Expression Of Interleukin-1 Receptor-associated Kinase Research Articles

The effect of transfected microRNA-146a on expression of interleukin-1 receptor-associated kinase 1 and tumor necrosis factor receptor-associated factor 6 in alveolar macrophages

To observe the effect of transfected microRNA-146a (miR-146a) on expression of interleukin-1 receptor-associated kinase 1 (IRAK-1) and tumor necrosis factor receptor-associated factor 6 (TRAF-6) in alveolar macrophages, and to explore the regulatory mechanism of miR-146a in the inflammatory response of alveolar macrophages. Alveolar macrophages NR8383 were cultured and divided into two groups: transfected miR-146a mimic group was transfected 50 nmol/L Pre-miR miR-146a precursors and the negative control group was transfected Cy3-labeled Pre-miR negative control. Cells were collected at 24 hours after transfection. The miR-146a and the mRNA expression of IRAK-1 and TRAF-6 were detected by real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-qPCR), and the protein expression of IRAK-1 and TRAF-6 was assayed by Western Blot. Compared with negative control group, the expression of miR-146a was upregulated by (24.55±6.14) fold compared with miR-146a mimic group (t=-9.353, P=0.001). The mRNA expressions of IRAK-1 and TRAF-6 in miR-146a mimic group were upregulated by (1.16±0.10) fold (t=2.701, P=0.054) and (1.19±0.16) fold (t=2.032, P=0.112) , respectively, compared with that of negative control group, but the protein levels of IRAK-1 and TRAF-6 were decreased by 73.0% (t=-9.353, P=0.001) and 64.1% (t=-6.839, P=0.002), respectively . miR-146a mimic was successfully transfected into the alveolar macrophage NR8383. The overexpression of miR-146a in alveolar macrophages can down-regulate the expression of IRAK-1 and TRAF-6 in protein translation levels, and its mechanism may be related with inhibition of protein translation.

Valproic Acid Downregulates NF-κB p50 Activity and IRAK-1 in a Progressive Thyroid Carcinoma Cell Line

Histone deacetylase inhibitor (HDACI) valproic acid (VPA) is a promising drug, currently in clinical phase 2, for the therapy of advanced/poorly differentiated thyroid cancer. The nuclear factor-κB (NF-κB) pathway is constitutively activated in most tumors, including thyroid carcinomas; this often contributes to aggressive tumor growth and therapeutic resistance. We hypothesized that VPA could be useful to decrease NF-κB activity in human thyroid cancer cells. To clarify this, we treated the highly progressive thyroid cancer cell line BHT-101 with VPA (1.0-3.0 mM) for 48 h. Real-time polymerase chain reaction (PCR) and Western blot were used to measure expression of NF-κB-regulatory genes and proteins. NF-κB p50 activity was measured using an ELISA-based colorimetric transcription factor assay kit. We found that VPA significantly and dose-dependently impaired NF-κB activity reducing DNA binding activity of NF-κB p50 subunit by 30% at 1 mM, 40% at 1.5 mM, and 70% at 3 mM. Expression of interleukin-1 receptor-associated kinase-1 (IRAK-1) protein, an upstream mediator of NF-κB activation, was reduced by ̴30% at 1 and 1.5 mM. Furthermore, 3 mM VPA treatment significantly decreased expressions of IRAK-1, phospho-IκBα and NF-κB p50 subunit protein by ̴ 50%. This is the first study to demonstrate that VPA decreases NF-κB activity in a progressive thyroid cancer cell line. Intriguingly, 1mM of VPA, a clinically safe dose in the therapeutic range for epilepsy, was sufficient to reduce NF-κB activity. Thus, VPA may be a promising agent to overcome chemoresistance in cancer therapy and to improve therapeutic efficiency.

Molecular cloning and expression of IRAK-4, IL-17 and I-κB genes in Haliotis rufescens challenged with Vibrio anguillarum

The candidate genes interleukin-1 receptor associated kinase 4 (IRAK-4), Interleukin 17 (IL-17) and Inhibitor of NF-κB (I-κB) were cloned and evaluated in Californian abalone (Haliotis rufescens) hemocytes in response to Vibrio anguillarum. Molecular characterization evidenced that HrI-κB has a full cDNA sequence of 3027 bp with an encoding region of 401 amino acids (aa), HrIRAK-4 comprised 1969 bp that encoded for 516 aa, and Hr-IL17 had a full sequence of 806 bp encoding for 165 aa. qPCR analysis showed the higher constitutive expression level of Hr-IL17 in hemocytes; meanwhile Hr-IκB and Hr-IRAK4 gene expression levels were higher in gills and mantle. The assessment of gene expression in hemocytes after infection with V. anguillarum evidences the immune responses of Hr-IκB, Hr-IRAK4, and Hr-IL17 and their relationships through the NF-κB signaling pathway.

Distinct IRAK4 Expression in Normal and B-CLL Lymphocytes

Toll-like receptors (TLR) signaling plays an important role in the B cell biology. It induces the maturation, proliferation and antibody production in response to pathogen recognition. Interleukin-1 Receptor-associated Kinase-4 (IRAK-4) is TLR downstream molecule which is of strategic importance in starting a cascade of NF-kappaB (Nuclear factor-kappaB) and MAPK (Mitogen-activated protein kinase) intracellular signaling pathways. The aim of the study was to identify the differences in IRAK-4 expression in normal and chronic lymphocytic leukemia (CLL) lymphocytes. We have also studied the IRAK-4 expression alteration upon TLR2/4 ligand stimulation in leukemic and normal B cells. We carried out Real-Time PCR and Western blot analysis of IRAK4 mRNA (RQ) and protein expression. The study were performed on the isolated CD19+ normal B lymphocytes and CLL cells (CD5+CD19+) before and 30 min after (Lipopolysaccharide) LPS and (Staphylococcus aureus strain Cowan I) SAC stimulation. IRAK-4 mRNA expression was significantly higher in normal CD19+ lymphocytes as compared to leukemic CD5+CD19+ subpopulation (RQ median 0.90 vs 0.54; p=0.012). In normal lymphocytes, LPS and SAC treatment led to significant IRAK-4 down-regulation both at mRNA (RQ median 0.90 vs 0.53 vs 0.40; p=0.03, p=0.01) and protein level, whereas generally there were no significant changes in IRAK-4 expression neither on mRNA nor protein level. No trend or significant differences in IRAK-4 expression in leukemic lymphocytes after LPS and SAC stimulation may imply the presence of alleged defect of immune tolerance in CLL.

Increased IRAK-4 Kinase Activity in Alzheimer’s Disease; IRAK-1/4 Inhibitor I Prevents Pro-inflammatory Cytokine Secretion but not the Uptake of Amyloid Beta by Primary Human Glia

Alzheimer’s disease (AD) is characterized by the deposition of amyloid-β (Aβ), which is associated with a neuroinflammatory response involving microglia and astrocytes. This neuroinflammatory response has detrimental effects on disease progression but also has a beneficial function on removal of excess Aβ. Microglia and astrocytes are involved in the clearance of Aβ from the brain, but neuroinflammation also promotes neurodegeneration. In order to identify signal transduction pathways critically involved in AD we analysed human brain tissue using protein kinase activity profiling. We identified increased activity of the Interleukin 1 Receptor Associated Kinase 4 (IRAK-4) in AD compared to control brain tissue. IRAK-4 is a component of the signal transduction pathway that functions downstream of the Toll-like receptors and the interleukin-1 receptor. Immunohistochemical analysis of human brain tissue revealed the presence of IRAK-4 in astrocytes and microglia. Quantification of IRAK-4 and the phosphorylated form of IRAK-1, a specific substrate for IRAK-4, revealed increased expression and activity of IRAK-4 in AD. Interestingly, IRAK-1/4 inhibitor I reduces the lipopolysaccharide-induced secretion of monocyte chemotactic protein-1 (MCP-1) by primary human microglia and the interleukin-1β-induced secretion of MCP-1 and interleukin 6 by primary human astrocytes. In contrast, the uptake of Aβ by astrocytes and microglia is not affected by IRAK-1/4 inhibition. Our data show that IRAK-4 protein kinase activity is increased in AD and selective inhibition of IRAK-1/4 inhibits a pro-inflammatory response without affecting the uptake of Aβ by glial cells, indicating that the IRAK signalling pathway is a potential target for modulating neuroinflammation in AD.

Characterization of the interleukin 1 receptor-associated kinase 4 (IRAK4)-encoding gene in salmonid fish: The functional copy is rearranged in Oncorhynchus mykiss and that factor can impair TLR signaling in mammalian cells

The interleukin 1 receptor-associated kinase 4 (IRAK4) is an essential factor for TLR-mediated activation of the host's immune functions subsequent to pathogen contact. We have characterized the respective cDNA and gene sequences from three salmonid species, salmon, rainbow trout and maraena whitefish. The gene from salmon is structured into eleven exons, as is the mammalian homologue, while exons have been fused in the genes from the two other salmonid species. Rainbow trout expresses also a pseudogene at low levels. Its basic structure resembles more closely the primordial gene than the functional copy does. The N-terminal death domain and the C-terminal protein kinase domain of the factors are better conserved throughout evolution than the linker domain. The deduced amino acid sequences of the factors from all three species group together in an evolutionary tree of IRAK4 factors. Scrutinizing expression and function of IRAK4 from rainbow trout, we found its highest expression in head kidney and spleen and lowest expression in muscle tissue. Infecting fish with Aeromonas salmonicida did not modulate its expression during 72 h of observation. Expression of a GFP-tagged trout IRAK4 revealed, expectedly, its cytoplasmic localization in human HEK-293 cells. However, this factor significantly quenched in a dose-dependent fashion not only the pathogen-induced stimulation of NF-κB factors in the HEK-293 reconstitution system of TLR2 signaling, but also the basal NF-κB levels in unstimulated control cells. Our data unexpectedly imply that IRAK4 is involved in establishing threshold levels of active NF-κB in resting cells.

Decreased Expressions of Toll-Like Receptor 9 and Its Signaling Molecules in Chronic Hepatitis B Virus–Infected Patients

Toll-like receptors (TLRs) play crucial roles in immune responses, especially innate immunity, against viral infections. Toll-like receptor 9 recognizes intracellular viral double-strand DNA, which leads to the activation of nuclear factor B (NF-κB) through the myeloid differentiation primary response 88 (MYD88) pathway. Defects in the expression of TLR9 and its signaling molecules may cause attenuated immune responses against hepatitis B virus. To determine expression levels of TLR9 messenger RNA along with MYD88, interleukin 1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), and NF-κB in the peripheral blood mononuclear cells obtained from chronic hepatitis B virus (CHB)-infected patients. In this study, 60 CHB patients and 60 healthy controls were recruited and the expression of TLR9 and its downstream signaling molecules was examined by real-time polymerase chain reaction techniques using β-actin as a housekeeping gene. Our results showed that expression of TLR9, MYD88, IRAK1, TRAF6, and NF-κB in peripheral blood mononuclear cells of CHB patients was significantly decreased in comparison with healthy controls. According to our results, it appears that CHB patients are unable to appropriately express genes in the TLR9 pathway, which may impede immune responses against hepatitis B virus infection. These results suggest a mechanism that may partially explain the fact that immune responses are disrupted in CHB patients.

Histone deacetylase inhibitors increase microRNA-146a expression and enhance negative regulation of interleukin-1β signaling in osteoarthritis fibroblast-like synoviocytes

MiR-146a exerts negative control on inflammatory responses by suppressing cytokine-induced expression of interleukin-1 receptor-associated kinase-1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) by impairing NF-κB activity and inhibiting the expression of target genes. Recent study suggests that histone deacetylases (HDACs) are involved in the regulation of microRNA (miRNA) expression. Therefore, we determined whether HDAC inhibitors can increase miR-146a expression, thereby inhibiting interleukin-1β (IL-1β)-induced signaling in osteoarthritis fibroblast-like synoviocytes (OA-FLS). MiRNA expression was analyzed using real-time PCR. IL-1β-induced downstream signals and cytokine expression were evaluated using Western blotting and ELISA. Transcription factors regulating promoter activation were identified using chromatin immunoprecipitation assays. IL-1β treatment of OA-FLS induced a mild (1.7-fold) increase in miR-146a expression that was unable to appropriately downregulate IRAK1 and TRAF6 expression. HDAC inhibitors, SAHA (vorinostat), and LBH589 (panobinostat) significantly (6.1- and 5.4-fold) elevated miR-146a expression by increasing the binding of the transcription factor NF-κB to the miR-146a promoter, and negatively regulated IL-1β-induced IKK/IκB/p65 phosphorylation signaling and IL-6 secretion. The increase in miR-146a expression induced by the HDAC inhibitors was prevented by transfection of miR-146a inhibitor or HDAC1 (class I HDAC), HDAC4 (class IIa HDAC), and HDAC6 (class IIb HDAC) overexpression, suggesting that they were due to inhibition of HDAC activity. Our study demonstrated that HDAC inhibitor treatment in OA-FLS significantly increased miR-146a expression and mediated markedly negative regulation to inhibit IL-1β-induced signaling and cytokine secretion. Our results indicate the potential rationale of anti-inflammatory effects for HDAC inhibitors.

The expression and significance of interleukin-1 receptor associated kinase-4 in inferior mesenteric vein of rats with schistosomal cirrhosis

Objective To explore the expression and significance of interleukin-1 receptor associated kinase-4 (IRAK-4) in mesenteric vein of rats with schistosomal cirrhosis.Methods Fifty-five rats were randomly divided into two groups:normal control group (N group,n =15)) and model group (M group,n =40).The rats in M group were infected percutaneously with cercariae of schistosomiasis japonica,and divided into post-infection 12-week subgroup and 20-week subgroup.The mRNA expression levels of IRAK-4 and tumor necrosis factor (TNF)-α were detected by using reverse transcriptase polymerase chain reaction (RT-PCR).Western blotting was applied to examined the protein expression of IRAK-4.Results The expression of IRAK-4 mRNA in mesenteric vein in M groups (12th week:0.734 ± 0.081 ;20th week:0.682 ±0.069) was significantly stronger than in N group (0.163 ±0.031).The expression of IRAK-4 protein in M group (12th week:1034.52 ± 89.72 ; 20th week:989.56 ± 69.35) was significantly stronger than in N group (667.45 ± 72.22).No significant differences were found in the level of IRAK-4 between post-infection 20-week group and post-infection 12-week group (P ＞ 0.05).The expression of IRAK-4 mRNA was significantly positively correlated with the level of TNF-α mRNA (r =5.56,P ＜0.01).Conclusion The high expression of IRAK-4 and activation of downstream inflammatory factor play an important role in vasculopahty of rats with schistosomal cirrhosis. Key words: Portal hypertension; Endotoxemia; Vasculopahty; Interleukin-1 receptor associated kinase-4

MiR-146a Polymorphism Influences Levels of miR-146a, IRAK-1, and TRAF-6 in Young Patients with Coronary Artery Disease

Modulation of nuclear factor KappaB (NF-κB) activation may play a role in regulating inflammatory conditions associated with coronary artery disease (CAD). MicroRNA-146a (miR-146a) primarily targets interleukin-1 receptor-associated kinase 1 (IRAK-1) and tumour necrosis factor receptor associated factor 6 (TRAF-6), which results in inhibition of NF-κB via the TLR pathway. This study investigated the influence of the miR-146a GC rs2910164 on miR-146a expression in young South African Indians with CAD. CAD patients and controls were genotyped by PCR-RFLP and miRNA-146a levels were measured by qPCR. IRAK-1, TRAF-6 and NF-κB expression was determined by Western blot. No differences in genotypic frequency was found (GG: 45 vs. 47%, GC: 46 vs. 41%, CC: 9 vs. 12%) in controls and patients respectively (odds ratio = 1.025; 95% confidence interval 0.6782-1.550; p = 0.9164). Significantly higher levels of miR-146a was associated with CAD patients with the CC genotype (6.25-fold increase relative to controls and patients with the wildtype variant, p < 0.0001). Significantly lower levels of IRAK-1 (0.38 ± 0.02; p = 0.0072) and TRAF-6 (0.44 ± 0.02; p = 0.0146) was found in CAD patients with the CC genotype. The lowest levels of NF-κB and C-reactive protein were found in patients with the homozygous C allele compared to the heterozygous GC and wildtype variants. We propose a role for miR-146a in TLR signalling through a negative feedback mechanism involving the attenuation of NF-κB by down-regulation of IRAK-1 and TRAF-6. Our observations implicate miR-146a as a target for lowering inflammation in CAD patients.

Evaluation of Expression Pattern of Mirna146a in Pakistani Rheumatoid Arthritis Patients

Event Abstract Back to Event Evaluation of Expression Pattern of Mirna146a in Pakistani Rheumatoid Arthritis Patients Sami Ullah1*, Peter John1 and Attya Bhatti1 1 National University of Science and Technology, Pakistan Objectives: The objectives of the present study were to identify the role of miRNA146a in rheumatoid arthritis (RA) pathogenesis and to evaluate expression of miRNA146a and its target genes in RA patients. Methods: In the present study, expression of microRNA146a (miRNA146a) and its target genes were analyzed using Real time PCR and Semi-quantitative Reverse transcription PCR in blood and synovial fluid of rheumatoid arthritis patients. Results: An average of 0.7 fold increase was observed in expression of miRNA146a in blood and 14 fold in synovial fluid in rheumatoid arthritis patients as compared to healthy controls. Tumor necrosis factor receptor-associated factor 6 (TRAF6) was down-regulated and its expression correlated with miRNA146a in blood but not in synovial fluid. Interleukin-1 receptor-associated kinase 1 (IRAK-1) and Tumor Necrosis factor alpha (TNF-α) was up-regulated in both blood and synovial fluid. IRAK-1 expression did not correlate with miRNA16a in both blood and synovial fluid while TNF-α correlated with miRNA146a expression in synovial fluid only (R square = 1.4). Conclusion: Increased expression of miRNA146a and its statistically significant correlation with expression of TNF-α means that miRNA146a can be used as a potential diagnostic biomarker in rheumatoid arthritis. Acknowledgements Higher Education Commission of Pakistan is acknowledged for the funds provided to conduct the research Keywords: MicroRNA146a, real time PCR, Rheumatoid arthritis, biomarkers, expression analysis Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Ullah S, John P and Bhatti A (2013). Evaluation of Expression Pattern of Mirna146a in Pakistani Rheumatoid Arthritis Patients. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00037 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Mr. Sami Ullah, National University of Science and Technology, Isl, Pakistan, samibiotech@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sami Ullah Peter John Attya Bhatti Google Sami Ullah Peter John Attya Bhatti Google Scholar Sami Ullah Peter John Attya Bhatti PubMed Sami Ullah Peter John Attya Bhatti Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Increased expression of microRNA-146a decreases myocardial ischaemia/reperfusion injury

We have reported that either toll-like receptor 4 deficiency (TLR4(-/-)) or TLR2 modulation protects against myocardial ischaemia/reperfusion (I/R) injury. The mechanisms involve attenuation of I/R-induced nuclear factor KappaB (NF-κB) activation. MicroRNA-146a (miR-146a) has been reported to target interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), resulting in inhibiting NF-κB activation. This study examined the role of microRNA-146a in myocardial I/R injury. We constructed lentivirus expressing miR-146a (LmiR-146a). LmiR-146a was transfected into mouse hearts through the right common carotid artery. The lentivirus vector (LmiR-Con) served as vector control. Untransfected mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (60 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by triphenyltetrazolium chloride (TTC) staining. In separate experiments, the hearts were subjected to ischaemia (60 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography prior to I/R, 3 and 7 days after myocardial I/R. LmiR-146a transfection significantly decreased I/R-induced myocardial infarct size by 55% and prevented I/R-induced decreases in ejection fraction (EF%) and fractional shortening (%FS). LmiR-146a transfection attenuated I/R-induced myocardial apoptosis and caspase-3/7 and -8 activities. LmiR-146a transfection suppresses IRAK1 and TRAF6 expression in the myocardium. In addition, transfection of LmiR-146a prevented I/R-induced NF-κB activation and inflammatory cytokine production. MicroRNA-146a protects the myocardium from I/R injury. The mechanisms may involve attenuation of NF-κB activation and inflammatory cytokine production by suppressing IRAK1 and TRAF6.

Lipopolysaccharide-induced gene expression of interleukin-1 receptor-associated kinase 4 and interleukin-1β in roughskin sculpin (Trachidermus fasciatus)

Toll-like receptor (TLR) signaling pathway plays a crucial role in innate immune responses. In the present study, we first identified and characterized a key TLR pathway signal transduction molecule interleukin-1 receptor-associated kinase 4 (IRAK-4), and an important signal-out molecule interleukin-1β (IL-1β) from roughskin sculpin (Trachidermus fasciatus). IRAK-4 had an open reading frame (ORF) of 1401 bp, which encoded a protein of 467 amino acids with a highly conserved death domain (DD) and a serine/threonine/tyrosine protein kinase domain (STYKc). The full-length cDNA of IL-1β was 1242 bp with a 756 bp ORF, encoding a protein of 252 amino acids. Neither a signal peptide nor an IL-1β-converting enzyme (ICE) cleavage site was detected in this protein. Both genes were broadly expressed in all the ten examined tissues, with the highest transcript level in the skin, indicating that the host could trigger rapid immune responses in infected tissues through TLR signaling pathway. Quantitative real-time polymerase chain reaction was employed to investigate their temporal expression profiles post lipopolysaccharide challenge. The transcripts of both genes were significantly increased in the skin, blood, liver, spleen, and brain. It was shown that the transcript of IL-1β was dramatically induced to 700 times higher than that of the control group in the blood and liver. These results indicate that TLR signaling process may play an important role in fish immune response against microbial infections.

Endotoxin Tolerance Attenuates Liver Ischemia/Reperfusion Injury by Down-Regulation of Interleukin-1 Receptor-Associated Kinase 4 in Kupffer Cells

Aim The aim of this study was to study the role of interleukin-1 receptor-associated kinase 4 (IRAK-4) in the formation of endotoxin tolerance (ET) in liver ischemia/reperfusion (I/R) injury. Methods Animals were randomly divided into 3 groups: control group, I/R group, and ET group. Liver morphological changes were observed using optical microscopy with hematoxylin eosin (HE) staining. Alanine aminotransferase (ALT) was quantified to measure liver functional injury. The messenger RNA (mRNA) and protein expressions of IRAK-4 in Kupffer cells (KCs) isolated from recipients were detected using real-time polymerase chain reaction (PCR) and Western blot, respectively. The activities of NF-κB and the supernatant levels of tumor necrosis factor-alpha (TNF-α), IL-10 were assayed using enzyme-linked immunosorbent assay (ELISA). Results Endotoxin preconditioning improved hepatic tissue injury as indicated by morphological analysis, whereas serum ALT levels were significantly decreased at various times ( P < .05); concurrently, the expression of IRAK-4 and TNF-α in KCs was down-regulated ( P < .05) and the secretion of IL-10 was enhanced ( P < .05); NF-κB DNA-binding activity of KCs was also significantly inhibited by endotoxin preconditioning ( P < .05). Conclusion Endotoxin preconditioning attenuated the liver I/R injury caused by transplantation. The expression of IRAK-4 in KCs may play an important role in the formation of ET.

Differential expression of miRNA-146a-regulated inflammatory genes in human primary neural, astroglial and microglial cells

Micro RNA-146a (miRNA-146a) is an inducible, 22 nucleotide, small RNA over-expressed in Alzheimer's disease (AD) brain. Up-regulated miRNA-146a targets several inflammation-related and membrane-associated messenger RNAs (mRNAs), including those encoding complement factor-H (CFH) and the interleukin-1 receptor associated kinase-1 (IRAK-1), resulting in significant decreases in their expression (p<0.05, ANOVA). In this study we assayed miRNA-146a, CFH, IRAK-1 and tetraspanin-12 (TSPAN12), abundances in primary human neuronal-glial (HNG) co-cultures, in human astroglial (HAG) and microglial (HMG) cells stressed with Aβ42 peptide and tumor necrosis factor alpha (TNFα). The results indicate a consistent inverse relationship between miRNA-146a and CFH, IRAK-1 and TSPAN12 expression levels, and indicate that HNG, HAG and HMG cell types each respond differently to Aβ42-peptide+TNFα-triggered stress. While the strongest miRNA-146a-IRAK-1 response was found in HAG cells, the largest miRNA-146a-TSPAN12 response was found in HNG cells, and the most significant miRNA-146a-CFH changes were found in HMG cells, the ‘resident scavenging macrophages’ of the brain.

Molecular cloning and expression of interleukin-1 receptor-associated kinase 4, an important mediator of Toll-like receptor signal pathway, from small abalone Haliotis diversicolor

Mammal interleukin-1 receptor-associated kinases (IRAKs) have been demonstrated to play important functions in TLRs (Toll-like receptor) signal pathway and T cell proliferation, but there is less knowledge available on mollusc IRAKs. In this study, a molluscan IRAK-4 gene, saIRAK-4, was cloned for the first time from the small abalone ( Haliotis diversicolor). Its full-length cDNA sequence was 2062 bp, with a 1548 bp open reading frame encoding a protein of 516 aa. The molecular mass of the deduced protein was approximately 57.8 kDa with an estimated pI of 5.23, and showed highest identity (47%) to acorn worm Saccoglossus kowalevskii. Amino acid sequence analysis revealed saIRAK-4 shares conserved signature motifs with other IRAK-4 proteins, including the death domain (DD), serine/threonine/tyrosine protein kinase domain (STYKc), protein kinases ATP-binding region signature, serine/threonine protein kinases active-site signature and prokaryotic membrane lipoprotein lipid attachment site. Quantitative real-time PCR was employed to investigate the tissue distribution of saIRAK-4 mRNA, and its expression in abalone under bacteria challenge and larvae at different developmental stages. The saIRAK-4 mRNA could be detected in all examined tissues, with the highest expression level in gills, and was up-regulated in hemocytes and gills after bacteria injection. Additionally, saIRAK-4 was constitutively expressed at all examined developmental stages. These results indicate that saIRAK-4 could respond to pathogenic infection and may play an important role in the adult abalone immune system and early innate immunity in the process of abalone larval development.

603 Baseline circulating tumor cell (CTC) counts enhance the performance of the Royal Marsden Hospital (RMH) Prognostic Score and improve patient selection for phase 1 clinical trials

MiR-146a exerts negative control on inflammatory responses by suppressing cytokine-induced expression of interleukin-1 receptor-associated kinase-1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) by impairing NF-κB activity and inhibiting the expression of target genes. Recent study suggests that histone deacetylases (HDACs) are involved in the regulation of microRNA (miRNA) expression. Therefore, we determined whether HDAC inhibitors can increase miR-146a expression, thereby inhibiting interleukin-1β (IL-1β)-induced signaling in osteoarthritis fibroblast-like synoviocytes (OA-FLS).MiRNA expression was analyzed using real-time PCR. IL-1β-induced downstream signals and cytokine expression were evaluated using Western blotting and ELISA. Transcription factors regulating promoter activation were identified using chromatin immunoprecipitation assays.IL-1β treatment of OA-FLS induced a mild (1.7-fold) increase in miR-146a expression that was unable to appropriately downregulate IRAK1 and TRAF6 expression. HDAC inhibitors, SAHA (vorinostat), and LBH589 (panobinostat) significantly (6.1- and 5.4-fold) elevated miR-146a expression by increasing the binding of the transcription factor NF-κB to the miR-146a promoter, and negatively regulated IL-1β-induced IKK/IκB/p65 phosphorylation signaling and IL-6 secretion. The increase in miR-146a expression induced by the HDAC inhibitors was prevented by transfection of miR-146a inhibitor or HDAC1 (class I HDAC), HDAC4 (class IIa HDAC), and HDAC6 (class IIb HDAC) overexpression, suggesting that they were due to inhibition of HDAC activity.Our study demonstrated that HDAC inhibitor treatment in OA-FLS significantly increased miR-146a expression and mediated markedly negative regulation to inhibit IL-1β-induced signaling and cytokine secretion. Our results indicate the potential rationale of anti-inflammatory effects for HDAC inhibitors.

The influence of over expression of interleukin-1 receptor-associated kinase 1 on bacterial lipoprotein-induced tolerance

To investigate Toll-like receptor 2 (TLR2) and interleukin-1 receptor-associated kinase 1 (IRAK-1) in bacterial lipoprotein (BLP) tolerance. Western blotting was used to confirm the over expression of TLR2 and IRAK-1 in human embryo kidney 293 (HEK293) cells. Plasmids for dual luciferase reporter gene with nuclear factor-KappaB promoter (pNF-KappaB-Luc) or CMV promoter (phRL-CMV internal control vector) were used to detect the NF-KappaB activation and the induction of BLP tolerance in HEK-TLR2 cells. BLP stimulation resulted in dose-dependent NF-KappaB activation in HEK293 cells stably expressing TLR2. And BLP pretreatment could reduce NF-KappaB activation and induce BLP tolerance in HEK-TLR2 cells. The NF-KappaB activation was 0.329+/-0.010 and 0.168+/-0.010 in BLP-activated and BLP-tolerant HEK-TLR2 cells, respectively. After transfection with 0.02 microg IRAK-1 plasmid, NF-KappaB activation in the two groups was 0.493+/-0.010 and 0.427+/-0.035, respectively (both P<0.01). So over expression of IRAK-1 could increase NF-KappaB activation in a dose-dependent manner. These results demonstrated that over expression of IRAK-1 could reverse BLP tolerance, whereas over expression of TLR2 failed to prevent the induction of BLP tolerance. Therefore reduced IRAK-1 protein expression is an important mechanism in the development of BLP-induced tolerance, suggesting that it could be a potentially important target for future therapeutic strategies in bacterial infection and sepsis.

Thalidomide inhibits lipopolysaccharide-induced tumor necrosis factor-α production via down-regulation of MyD88 expression

The effect of thalidomide on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha production was studied by using RAW 264.7 murine macrophage-like cells. Thalidomide significantly inhibited LPS-induced TNF-alpha production. Thalidomide prevented the activation of nuclear factor (NF)-KB by down-regulating phosphorylation of inhibitory KB factor (IKB), and IKB kinase (IKK)-alpha and IKK-beta Moreover, thalidomide inhibited LPS-induced phosphorylation of AKT, p38 and stress-activated protein kinase (SAPK)/JNK. The expression of myeloid differentiation factor 88 (MyD88) protein and mRNA was markedly reduced in thalidomide-treated RAW 264.7 cells but there was no significant alteration in the expression of interleukin-1 receptor-associated kinase (IRAK) 1 and TNF receptor-associated factor (TRAF) 6 in the cells. Thalidomide did not affect the cell surface expression of Toll-like receptor (TLR) 4 and CD14, suggesting the impairment of intracellular LPS signalling in thalidomide-treated RAW 264.7 cells. Thalidomide significantly inhibited the TNF-alpha production in response to palmitoyl-Cys(RS)-2,3-di(palmitoyloxy) propyl)-Ala-Gly-OH (Pam(3)Cys) as a MyD88-dependent TLR2 ligand. Therefore, it is suggested that thalidomide might impair LPS signalling via down-regulation of MyD88 protein and mRNA and inhibit LPS-induced TNF-alpha production. The putative mechanism of thalidomide-induced MyD88 down-regulation is discussed.

Interleukin-1 regulates keratinocyte expression of T cell targeting chemokines through interleukin-1 receptor associated kinase-1 (IRAK1) dependent and independent pathways

IL-1 is a potent pro-inflammatory cytokine that activates intracellular signaling cascades some of which may involve IL-1 receptor associated kinase-1 (IRAK1). Psoriasis is a T cell dependent chronic inflammatory condition of the skin of unknown cause. IL-1 has been implicated in psoriasis pathology, but the mechanism has not been elucidated. Interestingly, expression of IRAK1 is elevated in psoriatic skin. To identify a potential link between IL-1, keratinocytes and T cells in skin inflammation we employed pathway-focused microarrays to evaluate IL-1 dependent gene expression in keratinocytes. Several candidate mRNAs encoding known T cell chemoattractants were identified in primary keratinocytes and the stable keratinocyte cell line HaCaT. CCL5 and CCL20 mRNA and protein levels were confirmed up-regulated by IL-1 in concentration and time-dependent manners. Furthermore IL-1 synergized with IFN-γ and TNF-α. Expression of CXCL9, CXCL10 and CXCL11 mRNAs was also increased in response to IL-1, but protein could only be detected in medium from cells treated with IFN-γ alone or in combination with IL-1. Over-expression of IRAK1 led to increased constitutive and cytokine induced production of CCL5 and CCL20. Inhibition of IRAK1 activity through RNAi or expression of a dominant negative mutant blocked production of CCL5 and CCL20 but had no effect upon the IL-1 enhancement of IFN-γ induced CXCL9, CXCL10 and CXCL11 production. In conclusion IL-1 regulates T cell targeting chemokine production in keratinocytes through IRAK1 dependent and independent pathways. These pathways may contribute to acute and chronic skin inflammation.