Dextran Sodium Sulfate Exposure Research Articles

FGL2 improves experimental colitis related to gut microbiota structure and bile acid metabolism by regulating macrophage autophagy and apoptosis

Inflammatory bowel disease (IBD) is a refractory disease with immune abnormalities and pathological changes. Intestinal macrophages are considered to be the main factor in establishing and maintaining intestinal homeostasis. The immunoregulatory and anti-inflammatory activity of fibrinogen-like protein 2 (FGL2) can regulate macrophage polarization. However, its function in IBD is unclear. In this study, we explored the effect of FGL2 on macrophage polarization, autophagy, and apoptosis in bone marrow-derived macrophages (BMDMs) treated with lipopolysaccharide (LPS) and further investigated changes in the intestinal barrier, flora, and bile acid in dextran sodium sulfate (DSS)-treated mice. Our results demonstrated that FGL2−/− weakened ERK signaling to promote M1 polarization and upregulate inflammation, autophagy, and apoptosis in LPS-stimulated BMDMs. rFGL2 treatment reversed these effects. FGL2−/− mice exhibited higher sensitivity to DSS exposure, with faster body weight loss, shorter colon lengths, and higher disease activity index (DAI) values. rFGL2 treatment protected against experimental ulcerative colitis (UC), restrained excessive autophagy, apoptosis, and improved gut barrier impairment. Gut microbiota structure and bile acid homeostasis were more unbalanced in FGL2−/− DSS mice than in wild-type (WT) DSS mice. rFGL2 treatment improved gut microbiota structure and bile acid homeostasis. Altogether, our results established that FGL2 is a potential therapeutic target for IBD.

Neuropeptide substance P attenuates colitis by suppressing inflammation and ferroptosis via the cGAS-STING signaling pathway.

Neuropeptide substance P (SP) belongs to a family of bioactive peptides and regulates many human diseases. This study aims to investigate the role and underlying mechanisms of SP in colitis. Here, activated SP-positive neurons and increased SP expression were observed in dextran sodium sulfate (DSS)-induced colitis lesions in mice. Administration of exogenous SP efficiently ameliorated the clinical symptoms, impaired intestinal barrier function, and inflammatory response. Mechanistically, SP protected mitochondria from damage caused by DSS or TNF-α exposure, preventing mitochondrial DNA (mtDNA) leakage into the cytoplasm, thereby inhibiting the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway. SP can also directly prevent STING phosphorylation through the neurokinin-1 receptor (NK1R), thereby inhibiting the activation of the TBK1-IRF3 signaling pathway. Further studies revealed that SP alleviated the DSS or TNF-α-induced ferroptosis process, which was associated with repressing the cGAS-STING signaling pathway. Notably, we identified that the NK1R inhibition reversed the effects of SP on inflammation and ferroptosis via the cGAS-STING pathway. Collectively, we unveil that SP attenuates inflammation and ferroptosis via suppressing the mtDNA-cGAS-STING or directly acting on the STING pathway, contributing to improving colitis in an NK1R-dependent manner. These findings provide a novel mechanism of SP regulating ulcerative colitis (UC) disease.

Microalgae polysaccharides exert antioxidant and anti-inflammatory protective effects on human intestinal epithelial cells in vitro and dextran sodium sulfate-induced mouse colitis in vivo

Microalgae polysaccharides (MAPS) have emerged as novel prebiotics, but their direct effects on intestinal epithelial barrier are largely unknown. Here, MAPS isolated from Chlorella pyrenoidosa, Spirulina platensis, and Synechococcus sp. PCC 7002 were characterized as mainly branched heteropolysaccharides, and were bioavailable to Caco-2 cells based on fluorescein isothiocyanate labeling and flow cytometry analysis. These MAPS were equally effective to scavenge hydroxyl and superoxide radicals in vitro and to attenuate the H2O2-, dextran sodium sulfate-, tumor necrosis factor α-, and interleukin 1β-induced burst of intracellular reactive oxygen species and mitochondrial superoxide radicals, interleukin-8 production, cyclooxygenase-2 and inducible nitric oxide synthase expression, and/or tight junction disruption in polarized Caco-2 cells. MAPS and a positive drug Mesalazine were intragastrically administered to C57BL/6 mice daily for 7 d during and after 4-d dextran sodium sulfate exposure. Clinical signs and colon histopathology revealed equivalent anti-colitis efficacies of MAPS and Mesalazine, and based on biochemical analysis of colonic tight junction proteins, goblet cells, mucin 2 and trefoil factor 3 transcription, and colonic and peripheral pro-inflammatory cytokines, MAPS alleviated dextran sodium sulfate-induced intestinal epithelial barrier dysfunction, and their activities were even superior than Mesalazine. Overall, MAPS confer direct antioxidant and anti-inflammatory protection to intestinal epithelial barrier function.

Chronic gut inflammation impairs contextual control of fear

Chronic inflammatory diseases are highly comorbid with anxiety in humans. The extent to which chronic inflammation is responsible for this relationship remains to be determined. We therefore tested the hypothesis that prolonged, but not brief, gut inflammation is sufficient to evoke anxiety-related behaviours in mice. We used the discriminative fear to context conditioning paradigm to assess fear generalization, which is a prominent feature of anxiety disorders. Gut inflammation was induced by exposure to dextran sodium sulfate (DSS) in the drinking water, a well-established rodent model of ulcerative colitis evoking prolonged inflammation. Neither acute (1 × 5 day cycle) nor chronic (3 × 5 day cycles) exposure to DSS affected fear responses when tested shortly after conditioning. Mice in all groups generated more fear responses (freezing) in a chamber previously paired with mild shock, as compared to a chamber with no pairing. This suggests DSS exposure had no effect on acquisition or expression of conditioned fear. Acute and control animals showed this same contextual control of freezing when tested 9 days later. In contrast, at this remote time point, the chronically treated animals exhibited increased freezing in the unpaired chamber such that freezing was equivalent in both contexts. These animals, however, showed intact preference for the unpaired chamber when allowed to freely move between chambers. These data suggest that some mnemonic process engaged after training, such as memory consolidation, is affected by past chronic inflammation so as to generalize negative associations and engage fearful responding in inappropriate contexts, despite intact knowledge that the chambers have different affective associations sufficient for place preference.

Effects of non‐caloric artificial sweeteners on naïve and dextran sodium sulfate‐exposed Drosophila melanogaster

AbstractSaccharin and sucralose are worldwide used tabletop non‐caloric artificial sweeteners (NAS), and although they are considered metabolically inert and safe for consumption, recent studies observed an NAS‐mediated modulation of the intestinal microbiota, which may result in the development of intestinal inflammatory diseases. The present study investigated the effects of NAS alone and in combination with dextran sodium sulfate (DSS), a chemical known to induce gut inflammation, on parameters of overall fitness including longevity and climbing ability of female Drosophila melanogaster. DSS‐exposed flies were subjected to saccharin or sucralose, and their effects on survival and the gene expression levels of different antimicrobial peptides (AMPs) were assessed. Saccharin supplementation significantly increased the lifespan of D. melanogaster, while DSS significantly decreased the survival in a dose‐dependent manner. Additionally, DSS exposure of flies resulted in a trend toward an up‐regulation of the mRNA levels of the AMPs, drosocin and metchnikowin. This effect was more pronounced in the NAS‐supplemented group reaching significance. This may indicate that NAS supplementation improves intestinal immune reactions during an inflammatory metabolic state.

A High-Fat Western Diet Attenuates Intestinal Changes in Mice with DSS-Induced Low-Grade Inflammation

BackgroundA Western diet (WD) is associated with increased inflammation in the large intestine, which is often ascribed to the high dietary fat content. Intestinal inflammation in rodents can be induced by oral administration of dextran sodium sulfate (DSS). However, most studies investigating effects of WD and DSS have not used appropriate low-fat diets (LFDs) as control. ObjectivesTo compare the effects of a WD with those of an LFD on colon health in a DSS-induced low-grade colonic inflammation mouse model. MethodsSix-week-old male C57BL/6JRj mice were fed an LFD (fat = 10.3% energy, n = 24) or a WD (fat = 41.2% energy, n = 24) for 15 wk [Experiment 1 (Exp.1)]. Half the mice on each diet (n = 12) then received 1% DSS in water for 6 d with the remainder (n = 12 in each diet) administered water. Disease activity, proinflammatory genes, inflammatory biomarkers, and fecal microbiota (16S rRNA) were assessed (Exp.1). Follow-up experiments (Exp.2 and Exp.3) were performed to investigate whether fat source (milk or lard; Exp.2) affected outcomes and whether a shift from LFD to WD 1 d prior to 1% DSS exposure caused an immediate effect on DSS-induced inflammation (Exp.3). ResultsIn Exp.1, 1% DSS treatment significantly increased disease score in the LFD group compared with the WD group (2.7 compared with 0.8; P < 0.001). Higher concentrations of fecal lipocalin (11-fold; P < 0.001), proinflammatory gene expression (≤82-fold), and Proteobacteria were observed in LFD-fed mice compared with the WD group. The 2 fat sources in WDs (Exp.2) revealed the same low inflammation in WD+DSS mice compared with LFD+DSS mice. Finally, the switch from LFD to WD just before DSS exposure resulted in reduced colonic inflammation (Exp.3). ConclusionsHerein, WDs (with milk or lard) protected mice against DSS-induced colonic inflammation compared with LFD-fed mice. Whether fat intake induces protective mechanisms against DSS-mediated inflammation or inhibits establishment of the DSS-induced colitis model is unclear.

Dandelion root extracts abolish MAPK pathways to ameliorate experimental mouse ulcerative colitis.

Dextran sodium sulfate (DSS)-triggered ulcerative colitis (UC) model in animals provides a valuable platform to preclinically evaluate the outcome of drug candidates for UC. Dandelion root extracts (DRE) have a therapeutic effect on UC. However, the protective mechanism of DRE against UC remains unknown. To discover the targeting pathway involved in DRE-induced protection against UC. The UC model was developed in C57BL/6 mice by oral administration of DSS. Following DSS exposure, sulfasalazine (SASP), low dose of DRE (DRE-L), moderate dose of DRE (DRE-M), high dose of DRE (DRE-H), and DRE-H plus mitogen-activated protein kinases (MAPK) agonist (DRE-H+MA) were administered to the mice. Colon Mucosal Damage Index (CMDI) and histopathological analysis were used to evaluate the colonic mucosal damage. The cytokine levels were detected using commercial enzyme-linked immunosorbent assay (ELISA) kits. The MAPK pathway activation was determined with western blotting. We found that DRE-H attenuated DSS-triggered colonic mucosal damage. The DSS-induced inflammatory responses and oxidative stress in the bloodstream and colon tissues were dramatically inhibited by DRE-H administration. Also, this plant impaired DSS-provoked phosphorylation levels of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), p38 mitogen-activated protein kinases (p38), p65, and IκB. More importantly, MAPK agonist, BIM-23A760, removed the protective effect of DRE-H on the bloodstream and colon tissues. The DRE-H is capable of relieving DSS-induced UC, and its mechanism links to the MAPK pathways.

Intestinal Epithelial AMPK Deficiency Causes Delayed Colonic Epithelial Repair in DSS-Induced Colitis.

Dysfunctions in the intestinal barrier, associated with an altered paracellular pathway, are commonly observed in inflammatory bowel disease (IBD). The AMP-activated protein kinase (AMPK), principally known as a cellular energy sensor, has also been shown to play a key role in the stabilization and assembly of tight junctions. Here, we aimed to investigate the contribution of intestinal epithelial AMPK to the initiation, progression and resolution of acute colitis. We also tested the hypothesis that protection mediated by metformin administration on intestinal epithelium damage required AMPK activation. A dextran sodium sulfate (DSS)-induced colitis model was used to assess disease progression in WT and intestinal epithelial cell (IEC)-specific AMPK KO mice. Barrier integrity was analyzed by measuring paracellular permeability following dextran-4kDa gavage and pro-inflammatory cytokines and tight junction protein expression. The deletion of intestinal epithelial AMPK delayed intestinal injury repair after DSS exposure and was associated with a slower re-epithelization of the intestinal mucosa coupled with severe ulceration and inflammation, and altered barrier function. Following intestinal injury, IEC AMPK KO mice displayed a lower goblet cell counts with concomitant decreased Muc2 gene expression, unveiling an impaired restitution of goblet cells and contribution to wound healing process. Metformin administration during the recovery phase attenuated the severity of DSS-induced colitis through improvement in intestinal repair capacity in both WT and IEC AMPK KO mice. Taken together, these findings demonstrate a critical role for IEC-expressed AMPK in regulating mucosal repair and epithelial regenerative capacity following acute colonic injury. Our studies further underscore the therapeutic potential of metformin to support repair of the injured intestinal epithelium, but this effect is conferred independently of intestinal epithelial AMPK.

P080 ADS024 attenuates weight loss and decreases disease activity in DSS-induced colitis

Abstract Background Ulcerative colitis (UC) is a chronic, idiopathic inflammatory disease characterised by a dysregulated innate and adaptive immune response to gut microbiota that contributes to the perpetuation of colonic inflammation. ADS024 is an orally administered live biotherapeutic product (LBP), comprised of a single strain of Bacillius velezensis, being developed for potential application in the treatment of patients with UC. Methods Colitis was induced in 39 mice by exposure to 3% dextran sodium sulfate (DSS)-treated drinking water from Day 0 to Day 5; 6 control animals did not receive DSS. Animals were then dosed with vehicle (PBS; n=15) or ADS024 (n=12) twice daily for 14 days via oral gavage or with comparator anti-p40 mAb (n=12) once daily (IP) on Days 6, 9, 12, 15. All animals were monitored daily for body weight loss, diarrhea, blood in stool, and activity level. Each of these parameters was individually scored according to the disease activity index (DAI) scoring scheme. Animals underwent video endoscopy on Day 19 to assess colitis severity and stool consistency was scored during endoscopy. Following endoscopy on Day 19, all surviving animals were sacrificed, and blood was collected and processed for serum and analysed for metabolomic profile. Results Animals in which colitis was induced by exposure to 3% DSS demonstrated significant weight loss, most profoundly from Days 8 to 11, an increase in the composite DAI and increased mean colon weight:length ratios as compared to control animals. Animals treated with ADS024 had improved body weight, starting at 72 hours post-treatment and decreased DAI scoring over the course of the study compared to the vehicle control group (p&amp;lt;0.01 and p&amp;lt;0.05 respectively), comparable to treatment with anti-p40 mAb which has a mechanism of action similar to ustekinumab (Stelara®) approved for the treatment of UC. A trend towards a decrease in the mean colon weight:length ratio was observed in animals treated with ADS024, but there was no difference in mean endoscopy scoring compared to the vehicle control group. DSS treatment altered the serum biochemical profile at day 19 and ADS024 induced changes suggesting a reversion towards the control state. Conclusion Oral treatment with ADS024 improved body weight loss, reduced DAI, and resulted in a trend towards reduction in mean colon weight:length ratios in the DSS-induced mouse colitis model. In addition, ADS024 treatment led to a rebalancing of the systemic metabolomic shift caused by DSS exposure.

A Novel Combination Therapy Using Rosuvastatin and Lactobacillus Combats Dextran Sodium Sulfate-Induced Colitis in High-Fat Diet-Fed Rats by Targeting the TXNIP/NLRP3 Interaction and Influencing Gut Microbiome Composition.

Inflammasome targeting and controlling dysbiosis are promising therapeutic approaches to control ulcerative colitis. This report is the first to investigate the mechanisms underlying the coloprotective effects of rosuvastatin and Lactobacillus and their combined therapy on dextran sodium sulfate (DSS)-induced colitis in high-fat diet (HFD)-fed rats. Our results demonstrate the aggravation of intestinal inflammation as a consequence of an HFD following DSS administration. An association between dyslipidemia, LDL oxidation, CD36 expression, ROS generation, thioredoxin-interacting protein (TXNIP) upregulation, and NLRP3 inflammasome activation was demonstrated by DSS exposure in HFD-fed rats. We demonstrated that rosuvastatin/Lactobacillus significantly suppressed the DSS/HFD-induced increase in colon weight/length ratio, DAI, MDI, and myeloperoxidase, as well as corrected dysbiosis and improved histological characteristics. Additionally, caspase-1 activity and IL-1β-driven pyroptotic activity was significantly reduced. Rosuvastatin/Lactobacillus showed prominent anti-inflammatory effects as revealed by the IL-10/IL-12 ratio and the levels of TNF-α and IL-6. These latter effects may be attributed to the inhibition of phosphorylation-induced activation of NF-κB and a concomitant reduction in the expression of NLRP3, pro-IL-1β, and pro-IL-18. Furthermore, rosuvastatin/Lactobacillus reduced Ox-LDL-induced TXNIP and attenuated the inflammatory response by inhibiting NLRP3 inflammasome assembly. To conclude, rosuvastatin/Lactobacillus offers a safe and effective strategy for the management of ulcerative colitis.

Pharmacokinetic Analysis of Carnosic Acid and Carnosol in Standardized Rosemary Extract and the Effect on the Disease Activity Index of DSS-Induced Colitis.

Rosemary extract (RE) is an approved food preservative in the European Union and contains dietary phytochemicals that are beneficial for gastrointestinal health. This study investigated the effects of RE on dextran sodium sulfate (DSS)-induced colitis and also determined the pharmacokinetics of dietary phytochemicals administered to mice via oral gavage. Individual components of rosemary extract were separated and identified by LC–MS/MS. The pharmacokinetics of two major diterpenes from RE, carnosic acid (CA) and carnosol (CL), administered to mice via oral gavage were determined. Then, the effect of RE pre-treatment on the disease activity index (DAI) of DSS-induced colitis in mice was investigated. The study determined that 100 mg/kg RE significantly improved DAI in DSS-induced colitis compared to negative control. Sestrin 2 protein expression, which increased with DSS exposure, was reduced with RE treatment. Intestinal barrier integrity was also shown to improve via fluorescein isothiocyanate (FITC)–dextran administration and Western blot of zonula occludens-1 (ZO-1), a tight junction protein. Rosemary extract was able to improve the DAI of DSS-induced colitis in mice at a daily dose of 100 mg/kg and showed improvement in the intestinal barrier integrity. This study suggests that RE can be an effective preventative agent against IBD.

Rapamycin Extends Life Span in ApcMin/+ Colon Cancer FAP Model

BackgroundWe previously showed that lifelong rapamycin treatment of short-lived ApcMin/+ mice, a model for familial adenomatous polyposis, resulted in a normal lifespan. ApcMin/+ mice develop colon polyps with a low frequency but can be converted to a colon cancer model by dextran sodium sulfate (DSS) treatments (ApcMin/+-DSS model). Materials and MethodsWe asked, what effect would pretreatment of ApcMin/+ mice with chronic rapamycin prior to DSS exposure have on survival and colonic neoplasia? ResultsForty-two ppm enteric formulation of rapamycin diet exacerbated the temporary weight loss associated with DSS treatment in both sexes. However, our survival studies showed that chronic rapamycin treatment significantly extended lifespan of ApcMin/+-DSS mice (both sexes) by reductions in colon neoplasia and prevention of anemia. Rapamycin also had prophylactic effects on colon neoplasia induced by azoxymethane and DSS in C57BL/6 males and females. Immunoblot assays showed the expected inhibition of complex 1 of mechanistic or mammalian target of rapamycin (mTORC1) and effectors (S6K→rpS6 and S6K→eEF2K→eEF2) in colon by lifelong rapamycin treatments. To address the question of cell types affected by chronic enteric rapamycin treatment, immunohistochemistry analyses demonstrated that crypt cells had a prominent reduction in rpS6 phosphorylation and increase in eEF2 phosphorylation relative controls. ConclusionThese data indicate that enteric rapamycin prevents or delays colon neoplasia in ApcMin/+-DSS mice through inhibition of mTORC1 in the crypt cells.

Microalgae aqueous extracts exert intestinal protective effects in Caco-2 cells and dextran sodium sulphate-induced mouse colitis.

Microalgae are emerging as a good source of natural nutraceuticals. Here, we examined the intestinal protective effects of microalgae aqueous extracts (MAEs) from Chlorella pyrenoidosa, Spirulina platensis, and Synechococcus sp. PCC 7002 in human intestinal epithelial Caco-2 cells and dextran sodium sulphate (DSS)-induced colitis in C57BL/6 mice. MAEs displayed intestinal barrier-protective activities in Caco-2 cells by increasing the expression of heat shock protein (Hsp)-27 and tight junction proteins of occludin and claudin-4 and attenuating the H2O2-induced intracellular reactive oxygen species production, plasma membrane impairment and apoptosis. They also showed anti-inflammatory potential in tumor necrosis factor (TNF)-α-, interleukin (IL)-1β- and H2O2-stimulated Caco-2 cells by suppressing the secretion of IL-8 and the expression of cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS). The 8 d daily intragastric administration of MAEs during and after 4 d DSS exposure effectively alleviated colitis symptoms of weight loss, diarrhea, rectal bleeding, and colon shortening and histopathology, protected intestinal barrier function by increasing colonic Hsp-25, occludin and claudin-4, and attenuated colonic and systemic inflammation by suppressing colonic myeloperoxidase activity, production of TNF-α, IL-1β and IL-6, expression of COX-2 and iNOS, and peripheral leukocytosis, monocytosis and granulocytosis. Microalgae can thus serve as a functional food to maintain gut health.

The impact of gut microbiota manipulation with antibiotics on colon tumorigenesis in a murine model.

It has been suggested that manipulation of gut microbiota using antibiotics can inhibit colitis-associated colorectal cancer (CAC) in a mouse model. We investigated whether timing of gut microbial manipulation using antibiotics affects colon tumorigenesis in the azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CAC model. CAC was induced in C57BL/6 mice by injection of 12.5 mg/kg AOM followed by three rounds of 1.7% DSS exposure. There were six groups based on timing of antibiotic administration. Colonic inflammation, proliferation, and tumorigenesis were evaluated after animal sacrifice. High-throughput sequencing of the mice feces was performed to characterize changes in gut microbiota. Full-time antibiotic treatment significantly decreased the number and size of tumors, histological scores, and expression of pro-inflammatory cytokines compared to the AOM/DSS group without antibiotic treatment. The early and late antibiotic groups, antibiotic administration from the first and second rounds of DSS to the end of the study, showed significantly lower histological scores and tumor burden. In contrast, the pretreatment antibiotic group, antibiotic administration from 3 weeks prior to AOM to the first round of DSS, did not exhibit decreased tumorigenesis. Principal coordinate analysis showed similar gut microbial community structures among the full-time, early, and late antibiotic groups, whereas other groups showed distinct gut microbial profiles. There was a positive correlation between number of tumors and number of operational taxonomic units. Colonic tumorigenesis was attenuated by antibiotic administration, except for that only prior to DSS administration, suggesting that gut microbial changes should be maintained throughout the entire period of inflammation to suppress tumorigenesis.

Cytomegalovirus Infection Exacerbates Experimental Colitis by Promoting IL-23 Production.

Many studies have demonstrated an association between cytomegalovirus (CMV) infection and inflammatory bowel disease (IBD). Moreover, CMV infection is more common in patients with severe or steroid-refractory IBD. However, it is not clarified whether CMV worsens IBD or if it is merely a surrogate marker for IBD. Here, we used the dextran sodium sulfate (DSS)-induced colitis model to investigate if CMV infection exacerbates colitis. The mice were injected intraperitoneally with 10 MOI of murine CMV (MCMV) and thereafter, chronic colitis was induced by one cycle of DSS exposure. Anti-IL-23R mAb at 20 μg/mice and pyrrolidine dithiocarbamate (PDTC), an effective NF-κB inhibitor, at 50 mg/kg were administrated to the mice. The MCMV-infected mice had a shorter colon length and a higher histopathology score than the mock inoculum-treated mice, while anti-IL-23R mAb administration ameliorated the pathological changes. Expression of IL-23, phospho-NF-κB p65, and phospho-IκBα was upregulated in colon tissues of the MCMV-infected mice compared to mock inoculum-treated mice, while treatment with PDTC attenuated colonic IL-23 production. These data demonstrated that CMV infection could accelerate IBD development. This effect may be due to its activation on NF-κB signaling pathway and subsequently IL-23 production.

The impact of dietary fermentable carbohydrates on a postinflammatory model of irritable bowel syndrome

A low fermentable carbohydrate (FODMAP) diet is used in quiescent inflammatory bowel disease when irritable bowel syndrome-like symptoms occur. There is concern that the diet could exacerbate inflammation by modifying microbiota and short-chain fatty acid (SCFA) production. We examined the effect of altering dietary FODMAP content on inflammation in preclinical inflammatory models. C57BL/6 mice were given 3% dextran sodium sulfate (DSS) in drinking water for 5days and recovered for 3weeks (postinflammatory, n=12), or 5days (positive-control, n=12). Following recovery, DSS-treated or control mice (negative-control, n=12) were randomized to 2-week low- (0.51g/100g total FODMAP) or high-FODMAP (4.10g) diets. Diets mimicked human consumption containing fructose, sorbitol, galacto-oligosaccharide, and fructan. Colons were assessed for myeloperoxidase (MPO) activity and histological damage. Supernatants were generated for perforated patch-clamp recordings and cytokine measurement. Cecum contents were analyzed for microbiota, SCFA, and branched-chain fatty acids (BCFA). Data were analyzed by two-way ANOVA with Bonferroni. Inflammatory markers were higher in the positive-control compared with negative-control and postinflammatory groups, but no differences occurred between the two diets within each treatment (MPO P>.99, histological scores P>.99, cytokines P>.05), or the perforated patch-clamp recordings (P>.05). Microbiota clustered mainly based on DSS exposure. No difference in SCFA content occurred. Higher total BCFA occurred with the low-FODMAP diet in positive-control (P<.01) and postinflammatory groups (P<.01). In this preclinical study, reducing dietary FODMAPs did not exacerbate nor mitigate inflammation. Microbiota profile changes were largely driven by inflammation rather than diet. Low FODMAP intake caused a shift toward proteolytic fermentation following inflammation.

Inhibition of myeloid differentiation 1 specifically in colon with antisense oligonucleotide exacerbates dextran sodium sulfate-induced colitis.

Myeloid differentiation 1 (MD-1), also known as lymphocyte antigen 86 (Ly86), is a soluble protein homologous to MD-2 and forms a complex with radioprotective 105 (RP105). RP105/MD-1 complex negatively regulates toll-like receptor 4 (TLR4) signaling and is involved in several immune disorders. However, the precise role of MD-1 in inflammatory bowel diseases (IBD) remains poorly understood. To further investigate the involvement of MD-1 in IBD, we inhibited MD-1 in colon with antisense oligonucleotide (AS-ODN) and assessed the effect of MD-1 inhibition on dextran sodium sulfate (DSS)-induced colitis. We discovered that MD-1 protein expression was remarkably decreased in both patients with ulcerative colitis and mice with DSS-induced colitis. For the first time, we showed that oral administration of MD-1 AS-ODN to mice significantly suppressed the MD-1 protein levels in colon rather than systemic tissues. Subsequently, we found that MD-1 AS-ODN treated mice were more susceptible to DSS-induced colitis based on loss of body weight, colon length, histological scores, and disease activity index. MD-1 inhibition also significantly enhanced inflammatory cytokines production such as IL-6 and IL-1β in colons. Finally, mice treated with MD-1 AS-ODN exhibited increased messenger RNA levels of TLR4 and MyD88 after DSS exposure and showed enhanced nuclear factor (NF)-κB activation compared with the control. Taken together, specifically suppression of MD-1 in colon tissues with AS-ODN exacerbates DSS-induced experimental colitis in mice, which is possibly related to activation of TLR4/NF-κB signaling.

Intestinal NCoR1 Protects against Dextran Sodium Sulfate‐Induced Ulcerative Colitis

Ulcerative colitis (UC), one of the principal types of inflammatory bowel disease (IBD), is characterized as a long‐term condition that results in colon and rectum inflammation. UC affects approximately 1 in 400 people worldwide, with the highest incidence and prevalence in Northern Europe and North America. Most recently, the introduction of genetically modified mouse models and high‐throughput sequencing technology have led to key advances in identifying and characterizing genetic markers and environmental factors associated with the risk for UC. However, the precise, disease‐causing genes and their functions that link gene‐environment interactions in UC initiation have not been identified. The importance of intrinsic NCoR1 in intestinal homeostasis was first discovered in our laboratory when we generated intestinal epithelial cell (IEC) specific NCoR1 deletion mice (NCoR1ΔIEC) and identified the role of NCoR1 in regulating IEC proliferation and enterocyte maturation during neonatal development. To explore NCoR1 function in the context of gastrointestinal disease, we employed dextran sodium sulfate (DSS), a standard agent used to induce experimental UC in both NCoR1F/F (control) and NCoR1ΔIEC mice. Eight‐week old NCoR1F/F and NCoR1ΔIEC mice were given 2.5% (w/v) DSS (40–50 kDa, Affymetrix) in their drinking water for six days, and body weights (BW) were monitored. It is shown that NCoR1F/F mice were minimally affected, while NCoR1ΔIEC mice exhibited profound body weight (BW) loss (p&lt;0.0001, two‐way ANOVA, n=10). The BW difference was observed initially at day 5 after DSS exposure (p&lt;0.01, student's t test, n=10). The greatest weight loss was observed on day 8 (two days after DSS withdrawal) with 18.7% weight loss in NCoR1ΔIEC vs 8.0% in NCoR1F/F mice. After day 8, BW began to recover but NCoR1ΔIEC mice showed slower recovery compared to controls. In H&amp;E staining, the water‐NCoR1ΔIEC mice showed no obvious histological difference from the water‐NCoR1F/F mice. In the DSS treated group, NCoR1ΔIEC mice demonstrated increased disease severity as measured by the histopathological colitis score quantitated based on the presence and severity of ulcerative lesions, disrupted epithelial structure, damaged crypt architecture, and increased inflammatory cell infiltration. Consistently, RT‐qPCR also demonstrated increased mRNA expression of pro‐inflammatory cytokines TNFα, IL‐1β, IL‐6, and IL‐8 in NCoR1ΔIEC mice. To further explore the potential mechanisms, we have carried out BrdU cell proliferation assays. Results demonstrated that BrdU‐positive (BrdU+) cells had increased by approximately 70% in water‐NCoR1ΔIEC mice (n = 5, p&lt;0.05). Following DSS exposure for two days, no significant changes were observed in DSS‐NCoR1F/F mice. However, BrdU+ cells were significantly decreased in NCoR1ΔIEC mice, which maintained approximately 35% BrdU+ cells in comparison to DSS‐NCoR1F/F mice. These results suggest that intrinsic NCoR1 plays an important role in protecting against DSS‐induced UC, particularly the intestinal proliferative cells in mice.Support or Funding InformationSupported by ES024818 and AI135677.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

10 A VARIANT OF CHOLERA TOXIN B SUBUNIT ENHANCES COLON EPITHELIAL WOUND HEALING VIA AN UNFOLDED PROTEIN RESPONSE

Cholera toxin B subunit (CTB) exhibits multifaceted biological activity in the mucosa via binding to GM1 ganglioside on intestinal epithelial cells. We recently found that a recombinant variant of CTB, which contains an artificial KDEL endoplasmic reticulum (ER) retention signal (CTB-KDEL), induces mucosal healing in a mouse dextran sodium sulfate (DSS) acute colitis model. The objectives of this study are to investigate CTB-KDEL’s epithelial wound repair mechanism and characterize its efficacy in preclinical models. We hypothesize that CTB-KDEL’s wound healing effect is mediated through ER retention within epithelial cells, thereby facilitating mucosal healing in IBD. The effects of CTB-KDEL, CTB and G33D-CTB-KDEL (a non-GM1-binding variant) were analyzed for cell migration, subcellular localization, an unfolded protein response (UPR) and TGFβ expression in Caco2 cells. In a DSS acute colitis model, C57BL/6J mice were gavaged with a single dose of CTB-KDEL, CTB or PBS at the end of 7-day DSS exposure. An optimal dosing regimen of CTB-KDEL was evaluated in mice exposed to repeated DSS cycles. Efficacy was measured on the basis of colon length, Disease Activity Index, histopathological scoring, E-cadherin immunohistochemistry and wound healing gene/cytokine expression in the colon. For ex vivohuman colonic explant experiments, colon tissues were obtained from consenting IBD patients who underwent total colectomy, which were cultured with CTB-KDEL, CTB or a PBS for 24 hours. Caco2 cell migration was enhanced by CTB-KDEL at concentrations of 0.3 – 3 μM, while CTB and G33D-CTB-KDEL failed to show such an effect. CTB-KDEL showed longer residence in the ER than CTB, leading to the activation of UPRs, particularly the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) arm, and subsequent TGFβ signaling in Caco2 cells. In the acute colitis model, CTB-KDEL, but not CTB, facilitated recovery from colonic damage as evident in macroscopic and histological evaluations, with significant upregulation of wound healing genes and E-cadherin positive staining in the colon (Fig. 1). Weekly oral administration of 3 μg CTB-KDEL showed significant efficacy against established colitis in the chronic DSS model. In human IBD colon explants, CTB-KDEL at 1 μM induced significant upregulation of the IRE1/Xbp1 pathway as well as wound healing genes including TGFB1and the E-cadherin gene CDH1, along with the retention of viable crypts in the colon. The results demonstrated CTB-KDEL’s unique colon epithelial wound healing activity via a UPR, providing implications for new IBD treatment strategies.

Orally administered gold nanoparticles protect against colitis by attenuating Toll-like receptor 4- and reactive oxygen/nitrogen species-mediated inflammatory responses but could induce gut dysbiosis in mice

BackgroundGold nanoparticles (AuNPs) are attracting interest as potential therapeutic agents to treat inflammatory diseases, but their anti-inflammatory mechanism of action is not clear yet. In addition, the effect of orally administered AuNPs on gut microbiota has been overlooked so far. Here, we evaluated the therapeutic and gut microbiota-modulating effects, as well as the anti-inflammatory paradigm, of AuNPs with three different coatings and five difference sizes in experimental mouse colitis and RAW264.7 macrophages.ResultsCitrate- and polyvinylpyrrolidone (PVP)-stabilized 5-nm AuNPs (Au-5 nm/Citrate and Au-5 nm/PVP) and tannic acid (TA)-stabilized 5-, 10-, 15-, 30- and 60-nm AuNPs were intragastrically administered to C57BL/6 mice daily for 8 days during and after 5-day dextran sodium sulfate exposure. Clinical signs and colon histopathology revealed more marked anti-colitis effects by oral administration of Au-5 nm/Citrate and Au-5 nm/PVP, when compared to TA-stabilized AuNPs. Based on colonic myeloperoxidase activity, colonic and peripheral levels of interleukin-6 and tumor necrosis factor-α, and peripheral counts of leukocyte and lymphocyte, Au-5 nm/Citrate and Au-5 nm/PVP attenuated colonic and systemic inflammation more effectively than TA-stabilized AuNPs. High-throughput sequencing of fecal 16S rRNA indicated that AuNPs could induce gut dysbiosis in mice by decreasing the α-diversity, the Firmicutes/Bacteroidetes ratio, certain short-chain fatty acid-producing bacteria and Lactobacillus. Based on in vitro studies using RAW264.7 cells and electron spin resonance oximetry, AuNPs inhibited lipopolysaccharide (LPS)-triggered inducible nitric oxide (NO) synthase expression and NO production via reduction of Toll-like receptor 4 (TLR4), and attenuated LPS-induced nuclear factor kappa beta activation and proinflammatory cytokine production via both TLR4 reduction and catalytic detoxification of peroxynitrite and hydrogen peroxide.ConclusionsAuNPs have promising potential as anti-inflammatory agents; however, their therapeutic applications via the oral route may have a negative impact on the gut microbiota.