IL-8 Promoter Activity Research Articles

Nuclear factor 45 of half smooth tongue sole Cynoglossus semilaevis: Gene structure, expression profile, and immunoregulatory property

Nuclear factor 45 (NF45) is a component of the protein complex called nuclear factor of activated T-cells (NFAT), which in mammals regulates interleukin (IL)-2 expression. To date very little is known about fish NF45. In this study, we identified a NF45 (named CsNF45) from half smooth tongue sole Cynoglossus semilaevis and examined its gene organization, expression profile, and regulatory function. We found that CsNF45 is composed of 387 residues and shares 90.3%–97.9% overall sequence identities with the NF45 of human and teleosts. Genetic analysis showed that the genomic sequence of the ORF region of CsNF45 consists of 14 exons and 13 introns. Constitutive expression of CsNF45 occurred in multiple tissues including gill, muscle, brain, heart, liver, head kidney, spleen, and gut. Experimental infection with viral and bacterial pathogens upregulated the expression of CsNF45 in head kidney and spleen in a time-dependent manner. Transient transfection analysis showed that CsNF45 was localized in the nucleus and able to stimulate the activity of mouse IL-2 promoter. These results indicate that CsNF45 possesses immunoregulatory property and is possibly involved in host immune defense against bacterial and viral infection.

THU0076 Differential Dna Methylation of TBX5 Transcription Factor in Synovial Fibroblasts from Resolving Arthritis and Rheumatoid Arthritis at Different Stages of Evolution

ObjectivesChanges in DNA methylation and histone marks have been associated with diseases such as cancer, rheumatoid arthritis (RA) and systemic lupus erythematosus. Previously, we reported global genomic hypomethylation in late...

Viral Infection Increases Glucocorticoid-Induced Interleukin-10 Production through ERK-Mediated Phosphorylation of the Glucocorticoid Receptor in Dendritic Cells: Potential Clinical Implications

The hypothalamic-pituitary-adrenal axis plays a central role in the adaptive response to stress including infection of pathogens through glucocorticoids. Physical and/or mental stress alter susceptibility to viral infection possibly by affecting this regulatory system, thus we explored potential cellular targets and mechanisms that underlie this phenomenon in key immune components dendritic cells (DCs). Dexamethasone (DEX) treatment and subsequent Newcastle disease virus (NDV) infection most significantly and cooperatively stimulated mRNA expression of the interleukin (IL)-10 in murine bone marrow-derived DCs among 89 genes involved in the Toll-like receptor signaling pathways. NDV increased DEX-induced IL-10 mRNA and protein expression by 7- and 3-fold, respectively, which was observed from 3 hours after infection. Conventional DCs (cDCs), but not plasmacytoid DCs (pDCs) were major sources of IL-10 in bone marrow-derived DCs treated with DEX and/or infected with NDV. Murine cytomegalovirus and DEX increased serum IL-10 cooperatively in female mice. Pre-treatment of DCs with the extracellular signal-regulated kinase (ERK) inhibitor U0126 abolished cooperative induction of IL-10 by DEX and NDV. Further, ERK overexpression increased IL-10 promoter activity stimulated by wild-type human GR but not by its mutant defective in serine 203, whereas ERK knockdown abolished NDV/DEX cooperation on IL-10 mRNA and phosphorylation of the mouse GR at serine 213. NDV also increased DEX-induced mRNA expression of three known glucocorticoid-responsive genes unrelated to the Toll-like receptor signaling pathways in DCs. These results indicate that virus and glucocorticoids cooperatively increase production of anti-inflammatory cytokine IL-10 by potentiating the transcriptional activity of GR in DCs, through which virus appears to facilitate its own propagation in infected hosts. The results may further underlie in part known exacerbation of IL-10/T helper-2-related allergic disorders by stress and viral infection.

Skeletal muscle interleukin-6 regulation in hyperthermia

We previously reported that IL-6 production is acutely elevated in skeletal muscles exposed to ≥41°C, but the regulatory pathways are poorly understood. The present study characterizes the heat-induced transcriptional control of IL-6 in C2C12 muscle fibers. Hyperthermia exposure (42°C for 1 h) induced transcription from an IL-6 promoter-luciferase reporter plasmid. Heat shock factor-1 (HSF-1), a principal mediator of the heat shock response, was then tested for its role in IL-6 regulation. Overexpression of a constitutively active HSF-1 construct increased basal (37°C) promoter activity, whereas overexpression of a dominant negative HSF-1 reduced IL-6 promoter activity during basal and hyperthermia conditions. Since hyperthermia also induces stress-activated protein kinase (SAPK) signaling, we tested whether mutation of a transcription site downstream of SAPK, (i.e., activator protein-1, AP-1) influences IL-6 transcription in hyperthermia. The mutation had no effect on baseline reporter activity but completely inhibited heat-induced activity. We then tested whether pharmacologically induced states of protein stress, characteristic of cellular responses to hyperthermia and known to induce SAPKs and HSF-1, would induce IL-6 production in the absence of heat. The proteasome was inhibited with MG-132 in one set of experiments, and the unfolded protein response was stimulated with dithiothreitol, thapsigargin, tunicamycin, or castanospermine in other experiments. All treatments stimulated IL-6 protein secretion in the absence of hyperthermia. These studies demonstrate that IL-6 regulation in hyperthermia is directly controlled by HSF-1 and AP-1 signaling and that the IL-6 response in C2C12 myotubes is sensitive to categories of protein stress that reflect accumulation of damaged or unfolded proteins.

EpCAM Modulates NF-κB Signaling and Interleukin-8 Expression in Breast Cancer

The epithelial cell adhesion molecule (EpCAM) is a 40-kD type I transmembrane protein that is overexpressed in human epithelial cancers and is currently the target of molecular therapy based on its overexpression at the cell surface. Recently, we and others have shown a role for EpCAM in cell signaling and carcinogenesis, and EpCAM expression seems to promote breast cancer invasion. Interleukin-8 (IL-8/CXCL-8) is an inflammatory cytokine that has recently been shown to modulate breast cancer invasion and angiogenesis. In preliminary experiments, we identified a correlation between EpCAM and IL-8 expression in primary human breast cancers. Specific ablation of EpCAM in breast cancer cell lines results in decreased IL-8 expression, and IL-8 contributes to EpCAM-dependent breast cancer invasion. Specific ablation of EpCAM is also associated with decreased NF-κB transcription factor activity, decreased phosphorylation of the NF-κB family member RELA, and increased IκBα protein expression. EpCAM modulates IL-8 expression at baseline, and following IL-1β stimulation, which is known to be a potent inducer of NF-κB in breast cancer. In functional rescue experiments, specific ablation of RELA or forced expression of the NF-κB inhibitor protein IκBα prevented EpCAM-dependent rescue of IL-8 promoter activity. These studies show for the first time that EpCAM can modulate NF-κB transcription factor activity and IL-8 expression in breast cancer and confirm the role of EpCAM signaling in modulating breast cancer invasion. Further study is required to define the molecular mechanism(s) of EpCAM signaling in breast cancer and to direct the rational development of molecular therapies targeting EpCAM.

NF‐κB‐dependent IL‐8 induction by prostaglandin E2 receptors EP1 and EP4

Recent studies suggested a role for PGE(2) in the expression of the chemokine IL-8. PGE(2) signals via four different GPCRs, EP(1) -EP(4) . The role of EP(1) and EP(4) receptors for IL-8 induction was studied in HEK293 cells, overexpressing EP(1) (HEK-EP(1) ), EP(4) (HEK-EP(4) ) or both receptors (HEK-EP(1) + EP(4) ). IL-8 mRNA and protein induction and IL-8 promoter and NF-κB activation were assessed in EP expressing HEK cells. In HEK-EP(1) and HEK-EP(1) + EP(4) but not HEK or HEK-EP(4) cells, PGE(2) activated the IL-8 promoter and induced IL-8 mRNA and protein synthesis. Stimulation of HEK-EP(1) + EP(4) cells with an EP(1) -specific agonist activated IL-8 promoter and induced IL-8 mRNA and protein, whereas a specific EP(4) agonist neither activated the IL-8 promoter nor induced IL-8 mRNA and protein synthesis. Simultaneous stimulation of HEK- EP(1) + EP(4) cells with both agonists activated IL-8 promoter and induced IL-8 mRNA to the same extent as PGE(2) . In HEK-EP(1) + EP(4) cells, PGE(2) -mediated IL-8 promoter activation and IL-8 mRNA induction were blunted by inhibition of IκB kinase. PGE(2) activated NF-κB in HEK-EP(1) , HEK-EP(4) and HEK-EP(1) + EP(4) cells. In HEK-EP(1) + EP(4) cells, simultaneous activation of both receptors was needed for maximal PGE(2) -induced NF-κB activation. PGE(2) -stimulated NF-κB activation by EP(1) was blocked by inhibitors of PLC, calcium-signalling and Src-kinase, whereas that induced by EP(4) was only blunted by Src-kinase inhibition. These findings suggest that PGE(2) -mediated NF-κB activation by simultaneous stimulation of EP(1) and EP(4) receptors induces maximal IL-8 promoter activation and IL-8 mRNA and protein induction.

MiR-31 regulates interleukin 2 and kinase suppressor of ras 2 during T cell activation

MicroRNA (miRNA) has an important role as a master regulator of gene expression in immune system and is upregulated during T cell differentiation, however its function is not clear yet. In this study, the contribution of miR-31 in T cell activation was investigated. miR-31 was upregulated during the activation of primary T lymphocytes upon T-cell receptor (TCR) stimulation. Ectopic expression of miR-31 increased the expression of interleukin (IL)-2, while knockdown of endogenous miR-31 decreased the IL-2 expression. To gain more insights into the regulatory mechanism, we performed a bioinformatic analysis and found miR-31 potentially targeted kinase suppressor of ras 2 (KSR2), a repression factor of Ras2 kinase. Using reporter gene and western blotting assays, we confirmed that miR-31 could inhibit KSR2 by directly targeting its 3' untranslated region (UTR). Moreover, miR-31 enhanced nuclear factor of activated T cells (NF-AT) activity in Jurkat T cells, and increased transcription activity of IL-2 promoter in primary T cells. In conclusion, our study demonstrated that miR-31 upregulated IL-2 expression via reduction of its up-stream kinase suppressor, KSR2, and is a component of T cell activation.

CCN4 induces IL-6 production through αvβ5 receptor, PI3K, Akt, and NF-κB singling pathway in human synovial fibroblasts

IntroductionOsteoarthritis (OA) is the most common degenerative joint disease that is involved in the degradation of articular cartilage. The exact etiology of OA is not completely understood. CCN4 is related to up-regulation in the cartilage of patients with osteoarthritis. Previous studies have shown that CCN4 might be associated with the pathogenesis of OA, but the exact signaling pathways in CCN4-mediated IL-6 expression in synovial fibroblasts (SF) are largely unknown. Therefore, we explored the intracellular signaling pathway involved in CCN4-induced IL-6 production in human synovial fibroblast cells.MethodsCCN4-induced IL-6 production was assessed with quantitative real-time qPCR and ELISA. The mechanisms of action of CCN4 in different signaling pathways were studied by using Western blotting. Neutralizing antibodies of integrin were used to block the integrin signaling pathway. Luciferase assays were used to study IL-6 and NF-κB promoter activity. Immunocytochemistry was used to examine the translocation activity of p65.ResultsOsteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCN4 and the expression was higher than in normal SFs. OASF stimulation with CCN4 induced concentration- and time-dependent increases in IL-6 production. Pretreatment of OASFs with αvβ5 but not α5β1 and αvβ3 integrin antibodies reduced CCN4-induced IL-6 production. CCN4-mediated IL-6 production was attenuated by PI3K inhibitor (LY294002 and Wortmannin), Akt inhibitor (Akti), and NF-κB inhibitor (PDTC and TPCK). Stimulation of cells with CCN4 also increased PI3K, Akt, and NF-κB activation.ConclusionsOur results suggest that CCN4 activates αvβ5 integrin, PI3K, Akt, and NF-κB pathways, leading to up-regulation of IL-6 production. According to our results, CCN4 may be an appropriate target for drug intervention in OA in the future.

Transcriptional Regulation of IL-10 Expression by Peroxisome Proliferator-Activated Receptor (Pparγ) in Human Intestinal Epithelial Cells

IL-10 is an important immunomodulatory cytokine in the regulation of intestinal inflammation in inflammatory bowel disease (IBD). PPARγ is a nuclear receptor known to modulate transcriptional activities of many cytokines and is highly expressed in intestinal epithelial cells (IECs). Human IECs have been shown to express IL-10, but how its expression is regulated remains obscure especially in the setting of inflammation. We have demonstrated that intestinal 15d-PGJ2 (an endogenous PPARγ ligand) is predominantly induced by infiltrated macrophages (Mφs) through toll-like receptor 4 (TLR4)-mediated Cox-2 induction during colitis. In this study, we addressed whether PPARγ activation regulates expression of IL-10 in IECs. Human IEC cell line SW480-APC expressing TLR4 (top) and mouse peritoneal Mφs (bottom) were co-cultured in transwells. 15d-PGJ2 production and IL-10 mRNA expression were measured by enzyme linked assay and real-time PCR in the presence or absence of lipopolysaccharide (LPS). In order to examine whether the IL-10 promoter has PPARγ responsive elements, a 2,000 bp of IL-10 promoter was cloned from SW480-APC genomic DNA into the luciferase reporter vector. Expression and activity of PPARγ were blocked by siRNA and a specific inhibitor SR202, respectively. The IL-10 promoter activity was analyzed by luciferase reporter assay. PPARγ activity was confirmed using a reporter vector PPRE X3-TK-luc. Co-culturing SW480-APC with Mφs increased IL-10 expression in IECs within 24 hours, which was significantly enhanced by LPS stimulation in later time point (72 hours). LPS stimulation of SW480-APC resulted in PPARγ activation peaked at 48 hours. During SW480-APC/Mφ co-culture, LPS stimulation induced 15d-PGJ2 production, which is abolished when TLR4-deficient Mφs were used. Knocking down the expression of PPARγ suppressed IL-10 promoter activity and mRNA expression in SW480-APC in baseline as well as after LPS stimulation. These results suggest that PPARγ positively regulates IL-10 expression in IECs at the transcriptional level. During inflammation, mucosal Mφs induce PPARγ ligand (15d-PGJ2) in a TLR4-dependent manner that contributes to PPARγ activation in IECs and thus IL-10 expression. This may be an intrinsic immunoregulatory program that is mediated through host-bacterial interactions, which dampens on-going inflammation during intestinal inflammation and thus can be a novel therapeutic target for IBD.

Identification of microRNA‐31 as a novel regulator contributing to impaired interleukin‐2 production in T cells from patients with systemic lupus erythematosus

MicroRNAs (miRNAs) function to fine-tune the control of immune cell signaling. It is well established that there are abnormalities in the interleukin-2 (IL-2)-related signaling pathways in systemic lupus erythematosus (SLE). The miR-31 microRNA has been found to be markedly underexpressed in patients with SLE, and thus the present study was undertaken to investigate the role of miR-31 in IL-2 defects in lupus T cells. Expression levels of miR-31 were quantitated using TaqMan miRNA assays. Transfection and stimulation of cultured cells followed by TaqMan quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and reporter gene assays were conducted to determine the biologic function of miR-31. NF-AT nuclear translocation and expression were quantitatively measured using an ImageStream cytometer. Bioinformatics analysis, small interfering RNA (siRNA) knockdown, and Western blotting were performed to validate miR-31 targets and effects. The expression of miR-31 was significantly decreased in lupus T cells, and this was positively correlated with the expression of IL-2. Overexpression of miR-31 in T cells increased the production of IL-2 by altering NF-AT nuclear expression and IL2 promoter activity, while knockdown of endogenous miR-31 reduced IL-2 production. RhoA expression was directly repressed by miR-31 in T cells. Of note, siRNA-mediated knockdown of RhoA enhanced IL2 promoter activity and, consequently, up-regulated IL-2 production. RhoA expression was consistently up-regulated and negatively correlated with the levels of miR-31 in lupus T cells. Manipulation of miR-31 expression in lupus T cells restored the expression of IL-2 at both the messenger RNA and protein levels. MicroRNA-31 is a novel enhancer of IL-2 production during T cell activation. Dysregulation of miR-31 and its target, RhoA, could be a novel molecular mechanism underlying the IL-2 deficiency in patients with SLE.

TLR 2 Induces Vascular Smooth Muscle Cell Migration Through cAMP Response Element−Binding Protein−Mediated Interleukin-6 Production

Migration of vascular smooth muscle cells (VSMCs) from the media into intima contributes to the development of atherosclerosis. Gene deletion experiments implicate a role for toll-like receptor 2 (TLR2) in atherogenesis. However, the underlying mechanisms remain unclear. We postulate that TLR2 promotes VSMC migration by enhancing interleukin (IL)-6 production. Migration assays revealed that TLR2 agonists promoted VSMC migration but not cell proliferation or viability. TLR2 deficiency or inhibition of TLR2 signaling with anti-TLR2 antibody suppressed TLR2 agonist-induced VSMC migration and IL-6 production, which was mediated via p38 mitogen-associated protein kinase and extracellular signal-regulated kinase 1/2 signaling pathways. Neutralizing anti-IL-6 antibodies impaired TLR2-mediated VSMC migration and formation of filamentous actin fiber and lamellipodia. Blockade of p38 mitogen-associated protein kinase or extracellular signal-regulated kinase 1/2 activation inhibited TLR2 agonist pam3CSK4-induced phosphorylation of cAMP response element-binding protein, which regulates IL-6 promoter activity through the cAMP response element site. Moreover, cAMP response element-binding protein small interfering RNA inhibited pam3CSK4-induced IL-6 production and VSMC migration. Additionally, Rac1 small interfering RNA inhibited pam3CSK4-induced VSMC migration but not IL-6 production. Our results suggest that on ligand binding, TLR2 activates p38 mitogen-associated protein kinase and extracellular signal-regulated kinase 1/2 signaling in VSMCs. These signaling pathways act in concert to activate cAMP response element-binding protein and subsequent IL-6 production, which in turn promotes VSMC migration via Rac1-mediated actin cytoskeletal reorganization.

Dendritic Cells Control CD4+CD25+ Treg Cell Suppressor Function In Vitro through Juxtacrine Delivery of IL-2

CD4+CD25+Foxp3+ regulatory T cells (Tregs) restrict inflammatory responses to self and nonself. Aberrant Treg activity is pathologic: Insufficient Treg activity is implicated in autoimmunity, allergy, and graft-versus-host-disease; overabundant activity is implicated in chronic infection and cancer. Tregs require IL-2 for their expansion and acquisition/execution of suppressor function; however, because Tregs cannot produce IL-2, they depend on IL-2 from an exogenous source. Until now, that IL-2 source had not been established. We asked whether dendritic cells (DCs) could supply IL-2 to Tregs and, if so, what was required for that delivery. We used flow cytometry, IL-2 ELISPOT, RT-qPCR, and IL-2 promoter-driven reporter assays to measure intracytoplasmic IL-2, secreted protein, IL-2 message and IL-2 promoter activity in bone marrow-derived (BMDC) and splenic DCs. We examined conjugate formation between Tregs, conventional CD4+ cells, and IL-2-expressing DCs. We measured Treg levels of CD25, Foxp3, and suppressor function after co-culture with IL-2 sufficient and IL-2−/− DCs. We generated IL-2-mCherry-expressing DCs and used epifluorescence microscopy and flow cytometry to track IL-2 transfer to Tregs and test requirements for transfer. Between 0.7 to 2.4% of DCs constitutively produced IL-2 and diverted IL-2 secretion to Tregs by preferentially forming conjugates with them. Uptake of DC IL-2 by Tregs required cell-cell contact and CD25. Tregs increased levels of CD25 and Foxp3 from baseline and showed greater suppressor function when co-cultured with IL-2-sufficient DCs, but not when co-cultured with IL-2−/− DCs. Exogenous IL-2, added in excess of 500 U/ml to co-cultures with IL-2−/− DCs, restored Treg suppressor function. These data support a model of juxtacrine delivery of IL-2 from DCs to Tregs and suggest that a subset of DCs modulates Treg function through controlled, spatial delivery of IL-2. Knowledge of how DCs regulate Tregs should be integrated into the design of interventions intended to alter Treg function.

Calcium/calmodulin-dependent protein kinase II regulates IL-10 production by human T lymphocytes: A distinct target in the calcium dependent pathway

Calcium (Ca2+) plays an essential role in lymphocyte activation and differentiation by affecting signaling pathways leading to cytokine production. Among the enzymes responding to calcium increase, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been involved in anergy with a still poorly characterized role. IL-10 produced by different T lymphocyte subpopulations is critical mediator of tolerance. We tested the hypothesis that CaMKII may be involved in IL-10 production. We report that CaMKII upregulates IL-10 production by primary human T lymphocytes stimulated through the antigen receptor or bypassing that. Overexpression of constitutively active mutant forms of Calcineurin or CaMKII specifically increase IL-10 protein product and IL-10 mRNA accumulation in T lymphocytes. By cotransfecting constitutively active CaMKII with luciferase reporter plasmids carrying specific fragments or the whole IL-10 promoter, we show that CaMKII specifically activates IL-10 promoter activity, whereas it inhibits IL-2 and IL-4 promoter. This effect is mediated by the first 500bp fragment, which contains binding sites for Myocyte Enhancer Factor-2 (MEF2). A constitutively active mutant of CaMKII activated a luciferase reporter plasmid under the control of MEF2, when cotransfected in T lymphocytes stimulated by Ionomycin and PMA, whereas its inhibitor KN-62 inhibited MEF2 binding in cell lysates of the same cells. Moreover, overexpression of MEF2 enhanced by 2.5-fold IL-10 promoter activity. Our data for the first time suggest a distinct role of CaMKII in the induction of anergy in T lymphocytes, by differential regulation of IL-10 and IL-2 gene transcription suggest MEF2 as a molecular target which can integrate different calcium signals.

Chitin‐binding protein GbpA of Vibrio cholerae induces interleukin‐8 gene expression in intestinal cells through a TLR2/TLR1/CD14 complex

Vibrio cholerae N‐acetylglucosamine binding protein (GbpA) is a secretory protein which is essential for bacterial survival in environment as well as in human gut. Here we show that GbpA binds to toll‐like receptor 2 (TLR2) and initiates downstream cellular inflammatory signaling pathway by IL8 secretion. GbpA triggered formation of lipid raft microdomain on intestinal cell surface by interacting with TLR2 with a Kd of 76 nM. The microdomain contains TLR1, CD14 and flotilin in addition to TLR2. As a downstream effect, the lipid raft complex triggered IL8 secretion and the effect was specific in HT29 intestinal cells. The critical involvement of the GbpA‐induced lipid raft microdomain complex in IL8 secretion was indicated by the decrease in secretion caused by disruption of the raft by extraction of cholesterol by methyl‐β‐D‐cyclodextrin and by its restoration by extraneous addition of cholesterol. The GbpA‐mediated elevation in IL8 secretion involved activation of NFκB and formation of AP1 complex which eventually increased IL8 promoter activity. Taken together, the results suggest that GbpA is a key player in pathogenesis of cholera by triggering pro‐inflammatory response in infection by inducing downstream activation through clustering of GbpA‐TLR1/2‐CD14 complex in lipid raft microdomain. [Supported in part by grant from CSIR, Govt. of India No. 37(1410)/10/EMR‐II]

Lysophosphatidic Acid Induces Lymphangiogenesis and IL-8 Production in Vitro in Human Lymphatic Endothelial Cells

The bioactive phospholipid lysophosphatidic acid (LPA) and its receptors LPA(1-3) are aberrantly expressed in many types of human cancer. LPA has been reported to induce tumor cell proliferation, migration, and cytokine production. However, whether LPA exerts an effect on lymphatic endothelial cells (LECs) or on lymphangiogenesis, a process of new lymphatic vessel formation that is associated with increased metastasis and poor prognosis in cancer patients, has been unknown. Here, we show that LPA induces cell proliferation, survival, migration, and tube formation, and promotes lymphangiogenesis in vitro in human dermal LECs. In addition, LPA induces IL-8 expression by enhancing IL-8 promoter activity via activation of the NF-κB pathway in LECs. Using IL-8 siRNA and IL-8 neutralizing antibody, we revealed that IL-8 plays an important role in LPA-induced lymphangiogenesis in vitro. Moreover, using siRNA inhibition, we discovered that LPA-induced lymphangiogenesis in vitro and IL-8 production are mediated via the LPA(2) receptor in LECs. Finally, using human sentinel afferent lymphatic vessel explants, we demonstrated that LPA up-regulates IL-8 production in the LECs of lymphatic endothelia. These studies provide the first evidence that LPA promotes lymphangiogenesis and induces IL-8 production in LECs; we also reveal a possible new role of LPA in the promotion of tumor progression, as well as metastasis, in different cancer types.

Interleukin‐8 gene regulation in epithelial cells by Vibrio cholerae: role of multiple promoter elements, adherence and motility of bacteria and host MAPKs

Interleukin (IL)-8 is an important mediator in neutrophil-mediated acute inflammation. We previously demonstrated that incubation of intestinal epithelial cells with Vibrio cholerae O395 resulted in increased IL-8 mRNA expression and IL-8 secretion, which was associated with the adherence and motility of bacteria. However, the mechanisms responsible for transcriptional regulation of the IL-8 gene in epithelial cells during V. cholerae infections were not explored. Transient transfection analysis of 5' deletions and mutations of the IL-8 promoter driving expression of the luciferase reporter gene indicates that multiple binding sites contribute to IL-8 promoter induction in response to V. cholerae and that cooperation among these different sites is required for full activation of the promoter. Stimulation with V. cholerae O395 insertional mutants, defective in adherence and motility, significantly reduced IL-8 promoter activity compared with the wild-type strain. We further demonstrate maximal involvement of extracellular signal-regulated kinase 1/2/mitogen-activated protein kinase pathways to regulate IL-8 gene transcription. This study will help to design agents which could reduce the V. cholerae-induced inflammatory response and in the generation of safe vaccines.

Polyubiquitination of Transforming Growth Factor β (TGFβ)-associated Kinase 1 Mediates Nuclear Factor-κB Activation in Response to Different Inflammatory Stimuli

The transcription factor nuclear factor κB (NF-κB) plays a central role in regulating inflammation in response to several external signals. The TGFβ-associated kinase 1 (TAK1) is an upstream regulator of NF-κB signaling. In TGFβ-stimulated cells, TAK1 undergoes Lys-63-linked polyubiquitination at Lys-34 by TNF receptor-associated factor 6 and is thereby activated. The aim of this study was to investigate whether TAK1 polyubiquitination at Lys-34 is also essential for NF-κB activation via TNF receptor, IL-1 receptor and toll-like receptor 4. We observed that TAK1 polyubiquitination occurred at Lys-34 and required the E3 ubiquitin ligase TNF receptor-associated factor 6 after stimulation of cells with IL-1β. Polyubiquitination of TAK1 also occurred at Lys-34 in cells stimulated by TNF-α and LPS, which activates TLR4, as well as in HepG2 and prostate cancer cells stimulated with TGFβ, which in all cases resulted in NF-κB activation. Expression of a K34R-mutant TAK1 led to a reduced NF-κB activation, IL-6 promoter activity, and proinflammatory cytokine secretion by TNF-α-stimulated PC-3U cells. Similar results were obtained in the mouse macrophage cell line RAW264.7 after LPS treatment. In conclusion, polyubiquitination of TAK1 is correlated with activation of TAK1 and is essential for activation of NF-κB signaling downstream of several receptors.

Native X-DING-CD4 protein secreted by HIV-1 resistant CD4+ T cells blocks activity of IL-8 promoter in human endothelial cells infected with enteric bacteria

Onsets of bacterial infections devastate the compromised immune system in AIDS patients. Damaged gut mucosa permits dissemination of bacterial toxins into deeper layers and hyper-activation of the immune system. We previously reported that the unfractionated supernatants of HIV-resistant CD4(+) T cells impeded the NF-κB/DNA binding in macrophages induced by either HIV-1 or LPS. The active component of this soluble material was identified as X-DING-CD4 (extracellular DING from CD4 T cells). We hypothesized that the anti-inflammatory effect of the X-DING-CD4 protein might extend to non-immune cells, for example endothelial cells, undergoing persistent endotoxin stimulation in the course of advanced HIV disease. To test this proposition, we evaluated the efficiency of NF-κB and Ap-1 binding to the IL-8 promoter in LPS-activated endothelial cells and control human macrophages exposed to native X-DING-CD4 protein. We found a deficiency of NF-κB- but not AP-1-DNA binding in the systems where cells were treated with native soluble X-DING-CD4 protein. The X-DING-CD4-mediated inhibition of the IL-8 promoter also resulted in a reduction of the soluble IL-8 protein in endothelial cells and human macrophages infected with a subset of enteric bacteria frequently causing diarrhea in progressive HIV disease. Bacterial endotoxin did not induce the endogenous X-DING-CD4 mRNA activity in human macrophages and transformed CD4(+)T cells, indicating that the reduction of LPS-mediated IL-8 promoter activation was not related to de novo X-DING-CD4 protein synthesis, but depended on function of the exogenous X-DING-CD4 protein. This study provides evidence that the X-DING-CD4 protein might be developed as a novel biotherapeutic to control LPS-mediated inflammation in advanced HIV disease.

Activation of Toll-like receptor 9 and production of epitope specific antibody by liposome-encapsulated CpG-DNA

Several investigators have shown that CpG-DNA has outstanding effects as a Th1-responsive adjuvant and that its potent adjuvant effects are enhanced by encapsulation with a liposome of proper composition. In this study, we showed that encapsulation with phosphatidyl-Β-oleoyl-γ-palmitoyl ethanolamine (DOPE): cholesterol hemisuccinate (CHEMS) complex enhances the immunostimulatory activity of CpG DNA and the binding of CpG-DNA to TLR9. We also examined involvement of myeloid differentiation protein (MyD88) and NF-κB activation in liposome-encapsulated CpG-DNA-induced IL-8 promoter activation. In this manuscript, the natural phosphodiester bond CpG-DNA encapsulated by DOPE : CHEMS complex is designated as Lipoplex(O). Importantly, we successfully screened B cell epitopes of envelope protein (E protein) of hepatitis C virus (HCV-E) and attachment glycoprotein G of human respiratory syncytial virus (HRSV-G) by immunization with complexes of several peptides and Lipoplex(O) without carriers. Therefore, Lipoplex(O) is potentially applicable as a universal adjuvant for peptide-based epitope screening and antibody production.

Regulation of IL-2 gene expression by Siva and FOXP3 in human T cells

BackgroundSevere autoinflammatory diseases are associated with mutations in the Foxp3 locus in both mice and humans. Foxp3 is required for the development, function, and maintenance of regulatory T cells (Tregs), a subset of CD4 cells that suppress T cell activation and inflammatory processes. Siva is a pro-apoptotic gene that is expressed across a range of tissues, including CD4 T cells. Siva interacts with three tumor necrosis factor receptor (TNFR) family members that are constitutively expressed on Treg cells: CD27, GITR, and OX40.ResultsHere we report a biophysical interaction between FOXP3 and Siva. We mapped the interaction domains to Siva's C-terminus and to a central region of FOXP3. We showed that Siva repressed IL-2 induction by suppressing IL-2 promoter activity during T cell activation. Siva-1's repressive effect on IL-2 gene expression appears to be mediated by inhibition of NFkappaB, whereas FOXP3 repressed both NFkappaB and NFAT activity.ConclusionsIn summary, our data suggest that both FOXP3 and Siva function as negative regulators of IL-2 gene expression in Treg cells, via suppression of NFAT by FOXP3 and of NFkappaB by both FOXP3 and Siva. Our work contributes evidence for Siva's role as a T cell signalling mediator in addition to its known pro-apoptotic function. Though further investigations are needed, evidence for the biophysical interaction between FOXP3 and Siva invites the possibility that Siva may be important for proper Treg cell function.