LPS-stimulated TNF-α Expression Research Articles

Identification of ribosomal protein L9 as a novel regulator of proinflammatory damage-associated molecular pattern molecules.

We previously reported that advanced glycation endproducts (AGEs) increase the proinflammatory activity of high mobility group box-1 (HMGB1), a representative damage-associated molecular pattern molecule (DAMP), through their direct interaction. This suggested that AGEs activate other DAMPs and led us to search for novel DAMPs capable of interacting with AGEs. The chromatographic analysis using AGE-immobilized gel revealed the ribosomal protein family to be a factor with binding activity to AGEs. Ribosomal protein L9 (RPL9), a member of the ribosomal protein family, was found in the centrifugal supernatant of ruptured cells and in the serum of lipopolysaccharide (LPS)-stimulated sepsis model mice, exhibiting similar characteristic properties to HMGB1. Although HMGB1 potentiated LPS-stimulated TNF-α expression in macrophage-like RAW264.7 cells, RPL9 hardly exhibited this activity. Of note, RPL9 significantly suppressed the potentiated mRNA expression and protein production of TNF-α by HMGB1 plus LPS stimulation, suggesting its regulatory roles in DAMP-induced proinflammatory activity. Based on the differential scanning fluorimetric analysis, the direct interaction between RPL9 and HMGB1 may play a role in the suppressive effects of RPL9. This study suggested that RPL9 is a novel type of DAMP with a regulatory role in the proinflammatory response and provided insight into the pathophysiology of inflammatory diseases.

Heparin-Binding Protein Enhances NF-κB Pathway-Mediated Inflammatory Gene Transcription in M1 Macrophages via Lactate.

In early-stage sepsis, glucose metabolism is increased primarily through glycolysis in the inflammatory response of M1 macrophages. Heparin-binding protein (HBP) has been linked to sepsis, which can promote macrophage activation and inflammatory factor release. However, the mechanism by which glucose metabolism regulates the inflammatory response is unclear. We show that HBP contributes to sepsis by modulating the inflammatory response via lactate-dependent glycolysis in macrophages. Peritoneal macrophages from BALB/c mouse were treated with lipopolysaccharide (LPS). The expression of M1-related proinflammatory genes was investigated by PCR array. IL-1β, iNOS, TNF-α, and IL-6 mRNA expression was determined by qRT-PCR. Intracellular lactate levels were measured using lactate assays. Nuclear factor-kappaB (NF-κB) activity was determined by electrophoretic mobility shift assays (EMSAs). TNF-α levels were measured by qRT-PCR. HBP enhanced inflammatory gene expression in mouse peritoneal macrophages and intracellular lactate accumulation and significantly increased LPS-stimulated NF-κB transcriptional activity and TNF-α expression through lactate. Lactate was essential for the HBP-induced increase in LPS-stimulated TNF-α expression. The critical role of lactate in HBP-induced NF-κB signaling was confirmed, as α-CHCA-mediated (MCT) suppression significantly inhibited NF-κB activity and TNF-α expression. HBP plays an important role in the initial inflammatory reaction, presumably by activating M1 macrophages, increasing lactate levels, and regulating proinflammatory factor release via NF-κB pathway activation.

Identification of DNMT3B2 as the Predominant Isoform of DNMT3B in Porcine Alveolar Macrophages and Its Involvement in LPS-Stimulated TNF-α Expression.

DNA methyltransferase 3B (DNMT3B) as one member of the DNMT family functions as a de novo methyltransferase, characterized as more than 30 splice variants in humans and mice. However, the expression patterns of DNMT3B in pig as well as the biological function of porcine DNMT3B remain to be determined. In this study, we first examined the expression patterns of DNMT3B in porcine alveolar macrophages (PAM). We demonstrated that only DNMT3B2 and DNMT3B3 were the detectable isoforms in PAM. Furthermore, we revealed that DNTM3B2 was the predominant isoform in PAM. Next, in the model of LPS (lipopolysaccharide)-activated PAM, we showed that in comparison to the unstimulated PAM, (1) expression of DNTM3B is reduced; (2) the methylation level of TNF-α gene promoter is decreased. We further establish that DNMT3B2-mediated methylation of TNF-α gene promoter restricts induction of TNF-α in the LPS-stimulated PAM. In summary, these findings reveal that DNMT3B2 is the predominant isoform in PAM and its downregulation contributes to expression of TNF-α via hypomethylation of TNF-α gene promoter in the LPS-stimulated PAM.

Role of the DNA repair glycosylase OGG1 in the activation of murine splenocytes

OGG1 (8-oxoguanine-DNA glycosylase) is the major DNA repair glycosylase removing the premutagenic DNA base modification 8-oxo-7,8-dihydroguanine (8-oxoG) from the genome of mammalian cells. In addition, there is accumulating evidence that OGG1 and its substrate 8-oxoG might function in the regulation of certain genes, which could account for an attenuated immune response observed in Ogg1−/− mice in several settings. Indications for at least two different mechanisms have been obtained. Thus, OGG1 could either act as an ancillary transcription factor cooperating with the lysine-specific demethylase LSD1 or as an activator of small GTPases. Here, we analysed the activation by lipopolysaccaride (LPS) of primary splenocytes obtained from two different Ogg1−/− mouse strains. We found that the induction of TNF-α expression was reduced in splenocytes (in particular macrophages) of both Ogg1−/− strains. Notably, an inhibitor of LSD1, OG-L002, reduced the induction of TNF-α mRNA in splenocytes from wild-type mice to the level observed in splenocytes from Ogg1−/− mice and had no influence in the latter cells. In contrast, inhibitors of the MAP kinases p38 and JNK as well as the antioxidant N-acetylcysteine attenuated the LPS-stimulated TNF-α expression both in the absence and presence of OGG1. The free base 8-oxo-7,8-dihydroguanine had no influence on the TNF-α expression in the splenocytes. The data demonstrate that OGG1 plays a role in an LSD1-dependent pathway of LPS-induced macrophage activation in mice.

Dibutyltin Compounds Effects on PPARγ/RXRα Activity, Adipogenesis, and Inflammation in Mammalians Cells.

Organotins are a group of chemical compounds that have a tin atom covalently bound to one or more organic groups. The best-studied organotin is tributyltin chloride, which is an environmental pollutant and an endocrine disruptor. Tributyltin chloride has been shown to bind to PPARγ/RXRα and induces adipogenesis in different mammalian cells. However, there are few studies with other organotin compounds, such as dibutyltins. The aim of this study was to investigate the effect of dibutyltins diacetate, dichloride, dilaurate, and maleate on the transcriptional activity of the nuclear PPARγ and RXRα receptors, and on adipogenesis and inflammation. Analogous to tributyltin chloride, in reporter gene assay using HeLa cells, we observed that dibutyltins diacetate, dichloride, dilaurate, and maleate are partial agonists of PPARγ. Unlike tributyltin chloride, which is a full agonist of RXRα, dibutyltins dichloride and dilaurate are partial RXRα agonists. Additionally, the introduction of the C285S mutation, which disrupts tributyltin chloride binding to PPARγ, abrogated the dibutyltin agonistic activity. In 3T3-L1 preadipocytes, all dibutyltin induced adipogenesis, although the effect was less pronounced than that of rosiglitazone and tributyltin chloride. This adipogenic effect was confirmed by the expression of adipogenic markers Fabp4, Adipoq, and Glut4. Exposure of 3T3-L1 cells with dibutyltin in the presence of T0070907, a specific PPARγ antagonist, reduced fat accumulation, suggesting that adipogenic effect occurs through PPARγ. Furthermore, dibutyltins dichloride, dilaurate, and maleate inhibited the expression of proinflammatory genes in 3T3-L1 cells, such as Vcam1, Dcn, Fn1, S100a8, and Lgals9. Additionally, in RAW 264.7 macrophages, tributyltin chloride and dibutyltin dilaurate reduced LPS-stimulated TNFα expression. Our findings indicate that dibutyltins diacetate, dichloride, dilaurate, and maleate are PPARγ partial agonists and that dibutyltins dichloride and dilaurate are also partial RXRα agonists. Furthermore, dibutyltins induce adipogenesis in a PPARγ-dependent manner and repress inflammatory genes in 3T3-L1 and RAW 264.7 cells. Although dibutyltins display some partial PPARγ/RXRα agonistic effects, the translation of cell-based results assays into in vivo effects on inflammation and insulin resistance is not entirely known. Nevertheless, further studies are necessary to address their effects in different periods of life and to elucidate the actions of organostanic compounds in whole-body context.

Role of p38 MAPK and STAT3 in lipopolysaccharide-stimulated mouse alveolar macrophages.

Excessive production of inflammatory mediators is an important feature of inflammatory lung disease. In macrophages, mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription-3 (STAT3) are crucial mediators for the production of proinflammatory cytokines. In the present study, the role of MAPK and STAT3 on tumor necrosis factor (TNF)-α and interleukin (IL)-10 production was investigated in mouse alveolar macrophages. The levels of TNF-α and IL-10 in lipopolysaccharide (LPS; 100 ng/ml)-stimulated MH-S cell lines were measured by an enzyme-linked immunosorbent assay, with or without p38 inhibitor (SB203580; 5, 10 or 15 μM) intervention. Phosphorylated STAT3 (p-STAT3) expression was examined by western blot analysis and immunocytochemistry following LPS stimulation for 15 or 30 min. Antibodies against STAT3 were used to verify comparable sample loading. Cells stimulated with LPS showed significantly increased levels of p-STAT3 protein (P<0.05) when compared with the baseline levels. TNF-α and IL-10 protein levels also increased following LPS stimulation (P<0.05). By contrast, treatment with the p38 inhibitor, SB203580, decreased the levels of p-STAT3, TNF-α and IL-10 (P<0.05) following LPS stimulation. SB203580 was shown to inhibit LPS-stimulated TNF-α expression (P<0.05) in a concentration-dependent manner, reaching significance at a concentration of 10 μM. However, the inhibition of IL-10 expression was not concentration-dependent. Therefore, LPS-stimulated overproduction of TNF-α and IL-10 is mediated at least partially by the MAPK pathway. Inhibition of p38 prevented LPS-induced STAT3 phosphorylation, indicating an interaction between the STAT3 and MAPK signaling pathways.

Rat Kupffer cells: Isolation, identification and LPS-stimulated TNF-α expression and secretion

Rat Kupffer cells: Isolation, identification and LPS-stimulated TNF-α expression and secretion

Molecular Mechanism for Adiponectin-dependent M2 Macrophage Polarization: LINK BETWEEN THE METABOLIC AND INNATE IMMUNE ACTIVITY OF FULL-LENGTH ADIPONECTIN

The anti-inflammatory effects of globular adiponectin (gAcrp) are mediated by IL-10/heme oxygenase 1 (HO-1)-dependent pathways. Although full-length (flAcrp) adiponectin also suppresses LPS-induced pro-inflammatory signaling, its signaling mechanisms are not yet understood. The aim of this study was to examine the differential mechanisms by which gAcrp and flAcrp suppress pro-inflammatory signaling in macrophages. Chronic ethanol feeding increased LPS-stimulated TNF-α expression by Kupffer cells, associated with a shift to an M1 macrophage polarization. Both gAcrp and flAcrp suppressed TNF-α expression in Kupffer cells; however, only the effect of gAcrp was dependent on IL-10. Similarly, inhibition of HO-1 activity or siRNA knockdown of HO-1 in RAW264.7 macrophages only partially attenuated the suppressive effects of flAcrp on MyD88-dependent and -independent cytokine signatures. Instead, flAcrp, acting via the adiponectin R2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype. gAcrp, acting via the adiponectin R1 receptor, was much less effective at eliciting an M2 pattern of gene expression. M2 polarization was also partially dependent on AMP-activated kinase. flAcrp polarized RAW264.7 macrophages to an M2 phenotype in an IL-4/STAT6-dependent mechanism. flAcrp also increased the expression of genes involved in oxidative phosphorylation in RAW264.7 macrophages, similar to the effect of flAcrp on hepatocytes. In summary, these data demonstrate that gAcrp and flAcrp utilize differential signaling strategies to decrease the sensitivity of macrophages to activation by TLR4 ligands, with flAcrp utilizing an IL-4/STAT6-dependent mechanism to shift macrophage polarization to the M2/anti-inflammatory phenotype.

Rac1 mediates sex difference in cardiac tumor necrosis factor-α expression via NADPH oxidase–ERK1/2/p38 MAPK pathway in endotoxemia

The purpose of this study was to investigate the role of Rac1 and estrogen in sex difference of cardiac tumor necrosis factor-alpha (TNF-α) expression during endotoxemia. Endotoxemia was induced in male and female mice by peritoneal injection of lipopolysaccharide (LPS, 4 mg/kg). Compared with female mice, male mice produced more TNF-α in the heart 4 h after LPS treatment, which were correlated with higher Rac1 and NADPH oxidase activity, more phosphorylation of ERK1/2 and p38 MAPK, and up-regulation of toll-like receptor-4 (TLR-4) expression in male mice. Cardiac specific Rac1 knockout or administration of 17β-estradiol down-regulated Rac1 expression, attenuated gp91 phox -NADPH oxidase expression and activity, decreased phosphorylation of ERK1/2/p38 MAPK and inhibited cardiac TNF-α expression induced by LPS, suggesting an important role of Rac1 and estrogen in LPS-stimulated TNF-α expression in the heart. More importantly, the sex difference in TNF-α expression was abrogated by Rac1 knockout or gp91 phox knockout and by administration of apocynin or N-acetylcysteine in LPS-stimulated mice. To investigate the functional significance of sex difference in endotoxemia, heart function was measured in isolated hearts with a Langendorff system. Male mice exhibited worse myocardial dysfunction compared with female in endotoxemia. Treatment of male mice with 17β-estradiol attenuated myocardial dysfunction during endotoxemia. In conclusion, LPS induces Rac1 activation, which contributes to NADPH oxidase activity and phosphorylation of ERK1/2/p38 MAPK, leading to TNF-α expression in the heart. The sex difference in TNF-α expression is estrogen-dependent and mediated via Rac1 dependent NADPH oxidase/ERK1/2 and p38 MAPK pathway in LPS-stimulated hearts.

Higher body mass index (BMI) is associated with reduced glucocorticoid inhibition of inflammatory cytokine production following acute psychosocial stress in men

Body mass index (BMI) and mental stress seem to exert part of their cardiovascular risk by eliciting inflammation. However, the adverse effects of stress on inflammatory activity with BMI are not fully understood. We investigated whether higher BMI is associated with reduced glucocorticoid inhibition of inflammatory cytokine production following stress in men while controlling for age and blood pressure. We measured glucocorticoid inhibition of lipopolysaccharide (LPS)-stimulated release of the proinflammatory cytokine tumor necrosis factor (TNF)-alpha. Forty-two men (age range 21-65 years; BMI range 21-34 kg/m(2)) underwent the Trier Social Stress Test (combination of mock job interview and mental arithmetic task). Whole blood samples were taken immediately before and after stress, and during recovery up to 60 min post-stress. Glucocorticoid sensitivity of LPS-stimulated TNF-alpha expression was assessed in vitro with and without coincubating increasing doses of dexamethasone. Moreover, salivary cortisol was measured during the experiment and on a normal day for assessment of baseline circadian cortisol. Higher BMI was associated with lower glucocorticoid sensitivity of monocyte TNF-alpha production after stress (main effect of BMI: p<0.001) and with more pronounced decreases of glucocorticoid sensitivity following stress (interaction of stress-by-BMI: p=0.002). Neither LPS-stimulated TNF-alpha release nor baseline glucocorticoid sensitivity were associated with BMI. Similarly, BMI was not associated with salivary cortisol, either in reaction to stress or in circadian cortisol secretion. Our data suggest that with increasing BMI, glucocorticoids are less able to inhibit TNF-alpha production following stress. This might suggest a new mechanism linking BMI with elevated risk for adverse cardiovascular outcomes following stress.

Short-term Treatment of RAW264.7 Macrophages with Adiponectin Increases Tumor Necrosis Factor-α (TNF-α) Expression via ERK1/2 Activation and Egr-1 Expression

Short-term Treatment of RAW264.7 Macrophages with Adiponectin Increases Tumor Necrosis Factor-α (TNF-α) Expression via ERK1/2 Activation and Egr-1 Expression

Activation of GSK‐3β inhibited TNF‐α expression in cardiomyocytes during LPS stimulation

This study was to investigate the role of glycogen synthase kinase (GSK)-3β in cardiomyocyte TNF-α expression induced by lipopolysaccharides (LPS). Neonatal mouse cardiomyocytes were cultured and incubated with LPS in the presence of SB216763, a specific inhibitor of GSK-3β, or DMSO for 4 hours. SB216763 increased both TNF-α protein and mRNA expression, suggesting an inhibitory role of GSK-3β. To substantiate the role of GSK-3β, cardiomyocytes were infected with recombinant adenoviruses containing GSK-3β(Ad-GSK) or dominant active mutant of GSK-3β (Ad-AC-GSK) or dominant negative mutant of GSK-3β(Ad-DN-GSK) or Green Fluorescent Protein (GFP) (Ad-GFP) for 24 hours, followed by incubation with LPS for 4 hours. Both Ad-GSK and Ad-AC-GSK infection decreased whereas Ad-DN-GSK increased TNF-α protein and mRNA expression, compared to Ad-GFP. This result supported the inhibitory role of GSK-3β in TNF-α expression in LPS-stimulated cardiomyocytes. In addition, inhibition of protein kinase B activation (AKT) by either LY294002 or over-expression of dominant negative mutant of AKT significantly decreased TNF-α protein and mRNA expression in LPS-stimulated cardiomyocytes. As AKT suppresses GSK-3β activation, our data further supports the notion that GSK-3β inhibits TNF-α expression. In conclusion, our study demonstrated an inhibitory role of GSK-3β in LPS-stimulated TNF-α expression, suggesting that GSK-3β may be a therapeutic target for sepsis.

Early Growth Response-1 Transcription Factor Is Essential for Ethanol-Induced Fatty Liver Injury in Mice

Early growth response-1 (Egr-1), an immediate early gene/zinc-finger transcription factor, is required for maximal stimulation of tumor necrosis factor alpha (TNF-alpha) transcription in response to lipopolysaccharide (LPS). Because chronic ethanol exposure sensitizes macrophages to LPS-stimulated TNF-alpha expression, we have investigated the role of Egr-1 in mediating increased LPS-stimulated TNF-alpha expression after chronic ethanol feeding. Furthermore, because TNF-alpha contributes to alcoholic liver injury, we tested the hypothesis that Egr-1 is required for the development of ethanol-induced fatty liver injury in wild type and egr-1 -/- mice. Wild-type and egr-1 -/- mice were fed ethanol-containing diets or pair-fed control diets for 6 weeks. Wild-type mice fed the ethanol diet developed hepatic steatosis characterized by micro- and macrovesicular lipid accumulation. However, egr-1 -/- mice did not develop steatosis after ethanol feeding. Alanine transferase and TNF-alpha concentrations in serum were increased after ethanol feeding in wild-type but not egr-1 -/- mice. In wild-type mice, challenge with LPS increased Egr-1 messenger RNA (mRNA) and DNA binding activity in liver; this response to LPS was enhanced after chronic ethanol feeding. LPS challenge also increased hepatic TNF-alpha mRNA and serum TNF-alpha to a greater extent after ethanol feeding compared with pair-fed wild-type mice. However, chronic ethanol feeding did not enhance LPS-stimulated TNF-alpha mRNA or serum TNF-alpha in egr-1 -/- mice. These data show that Egr-1 contributes to increased LPS-mediated TNF-alpha expression after chronic ethanol and that the absence of Egr-1 prevents chronic ethanol-induced fatty liver, as well as increased sensitivity to LPS.

The relation of aggression, hostility, and anger to lipopolysaccharide-stimulated tumor necrosis factor (TNF)-α by blood monocytes from normal men

Aggression, hostility, and anger significantly predict morbidity and mortality from atherosclerotic cardiovascular disease (ACVD). ACVD is believed to be an inflammatory disease characterized by increased expression of a number of proinflammatory cytokines, such as tumor necrosis factor (TNF)-α. This study examined the relation of aggression, hostility, and anger to monocyte-associated TNF-α expression following lipopolysaccharide (LPS) stimulation. Participants were 62 healthy, non-smoking men (aged 18–45 years). Hostility, anger, verbal, and physical aggression were assessed using the Buss–Perry aggression questionnaire (BPAQ). LPS-stimulated TNF-α expression was determined using dual-color flow cytometry gating for CD14 + cells. After controlling for age, race, education, and alcohol use, scores on the hostility ( p = .013), physical aggression ( p = .010), and verbal aggression ( p = .034) subscales, and the total score ( p = .007) on the BPAQ were positively associated with LPS-stimulated TNF-α expression. The results suggest that hostility and aggression are associated with an increased expression of TNF-α, a cytokine implicated in ACVD.