Koanhoi Kim

Roles of MAPK and NF-κB in Interleukin-6 Induction by Lipopolysaccharide in Vascular Smooth Muscle Cells

Toll-like receptor (TLR)-4 signaling promotes cytokine synthesis in vascular smooth muscle cells (VSMC). However, it is unknown how TLR-4 regulates interleukin-6 (IL-6) in VSMC. Therefore, the present study investigated cellular factors involved in TLR-4-mediated IL-6 in VSMC in terms of MAPK and transcription elements. Exposure of aortic smooth muscle cells to TLR4-specific lipopolysaccharide (LPS) not only enhanced IL-6 release but also induced IL-6 transcript via promoter activation. The promoter activation was attenuated by dominant-negative MKK1 and to a lesser extent by dominant-negative MKK3, but not by dominant-negative MKK4. IL-6 promoter activity was diminished by U0126 or SB202190, but not by SP600125. Co-transfection with dominant negative CCAAT/enhancer binding protein or with IκB suppressed LPS-induced promoter activation, whereas the promoter activity was not influenced by dominant negative c-Jun. Mutation in the IL-6 promoter region at the binding site of NF-κB or C/EBP impaired promoter activation in response to LPS. Further impairment occurred when both NF-κB- and C/EBP-binding sites were mutated. LPS-induced IL-6 promoter activation was also prevented by pretreatment with epigallocatechin 3-gallate, curcumin, and resveratrol. The present study reports that TLR4-agonistic LPS induces IL-6 through transcriptional activation in VSMC and ERK1/2, p38 MAPK, NF-κB, and C/EBP play active roles in that process.

Functional Modulation of Dendritic Cells and Macrophages by Japanese Encephalitis Virus through MyD88 Adaptor Molecule-Dependent and -Independent Pathways

Dendritic cells (DCs) are potent initiators of T cell-mediated immunity that undergo maturation during viral infections. However, few reports describing the interactions of DCs with Japanese encephalitis virus (JEV), which remains the most frequent cause of acute and epidemic viral encephalitis, are available. In this study, we investigated the interaction of JEV with DCs and macrophages. JEV replicated its viral RNA in both cells with different efficiency, and JEV infection of macrophages followed the classical activation pathway of up-regulation of tested costimulatory molecules and proinflammatory cytokine production (IL-6, TNF-α, and IL-12). On the contrary, JEV-infected DCs failed to up-regulate costimulatory molecules such as CD40 and MHC class II. Of more interest, along with production of proinflammatory cytokines, DCs infected by JEV released antiinflammatory cytokine IL-10, which was not detected in macrophages. Moreover, signaling through MyD88 molecule, a pan-adaptor molecule of TLRs, and p38 MAPK in JEV-infected DCs was found to play a role in the production of cytokines and subversion of primary CD4+ and CD8+ T cell responses. We also found that IL-10 released from JEV-infected DCs led to a reduction in the priming of CD8+ T cells, but not CD4+ T cells. Taken together, our data suggest that JEV induces functional impairment of DCs through MyD88-dependent and -independent pathways, which subsequently leads to poor CD4+ and CD8+ T cell responses, resulting in boosting viral survival and dissemination in the body.

Distinct dictation of Japanese encephalitis virus-induced neuroinflammation and lethality via triggering TLR3 and TLR4 signal pathways.

Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3−/− and TLR4−/− mice, i.e. TLR3−/− mice were highly susceptible to JE, whereas TLR4−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b+Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4−/− mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3−/− myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4+ and CD8+ T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b+Ly-6Chigh monocytes) and CD4+Foxp3+ Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors.

Multifront assault on antigen presentation by Japanese encephalitis virus subverts CD8+ T cell responses.

Japanese encephalitis virus (JEV) is a frequent cause of acute and epidemic viral encephalitis. However, there is little information describing the mechanisms by which JEV subverts immune responses that may predispose the host to secondary infections. In this study, we found that JEV induced the subversion of CD8+ T cell responses in a transient manner that was closely correlated with viral multiplication. Subsequently, analysis using a TCR-transgenic system revealed that CD8+ T cells purified from JEV-infected mice showed impaired responses, and that naive CD8+ T cells adoptively transferred into JEV-infected recipients showed less expanded responses. Furthermore, JEV altered the splenic dendritic cell (DC) subpopulation via preferential depletion of CD8α+CD11c+ DCs without changing the plasmacytoid DCs and induced a significant reduction in the surface expression of MHC class II and CD40, but not MHC class I, CD80, and CD86 molecules. Finally, JEV was shown to inhibit the presentation of MHC class I-restricted Ag in DCs, and this immune suppression was ameliorated via the activation of DCs by TLR agonists. Collectively, our data indicate that JEV precludes the functions of Ag-presenting machinery, such as depletion of CD8α+CD11c+ DCs and downregulation of MHC class I-restricted Ag presentation, thereby leading to immune subversion of CD8+ T cells.

7-Ketocholesterol Upregulates Interleukin-6 via Mechanisms That Are Distinct from Those of Tumor Necrosis Factor-α, in Vascular Smooth Muscle Cells

This study investigated the effects of 7-ketocholesterol on interleukin (IL)-6 expression in vascular smooth muscle cells (VSMC). Among the 7 IL examined, only IL-6 transcript was increased by 7-ketocholesterol treatment in human aorta smooth muscle cells. IL-6 transcripts increased up to 24 h after treatment with 7-ketocholesterol, and this effect was profoundly repressed by treatment with p38 MAPK inhibitors and to a lesser extent JNK inhibitors. 7α-Hydroxycholesterol, 27-hydroxycholesterol or cholesterol, however, did not induce IL-6 expression. Mechanisms of IL-6 induction by 7-ketocholesterol were investigated in comparison with tumor necrosis factor (TNF)-α. Whereas TNF-α activated IL-6 promoter, which was impaired by p38 MAPK inhibitors or by mutation in the NF-κB-binding site within the promoter region, 7-ketocholesterol did not affect IL-6 promoter activity. Instead, this oxysterol slowed degradation of IL-6 mRNA and increased the amount of cytoplasmic HuR. 7-ketocholesterol significantly increased the amount of intracellular IL-6 protein in the presence of brefeldin A. 7-Ketocholesterol also enhanced IL-6 release from VSMC. IL-6 release by 7-ketocholesterol, although significant, was not as remarkable as that induced by TNF-α. These data suggest that 7-ketocholesterol upregulates IL-6 via mechanisms distinct from TNF-α and contributes to the intra- and extracellular IL-6 deposits within the vasculature.

Extracellular heat shock protein 90 induces interleukin-8 in vascular smooth muscle cells

Oxidative stress results in sustained release of heat shock protein 90 (HSP90) from vascular smooth muscle cells (VSMCs). The aim of this article is to investigate whether extracellular HSP90 predisposes VSMCs to pro-inflammatory phenotype. Exposure of aortic smooth muscle cells to HSP90 elevated IL-8 release and IL-8 transcript via promoter activation. HSP90-induced IL-8 promoter activation was suppressed by dominant-negative forms of Toll-like receptor (TLR)-4 and MyD88, but not by dominant-negative-forms of TLR-3, TLR-2, and TRIF. IL-8 up-regulation in response to HSP90 was also attenuated by IkappaB, rasveratrol, curcumin, diphenyleneiodium, N-acetylcystein, U0126, and SB202190. Mutation at the NF-kappaB- or C/EBP-binding site, but not at the AP-1-binding site, in the IL-8 promoter region suppressed the promoter activation by HSP90. This study proposes that extracellular HSP90 would contribute to IL-8 elevation in the stressed vasculature, and that TLR-4, mitogen-activated protein kinases, NF-kappaB, and reactive oxygen species are involved in that process.

Thrombin promotes proinflammatory phenotype in human vascular smooth muscle cell

Expression of PAR, the thrombin receptor, is elevated in smooth muscle cell-rich areas in atherosclerotic plaques, where the cells change to proinflammatory or synthetic phenotype. In this study we investigated whether thrombin promotes a proinflammatory phenotype in vascular smooth muscle cell (VSMC), characterized by increased cytokine and chemokine synthesis. Thrombin not only elevated transcripts for IL-6, CXCL8, and CCL11 genes but also enhanced release of IL-6 and CXCL8 protein from human aortic smooth muscle cell (HAoSMC). Thrombin activated Akt, PKC and MAPK in HAoSMC, and thrombin-mediated expression of IL-6 and CXCL8 was significantly inhibited by LY294002, AKT IV, RO318220, and GF109203X as well as by diphenyleneiodium at the messenger RNA and the protein levels. SB202129 and U0126 also significantly attenuated thrombin-mediated release of IL-6 and CXCL8 proteins from HAoSMC. These results indicate that thrombin promotes proinflammatory phenotype in human VSMC and that PI3K, Akt, PKC, NADPH oxidase, and MAPK are involved in that process. We propose that activation VSMC in response to thrombin after endothelial injury and/or thrombus formation will enhance inflammation in vasculature.

27-hydroxycholesterol induces production of tumor necrosis factor-alpha from macrophages.

Enhanced production of TNF-α from macrophages promotes development and instability of atherosclerotic plaques, but involvement of lipid component in TNF-α production has not been clarified in atherosclerosis. We attempted to determine whether cholesterol oxidation products (oxysterols) could modify TNF-α production. Treatment of THP-1 cells with 27-hydroxycholesterol (27OHChol) or 7α-hydroxycholesterol (7αOHChol) resulted in a profound increase in TNF-α transcription, while treatment with an identical concentration of cholesterol and 7-ketochoelsterol did not lead to any change in TNF-α expression. Treatment with 27OHChol resulted in increased synthesis, as well as secretion, of TNF-α, while 7αOHChol led to increased synthesis of TNF-α without affecting secretion of the cytokine. Co-treatment with 7αOHChol or 27OHChol and LPS resulted in synergistically enhanced or augmented secretion of TNF-α. Treatment with TO-901317, pertussis toxin, PP2, and LY294002 resulted not only in attenuated transcription of TNF-α induced by 27OHChol and 7αOHChol, but also secretion of TNF-α enhanced by 27OHChol. This is the first report demonstrating enhanced production of TNF-α in macrophages by treatment with oxysterols which are detected in abundance in atheromatous lesions; in addition, results of the current study provide evidence indicating that certain types of oxysterols contribute to development of atherosclerosis by promoting production of proinflammatory cytokines.

A pertussis toxin sensitive G-protein-independent pathway is involved in serum amyloid A-induced formyl peptide receptor 2-mediated CCL2 production

Serum amyloid A (SAA) induced CCL2 production via a pertussis toxin (PTX)-insensitive pathway in human umbilical vein endothelial cells (HUVECs). SAA induced the activation of three MAPKs (ERK, p38 MAPK, and JNK), which were completely inhibited by knock-down of formyl peptide receptor 2 (FPR2). Inhibition of p38 MAPK and JNK by their specific inhibitors (SB203580 and SP600125), or inhibition by a dominant negative mutant of p38 MAPK dramatically decreased SAA-induced CCL2 production. Inactivation of Gi protein(s) by PTX inhibited the activation of SAA-induced ERK, but not p38 MAPK or JNK. The results indicate that SAA stimulates FPR2-mediated activation of p38 MAPK and JNK, which are independent of a PTX-sensitive G-protein and are essential for SAA-induced CCL2 production.

Thromboxane A2 increases endothelial permeability through upregulation of interleukin-8

Thromboxane A(2) (TXA(2)), a major prostanoid formed from prostaglandin H(2) by thromboxane synthase, is involved in the pathogenesis of a variety of vascular diseases. In this study, we report that TXA(2) mimetic U46619 significantly increases the endothelial permeability both in vitro and in vivo. U46619 enhanced the expression and secretion of interleukin-8 (IL-8), a major inducer of vascular permeability, in endothelial cells. Promoter analysis showed that the U46619-induced expression of IL-8 was mainly regulated by nuclear factor-kappaB (NF-kappaB). U46619 induced the activation of NF-kappaB through IkappaB kinase (IKK) activation, IkappaB phosphorylation and NF-kappaB nuclear translocation. Furthermore, the inhibition of IL-8 or blockade of the IL-8 receptor attenuated the U46619-induced endothelial cell permeability by modulating the cell-cell junctions. Overall, these results suggest that U46619 promotes vascular permeability through the production of IL-8 via NF-kappaB activation in endothelial cells.