LPS-induced Phosphorylation Of P38 MAPK Research Articles

MKP-3 suppresses LPS-induced inflammatory responses in HUVECs via inhibition of p38 MAPK/NF-κB pathway

ABSTRACT Endothelial cell dysfunction and inflammatory responses play critical roles in the development of atherosclerosis. Recent data on the processes underlying atherogenesis indicate the substantial role of endotoxins (lipopolysaccharides; LPS) of the intestinal microflora in the initiation and progression of atherosclerosis. Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a cytoplasmic dual-specificity protein phosphatase that specifically binds to and inactivates MAP kinases in mammalian cells, but its biological function in endothelial cell dysfunction and inflammatory responses remains largely unknown. The aim of the present study was to investigate the role of MKP-3 in endotoxin-induced endothelial inflammation by western blotting, quantitative polymerase chain reaction, and immunofluorescence. The results of our study demonstrated that MKP-3 overexpression markedly inhibited the adhesion of human monocytic THP-1 cells to human umbilical vein endothelial cells (HUVECs) by downregulating the expression of vascular cell adhesion protein 1 (VCAM-1) and pro-inflammatory cytokines. In contrast, MKP-3-encoding gene knockdown by small interfering RNA (siRNA) exacerbated LPS-induced endothelial dysfunction. Additionally, we found that MKP-3 overexpression inhibited LPS-induced p38 MAPK phosphorylation and decreased the nuclear translocation of nuclear factor kappa B (NF-κB) after LPS treatment, suggesting its implication in the LPS/Toll-like receptor 4 (TLR4)/p38/NF-κB pathway. Overall, these observations suggest that MKP-3 plays a protective role in endothelial dysfunction and may be a therapeutic target.

Glia Maturation Factor Gene Editing Improves Neurocognitive Function in an Alzheimer's Disease Mouse Model

Alzheimer's Disease (AD) is a progressive neurodegenerative disorder that causes an irreversible cognitive decline in an estimated 5.5 million Americans. If the present trend continues, by 2050 the number of AD patients will surpass 16 million with ~$1.1 trillion in healthcare costs thereby necessitating the development of novel therapeutic strategies to effectively treat AD. Towards fulfillment of the unmet need for an effective AD therapy, our ultimate goal is to develop a robust AD patient‐specific personalized precision‐guided targeted gene editing and stem cell therapy. Glia maturation factor (GMF), a brain specific pro‐inflammatory molecule discovered in our laboratory plays a crucial role in neuroinflammation and AD pathophysiology. We hypothesized that CRISPR/Cas9‐mediated GMF gene editing in microglia is a novel approach to reduce neuroinflammation, neurodegeneration, AD pathophysiology and improve cognitive function. To achieve GMF gene editing, we developed recombinant adeno‐associated viral (AAV) and lentiviral vectors (LVs) expressing CRISPR/Cas9 and GMF‐specific sgRNAs. Confocal microscopy of BV2 cells transduced with an AAV simultaneously co‐expressing Staphylococcus aureus CRISPR/Cas9 (SaCas9) and a GMF specific sgRNA revealed a small subset of BV2 cells expressing SaCas9 while completely lacking GMF expression, thereby confirming successful biallelic GMF gene editing. To further improve GMF gene editing efficiency, we generated LVs coexpressing either 1) Streptococcus pyogenes CRISPR/Cas9 (SpCas9), eGFP and Neor or 2) GMF‐specific individual sgRNAs1‐3, mCherry and Puror. BV2 cells were sequentially transduced with LV‐EF1α‐SpCas9 and LV‐GMF‐sgRNAs1‐3 to generate stable clones. Confocal microscopy revealed reduced GMF expression in GMF‐edited BV2 cells as compared to non‐edited BV2 cells. DNA sequencing of GMF edited clones revealed indels in the exons 2–3 of the GMF coding sequence thereby conclusively proving SpCas9‐mediated GMF gene editing. Treatment of wild type non‐edited and GMF‐edited BV2 cells with LPS revealed differences in basal p38 MAPK as well as LPS‐induced phosphorylation of p38 MAPK. In wild type non‐edited BV2 cells LPS treatment induced significant upregulation of pp38 MAPK, however in GMF‐edited BV2 cells pp38 MAPK levels were significantly lower, thereby indicating that GMF gene editing leads to attenuation of microglial activation. Further, GMF gene editing in BV2 cells leads to regulation of mitochondrial dynamics by attenuation of NRF2/HO‐1 dependent ferritin activation. Our in vivo GMF gene editing data in the 5XFAD mouse model of AD revealed improved neurocognitive function. Overall, our data suggest that GMF gene editing represents a novel approach to develop precision‐targeted AD patient‐specific therapy.Support or Funding InformationVA Merit Award I01BX002477 and NIH Grants AG048205 and NS073670 to AZThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Eupatoriopicrin Inhibits Pro-inflammatory Functions of Neutrophils via Suppression of IL-8 and TNF-alpha Production and p38 and ERK 1/2 MAP Kinases

During chronic inflammation, neutrophils acting locally as effector cells not only activate antibacterial defense but also promote the inflammatory response. Interleukin 8 (IL-8), the main cytokine produced by activated neutrophils, positively correlates with the severity of respiratory tract diseases. By screening European plants traditionally used for treating respiratory tract diseases, we found that extracts of aerial parts of Eupatorium cannabinum inhibit IL-8 release from neutrophils. Using bioassay-guided fractionation, we identified five sesquiterpene lactones, eupatoriopicrin (1), 5'-deoxyeupatoriopicrin (2), hiyodorilactone A (3), 3-hydroxy-5'- O-acetyleupatoriopicrin = hiyodorilactone D (4), and hiyodorilactone B (5), that efficiently (IC50 < 1 μM) inhibited IL-8 and TNF-α release in lipopolysaccharide (LPS)-stimulated human neutrophils. Moreover, all these sesquiterpene lactones suppressed the adhesion of human neutrophils to an endothelial monolayer by downregulating the expression of the β2 integrin CD11b/CD18 on the neutrophil surface. Furthermore, eupatoriopicrin efficiently suppressed LPS-induced phosphorylation of p38 MAPK and ERK and attenuated neutrophil infiltration in the thioglycolate-induced peritonitis model in mice. Altogether, these results demonstrate the potential of the sesquiterpene lactone eupatoriopicrin as a lead substance for targeting inflammation.

Molecular cloning and functional characterization of TRAF6 and TAK1 in rainbow trout, Oncorhynchus mykiss

TRAF6 and TAK1 are known to play important roles in vertebrate innate immunity as molecular bridge, linking upstream toll-like receptors (TLRs) with the downstream MAPK and NF-κB signalling pathways. However, their roles in TLR signalling pathway have yet to be fully described in fish. Here we identified genes encoding TRAF6 (OmTRAF6) and TAK1 (OmTAK1) from rainbow trout, Oncorhynchus mykiss, and examined their roles during pathogenic infections. The deduced amino acid sequences of OmTRAF6 and OmTAK1 contained the characteristic domains conserved in the TRAF and TAK1 families, respectively (OmTRAF6: RING, two TRAF-type zinc fingers, CCR and MATH domains; OmTAK1: STKc and CCR domains). In RTH-149 cells, the expression of OmTRAF6 and OmTAK1 was increased by stimulation with Edwardsiella tarda and LPS. Silencing of OmTRAF6 and OmTAK1 in RTH-149 cells negatively regulated the LPS-induced phosphorylation of p38 MAPK and JNK. TAK1 inhibitor (5z)-7-Oxozeaenol significantly decreased the LPS-induced activation of NF-κB in RTH-149 cells. In addition, silencing of OmTRAF6 and OmTAK1 significantly decreased the expression of MAPKs and NF-κB downstream target genes induced by LPS in RTH-149 cells. These findings suggest that OmTRAF6 and OmTAK1 might function like those of mammals to regulate bacteria-triggered signalling pathway in fish.

Protostemonine effectively attenuates lipopolysaccharide-induced acute lung injury in mice.

Protostemonine (PSN) is the main anti-inflammatory alkaloid extracted from the roots of Stemona sessilifolia (known as "Baibu" in traditional Chinese medicine). Here, we reported the inhibitory effects of PSN on lipopolysaccharide (LPS)-induced macrophage activation in vitro and LPS-induced acute lung injury in mice. Macrophage cell line RAW264.7 cells and mouse bone marrow-derived macrophages (BMDMs) were treated with PSN (1, 3, 10, 30 and 100 μmol/L) for 0.5 h and then challenged with LPS (0.1 μg/mL) for 24 h. Pretreatment with PSN significantly inhibited LPS-induced phosphorylation of MAPKs and AKT, iNOS expression and NO production in the macrophages. C57BL/6 mice were intratracheally injected with LPS (5 mg/kg) to induce acute lung injury (ALI). The mice were subsequently treated with PSN (10 mg/kg, ip) at 4 and 24 h after LPS challenge. PSN administration significantly attenuated LPS-induced inflammatory cell infiltration, reduced pro-inflammatory cytokine (TNF-α, IL-1β and IL-6) production and eliminated LPS-mediated lung edema. Furthermore, PSN administration significantly inhibited LPS-induced pulmonary MPO activity. Meanwhile, LPS-induced phosphorylation of p38 MAPK, iNOS expression and NO production in the lungs were also suppressed. The results demonstrate that PSN effectively attenuates LPS-induced inflammatory responses in vitro and in vivo; the beneficial effects are associated with the decreased phosphorylation of MAPK and AKT and the reduced expression of pro-inflammatory mediators, such as iNOS, NO and cytokines. These data suggest that PSN may be a potential therapeutic agent in the treatment of ALI.

Adenosine effectively restores endotoxin-induced inhibition of human neutrophil chemotaxis via A1 receptor-p38 pathway.

Neutrophil chemotaxis plays an essential role in recruiting neutrophils to sites of inflammation. Neutrophil chemotaxis is suppressed both after exposure to lipopolysaccharide (LPS) in vitro and during clinical and experimental endotoxemia, leading to serious consequences. Adenosine (ADO) is a potent anti-inflammatory agent that acts on a variety of neutrophil functions. However, its effects on human neutrophil chemotaxis during infection have been less well characterized. In the present study, we investigated the effect of ADO and its receptor-specific antagonist and agonist on neutrophil chemotaxis in an in vitro LPS-stimulated model. The results showed that increasing the concentration of ADO effectively restored the LPS-inhibited neutrophil chemotaxis to IL-8. A similar phenomenon occurred after intervention with a selective A1 receptor agonist but not with a selective antagonist. Pre-treatment with cAMP antagonist failed to restore LPS-inhibited chemotaxis. Furthermore, protein array and western blot analysis showed that the activation of A1 receptor significantly decreased LPS-induced p38 MAPK phosphorylation. However, the surface expression of the A1 receptor in LPS-stimulated neutrophils was not significantly changed. Taken together, these data indicated that ADO restored the LPS-inhibited chemotaxis via the A1 receptor, which downregulated the phosphorylation level of p38 MAPK, making this a promising new therapeutic strategy for infectious diseases.

The Anti-Inflammatory Effects and Mechanisms of Eupafolin in Lipopolysaccharide-Induced Inflammatory Responses in RAW264.7 Macrophages.

Eupafolin is a flavone isolated from Artemisia princeps Pampanini (family Asteraceae). The aim of this study was to examine the anti-inflammatory effects of eupafolin in lipopolysaccharide (LPS)-treated RAW264.7 macrophages and LPS-induced mouse skin and lung inflammation models and to identify the mechanism underlying these effects. Eupafolin decreased the LPS-induced release of inflammatory mediators (iNOS, COX-2 and NO) and proinflammatory cytokines (IL-6 and TNF-α) from the RAW264.7 macrophages. Eupafolin inhibited the LPS-induced phosphorylation of p38 MAPK, ERK1/2, JNK, AKT and p65 and the nuclear translocation of p65 and c-fos. These effects were mainly mediated by the inhibition of JNK. In the mouse paw and lung models, eupafolin effectively suppressed the LPS-induced edema formation and down-regulated iNOS and COX-2 expression. These results demonstrated that eupafolin exhibits anti-inflammatory properties and suggested that eupafolin can be developed as an anti-inflammatory agent.

A novel synthetic compound MCAP suppresses LPS-induced murine microglial activation in vitro via inhibiting NF-kB and p38 MAPK pathways.

To investigate the anti-neuroinflammatory activity of a novel synthetic compound, 7-methylchroman-2-carboxylic acid N-(2-trifluoromethyl) phenylamide (MCAP) against LPS-induced microglial activation in vitro. Primary mouse microglia and BV2 microglia cells were exposed to LPS (50 or 100 ng/mL). The expression of iNOS and COX-2, proinflammatory cytokines, NF-κB and p38 MAPK signaling molecules were analyzed by RT-PCR, Western blot and ELISA. The morphological changes of microglia and nuclear translocation of NF-ĸB were visualized using phase contrast and fluorescence microscopy, respectively. Pretreatment with MCAP (0.1, 1, 10 μmol/L) dose-dependently inhibited LPS-induced expression of iNOS and COX-2 in BV2 microglia cells. Similar results were obtained in primary microglia pretreated with MCAP (0.1, 0.5 μmol/L). MCAP dose-dependently abated LPS-induced release of TNF-α, IL-6 and IL-1β, and mitigated LPS-induced activation of NF-κB by reducing the phosphorylation of IκBα in BV2 microglia cells. Moreover, MCAP attenuated LPS-induced phosphorylation of p38 MAPK, whereas SB203580, a p38 MAPK inhibitor, significantly potentiated MCAP-caused inhibition on the expression of MEF-2 (a transcription factor downstream of p38 MAPK). MCAP exerts anti-inflammatory effects in murine microglia in vitro by inhibiting the p38 MAPK and NF-κB signaling pathways and proinflammatory responses. MCAP may be developed as a novel agent for treating diseases involving activated microglial cells.

Hypothemycin inhibits tumor necrosis factor-α production by tristetraprolin-dependent down-regulation of mRNA stability in lipopolysaccharide-stimulated macrophages

Hypothemycin, a resorcylic acid lactone polyketide, has been shown to inhibit oncogenic ras-transformation and T cell activation. In the present study, we investigated the effect of hypothemycin on tumor necrosis factor-α (TNF-α) production in macrophages and the molecular mechanisms involved in this effect. Hypothemycin potently suppressed the TNF-α production without affecting nitric oxide production in lipopolysaccharide (LPS)-stimulated macrophages. However, hypothemycin had no effect on the activity of TNF-α-converting enzyme, a key enzyme for converting membrane-bound pro-TNF-α into soluble TNF-α. Further study demonstrated that the stability of TNF-α mRNA was decreased by hypothemycin treatment. In addition, hypothemycin suppressed LPS-induced phosphorylation of p38 MAPK and ERK. Moreover, knockdown of tristetraprolin (TTP), which is an important trans-acting regulator of TNF-α mRNA stability and downstream target of p38 MAPK and ERK, reversed hypothemycin-mediated inhibition of TNF-α mRNA expression. Collectively, our results suggest that hypothemycin suppresses TNF-α production by TTP-dependent destabilization of TNF-α mRNA and this is mediated, at least in part, by blocking the activation of p38 MAPK and ERK.

Berteroin present in cruciferous vegetables exerts potent anti-inflammatory properties in murine macrophages and mouse skin.

Berteroin (5-methylthiopentyl isothiocyanate) is a sulforaphane analog present in cruciferous vegetables, including Chinese cabbage, rucola salad leaves, and mustard oil. We examined whether berteroin exerts anti-inflammatory activities using lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophages and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin inflammation models. Berteroin decreased LPS-induced release of inflammatory mediators and pro-inflammatory cytokines in Raw 264.7 macrophages. Berteroin inhibited LPS-induced degradation of inhibitor of κBα (IκBα) and nuclear factor-κB p65 translocation to the nucleus and DNA binding activity. Furthermore, berteroin suppressed degradation of IL-1 receptor-associated kinase and phosphorylation of transforming growth factor β activated kinase-1. Berteroin also inhibited LPS-induced phosphorylation of p38 MAPK, ERK1/2, and AKT. In the mouse ear, berteroin effectively suppressed TPA-induced edema formation and down-regulated iNOS and COX-2 expression as well as phosphorylation of AKT and ERK1/2. These results demonstrate that berteroin exhibits potent anti-inflammatory properties and suggest that berteroin can be developed as a skin anti-inflammatory agent.

Disruption of transient receptor potential melastatin 2 attenuate lipopolysaccharide-induced heme oxygenase-1 expression in mouse macrophages through p38 mitogen-activated protein kinase (P5202)

Abstract Transient Receptor Potential Melastatin 2 (TRPM2) is a member of transient receptor potential channels. TRPM2 mediated Ca2+ entry is an important mechanism for lipopolysaccharide (LPS)-induced cytokine and reactive oxygen species (ROS)-induced chemokine production in monocytes/macrophages. However, whether TRPM2-mediated Ca2+ entry contributes to intracellular protective protein Homo oxygenase-1 (HO-1) production in response to LPS remained unknown. Bone marrow-derived macrophages were isolated from TRPM2 knock-out (KO) mice and wild-type (WT) mice and cultured. After 100ng/ml LPS stimulation, the protein levels of HO-1 and its related signaling molecules p38 MAPK and Nrf2 were measured using immunobloting. Furthermore, in LPS stimulated WT macrophages, the p38 MAPK levels were detected after chelating Ca2+, and the HO-1 levels were evaluated after inhibition of p38 MAPK signaling. In TRPM2 KO macrophages, the expression levels of HO-1 were significantly reduced after LPS stimulation. The levels of phosphorylation of p38 MAPK and Nrf2 nuclear translocation showed the same manner when compared to those in WT macrophages. Chelation of calcium obviously inactivated LPS-induced phosphorylation of p38 MAPK. Inhibition of p38 MAPK signaling completely suppressed HO-1 expression. TRPM2 plays an important role in controlling HO-1 expression in macrophages and thereby may protect against inflammation and tissue oxidant injury.

Berteroin Suppresses Inflammatory Responses via NF‐κB signaling in Macrophages and Mouse Skin

Berteroin (5‐methylthiopentyl isothiocyanate) is a sulforaphane analog found in cruciferous vegetables including Chinese cabbage, rucola salad leaves, and mustard oil. The present study examined in vitro and in vivo anti‐inflammatory effects of berteroin. Berterion decreased LPS‐induced release of nitric oxide and prostaglandin E2 in Raw264.7 macrophages, which was accompanied by decreases in the protein expression and transcriptional activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2), respectively. Additionally, berteroin decreased LPS‐induced increases in the protein and mRNA levels of tumor necrosis factor‐α (TNF‐α), interleukin (IL)‐6 and IL‐1β. Berteroin inhibited LPS‐induced degradation of inhibitor of κBα and translocation of p65 subunit of NF‐κB to the nucleus. In addition, berteroin inhibited LPS‐induced phosphorylation of p38 MAPK, ERK1/2, and Akt. Furthermore, berteroin suppressed TPA‐induced inflammatory responses in the mouse ear and down‐regulated the expression of iNOS and COX‐2 as well as the phosphoryation of ERK1/2 in mouse skin. We demonstrated that berteroin exhibits potent anti‐inflammatory properties that are associated with the inhibition of transcription and protein expression of iNOS, COX‐2, TNF‐α, IL‐6 and IL‐1β, which is accompanied by decrease activation of NF‐κB as well as signaling molecules including ERK1/2, Akt and p38 MAPK.

Chitosan oligosaccharides suppress production of nitric oxide in lipopolysaccharide-induced N9 murine microglial cells in vitro

Chitosan oligosaccharides (COS) have been reported to exert many biological activities, such as antioxidant, antitumor and anti-inflammatory effects. In the present study, we examined the effect of COS on nitric oxide (NO) production in LPS induced N9 microglial cells. Pretreatment with COS (50~200 μg/ml) could markedly inhibit NO production by suppressing inducible nitric oxide synthase (iNOS) expression in activated microglial cells. Signal transduction studies showed that COS remarkably inhibited LPS-induced phosphorylation of p38 MAPK and ERK1/2. COS pretreatment could also inhibit the activation of both nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). In conclusion, our results suggest that COS could suppress the production of NO in LPS-induced N9 microglial cells, mediated by p38 MAPK and ERK1/2 pathways.

Lysophosphatidic acid receptor 1 modulates lipopolysaccharide-induced inflammation in alveolar epithelial cells and murine lungs

Lysophosphatidic acid (LPA), a bioactive phospholipid, plays an important role in lung inflammation by inducing the release of chemokines and lipid mediators. Our previous studies have shown that LPA induces the secretion of interleukin-8 and prostaglandin E(2) in lung epithelial cells. Here, we demonstrate that LPA receptors contribute to lipopolysaccharide (LPS)-induced inflammation. Pretreatment with LPA receptor antagonist Ki16425 or downregulation of LPA receptor 1 (LPA(1)) by small-interfering RNA (siRNA) attenuated LPS-induced phosphorylation of p38 MAPK, I-κB kinase, and I-κB in MLE12 epithelial cells. In addition, the blocking of LPA(1) also suppressed LPS-induced IL-6 production. Furthermore, LPS treatment promoted interaction between LPA(1) and CD14, a LPS coreceptor, in a time- and dose-dependent manner. Disruption of lipid rafts attenuated the interaction between LPA(1) and CD14. Mice challenged with LPS increased plasma LPA levels and enhanced expression of LPA receptors in lung tissues. To further investigate the role of LPA receptors in LPS-induced inflammation, wild-type, or LPA(1)-deficient mice, or wild-type mice pretreated with Ki16425 were intratracheally challenged with LPS for 24 h. Knock down or inhibition of LPA(1) decreased LPS-induced IL-6 release in bronchoalveolar lavage (BAL) fluids and infiltration of cells into alveolar space compared with wild-type mice. However, no significant differences in total protein concentration in BAL fluids were observed. These results showed that knock down or inhibition of LPA(1) offered significant protection against LPS-induced lung inflammation but not against pulmonary leak as observed in the murine model for lung injury.

Inhibitory mechanism of MMP-9 gene expression by ethyl pyruvate in lipopolysaccharide-stimulated BV2 microglial cells

Ethyl pyruvate (EP) is a stable derivative of pyruvate and has been identified as a therapeutic agent for various inflammatory diseases. In the present study, we showed that EP and sodium pyruvate (SP) inhibited the production of TNF-α, nitric oxide (NO), or reactive oxygen species (ROS) in LPS-stimulated BV2 microglial cells. The inhibitory effects of EP were more potent than SP. Because matrix metalloproteinase-9 (MMP-9) plays a key role in neuroinflammation, as well as in neuronal cell death, we examined the effect of EP on MMP-9 expression. RT-PCR and Western blot analyses revealed that EP inhibits MMP-9 expression at mRNA and protein levels in LPS-stimulated BV2 cells. In addition, EP suppressed MMP-9 secretion, as demonstrated by gelatin zymography analysis. In contrast, SP did not affect MMP-9 expression at an equivalent concentration of EP. Further mechanistic studies revealed that EP inhibits MMP-9 promoter activity by reducing the binding of NF-κB and AP-1 to its cognitive binding sites. In addition, EP suppressed LPS-induced phosphorylation of p38 MAPK, ERK, and Akt, which are upstream signaling molecules in MMP-9 gene expression. Taken together, our data suggest that the inhibition of MMP-9 may be one of the factors contributing to anti-inflammatory activity of EP in LPS-stimulated microglia.

The FGL2/fibroleukin prothrombinase is involved in alveolar macrophage activation in COPD through the MAPK pathway

Fibrinogen-like protein 2 (FGL2)/fibroleukin has been reported to play a vital role in the pathogenesis of some critical inflammatory diseases by possessing immunomodulatory activity through the mediation of “immune coagulation” and the regulation of maturation and proliferation of immune cells. We observed upregulated FGL2 expression in alveolar macrophages from peripheral lungs of chronic obstructive pulmonary disease (COPD) patients and found a correlation between FGL2 expression and increased macrophage activation markers (CD11b and CD14). The role of FGL2 in the activation of macrophages was confirmed by the detection of significantly decreased macrophage activation marker (CD11b, CD11c, and CD71) expression as well as the inhibition of cell migration and inflammatory cytokine (IL-8 and MMP-9) production in an LPS-induced FGL2 knockdown human monocytic leukemia cell line (THP-1). Increased FGL2 expression co-localized with upregulated phosphorylated p38 mitogen-activated protein kinase (p38-MAPK) in the lung tissues from COPD patients. Moreover, FGL2 knockdown in THP-1 cells significantly downregulated LPS-induced phosphorylation of p38-MAPK while upregulating phosphorylation of c-Jun N-terminal kinase (JNK). Thus, we demonstrate that FGL2 plays an important role in macrophage activation in the lungs of COPD patients through MAPK pathway modulation.

Role of matrix metalloproteinase-9 in lipopolysaccharide-induced mucin production in human airway epithelial cells

Matrix metalloproteinases (MMPs) have been found to be involved in the pathogenesis of inflammatory airway diseases. However, the role of MMPs in lipopolysaccharide (LPS)-induced mucin overproduction remains unclear. We explored the role of MMP-9 in LPS-induced MUC5AC production and the effect of doxycycline on MUC5AC production. The study showed that LPS induced transcription and protein expression of both MMP-9 and MUC5AC in NCI-H292 cells and in primary human epithelial cells, and the increased MUC5AC level were associated with increased MMP-9 transcripts, protein and activity. However, the increase of MUC5AC transcripts and protein were diminished after cells had been treated with doxycycline, MMP-9 siRNA or EGFR inhibitor. Doxycycline inhibited MMP-9 transcription, protein production and activity, while LPS-induced increase of MMP-9 transcription was inhibited by EGFR inhibitor, p38 MAPK and JNK inhibitor. The LPS-induced phosphorylation of p38 MAPK and JNK were inhibited by EGFR inhibitor. These results suggested that LPS-induced MUC5AC production may be partially mediated by MMP-9 activation and EGFR-p38 MAPK/JNK signaling pathway. Doxycycline may play a therapeutic role in LPS-induced mucus hypersecretion.

Adenylate cycalse toxin ofBordetella pertussisinhibits TLR-induced IRF-1 and IRF-8 activation and IL-12 production and enhances IL-10 through MAPK activation in dendritic cells

Adenylate cyclase toxin (CyaA) of Bordetella pertussis binds to CD11b/CD18 on macrophages and dendritic cells (DC) and confers virulence to the bacteria by subverting innate immune responses of the host. We have previously demonstrated that CyaA promotes the induction of IL-10-secreting regulatory T cells in vivo by modulating DC activation. Here, we examine the mechanism of immune subversion, specifically, the modulation of TLR signaling pathways in DC. We found that CyaA synergized with LPS to induce IL-10 mRNA and protein expression in DC but significantly inhibited IL-12p70 production. CyaA enhanced LPS-induced phosphorylation of p38 MAPK and ERK in DC, and inhibitors of p38 MAPK, MEK, or NF-kappaB suppressed IL-10 production in response to LPS and CyaA. However, inhibition of p38 MAPK, MEK, and NF-kappaB did not reverse the inhibitory effect of CyaA on TLR agonist-induced IL-12 production. Furthermore, CyaA suppression of IL-12 was independent of IL-10. In contrast, CyaA suppressed LPS- and IFN-gamma-induced IFN-regulatory factor-1 (IRF-1) and IRF-8 expression in DC. The modulatory effects of CyaA were dependent on adenylate cyclase activity and induction of intracellular cAMP, as an enzyme-inactive mutant of CyaA failed to modulate TLR-induced signaling in DC, whereas the effects of the wild-type toxin were mimicked by stimulation of the DC with PGE2. Our findings demonstrate that CyaA modulates TLR agonist-induced IL-10 and IL-12p70 production in DC by, respectively, enhancing MAPK phosphorylation and inhibiting IRF-1 and IRF-8 expression and that this is mediated by elevation of intercellular cAMP concentrations.

Pioglitazone inhibition of lipopolysaccharide-induced nitric oxide synthase is associated with altered activity of p38 MAP kinase and PI3K/Akt

BackgroundPrevious studies have suggested that peroxisome proliferator activated receptor-gamma (PPAR-γ)-mediated neuroprotection involves inhibition of microglial activation and decreased expression and activity of inducible nitric oxide synthase (iNOS); however, the underlying molecular mechanisms have not yet been well established. In the present study we explored: (1) the effect of the PPAR-γ agonist pioglitazone on lipopolysaccharide (LPS)-induced iNOS activity and nitric oxide (NO) generation by microglia; (2) the differential role of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun NH(2)-terminal kinase (JNK), and phosphoinositide 3-kinase (PI3K) on LPS-induced NO generation; and (3) the regulation of p38 MAPK, JNK, and PI3K by pioglitazone.MethodsMesencephalic neuron-microglia mixed cultures, and microglia-enriched cultures were treated with pioglitazone and/or LPS. The protein levels of iNOS, p38 MAPK, JNK, PPAR-γ, PI3K, and protein kinase B (Akt) were measured by western blot. Different specific inhibitors of iNOS, p38MAPK, JNK, PI3K, and Akt were used in our experiment, and NO generation was measured using a nitrite oxide assay kit. Tyrosine hydroxylase (TH)-positive neurons were counted in mesencephalic neuron-microglia mixed cultures.ResultsOur results showed that pioglitazone inhibits LPS-induced iNOS expression and NO generation, and inhibition of iNOS is sufficient to protect dopaminergic neurons against LPS insult. In addition, inhibition of p38 MAPK, but not JNK, prevented LPS-induced NO generation. Further, and of interest, pioglitazone inhibited LPS-induced phosphorylation of p38 MAPK. Wortmannin, a specific PI3K inhibitor, enhanced p38 MAPK phosphorylation upon LPS stimulation of microglia. Elevations of phosphorylated PPAR-γ, PI3K, and Akt levels were observed with pioglitazone treatment, and inhibition of PI3K activity enhanced LPS-induced NO production. Furthermore, wortmannin prevented the inhibitory effect of pioglitazone on the LPS-induced NO increase.ConclusionWe demonstrate that pioglitazone protects dopaminergic neurons against LPS insult at least via inhibiting iNOS expression and NO generation, which is potentially mediated via inhibition of p38 MAPK activity. In addition, the PI3K pathway actively participates in the negative regulation of LPS-induced NO production. Our findings suggest that PPAR-γ activation may involve differential regulation of p38 MAPK and of the PI3K/Akt pathway in the regulation of the inflammatory process.

Selective modulation of lipopolysaccharide-stimulated cytokine expression and mitogen-activated protein kinase pathways by dibutyryl-cAMP in BV2 microglial cells

Cyclic AMP is a very important regulator in a wide range of biological processes, including inflammatory reactions. To investigate the role of cAMP in microglia, we examined the effect of dibutyryl-cAMP (dbcAMP) on lipopolysaccharide (LPS)-stimulated cytokine expression and signaling pathways in murine BV2 microglial cells. DbcAMP strongly suppressed LPS-induced TNF-α expression, without affecting NO, IL-6 or TGF-β1 expression. In contrast, LPS-induced IL-1β or IL-10 expressions were dramatically increased by dbcAMP. We further examined the effect of elevated cAMP on signaling molecules such as MAP kinases (p38 MAPK, ERK and JNK), NF-κB and AP1, which are involved in the regulation of inflammatory responses. DbcAMP decreased the LPS-induced phosphorylation of p38 MAPK, while it modestly enhanced the ERK activity. JNK phosphorylation was slightly reduced by dbcAMP only at the later time point. Electrophoretic mobility shift assay revealed that the elevated cAMP potentiated AP-1 binding activity by enhancing c-fos binding. On the other hand, dbcAMP repressed NF-κB-mediated transcription without affecting NF-κB binding. Treatment with H89, a selective inhibitor of protein kinase A, completely reversed cAMP-induced IL-10 and IL-1β upregulation but only partially reversed the cAMP-induced repression of TNF-α. Thus, the effect of dbcAMP in BV2 cells appears to be mediated through both protein kinase A-dependent and -independent pathways. Taken together, our results demonstrate that cAMP modulates microglia activation in a diverse and complex manner.