Plasminogen activator inhibitor type 1 expression induced by lipopolysaccharide of Porphyromonas gingivalis in human gingival fibroblast

In the gingival tissues of patients with periodontitis, inflammatory responses are mediated by a wide variety of genes. In our previous screening study, plasminogen activator inhibitor type 1 (PAI-1) mRNA binding protein expression was increased in gingiva from periodontitis patients. In this study, we further investigated the signaling pathway involved in PAI-1 expression induced by Porphyromonas gingivalis LPS (Pg LPS) in human gingival fibroblasts (HGF). When HGFs were treated with Pg LPS, both PAI-1 mRNA expression and PAI-1 protein were induced in a dose-dependent manner. Pg LPS induced NF-κB activation and the expressions of PAI-1 mRNA and protein were suppressed by pretreating with a NF-κB inhibitor. Pg LPS also induced ERK, p38, and JNK activation, and Pg LPS-induced PAI-1 expression was inhibited by ERK/p38/JNK inhibitor pretreatment. In conclusion, Pg LPS induced PAI-1 expression through NF-κB and MAP kinases activation in HGF.     

Gadd45a and Gadd45b modulate innate immune functions of granulocytes and macrophages by differential regulation of p38 and JNK signaling

Gadd45 proteins function as stress sensors in response to various physiological and environmental stressors, interacting with other cellular proteins implicated in cellular stress responses, including p38 and JNK. This study shows that mice lacking either Gadd45a or Gadd45b are defective in the recruitment of granulocytes and macrophages to the intra-peritoneal cavity following intra-peritoneal administration of the bacterial cell wall pathogen-associated molecular pattern lipopolysaccharide (LPS). Bone marrow derived granulocytes and macrophages lacking either Gadd45a or Gadd45b are shown to be impaired in their chemotactic response to LPS, as well as other inflammatory stimuli such as N-formyl-methionine-leucine-phenylalanine and IL-8. Evidence was obtained also implicating Gadd45a and Gadd45b in other myeloid innate immune functions, including reactive oxygen species production, phagocytosis, and adhesion. Gadd45a and Gadd45b activation of p38 kinase was implicated in the response of granulocytes to LPS mediated chemotaxis, whereas Gadd45a and Gadd45b curtailment of JNK activation was linked to chemotaxis of macrophages in response to LPS. Collectively, these data highlight a novel role for both Gadd45a and Gadd45b in myeloid innate immune functions by differential modulation of p38 and JNK signaling in granulocytes compared to macrophages.     

Inhibition of p38 MAPK during cellular activation modulate gene expression of head kidney leukocytes isolated from Atlantic salmon (Salmo salar) fed soy bean oil or fish oil based diets

Head kidney leukocytes isolated from Atlantic salmon fed either a diet based on fish oil (FO) or soy bean oil (VO) were used in order to evaluate if different lipid sources could contribute to cellular activation of the salmon innate immune system. A specific inhibitor of p38 MAPK, SB202190, was used to investigate the effect of lipopolysaccharide (LPS) signalling in the head kidney leukocytes. The results show that LPS up regulate IL-1β, TNF-α, Cox2 expression in leukocytes isolated from fish fed either diet. The p38 MAPK inhibitor, SB202190, reduced the LPS induced expression of these genes in both dietary groups. In LPS stimulated leukocytes isolated from VO fed fish, SB202190 showed a clear dose dependent inhibitory effect on IL-1β, TNF-α and Cox2 expression. This effect was also observed for Cox2 in leukocytes isolated from FO fed fish. Furthermore, there was a stronger mean induction of Cox2 in LPS stimulated leucocytes isolated from the VO-group compared to LPS stimulated leukocytes isolated from the FO-group. In both dietary groups, LPS stimulation of salmon head kidney leukocytes increased the induction of CD83, a dendrite cell marker, while the inhibitor reduced CD83 expression in the VO fed fish only. The inhibitor also clearly reduced hsp27 expression in VO fed fish. Indicating a p38 MAPK feedback loop, LPS significantly inhibited the expression of p38MAPK itself in both diets, while SB202190 increased p38MAPK expression especially in the VO diet group. hsp70 expression was not affected by any treatment or feed composition. There were also differences in p38MAPK protein phosphorylation comparing treatment groups but no obvious difference comparing the two dietary groups. The results indicate that dietary fatty acids have the ability to modify signalling through p38 MAPK which may have consequences for the fish's ability to handle infections and stress. Signalling through p38MAPK is ligand dependent and affects gene and protein expression differently.     

RNA-binding protein AUF1 regulates lipopolysaccharide-induced IL10 expression by activating IkappaB kinase complex in monocytes

Exposure of monocytes and macrophages to endotoxin/lipopolysaccharide (LPS) from Gram-negative bacteria activates the NF-κB signaling pathway. At early times, this leads to their production of proinflammatory cytokines, but subsequently, they produce anti-inflammatory interleukin-10 (IL-10) to quell the immune response. LPS-mediated induction of IL10 gene expression requires the p40 isoform of the RNA-binding protein AUF1. As LPS exerts modest effects upon IL10 mRNA stability, we hypothesized that AUF1 controls the expression of signaling proteins. Indeed, knockdown of AUF1 impairs LPS-mediated p38 mitogen-activated protein kinase (MAPK) and NF-κB signaling, and the expression of an RNA interference-refractory p40(AUF1) cDNA restores both signaling pathways. To define the molecular mechanisms by which p40(AUF1) controls IL10 expression, we focused on the NF-κB pathway in search of AUF1-regulated targets. Here, we show that p40(AUF1) serves to maintain proper levels of the kinase TAK1 (transforming growth factor-β-activated kinase), which phosphorylates the IKKβ subunit within the IκB kinase complex to activate NF-κB-regulated genes. However, p40(AUF1) does not control the TAK1 mRNA levels but instead promotes the translation of the mRNA. Thus, p40(AUF1) regulates a critical node within the NF-κB signaling pathway to permit IL10 induction for the anti-inflammatory arm of an innate immune response.     

Lipopolysaccharide-induced c-Jun NH2-terminal kinase activation in human neutrophils: role of phosphatidylinositol 3-Kinase and Syk-mediated pathways

Polymorphonuclear leukocytes (neutrophils) respond to lipopolysaccharide (LPS) through the up-regulation of several pro-inflammatory mediators. We have recently shown that LPS-stimulated neutrophils express monocyte chemoattractant protein 1 (MCP-1), an AP-1-dependent gene, suggesting that LPS activates the c-Jun N-terminal kinase (JNK) pathway in neutrophils. Previously, we have shown the activation of p38 MAPK, but not JNK, in suspended neutrophils stimulated with LPS but have recently shown activation of JNK by TNF-alpha in an adherent neutrophil system. We show here that exposure to LPS activates JNK in non-suspended neutrophils and that LPS-induced MCP-1 expression, but not tumor necrosis factor-alpha (TNF-alpha) or interleukin-8 (IL-8), is dependent on JNK activation. In addition, LPS stimulation of non-suspended neutrophils activates Syk and phosphatidylinositol 3-kinase (PI3K). Inhibition of Syk with piceatannol or PI3K with wortmannin inhibited LPS-induced JNK activation and decreased MCP-1 expression after exposure to LPS, suggesting that both Syk and PI3K reside in a signaling pathway leading to LPS-induced JNK activation in neutrophils. This Syk- and PI3K-dependent pathway leading to JNK activation after LPS exposure in non-suspended neutrophils is specific for JNK, because inhibition of neither Syk nor PI3K decreased p38 activation after LPS stimulation. Furthermore we show that PI3K inhibition decreased LPS-induced Syk activation suggesting that PI3K resides upstream of Syk in this pathway. Finally, we show that Syk associates with Toll-like receptor 4 (TLR4) upon LPS stimulation further implicating Syk in the LPS-induced signaling pathway in neutrophils. Overall our data suggests that LPS induces JNK activation only in non-suspended neutrophils, which proceeds through Syk- and PI3K-dependent pathways, and that JNK activation is important for LPS-induced MCP-1 expression but not for TNF-alpha or IL-8 expression.     

Early expression of SCIN and CHIPS drives instant immune evasion by Staphylococcus aureus

Chemotaxis inhibitory protein of staphylococci (CHIPS) and Staphylococcal complement inhibitor (SCIN) are small, excreted molecules that play a crucial role in the staphylococcal defence against the human innate immune system. Here we show that they both counteract crucial acute responses of our immune system such as complement activation, neutrophil chemotaxis and neutrophil activation. By studying gene expression via promoter-green fluorescent protein fusions, Northern blots and protein expression analyses, we show that SCIN and CHIPS are produced during the early (exponential) growth stages. Although the SCIN and CHIPS genes are expressed simultaneously, they are differently regulated by various Staphylococcus aureus regulatory loci. However, the sae locus is crucial for upregulation of both SCIN and CHIPS. This is the first study that presents the expression of two extracellular S. aureus proteins early during growth. Because SCIN and CHIPS are both efficient modulators of neutrophil chemotaxis, phagocytosis and killing, their early expression is necessary for efficient modulation of the early immune response.     

Upregulation of lipopolysaccharide-induced interleukin-10 by prostaglandin A1 in mouse peritoneal macrophages

The cyclopentenone prostaglandins (cyPGs) prostaglandin A1 (PGA1) and 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) have been reported to exhibit antiinflammatory activity in activated monocytes/macrophages. However, the effects of these two cyPGs on the expression of cytokine genes may differ. In this study, we investigated the mechanism of action of PGA1 in lipopolysaccharide (LPS)-induced expression of interleukin (IL)-10 mRNA in mouse peritoneal macrophages. 15d-PGJ2 inhibited expression of LPSinduced IL-10, whereas PGA1 increased LPS-induced IL-10 expression. This synergistic effect of PGA1 on LPS-induced IL-10 expression reached a maximum as early as 2 h after simultaneous PGA1 and LPS treatment (PGA1/LPS), and did not require new protein synthesis. The synergistic effect of PGA1 was inhibited by GW9662, a specific peroxisome proliferator-activated receptor (PPAR) antagonist, and Bay-11-7082, a NF-kappaB inhibitor. The extracellular signalregulated kinases (ERK) inhibitor PD98059 increased the expression of PGA1/LPS-induced IL-10 mRNA, rather than inhibiting the IL-10 expression. Moreover, PGA1 inhibited LPS-induced ERK phosphorylation. The synergistic effect of PGA1 on LPS-induced IL-10 mRNA and protein production was inhibited by p38 inhibitor PD169316, and PGA1 increased LPS-induced p38 phosphorylation. In the case of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), the SAPK/JNK inhibitor SP600125 did not inhibit IL-10 mRNA synthesis but inhibited the production of IL-10 protein remarkably. These results suggest that the synergistic effect of PGA1 on LPS-induced IL-10 expression is NF-kappaB-dependent and mediated by mitogen-activated protein (MAP) kinases, p38, and SAPK/ JNK signaling pathways, and also associated with the PPARgamma pathway. Our data may provide more insight into the diverse mechanisms of PGA1 effects on the expression of cytokine genes.     

Effects of CXCL4/CXCR3 on the lipopolysaccharide-induced injury in human umbilical vein endothelial cells

Chemokines and inflammatory response of endothelial cells is crucial in the development and progression of inflammatory disease. Lipopolysaccharide (LPS) is a well-known factor to trigger inflammatory response and induce damage of endothelial cells. The present study used lipopolysaccharide (LPS)-treated human vascular endothelial cells (HUVECs) to investigate the function of chemokine CXC chemokine ligand 4 (CXCL4) and its receptor CXC chemokine receptor 3 (CXCR3) in inflammatory-induced endothelial injury. LPS exposure (50, 100, 200 ng/ml) to HUVECs induced a dose- and time-dependent increase in CXCL4 and CXCR3 expression at both mRNA and protein levels. The LPS-induced endothelium hyperpermeability was inhibited by the addition of CXCL4 neutralizing antibody. Moreover, the addition of CXCL4 neutralizing antibody abolished the effects of LPS on tight junction (TJ) protein expression (occludin claudin-4 and Zonula occluden-1[ZO-1]) and p38 phosphorylation, which is supported by the observation of increased TJ protein expression and decreased p38 phosphorylation in LPS-treated HUVECs. SB203580, a p38 inhibitor, protected HUVECs from CXCL4-stimulated damage. In conclusion, CXCL4/CXCR3, which was enhanced by LPS, may be involved in endothelial proliferation, apoptosis, and permeability via the p38 signaling pathway.