Expression Of IL-6 Research Articles

Cardiac dilation, energy stress and ventricular remodeling: insights from prolonged voluntary exercise in male mice with TAC-induced HFpEF.

Exercise in heart failure with preserved ejection fraction (HFpEF) remains a hot topic, although current treatment strategies have not been shown to improve the long-term prognosis of HFpEF. Previous studies have mostly focused on the roles of endurance training, the mechanisms underlying long-term voluntary exercise have not been elucidated. The purpose of the present analysis was to evaluate alterations in cardiac function in HFpEF mice (HFpEF-Sed) after 6 weeks of voluntary running (HFpEF-Ex), investigate mechanisms, and compare the effects with fluoxetine (HFpEF-FLX). We found that voluntary exercise, instead of fluoxetine intervention, significantly improved left ventricular end-diastolic internal diameter (LVIDd) and the rate of change in anterior wall thickness (ATW) in HFpEF mice. The exercise capacity of HFpEF-Sed mice was significantly reduced, but prolonged voluntary running significantly reversed the expression of myocardial BNP, TNF-α, and IL-6, α-MHC, and β-MHC in HFpEF-Sed mice, along with myocardial fiber disorders accompanied by massive inflammatory cell infiltrates. Importantly, myocardial Complex III and Complex V, Mfn2, Drp1, p62, and LC3 II/I expression in HFpEF-Sed mice were all significantly different from those of normal mice, whereas voluntary exercise significantly reversed these expressions. These findings strongly suggest that long-term voluntary exercise is effective in avoiding acute and chronic energy stress in HFpEF-Sed mice, which is consistent with the mechanism of current first-line treatment for HFpEF. This notion was further supported by electron microscopy results, which showed no pathological features in cardiomyocyte mitochondrial morphology after prolonged voluntary exercise. Additionally, fluoxetine was found to inhibit depressive-like behavior in HFpEF mice.

The cellular response of lipopolysaccharide-induced inflammation in keratoconus human corneal fibroblasts to RB-PDT: Insights into cytokines, chemokines and related signaling pathways.

Rose Bengal Photodynamic Therapy (RB-PDT) offers dual therapeutic benefits by enhancing corneal stiffness and providing antibacterial activity, presenting significant potential for patients with keratoconus complicated by keratitis. Our purpose was to assess the effect of rose bengal photodynamic therapy (RB-PDT) on the expression of pro-inflammatory cytokines and chemokines, as well as on extracellular matrix (ECM)-related molecules, in lipopolysaccharide (LPS)-induced inflammation of keratoconus human corneal fibroblasts (KC-HCFs). Additionally, the involvement of the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways which are downstream of the Toll-like receptor 4 (TLR4) pathway were examined. KC-HCFs were stimulated with varying concentrations of LPS (0-10 μg/ml), which was followed by RB-PDT. The expression levels of interleukin-1β (IL-1β), IL-6, IL-8, interferon alpha 2 (IFNA2), IFNB1, intercellular adhesion molecule 1 (ICAM-1), chemokine (C-C motif) ligand 4 (CCL-4), collagen I, collagen V, lysyl oxidase (LOX), transforming growth factor β 1(TGF-β1) were measured using qPCR, ELISA, or western blot. The activation of the NF-κB and MAPK pathways was assessed using qPCR and western blot. In LPS-induced inflammation of KC-HCFs, the expression of IL-6 was further amplified by the treatment with RB-PDT (p = 0.001). However, the activation of the MAPK and NF-κB pathways did not increase following RB-PDT. Additionally, RB-PDT reduced the transcription of collagen I and collagen V (p≤0.03), while the transcription of LOX and TGF-β1 secretion remained unchanged in KC-HCFs exposed to LPS. In LPS-induced inflammation of KC-HCFs treated with RB-PDT, despite the increased expression of pro-inflammatory cytokines, the activation of the TLR4 signaling pathways is lacking. RB-PDT may have no adverse effects on corneal scar formation of keratoconus corneas in the short term.

Pharmacokinetic and Pharmacodynamic Study of Folic Acid-Modified Chitosan–Stearic Acid Nanomicelles Loaded with Tetrandrine for Rheumatoid Arthritis

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease, and it is currently incurable. Tetrandrine (TET) has an obvious curative effect with therapeutic efficacy on RA, but its use is limited due to its poor water-solubility and bioavailability. Therefore, TET-loaded nanomicelles modified with chitosan, stearic acid, and folic acid (FCST) was prepared in the study, and the pharmacokinetics and pharmacodynamics were studied. Methods: The plasma concentrations of FCST and TET were measured by the PLC-MS/MS method at different times, and the pharmacokinetic parameters were calculated. A collagen-induced arthritis (CIA) model was established with rats. On the 16th day after the first immunization, 50 rats were randomized into five groups with 10 rats in each group according to the arthritis score. The drugs were administered by intraperitoneal injection for 30 days. The swelling degree and joint score of the rats were tested during each administration. In addition, the pro-inflammatory factors IL-1β, IL-6, IL-17, and TNF-α in the serum of the rats were tested by an ELISA kit, and their joints were examined by histopathology. Results: Pharmacokinetic studies showed that the AUC0–72h of FCST was 1.93 times that of TET. FCST demonstrated higher bioavailability compared to TET (p < 0.05). Pharmacodynamic studies demonstrated that FCST had significant anti-inflammatory effects, and its anti-inflammatory activity was stronger compared to the same dose of TET, as evidenced by measuring toe thickness and observing toe appearance. It significantly reduced the expression of IL-1, IL-6, IL-17, and TNF-α in rats with rheumatoid arthritis (p < 0.05). Conclusions: FCST can significantly improve bioavailability and has a significant therapeutic effect on rheumatoid arthritis.

The Potential Roles of IL-1β, IL-6, and RIPK3 in the Pathogenesis of Stevens–Johnson Syndrome/Toxic Epidermal Necrolysis

Background/Objectives: Stevens–Johnson Syndrome and Toxic Epidermal Necrolysis (SJS/TEN) are rare but severe skin conditions, often triggered by medications, that can be life-threatening. These conditions frequently affect the eyes, causing ocular surface disease, which can result in visual impairment or blindness. Although the exact mechanisms behind SJS/TEN remain unclear, key inflammatory mediators such as IL-1β, IL-6, and RIPK3 are believed to play critical roles in inflammation, necroptosis, and regulatory processes. Investigating these factors offers new insights into the disease’s underlying mechanisms and potential targets for treatment. This study aims to determine the roles of IL-1β, IL-6, and RIPK3 in the pathogenesis of SJS/TEN. Methods: The study examined the expression levels of IL-1β, IL-6, and RIPK3 in skin biopsies from patients with biopsy-confirmed SJS/TEN, using lichen planus as a positive control and normal skin as a baseline control. Immunohistochemistry was employed for this analysis. Additionally, the impact of SJS/TEN patient plasma on mitochondrial function was assessed in platelets and human corneal epithelial (H-CET) cells. Using a fluorescent plate reader, mitochondrial activity and superoxide ion levels were measured, comparing plasma from SJS/TEN patients to normal human plasma. Results: Skin biopsies from SJS/TEN patients showed a significantly higher expression of IL-1β, IL-6, and RIPK3 compared to both lichen planus and normal controls. Furthermore, plasma from SJS/TEN patients significantly reduced platelet viability and increased mitochondrial and total cellular superoxide ions, as demonstrated by elevated levels of MitoSOX Red and CellROX Red. Conclusions: These findings suggest that IL-1β, IL-6, and RIPK3 may contribute to the pathogenesis of SJS/TEN and highlight their potential as targets for therapeutic intervention.

Expression of interleukin-17 in oral tongue squamous cell carcinoma and its effect on biological behavior

Tongue squamous cell carcinoma (TSCC) is a common malignant oral cancer characterized by substantial invasion, a high rate of lymph node and distant metastasis, and a high recurrence rate. This study aims to provide new ideas for the diagnosis and treatment of TSCC patients by exploring the related mechanisms that affect the migration and invasion of TSCC and inhibit the migration and spread of cancer cells. The results indicated the rate of high expression of IL-17 in cancer tissues was greater than that in tongue tissues, and the expression of IL-17 was related to the TNM stage. The expression of IL-17 in Cal-27 cells was greater than that in HOEC. With increasing IL-17 concentration, cell proliferation, migration, and invasion increased, and the apoptosis rate decreased. After adding the IL-17 inhibitor, the cell proliferation, invasion, and migration abilities decreased, the apoptosis rate increased, and the expression of JAK1and p-STAT3 decreased.IL-17 is highly expressed in oral tongue squamous cell carcinoma and is involved in the occurrence and development of TSCC, possibly through the JAK‒Stat signaling pathway. This study provides a new target and theoretical basis for treating tongue squamous cell carcinoma.

Amelioration of premature aging in Werner syndrome stem cells by targeting SHIP/AKT pathway

BackgroundPathogenic or null mutations in WRN helicase is a cause of premature aging disease Werner syndrome (WS). WRN is known to protect somatic cells including adult stem cells from premature senescence. Loss of WRN in mesenchymal stem cells (MSCs) not only drives the cells to premature senescence but also significantly impairs the function of the stem cells in tissue repair or regeneration.ResultsIn this study, we profiled the signaling pathways altered in WRN-deficient MSC and applied pharmacological method to activate the AKT signaling in these cells and examined their cellular phenotype related to aging. We found that the AKT signaling in WRN-deficient MSCs was significantly suppressed while the AKT upstream phosphatases (SHIP1/2) were upregulated. Knockdown or inhibition of SHIP1/2 could ameliorate premature senescence in WRN-deficient MSCs. Moreover, SHIP inhibition stimulated MSC proliferation and suppressed expression of pro-inflammatory cytokines IL-6 and IL-8. The stemness of WRN-deficient MSC was also improved upon pharmacological treatments with the inhibitors.ConclusionsThese results suggested that targeting the SHIP/AKT signaling pathway is beneficial to WRN-deficient stem cells and fibroblasts, which might be applied for improving the trophic function of MSC in, for instance, promoting angiogenesis.

Pericytes mediate neuroinflammation via Fli-1 in endotoxemia and sepsis in mice

BackgroundSepsis-associated encephalopathy (SAE) often results from neuroinflammation. Recent studies have shown that brain platelet-derived growth factor receptor β (PDGFRβ) cells, including pericytes, may act as early sensors of infection by secreting monocyte chemoattractant protein-1 (MCP-1), which transmits inflammatory signals to the central nervous system. The erythroblast transformation-specific (ETS) transcription factor Friend leukemia virus integration 1 (Fli-1) plays a critical role in inflammation by regulating the expression of key cytokines, including MCP-1. However, the role of pericyte Fli-1 in neuroinflammation during sepsis remains largely unknown.MethodsWT and pericyte-specific Fli-1 knockout mice were subjected to endotoxemia through LPS injection or sepsis via cecal ligation and puncture (CLP). In vitro, Fli-1 was knocked down using small interfering RNA in cultured mouse brain pericytes, followed by LPS stimulation.ResultsElevated Fli-1 levels were observed in isolated brain pericytes 2 h after LPS administration, in brain tissues 4 h after CLP, and in cultured mouse brain pericytes 2 h after LPS stimulation in vitro. In endotoxemic mice, pericyte-specific Fli-1 knockout reduced expression of MCP-1 and IL-6 in brain tissue 2 h after LPS injection. At 24 h post-LPS administration, protein levels of MCP-1 and IL-6, and microglia activation were suppressed in pericyte-Fli-1 knockout mice. Additionally, Fli-1 deficiency in pericytes significantly reduced MCP-1 and IL-6 mRNA levels in the brain tissue 4 h after CLP. Moreover, in cultured brain pericytes, Fli-1 knockdown markedly decreased MCP-1 and IL-6 levels after LPS stimulation. Notably, LPS stimulation increased Fli-1 levels via TLR4-Myd88 signaling, which subsequently led to elevated production of MCP-1 in brain pericytes.ConclusionsFli-1 in pericytes may serve as a crucial mediator of neuroinflammation during sepsis by directly regulating pivotal cytokines such as MCP-1 and IL-6. Therefore, Fli-1 has the potential to serve as a therapeutic target in SAE and other neuroinflammatory disorders.

Adipose Tissue-Derived Mesenchymal Stem Cells Regulate Th17/Treg in a Rat Model of Allergic Rhinitis by Activating IL-2/JAK3/STAT5 Signaling Pathway.

To investigate how adipose-derived mesenchymal stem cells (ADSCs) regulate the balance between regulatory T cells (Treg) and Th17 cells through the IL-2/JAK3/STAT5 signaling pathway in a rat model of allergic rhinitis (AR). Adipose-derived stem cells (ADSCs) were used to treat an ovalbumin (OVA)-induced AR rat model. The pathological changes and nasal symptoms were observed by HE staining and scanning electron microscopy. The concentrations of OVA-sIgE, IL-10, and IL-17 in serum were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of JAK-3, STAT-5, forkhead box P3 (Foxp3), and retinoic acid-associated orphan receptor γt (RORγt) in nasal mucosa were detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of IL-2, JAK-3, and STAT-5 proteins in nasal mucosa was detected by Western blotting, and the expression patterns of these proteins were analyzed. ADSCs significantly reduced nasal symptoms and histological abnormalities by significantly reducing the levels of OVA-sIgE and IL-10. The expressions of IL-2, JAK-3, STAT-5, IL-2, JAK-3, STAT-5, Foxp3 mRNA, and RORγ t mRNA in the nasal mucosa of AR rats were significantly increased. ADSC can control the development of Treg/Th17 cells by the IL-2/JAK-3/STAT-5 signaling pathway and reduce OVA-induced allergic inflammation.

Human 3D Lung Cancer Tissue Photothermal Therapy Using Zn- and Co-Doped Magnetite Nanoparticles.

Iron oxide-based nanoparticles are promising materials for cancer thermal therapy and immunotherapy. However, several proofs of concept reported data with murine tumor models that might have limitations for clinical translation. Magnetite is nowadays the most popular nanomaterial, but doping with distinct ions can enhance thermal therapy, namely, magnetic nanoparticle hyperthermia (MNH) and photothermal therapy (PTT). In this study, we used a 3D alveolar reconstructed A549 lung cancer tissue model and investigated the thermal properties, toxicity, and impact of the thermal dose on tissue viability and inflammatory response using magnetite codoped with 40% Zn and 2% Co divalent ions. The ZnCo-doped magnetite nanoparticles are not toxic up to an NP concentration of 30 mg/mL. PTT showed a better heat generation response than MNH under the evaluated conditions, while NP showed a high external photothermal conversion efficiency of ∼1.3 g·L-1·cm-1 at 808 nm. PTT study is carried out at different temperatures, 43 and 47 °C, for 15 min. Tissue viability decreased with increasing thermal dose, while intracelullar ROS levels increased, mitochondrial activity decreased, and active caspase-3 increased, suggesting cell death via apoptosis. Nanoparticles and PTT did not influence the cytokine TNF, IL-10, IL-1B, and IL-12p70. In contrast, IL-6 and IL-8 were triggered by NP and PTT. Increased expression of IL-6 and IL-8 with higher thermal doses is correlated with tissue injury results, suggesting the potential role in activating and attracting immune cells to the site of thermal-mediated tissue injury.

Exploring the therapeutic effect of human recombinant IL11 on lesioned OA human osteochondral explants

ObjectiveTo explore IL11 co-expression profiles in our previously reported RNA-sequencing dataset of OA articular cartilage, in interaction with IL6, and to investigate the effects of hrIL11 administration as potential therapeutic strategy for OA articular cartilage using our biomimetic aged human osteochondral explant model of OA.MethodsWe used RNA-sequencing datasets of macroscopically preserved and lesioned OA articular cartilage (N = 35 patients). Spearman correlations were calculated between IL11 and IL6 expression levels and genes expressed in cartilage (N = 20048 genes). Osteochondral explants were isolated from macroscopically preserved and lesioned areas of the joint and were kept in culture for two weeks, with or without exposure to 200ng/ml hrIL11.ResultsWe found no overlap in correlating genes between IL11 and IL6, indicating their distinct roles in articular cartilage. Moreover, we identified more genes being correlated to IL11 in the lesioned compared to preserved articular cartilage (N = 203 and 106 genes, respectively). Upon treatment of ex vivo OA articular cartilage with hrIL11, we overall observed unbeneficial effects on chondrocyte phenotype, as illustrated by upregulation of MMP13, EPAS1, RUNX2, and POSTN. We did not observe significant differences in Mankin scores upon addition of hrIL11.ConclusionThe current study showed that treatment of OA articular cartilage with hrIL11 is unlikely to be beneficial despite previous indications of hrIL11 as potential druggable target. These findings underscore the importance of functionally investigating OA risk genes. Better understanding of IL11 signaling and the underlying pathways is necessary towards the development of OA treatment strategy.

PARP inhibitors differentially regulate immune responses in distinct genetic backgrounds of high-grade serous tubo-ovarian carcinoma models.

Immune checkpoint inhibitors (ICIs) have revolutionized treatment for several tumor indications without demonstrated benefit for ovarian cancer patients. To improve the therapeutic ratio of ICIs in ovarian cancer patients, several different clinical trials are testing combinations with poly (ADP-ribose) polymerase (PARP) inhibitors. Comparing the immunomodulatory effects of clinically advanced PARP inhibitors may help to identify the best partner to combine with ICIs. We examined the treatment effect of talazoparib (a PARP trapper) and veliparib (a solely PARP enzymatic inhibitor) in homologous recombination deficient (HRD) and homologous recombination proficient (HRP) high-grade serous tubo-ovarian carcinoma (HGSC) cell lines on immune-related gene expression. We discovered and validated that CXCL8, IL-6, and TNF gene expression were upregulated after talazoparib treatment in both OVCAR3 (HRD) and CAOV3 (HRP) HGSC cell lines. In contrast, veliparib treatment slightly elevated similar genes exclusively in a HRD HGSC cell line model. We expanded these studies to include olaparib, a PARP trapper less potent than talazoparib, and found effects specific to COV361 (BRCA1 mutant) and OVCAR8 (BRCA1 methylated) HGSC cells but not all HRD HGSC cell lines. Our studies also identified differences among PARP trappers versus veliparib on augmenting CXCL10 expression. Finally, we show that talazoparib modulates the CXCL10 response in cGAS-defective cell lines, independent of the cGAS-STING pathway. These mechanistic studies advance our understanding of how different PARP inhibitors affect the immune system in various genetic backgrounds.

Expression and correlation analysis of VISTA in peripheral blood mononuclear cells of myasthenia gravis patients.

Myasthenia gravis (MG) is a T cell-dependent, B cell-mediated disorder strongly associated with antigen presentation by dendritic cells (DCs). In MG, mucosal tolerance is linked to increased expression of TGF-β mRNA in monocytes. Additionally, monocytic myeloid-derived suppressor cells (M-MDSCs) exhibit negative immunomodulatory effects by suppressing autoreactive T and B cells. In short, all these cells play an important role in the occurrence of MG. V domain-containing Ig suppressor of T-cell activation (VISTA) is an immune checkpoint molecule constitutively expressed on dendritic cells (DCs), CD4+, CD8+ T cells, monocytes and M-MDSCs. Similar to CTLA-4 and PD-1, VISTA controls peripheral tolerance and autoimmune disorder. VISTA expressed on APCs acts as a ligand to suppress the proliferation and cytokine production of both CD4+ and CD8+ T cells. VISTA expressed on CD4+ T cells also suppresses T cell activation in a T-cell autonomous manner. Thus, VISTA may also play an important role in the immune regulation of MG disease. To investigate the role of VISTA in MG pathogenesis, we collected peripheral blood mononuclear cells (PBMCs) from MG patients and detected VISTA expression in different immune cell populations by FACs. VISTA expression was increased in CD4+ T cells, CD8+ T cells, B cells, monocytes, DCs, and M-MDSCs of PBMCs from MG patients. Correlation analysis further demonstrated that VISTA expression in monocytes was correlated with anti-AChR-IgG levels and MG-ADL scores. VISTA expression was positively associated with IL-4, TGF-β, Foxp3, IL-10, and TNF-α mRNA and negatively associated with IL-1β and IL-6 mRNA in MG PBMCs. VISTA can inhibit expression of IL-6 and TNF-α in vitro. These results suggest that VISTA may modulate MG disease progression and indicate the potential utility of VISTA agonists in MG treatment.

A Therapeutic Approach of Chitosan-loaded p-Coumaric Acid Nanoparticles to Alleviate Diabetic Nephropathy in Wister Rats.

This study evaluated the renoprotective effects of p-Coumaric acid nanoparticles (PCNPs) in nephropathic rats. Six groups of male Albino Wistar rats were randomly assigned. Group 1 was the control, while Group 2 received 45 mg/kg of streptozotocin (STZ) to induce diabetic nephropathy. Groups 3, 4, and 5 received STZ (45 mg/kg) along with PCNPs at doses of 20, 40, and 80 mg/kg, respectively. Group 6 received 80 mg/kg of PCNPs without STZ. Body weight, blood glucose, insulin, hemoglobin (Hb), and glycosylated hemoglobin (HbA1c) levels were measured. Blood urea, serum creatinine, kidney antioxidant enzymes, and lipid peroxidation levels were also analyzed. Histological and immunohistochemical studies of kidney tissues were performed. PCNPs (80 mg/kg) significantly reduced serum glucose, creatinine, and urea levels while increasing insulin levels and antioxidant activity in the kidneys. Histological analysis revealed that nephropathic rats exhibited cellular damage, including shrinkage of Bowman's capsule and lesions in the kidneys, along with degeneration in the Islets of Langerhans in the pancreas. PCNPs treatment restored these morphological alterations in the pancreas, liver, and kidneys to near-normal. Furthermore, nephropathic rats had elevated IL-6 and TNF-α expression in the renal tubules and glomeruli, which was reduced following PCNPs treatment. The findings suggest that PCNPs exhibit antihyperglycemic, antioxidant, antiglycation, and renoprotective effects in STZ-induced diabetic nephropathy.

P0033 GPR84, which is increased in Ulcerative Colitis patients, regulates the cytokine secretion of macrophages

Abstract Background Inflammatory Bowel Disease (IBD) is a gastrointestinal disorder comprising Ulcerative Colitis (UC) and Crohn´s Disease (CD) characterized by an exacerbated and chronic immune response. Although GPR84 has emerged as a proinflammatory receptor in some pathologies such as atherosclerosis or reflux esophagitis, its role in IBD remains unclear. We aim to analyze the protein expression of GPR84 in IBD patients and its role in the cytokine secretion in macrophages. Methods Surgical resections from UC (n=5) and CD patients (n=5) were obtained. Healthy colon and ileum from colon-carcinoma patients (n=10) were used as controls. Protein expression of GPR84 and CD68 was analyzed by WesternBlot and immunohistochemistry. U937 monocytes were differentiated into macrophages with PMA (0,01μM) during 48 hours and treated with the GPR84 agonist decanoic acid (50-250μM) and a proinflammatory cocktail (0,1μM LPS + 20ng/mL IFNγ) during 24 hours. Some of them were treated with the GPR84 antagonist (10μM) or transiently silenced with a specific siRNA-GPR84. RNA was isolated and gene expression of pro- and anti-inflammatory cytokines was quantified by qPCR. Cytokine levels in supernatant were also quantified by Luminex. Data were expressed as fold induction vs control (mean±SEM) and compared by t-test. A p-value<0.05 was considered statistically significant. Results Protein levels of GPR84 were significantly increased in UC patients (150.3±11.75 vs 100.0±11.85), while no differences were observed in CD patients vs healthy ileums. Immunohistochemistry revealed a slight staining of this receptor in intestinal epithelial cells, whereas several cells of the lamina propria were strongly stained. Double immunohistochemistry confirmed that intestinal CD68 macrophages expressed GPR84 in both UC and CD patients. U937-derived macrophages treated with decanoic acid showed a significant increased expression of IL1b (2.68±0.86) and IL8 (2.44±0.48) vs vehicle-treated macrophages (0.998±0.23 and 1.05±0.17, respectively). In addition, IL1b levels in supernatant from macrophages treated with decanoic acid were significantly increased compared with the supernatant from vehicle-treated macrophages (4.50±1.05 vs 1.00±0.19). Moreover, the cotreatment with decanoic acid and GPR84 antagonist significantly reduced the expression of IL1b (0.99±0.29) vs vehicle-treated macrophages (1.94±0.26) and the transiently silencing of this receptor significantly reduced the expression of IL8 (0.89±0.25) vs siCtrl macrophages (4.02±1.59). Conclusion GPR84 is increased in UC patients and expressed in intestinal CD68+ macrophages. It regulates the expression of several cytokines pointing GPR84 as a promising pharmacological target for UC patients.

P0176 Single-cell RNA sequencing reveals the immunological mechanisms underlying Anti-saccharomyces cerevisiae antibody positivity in both non-inflamed and inflamed mucosal tissues of IBD patients

Abstract Background Previous studies have shown anti-saccharomyces cerevisiae antibody (ASCA) positivity in inflammatory bowel disease (IBD) patients is associated with increased risk of developing IBD and poor prognosis. However, no comprehensive study has elucidated the ASCA positive-related immunological phenotype in non-inflamed and inflamed colonic mucosa and its clinical implications. Methods We collected endoscopic biopsies from 43 ulcerative colitis [28 non-inflamed sites and 43 inflamed sites] and 20 Crohn’s disease at Seoul National University Hospital. Single-cell RNA sequencing (scRNA-seq) was performed on these samples according to the ASCA positivity. First, we compared ASCA-positive and -negative samples obtained from non-lesion sites of UC patients. Second, we investigated immunologic phenotypes in inflamed mucosa based on the disease activity (remission vs active). Results We conducted scRNA-seq analysis of over 130,000 single cells from 28 biopsies obtained from non-inflamed tissues of UC patients. Substantial reduction in the frequency of type 3 innate lymphoid cells (ILC3) and expression of IL18 and IL22 in ILC3s, along with a significant decrease in tight junction-associated genes (TJP1, CLDN3, CLDN4) within epithelial cells were observed in the positive group[P <0.05]. In addition, data from positive group exhibited decreased levels of CSF2 in ILC3s, which is likely contributing to reduced M2 macrophage polarization. We performed scRNA-seq analysis of over 123,673 single cells from 63 biopsies obtained from inflamed tissues. In active inflamed status, the expression of IL-17A - a cytokine that helps maintain tight-junction barrier in the intestinal epithelium during inflammation was decreased in tissue-resident memory T cells (Tc17 TRM) [P 0.026]. In addition, ASCA positive resulted in decreased CD4 central memory T cells (CD4 TCM) [P 0.046], suggesting impaired gut barrier function and subsequent rapid response to reencountered antigens. Cytotoxic gene expression, including granzyme and perforin 1, was increased in CD8 T effector memory cells (CD8 TEM) [P<0.05] during remission, suggesting that ongoing subclinical inflammation may develop and contribute to tissue damage. Conclusion These findings suggest that Saccharomyces cerevisiae infection induces immunological changes characterized by gut barrier dysfunction, rapid responses to commensal antigens, and increased cytotoxic gene expression. Based on these findings, we believe that Saccharomyces cerevisiae may serve as a potential target for preventing disease progression in patients with IBD.

P0055 Impact of Indole-3-Propionic Acid on Bacterial Invasion Potential of Klebsiella Strains Isolated from Pediatric Ulcerative Colitis Patients

Abstract Background Indole-3-propionic acid (IPA), a tryptophan metabolite produced by microbes, has demonstrated beneficial effects, including reversing dysbiosis, balancing bacterial populations, and reducing inflammation.1,2,3 However, the mechanisms behind these effects and their impact on pathogenic microbes remain unclear. This study aimed to evaluate the effects of IPA on bacterial invasion in vitro, using potential pathobionts (Klebsiella variicola and Klebsiella quasipneumoniae) isolated from non-inflamed sections of the colon in pediatric ulcerative colitis (UC) patients. We hypothesized that IPA could reduce virulence traits, promoting a more homeostatic environment, which could lower inflammation in pediatric UC patients. Methods Caco-2 cells (colonic epithelial cell line) were infected with Klebsiella strains, with or without 0.5 mM IPA. Bacterial invasion was assessed via gentamicin protection assay, and qPCR measured Interleukin-8, IL-10, and TNF-α expression in infected cells. Transepithelial electrical resistance (TEER) assay evaluated barrier integrity, and qPCR analyzed the expression of bacterial virulence genes, including entB (siderophores), manC (capsule), and hcp1 (type VI secretion) in IPA-treated and untreated cultures. Additionally, siderophore production was quantified using a Chrome-Azurol Sulfonate (CAS) assay, and Anthony’s stain visualized the bacterial capsule. Results IPA-treated Caco-2 cells showed significantly reduced bacterial invasion, with further reductions when bacteria were also exposed to IPA. qPCR revealed a significant decrease in IL-8 expression in IPA-treated cells during infection. IL-10 levels decreased non-significantly, and TNF-α expression showed variable results. A trend toward improved tight junction integrity was observed in the TEER assay with IPA treatment, although no significant differences were detected. IPA treatment increased the expression of entB, manC, and hcp1 genes, except for K. variicola, where manC expression remained unchanged. IPA significantly reduced the capsule thickness of K. variicola and resulted in a significant reduction in K. quasipneumoniae siderophore production. Conclusion IPA reduces bacterial invasion and modulates immune responses in Caco-2 cells infected with Klebsiella pathobiont strains. It reduces the virulence of K. quasipneumoniae by targeting siderophore production and diminishes the virulence of K. variicola by reducing capsule formation, suggesting its potential as an anti-virulence agent against these pathobionts. These findings highlight IPA’s potential as an immunomodulatory agent for managing IBD. However, further research is needed into other Klebsiella virulence pathways and host-microbiota interactions.

DOP114 LDN-071, the novel amino acid-based PAI-1 inhibitor, significantly reduces the severity of acute and chronic inflammation in colitis models

Abstract Background Over recent decades, significant progress has been made in the pharmacological management of Inflammatory Bowel Diseases (IBD). However, the sustained efficacy of existing anti-inflammatory therapies remains suboptimal, highlighting the urgent need for novel treatments. Recent studies have implicated the coagulation pathway, particularly Plasminogen Activator Inhibitor 1 (PAI-1), as a crucial link between epithelium and inflammation in IBD. Genetic deletion of PAI-1 has been shown to reduce experimental colitis severity in mice. Based on these findings, we aimed to develop and evaluate novel PAI-1 inhibitors for IBD treatment. Methods Using the A3SMO® platform, we synthesized LDN-071, an amino acid-based PAI-1 inhibitor, and assessed its inhibitory potential and cytotoxicity in vitro. Acute and chronic colitis models were induced in mice using DSS, with 10 mg/bwkg LDN-071 administered orally. Body weight, spleen weight, and colon length were monitored, while colon histology was analysed. Cytokine expression was measured via ELISA and qRT-PCR, and collagen deposition was quantified using a hydroxyproline assay. Human colon organoid (HCO) and fibroblast (FB) cultures from IBD patients were used to evaluate LDN-071’s effects. Toxicity was assessed in vivo in mice. Results LDN-071 effectively inhibited PAI-1 in vitro without cytotoxicity (1µM–1mM). In DSS-treated mice, 10 mg/bwkg LDN-071 prevented body weight loss, bloody diarrhea, colon shortening, and spleen enlargement. Histological analysis showed that PAI-1 inhibition significantly reduced severe inflammation, ulceration, crypt structure loss, and goblet cell depletion in acute and chronic colitis models. Additionally, PAI-1 inhibition reduced mucosal TNF-α, IL-1β, and IL-6 expression during acute inflammation. In chronic colitis, LDN-071 decreased inflammation, epithelial erosion, and fibrosis while significantly lowering collagen deposition. Importantly, no toxicity was observed at 100 mg/bwkg LDN-071 for seven days. In vitro, LDN-071 reduced pro-inflammatory cytokine expression in HCOs from IBD patients and significantly decreased fibrosis-related gene expression in patient-derived FB cultures. Conclusion Our results showed that LDN-071, a novel amino acid-based inhibitor of PAI-1, significantly ameliorated the severity of acute and chronic colitis in mice. However, the LDN-071 reduced the expression of inflammation- and fibrosis-related genes and proteins in vitro human models. We propose that the inhibition of PAI-1 could be a potential novel therapeutic strategy in IBD.

P0098 Effect of the dietary emulsifier κ-carrageenan on the inflammatory state and permeability of intestinal epithelial cells from patients with Crohn’s disease

Abstract Background The consumption of ultra-processed foods has increased worldwide and is associated with the rise in inflammatory bowel disease (IBD)1. However, any causative factors and their underlying mechanisms are yet to be identified. Interestingly, our group found a higher consumption of the dietary emulsifier carrageenan (CGN) in Belgian IBD patients, compared to healthy controls2. This study aimed to elucidate whether different types of CGN can alter the permeability and inflammatory state of the intestinal epithelium in patients with Crohn’s disease (CD). Methods Confluent Caco-2/HT29-MTX cocultures (n=4) were exposed to either κ-, ι-, or λ-CGN (100 µg/ml) for 24 hours. Confluent epithelial monolayers derived from colonic organoids3 of 5 CD patients were exposed to κ-CGN (100 µg/ml), the most abundant subtype in foods, for 48 hours. Two independent experiments were performed per patient (n = 10). In both models, inflammation was established by adding an inflammatory mix (100 ng/mL TNF-α, 20 ng/mL IL-1β, and 1 µg/mL flagellin)4 to the basolateral side, 24 hours prior to CGN exposure. Changes in permeability were measured by transepithelial electrical resistance (TEER) at predetermined timepoints (0, 12, 24, 48 hours). In organoid-derived monolayers, cytokines were quantified in the apical and basolateral supernatant using the V-PLEX Proinflammatory Panel 1 kit (Meso Scale Diagnostics) and gene expression was analysed with RT-qPCR (Taqman probes, run on Viia7) (n = 8; 2 samples excluded due to low RNA concentrations). Results In organoid-derived monolayers, κ-CGN increased the expression of TNF, IL8 and IL1B, both in inflamed and non-inflamed monolayers (n=8; Repeated measures (RM) one-way ANOVA), compared to the control (Fig. 1A). Release of the cytokines IL-6, IL-13, IL-4, IL-2, and IL-10 in the apical supernatant was increased after 48 hours of κ-CGN stimulation (n=8; RM one-way ANOVA) (Fig. 1B). κ-CGN exposure did not affect TEER (n=10; RM one-way ANOVA) (Fig. 2B+C), but induced an upregulation of tight junction markers ZO1 and OCLN, and resulted in a downregulation of MUC5AC and upregulation of MUC5B (n=8; RM one-way ANOVA or Friedman test) (Fig. 2D). Additionally, none of the CGN subtypes altered permeability of non-inflamed or inflamed Caco-2/HT29-MTX cocultures (n=4; Kruskal-Wallis test), compared to the unexposed control (Fig. 2A). Conclusion Dietary κ-CGN caused upregulation of inflammatory markers and affected cytokine release of intestinal epithelial cells from patients with CD, while permeability remained unaltered. When inflammation was already present, this pro-inflammatory effect was more pronounced, suggesting a role for dietary CGN in perpetuating inflammation during active CD.

P0170 The presence of creeping fat in a TNBS-induced colitis model is associated with greater acute inflammation and bacterial translocation

Abstract Background The hyperplasia of the mesenteric adipose tissue in Crohn’s disease, known as creeping fat, is associated with transmural lesions and the translocation of microbial antigens. In a rat model of TNBS-induced colitis, 50% of the animals presented adipose hyperplasia. Macroscopic evaluations showed that these animals had a more advanced progression of the disease, and colonoscopy could predict the presence of fat two days before euthanasia (L Clua et al, ECCO, 2024). Methods Colitis was induced using TNBS (n=10; half female), with the animals being sacrificed after five days. Tissue from the lesion along with mesenteric adipose tissue was obtained for histopathological staining and evaluation (haematoxylin-eosin, Masson’s trichrome, and Gram). Samples of intestinal mucosa and mesenteric fat were preserved in RNAlater to assess the expression of inflammatory markers by qRT-PCR. Results were statistically compared between animals with and without creeping fat, including sex. Results In histopathological analysis, using a validated scoring system, animals with creeping fat showed significantly greater severity of acute inflammation at the transmural level, with an increase in cryptitis and a more pronounced and extensive fibrosis pattern. Additionally, colonies of Gram-positive cocci were detected in all animals with creeping fat. In contrast, animals without fat hyperplasia exhibited appreciable adipogenesis in only two individuals, with an average of 105 adipocytes per field, compared to 185 in animals with creeping fat. qRT-PCR revealed higher expression of Il1b, Il6, Il10, Nod2, and Tlr2 in the intestinal mucosa and hyperplastic mesenteric fat of animals with creeping fat. A significant increase in Tnfa was also observed in hyperplastic adipose tissue, while the expression of Tgfbr1 was higher in the intestinal mucosa of animals without creeping fat. No differences were found between sexes. Conclusion The results indicated a more intense acute inflammatory and fibrotic component in animals with creeping fat, consistent with the macroscopic observations. The hyperplasia and inflammation observed in the mesenteric adipose tissue may be provoked by bacterial translocation, whereas animals without hyperplasia present a component influenced by TGF-β1 activity that could help counteract the inflammation. References L Clua, R Suau, M Guasch, D Monfort-Ferré, I Vañó, C Serena, E Domènech, R Bartolí, J Manyé, P022 Development of a novel experimental model for studying creeping fat in Crohn’s Disease, Journal of Crohn’s and Colitis, Volume 18, Issue Supplement_1, January 2024, Pages i275–i276, https://doi.org/10.1093/ecco-jcc/jjad212.0152

DOP076 Translational and preclinical study validating anterior gradient 2 as a novel epithelial therapeutic target in Inflammatory Bowel Disease (IBD) involved in inflammation, fibrosis, and intestinal barrier integrity

Abstract Background Anterior Gradient 2 (AGR2) is an endoplasmic reticulum (ER) resident protein involved in protein folding, predominantly expressed in mucin-secreting epithelial cells. Under stress, AGR2 is secreted into the extracellular milieu (eAGR2), where it disrupts epithelial integrity, attracts monocytes, and activates fibroblasts. We explored the relationship between AGR2 tissue expression in IBD patients and the efficacy of a neutralizing anti-eAGR2 antibody (TH-009) in murine models of colonic inflammation and fibrosis Methods AGR2 expression was analyzed by immunohistochemistry on ileal and colonic biopsies from IBD patients and controls. In mice, the efficacy of TH-009 was evaluated in models of colitis (3% DSS) and digestive fibrosis (3 cycles of 1.5% DSS) Results 132 ileal and colonic endoscopic biopsies from 66 patients with Crohn’s disease (CD), 50 patients with ulcerative colitis (UC), and 16 controls, and 96 surgical samples from 11 CD patients, 24 UC patients, and 11 controls were included. AGR2 expression correlated with mucosal inflammation (R² = 0.416, p<0.0001) and tissue damage (R² = 0.329, p<0.0001). All patients with mucosal inflammation showed AGR2 overexpression, while 38% of histologically healed patients had high AGR2, with a relapse rate at 18 months three times higher than those whose AGR2 levels were comparable to controls.In a murine acute colitis model (3% DSS), intravenous (IV) treatment with TH-009 at doses of 5 and 20 µg showed an improvement in weight (p<0.005 and p=0.0001, respectively), in the global colitis DAI score (p=0.002 and p=0.001), and in the histological colitis score (p=0.04 for 5 µg) compared to controls. Intraperitoneal and subcutaneous administration were also effective. There was a significant reduction in tissue infiltration by macrophages (p<0.01) and T lymphocytes (p<0.001), in myeloperoxidase activity, and in the tissue and serum expression of pro-inflammatory cytokines TNFα and IL-6. TH-009-treated mice restored intestinal permeability, measured by FITC-Dextran blood passage, to levels comparable to unexposed DSS mice, unlike DSS-only exposed mice (p<0.01). In a fibrosis model (3 cycles of 1.5% DSS), preventive and curative treatment with TH-009 reduced digestive fibrosis by 49% (p<0.0001), and reduced submucosal fibrosis thickness by 59% (p<0.0001). There was a significant decrease in the colonic expression of fibrosis-related genes (Collagen A1, αSMA, fibrinogen, and TGFβ). Conclusion AGR2 is a novel therapeutic target in IBD, correlating with inflammation and complications. Its expression is not affected by major therapies and is overexpressed in 40% of patients with deep histological healing. TH-009 improves inflammation, reverses fibrosis, and restores epithelial barrier functions.