Release Of IL-6 Research Articles

Cannabinoid Receptor type 2 agonist GP1a attenuates macrophage activation induced by M. bovis-BCG by inhibiting NF-ĸB signaling.

Tuberculosis (TB) is one of the leading causes of death worldwide and a major public health problem. Immune evasion mechanisms and antibiotic resistance highlight the need to better understand this disease and explore alternative treatment approaches. Mycobacterial infection modulates the macrophage response and metabolism to persist and proliferate inside the cell. Cannabinoid receptor type 2 (CB2) is expressed mainly in leukocytes and modulates the course of inflammatory diseases. Therefore, our study aimed to evaluate the effects of the CB2-selective agonist GP1a on irradiated M. bovis-BCG (iBCG)-induced J774A.1 macrophage activation. We observed increased expression of CB2 in macrophages after iBCG stimulation. The pretreatment with CB2-agonists, GP1a, JWH-133, and GW-833972A (10 µM), reduced iBCG-induced TNF-α and IL-6 release by these cells. Moreover, the CB2-antagonist AM630 (200nM) treatment confirmed the activity of GP1a on CB2 by scale down its effect on cytokine production. GP1a pretreatment (10 µM) also inhibited the iBCG-induced production of inflammatory mediators as prostaglandin (PG)E2 and nitric oxide (NO) by macrophages. Additionally, GP1a pretreatment also reduced the transcription of proinflammatory genes (inos, il1b, cox2) and genes related to lipid metabolism (dgat1, acat1, plin2, atgl, cd36). Indeed, lipid droplet accumulation was reduced by GP1a treatment which was partially blockade by AM630 pretreatment. Finally, GP1a pretreatment reduced the activation of the NF-κB signaling pathway. In conclusion, the activation of CB2 by GP1a modulated the macrophage response to iBCG by reducing inflammatory mediator levels and metabolic reprogramming.

Functional characterization of OR51B5 and OR1G1 in human lung epithelial cells as potential drug targets for non-type 2 lung diseases

Abstract Background Hypersensitivity to odorants like perfumes can induce or promote asthma with non-type 2 inflammation for which therapeutic options are limited. Cell death of primary bronchial epithelial cells (PBECs) and the release of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 are key in the pathogenesis. Extra-nasal olfactory receptors (ORs) can influence cellular processes involved in asthma. This study investigated the utility of ORs in epithelial cells as potential drug targets in this context. Methods We used the A549 cell line and primary bronchial epithelial cells using air–liquid interface culture system (ALI-PBECs). OR expression was investigated by RT-PCR, Western blot, and Immunofluorescence. Effects of OR activation by specific ligands on intracellular calcium concentration, cAMP, Phospholipase C (PLC), cell viability, and IL-6 and IL-8 secretion were analyzed by calcium imaging, enzyme immunoassays, Annexin V/ propidium iodide -based fluorescence-activated cell staining or by ELISA, respectively. Results By screening A549 cells, the OR51B5 agonists Farnesol and Isononyl Alcohol and the OR1G1 agonist Nonanal increased intracellular Ca2 + . OR51B5 and OR1G1 mRNAs and proteins were detected. Both receptors showed a preferential intracellular localization. OR51B5- but not OR1G1-induced Ca2 + dependent on both cAMP and PLC signaling. Farnesol, Isononyl Alcohol, and Nonanal, all reduced cell viability and induced IL-8 and IL-6 release. The data were verified in ALI-PBECs. Conclusion ORs in the lung epithelium might be involved in airway-sensitivity to odorants. Their antagonism could represent a promising strategy in treatment of odorant-induced asthma with non-type 2 inflammation. Graphical Abstract

Dengue Virus Infection: Immune Response and Therapeutic Targets.

Flavivirus infection, especially dengue virus infection caused by DENV, is known to be a significant health concern globally owing to the high incidence and mortality rate. The expanding and increasing disease burden calls for the need to develop an effective treatment and prevent the event of fatal complications, including dengue hemorrhagic fever/dengue shock syndrome. The DENV-induced immune response has been described as paradoxical because it has a protective role in viral clearance but, at the same time, causes more severe infection through viral-specific immunity. This is further complicated by high homology and cross-reactivity between different serotypes of DENV, causing a more severe disease presentation during secondary infection by a heterologous serotype. This serotype complexity poses a challenge for the development of a universal flavivirus vaccine. This review highlights the significance of high motility group box 1 (HMGB1) and nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 (NLRP3) inflammasome activation pathways in initiating an inflammatory response through the downstream activation of nuclear factor κB and proinflammatory cytokine Interleukin (IL)-1B, IL-18 release in DENV infection. It also discusses the role of NLRP3 in activating cellular apoptosis and pyroptosis leading to systemic failure, especially in peripheral tissues. Over the decades, there has been much progress in understanding the immunopathogenesis of DENV infection. Researchers have been studying key pathogenic molecules for potential therapeutic targets including HMGB1 and NLRP3 inflammasome inhibitors, which is explored in this review. Ultimately, although there is not yet an effective antiviral or vaccine for DENV, immunomodulators continue to pave the way to decrease disease severity in infected individuals.

Abstract 4116603: Interleukin-33 from Necrotic Tunica Media Plays a Key Role in the Exacerbations of Coronary Arteritis in Kawasaki Disease

Objective: Alarmins resulting from cell death or oxidative stress have been shown to be involved in the development of Kawasaki disease (KD) vasculitis. In our previous study, we demonstrated the potential role of (IL)-33 as an alarmin in the development of KD vasculitis. Although edematous dissociation (necrotic change) of the tunica media is thought to be a major source of IL-33 in KD vasculitis, it has not yet been elucidated. Methods: We investigated the impact of IL-33 released from necrotic human coronary artery smooth muscle cells (HCASMCs) on human coronary artery endothelial cells (HCAECs) by a co-culture assay using the Transwell ® cell culture insert system. Subsequently, we evaluated the anti-inflammatory effects of anti-IL-33 and anti-ST2 antibodies compared to the conventional therapies of KD, such as high-dose IgG or anti-tumor necrosis factor (TNF)-α antibody. Results: Primary necrosis of HCASMCs induced significant release of IL-33. In co-cultures of necrotic HCASMCs with HCAECs, necrotic HCASMCs significantly induced the production of various proinflammatory cytokines in HCAECs. Anti-IL-33 and anti-ST2 antibodies exhibited unique inhibitory effects on the production of platelet-derived growth factor-BB or IL-12(p70) in HCAECs. Conclusion: The results of the present study suggest the potential involvement of edematous dissociation of the tunica media in the development of KD vasculitis. Furthermore, the distinctive anti-inflammatory effects of the anti-IL-33/ST2 axis drugs suggest novel therapeutic options for patients with refractory KD.

Inflammation-Responsive Functional Core-Shell Micro-Hydrogels Promote Rotator Cuff Tendon-To-Bone Healing by Recruiting MSCs and Immuno-Modulating Macrophages in Rats.

Rotator cuff injuries often necessitate surgical intervention, but the outcomes are often unsatisfactory. The underlying reasons can be attributed to multiple factors, with the intricate inflammatory activities and insufficient presence of stem cells being particularly significant. In this study, an innovative inflammation-responsive core-shell micro-hydrogel is designed for independent release of SDF-1 and IL-4 within a single delivery system to promote tendon-to-bone healing by recruiting MSCs and modulating M2 macrophages polarization. First, a MMP-2 responsive hydrogel loaded with IL-4 (GelMA-MMP/IL-4) is synthesized by cross-linking gelatin methacrylate (GelMA) with MMP-2 substrate peptide. Then, the resulting core particles are coated with a shell of chitosan /SDF-1/hyaluronic acid (CS/HA/SDF-1) using the layer-by-layer electrostatic deposition method to form a core-shell micro-hydrogel composite. The core-shell micro-hydrogel shows sustained release of SDF-1 and MMP-2-responsive release of IL-4 associated in situ MSCs homing and smart inflammation regulation by promoting M2 macrophages polarization. Additionally, by injecting these micro-hydrogels into a rat rotator cuff tear and repair model, notable improvements of fibrocartilage layer are observed between tendon and bone. Notably, this study presents a new and potentially powerful environment-responsive drug delivery strategy that offers valuable insights for regulating the intricate micro-environment associated with tissue regeneration.

TMOD-02. PATIENT-DERIVED GLIOBLASTOMA ORGANOIDS AS REAL-TIME AVATARS FOR ASSESSING RESPONSES TO CLINICAL CAR-T CELL THERAPY

Abstract Patient-derived tumor organoids have been increasingly leveraged for disease modeling and preclinical studies, but rarely in real-time to aid with the interpretation of patient treatment responses in the clinical setting. Whether tumor organoids can be applied to mirror clinical activity in patients and even to predict responses for personalized medicine remains uncertain. We recently demonstrated promising early efficacy signals in a first-in-human, phase 1 study of dual-targeting chimeric antigen receptor T cells (EGFR-IL13Rα2 CAR-T cells) in patients with recurrent glioblastoma (clinical trial identifier: NCT05168423). Determination of meaningful clinical efficacy, in particular for CAR-T cell therapy, may take months to manifest. Given this challenge, real-time analysis of patient-derived glioblastoma organoids (GBOs) could help to provide early assessment of clinical efficacy and guide patient management. We analyzed six sets of GBOs generated on the same timeline as the production of patients’ CAR-T cell products. GBOs demonstrated the presence and retention of both targeted tumor-associated antigens EGFR and IL13Rα2 by immunohistochemistry. These GBOs were then treated with the same autologous CAR-T cell products received by patients in our phase 1 study in parallel with patient treatment. CAR-T cell treatment led to target antigen reduction assessed by immunohistochemistry and flow cytometry. Furthermore, we confirmed cytolysis of tumor cells in GBOs by cleaved caspase-3 immunostaining and by co-culture in the Axion Maestro Cellular Impedance platform. Importantly, the degree of cytolysis ex vivo significantly correlated with CAR-T cell engraftment detected in patients’ cerebrospinal fluid (CSF) for all six patients. Furthermore, cytotoxic activity of CAR-T cells as measured by TNFα, IL-2, and IFNγ release kinetics in GBOs mirrored cytokine levels in patient CSF samples over time. Our findings highlight a unique trial design and suggest GBOs as a valuable platform for real-time assessment of CAR-T cell bioactivity and for insights into immunotherapy efficacy.

IMMU-08. PRECLINICAL EVALUATION OF GAL-1-SPECIFIC CAR-T CELL FUNCTION IN AN IN VITRO AND EX VIVO MODEL OF GLIOBLASTOMA

Abstract BACKGROUND Human glioblastoma (GBM), especially those exhibiting anti-VEGF resistance, overexpress the carbohydrate-binding protein galectin-1 (Gal-1). To develop novel anti-GBM approaches, we developed Gal-1 targeted CAR-T cells to investigate potential antitumor effect with in vitro and ex vivo slice culture models. METHODS Uniform GBM brain slice cultures were collected using NICO’s Myriad resection tool and their Automated Preservation System (APS), designed to obtain fresh brain tumor samples maintaining tumor cytoarchitecture and microenvironment. To prepare Gal-1 CAR-T cells, we used creative laboratory to utilize a third-generation Gal-1-CAR construct with high binding and codon optimized scFv-Gal-1 fragment fused with CD3ζ and co-stimulatory cytoplasmic domains of CD28 and 4-1BB (Gal-1 scFv-CD28-4-1BB-CD3ζ). We then transfected into the T cells, from peripheral blood, mononuclear cells using a lentiviral system. The anti-tumor capabilities of CAR-T cells were evaluated by co-culturing with GBM cells and ex vivo APS-GBM slices. RESULTS The collected APS- GBM slices samples were clear of debris and blood product, not requiring post-surgical mechanical slices, and exhibited viability up to 12 days over traditionally prepared ex vivo cultures. With this, we found Gal-1-CAR-T cells grew exponentially in the presence of cytokines and maintained phenotypic and biological attributes such as cell viability, potency, migration, and T cell activation. Gal-1 scFv-CD28-4-1BB-CD3ζ -CAR-T cells killed more than 80% of GBM tumor cells within 24 hours in Gal-1+ve, but not Gal-1-ve GBM cells in vitro. Additionally, these CAR-T cells selectively caused significant release of IL-2, IFN-γ and TNF-α only from Gal-1+ve co-cultures and induced significant Gal-1+ve GBM cell death in an ex vivo model without any general toxicity. CONCLUSION Based on in vitro cell culture and ex vivo APS-brain slice culture results, we conclude that the novel Gal-1 scFv-CD28-4-1BB-CD3ζ CAR-T cell therapy may offer an effective therapeutic option. Further pre-clinical and clinical studies need to be performed to validate this novel approach

Sorafenib-induced macrophage extracellular traps via ARHGDIG/IL4/PADI4 axis confer drug resistance through inhibiting ferroptosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common as well as leading causes of mortality worldwide, and sorafenib is the first-line treatment in HCC patients. Unfortunately, drug resistance to sorafenib often develops. However, the underlying mechanism remains unclear. Here, we reveal the important role of macrophage extracellular traps (METs)-mediated crosstalk between macrophages and tumor cells in sorafenib resistance. METs in HCC tumor tissues were detected using immunofluorescence. The concentrations of MPO-DNA, elastase and cytokines were measured using ELISA. The mRNA expression levels of genes were confirmed by qRT-PCR. The siRNAs were conducted to knock ARHGDIG in Hepa1-6 and Hep3B cells. Western Blot assay was performed to determine protein expression of Rho GDP dissociation inhibitor gamma (ARHGDIG, or RHOGDI-3), PADI2, and PADI4. Cell viability and migration were evaluated by CCK-8 assay and transwell assay, respectively. Cell ferroptosis was assessed by measurement of Fe2+ concentration, flow cytometry assay of lipid ROS, and western blot assay of GPX4. The functions of sorafenib, DNase I, IL4 neutralization antibody and GPX4 in tumor growth were explored through in vivo experiments. Sorafenib induced MET formation in M2 macrophages rather than M1 macrophages derived from both human and mice. In Hepa1-6 HCC mice, METs clearance by DNase I improved response to sorafenib therapy, detected by tumor weight, tumor growth curve, tumor volume, and survival. By screening candidate cytokines that affect macrophage function, we found that sorafenib-promoting IL4 secretion by HCC cells plays a crucial role in sorafenib-induced MET formation. Understanding the critical role of IL4 in sorafenib-induced MET formation led us to find that IL4 neutralization significantly improved the efficiency of sorafenib in HCC models. Mechanistically, we discovered that sorafenib increased the expression of ARHGDIG in HCC cells, which led to the release of IL4. In M2 macrophages, IL4 triggered MET formation by elevating the mRNA and protein expression of peptidyl arginine deiminase 4 (PADI4) rather than PADI2. In HCC models, GSK484 inhibition of PADI4 could consistently weaken sorafenib resistance and improve sorafenib efficiency. Importantly, we discovered that METs contribute to sorafenib resistance by inhibiting the ferroptosis of HCC cells. Meanwhile, PADI4 inhibition or DNase I could reverse the sorafenib resistance caused by METs-inhibiting ferroptosis of HCC cells. Our study concludes that sorafenib-induced METs inhibit the ferroptosis of tumor cells, suggesting that targeting the IL4/PADI4/METs axis in HCC could reduce or prevent sorafenib resistance.

CTIM-21. FIRST-IN-HUMAN PHASE I CLINICAL TRIAL USING 8R-70CAR T CELLS FOR NEWLY DIAGNOSED ADULT GLIOBLASTOMA

Abstract BACKGROUND To address the challenges of chimeric antigen receptor (CAR) T cell therapy in GBM, we utilized IL-8 release from tumor cells as a ‘GPS homing signal’ to enhance T-cell trafficking with a novel CD70-specific CAR design (8R-70CAR) that targets GBM. The 8R-70CAR offers several advantages: 1) Improved CAR T-cell trafficking, enhancing antitumor efficacy; 2) Reduced CD70, reshaping the tumor microenvironment (TME) and boosting endogenous immunity; 3) Utilized CD27 as the CAR, a co-stimulatory molecule that enhances T-cell functions; 4) Enabled CAR T cells to act as a sink to neutralize or remove IL-8, a pro-cancer chemokine, from the TME; and 5) Required only 120 ml of whole blood (not leukapheresis) for CAR T cell production. Preclinically, 8R-70CAR significantly enhanced CAR-T cell tumor migration and persistence, leading to complete tumor regression and sustained immune memory in a chemo/radiation/PD-1-blockade-resistant GBM model. These findings led to a phase-I trial (IMPACT trial: FDA-IND#23881, NCT05353530) using 8R-70CAR T cells for CD70 -positive newly diagnosed adult GBM. METHODS We aim to recruit 18 adult patients (3 + 3 dose-escalation design) with CD70-positive (>20%) tumors. The CAR T cells will be given intravenously after completing standard-of-care (SOC) therapy. Primary endpoints: Evaluate safety, tolerability, and dose-limiting toxicities (DLTs) to determine the maximum tolerated dose. Secondary endpoints: Assess preliminary efficacy. Immune monitoring and correlative studies will also be conducted. RESULTS To date, four patients have been enrolled in the IMPACT trial, with 2 eligible patients participating in treatment at dose level 1 (1x10^6/kg). One patient has completed treatment with 8R-70CAR T cells, experienced no DLTs, and is currently in follow-up. The remaining patient is under SOC therapy before receiving CAR T cell treatment. CONCLUSIONS This study aims to address current therapeutic gaps and offers options for implementing a novel CAR design for the development of a more effective therapeutic modality for GBM.

Haloxylon salicornicum Phytochemicals Suppress NF-κB, iNOS and Pro-inflammatory Cytokines in Lipopolysaccharide-induced Macrophages.

Haloxylon salicornicum is traditionally used for the treatment of several disorders associated with inflammation. Despite it is a defense response against tissue injury and infections, inflammation can become a chronic condition that can negatively impact the body. This study investigated the effect of H. salicornicum phytochemicals nuclear factor-kappaB (NF-κB), inducible nitric oxide synthase (iNOS) and cytokines release by lipopolysaccharide (LPS)-challenged macrophages in vitro. The binding affinity of the tested phytochemical towards NF-κB and iNOS was investigated using molecular docking. Ten compounds (four coumarins, three sterols and three flavonoids) were isolated from the ethanolic extract of H. salicornicum. Treatment of LPS-challenged macrophages with the compounds resulted in remarkable decrease in NF-κB p65 and iNOS mRNA abundance. All compounds suppressed the production of nitric oxide (NO) and the pro-inflammatory cytokines (tumor necrosis factor (TNF)-α and interleukin (IL)-6) from macrophages challenged with LPS. Molecular docking revealed the ability of the isolated phytochemicals to bind NF-κB p65 and iNOS. In conclusion, H. salicornicum is a rich source of phytochemicals with anti-inflammatory properties. The anti-inflammatory efficacy of H. salicornicum phytoconstituents is mediated via their ability to modulate NF-κB and iNOS, and suppress the release of NO, TNF-α, and IL-6 from macrophages.

Unraveling the role of exercise in cancer suppression: insights from a mathematical model

Recent experimental studies have shown that physical exercise has the potential to suppress tumor progression. Such suppression has been reported to be mediated by the exercise-induced activation of natural killer (NK) cells through the release of IL-6, a cytokine. Aimed at shedding light on how exercise-induced NK cell activation helps in the suppression of cancer, we developed a coarse-grained mathematical model based on a system of ordinary differential equations describing the interaction between IL-6, NK-cells, and tumor cells. The model is then used to study how exercise duration and exercise intensity affect tumor suppression. Our results show that increasing exercise intensity or increasing exercise duration leads to greater and sustained tumor suppression. Furthermore, multi-bout exercise patterns hold promise for improving cancer treatment strategies by adjusting exercise intensity and frequency. Thus, the proposed mathematical model provides insights into the role of exercise in tumor suppression and can be instrumental in guiding future experimental studies, potentially leading to more effective exercise interventions.

Chemical analysis, antibacterial and anti-inflammatory effect of Achillea fragrantissima essential oil growing wild in Egypt

BackgroundAchillea fragrantissima (F. Asteraceae) is traditionally used to treat skin infections and inflammation. The present work intended to prepare essential oils (EOs) from A. fragrantissima aerial parts growing widely in Egypt and investigate its antibacterial activity against skin-related pathogens and in vitro cell-based anti-inflammatory activity.MethodsEOs of the fresh aerial parts were extracted by hydrodistillation (HD), microwave-assisted hydrodistillation (MAHD), and head-space (HS), while those of the dried ones were prepared by supercritical fluid (SF). The result EOs were analyzed using GC/MS. The antibacterial activity was evaluated alongside Pseudomonas aeruginosa ATCC 9027, Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 25923, Streptococcus pyogenes ATCC 12344, Clostridium perfringens ATCC 13124 by agar diffusion, microwell dilution, and biofilm formation tests. The anti-inflammatory activity was evaluated by measuring tumor necrosis factor-alpha (TNF-α), interleukin 2 (IL-2), and 6 (IL-6) in lipopolysaccharides (LPS)- stimulated RAW 264.7 cells using ELISA assays in addition, expression of nitric oxide synthase (iNOS) was measured via western blot.ResultsThe SF method gave the highest EO yield (1.50 mL v/w). Oxygenated components constituted the highest percentage in the four methods, 84.14, 79.21, 73.29 and 33.57% in the HS, HD, MAHD, and SF, respectively. Moreover, variation in the amount of identified compounds was apparent; in HS EO α-thujone (29.37%), artemisia ketone (19.59%), and santolina alcohol (14.66%) are major components, while α-thujone (20.38%) and piperatone (12.09%) were significant in HD. Moreover, ( +)-spathulenol (12.22%) and piperatone (10.48%) were significant in MAHD, while piperatone (14.83%) and β-sitosterol (11.07%) were significant in SF EO. HD, MAHD, and SF EOs exhibited susceptibility against P. aeruginosa (IZ = 9–14 mm), E. coli (11–13 mm), and C. perfringens (IZ = 10–14 mm) in agar diffusion assay. MAHD EOs demonstrated potent growth inhibition (MICs = 0.25–2 mg/mL), followed by HD EOs (MICs = 13–52 mg/mL) to all tested microorganisms in well microdilution assay. Also, they exert MBC values equal to or higher than the MICs. Furthermore, SF EOs inhibited the biofilm formation of all tested microorganisms by 65.12—80.84%. Specifically, MAHD and HD EOs efficiently suppress the biofilm of S. pyogenes (77.87%) and P. aeruginosa (60. 29%), respectively. Ultimately, HD and SF EOs showed anti-inflammatory activity by suppressing the TNF-α, IL-2, and IL-6 release and iNOS expression in LPS-stimulated RAW 264.7 macrophages.ConclusionA. fragrantissima EO is rich in oxygenated volatile compounds with antibacterial and anti-inflammatory activities. It is encouraged as a bioactive agent for adjusting skin infections, though additional studies are essential for their safety in clinical settings.

Keep neuroinflammation in mind when addressing Alzheimer's disease: A microglia perspective.

This commentary offers a detailed examination of a newly published paper on the effects of small molecule decoys of amyloid-β (Aβ) aggregation on microglial activation. It was discovered that the NSC16224 decoy peptide inhibited proinflammatory cytokines TNFα and IL6 release from microglia in response to Aβ40 and Aβ42 treatment. The research addresses the potential of blocking a sequence of events that lead to the progression of Alzheimer's disease (AD). Here, we discuss the significance of these results in neuroinflammation, highlighting the greater implications for how decoy peptides would be interesting for the research and development of new drugs for AD therapy.

Sevoflurane Postconditioning Mitigates Neuronal Hypoxic-Ischemic Injury via Regulating Reactive Astrocytic STAT3 Protein Modification

Astrocyte activation plays a pivotal role in accelerating the cascade of neuroinflammation associated with the development of hypoxic-ischemic brain injury. This study aimed to investigate the mechanism by which sevoflurane postconditioning mitigates neuronal damage through astrocytes by regulating reactive astrocytic Signal Transducer and Activator of Transcription 3 (STAT3) modifications. A modified Rice‒Vannucci model in rats and a conditioned culture system established by subjecting primary astrocytes to oxygen glucose deprivation, followed by using the conditioned medium to culture the neuron cell line SH-SY5Y were used to simulate HI insult in vivo and in vitro, respectively. These models were followed by 30 minutes of 2.5% sevoflurane treatment. Stattic was used to inhibit STAT3 phosphorylation, and (Z)-PUGNAc or OSMI-1 was added to regulate O-linked-β-N-acetylglucosamine modification (O-GlcNAcylation) in primary astrocytes in vitro. Neurobehavioral tests, Nissl staining, CCK8 assay, and flow cytometry for apoptosis were used to assess neuronal function. Immunofluorescence staining was used to detect astrocyte reactivity and the intracellular distribution of STAT3. Immunoprecipitation combined with Western blotting was used to evaluate the O-GlcNAcylation of STAT3. Protein expression and phosphorylation levels were detected by Western blotting. ELISA was conducted to detect the detrimental cytokines IL-6 and IL-1β in astrocyte-conditioned medium. Sevoflurane postconditioning enhanced the O-GlcNAcylation of astrocytic STAT3 following HI insult via the manner of OGT. Crosstalk between O-GlcNAcylation and phosphorylation of STAT3 showed that O-GlcNAcylation inhibited STAT3 phosphorylation. The inhibitory effect on astrocytes suppressed STAT3 nuclear translocation, reduced astrocyte reactivity, decreased the release of the inflammatory cytokines IL6 and IL-1β, attenuated neuronal apoptosis following HI insult, and improved neuron viability. Sevoflurane postconditioning increased astrocytic STAT3 O-GlcNAcylation level to competitively inhibit STAT3 phosphorylation. This deactivated downstream inflammation pathways and reduced astrocyte reactivity, thereby mitigating HI insult in neurons both in vivo and in vitro.

Transcriptomic Analysis of THP-1 Cells Exposed by Monosodium Urate Reveals Key Genes Involved in Gout.

Gout is a common inflammatory arthritis, which is mainly caused by the deposition of monosodium urate (MSU) in tissues. Transcriptomics was used to explore the pathogenesis and treatment of gout in our work. The objective of the study was to analyze and validate potential therapeutic targets and biomarkers in THP-1 cells that were exposed to MSU. THP-1 cells were exposed to MSU. The inflammatory effect was characterized, and RNA-Seq analysis was then carried out. The differential genes obtained by RNA-Seq were analyzed with gene expression omnibus (GEO) series 160170 (GSE160170) gout-related clinical samples in the GEO database and gout-related genes in the GeneCards database. From the three analysis approaches, the genes with significant differences were verified by the differential genes' transcription levels. The interaction relationship of long non-coding RNA (lncRNA) was proposed by ceRNA network analysis. MSU significantly promoted the release of IL-1β and IL-18 in THP-1 cells, which aggravated their inflammatory effect. Through RNA-Seq, 698 differential genes were obtained, including 606 differential mRNA and 92 differential `LncRNA. Cross-analysis of the RNA-Seq differential genes, the GSE160170 differential genes, and the gout-related genes in GeneCards revealed a total of 17 genes coexisting in the tripartite data. Furthermore, seven differential genes-C-X-C motif chemokine ligand 8 (CXCL8), C-X-C motif chemokine ligand 2 (CXCL2), tumor necrosis factor (TNF), C-C motif chemokine ligand 3 (CCL3), suppressor of cytokine signaling 3 (SOCS3), oncostatin M (OSM), and MIR22 host gene (MIR22HG)-were verified as key genes that analyzed the weight of genes in pathways, the enrichment of inflammationrelated pathways, and protein-protein interaction (PPI) nodes combined with the expression of genes in RNA-Seq and GSE160170. It is suggested that MIR22HG may regulate OSM and SOCS3 through microRNA 4271 (miR-4271), OSM, and SOCS3m; CCL3 through microRNA 149-3p (miR-149-3p); and CXCL2 through microRNA 4652-3p (miR-4652-3p). The potential of CXCL8, CXCL2, TNF, CCL3, SOCS3, and OSM as gout biomarkers and MIR22HG as a therapeutic target for gout are proposed, which provide new insights into the mechanisms of gout biomarkers and therapeutic methods.

Development of optimized standardized extracts of Echinodorus macrophyllus for arthritis management

Ethnopharmacological relevanceEchinodorus macrophyllus is a medicinal plant traditionally used in Brazil to treat rheumatic diseases. It is listed as a priority species for the development of herbal preparations for the Brazilian Unified Health System (SUS). Previous studies have demonstrated the anti-inflammatory and antiedematogenic properties of extracts and fractions from this species, but these preparations were neither standardized nor optimized for anti-arthritis effects. Aim of studyThis study aimed to investigate the anti-inflammatory and antiarthritic effects of various standardized extracts from the aerial parts of E. macrophyllus and to outline their mechanisms of action. The goal was to define an extract suitable for future scientific validation and clinical use. Material and methodsExtracts from the aerial parts of E. macrophyllus were prepared through percolation with 96°GL ethanol (EtOH) and hydroethanolic solutions at 90%, 70%, and 50% (v/v). They were standardized based on the contents of six chemical markers quantified by UPLC-DAD. Mice with acute or chronic antigen-induced arthritis (AIA) were treated with the extracts orally. The effects were evaluated by counting total and differential inflammatory cells, measuring cytokines by ELISA, and by histological analysis with toluidine blue staining. The mechanism of selected extracts was investigated in LPS-stimulated murine chondrocytes. ResultsAll extracts, except for the 90% EtOH extract, exhibited significant anti-inflammatory activity in acute AIA, reducing the migration of total inflammatory cells, neutrophils, and monocytes to the intra-articular cavity. In chronic AIA, both the 96°GL EtOH and 70% EtOH extracts markedly reduced the migration of total inflammatory cells, neutrophils, and monocytes to the intra-articular cavity, decreased IL-1β and CXCL1 levels in the periarticular tissue, and diminished proteoglycan loss in the articular cartilage. Both extracts reduced IL-6 release and MMP-3 expression in LPS-stimulated chondrocytes. The 70% EtOH and 50% EtOH extracts showed heightened activity and higher levels of chemical markers, notably cinnamoyl-tartaric acid derivatives. ConclusionsThe standardized 70% EtOH and 50% EtOH extracts from the aerial parts of E. macrophyllus, which contain higher levels of chemical markers, exhibit potent anti-inflammatory activity and reduce proteoglycan degradation. These extracts are potentially useful for developing herbal preparations to manage arthritic conditions and should be prioritized for further studies.

Comparison of n-3 PUFA-Enriched vs. Olive-Oil-Based Lipid Emulsion on Oxidative Stress and Inflammatory Response in Critically Ill Post-Surgery Adults: Secondary Analysis of a Randomized Controlled Trial.

Malnutrition in critically ill patients represents a major concern as it can lead to adverse outcomes including increased morbidity and mortality. These patients exhibit an impaired immune response accompanied by increased oxidative stress. Nutritional support, including parenteral nutrition (PN), is critical in these patients. Intravenous lipid emulsions (ILEs), a key component of PN, provide energy and intervene in the modulation of inflammation. This was a secondary study of a randomized clinical trial at the Reina Sofia University Hospital (Murcia, Spain) for critically ill patients following major abdominal surgery that were administered PN supplemented with olive-oil-based ILE (OO-ILE, n = 29) or a mixed-lipid ILE (soybean oil, medium chain triglycerides, OO and fish oil, SMOF-ILE, n = 25). The effects on clinical outcomes, metabolic markers, oxidative stress, and inflammation were evaluated. No significant differences were observed between groups in the clinical parameters and outcomes, oxidative stress, or inflammatory markers. The within-group evaluation demonstrated an increase in total antioxidant capacity in both groups, while OO-ILE increased the levels of 15-F2t-isoprostane. In addition, the results showed that both mixtures reduced the release of IL-1β and IL-6. These findings suggest that both treatments had similar effects on oxidative stress and inflammatory response in this type of patient.

Malondialdehyde-specific natural IgM inhibit toll-like receptor 4 and peptidyl-arginine deiminase 4-dependent NETosis triggered by coronary extracellular vesicles of myocardial infarction patients

Abstract Background Neutrophil extracellular traps (NETs) are critical mediators of thromboinflammation during acute myocardial infarction (AMI). However, triggers and signalling pathways of NETosis in AMI remain incompletely understood. Levels of extracellular vesicles (EV) carrying oxidation-specific epitopes (OSE) originating from lipid peroxidation are increased at the culprit site in AMI. Importantly, natural IgM antibodies with specificity for OSE, such as malondialdehyde (MDA), have been shown to modulate functional effects of EV, and are associated with reduced cardiovascular risk. Purpose We investigated the stimulatory capacity of EV on neutrophil effector functions and specifically targeted key mediators of NET formation using pharmacological inhibitors and atheroprotective natural IgM to inhibit this process. Methods Patients were included after diagnosis of ST-segment elevation myocardial infarction and blood was aspirated from the culprit site and peripheral arterial site (n=28) during primary percutaneous coronary intervention (pPCI). Myocardial function was documented by cardiac magnetic resonance imaging 4±2 days and 195±15 days after pPCI. EV were isolated from cell culture supernatants and culprit site plasma for neutrophil stimulation in vitro. Pharmacological inhibitors were used to map NET signalling pathways of EV. Isolated EV were used for neutrophil stimulation in a murine injection model in the presence of the MDA-specific IgM LR04 or an isotype control. NET formation and other neutrophil functions were assessed by flow cytometry, ELISA and fluorescence microscopy. Results Levels of NET surrogate markers and CD45+ MDA+ EV were higher at the culprit site than in the peripheral circulation. EV generated by LPS-activated THP-1 monocytic cells induced in vitro NET formation, release of neutrophil elastase and IL-8, and degranulation of MPO and NGAL in primary human neutrophils, but not reactive oxygen species. Toll-like receptor 4 (TLR4) and peptidyl-arginine deiminase 4 (PAD4) were identified as key mediators of NET formation induced by in vitro-generated EV and EV isolated from the culprit site of AMI patients. Functionally, the MDA-specific monoclonal IgM antibody LR04, but not an isotype control, inhibited the ability of patient-derived and in vitro-generated EV to trigger release of NETs in primary human neutrophils and in mice. Titres of MDA-specific IgM antibodies were inversely associated with the NET marker citH3 in blood of AMI patients. Finally, we found that the concentration of CD45+ MDA+ EV per protective MDA-specific IgM at the culprit site showed an inverse association with left ventricular ejection fraction 72 hours and 6 months after AMI. Conclusions We show that EV can trigger several neutrophil effector functions. EV-induced NET formation is TLR4- and PAD4-dependent and can be inhibited by OSE-specific natural IgM potentially influencing cardiovascular outcomes after an acute event.

Transpulmonary proteome gradients identify inflammatory pathways involved in the development of pulmonary vasculopathy in heart failure

Abstract Background Some, but not all, patients with heart failure (HF) develop pulmonary vascular disease (PVD), which contributes to poor prognosis. Mechanisms leading to PVD in HF are poorly understood. Unbiased analysis of transpulmonary gradients may identify not only markers but also mediators of PVD by identifying proteins that may be consumed or elaborated across the lungs. Purpose Examine hemodynamics and transpulmonary proteome gradients in HFrEF and controls. Methods 21 non-HF controls and 160 patients with advanced HFrEF underwent pulmonary artery (PA) catheterization with blood sampling from the PA catheter in the wedged position (02sat > 90%, indicating pulmonary venous blood) and the un-wedged position to obtain transpulmonary proteome gradients. Samples from controls and HF from the highest quartile (Q4, n=40) and lowest quartile (Q1, n=40) of pulmonary vascular resistance (PVR) were analyzed using proteomic PEA assay of 275 proteins (Olink Target 96, panels CVII, CVIII, Inflammation). Pulmonary artery concentrations ("markers") or transpulmonary fold-change gradients (i.e. putative "mediators") of normalized protein expression (NPX) were plotted against significance; Benjamini–Hochberg FDR<0.05 considered as significant. Results Patients displayed typical characteristics of HFrEF (age 56±8y, NYHA 3.0 ±0.6, 87% males, LVEF 24±9.6%), Controls had similar anthropometrics, gender and age. Comparing all HF to Con, 38 markers of HF were identified (NPX fold-change>2), including 33 up (top 5: NTproBNP, BNP, GDF15, FGF23, IL8) and 5 down. HF patients in Q1 had median PVR 1.5 (IQR: 0.3-1.7) WU and those in Q4 had PVR 5.4 (4.3-6.6) WU. Comparison of Q1 and Q4 identified 3 protein markers of high PVR in PA blood from HF: chemokine CCL3, TNFRSF13B and surfactant protein-D (PSP-D). Examination of protein gradients from HF lungs (Figure) showed significant uptake of 14 protein mediators, most of which were associated with inflammatory responses (top 5: OSM, MMP9, CCL19, BNP, TR), and release of 8 mediators (top 4: IL-6, IL33, CCL4, CXCL10). In contrast, protein gradients were negligible in controls. Patients in Q4 PVR group were characterized by the highest pulmonary uptake of OSM (Oncostatin-M), lymphatic chemokine CCL19, BNP, MMP9, and had more significant transpulmonary release of IL6 and IL33. Across all patients, OSM lung uptake was the strongest correlate of transpulmonary pressure gradient (r=-0.3, p<0.001). Conclusions Lungs of patients with HF, and particularly those with high PVR, display abnormal uptake and release of proinflammatory CC and CXC chemokines and cytokines from IL1 and IL6 family, along with increased pulmonary uptake of IL6-type cytokine Oncostatin-M, a known mediator of lung inflammation and fibrosis, that acts via gp130/JAK/STAT, suggesting a novel role in PVD due to HF.Transpulmonary proteome in HF patients

Sinomenine hydrochloride alleviates autoimmune myocarditis via suppressing Th1 cell induced-M1 macrophage pyroptosis

Abstract Background Myocarditis is typical of the complex inflammatory response in the heart with high morbidity and mortality, accompanied by tissue damage and cardiac fibrosis. Previous studies have demonstrated that CD4+ T cells are crucial to the cardiac autoimmunity of myocarditis, but the role of different CD4+ T cell subsets remains unclear. Recently, sinomenine hydrochloride, a natural compound from the traditional Chinese herb Sinomenium acutum, is reported to inhibit T cell proliferation in some systemic autoimmune diseases such as rheumatoid arthritis. Given that autoimmune myocarditis is an organ-specific autoimmune disease, the effect of sinomenine hydrochloride on autoimmune myocarditis demands further study. Purpose This study aims to elucidate the role of CD4+ T-helper 1 (Th1) cells in regulating inflammatory cell death in autoimmune myocarditis and investigate the pharmacological effect of sinomenine hydrochloride on CD4+ Th1 cells and autoimmune myocarditis. Methods Male Balb/c mice were immunized with myosin heavy chain-α peptides to establish the experimental autoimmune myocarditis (EAM) model, followed by the treatments with sinomenine hydrochloride by gavage. Tohoku Hospital Pediatrics-1 (THP-1) cell line and murine T cells were used for in vitro experiments. Results In the acute phase of EAM, we observed plenty of CD4+ T cells infiltrating the heart tissue. RNA sequencing revealed that among the genes encoding typical transcription factors of CD4+ T cell subset, only Tbx21 and Stat4, both representing Th1 cells, were upregulated, which consisted with immunofluorescence staining. Further studies indicated that Th1 cells not only mediated classical activated macrophage (M1 macrophage) polarization by secreting interferon-γ (IFN-γ) but also associated with pro-inflammatory macrophage pyroptosis in the EAM, with the cleavage of gasdermin D (GSDMD), caspase-1 and IL-1β. Subsequent in vitro experiments confirmed that IFN-γ could dramatically promote macrophage pyroptosis combined with ATP, leading to the release of IL-1β and IL-18(Figure 1). To find an effective method for Th1 cell suppression and myocarditis remission, we identified that sinomenine hydrochloride impeded Th1 cell activation, differentiation and proliferation in vitro. Furthermore, sinomenine hydrochloride inhibited Th1 cell infiltration and M1 macrophage pyroptosis in the EAM heart, In the chronic phase, cardiac remodeling and fibrosis were suppressed, and cardiac systolic function was significantly improved in the treatment group (Figure 2). Conclusion Our study demonstrates that Th1 cells-induced M1 macrophage pyroptosis is an essential pathogenic mechanism of EAM. Sinomenine hydrochloride can alleviate the severity and progression of EAM, which may become a novel therapeutic strategy for treating myocarditis.Figure 1Figure 2