NF-κB Nuclear Translocation Research Articles

Exposure to Secreted Bacterial Factors Promotes HIV-1 Replication in CD4+ T Cells.

Microbial translocation is associated with systemic immune activation in HIV-1 disease. Circulating T cells can encounter microbial products in the bloodstream and lymph nodes, where viral replication takes place. The mechanisms by which bacteria contribute to HIV-associated pathogenesis are not completely deciphered. Here, we examined how bacteria may impact T cell function and viral replication. We established cocultures between a panel of live bacteria and uninfected or HIV-1-infected activated peripheral blood CD4-positive (CD4+) T cells. We show that some bacteria, such as Escherichia coli and Acinetobacter baumannii, sustain lymphocyte activation and enhance HIV-1 replication. Bacteria secrete soluble factors that upregulate CD25 and ICAM-1 cell surface levels and activate NF-κB nuclear translocation. Our data also demonstrate that CD25 polarizes at the virological synapse, suggesting a previously unappreciated role of CD25 during viral replication. These findings highlight how interactions between bacterial factors and T cells may promote T cell activation and HIV-1 replication. IMPORTANCE People living with HIV suffer from chronic immune activation despite effective antiretroviral therapy. Early after infection, HIV-1 actively replicates in the gut, causing the breakage of the intestinal epithelial barrier and microbial translocation. Microbial translocation and chronic immune activation have been proven linked; however, gaps in our knowledge on how bacteria contribute to the development of HIV-related diseases remain. Whether T cells in the peripheral blood react to bacterial products and how this affects viral replication are unknown. We show that some bacteria enriched in people living with HIV activate T cells and favor HIV-1's spread. Bacteria release soluble factors that cause the overexpression of cellular molecules related to their activation state. T cells overexpressing these molecules also replicate HIV-1 more efficiently. These results help us learn more about how HIV-1, T cells, and bacteria interact with each other, as well as the mechanisms behind chronic immune activation.

(S)-2-(Cyclobutylamino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)isonicotinamide Attenuates RANKL-Induced Osteoclast Differentiation by Inhibiting NF-κB Nuclear Translocation.

Osteoporosis is a common skeletal disease; however, effective pharmacological treatments still need to be discovered. This study aimed to identify new drug candidates for the treatment of osteoporosis. Here, we investigated the effect of EPZ compounds, protein arginine methyltransferase 5 (PRMT5) inhibitors, on RANKL-induced osteoclast differentiation via molecular mechanisms by in vitro experiments. EPZ015866 attenuated RANKL-induced osteoclast differentiation, and its inhibitory effect was more significant than EPZ015666. EPZ015866 suppressed the F-actin ring formation and bone resorption during osteoclastogenesis. In addition, EPZ015866 significantly decreased the protein expression of Cathepsin K, NFATc1, and PU.1 compared with the EPZ015666 group. Both EPZ compounds inhibited the nuclear translocation of NF-κB by inhibiting the dimethylation of the p65 subunit, which eventually prevented osteoclast differentiation and bone resorption. Hence, EPZ015866 may be a potential drug candidate for the treatment of osteoporosis.

Sustained Hyperammonemia Activates NF-κB in Purkinje Neurons Through Activation of the TrkB-PI3K-AKT Pathway by Microglia-Derived BDNF in a Rat Model of Minimal Hepatic Encephalopathy.

Chronic hyperammonemia is a main contributor to the cognitive and motor impairment in patients with hepatic encephalopathy. Sustained hyperammonemia induces the TNFα expression in Purkinje neurons, mediated by NF-κB activation. The aims were the following: (1) to assess if enhanced TrkB activation by BDNF is responsible for enhanced NF-κB activation in Purkinje neurons in hyperammonemic rats, (2) to assess if this is associated with increased content of NF-κB modulated proteins such as TNFα, HMGB1, or glutaminase I, (3) to assess if these changes are due to enhanced activation of the TNFR1-S1PR2-CCR2-BDNF-TrkB pathway, (4) to analyze if increased activation of NF-κB is mediated by the PI3K-AKT pathway. It is shown that, in the cerebellum of hyperammonemic rats, increased BDNF levels enhance TrkB activation in Purkinje neurons leading to activation of PI3K, which enhances phosphorylation of AKT and of IκB, leading to increased nuclear translocation of NF-κB which enhances TNFα, HMGB1, and glutaminase I content. To assess if the changes are due to enhanced activation of the TNFR1-S1PR2-CCR2 pathway, we blocked TNFR1 with R7050, S1PR2 with JTE-013, and CCR2 with RS504393. These changes are reversed by blocking TrkB, PI3K, or the TNFR1-SP1PR2-CCL2-CCR2-BDNF-TrkB pathway at any step. In hyperammonemic rats, increased levels of BDNF enhance TrkB activation in Purkinje neurons, leading to activation of the PI3K-AKT-IκB-NF-κB pathway which increased the content of glutaminase I, HMGB1, and TNFα. Enhanced activation of this TrkB-PI3K-AKT-NF-κB pathway would contribute to impairing the function of Purkinje neurons and motor function in hyperammonemic rats and likely in cirrhotic patients with minimal or clinical hepatic encephalopathy.

Exosome-Based Delivery of Super-Repressor IκBα Alleviates Alcohol-Associated Liver Injury in Mice.

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) instigates nuclear factor-κB (NF-κB)-mediated inflammatory responses in alcohol-associated liver diseases (ALD). Here, we utilized a novel optogenetically engineered exosome technology called 'exosomes for protein loading via optically reversible protein-protein interactions (EXPLOR)' to efficiently deliver the super-repressor IκB-loaded exosomes (Exo-srIκB) to the liver and examined its therapeutic potential in acute-on-chronic alcohol-associated liver injury. We detected enhanced uptake of DiI-labeled Exo-srIκB by LPS-treated inflammatory KCs, which suppressed LPS-induced inflammatory gene expression levels. In animal experiments, a single intravenous injection of Exo-srIκB prior to alcohol binge drinking significantly attenuated alcohol-associated hepatic steatosis and infiltration of neutrophils and macrophages but not a liver injury. Notably, three consecutive days of Exo-srIκB injection remarkably reduced alcohol-associated liver injury, steatosis, apoptosis of hepatocytes, fibrosis-related gene expression levels in hepatic stellate cells, infiltration of neutrophils and macrophages, and inflammatory gene expression levels in hepatocytes and KCs. In particular, the above effects occurred with inhibition of nuclear translocation of NF-κB in liver tissues, and these beneficial effects of Exo-srIκB on ALD were shown regardless of doses. Our results suggest an exosome-based modulation of NF-κB activity in KCs by Exo-srIκB as a novel and efficient therapeutic approach in ALD.

Dendrobium officinale alleviates high-fat diet-induced nonalcoholic steatohepatitis by modulating gut microbiota.

The gut microbiota plays an important role in the development of nonalcoholic steatohepatitis (NASH). This study investigated the preventive effect of Dendrobium officinale (DO), including whether its effect was related to the gut microbiota, intestinal permeability and liver inflammation. A NASH model was established in rats using a high-fat diet (HFD) and gavage with different doses of DO or Atorvastatin Calcium (AT) for 10 weeks. Body weight and body mass index along with liver appearance, weight, index, pathology, and biochemistry were measured to assess the preventive effects of DO on NASH rats. Changes in the gut microbiota were analyzed by 16S rRNA sequencing, and intestinal permeability and liver inflammation were determined to explore the mechanism by which DO treatment prevented NASH. Pathological and biochemical indexes showed that DO was able to protect rats against HFD-induced hepatic steatosis and inflammation. Results of 16S rRNA sequencing showed that Proteobacteria, Romboutsia, Turicibacter, Lachnoclostridium, Blautia, Ruminococcus_torques_group, Sutterella, Escherichia-Shigella, Prevotella, Alistipes, and Lactobacillus_acidophilus differed significantly at the phylum, genus, and species levels. DO treatment modulated the diversity, richness, and evenness of gut microbiota, downregulated the abundance of the Gram-negative bacteria Proteobacteria, Sutterella, and Escherichia-Shigella, and reduced gut-derived lipopolysaccharide (LPS) levels. DO also restored expression of the tight junction proteins, zona occludens-1 (ZO-1), claudin-1, and occludin in the intestine and ameliorated the increased intestinal permeability caused by HFD, gut microbiota such as Turicibacter, Ruminococcus, Escherichia-Shigella, and Sutterella, and LPS. Lower intestinal permeability reduced LPS delivery to the liver, thus inhibiting TLR4 expression and nuclear factor-kappaB (NF-κB) nuclear translocation, improving liver inflammation. These results suggest that DO may alleviate NASH by regulating the gut microbiota, intestinal permeability, and liver inflammation.

Total flavone of flowers of Abelmoschus manihot (L.) Medic inhibits the expression of adhesion molecules in primary mesenteric arterial endothelial cells and ameliorates dextran sodium sulphate-induced ulcerative colitis in mice

BackgroundFlowers of Abelmoschus manihot (L.) medic (AM) is a traditional Chinese medicine used to treat chronic nephritis, nephrotic syndrome, diabetic nephropathy, and colonic inflammation. PurposeThis study aimed to explore the influence of the total flavone of AM flowers (TFA) on acute ulcerative colitis (UC) and the potential underlying mechanism. MethodsEfficacy of TFA (30, 60, 120 mg/kg) on UC was evaluated in a dextran sodium sulphate (DSS)-induced colonic inflammatory mouse model by analyzing disease activity index (DAI), histopathological score, colon length, and cytokine expression. Expression levels of critical adhesion molecules and nuclear factor kappa B (NF-κB) were examined by qRT-PCR, Western blotting, or immunofluorescence labeling. Myeloperoxidase activity was examined using ELISA. In vitro THP-1 adhesion assay was used to evaluate monocyte adhesion. ResultsTFA significantly reduced DAI score, prevented colon shortening, and ameliorated histological injuries of colons in DSS-treated mice. TFA inhibited the expression of cytokines (IL-1β and TNF-α) and adhesion molecules (ICAM-1, VCAM-1, and MAdCAM-1) in colon tissues of DSS mice. In vitro studies on mesenteric arterial endothelial cells (MAECs) showed that TFA attenuated TNF-α-induced upregulation of ICAM-1, VCAM-1, and MAdCAM-1, as well as THP-1 cell adhesion to MAECs. TFA also suppressed the phosphorylation and nuclear translocation of NF-κB in MAECs. ConclusionTFA efficaciously ameliorates UC possibly by inhibiting monocyte adhesion through blocking TNF-α-induced NF-κB activation, which in turn suppresses the upregulation of adhesive molecules in colon endothelial cells. Inhibiting the expression of adhesion molecule in MAECs may represent a useful strategy for therapeutic development to treat UC, with TFA being a safe and efficacious therapeutic agent.

7,8-Dihydroxyflavone Attenuates Inflammatory Response and Insulin Resistance Induced by the Paracrine Interaction between Adipocytes and Macrophages.

Obesity-induced inflammation and insulin resistance are mediated by macrophage infiltration into adipose tissue. We investigated the effects of 7,8-dihydroxyflavone (7,8-DHF), a flavone found in plants, on the inflammatory response and insulin resistance induced by the interaction between adipocytes and macrophages. Hypertrophied 3T3-L1 adipocytes were cocultured with RAW 264.7 macrophages and treated with 7,8-DHF (3.12, 12.5, and 50 μM). The inflammatory cytokines and free fatty acid (FFA) release were evaluated by assay kits, and signaling pathways were determined by immunoblotting. Coculture of adipocytes and macrophages increased inflammatory mediators, such as nitric oxide (NO), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) and FFA secretion but suppressed the production of anti-inflammatory adiponectin. 7,8-DHF counteracted the coculture-induced changes (p < 0.001). 7,8-DHF also inhibited c-Jun N-terminal kinase (JNK) activation and blocked nuclear factor kappa B (NF-κB) nuclear translocation in the coculture system (p < 0.01). In addition, adipocytes cocultured with macrophages did not increase glucose uptake and Akt phosphorylation in response to insulin. However, 7,8-DHF treatment recovered the impaired responsiveness to insulin (p < 0.01). These findings show that 7,8-DHF alleviates inflammation and adipocyte dysfunction in the coculture of hypertrophied 3T3-L1 adipocytes and RAW 264.7 macrophages, indicating its potential as a therapeutic agent for obesity-induced insulin resistance.

Alpha‐kinase1 promotes tubular injury and interstitial inflammation in diabetic nephropathy by canonical pyroptosis pathway

BackgroundAlpha‐kinase 1 (ALPK1) is a master regulator in inflammation and has been proved to promote renal fibrosis by promoting the production of IL-1β in diabetic nephropathy (DN) mice. Pyroptosis is involved in high glucose (HG)-induced tubular cells injury, characterized by activation of Gasdermin D (GSDMD) and the release of IL-1β and IL-18, resulting in inflammatory injury in DN. It is reasonable to assume that ALPK1 is involved in pyroptosis-related tubular injury in DN. However, the mechanism remains poorly defined.MethodsImmunohistochemistry (IHC) staining was performed to detect the expression of pyroptosis- and fibrosis-related proteins in renal sections of DN patients and DN mice. DN models were induced through injection of streptozotocin combined with a high-fat diet. Protein levels of ALPK1, NF-κB, Caspase-1, GSDMD, IL-1β, IL-18 and α-SMA were detected by Western blot. HK-2 cells treated with high-glucose (HG) served as an in vitro model. ALPK1 small interfering RNA (siRNA) was transfected into HK-2 cells to down-regulate ALPK1. The pyroptosis rates were determined by flow cytometry. The concentrations of IL-1β and IL-18 were evaluated by ELISA kits. Immunofluorescence staining was used to observe translocation of NF-κB and GSDMD.ResultsThe heat map of differentially expressed genes showed that ALPK1, Caspase-1 and GSDMD were upregulated in the DN group. The expression levels of ALPK1, Caspase-1, GSDMD and CD68 were increased in renal biopsy tissues of DN patients by IHC. ALPK1expression and CD68+ macrophages were positively correlated with tubular injury in DN patients. Western blot analysis showed increased expressions of ALPK1, phospho-NF-κB P65, GSDMD-NT, and IL-1β in renal tissues of DN mice and HK-2 cells, accompanied with increased renal fibrosis-related proteins (FN, α-SMA) and macrophages infiltration in interstitial areas. Inhibition of ALPK1 attenuated HG-induced upregulation expressions of NF-κB, pyroptosis-related proteins Caspase-1, GSDMD-NT, IL-1β, IL-18, α-SMA, and pyroptosis level in HK-2 cells. Also, the intensity and nuclear translocation of NF-κB and membranous translocation of GSDMD were ameliorated in HG-treated HK-2 cells after treatment with ALPK1 siRNA.ConclusionsOur data suggest that ALPK1/NF-κB pathway initiated canonical caspase-1-GSDMD pyroptosis pathway, resulting in tubular injury and interstitial inflammation of DN.

Acute respiratory distress syndrome enhances tumor metastasis into lungs: Role of BRD4 in the tumor microenvironment.

Acute respiratory distress syndrome (ARDS) is associated with severe lung inflammation, edema, hypoxia, and high vascular permeability. The COVID-19-associated pandemic ARDS caused by SARS-CoV-2 has created dire global conditions and has been highly contagious. Chronic inflammatory disease enhances cancer cell proliferation, progression, and invasion. We investigated how acute lung inflammation activates the tumor microenvironment and enhances lung metastasis in LPS induced in vitro and in vivo models. Respiratory illness is mainly caused by cytokine storm, which further influences oxidative and nitrosative stress. The LPS-induced inflammatory cytokines made the conditions suitable for the tumor microenvironment in the lungs. In the present study, we observed that LPS induced the cytokine storm and promoted lung inflammation via BRD4, which further caused the nuclear translocation of p65 NF-κB and STAT3. The transcriptional activation additionally triggers the tumor microenvironment and lung metastasis. Thus, BRD4-regulated p65 and STAT3 transcriptional activity in ARDS enhances lung tumor metastasis. Moreover, LPS-induced ARDS might promote the tumor microenvironment and increase cancer metastasis into the lungs. Collectively, BRD4 plays a vital role in inflammation-mediated tumor metastasis and is found to be a diagnostic and molecular target in inflammation-associated cancers.

Cinnamomum verum extract inhibits NOX2/ROS and PKCδ/JNK/AP-1/NF-κB pathway-mediated inflammatory response in PMA-stimulated THP-1 monocytes

BackgroundCinnamomum verum J. Presl (Cinnamon) is widely used in the food and pharmaceutical industries. C. verum exhibits various biological activities. However, it is unclear whether C. verum can inhibit NOX, a major source of ROS generation, and exert anti-inflammatory and antioxidant effects in PMA-stimulated THP-1 cells. PurposeThis study investigates the anti-inflammatory and antioxidant effects of C. verum in PMA-stimulated THP-1 cells. MethodsThe MeOH extract of C. verum was analyzed using UPLC-QTOF/MS. Anti-inflammatory and antioxidant effects of C. verum extract were examined by DCF-DA staining, immunofluorescence staining, RT-PCR, and immunoblotting in PMA-stimulated THP-1 cells. ResultsC. verum and its components, cinnamic acid and coumarin, significantly attenuated the expression of IL-1β, IL-8, CCL5, and COX-2 in PMA-stimulated THP-1. C. verum decreased ROS levels via NOX2 downregulation, as well as ameliorated plasma membrane translocation of PKCδ and decreased JNK phosphorylation. Besides, C. verum suppressed the nuclear translocation of AP-1 and NF-κB, which modulates diverse pro-inflammatory genes. ConclusionC. verum effectively inhibits inflammation and oxidative stress during monocyte-macrophage differentiation and downregulates inflammatory mediators via NOX2/ROS and PKCδ/JNK/AP-1/NF-κB signaling.

ARMS-NF-κB signaling regulates intracellular ROS to induce autophagy-associated cell death upon oxidative stress

Ankyrin repeat-rich membrane spanning (ARMS) plays roles in neural development, neuropathies, and tumor formation. Such pleiotropic function of ARMS is often attributed to diverse ARMS-interacting molecules in different cell context. However, it might be achieved by ARMS' effect on global biological mediator like reactive oxygen species (ROS). We established ARMS-knockdown in melanoma cells (siARMS) and in Drosophila eyes (GMR>dARMS RNAi ) and challenged them with H2O2. Decreased ARMS in both systems compromises nuclear translocation of NF-κB and induces ROS, which in turn augments autophagy flux and confers susceptibility to H2O2-triggered autophagic cell death. Resuming NF-κB activity or reducing ROS by antioxidants in siARMS cells and GMR>dARMS RNAi fly decreases intracellular peroxides level concurrent with reduced autophagy and attenuated cell death. Conversely, blocking NF-κB activity in wild-type flies/melanoma enhances ROS and induces autophagy with cell death. We thus uncover intracellular ROS modulated by ARMS-NFκB signaling primes autophagy for autophagic cell death upon oxidative stress.

USP39 Regulates NF-κB-Mediated Inflammatory Responses through Deubiquitinating K48-Linked IκBα.

IκBα is a critical protein that inhibits NF-κB nuclear translocation and impairs NF-κB-mediated signaling. The abundance of IκBα determines the activation and restoration of the inflammatory response. However, posttranslational regulation of IκBα remains to be fully understood. In this study, we identified ubiquitin-specific protease 39 (USP39) as a negative regulator in the NF-κB inflammatory response by stabilizing basal IκBα. The expression of USP39 in macrophages was reduced under LPS-induced inflammation. Knockdown or knockout of USP39 in macrophages significantly increased the expression and secretion of proinflammatory cytokines upon exposure to LPS or Escherichia coli, whereas reexpression of exogenous USP39 in USP39-deficient macrophages rescued the effect. Moreover, USP39-defective mice were more sensitive to LPS or E. coli-induced systemic sepsis. Mechanistically, USP39 interacted with and stabilized IκBα by reducing K48-linked polyubiquination of IκBα. Taken together, to our knowledge, our study for the first time revealed the inhibitory function of USP39 in the NF-κB inflammatory response, providing a previously unknown mechanism for control of inflammatory cytokine induction in the cellular anti-inflammatory response.

Piperine as potential therapy of post-weaning porcine diarrheas: an in vitro study using a porcine duodenal enteroid model

Post-weaning diarrhea in piglets is a major problem, resulting in a significant loss in pig production. This study aimed to investigate the effects of piperine, an alkaloid abundantly found in black peppers, on biological activities related to the pathogenesis of post-weaning diarrhea using a porcine duodenal enteroid model, a newly established intestinal stem cell-derived in vitro model recapitulating physiology of porcine small intestinal epithelia. Porcine duodenal enteroid models were treated with disease-relevant pathological inducers with or without piperine (8 μg/mL and/or 20 μg/mL) before measurements of oxidative stress, mRNA, and protein expression of proinflammatory cytokines, nuclear factor-kappa B (NF-κB) nuclear translocation, barrier leakage, and fluid secretion. We found that piperine (20 μg/mL) inhibited H2O2-induced oxidative stress, TNF-α-induced mRNA, and protein expression of proinflammatory cytokines without affecting NF-κB nuclear translocation, and prevented TNF-α-induced barrier leakage in porcine duodenal enteroid monolayers. Importantly, piperine inhibited fluid secretion induced by both forskolin and heat-stable toxins (STa) in a three-dimensional model of porcine duodenal enteroids. Collectively, piperine possesses both anti-inflammatory and anti-secretory effects in porcine enteroid models. Further research and development of piperine may provide novel interventions for the treatment of post-weaning porcine diarrhea.

Extracellular vesicles from mesenchymal stem cells reduce neuroinflammation in hippocampus and restore cognitive function in hyperammonemic rats

Chronic hyperammonemia, a main contributor to hepatic encephalopathy (HE), leads to neuroinflammation which alters neurotransmission leading to cognitive impairment. There are no specific treatments for the neurological alterations in HE. Extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) reduce neuroinflammation in some pathological conditions. The aims were to assess if treatment of hyperammonemic rats with EVs from MSCs restores cognitive function and analyze the underlying mechanisms. EVs injected in vivo reach the hippocampus and restore performance of hyperammonemic rats in object location, object recognition, short-term memory in the Y-maze and reference memory in the radial maze. Hyperammonemic rats show reduced TGFβ levels and membrane expression of TGFβ receptors in hippocampus. This leads to microglia activation and reduced Smad7–IkB pathway, which induces NF-κB nuclear translocation in neurons, increasing IL-1β which alters AMPA and NMDA receptors membrane expression, leading to cognitive impairment. These effects are reversed by TGFβ in the EVs from MSCs, which activates TGFβ receptors, reducing microglia activation and NF-κB nuclear translocation in neurons by normalizing the Smad7–IkB pathway. This normalizes IL-1β, AMPA and NMDA receptors membrane expression and, therefore, cognitive function. EVs from MSCs may be useful to improve cognitive function in patients with hyperammonemia and minimal HE.

Functionalised penetrating peptide-chondroitin sulphate‑gold nanoparticles: Synthesis, characterization, and applications as an anti-Alzheimer's disease drug

The purpose of this study was to construct a transmembrane peptide-chondroitin sulphate‑gold nanoparticle (TAT-CS@Au) delivery system and investigate its activity as an anti-Alzheimer's disease (AD) drug. We successfully prepared TAT-CS@Au nanoparticles, investigated their anti-AD effects, and explored the possible mechanisms in in vitro models. TAT-CS@Au exhibited excellent cellular uptake and transport capacity, effectively inhibited the accumulation of Aβ1–40, and significantly reduced Aβ1–40-induced apoptosis in SH-SY5Y cells. Furthermore, TAT-CS@Au significantly reduced oxidative stress damage and cholinergic injury induced by Aβ1–40 by regulating intracellular concentrations of reactive oxygen species (ROS), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and acetylcholine (ACh). Western blotting results demonstrated that TAT-CS@Au inhibited aberrant tau phosphorylation (Ser199, Thr205, Ser404, and Ser396) through GSK3β inactivation. TAT-CS@Au decreased the levels of inflammatory factors, specifically TNF-α, IL-6, and IL-1β, by inhibiting NF-κB nuclear translocation by activating MAPK signalling pathways. Overall, these results indicate that TAT-CS@Au exhibits excellent transmembrane ability, inhibits Aβ1–40 accumulation, antagonises oxidative stress, reduces aberrant tau phosphorylation, and suppresses the expression of inflammatory factors. TAT-CS@Au may be a multi-target anti-AD drug with good cell permeability, providing new insights into the design and research of anti-AD therapeutics.

Turmeronols (A and B) from &lt;i&gt;Curcuma longa&lt;/i&gt; have anti-inflammatory effects in lipopolysaccharide-stimulated BV-2 microglial cells by reducing NF-κB signaling

Turmeronols (A and B), bisabolane-type sesquiterpenoids found in turmeric, reduce inflammation outside the brain in animals; however, their effects on neuroinflammation, a common pathology of various neurodegenerative diseases, are not understood. Inflammatory mediators produced by microglial cells play a key role in neuroinflammation, so this study evaluated the anti-inflammatory effects of turmeronols in BV-2 microglial cells stimulated with lipopolysaccharide (LPS). Pretreatment with turmeronol A or B significantly inhibited LPS-induced nitric oxide (NO) production; mRNA expression of inducible NO synthase; production of interleukin (IL)-1β, IL-6, and tumor necrosis factor α and upregulation of their mRNA expression; phosphorylation of nuclear factor-κB (NF-κB) p65 proteins and inhibitor of NF-κB kinase (IKK); and nuclear translocation of NF-κB. These results suggest that these turmeronols may prevent the production of inflammatory mediators by inhibiting the IKK/NF-κB signaling pathway in activated microglial cells and can potentially treat neuroinflammation associated with microglial activation.

Prevention of Bleomycin-Induced Pulmonary Inflammation and Fibrosis in Mice by Bilobalide.

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease. Bilobalide (BB) is a sesquiterpene isolated from Ginkgo biloba, and its role in IPF is poorly understood. Mice were intratracheally instilled with 2.5 mg/kg bleomycin (BLM) to induce IPF and then treated with 2.5, 5, and 10 mg/kg BB daily for 21 days. Treatment with BB ameliorated pathological injury and fibrosis of lung tissues in BLM-induced mice. BB suppressed BLM-induced inflammatory response in mice as demonstrated by reduced inflammatory cells counts (leukocytes, neutrophils, macrophages, and lymphocytes) and pro-inflammatory factors (CCL2 and TNF-α), as well as increased CXCL10 levels in BALF. The expression of BLM-induced hydroxyproline, LDH, and pro-fibrotic mediators including fibronectin, collagen I, α-smooth muscle actin (α-SMA), transforming growth factor (TGF)-β1, matrix metalloproteinase (MMP)-2, and MMP-9 in lung tissue was inhibited by BB treatment, and the tissue inhibitor of metalloproteinase-1 (TIMP-1) expression was increased. BB blocked the phosphorylation of JNK and NF-κB, and the nuclear translocation of NF-κB in the lung tissue of mice induced by BLM. Additionally, it abated the activation of NLRP3 inflammasome in lung tissue induced by BLM, which led to the downregulation of IL-18 and IL-1β in BALF. Our present study suggested that BB might ameliorate BLM-induced pulmonary fibrosis by inhibiting the early inflammatory response, which is probably via the inhibition of the JNK/NF-κB/NLRP3 signal pathway. Thus, BB might serve as a therapeutic potential agent for pulmonary inflammation and fibrosis.

PS-BPB09-3: CIGARETTE SMOKE EXTRACT INDUCES DNA DAMAGE AND TRIGGERS AN INNATE IMMUNE RESPONSE

Objective: Elevated blood pressure and tobacco smoking are causes of preventable mortality. The effects of tobacco smoking on blood pressure are complex and chronic effects are inconsistent. We have previously reported increased DNA damage in peripheral mononuclear cells of smokers compared to nonsmokers. In this study, we investigated the effects of cigarette smoke extract (CSE) on DNA damage and the physiological influence of DNA damage in endothelial cells. Design and method: The effect of CSE extracted from tobacco smoke was examined in human umbilical vein endothelial cells (HUVECs). CSE was added to HUVECs and the formation of DNA damage was quantified by fluorescent immunostaining. The mRNA expression of inflammatory cytokines was quantified by real-time RT PCR. Results: CSE increased double-strand breaks in HUVEC by 72 hours. CSE also increased oxidative DNA damage both in the nucleus and mitochondria, and decreased mitochondria membrane potential. Mitochondrial dysfunction partially induced mitochondrial outer membrane permeabilization, resulting in the partial activation of caspase-3 and nuclear translocation of caspase-activated deoxyribonuclease, which fragments DNA in the nucleus. It was suggested that this is one of the mechanisms of nuclear DNA double-strand breaks. CSE induced the accumulation of nuclear and mitochondrial DNA in the cytosol. We further examined whether accumulated cytosolic DNA activated cytosolic DNA-sensing pathways. The production of cGAMP, a second messenger in cGAS (a DNA-sensing receptor) signaling, was increased by CSE, followed by nuclear translocation of NF-kB and increased mRNA expression of IL-6. The increase in IL-6 expression was suppressed by si-cGAS. Conclusions: Our study revealed that continuous exposure to CSE induces DNA damages not only in the nucleus but also in the mitochondria, which leads to cytosolic DNA accumulation, and evokes chronic inflammation via the cGAS pathway.

Recombinant Klotho attenuates IFNγ receptor signaling and SAMHD1 expression through blocking NF-κB translocation in glomerular mesangial cells.

Interferon gamma (IFNγ) is a cytokine implicated in the pathogenesis of autoimmune diseases. SAM and HD domain-containing protein 1 (SAMHD1) is an IFNγ-inducible protein that modulates cellular dNTP levels. Mutations in the human SAMHD1 gene cause Aicardi-Goutières (AG) syndrome, an autoimmune disease sharing similar clinical features with systemic lupus erythematosus (SLE). Klotho is an anti-inflammatory protein which suppresses aging through multiple mechanisms. Implication of Klotho in autoimmune response is identified in rheumatologic diseases such as SLE. Little information exists regarding the effect of Klotho in lupus nephritis, one of the prevalent symptoms of SLE. The present study verified the effect of IFNγ on SAMHD1 and Klotho expression in MES-13 glomerular mesangial cells, a special cell type in glomerulus that is critically involved in lupus nephritis. IFNγ upregulated SAMHD1 expression in MES-13 cells through the Janus kinase-signal transducer and activator of transcription 1 (JAK-STAT1) and the nuclear factor kappa B (NFκB) signaling pathways. IFNγ decreased Klotho protein expression in MES-13 cells. Treatment of MES-13 cells with recombinant Klotho protein inhibited SAMHD1 expression by blocking IFNγ-induced NFκB nuclear translocation, but showed no effect on JAK-STAT1 signaling. Collectively, our findings support the protective role of Klotho in attenuating lupus nephritis through the inhibition of IFNγ-induced SAMHD1 expression and IFNγ downstream signaling in MES-13 cells.

MiR-582-5p targets Skp1 and regulates NF-κB signaling-mediated inflammation

A well-tuned inflammatory response is crucial for an effective immune process. Nuclear factor-kappa B (NF-κB) is a key mediator of inflammatory and innate immunity responses, and its dysregulation is closely associated with immune-related diseases. MicroRNAs (miRNAs) are important inflammation modulators. However, miRNA-regulated mechanisms that implicate NF-κB activity are not fully understood. This study aimed to identify a potential miRNA that could modulate the dysregulated NF-κB signaling during inflammation. We identified miR-582-5p that was significantly downregulated in inflamed murine adipose tissues and RAW264.7 cells. S-phase kinase-associated protein 1 (SKP1), a core component of an E3 ubiquitin ligase that regulates the NF-κB pathway, was proposed as a biological target of miR-582-5p by using TargetScan. The binding of miR-582-5p to a 3′-untranslated region site on Skp1 was confirmed using a dual-luciferase reporter assay; in addition, transfection with a miR-582-5p mimic suppressed SKP1 expression in RAW264.7 cells. Importantly, exogenous miR-582-5p attenuated the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 through suppressing the degradation of the NF-κB inhibitor alpha, followed by the nuclear translocation of NF-κB. Therefore, exogenously applied miR-582-5p can attenuate the NF-κB signaling pathway via targeting Skp1; this provides a prospective therapeutic strategy for treating inflammatory and immune diseases.