baker’s yeast (both p<0.05). Sb SDH1 showed higher disease activity compared to the Sb strains ENT, SbP and ENT3 (all p<0.05). At sacrifice, the macroscopic damage score in DSS was lower for SbP and ENT3 (both p<0.05) compared to Sb SDH1, and the colon weight/length-ratio was decreased for ENT and SbP compared to Sb SDH1 (resp. p=0.06 and p=0.08). Higher histologic inflammation was noted in the non- or transient-acetate producing strains on DSS compared to healthy PBS control (all p<0.05), whereas this increase was not observed for both high-acetate producing strains SbP and ENT3 on DSS (p=NS) indicating a production dependent response. Anti-inflammatory findings were confirmed by transcriptomic analysis of differentially expressed genes e.g. decreased expression of Tnf, Il1b and S100a8/9, predominantly expressed as calprotectin, upon stimulation with the highest acetate producing strain ENT3 (all adj.p<0.1; except PBS adj.p=0.16). Subsequent, microbial analysis showed a superior effect of ENT3 in maintaining a stable alpha diversity (p=0.58, Fig.E), which was not observed for the other strains. Conclusion Engineered high acetate producing Sb strains attenuated DSS-induced colitis, with its anti-inflammatory effects further supported by transcriptomic analysis and superiority in maintaining a stable microbiome composition. Together with our previous in vitro work in patient-derived organoids, these data indicate a role for high-acetate producing Sb- in attenuating intestinal inflammation.

ECCO Fellowship Effect of diet on disease development and faecal microbiota transplantation efficacy in humanized SAMP/Yit and DSS mice models

Abstract Background and Aims The gut microbiome and diet have been shown to be associated with IBD pathogenesis, and to be implicated as triggers in disease development. Increasing evidence shows that dysbiosis and Western diets may drive and sustain intestinal inflammation. Therefore, faecal microbiota transplantation (FMT) has been hypothesized to restore a balanced microbiome, leading to reduced inflammation and improved intestinal function. The mechanisms by which FMT exerts its therapeutic effects in IBD are not fully understood. Yet, FMT has shown promising effects in controlling disease in randomized controlled studies in UC. In contrast, data on FMT in CD are scarce and worsening of disease has even been described (Vermeire et al., 2016). Unfortunately, up to day, this different effect of FMT in UC versus CD remains unexplained. Therefore, the aim of this project is to evaluate the effect of FMT in a preclinical model of CD, and compare it to a UC-model and study the effect of ultra-processed Western diets. Methods The mice models which will be used include the acute DSS-induced colitis model for UC and the spontaneous terminal ileitis (SAMP/Yit) model for CD. Upon humanization of the mice with UC or CD patient’s stool, mice will be randomized to dietary interventions being a Western diet, a regular diet, or a Mediterranean diet. Subsequently, the mice will be randomized to receive either autologous (patient) FMT or one out of four healthy donor FMTs possessing different enterotypes. Anticipated Impact By evaluating, clinical endpoints, as well as microbiota and transcriptomic endpoints, this project will provide insights in the differences between FMT success in UC and CD, as well as in the importance of donor selection (enterotype matching) and additional effect of dietary interventions on inflammation. Herewith predicting that this project will aid to bring microbiota-targeted treatment strategies to the IBD clinic.

‘Ataulfo’ mango (Mangifera indica) bars mitigate colon inflammation and modulate intestinal microbiota in DSS-induced colitis in a mouse model

Anti-inflammatory and antioxidant properties have been associated with the bioactive compounds and dietary fiber found in ‘Ataulfo’ mango (Mangifera indica L.). However, the functionality of mango diet supplementation against intestinal dysbiosis during colitis has not been investigated. By use of 16S rRNA gene Illumina MiSeq, we analyzed the modulation of intestinal ecology with an ‘Ataulfo’ mango-enriched diet (DMango) in a dextran sodium sulphate DSS mice model. The histological analysis by haematoxylin and eosin (H&E) staining showed that the DMango diet depleted the immune cell infiltrations over D5001 standard diet. Also, antibody immunofluorescence analysis demonstrated a greater mucin-2 glycoprotein (MUC2) expression associated to DMango diet, even under induced colitis. Changes in the microbial profile demonstrated that a DMango diet promotes the formation of healthy of microbiota whilst attenuating the unfavourable dysbiosis produced by DSS. DMango diet significantly increased (p < 0.05) the abundance of Clostridium XIVa, Butirycicoccus, Kineothrix, Clostridium IV, Flintibacter, Intestinimonas, Mediterraneibacter and Schaedlerella genera. The phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) evidenced an increase of carbohydrate metabolism potential under the DMango diet. This is the first study showing the substantial health benefits resulting from supplementation with an ‘Ataulfo’ mango diet, mainly maintaining the integrity of the mucus barrier and restoring microbiota shape. This could be promissory as alternative treatment for the preventing or treating colitis.

Abstract P1165: Gut Microbial Peptide Induced Cardiac Inflammation Via Cyclin Dependent/NFkB Signaling In Macrophages

Background: Millions of people in the United States are affected by Inflammatory Bowel Disease (IBD). Recently, gut dysbiosis and impaired gut-barrier permeability are known to contribute to cardiovascular diseases in IBD patients. However, its mechanisms are poorly understood. Here, we hypothesized that gut derived bacterial metabolites (such a peptidoglycan in this case) in a mice colitis model, promotes cardiac inflammation and heart failure. Methods: To induced colitis WT mice were treated with control/DSS (dextran sodium sulfate). Heart and intestine were collected to evaluate inflammatory genes expression(qPCR), immune cell infiltration (FACS), and intestinal permeability (WB and immunohistochemistry). Blood was collected to quantify peptidoglycan (PGN) level. The effect of PGN on NFκB inflammatory pathways was evaluated in macrophages. Results: Echo-data showed cardiac dysfunction in DSS mice. Deteriorated intestinal villi in Masson’s trichrome staining and decreased Occludin and increased PV-1 in western data revealed leaky gut in DSS treated mice. Remarkably, PGN level was significantly increased in blood plasma 3 weeks following DSS treatment. Real-time gene expression data of pro-inflammatory cytokines (IL-6, IL-1 and TNFα) and fibrosis associated genes (col1α1, fibronectin) were increased in DSS mice heart. Increased CD68 expression in the immunostaining data of DSS mice heart indicated the recruitment of inflammatory macrophages. Intriguingly, we observed that CDK9 expression significantly increased upon PGN treatment leading to an increase in NFκB. Next, we inhibited CDK9 before PGN treatment and surprisingly we found that CDK9 inhibition reduces the effect of PGN. Conclusion: Taken together, data suggest that in gut dysbiosis state, PGN leaks into the blood and causes CDK9 mediated NFκB upregulation and cardiac inflammation.

Omentin-1 ameliorates experimental inflammatory bowel disease via Nrf2 activation and redox regulation

AimsOmentin-1 production is decreased in patients with IBD. However, the specific role of Omentin-1 in IBD has not been fully elucidated. This study aimed to investigate the expression and role of Omentin-1 in IBD and the potential mechanisms. Main methodsWe collected human serum and colon biopsy samples at the Wuhan Union Hospital. Omentin-1 recombinant protein was injected intraperitoneally in a DSS-induced experimental IBD mouse model. Omentin-1 levels were measured in IBD patients, colitis mice, and LPS-induced HT-29 cells. Omentin-1 and/or a Nrf2 specific inhibitor (ML385) were administered to DSS mice and LPS-induced HT-29 cells. The effects of Omentin-1 on inflammation, intestinal barrier function, Nrf2 pathway, oxidative stress, and NF-κB signaling were detected in vivo and in vitro. Key findingsSerum Omentin-1 levels were significantly reduced in UC and CD patients compared with controls (173.7 (IQR, 120.1–221.2) ng/ml, 80.8 (43.8–151.8) ng/ml, and 270.7 (220.7–306.5) ng/ml, respectively). The levels of Omentin-1 were also significantly lower in colitis mice and LPS-induced HT-29 cells. Omentin-1 treatment effectively ameliorated inflammation and impaired intestinal barrier, decreased ROS and MDA levels, and increased GSH and SOD production in the DSS-induced colitis mice and LPS-induced HT-29 cells. Mechanically, Omentin-1 repaired the intestinal barrier by activating Nrf2, then improving oxidative stress and inhibiting NF-κB signaling. Furthermore, the interaction between Omentin-1 and Nrf2 was identified. SignificanceOmentin-1 activates the Nrf2 pathway to regulate redox balance, ultimately protecting intestinal barrier function and reducing intestinal inflammation. In general, Omentin-1 can be used as a promising therapeutic target for IBD.

Perioperative Low-Dose Prednisolone Treatment Has Beneficial Effects on Postoperative Recovery and Anastomotic Healing in a Murine Colitis Model.

High-dose glucocorticoid treatment has been identified as a risk factor for anastomotic leakage in patients with inflammatory bowel disease [IBD] undergoing bowel resection surgery. By contrast, active disease during surgery is also associated with elevated morbidity. Perioperative low-dose treatment might be beneficial regarding postoperative outcomes by controlling disease activity. The present study is the first to investigate the dose-dependent effect of perioperative prednisolone therapy in a murine IBD model combining dextran sodium sulphate [DSS] colitis with intestinal anastomosis surgery. In 84 10-week-old wild-type mice, a colorectal anastomosis was performed using a microsurgical technique. Half the animals received induction of chemical colitis with 2% DSS via drinking water prior to surgery. In both groups, one-third of the animals received daily oral administration of high-dose [0.533 mg/kg] and one-third low-dose [0.133 mg/kg] prednisolone. Evaluation was performed on postoperative days 3 and 7. While high-dose prednisolone treatment led to an increased anastomotic leakage rate in mice under colitis, low-dose prednisolone treatment limited preoperative disease activity and did not influence the leakage rate. Histological examination showed a beneficial effect of low-dose prednisolone treatment on microscopic abscess formation at the anastomotic site in DSS mice as well as an increased anastomotic healing score. We demonstrate a beneficial effect of perioperative short-term low-dose prednisolone treatment on intestinal anastomotic healing in the context of colitis. Perioperative use of short-term low-dose prednisolone treatment might be beneficial in IBD patients who need to undergo surgery during active disease.

Effects of Huangqin Tang on NLRP3/Caspase-1 pathway in mice model of ulcerative colitis

The aim of this study was to explore the effects of Huangqin Tang(HQT) on the NLRP3/Caspase-1 signaling pathway in mice with DSS-induced ulcerative colitis(UC). C57BL/6J mice were randomly divided into a blank group, a model group(DSS group), and low-, medium-and high-dose HQT groups(HQT-L, HQT-M, and HQT-H), and western medicine mesalazine group(western medicine group). The UC model was induced in mice. Subsequently, the mice in the HQT-L, HQT-M, HQT-H groups, and the western medicine group were given low-, medium-, high-dose HQT, and mesalazine suspension by gavage, respectively, while those in the blank and DSS groups were given an equal volume of distilled water by gavage. After 10 days of administration, the body weight, DAI scores, and colonic histopathological score of mice in each group were determined. The levels of IL-6, IL-10, IL-1β, and TNF-α in serum were determined by ELISA. The mRNA expression of NLRP3 and Caspase-1 in colon tissues was determined by RT-qPCR. The protein expression of NLRP3 and Caspase-1 in colon tissues was detected by immunohistochemistry. The results showed that compared with the blank group, the DSS group showed decreased body weight of mice and increased DAI scores and intestinal histopathological score. Compared with the DSS group, the HQT groups and the western medicine group showed improved DAI scores, especially in the HQT-M, HQT-H, and the western medicine groups(P&lt;0.05). The intestinal histopathological scores of the HQT groups and the western medicine group significantly decreased, especially in the HQT-M, HQT-H, and the western medicine groups(P&lt;0.05). In addition, compared with the blank group, the DSS group showed elevated expression of NLRP3 and Caspase-1 in colon tissues, increased serum levels of IL-6, IL-1β, and TNF-α, and decreased IL-10 level. Compared with the DSS group, the HQT groups and the western medicine group displayed decreased expression of NLRP3 and Caspase-1 in colon tissues, reduced serum levels of IL-6, IL-1β, and TNF-α, and increased IL-10 level. The improvement was the most significant in the HQT-H group and the western medicine group(P&lt;0.01). In conclusion, HQT may reduce the expression of NLRP3 and Caspase-1 in colon tissues, reduce the se-rum levels of IL-6, IL-1β, and TNF-α, and increase the expression of IL-10 by regulating the classic pyroptosis pathway of NLRP3/Caspase-1, thereby improving the symptoms of intestinal injury and inflammatory infiltration of intestinal mucosa in DSS mice to achieve its therapeutic effect.

Inhibition of TLR4 Suppresses the Inflammatory Response in Inflammatory Bowel Disease (IBD) by Modulating the PDK1-Induced Metabolism Reprogramming via a m6A-Denpendent Manner.

Objective To investigate the role of TLR4 and PDK1 genes in IBD. Methods The DSS mouse model was established by inducing BALB/C with 5% DSS solution. The behavior of DSS mice was detected, and the m6A modification was detected by m6A methylation chip. At the same time, the expressions of TLR and PDK1 were detected by fluorescence real-time quantitative PCR. Results The results showed that the model of dextran sodium sulfate colitis in mice was successful, and the colon membrane of mice had obvious naked eye inflammation. Through comparison, it was found that there were differences in m6A modification between the blank group and the model group, and compared with the blank group, the expression of PKD1 in DSS group was significantly reduced and the expression of TLR4 was significantly increased. Conclusion TLR4 inhibition inhibits the inflammatory response in inflammatory bowel disease (IBD) in a m6A-dependent manner by regulating PDK1-induced metabolic reprogramming.

Candida Worsens Klebsiella pneumoniae Induced-Sepsis in a Mouse Model with Low Dose Dextran Sulfate Solution through Gut Dysbiosis and Enhanced Inflammation.

Klebsiella pneumoniae is an opportunistic pathogen and a commensal organism that is possibly enhanced in several conditions with gut dysbiosis, and frequently detectable together with Candida overgrowth. Here, K. pneumoniae with or without Candida albicans was daily orally administered for 3 months in 0.8% dextran sulfate solution-induced mucositis mice and also tested in vitro. As such, Candida worsened Klebsiella-DSS-colitis as demonstrated by mortality, leaky gut (FITC-dextran assay, bacteremia, endotoxemia, and serum beta-glucan), gut dysbiosis (increased Deferribacteres from fecal microbiome analysis), liver pathology (histopathology), liver apoptosis (activated caspase 3), and cytokines (in serum and in the internal organs) when compared with Klebsiella-administered DSS mice. The combination of heat-killed Candida plus Klebsiella mildly facilitated inflammation in enterocytes (Caco-2), hepatocytes (HepG2), and THP-1-derived macrophages as indicated by supernatant cytokines or the gene expression. The addition of heat-killed Candida into Klebsiella preparations upregulated TLR-2, reduced Occludin (an intestinal tight junction molecule), and worsened enterocyte integrity (transepithelial electrical resistance) in Caco-2 and enhanced casp8 and casp9 (apoptosis genes) in HepG2 when compared with heat-killed Klebsiella alone. In conclusion, Candida enhanced enterocyte inflammation (partly through TLR-2 upregulation and gut dysbiosis) that induced gut translocation of endotoxin and beta-glucan causing hyper-inflammatory responses, especially in hepatocytes and macrophages.

BAFF Blockade Attenuates DSS-Induced Chronic Colitis via Inhibiting NLRP3 Inflammasome and NF-κB Activation.

BackgroundBAFF production is increased in IBD patients. However, the specific role of BAFF in IBD is still uncovered. This study aimed to investigate the expression and function of BAFF in experimental colitis and the potential mechanisms.MethodsBAFF levels in the serum and colon tissues were measured by ELISA in DSS-induced colitis mice. Mouse-derived BAFF antibody was administered in DSS mice. The changes of body weight, disease activity index (DAI) scores, colon length, spleen weight, histopathological damage, inflammatory indicators, NF-κB signaling, and NLRP3 inflammasome were assayed in DSS mice and control. LPS-primed RAW264.7 cells and bone marrow derived macrophages (BMDMs) were treated with BAFF blockage and recombinant mouse BAFF. Inflammatory associated cytokines, NLRP3 inflammasomes and NF-κB signaling were detected among groups.ResultsBAFF production was elevated systemically and locally in colitis mice. BAFF blockade improved the body weight loss, DAI scores, colon length, spleen weight, and histopathological damage in colitis mice. Immunoflurescence analysis revealed that elevated macrophages in mucosal lamina propria were the primary source of BAFF in the colon. NLRP3 inflammasome and NF-κB signaling pathway activation were dramatically inhibited in DSS mice treated with BAFF blockage. In LPS-primed RAW264.7 cells/BMDMs, BAFF blockade decreased the activation of NLRP3 inflammasome (NLPR3, ASC, cleaved IL-1β, cleaved caspase 1) via inhibiting NF-κB signaling pathway. Moreover, LPS synergizes with BAFF to promote inflammatory factor secretion and expression of NF-κB signaling pathway in RAW264.7 cells.ConclusionsThese results suggested that BAFF blockade protected against colitis partially by relieving inflammation, inhibiting intestinal NLRP3 inflammasome and NF-κB signaling pathway from macrophages. BAFF plays an important role in inflammation regulation in IBD, thus providing a novel idea for further research on colitis and experimental evidences for novel potential therapeutic target in IBD.

NEW OXPHOS REGULATING COMPOUND – AUPHOS – INCREASES MITOCHONDRIAL COMPLEXES EXPRESSION IN MURINE AND HUMAN INTESTINAL EPITHELIAL CELLS

Abstract INTRODUCTION Mitochondrial dysfunction in intestinal epithelial cells (IEC) is a critical driver in the failure to heal chronic ulceration in IBD(1). OXPHOS is important for differentiation and cell polarity in repairing IEC(2,3). The development of new IBD drugs that improve mitochondria function would potentially accelerate mucosal repair and enhance overall clinical management of refractory patients. For this study, we tested the efficacy of a novel oral agent (AuPhos) that localized to mitochondria where it increases respiration (Wempe et al CCC, 2022) and mitochondrial energy production. METHODS Colonic IEC proteins from control and AuPhos-treated DSS mice were examined as described elsewhere (Wempe et al CCC, 2022). Both human biopsy and tissues from mice with reduced IEC mitochondria [VilCre/mTmG/TFAMfl/fl (IEC-TFAM-/-)](Abomhya et al CCC 2022) were incubated with AuPhos (0.2; 0.5; 1 μM for biopsies and 0.5 μM for mice) or vehicle for 3 hrs. at +4oC with rotation. N=4 mice/group. RESULTS In membranous IEC fractions of control mice, AuPhos increased protein expression of mitochondrial complexes (Mito-Cpx) (V – 50%, III – 52%, IV – 40%). DSS treatment decreased baseline expression dramatically (V – 62%, III – 56%, II - 69%; IV – 75%). AuPhos in DSS mice increased expression of complex V by 48%, III – 35%, II – 68%, IV – 55% {A,B}. Expression of transcriptional factors PGC1a and Nrf2, which regulate mitochondria biogenesis, were increased in nuclear IEC fractions of DSS mice by 50% and 43% respectively {C,D}. Mito-Cpx and TFAM levels increased in AuPhos-treated human biopsies in a dose dependent manner {E}. AuPhos normalized mitochondrial complex levels in tissue (3h) taken from mice with attenuated mitochondrial respiration (IEC-TFAM-/-). CONCLUSION AuPhos increases expression of mitochondrial complexes in human and in two different murine models. The results of biochemical signaling studies suggest increased mitochondrial respiration enhances PGC1a and Nrf2 levels which are upstream of TFAM, the major transcription factor for mitochondrial biogenesis. Our lab is currently investigating this novel compound as a means for improving mucosal metabolism and accelerating mucosal repair in IBD. (Wempe et al CCC, 2022) 1 Kugathasan, S. et al. Prediction of complicated disease course for children newly diagnosed with Crohn’s disease: a multicentre inception cohort study. Lancet389, 1710-1718, doi:10.1016/S0140-6736(1730317-3) (2017). 2 Rodriguez-Colman, M. J. et al. Interplay between metabolic identities in the intestinal crypt supports stem cell function. Nature543, 424-427, doi:10.1038/nature21673 (2017). 3 Schell, J. C. et al. Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism. Nat Cell Biol19, 1027-1036, doi:10.1038/ncb3593 (2017).

OXPHOS DRIVES CRYPT FISSIONING REQUIRED FOR HEALING OF IBD ULCERS

Abstract INTRODUCTION The failure of ulcers healing is a characteristic feature of IBD. Mitochondrial dysfunction in intestinal epithelial cells (IEC) occurs in IBD and is linked to worse clinical outcomes in pediatric Crohn’s and ulcerative colitis (Kugrathasan et al Lancet 2017, Haberman et al Nat Comm 2019). Cellular polarization and maintenance of cell polarity are energy-dependent and highly regulated by mitochondrial biogenesis (Hardie DG et al Genes Dev 2011). Yet, it is unclear how mitochondria participate in mucosal repair. METHODS To model mitochondrial deficiency, TFAM, a transcriptional factor needed for mitochondrial DNA (mtDNA)-encoded genes was silenced in human colonoids (shTFAM) and VilCre/mTmG/TFAMfl/fl (IEC-TFAM-/-) mice. Levels of crypt branching and intestinal stem cell (ISC) and mitochondrial gene mRNA levels were assessed in colonoids cultured in Wnt-containing (WENR) media. Effects on IEC mitochondrial complex levels and oxygen consumption rates (OCR) were assessed in control IEC-TFAM-/- mice and effects on disease activity and ulcer healing assessed in DSS colitis IEC-TFAM-/- mice (fed DSS x 7d, peak ulcer healing-D13-15). Dual florescence confocal microscopy examined crypt fissioning and IHC in WT (Tom+) and Tfamfl/fl (GFP+) crypts during windows of peak ulcer healing. RESULTS shTFAM-treated colonoids expressed 55-92% lower mitochondrial gene mRNA (Cox6A1, mt-CO1, ATP5A1) levels (p&amp;lt;0.01) {A} and displayed reduced size (35%) and budding (86%) compared to WT cells (p&amp;lt;0.005) {B}. Studies on IEC from IEC-TFAM-/- mice revealed lower mitochondrial complex protein levels {C i-iv}, and &amp;gt;65% lower mtDNA (p&amp;lt;0.01) {D} compared to controls that correlated with 43 and 65% lower IEC OCR (using Oroboros). In DSS mice, levels of ulceration were greater in IEC-TFAM-/- mice (25%) compared to WT (7.5%) (P&amp;lt;0.005). More importantly, levels of crypt fissioning were 85% lower in TFAM-deficient (GFP+) compared to WT (Tom+) crypts {E}. WT fissioning crypt stained for SOX9 {F} and nuclear pβCat552 {G}. CONCLUSION Together, these results suggest that IEC mitochondrial deficiency as seen in IBD patients, impairs crypt fissioning during ulcer healing through a process that impairs cooperation between Wnt and PI3K signaling pathways. The data argue that therapeutic improvement of mitochondrial function may enhance mucosal repair in IBD by promoting crypt fissioning needed to re-establish barrier function.

Ginsenosides Rd monomer inhibits proinflammatory cytokines production and alleviates DSS-colitis by NF-κB and P38MAPK pathways in mice

Background Ulcerative colitis (UC) is dramatically increasing worldwide, cannot be thoroughly cured, and reduces patients’ quality of life. Excessive activation of macrophages and over-production of cytokines play an important role in the pathogenesis of UC. Therefore, for its treatment, inhibiting macrophages' hyperactivation would be effective to develop new treatment approaches. Ginsenosides, extracted from ginseng, show an anti-inflammatory effect on the immunologic process. Our study used ginsenosides Rd monomer (GRd) to intervene in DSS-induced colitis mouse models and tested the immunological effect of macrophages. Method We observed body weights, weights of colons, colonic lengths, and inflammatory scores, as well as histological changes of DSS/DSS-GRd mice. We also isolated intestinal and peritoneal macrophages, performed qRT-PCR and ELISA to detect cytokines production by macrophages, and screened possible involved pathways by Western blotting. Results Administering 20 mg/Kg GRd to DSS mice for 7–14 days reduced colonic inflammation. Moreover, both in vivo and in vitro, levels of TNF-α, IFN-γ, IL-6, IL-12/23p40, and IL-17A were all inhibited by GRd at 14 days in intestinal macrophages, and 20 μmol/L GRd at 12 h in peritoneal macrophages, respectively, but longer time made no more benefit. Western blotting showed GRd could decrease expression of pJNK, p-p38, pIκBα, and P65 in nuclear. Conclusions Our data indicate that GRd could down-regulate cytokines production in macrophages and alleviate DSS-colitis in mice, which may be related to NF-κB and P38MAPK pathways. These results suggest that GRd has an anti-inflammatory effect on experimental colitis and may have potential efficacy in the treatment of UC alone or in combination.

A36 THE ROLE OF MICROBIAL INDOLE METABOLITES IN CONTROLLING INFLAMMATORY RESPONSES AND HEALING IN RESPONSE TO DSS-INDUCED COLITIS

Abstract Background Failure to resolve inflammation is often associate with the complications of Crohn’s Disease (CD). The pregnane X receptor (PXR), a xenobiotic receptor, is recognized for its role in suppressing inflammation and has recently been shown to influence fibrogenesis in the liver. In the intestine, PXR-signaling can be influenced by the microbial tryptophan metabolite indole-3- propionic acid (IPA), which can modulate intestinal inflammation, in turn influencing fibrogenesis, resolution and healing. This suggests that the gut microbiota could modulate mucosal homeostasis and resolution of inflammation via microbial metabolites Aims To understand and characterize the interplay between microbial complexity and the regulation of host inflammatory and healing responses, specifically focusing on the PXR and its microbial metabolite ligand IPA. Methods Intestinal inflammation was induced using DSS (1%, 1.5%, 2% and 3.5%) for 5 days followed by healing for 25 days in C57Bl/6 stably derived moderately diverse mouse microbiota 2 (sDMDMm2) colonized gnotobiotic and C57Bl/6 specific pathogen free (SPF) mice. Inflammation, architectural changes and fibrosis were assessed using Haemotoxylin and Eosin and Masson-Trichrome histological stains. Weight was recorded daily for the first 10 days and every other day after for 25 days, for a total of 30 days. Fecal lipocalin was quantified in samples collected throughout the study to assess inflammation. Innate immune cell influx was measured by flow cytometry, and the microbiota assessed via 16S rRNA sequencing. Results The gnotobiotic sDMDMm2 mice were exquisitely sensitive to DSS-induced colitis, exhibiting significantly increased mortality and morbidity at 2% and 3.5% w/v DSS compared to the SPF group. To elicit the same degree of disease to assess recovery, sDMDMm2 mice were exposed to 1.5% DSS and SPF mice to 3.5% DSS. Following 25 days recovery, sDMDMm2 colonized mice showed increased levels of fecal lipocalin 2, as compared to the SPF mice. DSS-treated sDMDMm2 mice supplemented with IPA during their recovery presented lower levels of fecal lipocalin, similar to colitic SPF mice. IPA supplemented sDMDMm2 mice also exhibited greater overall survival, with no significant differences in neutrophil count compared to mice given H20 during recovery. Conclusions A model system with a less complex microbiota (sDMDMm2) has a higher susceptibility to acute inflammation and a diminished capacity to resolve said inflammation. Addition of the microbial metabolite IPA normalized the recovery of the sDMDMm2 colonized mice, to a response indistinguishable from SPF mice, while also increasing survival. These data highlight the importance of microbial complexity in the regulation of intestinal mucosal homeostasis. Funding Agencies CAG, CCC, CIHR

A127 THE ROLE OF ENDOGENOUS OPIOIDS IN MITIGATING OPIOID TOLERANCE IN A PRE-CLINICAL MODEL OF IBD

Abstract Background While opioid drugs are used to treat pain in IBD patients, escalating doses can induce tolerance and paradoxical increased pain signaling. Endogenous opioids are released by immune cells during chronic colitis and have an analgesic action but it is unknown whether they can mitigate the development of tolerance to opioid drugs in IBD. Aims To evaluate whether endogenous opioids released during chronic inflammation prevent the development of tolerance and/or increased pain signaling following chronic morphine treatment. Methods Three groups of C57BL/6 mice were studied: 1) control, 2) chronic colitis induced with 3 cycles of 2% DSS (cDSS mice) and 3) chronic DSS mice injected with escalating doses of morphine on the last 7 days of DSS (10, 20, 30 mg/kg for the first 3 days, then 40mg/kg on 4 to 7 day) (cDSS-morphine mice). Ex vivo afferent nerve recordings were obtained from flat-sheet distal colon preparations (day 31) to determine their mechanosensitivity by probing with a 1g von Frey hair before and after acute morphine (1μM, 10 min perfusion). In parallel experiments, supernatants were obtained from the proximal colon and incubated overnight with isolated dorsal root ganglia (DRG) neurons from control mice. Neuronal excitability was then examined by measuring the rheobase using perforated patch clamp. Results In afferent nerve recordings, acute application of 1μM morphine inhibited the colonic afferent response to probing in control mice (17.86 vs 12.31 Hz; p&amp;lt;0.05), whereas no effect of morphine was seen in cDSS mice. In cDSS-morphine mice acute morphine perfusion paradoxically increased the mean afferent response to probing (13.99 vs 17.28 Hz, p&amp;lt;0.05). Single units were analyzed to identify how many mechanosensitive responses were increased, decreased or not affected by acute morphine perfusion. Compared to control mice, cDSS mice exhibited increased numbers of unaffected units (6/18 vs 1/14) whereas cDSS-morphine mice had decreased number of inhibited units (1/17 vs 11/14) and increased number of excited units (8/17 vs. 2/14) (Figure 1). In patch clamp recordings, colonic supernatants from cDSS mice reduced the excitability of DRG neurons (rheobase increased 28%; p&amp;lt;0.05), however, this effect was lost in neurons incubated with cDSS-morphine supernatants. Conclusions Endogenous opioids released during cDSS colitis do not mitigate tolerance to chronic morphine exposure and may diminish the response to acute morphine. Moreover, the paradoxical hyperexcitable response to acute morphine induced by chronic morphine was not blocked by endogenous opioids. Our patch clamp data suggest that endogenous opioid actions are lost following chronic morphine exposure and could result from inhibition of their release from immune cells. Funding Agencies CCC

TLR4‐driven Lymphatic Alterations during IBD are driven in PAMP/DAMP Specific Manners

BackgroundInflammatory bowel disease (IBD) is characterised by both acute and chronic phase inflammation of the gastro‐intestinal (GI) tract that affect a large and growing number of people worldwide with little to no effective treatments. This is in part due to the lack of understanding of the disease pathogenesis and also the currently poorly described involvement of other systems such as the lymphatics. During DSS induced colitis, mice also develop a severe inflammation of both the colon and terminal ileum with many features similar to IBD. As well as inflammation within the intestinal tract we have previously demonstrated lymphatic remodelling within the mesentery and mesenteric lymph nodes of DSS‐treated mice. The lymphatic remodelling includes lymphangiogenesis, lymphatic vessel dilation and leakiness, as well as cellular infiltration into the surrounding tissue and peripheral draining lymph nodes. The aim of the investigation was to delineate the contribution of PAMP‐ and/or DAMP‐driven lymphatic remodelling and inflammation during disease, in respect to Toll‐like receptor 4 (TLR4) activity.MethodsIntestinal inflammation was induced in C57BL/6 mice by administration of 2.5% DSS in drinking water for 7 days. Mice were treated with TLR4 blocker C34 or Polymyxin‐B (PMXB) daily from days 3–7 of DSS treatment via I.P. injection, and their effects on disease activity and lymphatic function were examined. TLR4 activity and subsequent effect on lymphangiogenesis, lymphadenopathy, and mesenteric lymph node (MLN) cellular composition were assessed.ResultsDSS Mice treated with TLR4 inhibitor, C34, had a significantly improved disease phenotype characterised by reduced ileal and colonic insult. The change correlated with significant reduction in colonic and mesenteric inflammation, resolved mesenteric lymphangiectasia, and CD103+ DC accumulation similar to that of healthy control. PMXB treatment did not resolve inflammation within the colon, or associated mesenteric lymphatic dysfunction, but did however prevent lymphadenopathy within the MLN through alteration of CCL21 gradients and CD103+DC migration.ConclusionsTLR4 appears to mediate several changes within the mesenteric lymphatics, more specifically it is shown to have different outcomes whether stimulation occurs through pathogen derived factors such as LPS or tissue derived DAMPs, a novel phenomenon.Support or Funding InformationThis study was supported by the Lymphedema Research and Education Program, Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary to P.‐Y.v.d.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.