Lipopolysaccharide Stimulation Research Articles

Inflammation induced PFKFB3-mediated glycolysis promoting myometrium contraction through the PI3K-Akt-mTOR pathway in preterm birth mice.

Inflammation is a significant risk factor for preterm birth. Inflammation enhances glycolytic processes in various cell types and contributes to the development of myometrial contractions. However, the potential of inflammation to activate glycolysis in pregnant murine uterine smooth muscle cells (mUSMCs) and its role in promoting inflammatory preterm birth remain unexplored. In this study, lipopolysaccharide was employed to establish both cell and animal inflammation models. We found that inflammation of mUSMCs during late pregnancy could initiate glycolysis and promoted cell contraction. Subsequently, the inhibition of glycolysis using the glycolysis inhibitor 2-deoxyglucose can reverse inflammation-induced cell contraction. The expression of 6-phosphofructokinase 2 kinase (PFKFB3) was significantly upregulated in mUSMCs following lipopolysaccharide stimulation. Additionally, lactate accumulation and enhanced contraction were observed. Inhibition of PFKFB3 reversed the lactate accumulation and enhanced contraction induced by inflammation. We also found that inflammation activated the phosphatidylinositol 3-kinase (PI3K) - protein kinase B (Akt) - mammalian target of rapamycin (mTOR) pathway, leading to the upregulation of PFKFB3 expression. The PI3K-Akt pathway inhibitor LY294002 and the mTOR pathway inhibitor Rapamycin effectively inhibited the upregulation of PFKFB3 protein expression, lactate production, and the enhancement of cell contraction induced by lipopolysaccharide. This study indicates that inflammation regulates PFKFB3 through the PI3K-Akt-mTOR pathway, which enhances the glycolytic process in pregnant mUSMCs, ultimately leading to myometrial contraction.

Cyanidin-3-glucoside reduces cell migration and inflammatory profile of acute leukemia cells.

Anthocyanins, water-soluble flavonoids found in fruits and vegetables, exhibit diverse biological activities, with cyanidin being the most common pigment. While cyanidin's chemo preventive and -antioxidant activity is well-studied, its impact on inflammatory profile and migration capacity of leukemic cells are less understood. This study evaluates the effects of Cyanidin-3-glucoside (C3G) on the inflammatory mechanisms influencing leukemic cell migration. Results show that C3G doses up to 50 µM do not affect cell metabolism, viability, or cell cycle phases. C3G significantly reduces TNF-α, IL-8, CCL2 production, and the p-NFκB/NFκB ratio in LPS (Lipopolysaccharide)-challenged cells. It also diminishes migration rates in response to LPS or fMLP (N-formyl-methionyl-leucyl-phenylalanine) stimulation and reduces Rho-GTP expression. Thus, C3G modifies the inflammatory and migration properties of leukemic cells, highlighting the potential of anthocyanins as a complementary therapy and an avenue for further therapeutic intervention.

Sex differences in the neuroinflammatory signaling pathway: effect of miRNAs on fatty acid synthesis in microglia

BackgroundSignificant sex differences exist in the prevalence and incidence of Alzheimer’s disease (AD). Notably, testosterone has been reported to regulate cognitive functions in the brain, with low serum testosterone levels correlating with increased AD risk. However, the specific mechanisms underlying this relationship remain unclear. Recent studies have demonstrated that microglia, the primary innate immune cells in the brain, play a crucial role in AD development. Therefore, this study aimed to explore sex differences in microglial function, specifically focusing on the role of testosterone in miRNA-mediated regulation of microglial gene expression.MethodsMicroglia were isolated from pooled hippocampal tissue of five 8-month-old male and female mice. Total RNA was extracted and subjected to miRNA microarray analysis. The mouse microglial cell line MG6 was used for in vitro experiments. Following testosterone treatment, miRNA, gene, and protein expression levels were investigated. An inflammatory response was induced using lipopolysaccharide (LPS) stimulation, and subsequent p65 phosphorylation was assessed.ResultsSex-dependent differences were observed in miRNA-mediated biological processes, with males exhibiting greater changes. Male-enriched miRNAs were associated with fatty acid synthesis and metabolism pathways. In MG6 cells, testosterone treatment upregulated the expression of several miRNAs enriched in male microglia, particularly those targeting genes related to fatty acid synthesis. Additionally, testosterone significantly reduced the gene expression of fatty acid synthase (FASN). This testosterone-induced inhibition of FASN expression attenuated NF-κB/p65 phosphorylation. Consequently, the suppression of FASN expression led to reduced expression and secretion of tumor necrosis factor-alpha following LPS stimulation in MG6 cells.ConclusionsThese findings suggest that testosterone modulates inflammation in male microglia by regulating fatty acid synthesis, potentially contributing to the observed sex differences in AD pathogenesis.

Activity-based chemical proteomics reveals caffeic acid ameliorates pentylenetetrazol-induced seizures by covalently targeting aconitate decarboxylase 1

BackgroundEpilepsy is a neurological disorder characterized by recurrent seizures, tightly associated with neuroinflammation. Activation of inflammatory cells and molecules in damaged nervous tissues plays a pivotal role in epilepsy. Caffeic acid, one of the most abundant polyphenols in coffee, has shown potent protective effects as a phytomedicine in various neurological disorders. However, the direct protein targets and exact molecular mechanisms of caffeic acid in epilepsy, remain largely elusive.PurposeThis study aimed to explore the protective effects of caffeic acid in epilepsy and elucidate its underlying mechanism.MethodsIn this study, we established pentylenetetrazol-induced acute and kindling models of seizures. Additionally, a BV2 microglial cellular inflammation model was established by lipopolysaccharide stimulation. The potential direct protein targets of caffeic acid in BV2 cells were analyzed using an activity-based protein profiling (ABPP) with a caffeic acid probe. Various methods such as pull-down assay, immunofluorescence and cellular heat transfer assays were used for experimental validation. The anti-inflammatory effects of caffeic acid in LPS-activated BV2 cells was proved by knocking down the target protein.ResultsHere, we found that caffeic acid exhibits antiepileptic effects in pentylenetetrazol-induced epilepsy mice and exerts anti-neuroinflammation effect in vivo and in vitro. Besides, we discovered that caffeic acid directly binds to aconitate decarboxylase 1 and influenced its enzymatic activity. Moreover, we indicated that caffeic acid exhibits anti-neuroinflammation effect through aconitate decarboxylase 1 mediated PERK-NF-κB pathway in vitro.ConclusionIn summary, this study elucidates, for the first time, the potential antiepileptic targets and mechanism of action of caffeic acid using the ABPP strategy. Our study provides evidence supporting the utilization of caffeic acid as a promising therapeutic agent for treating epilepsy and neuroinflammation-related disorders.

IFN alpha signaling drives hematopoietic stem cells malfunction under acute inflammation.

Inflammation stimulation regulates the activity of hematopoietic stem cells (HSCs) through direct-sensing and cytokine-mediation. It is known that HSCs directly sense lipopolysaccharide (LPS), a classical infection-related inflammatory signal, via toll like receptor 4 (TLR4) and subsequently become active. However, the mechanism underlying the activity change of HSCs induced by LPS remains incompletely disclosed. Here we explored that under LPS stimulation, the activation of interferon alpha (IFNα) signal pathway resulted in the activation and exhaustion of HSCs in vitro, indicating HSCs directly responded to LPS through the downstream IFNα signal pathway. We also discovered the increased production of IFNα in mice bone marrow and expression of interferon-α/β receptor (IFNAR) on mice HSCs after LPS stimulation. Creatine, an IFNα inhibitor, could reverse the activation and prevent the exhaustion of HSCs caused by LPS by suppressing the expressions of genes associated with the IFNα signal pathway both in vitro and in vivo. Furthermore, we found that the IFNAR deficiency in mice effectively protected HSCs from activation, elevated apoptosis and impaired reconstitution ability under LPS stimulation in vivo. This finding further supports the notion that LPS activates and injures HSCs indirectly via promoting IFNα secretion in the bone marrow environment. Overall, our findings reveal that LPS causes the injury to HSCs either through direct or cytokine-mediated indirect activation of the IFNα signal pathway.

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

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

Stigmasterol from Prunella vulgaris L. Alleviates LPS-induced mammary gland injury by inhibiting inflammation and ferroptosis.

Dairy mastitis, a prevalent condition affecting dairy cattle, represents a significant challenge to both animal welfare and the quality of dairy products. However, current treatment options remain limited. Stigmasterol (ST) is a bioactive component of Prunella vulgaris L. (PV) with various pharmacological functions such as anti-inflammatory and anti-oxidation. At present, the specific effects and underlying mechanisms of PV and ST on dairy mastitis are still not fully understood. The aim of this research was to evaluate the pharmacological effects of PV and its active component ST on lipopolysaccharide (LPS) -stimulated bovine mammary epithelial cells (BMECs) and a mouse mastitis model, and to elucidate the possible mechanisms of action. UPLC-Q-TOF-MS/MS was employed to identify the constituents of PV. BMECs and mice were used to establish in vitro and in vivo models of mastitis. Western Blotting, RT-qPCR, immunofluorescence and other techniques were used to explore the effects of PV and ST on inflammatory factors, blood-milk barrier integrity, ferroptosis related indicators and their potential molecular mechanisms. PV significantly attenuated the production of inflammatory mediators by LPS-stimulated BMECs. Subsequently, ST was found to be a potent anti-inflammatory agent in PV by inhibiting TLR4/NF-κB signaling pathway. This inhibition inhibits the myosin light chain (MLC)/MLC kinase signaling cascade and alleviates blood-milk barrier (BMB) disruption in BMECs. In addition, ferroptosis occurred in BMECs after LPS stimulation, and ST inhibited ferroptosis by stimulating Nrf2/GPX4 signaling pathway. Treatment of BMECs with the Nrf2 inhibitor ML385 significantly attenuated the therapeutic effect of ST. In vivo experiments further confirmed that both PV and ST attenuated LPS-induced breast tissue damage while reducing ferroptosis levels and restoring BMB. ST from PV exhibits substantial anti-inflammatory properties and is a promising candidate for the treatment of dairy mastitis.

Blueberry-derived exosome like nanovesicles carry RNA cargo into HIEC-6 cells and down-regulate LPS-induced inflammatory gene expression: A proof-of-concept study.

Exosome-like nanovesicles (ELNs) of food origin have received great attention in the last decade, due to the hypothesis that they contain bioactive molecules. ELNs purified from edible species have been shown to be protective and are able to regulate intestinal homeostasis. Despite ELNs being potential rising stars in modern healthy diets and biomedical applications, further research is needed to address underlying knowledge gaps, especially related to the specific molecular mechanism through which they exert their action. Here, we investigate the cellular uptake of blueberry-derived ELNs (B-ELNs) using a human stabilized intestinal cell line (HIEC-6) and assess the ability of B-ELNs to modulate the expression of inflammatory genes in response to lipopolysaccharide (LPS). Our findings show that B-ELNs are internalized by HIEC-6cells and transport labeled RNA cargo into them. Pretreatment with B-ELNs reduces LPS-induced ROS generation and cell viability loss, while modulating the expression of 28 inflammatory genes compared to control. Pathway analysis demonstrates their ability to suppress inflammatory responses triggered by LPS. In conclusion, our data indicate that B-ELNs are up taken by HIEC-6cells and can modulate inflammatory responses after LPS stimulation, suggesting a therapeutic potential. This study demonstrates the role of B-ELNs in regulating crucial biological processes, like anti-inflammatory responses, which could support intestinal health.

Characterization of tripartite motif containing 59 (TRIM59) in Epinephelus akaara: Insights into its immune involvement and functional properties in viral pathogenesis, macrophage polarization, and apoptosis regulation

The tripartite motif-containing (TRIM) superfamily is the largest family of RING-type E3 ubiquitin ligases that is conserved across the metazoan kingdom. Previous studies in mammals have demonstrated that TRIM59 possesses ubiquitin-protein ligase activity and acts as a negative regulator of NF-κB signaling. However, TRIM59 has rarely been characterized in fish. This study aimed to characterize TRIM59 from Epinephelus akaara (Eatrim59) and elucidate its structural features, expression patterns, and functional properties in innate immune responses and in the regulation of apoptosis. Eatrim59 is composed of 406 amino acids with a molecular weight of 45.84kDa and a theoretical isoelectric point of 5.25. It comprises a conserved RING domain, a B-box motif, and a coiled-coil region. Subcellular localization analysis revealed that Eatrim59 was localized in the endoplasmic reticulum. Eatrim59 was ubiquitously expressed in all tissues examined, with the highest relative expression detected in the blood, followed by the brain and spleen. Temporal expression of Eatrim59 was dynamically regulated in response to in vivo immune stimulation by Toll-like receptor ligands and nervous necrosis virus infection. In FHM cells overexpressing Eatrim59, an increase in viral replication was observed upon infection with the Viral hemorrhagic septicemia virus. This phenomenon is attributed to Eatrim59-mediated downregulation of interferon, pro-inflammatory cytokines, and other antiviral pathways. Moreover, macrophages stably overexpressing Eatrim59 exhibited a decrease in nitric oxide production and the formation of a filamentous actin structure upon lipopolysaccharide stimulation, indicating dampened M1 polarization. Furthermore, a decrease in apoptosis was observed in Eatrim59-overexpressing FHM cells under oxidative stress induced by H2O2. In conclusion, these findings demonstrate the multifaceted role of Eatrim59 as a regulator of innate immune response and apoptosis in E. akaara.

Impaired LPS Signaling in Macrophages Overexpressing the P2X7 C-Terminal Domain or Anti-P2X7 C-Terminal Domain Intrabody

The P2X7 receptor is involved in innate immune responses, with its intracellular C-terminal domain capable of interacting with signaling molecules to regulate immune cell activation; however, the mechanisms underlying the signaling complexes remain unclear. To elucidate the function of the P2X7 C-terminal domain, we established bone marrow-derived macrophage (BMDM) cell lines from transgenic (Tg) mice overexpressing the C-terminal domain of P2X7 or anti-P2X7 C-terminal domain single-chain variable fragment (scFv) intrabody. In contrast to wild-type mouse BMDMs, the Tg BMDMs showed impairment of inflammatory responses induced by lipopolysaccharide (LPS) stimulation, such as NF-κB activation and subsequent TNF-α, IL-1β, and IL-6 expression. Furthermore, P2X7 was specifically associated with myeloid differentiation primary response gene 88 (MyD88) in wild-type BMDMs; its specific interaction was strongly interfered with by overexpression of the P2X7 C-terminal domain or anti-P2X7 C-terminal domain scFv in Tg BMDMs. These observations strongly suggest that P2X7 may have pivotal roles in LPS signaling cascades and could modulate macrophage inflammatory responses through its C-terminal domain.

Ginsenoside Rd protects against acute liver injury by regulating the autophagy NLRP3 inflammasome pathway

Ginsenoside Rd (Rd) is a bioactive compound predominantly found in Panax ginseng C.A. Meyer and Panax notoginseng (Burkill) F.H. Chen ex C.H. Chow, both species belonging to genus Panax in the Araliaceae family. However, its hepatic protective effect against acute liver injury and related mechanistic action remain unexplored. To investigate the protective effect of Rd against thioacetamide (TAA)-induced acute liver injury and assess its underlying regulatory mechanisms related to autophagy and inflammation. Forty-eight 8 weeks old C57BL/6 mice were treated with saline (control or model group), Rd (12.5 mg/kg, 25 mg/kg or 50 mg/kg), and diammonium glycyrrhizinate (DG, 30 mg/kg) for three days. Then the mice were stimulated with TAA to establish acute liver injury model, excluding the control group. HSC-T6 cells were treated with Rd at concentrations of 2.5, 5, or 10 µM, for 12 h with or without Lipopolysaccharide (LPS) stimulation at 100 ng/mL. Immunofluorescence staining, qPCR and Western blot were employed to analyze the expressions of genes and proteins associated with inflammation and autophagy. To validate the role of Rd in regulating autophagy and inflammation, the autophagy inducers, rapamycin and GSK621, were utilised in reverse validation experiments in cells. Rd exhibited significant hepatic protective effects in mice by reducing the serum levels of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Glutathione S-transferase (GST) and Lactate dehydrogenase (LDH) with acute liver injury. It exhibited strong anti-inflammatory effect by reducing inflammation associated protein, such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), nod-like receptor protein 3 (NLRP3), associated speck-like protein containing a CARD (ASC), interleukin-18 (IL-18) and interleukin-1β(IL-1β) proteins and the mRNA expression levels of COX-2, Tumor Necrosis Factor α (TNF α), interleukin-6 (IL-6) and iNOS were decreased in liver tissue. And Rd inhibited LPS-induced inflammation by reducing the expression of COX-2 and NLRP3 in HSC-T6 cells. Moreover, not only in vivo but also in vitro, Rd downregulated the expression of LC3II, Beclin1, phosphorylation-AMP-activated protein kinase (p-AMPK), phosphorylation-ULK1 (p-ULK1) and upregulated the expression of p62 and phosphorylation-mechanistic target of rapamycin (p-mTOR) to suppress autophagy via the AMPK/mTOR/ULK1 pathway. Finally, the inhibitory effects of Rd on autophagy and inflammation in HSC-T6 cells were partially blocked by rapamycin and GSK621. Rd is a promising therapeutic agent to protect liver against TAA-induced acute liver injury by regulating the autophagy-NLRP3 inflammasome pathway.

Lipopolysaccharide (LPS) induces sclerostin secretion by extracellular vesicle via TLR4/miR-92a-3p/PTEN/NF-κB signalling pathway in murine macrophage.

Sclerostin (SOST) is traditionally regarded as an osteocyte-derived secreted glycoprotein that regulates bone mineralization. Recent studies reported that SOST is also released from non-skeletal sources, especially during inflammation. However, the cellular source and regulatory mechanisms governing SOST generation in inflammation remain unclear. This study investigated whether macrophages produce SOST in response to inflammatory stimuli and determined associated regulatory pathways. The effect of lipopolysaccharide (LPS)-induced inflammation in SOST generation and its underlying regulatory mechanism was examined on mouse macrophage RAW 264.7 by western blot and immunofluorescent staining. Transfection with miR-92a-3p mimic and inhibitor were used to validate its role in SOST production. The role of NF-κB and TLR4 were studied using pharmacological inhibitors BAY 11-7085 and TAK242, respectively. The involvement of NF-κB and TLR4 in LPS-induced SOST production was further validated through nuclear NF-κB p65 immunoprecipitation and TLR4 small interfering RNA (siRNA) experiments, respectively.GW4869 and manumycin A (extracellular vesicles (EV) biogenesis inhibitors) were used to examine the associated of SOST and EV. Finally, SOST expression and characteristics of the isolated EV were assessed by Western blot and nanoparticle tracking analysis (NTA). LPS significantly induced SOST protein expression and secretion in RAW 264.7. MiR-92a-3p was upregulated by LPS stimulation in macrophages. Transfection of miR-92a-3p mimic increased SOST generation in RAW 264.7. Inhibition of TLR4 and NF-κB signalling pathways using pharmacological inhibitors significantly suppressed LPS-induced SOST in RAW 264.7. Similarly, TLR4 siRNA effectively suppressed LPS-induced SOST level. However, the LPS-induced upregulation of miR-92a-3p was only regulated by TLR4, but not by NF-κB. NF-κB was found to directly bind to the mouse sost promoter, thereby activating sost transcription. Additionally, SOST secretion was found predominantly associated with EV from LPS-stimulated cells, and inhibition of EV biogenesis suppressed SOST production in RAW 264.7 cells. In conclusion, our study showed, for the first time, that LPS induced SOST generation and secretion via TLR4/miR-92a-3p/PTEN/NF-κB singling pathway in murine macrophage RAW 264.7 cells. Moreover, we showed that SOST is secreted from the RAW 264.7 cells in the form of extracellular vesicle. This study identified macrophage as a novel source of SOST, highlighting its potential role in inflammatory diseases.

Prenatal Lipopolysaccharide Stimulation Modulates Gastrointestinal Immunity and Oxidative Status in Weaned Pigs.

Gastrointestinal immunity and antioxidant defenses may be bolstered in young animals through prenatal immune system stimulation (PIS), but this is largely uninvestigated in swine. This study tested the hypothesis that PIS could regulate offspring's gastrointestinal immune response and oxidative stress profile. To this end, a PIS model was utilized in sows, delivering low-dose LPS during the final third of gestation to target the developing immune system. On day 78±1.8 of gestation, 14 Camborough sows (parity = 2.6±1.4) received either saline (CON) or LPS from E. coli O111:B4 (2.5 μg/kg of body weight). A subset of 34 weaned barrows (n=17 CON, PIS), weaned at 21±1.3 days, were anesthetized for subcutaneous temperature loggers and jugular catheter placement. Following recovery, all pigs received an intravenous injection of LPS (10 μg/kg body weight) from E. coli O111:B4. Our findings demonstrate that PIS enhances the gut immune response by upregulating key inflammatory cytokines, indicative of a proinflammatory profile. Consistently across the jejunum and ileum, SCF was modulated with heightened expression in PIS than CON (P≤ 0.05). In the ileum alone, PIS exhibited heightened expression of proinflammatory cytokines and chemokines, including TNFα, IL-6, IL-1β, and CCL3L1, compared to CON (P≤ 0.05). Exposure to PIS resulted in reduced systemic total antioxidant capacity (TAC) at hours 2-4 post-challenge (P= 0.004). Piglets exposed to PIS had decreased jejunal tissue malondialdehyde (MDA) concentrations (P= 0.049). Together, these data indicate that exposure to PIS alters the inflammatory profile of the GI immune response and oxidative status in weaned pigs.

CD14++CD16- classical monocyte subset secreting IL-1ß and IL-10 is associated with 'Tuberculosis Resisters' phenotype.

Mycobacterium tuberculosis (M.tb) infection can lead to various outcomes, including active tuberculosis or latent tuberculosis infection (LTBI). Household contacts of TB cases have a high risk of acquiring LTBI. However, some contacts exposed to M.tb remain negative for tuberculin skin test (TST) and interferon-gamma release assay (IGRA) tests and are called 'TB resisters'. Characterization of immune responses in 'TB resisters' may help to understand correlates of protection against M.tb. Based on the TST and IGRA tests, household contacts were divided into 'LTBI' and 'TB Resisters'. Peripheral blood mononuclear cells (PBMCs) of the study participants were isolated and processed to characterize the monocyte subsets based on CD14 and CD16 expression in flow cytometry. Monocyte intracellular cytokine expression (IL-10, IL-6, TNF-α and IL-1ß) was assessed after Lipopolysaccharide (LPS) stimulation. LTBI and active TB patients showed a higher frequency of intermediate and non-classical monocyte subsets depicting the infectious stage. Higher frequency of classical monocyte subsets was associated with 'TB resisters'. Marked expression of IL-1ß and a higher monocyte to lymphocytes (M/L) ratio was seen in PTB, LTBI and TB resister groups compared to healthy controls indicating active disease or exposure to M.tb. Classical Monocytes (CM) were further found to be associated with higher expression of IL-1ß and IL-10 in the 'TB resister group', which might help in the clearance of infection at an early stage. LTBI and PTB showed significantly higher TNF-α producing monocytes than healthy controls and 'TB Resisters'. IL6-producing monocytes were significantly higher in LTBI compared to other study groups. These findings could further be explored with follow-up in cohort of 'TB resisters'. Also, the role of IL-1ß and IL10 secreting classical monocytes in early clearance of infection could be explored with in vitro mechanistic studies.

P0085 Faecalibacterium prausnitzii induces an anti-inflammatory response and a metabolic reprogramming in human monocytes

Abstract Background Faecalibacterium prausnitzii, a highly abundant bacterium in the human gut microbiota, has been linked to overall health and is notably decreased in pathological conditions, such as Inflammatory Bowel Disease (IBD). F. prausnitzii has shown anti-inflammatory properties in human and mouse models, notably through the induction of IL-10 signaling. Here, we investigated which cell types from human blood and intestinal tissue are responsible for producing IL-10 induced by F. prausnitzii, and providing the first mechanistic insights. Methods Immune cells isolated from human blood and intestinal lamina propria of patients with IBD and non-inflamed controls, were stimulated with F. prausnitzii EXL01 strain or Escherichia coli lipopolysaccharide (LPS) and analysed by Legendplex, ELISA, flow cytometry, RNA-sequencing (RNAseq), and Seahorse analyser. Results F. prausnitzii EXL01 strain induced the direct and dose-dependent production of IL-10 in CD14+ monocytes from the systemic circulation and intestinal tissue of IBD patients and non-inflamed controls, without inducing a pro-inflammatory response as compared to LPS stimulation. RNAseq analysis corroborated these results and revealed that F. prausnitzii EXL01 strain differentially affects cell energy metabolism compared to LPS. The anti-inflammatory response induced by F. prausnitzii in monocytes was dependent on mitochondrial respiration. Conclusion F. prausnitzii induces an anti-inflammatory response and rewires energy metabolism in human monocytes, which might explain its beneficial impact on intestinal inflammation and human health in general. These results provide new insight into the mechanisms underlying the anti-inflammatory effects of F. prausnitzii and are crucial for a better understanding of its potential use in the treatment of IBD. The well-characterised F. prausnitzii EXL01 is already in clinical development in Crohn's disease (NCT05542355), and the results of the first phase 1/2A study involving 8 patients will be available in early 2025.

Sustainable Skincare Innovation: Cork Powder Extracts as Active Ingredients for Skin Aging.

Background: An emerging practice within the concept of circular beauty involves the upcycling of agro-industrial by-products. Cork processing, for instance, yields by-products like cork powder, which presents an opportunity to create value-added cosmetic ingredients. Building upon our previous research, demonstrating the antioxidant potential of hydroalcoholic extracts derived from two distinct cork powders (P0 and P1), in this work, aqueous extracts were prepared and analyzed. The safety and bioactivities of the newly obtained aqueous extracts, as well as the 30% ethanol extracts, previously reported to be the most promising for skin application, were also evaluated. Methods: Aqueous extracts were obtained from cork powders (P0 and P1) and the identification and quantification of some polyphenols was achieved by liquid chromatography (LC). Antioxidant potential was screened by DPPH method and the bioactivity and safety of extracts were further explored using cell-based assays. Results: All extracts exhibited a reduction in age-related markers, including senescence-associated beta-galactosidase (SA-β-gal) activity. Additionally, they demonstrated a pronounced anti-inflammatory effect by suppressing the production of several pro-inflammatory mediators in macrophages upon lipopolysaccharide stimulation. Moreover, the extracts upregulated genes and proteins associated with antioxidant activity, such as heme oxygenase 1. The aqueous extract from P1 powder was especially active in reducing pro-inflammatory mediators, namely the Nos2 gene, inducible nitric oxide protein levels, and nitric oxide production. Moreover, it did not induce skin irritation, as assessed by the EpiSkin test, in compliance with the OECD Test Guidelines. Conclusions: Overall, our findings underscore the potential of aqueous extracts derived from cork waste streams to mitigate various hallmarks of skin aging, including senescence and inflammaging, and their suitability for incorporation into cosmetics formulations. These results warrant further exploration for their application in the pharmaceutical and cosmetic industries and could foster a sustainable and circular bioeconomy.

The Mucilage From the Opuntia ficus-indica (L.) Mill. Cladodes Plays an Anti-Inflammatory Role in the LPS-Stimulated HepG2 Cells: A Combined In Vitro and In Silico Approach.

The effect of a mucilage extracted from Opuntia ficus-indica (L.) Mill (OFI) cladodes was tested in lipopolysaccharide (LPS)-challenged HepG2 hepatocarcinoma cells, through a combined in vitro-in silico approach. The OFI mucilage was characterized by gas chromatography-mass spectrometry and liquid chromatography-high resolution mass spectrometry. In cells treated with OFI (5-10µg/mL) prior to LPS (1 µg/mL, 24h), the gene expression profile of pro-inflammatory mediators, namely tumor necrosis factor alpha, interleukin-1 beta, interleukin-8, and cyclo-oxygenase-2, was significantly (p<0.01) reduced if compared to single LPS-challenged cells. The OFI-mediated cytokines reduction was also validated in polystyrene scaffold-grown 3D HepG2 cultures, undergoing treatment with the OFI mucilage (50µg/mL, 24h) and LPS stimulation (50µg/mL, 24h). We further demonstrated that OFI suppresses the LPS-triggered inflammatory response via impairment of the Toll-like receptor 4 (TLR4)/Myeloid differentiation protein-88/Nuclear factor-kappa B (NF-kB) pathway, by interfering with NF-kB phosphorylation at Serine 536. By molecular docking approach, we provided in silico demonstration of the direct molecular interaction between the mucilage monosaccharides and the TLR4 that interferes with the LPS receptor binding and down-stream inflammatory cascade activation. We also demonstrated that OFI cladodes mucilage downregulates the TLR4 pathway, showing an anti-inflammatory potential in HepG2 cells.

Comprehensive Metabolomics in Mouse Mast Cell Model of Allergic Rhinitis for Profiling, Modulation, Semiquantitative Analysis, and Pathway Analysis.

Allergic rhinitis affects millions globally, causing significant discomfort and reducing the quality of life. This study investigates the metabolic alterations in murine mast cells (MC/9) under allergic rhinitis conditions induced by lipopolysaccharide (LPS) stimulation, employing UHPLC-QTOF-MS-based untargeted and targeted metabolomics. The analysis identified 44 significantly regulated metabolites, including histamine, leukotrienes, prostaglandins, thromboxanes, and ceramides. Key metabolic pathways such as arachidonic acid, histidine, and sphingolipid metabolisms were notably modulated. The study further examined the therapeutic effects of triprolidine and zileuton, demonstrating their capacity to reverse LPS-induced metabolic shifts. Triprolidine primarily modulated histidine and sphingolipid metabolism, while zileuton targeted arachidonic acid and sphingolipid metabolism. These findings underscore the utility of metabolomics analysis in elucidating the complex biochemical pathways involved in allergic rhinitis and highlight the potential of metabolomics for evaluating therapeutic interventions. This study enhances our understanding of mast cell metabolism in allergic responses and provides a robust model for assessing the efficacy of anti-allergic agents, paving the way for more effective treatments.

Pterostilbene protects against lipopolysaccharide-induced inflammation and blood–brain barrier disruption in immortalized brain endothelial cell lines in vitro

Brain microvascular endothelial cells are connected by tight junction (TJ) proteins and interacted by adhesion molecules, which participate in the selective permeability of the blood–brain barrier (BBB). The disruption of BBB is associated with the progression of cerebral diseases. Pterostilbene is a natural compound found in blueberries and grapes with a wide range of biological activities, including anti-inflammatory, antioxidant, and anti-diabetic effects. In this study, we investigated the protective effects of pterostilbene on LPS-stimulated mouse brain endothelial (bEnd.3) cells and underlying mechanisms. The results showed that pterostilbene effectively upregulated the expressions of tight junction (TJ) proteins such as zonula occludens (ZO)-1 and claudin-5 and downregulated the expression of adhesion molecules such as intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, preventing BBB damage under LPS stimulation. Pterostilbene decreased the LPS-triggered expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 as well as the levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α and nitric oxide (NO). Meanwhile, we found that pterostilbene exerted an inhibitory effect on nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in bEnd.3 cells upon LPS stimulation. Additionally, pterostilbene exhibited antioxidant effects by activating heme oxygenase 1 (HO-1). These findings indicated that pterostilbene protected against lipopolysaccharide (LPS)-induced inflammation, oxidative stress and blood–brain barrier (BBB) disruption in bEnd.3 cells.

First Report on Microbial-Derived Polydeoxyribonucleotide: A Sustainable and Enhanced Alternative to Salmon-Based Polydeoxyribonucleotide.

Polydeoxyribonucleotide (PDRN) has emerged as a potent bioactive compound with proven efficacy in wound healing, tissue regeneration, and anti-inflammatory applications and is predominantly derived from salmonid gonads. However, this study presents a groundbreaking advancement by successfully extracting and characterizing PDRN from microbial sources, specifically Lactobacillus rhamnosus, marking the first report to utilize microbial-, biome-, or Lactobacillus-derived PDRN (L-PDRN). The findings demonstrate the enhanced biological properties of L-PDRN over traditional salmon-derived PDRN across several assays. L-PDRN exhibited superior antioxidant activity, with significantly higher SOD-like and DPPH radical scavenging activities compared to PDRN, particularly at higher concentrations. In wound-healing assays, L-PDRN demonstrated superior efficacy in promoting cell migration and wound closure, even under inflammatory conditions induced by tumor necrosis factor (TNF-α). Additionally, L-PDRN demonstrated the potential for enhanced immunostimulatory effects under non-inflammatory conditions while maintaining anti-inflammatory properties under lipopolysaccharide (LPS) stimulation. Electrophoretic analysis revealed that L-PDRN consists of smaller DNA fragments (under 100 bp) compared to salmon-derived PDRN (200-800 bp), suggesting greater bioavailability and skin absorption. Mechanistic studies confirmed that L-PDRN activates the focal adhesion kinase (FAK) and protein kinase B (AKT) signaling pathway through the A2A receptor, similar to PDRN, while also engaging alternative pathways for p38 and ERK phosphorylation, highlighting its signaling versatility. This study underscores the potential of L-PDRN as a multifunctional and sustainable alternative to salmon-derived PDRN, offering enhanced bioactivity, scalability, and environmental benefits. The novel approach of utilizing microbial-derived PDRN opens new avenues for therapeutic applications in oxidative stress management, tissue regeneration, and immune modulation, paving the way for a paradigm shift in PDRN sourcing and functionality.