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.     

Bacterial lipoprotein-induced tolerance is reversed by overexpression of IRAK-1

Tolerance to bacterial cell wall components including bacterial lipoprotein (BLP) represents an essential regulatory mechanism during bacterial infection. Reduced Toll-like receptor 2 (TLR2) and IL-1 receptor-associated kinase 1 (IRAK-1) expression is a characteristic of the downregulated TLR signaling pathway observed in BLP-tolerised cells. In this study, we attempted to clarify whether TLR2 and/or IRAK-1 are the key molecules responsible for BLP-induced tolerance. Transfection of HEK293 cells and THP-1 cells with the plasmid encoding TLR2 affected neither BLP tolerisation-induced NF-κB deactivation nor BLP tolerisation-attenuated pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) production, indicating that BLP tolerance develops despite overexpression of TLR2 in these cells. In contrast, overexpression of IRAK-1 reversed BLP-induced tolerance, as transfection of IRAK-1 expressing vector resulted in a dose-dependent NF-κB activation and TNF-α release in BLP-tolerised cells. Furthermore, BLP-tolerised cells exhibited markedly repressed NF-κB p65 phosphorylation and impaired binding of p65 to several pro-inflammatory cytokine gene promoters including TNF-α and interleukin-6 (IL-6). Overexpression of IRAK-1 restored the nuclear transactivation of p65 at both TNF-α and IL-6 promoters. These results indicate a crucial role for IRAK-1 in BLP-induced tolerance, and suggest IRAK-1 as a potential target for manipulation of the TLR-mediated inflammatory response during microbial sepsis.     

An NF-kappaB-sensitive micro RNA-146a-mediated inflammatory circuit in Alzheimer disease and in stressed human brain cells

Human brains retain discrete populations of micro RNA (miRNA) species that support homeostatic brain gene expression functions; however, specific miRNA abundance is significantly altered in neurological disorders such as Alzheimer disease (AD) when compared with age-matched controls. Here we provide evidence in AD brains of a specific up-regulation of an NF-kappaB-sensitive miRNA-146a highly complementary to the 3'-untranslated region of complement factor H (CFH), an important repressor of the inflammatory response of the brain. Up-regulation of miRNA-146a coupled to down-regulation of CFH was observed in AD brain and in interleukin-1beta, Abeta42, and/or oxidatively stressed human neural (HN) cells in primary culture. Transfection of HN cells using an NF-kappaB-containing pre-miRNA-146a promoter-luciferase reporter construct in stressed HN cells showed significant up-regulation of luciferase activity that paralleled decreases in CFH gene expression. Treatment of stressed HN cells with the NF-kappaB inhibitor pyrollidine dithiocarbamate or the resveratrol analog CAY10512 abrogated this response. Incubation of an antisense oligonucleotide to miRNA-146a (anti-miRNA-146a; AM-146a) was found to restore CFH expression levels. These data indicate that NF-kappaB-sensitive miRNA-146a-mediated modulation of CFH gene expression may in part regulate an inflammatory response in AD brain and in stressed HN cell models of AD and illustrate the potential for anti-miRNAs as an effective therapeutic strategy against pathogenic inflammatory signaling.     

Identification and characterization of murine IRAK-2

Interleukin-1 receptor-associated kinases (IRAKs) are pivotal signaling elements of the Toll/IL-1 receptor (TIL) family, which play a role in innate immune responses by coordinating host defence mechanisms. Presently four different forms of human IRAK molecules are cloned (hu-IRAK-1, hu-IRAK-2, hu-IRAK-M, and hu-IRAK-4). In the murine system, only three genes have been identified so far, mouse Pelle-Like Kinase (mPLK), which corresponds to human IRAK-1, mu-IRAK-M, and mu-IRAK-4. Here we report the molecular cloning and characterization of murine IRAK-2 (mu-IRAK-2), a mouse homolog to human IRAK-2 (hu-IRAK-2). Murine and human IRAK-2 molecules show 67% sequence identity, they are ubiquitiously expressed, and both practically lack autophoshorylation kinase activity. The murine molecule reveals two remarkable differences to its human counterpart: it shows a C-terminal extension and it has no stimulatory effect on IL-1 induced NF-kappa B activation when compared to hu-IRAK-2, suggesting subtle functional differences in signaling by IRAK-2 in human and mouse cells.     

Potential role of Hedgehog signaling and microRNA-29 in liver fibrosis of IKKβ-deficient mouse

Recent studies have reported that NF-κB mediated down-regulation of miRNA-29 and lower expression of miRNA-29 promoted the deposition of collagens in fibrotic liver. Our previous research demonstrated that the increased Hedgehog (Hh) signaling, a key regulator for hepatic fibrogenesis, induced the severe hepatic fibrosis in the livers with impaired NF-κB signaling. These findings led us to investigate the effect of Hh and miRNA-29 on the hepatic fibrosis under dysregulated NF-κB signaling. In this study, we used IKKβ^F/F and IKKβ-deficient IKKβ^ΔHEP mouse model with a defective NF-κB signaling pathway, and assessed the expression of the miRNA-29 family (miRNA-29a, miRNA-29b, and miRNA-29c), Hh, and proliferation of MF-HSCs in liver from IKKβ^F/F mice and IKKβ^ΔHEP mice both before and after MCDE treatment. The activation of NF-κB was significantly increased in MCDE diet-fed IKKβ^F/F mice compared to IKKβ^ΔHEP mice. Expression of miRNA-29 family was greater in MCDE diet-fed IKKβ^ΔHEP mice than IKKβ^F/F mice, demonstrating that the impaired NF-κB pathway was unable to suppress the expression of miRNA-29s after injury. However, expression of the Hh signaling pathway was greatly enhanced, and activation of Hh promoted the accumulation of MF-HSCs with impaired NF-κB, eventually increasing fibrogenesis in the damaged liver of IKKβ^ΔHEP mice. Therefore, these results demonstrated that Hh signaling regulates the proliferation of MF-HSCs irrespective of the action of miRNA-29, eventually contributing hepatic fibrosis, when the NF-κB pathway is disrupted.     

microRNA (miRNA) speciation in Alzheimer’s disease (AD) cerebrospinal fluid (CSF) and extracellular fluid (ECF)

Human cerebrospinal fluid (CSF), produced by the choroid plexus and secreted into the brain ventricles and subarachnoid space, plays critical roles in intra-cerebral transport and the biophysical and immune protection of the brain. CSF composition provides valuable insight into soluble pathogenic bio-markers that may be diagnostic for brain disease. In these experiments we analyzed amyloid beta (Aβ) peptide and micro RNA (miRNA) abundance in CSF and in short post-mortem interval (PMI <2.1 hr) brain tissue-derived extracellular fluid (ECF) from Alzheimer’s disease (AD) and age-matched control neocortex. There was a trend for decreased abundance of Aβ42 in the CSF and ECF in AD but it did not reach statistical significance (mean age ~72 yr; N=12; p~0.06, ANOVA). The most abundant nucleic acids in AD CSF and ECF were miRNAs, and their speciation and inducibility were studied further. Fluorescent miRNA-array-based analysis indicated significant increases in miRNA-9, miRNA-125b, miRNA-146a, miRNA-155 in AD CSF and ECF (N=12; p<0.01, ANOVA). Primary human neuronal-glial (HNG) cell co-cultures stressed with AD-derived ECF also displayed an up-regulation of these miRNAs, an effect that was quenched using the anti-NF-кB agents caffeic acid phenethyl ester (CAPE) or 1-fluoro-2-[2-(4-methoxy-phenyl)-ethenyl]-benzene (CAY10512). Increases in miRNAs were confirmed independently using a highly sensitive LED-Northern dot-blot assay. Several of these NF-кB-sensitive miRNAs are known to be up-regulated in AD brain, and associate with the progressive spreading of inflammatory neurodegeneration. The results indicate that miRNA-9, miRNA-125b, miRNA-146a and miRNA-155 are CSF- and ECF-abundant, NF-кB-sensitive pro-inflammatory miRNAs, and their enrichment in circulating CSF and ECF suggest that they may be involved in the modulation or proliferation of miRNA-triggered pathogenic signaling throughout the brain and central nervous system (CNS).     

尿酸性肾病中IL-1beta、IRAK-4 的表达及意义

目的：通过研究尿酸性肾病动物模型中白介素-1(IL-1)beta和白介素-1受体相关激酶4(IRAK-4) 表达的意义,了解IL-1beta信号 通路在尿酸性肾病中的作用。方法：Wistar 大鼠54 只随机分为高尿酸血症组30 只、正常组24 只,制备尿酸性肾病大鼠模型,检测 尿酸(UA)、尿素氮(BUN)、肌酐(CR)及肌酐清除率(Ccr)、24 h尿微量白蛋白(mA1b);取肾脏组织行HE 染色,观察形态学变化;免疫 组化测定IL-1beta的表达;荧光定量PCR 检测IRAK-4 mRNA的水平。结果：高尿酸组2、4、6 周时IL-1beta的表达均增加,免疫组化评 分(IHS)均明显升高(P<0.01);高尿酸血症组较正常组IRAK-4 mRNA 在2、4、6 周时均出现表达上调,4～6 周IRAK-4 mRNA表达 明显增加,与正常组比较有显著性差异(P<0.01)。结论：IL-1beta、IRAK-4 参与了尿酸性肾病炎症反应的过程,可能为尿酸性肾病治疗 提供新的可能。     

Common micro RNAs (miRNAs) target complement factor H (CFH) regulation in Alzheimer’s disease (AD) and in age-related macular degeneration (AMD)

Alzheimer’s disease (AD) and age-related macular degeneration (AMD) are complex and progressive inflammatory degenerations of the human neocortex and retina. Recent molecular, genetic and epigenetic evidence indicate that at least 4 micro RNAs (miRNAs) - including the NF-кB-regulated miRNA-9, miRNA-125b, miRNA-146a and miRNA-155 - are progressively up-regulated in both AD and AMD. This quartet of up-regulated miRNAs in turn down-regulate a small brain- and retinal-cell-relevant family of target mRNAs, including that encoding complement factor H (CFH), a major negative regulator of the innate immune and inflammatory response. Together miRNA-146a and miRNA-155 recognize an overlapping miRNA regulatory control (MiRC) region in the CFH 3’-untranslated region (3’- UTR; 5’-TTTAGTATTAA-3’) to which either of these miRNAs may interact. Progressive, pathogenic increases in specific miRNA binding to the entire 232 nucleotide CFH 3’-UTR appears to be a major regulator of CFH expression down-regulation, and the inflammatory pathology that characterizes both AMD and AD. The data presented in this report provides evidence that up-regulation of brain- and retinal- abundant miRNAs, including miRNA-9, miRNA-125b, miRNA-146a and miRNA-155, are common to the pathogenetic mechanism of CFH deficiency that drives inflammatory neurodegeneration, and for the first time indicates multiple, independent miRNA-mediated regulation of the CFH mRNA 3’-UTR.     

Divergent intracellular pathways regulate interleukin-1β-induced miR-146a and miR-146b expression and chemokine release in human alveolar epithelial cells

We have previously reported that IL-β-induced miR-146a and miR-146b expression negatively regulates IL-8 and RANTES release in human alveolar A549 epithelial cells. To determine the intracellular pathways that regulate this response, we demonstrate IL-1β-induced activation of the nuclear factor (NF)-κB, extracellular regulated kinase (ERK)-1/2, c-jun N-terminal kinase (JNK)-1/2 and p38 mitogen activated kinase (MAP) kinase pathways. Subsequent pharmacological studies show that IL-1β-induced miR-146a, IL-8 and RANTES production was regulated via NF-κB and JNK-1/2 whilst miR-146b expression was mediated via MEK-1/2 and JNK-1/2. These divergent intracellular pathways likely explain the differential expression and biological action of the miR-146 isoforms.     

Expression differences of miRNAs and genes on NF-κB pathway between the healthy and the mastitis Chinese Holstein cows

In order to discover the variation of microRNAs and genes associated with NF-κB signaling pathway between the healthy and the mastitis Chinese Holstein cows, Illumina Deep Sequencing and qRT-PCR are applied to detect 25 kinds of miRNAs (miR-16, miR-125b, miR-15, miR-29a, miR-23b, miR-146, miR-301a, miR-181b, let-7, miR-30b, miR-21, miR-223, miR-27b, miR-10a, miR-143, etc.) expression levels in blood samples and 14 genes (RelA, RelB, Rel, p105, p100, IκBα, IκBβ, IκBδ, IκBε, IκBζ, Bcl-3, IKKα, IKKβ, IKKγ/NEMO) relative expression levels in nine tissues. The total number of miRNAs is declining, and RelA, Rel, p105, p100, IκBα, IκBβ, IκBδ, IκBζ, Bcl-3, and IKKα expressions are rising in mastitis individuals. So, we suppose that NF-κB pathway is active in mastitis individuals as a result of the decrease inhibition of miRNAs. While in healthy ones, the NF-κB pathway is inactive, because of the miRNAs enhanced inhibition action. However, the specific regulatory mechanism of miRNAs on NF-κB pathway in mastitis Holstein cows needs further investigation. Moreover, due to obvious expression differences, some miRNAs, especially miR-16 and miR-223, may be used as new markers for the dairy mastitis prognosing.     

IRAK-1-mediated negative regulation of Toll-like receptor signaling through proteasome-dependent downregulation of TRAF6

TRAF6 plays a crucial role in signal transduction of the Toll-like receptor (TLR). It has been reported that TRAF6 catalyzes the formation of unique Lys63-linked polyubiquitin chains, which do not lead to proteasome-mediated degradation. Here we found that stimulation of J774.1 cells with various TLR ligands led to decreases in TRAF6 protein levels that occurred at a slower rate than IκBα degradation. The decrease in TRAF6 was inhibited by proteasome inhibitors MG-132, lactacystin and N-acetyl-leucyl-leucyl-norleucinal. Among intracellular TLR signaling molecules MyD88, IRAK-4, IRAK-1, TRAF6, and IKKβ, only IRAK-1 expression downregulated TRAF6 in HEK293 cells. The amount of TRAF6 expressed either transiently or stably was also reduced by co-expression of IRAK-1 and no TRAF6 cleavage products were detected. The levels of either a TRAF6 N-terminal deletion mutant or a ubiquitin ligase-defective mutant were not affected by IRAK-1 expression. Downregulation of TRAF6 required the TRAF6-binding site (Glu544, Glu587, Glu706) of IRAK-1 but not its catalytic site (Asp340). Upon IRAK-1 transfection, no significant TRAF6 ubiquitination was detected. Instead, TRAF6-associated IRAK-1 was ubiquitinated with both Lys48- and Lys63-linked polyubiquitin chains. TRAF6 downregulation was inhibited by co-expression of the E3 ubiquitin ligase Pellino 3, whose Lys63-linked polyubiquitination on IRAK-1 is reported to compete with Lys48-linked IRAK-1 polyubiquitination. Expression of IRAK-1 inhibited IκBα phosphorylation in response to TLR2 stimulation. These results indicate that stimulation of TLRs induces proteasome-dependent downregulation of TRAF6. We conclude that TRAF6 associated with ubiquitinated IRAK-1 is degraded together by the proteasome and that IRAK-1 possesses a negative regulatory role on TLR signaling.     

IRAK-4 in macrophages contributes to inflammatory osteolysis of wear particles around loosened hip implants

TLRs recognizing PAMPS play a role in local immunity and participate in implant-associated loosening. TLR-mediated signaling is primarily regulated by IL-1 receptor associated kinase-M (IRAK-M) negatively and IRAK-4 positively. Our previous studies have proved that wear particles promote endotoxin tolerance in macrophages by inducing IRAK-M. However, whether IRAK-4 is involved in inflammatory osteolysis of wear particles basically, and the specific mechanism of IRAK-4 around loosened hip implants, is still unclear. IRAK-4 was studied in the interface membranes from patients in vivo and in particle-stimulated macrophages to clarify its role. Also, IL-1β and TNF-α levels were measured after particle and LPS stimulation in macrophages with or without IRAK-4 silenced by siRNA. Our results showed that the interface membranes around aseptic and septic loosened prosthesis expressed more IRAK-4 compared with membranes from osteoarthritic patients. IRAK-4 in macrophages increased upon particle and LPS stimulation. In the former, IL-1β and TNF-α levels were lower compared with those of LPS stimulation, and IRAK-4 siRNA could suppress production of pro-inflammatory cytokines. These findings suggest that besides IRAK-M, IRAK-4 also plays an important role in the local inflammatory reaction and contributes to prosthesis loosening.     

The effect of oxidized low density lipoprotein (oxLDL)-induced heme oxygenase-1 on LPS-induced inflammation in RAW 264.7 macrophage cells

Macrophages take up oxidized low density lipoprotein (oxLDL) after being exposed to it in the blood vessels. oxLDL transforms macrophages into foam cells, which are a hallmark of atherosclerosis. The effects that oxLDL have on the inflammatory responses of foam cells are not clear. Here, we investigated how oxLDL modulates lipopolysaccharide (LPS)-induced inflammatory mediators in RAW 264.7 murine macrophages. Our results showed that oxLDL dramatically induced HO-1 expression, but did not increase pro-inflammatory mediators such as interleukin-1β, tumor necrosis factor-α, iNOS, and monocyte chemoattractant protein (MCP)-1. In RAW 264.7 macrophages, oxLDL markedly inhibited LPS-induced inflammatory mediators such as inducible nitric oxide synthase (iNOS), IL-1β, IL-6, granulocyte macrophage colony-stimulating factor and stromal cell-derived factor-1. Interestingly, however, the down-regulation of HO-1 by siRNA did not recover the inhibition of LPS-induced expression and/or the secretion of inflammatory mediators. oxLDL blocked LPS-induced NF-κB nuclear translocation by inhibiting inhibitory κB (IκB) degradation. Taken together, our results suggest that oxLDL could modulate LPS-induced inflammatory responses by inhibiting NF-κB signaling independently of HO-1 expression.