Release Of IL-6 Research Articles

REDD1 mediates HDM-induced nuclear-cytoplasmic translocation and release of IL-33 in airway epithelial cells by downregulating Nrf2

ObjectiveThis study aims to investigate whether REDD1 (Regulated in Development and DNA Damage Responses 1) mediates the nuclear-to-cytoplasmic translocation and release of IL-33 in airway epithelial cells induced by house dust mites (HDM).MethodsREDD1 expression levels in bronchial asthma patients were validated using public databases, followed by immunohistochemical analysis of REDD1 protein in airway epithelial cells from these patients. An asthma model was then established using HDM-induced 16HBE cell lines, with REDD1 gene knockout performed. The relationship between varying levels of REDD1 expression, Nrf2, and related inflammatory factors was assessed using Western blot and qPCR. To further investigate the role of the REDD1-Nrf2-IL-33 axis in the development of asthma, we employed Nrf2 activators and inhibitors to reassess the impact of REDD1 on IL-33.ResultsAt both mRNA and protein levels, we found that REDD1 was significantly overexpressed in samples from asthma patients (P < 0.05). In vitro, 24-hour exposure to HDM induced a notable nuclear-to-cytoplasmic translocation of IL-33 and increased its levels in the culture medium of 16HBE cells. In addition, HDM treatment significantly upregulated the expression of both REDD1 and Nrf2. Knockdown of REDD1 markedly suppressed HDM-induced IL-33 release and the expression of TNF-α, IL-6, and IL-1β, while enhancing Nrf2 expression. Moreover, treatment with the Nrf2 agonist curcumin inhibited HDM-induced nuclear-to-cytoplasmic translocation and extracellular secretion of IL-33, whereas the opposite effect was observed when using the Nrf2 antagonist ML385.ConclusionThis study reveals the crucial regulatory role of the REDD1-Nrf2-IL-33 axis in the pathological process of bronchial asthma. REDD1 modulates the expression of IL-33 and other inflammatory factors through the Nrf2 signaling pathway, thereby influencing the onset and progression of asthma.Clinical trial numberNot applicable.

Molecular responses to clove and oregano essential oils are associated with reduced inflammation and improved gut barrier function in broiler chickens.

In vitro tests were conducted to characterize the host-mediated responses of chickens to Clove Essential Oil (CEO) and Oregano Essential Oil (OEO). Chicken macrophage cells (CMCs), chicken intestinal epithelial cells (IECs), quail muscle cells (QMCs), and chicken embryonic muscle cells (EMCs) were utilized in these assays. EMCs were collected from the 13-day-old embryo during egg incubation and all cell lines were seeded at 2 × 105/mL in a 24-well plate. In CMCs, an inflammatory response was induced by stimulating with 1.0 µg/mL of Lipopolysaccharide (LPS). To induce muscle cell differentiation, 0.5 % FBS was used in QMCs and 2.0 % FBS in EMCs. Three different concentrations (1.0, 10.0, and 100 µg/mL) of CEO and OEO were administered. qRT-PCR was used to measure gene expression levels of IL-1β and IL-8 from CMCs, occludin, ZO-1, and MUC2 from IECs, and Pax7 and MyoG from QMCs and EMCs. Cytotoxic effects of CEO and OEO were determined using an MTT assay; CEO and OEO did not show cytotoxicity at concentrations below 0.1 mg/mL in CMCs, IECs, QMCs, and EMCs. CEO reduced (P < 0.05) the LPS-induced increase of IL-1β and IL-8 in CMCs and increased (P < 0.05) ZO-1 and MUC2 in IECs. OEO suppressed (P < 0.05) the release of IL-8, increased ZO-1, and Pax7. Both CEO and OEO demonstrated microbicidal activity against sporozoite of E. tenella and C. perfringens bacteria, but only at doses 10-100 × higher than those that would be used in feed. These findings support our previous findings on other phytochemicals; both CEO and OEO are promising candidates for improved resilience in chickens not due to their direct antimicrobial effects, but due to gut physiological responses that take place at the level of the host.

Unraveling toxicity of nanoparticles from different subway materials in lung epithelial cells and macrophages.

Nanoparticles (ultrafine particles) are prevalent in various environments and raise concerns due to their potential health effects. In this study, we aimed to enhance the understanding of the toxicity associated with nanoparticles generated within subway systems. Specifically, we investigated nanoparticles produced using spark discharge from electrodes made of the same material as the third rail (which provides electric power), rail, and wheel components in the Stockholm subway system. Characterization revealed that the generated nanoparticles typically had a primary size of 6-10 nm and exhibited high agglomeration. They consisted mainly of iron, along with varying amounts of manganese and silicon. Despite having low oxidative potential, they showed some cytotoxicity and clearly induced DNA strand breaks in both dTHP-1 cells (monocyte-derived macrophages) and A549 cells (lung epithelial cells). In addition, gene expression analysis showed an upregulation of the cytokine IL-8 in dTHP-1 cells. No increased release of IL-1β, IL-8, IL-6, and TNF-a was noted. Consistent differences in toxicity between the nanoparticles from different materials were not observed. In conclusion, the results show that subway-related nanoparticles can cause DNA damage in cultured lung cells, but the inflammatory potential in terms of cytokine release was limited.

The Anti-inflammatory Potential of Tricyclic Antidepressants (TCAs): A Novel Therapeutic Approach to Atherosclerosis Pathophysiology

Atherosclerosis, a chronic inflammatory disease, is driven by complex molecular mechanisms involving inflammatory cytokines and immune pathways. According to recent research, tricyclic antidepressants (TCAs), which are typically prescribed to treat depressive disorders, have strong anti-inflammatory effects. TCAs, including imipramine and amitriptyline, alter inflammatory signaling cascades, which include lowering the levels pro-inflammatory cytokines like TNF-α, IL-1β, and IL-6 and inhibiting NF-κB activation. By inhibiting the NLRP3 inflammasome and suppressing pathways including JAK/STAT, MAPK, and PI3K, these effects are produced, improving endothelial function and reducing oxidative stress. The intricacy of TCAs’ anti-inflammatory actions has demonstrated by the existence of contradictory findings about how they alter IL-6 levels. The dependence of the heterogeneity of the reaction on the use of particular TCAs and experimental settings is shown by the fact that some studies show reduced IL-6 production, while others indicate increases or no changes. This review explores the multifaceted mechanisms through which TCAs modulate inflammatory pathways. TCAs inhibit NF-κB activation, reduce oxidative stress, and suppress the production of key inflammatory mediators, including IL-6 and TNF-α. They also regulate Toll-like receptor (TLR) signaling and NOD-, LRR-, and NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome activation, reducing the release of IL-1β and IL-18, critical drivers of endothelial dysfunction and plaque instability. Given their capacity to target critical inflammatory molecules and pathways, TCAs provide great potential in the therapy of atherosclerosis, particularly for individuals with associated depression and cardiovascular risk factors. Nevertheless, further research is essential to clarify the precise molecular mechanisms, resolve inconsistencies in current findings, and establish the clinical applicability of TCAs as anti-inflammatory agents in atherosclerosis management.

Extracellular vesicle surface engineering with integrins (ITGAL & ITGB2) to specifically target ICAM-1-expressing endothelial cells.

Extracellular vesicles (EVs) are taken up by most cells, however specific or preferential cell targeting remains a hurdle. This study aims to develop an EV that targets cells involved in inflammation, specifically those expressing intercellular adhesion molecule-1 (ICAM-1). To target these cells, we overexpress the ICAM-1 binding receptor "lymphocyte function-associated antigen-1" (LFA-1) in HEK293F cells, by sequential transfection of plasmids of the two LFA-1 subunits, ITGAL and ITGB2 (CD11a and CD18). The LFA-1 receptor was strongly overexpressed on the EVs released by the transfected cells. We further loaded these EVs with a therapeutic peptide, targeting myeloid differentiation primary response 88 (Myd88; EVMyd88), through a developed EV open-and-close procedure. Myd88 is a downstream common intracellular messenger for most TLR-receptors. EV expression of LFA-1 increases EV binding to ICAM-1-expressing cells, an effect that was dose-dependently inhibited by a specific neutralizing ICAM-1 antibody. Further, activated human endothelial cells treated with LFA-1 EVMyd88 had increased uptake of these EVs, resulting in dose-dependent inhibition of induced release of IL-8, presumably by targeting Myd88. We conclude that LFA-1-expressing EVMyd88 may be a candidate suitable for delivering therapeutic peptides in inflammatory diseases associated with TLR-activation.

Intralesional injection of CpG ODNs complexed with glatiramer acetate mitigates systemic cytokine toxicities and synergistically advances checkpoint blockade efficacy.

PD-L1/PD-1 checkpoint inhibitors (CPIs) are mainstream agents for cancer immunotherapy, but the prognosis is unsatisfactory in solid tumor patients lacking preexisting T-cell reactivity. Adjunct therapy strategies including the intratumoral administration of immunostimulants aim to address this limitation. CpG oligodeoxynucleotides (ODNs), TLR9 agonists that can potentiate adaptive immunity, have been widely investigated to tackle PD-L1/PD-1 resistance, but clinical success has been hindered by inconsistent efficacy and immune-related toxicities caused by systemic exposure. Here, we utilized glatiramer acetate (GA), the FDA-approved, lysine-rich polypeptidesto complex CpG into polycationic nanoparticles (R4B) and investigated the safety and antitumor efficacy of CpG ODNs in the murine CT26 colorectal carcinoma model. In a maximum tolerated dose study, repetitive R4B treatment displayed comparable antitumor efficacy to CpG alone treatment within a dose range from 15µg to 150µg while significantly attenuating systemic proinflammatory cytokine IL-6 release. A pharmacokinetic and biodistribution analysis confirmed that R4B localized and gradually released CpG around the lesions within 96h while 'naked' CpG quickly diffused from the injection site. Genome-wide transcriptome analysis validated that R4B treatment activated prominent TLR9-driven immune system responses in both lesions and spleens. In a CT26 multiple tumor model, intratumoral administration of R4B generated systemic immune efficacy, evidenced by an abscopal effect on untreated tumors. Notably, R4B treatment accomplished these effects with mitigated systemic proinflammatory cytokines when compared with CpG alone. We further discovered that combining R4B with anti-PD-1 treatment led to the most pronounced effects on tumor growth and longest benefits to survival time. Our investigation into possible mechanisms underlying this phenomenon included increased recruitment of cytotoxic CD8+ T cells and natural killer (NK) cells to the tumor microenvironments and the reversal of PD-L1/PD-1 axis inhibition. In summary, these results warrant further investigation for safely improving clinical responses in CPI-resistant solid tumor patients with localized CpG ODN therapy.

Isorhamnetin Ameliorates Non-Esterified Fatty Acid-Induced Apoptosis, Lipid Accumulation, and Oxidative Stress in Bovine Endometrial Epithelial Cells via Inhibiting the MAPK Signaling Pathway

High concentrations of non-esterified fatty acids (NEFAs) in the blood contribute to various metabolic disorders and are linked to endometritis in dairy cows. Isorhamnetin (ISO), a flavonoid found in many plants, is known for its antioxidant, anti-inflammatory, and anti-obesity properties. This study systematically assessed NEFA-induced damage in bovine endometrial epithelial cells (bEECs) and investigated whether ISO alleviates NEFA-induced cell damage and its underlying molecular mechanisms. Our observations revealed that excessive NEFA inhibited proliferation and induced apoptosis in bEECs, accompanied by an increase in the expression of BAX and cleaved caspase-3. We further observed that NEFA could induce lipid accumulation, reactive oxygen species (ROS) generation, and the release of pro-inflammatory factors IL-1β, IL-6, and TNF-α in bEECs. RNA sequencing and Western blot analysis revealed that NEFA induced damage in bEECs by activating MAPK signaling pathway. Notably, ISO treatment ameliorated these effects induced by NEFA, as evidenced by decreased protein levels of BAX, cleaved caspase-3, and PPAR-γ, along with reductions in triglyceride content, ROS generation, and levels of IL-1β, IL-6, and TNF-α. Mechanistically, our experimental results demonstrated that ISO inhibited NEFA-induced activation of MAPK signaling. Overall, ISO shows promise for therapeutic development to address NEFA-related endometritis in dairy cows.

Mechanisms of wogonoside in the treatment of atherosclerosis based on network pharmacology, molecular docking, and experimental validation

BackgroundAtherosclerosis serves as the fundamental pathology for a variety of cardiovascular disorders, with its pathogenesis being closely tied to the complex interplay among lipid metabolism, oxidative stress, and inflammation. Wogonoside is a natural flavonoid extracted from Scutellaria baicalensis with a variety of biological activities, including anti-inflammatory, hypolipidemic, and cardiac function improvement properties. Despite these known effects, the specific role of wogonoside in the context of atherosclerosis remains to be elucidated.PurposeTo validate the efficacy of wogonoside in the treatment of atherosclerosis and to investigate its possible therapeutic mechanisms.MethodsNetwork pharmacology was used to obtain the core targets and signaling pathways that may be efficacious in the treatment of atherosclerosis with wogonoside, which were validated using molecular docking and molecular dynamics simulations. To further validate the core targets in the signaling pathway, we performed in vivo experiments using apolipoprotein E (ApoE)-/- mice. This included pathological morphology and lipid deposition analysis of mouse aorta, serum lipid level analysis, Elisa analysis, oxidative stress analysis, reactive oxygen species (ROS) fluorescence assay, immunohistochemical analysis and protein blot analysis.ResultsPredictions were obtained that wogonoside treatment of atherosclerosis has 31 core targets, which are mainly focused on pathways such as Toll-like receptor (TLR) signaling pathway and NF-kappa B (NF-κB ) signaling pathway. Molecular docking and molecular dynamics simulations showed that wogonoside has good binding properties to the core targets. In vivo experimental results showed that wogonoside significantly inhibited aortic inflammatory response and lipid deposition, significantly reduced the release levels of total cholesterol (TC), triglycerides (TG), low-density-lipoprotein cholesterol (LDL-C), oxidized low density (ox-LDL) and free fatty acid (FFA), and significantly inhibited the release of inflammatory factors TNF-α, IL-1β, IL-6 and oxidative stress in ApoE-/- mice. Further molecular mechanism studies showed that wogonoside significantly inhibited the activation of TLR4/NF-κB signaling pathway in ApoE-/- mice.ConclusionWogonoside may be an effective drug monomer for the treatment of atherosclerosis, and its mechanism of action is closely related to the inhibition of the activation of the TLR4/NF-κB signaling pathway.Graphical

Comprehensive chemical and bioactive investigation of Chinese peony flower: a case of valorization of by-products as a new food ingredient from Chinese herb

IntroductionIn the present study, the flower of Chinese peony (CPF), major waste by-product of Chinese Herb Radix paeoniae, was comprehensively investigated for the first time.MethodsA validated UHPLC Orbitrap Mass spectrometry combined a three-levels characterization strategy were used to analyze CPF samples from four representative cultivars. The anti-inflammatory and antioxidant activities were analyzed using RAW264.7 cells, and DPPH, ABTS, FRAP, and ORAC antioxidant assays.ResultsA total of 150 chemical components were identified in CPF, among them, more than 50 components were reported from this species for the first time, with potential new chemicals reported. 67 quantified or semi-quantified targeted metabolomics analysis indicated a clear distinction between flower parts and four cultivars. CPF demonstrated significant antioxidant activities and displayed anti-inflammatory effects by reducing nitric oxide, IL-6, and TNF-a release in LPS-induced macrophages. Correlation analysis highlighted a strong positive correlation between total phenolic content and DPPH ABTS, and FRAP antioxidant activities.DiscussionThe present study is the first to comprehensively investigate the chemical profile and bioactivities of CPF, which provide insights into further understanding of its health-promoting potential.

Mitochondria complex III-generated superoxide is essential for IL-10 secretion in macrophages.

Mitochondrial electron transport chain (ETC) function modulates macrophage biology; however, mechanisms underlying mitochondria ETC control of macrophage immune responses are not fully understood. Here, we report that mutant mice with mitochondria ETC complex III (CIII)-deficient macrophages exhibit increased susceptibility to influenza A virus (IAV) and LPS-induced endotoxic shock. Cultured bone marrow-derived macrophages (BMDMs) isolated from these mitochondria CIII-deficient mice released less IL-10 than controls following TLR3 or TLR4 stimulation. Unexpectedly, restoring mitochondrial respiration without generating superoxide using alternative oxidase (AOX) was not sufficient to reverse LPS-induced endotoxic shock susceptibility or restore IL-10 release. However, activation of protein kinase A (PKA) rescued IL-10 release in mitochondria CIII-deficient BMDMs following LPS stimulation. In addition, mitochondria CIII deficiency did not affect BMDM responses to interleukin-4 (IL-4) stimulation. Thus, our results highlight the essential role of mitochondria CIII-generated superoxide in the release of anti-inflammatory IL-10 in response to TLR stimulation.

Molecular and Immunological Characterization of Troponin C: An Allergen from Scylla paramamosain.

Scylla paramamosain, a crustacean of substantial importance, is a frequent trigger of food allergies. This study examined the molecular and immunological properties of troponin C from S. paramamosain (Scy p TnC) as an allergen. The findings indicated that thermal stability of Scy p TnC comprised 150 amino acids and facilitated the induction of CD63/CD203c in basophils from crab allergy patients. Furthermore, treatment of Scy p TnC with chemical denaturants caused structural degradation, which resulted in diminished IgG binding capacity. Subsequently, 6 linear epitopes and 4 conformational epitope regions of TnC were predicted, with epitopes at the C-terminal being conserved throughout 9 discovered TnCs. Concurrently, mice sensitized with Scy p TnC exhibited markedly increased levels of IgE and IL-4 release, provoking a Th2 immune response. The results reveal crab allergens and enhance existing knowledge regarding allergenic components in crabs, thereby facilitating the advancement of molecular diagnostics and targeted therapeutic strategies.

Allergen-induced activation of epithelial P2Y2 receptors promotes ATP exocytosis and type 2 immunity in airways.

Environmental allergens induce the release of danger signals from the airway epithelium that trigger type 2 immune responses and promote airway inflammation. To investigate the role of allergen-stimulated P2Y2 receptor activation in regulating ATP, IL-33 and DNA release by human bronchial epithelial (hBE) cells and mouse airways. hBE cells were exposed to Alternaria alternata extract and secretion of ATP, IL-33 and DNA were studied in vitro. Molecular and cellular mechanisms were examined by biochemical and genetic approaches. Mice were treated intranasally (i.n.) with pharmacologic agents and exposed to Alternaria extract. Exposure of hBE cells to Alternaria extract stimulated P2Y2 receptors coupled to phospholipase Cβ3, leading to activation of multiple PKC isoforms and an increase in intracellular Ca2+ concentration. Small interfering RNAs (siRNAs) targeting PKCδ or inhibiting PKCδ activity with delcasertib blocked exocytosis of ATP and reduced IL-33 and DNA secretion. Moreover, a peptide antagonist for MARCKS reduced vesicular ATP release. A proximity ligand assay showed that Alternaria extract stimulated MARCKS desorption from the plasma membrane and delcasertib prevented the response. Finally, the P2Y2 receptor antagonist AR-C118925XX and delcasertib blocked IL-33, DNA and type 2 cytokine secretion in vivo in mice exposed to Alternaria. P2Y2 receptor stimulation following allergen exposure promoted activation of PLCβ3, PKCδ and MARCKS protein desorption from the apical membrane, which facilitated ATP exocytosis and subsequent secretion of IL-33 and DNA. Epithelial P2Y2 receptors serve as primary sensors for aeroallergen-induced alarmin release by airway epithelial cells.

P0054 Human colonoid-derived monolayers and Caco-2: comparative analysis of inflammatory responses

Abstract Background Given the increasing incidence of inflammatory bowel diseases, there is a growing need for reliable preclinical models that replicate the inflamed intestinal epithelium. Immortalized Caco-2 cells are widely considered the standard in vitro model for intestinal studies, but human-derived colonoid monolayers (CDMs) could be proposed as a powerful alternative to more accurately mimic the pathophysiological features of intestinal inflammation. The present study aims to compare the functional and morphological responses induced by inflammatory cytokines TNFα and IFNγ on CDMs and Caco-2. Methods Human colonic crypts from biopsies of 5 healthy volunteers were embedded in Matrigel® and cultured for 5-7 days. IntestiCult™ OGM was used to expand cystic-like colonoids, and single cells were seeded onto coated 24-well inserts. Upon reaching confluence, the medium was switched to IntestiCult™ ODM. Caco-2 cells were cultured for 21 days on 24-well inserts. Differentiated CDMs and Caco-2 monolayers were exposed to 25 ng/mL TNFα-IFNγ for 24 hours. Cell differentiation was assessed by immunocytochemistry (ICC), and barrier integrity was evaluated by transepithelial electrical resistance (TEER) measurement. Cytotoxicity was assessed through the LDH assay. IL-8 and CCL20 release were quantified. Results Both models showed increasing TEER over the growth period, indicating the gradual development of epithelial barriers, up to 484±58 Ω*cm2 for CDMs and 700±184 Ω*cm2 for Caco-2. ICC staining for epithelial markers villin and MUC2 confirmed polarization and differentiation under basal conditions in both models, cytokines reduced the expression of epithelial markers. Inflammation reduced the barrier integrity of the monolayers (approximately 18%), although more uniformly in Caco-2 than in CDMs. IL-8 and CCL20 release was augmented by cytokines in both models, (for IL-8 of about 20 times in CDM and 2 times in Caco-2). As regards cell viability, CDMs (40% cytotoxicity) showed higher sensitivity to cytokines than Caco-2 (22% cytotoxicity). Conclusion Exposure to an inflammatory environment altered cell morphology and impaired epithelial barrier integrity in both CDMs and Caco-2 monolayers. These findings highlight the potential of CDMs as an advanced model for studying personalized responses in intestinal inflammation.

P0080 Thalidomide-Carbomer Gel for Rectal Administration Alleviates DSS-Induced Colitis and fibrosis in Mouse by Targeting S1pr5 and Il1f9 (Il36γ)

Abstract Background Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the digestive tract, including ulcerative colitis (UC) and Crohn's disease (CD). Thalidomide, an inhibitor of Tnf-α, is used in IBD treatment but is limited by systemic side effects when taken orally. This study aimed to develop a thalidomide-carbomer gel for rectal administration to reduce systemic side effects and explore new therapeutic targets. Methods An acute colitis model was established using DSS-induced wild-type C57 mice. Mice were treated with thalidomide-carbomer gel via rectal administration. Body weight, disease activity index (DAI), colon length, liver and spleen indices, and histological changes were evaluated. RNA-seq, qPCR, WB, and immunohistochemistry were performed to assess inflammation and fibrosis markers. In vitro, an LPS-induced inflammation model was constructed using NCM460 cells, and cell migration and inflammatory cytokine expression were analyzed following drug treatment. Results Thalidomide-carbomer gel significantly alleviated symptoms of DSS-induced colitis in mice, including reducing body weight loss, DAI score, colon shortening, and liver/spleen indices. Histological analysis showed decreased infiltration of inflammatory cells and reduced expression of Tnf-α, α-SMA, and Tgf-β. The gel also enhanced intestinal barrier function by increasing the expression of E-cadherin and Occludin. RNA-seq and qPCR analyses suggested that the therapeutic effect may involve the regulation of S1pr5 and Il1f9（Il36γ）pathways. Toxicity tests indicated no significant adverse effects on hematological or liver and kidney functions. In NCM460 cells, thalidomide inhibited LPS-induced cell migration and IL-6 release. Conclusion Thalidomide-carbomer gel, through the inhibition of Tnf-α, S1pr5, and Il1f9 (Il36γ), significantly improved DSS-induced colitis and fibrosis in mouse, showing promising therapeutic potential for 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.

P0640 Development and characterization of LQ080, a novel extended half-life bispecific TL1A/IL-23 p19 single domain antibody for the treatment of IBD

Abstract Background Despite the effectiveness of current anti-TL1A treatments, unmet needs remain for effective therapeutics for IBD. Therapies simultaneously neutralizing TL1A and IL-23 might be better choices owing to their synergistic anti-inflammatory effects in the pathogenesis of IBD. Methods TL1A and IL-23 single domain antibodies (sdAbs) were selected from immunized sdAb Phage display libraries. A human whole blood-based assay measuring IFNγ secretion was used to assess functional blockade of TL1A. Mouse splenocytes based assay measuring mouse IL-17 release was used to evaluate functional blockade of IL-23. Dextran Sulfate Sodium (DSS) induced mouse bowel inflammatory model was used to judge the in vivo effect of anti-TL1A antibodies. Synergistic anti-inflammatory effect was evaluated in mouse dermatitis model. Half-life extension was measured via pharmacokinetic analysis in non-human primate (NHP) given a single bolus of LQ080 by subcutaneous administration. Results The TL1A sdAb, which binds to both soluble and membrane-bound TL1A in a pH dependent pattern, potently blocked TL1A/DR3 while leaving the TL1A/DcR3 binding undisturbed and illustrated better IFNγ release in human whole blood induced by TL1A than RVT-3101 and PRA023. In DSS model, the TL1A sdAb also demonstrated better anti-inflammation effect than RVT-3101. The TL1A/IL-23 p19 biparatropic sdAb, LQ080, showed better IFNγ secretion inhibition than RVT-3101 and PRA023, and better inhibition of mouse IL-17 induction than Risankizumab and Guselkumab. Synergistic effects were found using LQ080 in both IFNγ release inhibition in human PBMCs induced by IL-23 and TL1A and in the mouse dermatitis model. The half-life of LQ080 significantly extended of 2-3 folds in non-human primate (NHP) compared to that of RVT-3101. Besides, LQ080 did not form large immune complex in vitro when mixed with TL1A and IL-23. Moreover, LQ080 did not stimulate the release of pro-inflammatory cytokines in the cytokines release assay. Conclusion The TL1A sdAb demonstrated better bioactivity than RVT-3101 and PRA023. LQ080, a TL1A/IL-23 p19 biparatropic sdAb, illustrated synergistic anti-inflammation effect both in vitro and iv vivo for its simultaneous neutralization of TL1A/DR3 and IL-23/IL-23R pathways, which would greatly improve the clinical results of IBD patients. The pH dependent TL1A binding makes LQ080 very different from other anti-TL1A mAbs. LQ080 demonstrated extended half-life in NHP making infrequent dosing possible for IBD treatment. No large immune complex formation when mixed with TL1A and IL-23 would make LQ080 safer when administrated by SC route. Overall, LQ080 is a promising TL1A/IL-23 p19 sdAb for IBD treatment.

Design and Synthesis of Hederagenin Derivatives for the Treatment of Sepsis by Targeting TAK1 and Regulating the TAK1-NF-κB/MAPK Signaling.

Sepsis is a systemic inflammatory response caused by infection and is a leading cause of death worldwide. We designed and synthesized a series of hederagenin analogues with anti-inflammatory activity. The most effective compound, 14, reduced the release of TNF-α and IL-6 in RAW264.7 cells induced by lipopolysaccharide by affecting NF-κB/MAPK signaling. It demonstrated significant protection against sepsis in vivo and ameliorated histopathological changes in the liver, lungs, and kidneys. It exhibited good safety in subacute toxicity assays. Western blotting results indicated that it reduced the generation of p-p65, p-IκB, p-p38, p-JNK, and p-ERK. Immunofluorescence assay results suggested that the compound inhibited nuclear translocation of p65 and c-Fos. It was found to target TAK1 with a novel molecular backbone, distinct from the few TAK1 inhibitors previously reported. This work provides a new lead structure for the study of TAK1 inhibitors and a potential target for TAK1 in sepsis therapy.

Long-term cultivation of retinal pigment epithelium cells on nanofiber scaffolds.

The retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age-related macular degeneration (AMD) and other retinal degenerative diseases. The introduction of healthy RPE cell cultures into the subretinal space offers a potential treatment strategy. The aim of this study was the long-term culture and characterisation of RPE cells on nanofiber scaffolds. Nanofiber scaffolds consisting of polycaprolactone (PCL) and collagen were prepared by electrospinning. Porcine RPE cell cultures were maintained on PCL scaffolds, PCL-collagen scaffolds, and controls at the bottom of 24-well plates. Cell culture analysis was performed by immunohistochemistry, while the release of inflammatory cytokines IL-6, IL-8, TNF-α, and PDGF-β was measured by ELISA and multiplex assays. Ultrastructural features were examined by transmission electron microscopy. The observation period averaged 42.7 weeks for controls, 38.7 weeks for PCL scaffold cultures, and 36.1 weeks for PCL-collagen scaffold cultures, with cell number and morphology remaining stable. TNF-α levels in the supernatants were minimal, IL-6 levels were consistently low, and IL-8 levels decreased from initially high to lower levels over time. RPE cells were stably cultured on nanofiber scaffolds for extended periods of time. The long-term physiological properties of RPE cells, including phagocytic ability and visual cycle enzyme activity, need to be further investigated before clinical application. In addition, controlling the expression of inflammatory mediators is a major challenge. Despite these hurdles, overcoming them is critical given the increasing prevalence of retinal degenerative diseases.

Nobiletin and Eriodictyol Suppress Release of IL-1β, CXCL8, IL-6, and MMP-9 from LPS, SARS-CoV-2 Spike Protein, and Ochratoxin A-Stimulated Human Microglia.

Neuroinflammation is involved in various neurological and neurodegenerative disorders in which the activation of microglia is one of the key factors. In this study, we examined the anti-inflammatory effects of the flavonoids nobiletin (5,6,7,8,3',4'-hexamethoxyflavone) and eriodictyol (3',4',5,7-tetraxydroxyflavanone) on human microglia cell line activation stimulated by either lipopolysaccharide (LPS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) full-length Spike protein (FL-Spike), or the mycotoxin ochratoxin A (OTA). Human microglia were preincubated with the flavonoids (10, 50, and 100 µM) for 2 h, following which, they were stimulated for 24 h. The inflammatory mediators interleukin-1 beta (IL-1β), chemokine (C-X-C motif) ligand 8 (CXCL8), IL-6, and matrix metalloproteinase-9 (MMP-9) were quantified in the cell culture supernatant by enzyme-linked immunosorbent assay (ELISA). Both nobiletin and eriodictyol significantly inhibited the LPS, FL-Spike, and OTA-stimulated release of IL-1β, CXCL8, IL-6, and MMP-9 at 50 and 100 µM, while, in most cases, nobiletin was also effective at 10 µM, with the most pronounced reductions at 100 µM. These findings suggest that both nobiletin and eriodictyol are potent inhibitors of the pathogen-stimulated microglial release of inflammatory mediators, highlighting their potential for therapeutic application in neuroinflammatory diseases, such as long COVID.

Anti-inflammatory effects of moxifloxacin and levofloxacin on cadmium-activated human astrocytes: Inhibition of proinflammatory cytokine release, TLR4/STAT3, and ERK/NF-κB signaling pathway.

Cadmium is a non-essential element and neurotoxin that causes neuroinflammation, which leads to neurodegenerative diseases and brain cancer. To date, there are no specific or effective therapeutic agents to control inflammation and alleviate cadmium-induced progressive destruction of brain cells. Fluoroquinolones (FQs), widely used antimicrobials with effective blood-brain barrier penetration, show promise in being repurposed as anti-inflammatory drugs. Therefore, we aimed to test the efficacy of repurposed FQs for the treatment of cadmium-induced inflammation using cultures of U-87 MG human astrocytes and primary human astrocytes. Both FQs abrogated cadmium-induced interleukin (IL)-6 and IL-8 release from human astrocytes in a concentration and time-dependent manner, although levofloxacin had a stronger inhibitory effect than moxifloxacin. The downregulation of inflammatory cytokine release occurred with a concomitant reduction in cadmium-induced elevations in p65 nuclear factor-κB (NF-κB) and extracellular signal-regulated kinases (ERKs) 1/2 phosphorylation. Additionally, levofloxacin treatment significantly alleviated cadmium-induced activation of phosphorylated NF-κB translocation and toll-like receptor (TLR)-4/signal transducer and activator of transcription (STAT) 3 signaling. Transcriptome analysis revealed that modulation of inflammation-related pathways was the most enriched after FQ treatment. Our data suggest that FQs, particularly levofloxacin, attenuate the inflammatory process mediated by cadmium in human astrocytes. These effects may be mediated, at least in part, by inhibition of immune pathways regulated by TLR4, STAT3, ERK MAPK, and NF-κB.