Tight Junction Proteins Research Articles

Genetically engineered bacteria expressing IL-34 alleviate DSS-induced experimental colitis by promoting tight junction protein expression in intestinal mucosal epithelial cells.

The intestinal mucosa of ulcerative colitis patients expresses high levels of interleukin 34, and mice lacking IL-34 have more severe DSS-induced experimental colitis. There are no studies on the effects of directly upregulating intestinal IL-34 on experimental colitis in mice. The bacteria EcN/CSF-1 and EcN/IL-34, which express CSF-1 and IL-34, respectively, were genetically engineered from Escherichia coli Nissle 1917 (EcN). Colitis mice received daily gavage of sterile PBS buffer, empty plasmid E. coli (EcN/WT), EcN/CSF-1, or EcN/IL-34. Each group of mice was assessed for body mass, clinical signs, DAI, intestinal mucosal permeability, pathological, and immunohistological changes. In vitro, NCM460 cells were treated with CSF-1 or IL-34 recombinant proteins in the presence of signaling pathway inhibitors to evaluate tight junction protein expression. Additionally, intestinal mucosal epithelial cells isolated from active UC patients were analyzed for IL-34 and tight junction protein levels. DSS-induced colitis mice are protected by EcN/IL-34 gavage. Pathological results showed that EcN/IL-34 group colonic histological injury was significantly improved and tight junction protein ZO-1 and Occludin expression increased. In NCM460 cells, IL-34 also increased tight junction protein expression. More importantly, expression of IL-34 was positively correlated with the level of tight junction protein expression in epithelial cells of UC patients. EcN/IL-34 can directly act on damaged intestinal mucosa, up-regulate IL-34 expression, and promote tight junction protein expression in intestinal mucosal epithelial cells to alleviate experimental colitis in mice. IL-34 may be a potential therapeutic target for ulcerative colitis, and genetically engineered bacteria carrying the cytokine may offer new ideas for treating UC.

Sodium butyrate prevents lipopolysaccharide induced inflammation and restores the expression of tight junction protein in human epithelial Caco-2 cells.

The gastrointestinal (GI) tract is susceptible to damage under high altitude hypoxic conditions, leading to gastrointestinal discomfort and intestinal barrier injury. Sodium butyrate, a short-chain fatty acid present as a metabolite in the gut, has emerged as a promising therapeutic agent due to its ability to act as an immunomodulatory agent and restore intestinal barrier integrity. This study aimed to explore the mechanism by which sodium butyrate exhibits anti inflammatory effect on intestinal epithelial cells. In vitro, Caco-2 epithelial cells and RAW 264.7 macrophages were used to investigate the protective role of sodium butyrate on Lipopolysaccharide (LPS) induced inflammation. Cell viability assays demonstrated that 1mM (110.86μg/mL) of sodium butyrate did not exhibit cytotoxicity on cells in vitro. Treatment with sodium butyrate suppressed reactive oxygen species levels and TNF-α production in LPS-stimulated macrophages, indicating its efficacy in mitigating inflammatory responses. Western blot analysis revealed that sodium butyrate attenuated the expression of iNOS in RAW 264.7 macrophage cells. Moreover, sodium butyrate also reversed the LPS induced over expression of HIF-1α, NLRP3, IL-1β as well as NF-kB in Caco-2 epithelial cells and also had a suppressive effect on IL-8 secretion after LPS stimulation. Immunocytochemistry demonstrated that sodium butyrate enhanced tight junction protein occludin expression in Caco-2 cells while also restoring the decreased permeability of the Caco-2 monolayer due to LPS. These results indicate that sodium butyrate may influence immune responses by suppressing inflammatory mediators and improving the integrity of the epithelial barrier. Understanding the intricate interactions between gut metabolites and host immune responses may help in the development of innovative therapeutic strategies to alleviate intestinal inflammation in high altitude environments.

The potential of supplementing compound organic trace elements at lower levels in Chinese yellow- feathered broiler diets, part II: Impacts on growth performance, gut health, intestinal microbiota, and fecal mineral excretion.

This study aimed to investigate the effects of reducing inorganic trace minerals (ITM) by supplementing compound organic trace minerals (OTM) chelates on growth performance, fecal mineral excretion, intestinal health, and cecal microbiota of yellow-feathered broilers. A total of 960 one day old male broilers were randomly assigned to 6 treatments, among which birds were fed with the basal diets (negative control, NC), or supplemented with 1,000 mg/kg (positive control, PC), 300, and 500 mg/kg ITM or OTM, respectively. Dietary supplementation of OTM significantly increased the average daily gain (ADG) during 22-53 d and 1-53 d, and reduced the fecal emissions of Fe, Cu, Zn, and Mn of Chinese yellow-feathered broilers (P < 0.05). Furthermore, the OTM300 group significantly reduced the crypt depth in the duodenum, and increased the ratio of villus height to crypt depth (V/C) in the duodenum and jejunum (P < 0.05). The mRNA expression of TGF-β, Bcl-2, CAT, and GPX4 as well as tight junction proteins (occludin, ZO-1, claudin-1, and claudin-5) in jejunum mucosa were significantly increased by compound OTM when comparing with ITM300 group (P < 0.05). Moreover, dietary compound OTM significantly changed the Chao1 index and β diversity index of cecal microbiota of Chinese yellow-feathered broilers. The abundances of Firmicutes (phylum), Eubacterium_coprostanoligenes_group (family) and Oscillibacter (genus) were increased, while the abundances of Bacteroidetes (phylum) and Rikenellaceae RC9 group (genus) were decreased by OTM treatment. Spearman correlation analysis showed that the mRNA of occludin and jejunal V/C ratio were positively correlated with the abundance of Firmicutes (phylum), but negatively correlated with the abundance of Bacteroidota (phylum). In addition, the abundance of Eubacterium_coprostanoligenes_group (family) was positively correlated with the mRNA of claudin-1, Bcl-2, and TGF-β. PICRUST prediction of microbial function revealed that OTM treatment enriched the pathways related to amino acid metabolism and DNA replication. In conclusion, dietary supplementation at lower levels of compound OTM to replace ITM could improve growth performance and intestinal health, and reduce the fecal excretion of trace elements by modulation of cecal microbiota community and diversity in Chinese yellow-feathered broilers.

Long-term effects of Nε-carboxymethyllysine intake on intestinal barrier permeability: Associations with gut microbiota and bile acids.

Advanced glycation end products (AGEs) in processed foods are closely linked to intestinal injury. However, the long-term effects of exposure to free Nɛ-carboxymethyl lysine (CML), a prevalent AGE molecule, on intestinal barrier integrity have been rarely evaluated. This study investigated the temporal effects of CML exposure on intestinal barrier permeability in C57BL/6N mice at diet-related doses over 12, 14, and 16weeks. No significant changes were observed at 12weeks, but CML exposure significantly increased intestinal permeability at 14 and 16weeks, accompanied by elevated serum LPS levels, colonic histological damage, and reduced tight junction protein expression at 16weeks. CML exposure also altered gut microbiota composition and intestinal bile acid (BA) profiles, specifically reducing TDCA, GDCA, and GCDCA levels. Given the important role of colonic BA receptor signaling in maintaining the intestinal barrier integrity, the impact of CML on BA receptor signaling was assessed. CML exposure significantly downregulated BA receptor TGR5-YAP signaling in mice, while no significant effects were observed in vitro, suggesting that the changes observed in TGR5-YAP signaling in vivo may not result from the direct effects of CML. Spearman's correlation analysis revealed strong associations between altered gut microbiota, BA levels, TGR5-YAP signaling, and intestinal barrier injury. This study highlighted the chronic health risks of long-term CML intake and provided new insights into the links between CML-induced intestinal toxicity, gut microbiota, BA profiles, and BA receptor signaling.

Understanding glucose metabolism and insulin action at the blood-brain barrier: Implications for brain health and neurodegenerative diseases.

The blood-brain barrier (BBB) is a highly selective, semipermeable barrier critical for maintaining brain homeostasis. The BBB regulates the transport of essential nutrients, hormones, and signaling molecules between the bloodstream and the central nervous system (CNS), while simultaneously protecting the brain from potentially harmful substances and pathogens. This selective permeability ensures that the brain is nourished and shielded from toxins. An exception to this are brain regions, such as the hypothalamus and circumventricular organs, which are irrigated by fenestrated capillaries, allowing rapid and direct response to various blood components. We overview the metabolic functions of the BBB, with an emphasis on the impact of altered glucose metabolism and insulin signaling on BBB in the pathogenesis of neurodegenerative diseases. Notably, endothelial cells constituting the BBB exhibit distinct metabolic characteristics, primarily generating ATP through aerobic glycolysis. This occurs despite their direct exposure to the abundant oxygen in the bloodstream, which typically supports oxidative phosphorylation. The effects of insulin on astrocytes, which form the glial limitans component of the BBB, show a marked sexual dimorphism. BBB nutrient sensing in the hypothalamus, along with insulin signaling, regulates systemic metabolism. Insulin modifies BBB permeability by regulating the expression of tight junction proteins, angiogenesis, and vascular remodeling, as well as modulating blood flow in the brain. The disruptions in glucose and insulin signaling are particularly evident in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, where BBB breakdown accelerates cognitive decline. This review highlights the critical role of normal glucose metabolism and insulin signaling in maintaining BBB functionality and investigates how disruptions in these pathways contribute to the onset and progression of neurodegenerative diseases.

A phase 1/2 study evaluating the safety and efficacy of autologous TAC T cells in subjects with claudin 18.2+ advanced solid tumors.

828 Background: Claudin18.2 (CLDN18.2) is a tight junction protein found in gastric epithelial cells. It can become abnormally expressed in gastric cancer and other solid tumors, rendering it a candidate for targeted therapy. There are no approved therapeutic agents against CLDN18.2. The T cell antigen coupler (TAC) technology modifies T cells ex vivo , allowing cytotoxicity of tumor cells by co-opting the natural T cell receptor. TAC T cells demonstrate a safer profile than chimeric antigen receptor T cells. TAC01-CLDN18.2 is an autologous T-cell product comprising T cells expressing CLDN18.2 TAC. Methods: In this first-in-human study (NCT05862324), subjects will undergo leukapheresis and may receive bridging anticancer therapy during cell manufacturing. Prior to TAC01-CLDN18.2 infusion, subjects will undergo lymphodepletion chemotherapy. In Phase I, TAC01-CLDN18.2 will be administered at increasing doses (3 cohorts) in adult subjects with ≥2 lines of prior therapy (1 for subjects with pancreatic ductal adenocarcinoma (PDAC)) using the classic 3+3 dose escalation study design. CLDN18.2 expression levels will be determined centrally using a validated clinical trial assay. Dose-limiting toxicities (DLTs) will be assessed up to 28 days after the infusion. A second dose may be administered according to preidentified clinical and safety criteria. In Phase II, dose expansion groups will evaluate the efficacy, safety, and pharmacokinetics of the optimal TAC01-CLDN18.2 dose, with the option of redosing. Indications will include gastric and esophageal adenocarcinoma (group A), PDAC (group B) and ovarian and non-small cell lung cancer (group C) in subjects with &lt;4 lines of prior therapy. Results: The first two dose cohorts of Phase I have been completed, with no reported dose-limiting toxicities (DLT). All of the TAC-related adverse events were low-grade. No cytokine release syndrome was reported. One subject in cohort 2 experienced a grade 1 neurotoxicity, which resolved the same day without intervention. Six subjects reported a total of 6 serious adverse events, none related to TAC01-CLDN18.2. An 83.3% disease control rate was observed at first tumor assessment (Day 29). One subject in cohort 1 with heavily pre-treated PDAC (3 prior lines) with high CLDN18.2 expression has an ongoing, confirmed partial response. The subject received a second dose and is still on treatment (6 months). Conclusions: Treatment with the lower doses of TAC01-CLDN18.2 is safe and shows promising clinical activity in a heavily pre-treated cancer population. Treatment of cohort 3 has begun. Clinical trial information: NCT05862324 .

Soybean bioactive peptide supplementation improves gut health and metabolism in broiler chickens.

This study aimed to investigate the effects of soybean bioactive peptide (SBP) on the growth performance and intestinal health of yellow-feathered broilers and to further elucidate the regulatory mechanisms of intestinal health using multi-omics analysis. A total of 320 1-day-old yellow-feathered broilers were randomly divided into two groups, with 10 replicates per group and 16 birds per replicate. Broilers in the control group received the basal diet, and those in the experimental group (SBPG) received the basal diet with 0.2 % SBP replacing the same amount of soybean meal. The experiment lasted for 70 d. The results showed that, compared with those in the control group, the final body weight and average daily gain of SBPG broilers were significantly higher (P < 0.05), and the feed conversion ratio was significantly lower (P < 0.05). Notably, SBP significantly improved gut health in chickens, including increased intestinal villus height, decreased levels of proinflammatory factors, such as IL-1β and interferon-γ, and upregulated expression of tight junction proteins, such as ZO-1 and occludin. In addition, transcriptome sequencing results revealed that broilers in the SBP group exhibited significant enrichment in multiple metabolic pathways, including fatty acid metabolism, fatty acid degradation, and the biosynthesis of unsaturated fatty acids (P < 0.05). Cecal 16S rRNA sequencing showed that SBPG increased the abundance of the butyrate-producing beneficial bacteria Muribaculaceae. Subsequent cecal metabolome analysis also revealed that SBPG enhanced lipid-related metabolic pathways, such as alpha-linolenic acid metabolism and GPI-anchor biosynthesis. In conclusion, SBP is a potential feed additive that can improve intestinal morphology, enhance intestinal immunity and barrier function, optimize the structure of the intestinal microbiota, and enhance metabolic function.

Lactic acid bacteria reduce polystyrene micro- and nanoplastics-induced toxicity through their bio-binding capacity and gut environment repair ability

Microplastics and nanoplastics (MNPs) are emerging environmental contaminants that have received significant attention in recent years. Currently, there are more studies on the toxic effects of MNPs exposure on animals (especially aquatic organisms and mammals), but data on the reduction of toxic effects caused by MNPs exposure are still very limited. Lactic acid bacteria (LAB), recognized as safe food-grade microorganisms, possess the capability to bioconjugate harmful substances. In this experiment, we chose lactic acid bacteria (LAB) with different binding capacities to MNPs in vitro to intervene in MNPs-exposed mice to investigate the reducing effect on the toxicity caused by MNPs exposure. Our study showed that LAB with a high intercalation capacity with MNPs in vitro were more effective in alleviating the toxicity caused by MNPs exposure. Notably, Lactobacillus plantarum DT22, despite its low inter-adsorption with MNPs, played a pivotal role in upregulating the relative expression of tight junction proteins and modulating the intestinal microbiota. Thus, LAB strains' mitigation of MNPs toxicity extends beyond bio-binding; their capacity to repair the damaged gut environment is also crucial. LAB strains are proposed as a dietary intervention to reduce MNPs-induced toxicity.

Abdominal LIPUS Stimulation Prevents Cognitive Decline in Hind Limb Unloaded Mice by Regulating Gut Microbiota.

Weightlessness usually causes disruption of the gut microbiota and impairs cognitive function. There is a close connection between gut microbiota and neurological diseases. Low-intensity pulsed ultrasound (LIPUS) has a beneficial effect on reducing intestinal inflammation. So we wondered if abdominal LIPUS stimulation can have a positive impact on weightlessness induced cognitive decline by reducing intestinal dysfunction. The findings revealed that the hind limb unloaded mice exhibited evident disruption in intestinal structure and gut microbial homeostasis, along with impairment in their learning and memory capabilities. However, 4-week abdominal LIPUS treatment improved intestinal function in hind limb unloaded mice, characterized by upregulation of tight junction proteins ZO-1 and Occludin expression in the colon, increased diversity and abundance of intestinal microbiota, decreased serum lipopolysaccharide (LPS), and increased short chain fatty acids in colon contents. The hind limb unloaded mice treated with LIPUS exhibited heightened activity levels, improved exploratory tendencies, and significantly enhanced learning and memory faculties, and elevated expression of neuroadaptation-related proteins such as PSD95, GAP43, P-CREB, BDNF, and its receptor TRKB in the hippocampus. Furthermore, the hind limb unloaded mice receiving fecal transplants from the mice whose abdomens were irradiated with LIPUS displayed enhanced cognitive abilities and improved intestinal structure, akin to the outcomes observed in hind limb unloaded mice who received LIPUS abdominal treatment directly. The above results indicate that LIPUS enhances intestinal structure and microbiota, which helps alleviate cognitive impairment caused by weightlessness. LIPUS could be a potential strategy to simultaneously improve gut dysfunction and cognitive decline in astronauts or bedridden patients.

Bisdemethoxycurcumin and Curcumin Alleviate Inflammatory Bowel Disease by Maintaining Intestinal Epithelial Integrity and Regulating Gut Microbiota in Mice.

Curcuminoids, found in turmeric (Curcuma longa L.), include curcumin (CUR), demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). Although CUR and DMC are well-studied, the anti-inflammatory effects of BDMC remain less explored. Recent studies highlight BDMC's stronger NF-κB inhibition compared to CUR and DMC in cell models, along with its ability to target pathways associated with inflammatory bowel disease (IBD) in DSS-induced colitis mice, reflected by lower disease activity scores and reduced inflammation. This study assessed CUR and BDMC in a DSS-induced colitis mouse model. Dietary administration of CUR or BDMC strengthened tight junction (TJ) proteins, reduced inflammatory cytokine secretion, and attenuated intestinal inflammatory protein expression, thereby alleviating DSS-induced IBD in mice. Furthermore, gut microbiota and short-chain fatty acid analyses revealed that CUR and BDMC effectively regulated gut microbial imbalance and promoted the relative abundance of butyrate-producing bacteria. Furthermore, CUR showed low absorption and was primarily excreted in feces, while BDMC had higher absorption levels. In conclusion, while both BDMC and CUR have potential as adjunct therapies for IBD, BDMC at a concentration of 0.1% showed strong anti-inflammatory effects and enhanced TJ proteins, suggesting that BDMC, even at lower concentrations than CUR, holds promising therapeutic potential and prospects.

Lactobacillus rhamnosus GG Combined with Metformin Alleviates Alcohol-Induced Liver Inflammation in Mice by Maintaining the Intestinal Barrier and Regulating Treg/Th1 Cells.

Disturbances of the intestinal barrier enabling bacterial translocation exacerbate alcoholic liver disease (ALD). Lactobacillus rhamnosus GG (LGG) has been shown to exert beneficial effects in gut dysbiosis and chronic liver disease. The current study assessed the combined effects of LGG and metformin, which play roles in anti-inflammatory and immunoregulatory processes, in alcohol-induced liver disease mice. A diet comprising 5% alcohol for 4 weeks was employed to develop an alcohol-induced liver injury model. Mice were orally administered LGG, metformin, or their combination on alternate days. Tight junction (TJ) proteins, gut microbiome composition, inflammatory cytokines, Jun N-terminal kinase (JNK), and p38 signals were assessed. When compared with treatment with LGG or metformin alone, combined LGG and metformin treatment substantially lowered the symptoms of inflammation, steatosis, and elevated liver enzymes caused by alcohol administration. Combination treatment significantly improved intestinal microecology, evidenced by the recovery of intestinal flora, TJ proteins, and intestinal villi. Combination treatment reduced hepatic inflammation by blocking p38 and JNK phosphorylation. The combination of LGG and metformin corrected immune-response dysregulation and improved ALD by enhancing the intestinal microbiome, restoring mucosal barrier integrity, modulating immune function, and decreasing liver injury. These results provide information for the development of intestinal microbiota-based preventive and therapeutic agents against ALD.

Tea Polyphenols Relieve the Fluoride-Induced Oxidative Stress in the Intestinal Porcine Epithelial Cell Model

Prolonged excessive intake of fluoride (F) can result in fluorosis, leading to a range of tissue oxidative damages. Therefore, mitigating the oxidative stress induced by fluorosis has become a significant research concern. Consequently, how to relieve oxidative stress caused by fluorosis is an urgent matter. In the present study, intestinal porcine epithelial (IPEC-J2) cells were chosen to explore the underlying mechanism of tea polyphenols (TPs) on F-induced oxidative stress. The results show that the cytotoxicity of IPEC-J2 cells induced by F presented a dose-dependent manner according to cell viability. Additionally, F treatment inhibited the activity of T-SOD, CAT, and GSH-Px as well as their transcription levels, increased the reactive oxygen (ROS) formation and cell damage rates, and then promoted cell apoptosis through the results of TUNEL and mitochondrial membrane potential detection when compared with the IPEC-J2 cells from the control group. As the main antioxidant ingredient in tea, TPs alleviated F-induced cell oxidation and apoptosis via blocking F-induced ROS generation and LDH’s release, as well as promoting the transcription of tight junction (TJ) proteins and the activities of antioxidant enzymes in IPEC-J2 cells. These results provide a new treatment strategy for F-induced intestinal oxidative impairment.

Effects of Methylglyoxal on Intestine and Microbiome Composition in Aged Mice.

Methylglyoxal (MGO), a highly reactive precursor of advanced glycation end products (AGEs), is endogenously produced and prevalent in various ultra-processed foods. MGO has emerged as a significant precursor implicated in the pathogenesis of type 2 diabetes and neurodegenerative diseases. To date, the effects of dietary MGO on the intestine have been limited explored. Thus, this study investigates the impact of prolonged oral administration of MGOs on gut health in aged mice. Aged mice received MGO chronically (100 mg/kg/day) for 4 weeks. Intestinal samples were analyzed using RT-PCR and immunohistochemistry for proinflammatory cytokines, permeability markers, and tight junction proteins. 16S rRNA gene-based microbiome analysis was also performed to characterize microbiome composition and its metabolic potential. MGO treatment induced notable alterations at the intestinal level, characterized by an increased formation of MGO-glycated proteins with a concurrent induction of a pro-inflammatory status and reduced expression and delocalization of zonulin-1 and occludin, tight junction proteins. Changes in intestinal morphology were also observed, including hyperproliferation of Paneth cells and an augmented thickness of the intestinal mucus layer, as indicated by immunohistochemical data from MGO-treated mice. Investigation into the microbiota composition revealed that MGO is effective in selectively modifying its composition and metabolic pathways. A decreased abundance of bacterial genera associated with the production of acetic and butyric acids (i.e. Harryflintia, Intestinimonas and Ruminococcaceae genera) and a substantial increase in Lachnospiraceae and Akkermansia genera were found in MGO-treated mice. These findings highlight how dietary MGO can affect intestinal balance, providing valuable insights into the potential links between glycotoxins, gut microbiota, and overall gut functionality.

P0221 The role of caffeine and its metabolites in the modulation of intestinal inflammation and epithelial barrier integrity

Abstract Background Methylxanthine caffeine and its metabolites are widely consumed bioactive compounds that are known to influence various physiological processes 1. However, their impact on gut health, particularly in modulating inflammation and epithelial permeability, remains underexplored. In this study we aimed to investigate the effects of caffeine and its primary metabolites paraxanthine, theobromine and theophylline on inflammatory responses and barrier integrity in gut epithelial models, providing insights into their potential role in gastrointestinal health in the context of Inflammatory bowel disease (IBD). Methods Primary screening of different concentrations of caffeine and its metabolites paraxanthine, theobromine and theophylline was performed in Caco-2 cells. Selected metabolites were further tested on inflamed 3D intestinal epithelial organoids derived from ulcerative colitis patient (n = 1) and non-IBD individual (n = 1). Inflammation was induced for 24 h using TNF-α and IFN-γ cytokine mix. The effects on inflammation status and epithelial barrier integrity were tested through targeted gene expression analysis using RT-PCR, while the effects on generation of reactive oxygen species were investigated by Flow cytometry and DHE assay. Results Primary screening revealed that both caffeine and its metabolite paraxanthine affected the expression of genes encoding inflammatory cytokines (TNF-α, CXCL9; p &amp;lt; 0.05) and tight junction proteins (OCLN, ZO-1, CLDN1; p &amp;lt; 0.05) and were selected for further analysis in 3D intestinal organoids. Neither caffeine nor paraxanthine reduced the expression of inflammatory cytokine-encoding genes in inflamed intestinal organoids. However, both metabolites significantly restored the expression of tight junction protein-coding genes OCLN, ZO-1 and CLDN1 (p &amp;lt; 0.05). A tendency of reduction in reactive oxygen species production (by 11 %) was also observed after exposure with paraxanthine. Conclusion Methylxanthine caffeine and its primary metabolite paraxanthine might have a positive effect on gut epithelial barrier function through restoration of the expression of tight junction protein-coding genes. Study was funded by the European Union and the State Secretariat for Education, Research and Innovation (SERI) (project: miGut-Health, grant no. 101095470). References Reddy V. S. et al. (2024) Pharmacology of caffeine and its effects on the human body. European Journal of Medicinal Chemistry Reports, 10 (100138). https://doi.org/10.1016/j.ejmcr.2024.100138.

DOP116 Preclinical characterization of IMU-856, an orally available epigenetic modulator of gut barrier function and regeneration

Abstract Background A dysfunctional intestinal mucosal barrier is a key pathophysiological hallmark and driver of a broad range of gastrointestinal diseases, including inflammatory bowel disease (IBD). To date, there are no adequate treatment strategies to ameliorate impaired barrier function. IMU-856 is an orally available and systemically acting small molecule modulator of sirtuin 6 (SIRT6), a histone/non-histone protein deacetylase and transcriptional regulator that targets the restoration of intestinal barrier function and regeneration of bowel epithelium without having any direct effect on the immune system. A double-blind, randomized, placebo-controlled phase 1/1b clinical trial of IMU-856 has been completed [1]. IMU-856 was shown to be safe and well-tolerated with a benign adverse event profile and pharmacokinetics that allow once-daily dosing. In Part C, a ‘proof of concept study’ in patients with celiac disease, IMU-856 demonstrated positive effects in four key dimensions of clinical outcome: protecting gut architecture, improving patients' symptoms severity, biomarker response and enhancing nutrient absorption. Methods In the presented preclinical studies, the target specificity and effects of IMU-856 on intestinal barrier function and regeneration were elucidated by in-vitro sirtuin activity assays, transepithelial electrical resistance (TEER) assays, dextran sulphate sodium (DSS)-induced colitis mouse models and gene expression analysis (bulk RNA sequencing). Moreover, potential immune suppressive effects of IMU-856 were assessed in stimulated human peripheral blood mononuclear cells (PBMCs). Multiple biomarkers related to health of intestinal epithelium and of enteric function were also assessed in preclinical studies and in the phase 1 trial. Results IMU-856 is a highly selective and potent modulator of SIRT6. In TEER assays, IMU-856 treatment enhanced the epithelial barrier recovery of Caco-2 monolayers after a destructive cytokine challenge and modulated tight junction protein levels. IMU-856 demonstrated activity in acute DSS-induced colitis models by reducing the severity of colitis, improving the mucosal architecture and preserving functional enterocytes (upregulation of enterocyte marker genes and the plasma biomarker citrulline). No effects of IMU-856 on T-cell proliferation and cytokine secretion (interferon-g (IFN-g)) were observed in human PBMCs. Conclusion IMU-856 data indicate restoration of intestinal barrier function and promotion of physiological mucosal regeneration without direct immunosuppressive effects and may therefore offer potential for the treatment of IBD and other gastrointestinal diseases with compromised barrier function.

Lentinan's effect on gut microbiota and inflammatory cytokines in 5-FU-induced mucositis mice

Chemotherapeutic therapies for cancer are frequently associated with cytotoxic side effects that can be harmful to human health, including the development of intestinal mucositis (IM). It mostly affects the gastrointestinal tract, causing ulceration, inflammation, and the formation of lesions in the colon. Surprisingly, despite the frequency of IM, therapeutic choices remain restricted. In our search for new intestinal mucositis therapies, we wanted to see how Lentinan (LT), derived from Lentinus edodes, would fare in mouse models of intestinal mucositis. To create the intestinal mucositis model in mice, we gave them intra-peritoneal doses of 5-fluorouracil (5-FU) (50 mg/kg) and then tested the effects of Lentinan on intestinal mucositis. This examination required constant monitoring of several factors, such as body weight fluctuations, food consumption, and diarrhea. In addition, we measured the levels of certain inflammatory cytokines (Tumour Necrosis Factor-alpha (TNF-α), Interleukin-1 (IL-1), Interleukin-6 (IL-6), and Interleukin-10 (IL-10), looked at the expression of tight junction proteins (Zonula Occludens-1(ZO-1), Claudin-1), measured mucin-2 levels, and looked into changes in the gut flora. In the mouse model of intestinal mucositis, our findings showed that LT effectively reduced weight loss, increased food intake, and relieved diarrhea. Concurrently, we saw a decrease in the expression of inflammatory cytokines such as TNF-α, IL-1, and IL-6, as well as a considerable increase in the concentration of IL-10. Furthermore, LT reduced intestinal mucositis by increasing the length and structural integrity of the colon. Furthermore, increased expression of tight junction proteins (ZO-1, Claudin-1), mucin-2, and an increase in the number of goblet cells all confirmed our previous findings. Notably, the makeup of beneficial bacteria in the stomach increased as well. Finally, our findings suggest that LT can effectively prevent 5-fluorouracil-induced intestinal mucositis in mice by improving immune function, restoring intestinal barrier integrity, and rebalancing gut microbial flora.

Genome analysis of Bifidobacterium adolescentis and investigation of its effects on inflammation and intestinal barrier function

Numerous studies have confirmed that gut microbiota is a key driver in the occurrence and progression of inflammatory bowel disease (IBD). Based on the bacterial collection constructed in our previous studies, we founded that Bifidobacterium adolescentis AF91-08b2A has the potential beneficial function. We designed cohort studies, genomic studies and animal experiments to further explore the probiotic function of Bifidobacterium adolescentis AF91-08b2A and its therapeutic effect on IBD. The depletion of B. adolescentis in individuals with IBD suggested its significance for intestinal health. Genomic analysis highlighted the probiotic attributes of B. adolescentis AF91-08b2A, including resistance to antibiotics and stress, and metabolic pathways related to energy and carbohydrate metabolism, which are likely to enhance its therapeutic efficacy. In DSS-induced mice colitis model, the strain significantly enhanced the disease activity index (DAI), curbed weight loss, and attenuated colonic damage. It effectively modulated the immune response by reducing the levels of pro-inflammatory cytokines such as IL-6, IL-1β, IL-17A, IFN-γ, and TNF-α, while promoting the secretion of anti-inflammatory cytokines like IL-4, IL-10, and TGF-β1. The restoration of tight junction proteins ZO-1, occludin, and claudin-2 by B. adolescentis AF91-08b2A demonstrated its capacity to safeguard the intestinal epithelial barrier. Collectively, our findings indicate B. adolescentis AF91-08b2A as a valuable therapeutic option for UC, with its multifaceted approach to reducing inflammation and fortifying the intestinal barrier.

P0132 ACSS2 inhibition alleviates inflammatory signaling and improves epithelial integrity in Inflammatory Bowel Disease in vitro models

Abstract Background Inflammatory bowel disease (IBD), encompassing Ulcerative Colitis (UC) and Crohn’s Disease (CD), is marked by chronic gastrointestinal inflammation stemming from genetic, environmental, immunological, and microbiota factors1. Acetyl-CoA Synthetase Short Chain Family Member 2 (ACSS2) converts acetate into acetyl-CoA and is primarily cytosolic, but translocates to the nucleus under metabolic stress, where it regulates gene transcription and cellular processes2. Although the gastrointestinal microbiota influences acetate levels, the role of ACSS2 in colitis and intestinal inflammation remains poorly understood3. Methods To evaluate ACSS2's role in IBD, we employed in vitro IBD models using Caco-2 and HT-29 intestinal epithelial cell lines. ACSS2 activity was inhibited with an ACSS2 inhibitor, followed by inflammatory stimulation with 100 ng/ml Tumor Necrosis Factor-α (TNF-α) for 30 minutes or 1% Dextran Sodium Sulfate (DSS) for 6 hours. Protein expression levels were assessed via Western blot and immunohistochemistry (IHC) on paraffin-embedded samples from UC patients. The effects of ACSS2 inhibition on inflammatory signaling pathways and cell integrity were assessed using RT-qPCR, immunoblotting, and transepithelial electrical resistance (TEER) measurements. Results Treatment of Caco-2 and HT-29 cells with DSS or TNF-α induced upregulation of ACSS2 protein levels compared to controls. IHC analysis of 30 paired paraffin-embedded colon samples from UC patients revealed higher ACSS2 expression and nuclear localization in inflamed sections compared to non-inflamed regions. Inhibition of ACSS2 activity in stimulated Caco-2 and HT29 cells led to a significant decrease in the gene expression levels of pro-inflammatory cytokines such as Interleukin 8 (IL-8), IL-1β and TNF-α. Additionally, immunoblotting demonstrated reduced phosphorylation of p65 and ERK/MEK, indicating suppression of NF-κB and MAPK inflammatory signaling pathways. To assess the effect of ACSS2 inhibition on cell integrity, TEER assays revealed that ACSS2 inhibition preserved cell integrity in DSS-treated monolayers, accompanied by increased expression of tight junction proteins Occludin and ZO-1, suggesting improved epithelial integrity in the inflammatory context. Conclusion Our findings demonstrate a pro-inflammatory role for ACSS2 in IBD, mediated through its involvement in inflammatory signaling and epithelial integrity. These results highlight ACSS2 as a potential therapeutic target for IBD and warrant further investigations in more advanced models, including human-derived intestinal organoids and in vivo systems, to validate its role in disease pathogenesis and therapeutic potential.

Blockade of A2AR improved brain perfusion and cognitive function in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder that affects more than 6.2 million Americans aged 65 and older, particularly women. Along with AD's main hallmarks (formation of β-amyloid plaques and tau neurofibrillary tangles), there are vascular alterations that occurs in AD pathology. Adenosine A2 receptor (A2AR) is one of the key factors of brain vascular autoregulation and is overexpressed in AD patients. Our previous findings suggest that protein arginine methyltransferase 4 (PRMT4) is overexpressed in AD, which leads to decrease in cerebral blood flow in aged female 3xTg mice. We aimed to investigate the mechanism behind A2AR signaling in the regulation of brain perfusion and blood-brain barrier integrity in age and sex-dependent 3xTg mice, and if it is related to PRMT4. Istradefylline, a highly selective A2AR antagonist, was used to modulate A2AR signaling. Aged female 3xTg and C57BL/6J mice were evaluated for brain perfusion (via laser speckle) and cognitive function (via open field, T-maze and novel object recognition). Our results suggest that modulation of A2AR signaling in aged female 3xTg increased cerebral perfusion by decreasing PRMT4 expression, restored the levels of APP and tau, maintained blood-brain barrier integrity by maintaining the expression of tight junction proteins, and preserved functional learning/memory.

P1350 The Role of oral Porphyromonas gingivalis and Its Outer Membrane Vesicles in Inflammatory Bowel Disease

Abstract Background The etiology of Inflammatory bowel disease (IBD) remains elusive, but emerging evidence suggests a potential link between oral microbiota and IBD. Porphyromonas gingivalis(Pg.), a major pathogen in periodontal disease, and its outer membrane vesicles(OMVs) may play significant roles in the development and progression of IBD. Methods Animal models of IBD were established to observe the impact of Pg. and its OMVs on intestinal inflammation. In vitro cell culture was conducted to explore the interaction between Pg.-OMVs and intestinal epithelial cells and immune cells. Results Through the analysis of salivary microbiota in IBD patients and healthy individuals, we observed a significant increase in the abundance of Pg. in the saliva of IBD patients. OMVs may serve as a novel medium for oral bacteria to communicate with the gut. Our findings indicate that Pg.-OMVs can promote the development of IBD through various mechanisms. Firstly, they can disrupt intestinal barrier function by promoting inflammation in intestinal epithelial cells and degrading tight junction proteins. Additionally, Pg.-OMVs carry pathogenic factors and pro-inflammatory molecules, which activate intestinal macrophages to polarize towards the M1 phenotype, inducing inflammatory responses. Beyond systemic effects, we discovered that the key virulence factor LPS of Pg. can stimulate oral epithelial cells to secrete exosomes. These exosomes exhibit pro-inflammatory properties, including the disruption of the intestinal mucosal barrier and the promotion of macrophage polarization towards the M1 phenotype, with subsequent secretion of inflammatory factors. Conclusion Porphyromonas gingivalis and its outer membrane vesicles play important roles in the pathogenesis of IBD. Understanding these mechanisms provides new insights into the relationship between oral health and IBD and may lead to the development of novel diagnostic and therapeutic strategies for IBD.