Systematic considerations for a multicomponent pharmacokinetic study of Epimedii wushanensis herba: From method establishment to pharmacokinetic marker selection

BackgroundPrenylflavonoids are major active components of Epimedii wushanensis herba (EWH). The global pharmacokinetics of prenylflavonoids are unclear, as these compounds yield multiple, often unidentified metabolites.PurposeThis study successfully elucidated the pharmacokinetic profiles of EWH extract and five EWH-derived prenylflavonoid monomers in rats.Study designThe study was a comprehensive analysis of metabolic pathways and pharmacokinetic markers.MethodsMajor plasma compounds identified after oral administration of EWH-derived prototypes or extract included: (1) prenylflavonoid prototypes, (2) deglycosylated products, and (3) glucuronide conjugates. To select appropriate EWH-derived pharmacokinetic markers, a high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was established to simultaneously monitor 14 major compounds in unhydrolyzed plasma and 10 potential pharmacokinetic markers in hydrolyzed plasma.ResultsThe pharmacokinetic profiles indicated that the glucuronide conjugates of icaritin were the principle circulating metabolites and that total icaritin accounted for ∼99% of prenylflavonoid exposure after administration of EWH-derived materials to rats. To further investigate icaritin as a prospective pharmacokinetic marker, correlation analysis was performed between total icaritin and its glucuronide conjugates, and a strong correlation (r > 0.5) was found, indicating that total icaritin content accurately reflected changes in the exposure levels of the glucuronide conjugates over time. Therefore, icaritin is a sufficient pharmacokinetic marker for evaluating dynamic prenylflavonoid exposure levels. Next, a mathematical model was developed based on the prenylflavonoid content of EWH and the exposure levels in rats, using icaritin as the pharmacokinetic marker. This model accurately predicted exposure levels in vivo, with similar predicted vs. experimental area under the curve (AUC)_0–96 h values for total icaritin (24.1 vs. 32.0 mg/L h).ConclusionIcaritin in hydrolyzed plasma can be used as a pharmacokinetic marker to reflect prenylflavonoid exposure levels, as well as the changes over time of its glucuronide conjugates.     

Simple and sensitive liquid chromatography–tandem mass spectrometry assay for simultaneous measurement of five Epimedium prenylflavonoids in rat sera

A rapid and sensitive method to separate and quantify icariin, icariside I, icariside II, icaritin and desmethylicaritin in rat sera was developed using liquid chromatography–tandem mass spectrometry. Serum samples were extracted with ethyl acetate without further derivatization. Using coumestrol as an internal standard, calibration curves with good linearity (r² > 0.99) within the concentration range of 0.78–12.5 nM for icariin, icaritin and desmethylicaritin, and 0.78–100 nM for icariside I and II, were obtained in the multiple reaction monitoring mode. For all analytes, the limits of detection and quantification were <1 nM and 1–2 nM, respectively. Inter- and intra-assay variabilities were <15% and accuracies were between 94% and 114%, respectively. This method was successfully applied to quantify levels of icariin, icariside I, icariside II, icaritin and desmethylicaritin in rat sera after oral administration of an Epimedium preparation.     

Sensitive and rapid method to quantify icaritin and desmethylicaritin in human serum using gas chromatography-mass spectrometry

The prenyl-flavones, icaritin and desmethylicaritin, are bioactive compounds from the traditional Chinese medicinal herb, Epimedium, extracts of which can enhance bone health in animal models. In order to examine their bioavailability in humans, we have developed and validated a sensitive method to quantify icaritin and desmethylicaritin in human sera, using gas chromatography-mass spectrometry. The serum samples were extracted with ethyl acetate and then derivatized with BSTFA in pyridine (4:1). With genistein as internal standard, calibration curves with good linearity (R(2)>0.99) within the concentration range of 0.15-10nM in the selective ion monitoring mode were obtained. The limits of detection and quantization were 11 and 33 pM for icaritin, and 23 and 70 pM for desmethylicaritin, respectively; inter- and intra-assay variabilities were <15%, and accuracies were between 89 and 110%. Icaritin, but not desmethylicaritin, was detected from 1h, increasing to a peak at 8h (1.51+/-1.6 nM) in sera of human volunteers after ingestion of an aqueous decoction of Epimedium. This sensitive method can be used to quantify serum levels of icaritin and desmethylicaritin for pharmacokinetic studies.     

Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodeling

Increasingly natural products particularly flavonoids are being explored for their therapeutic potentials in reducing bone loss and maintaining bone health. This study has reviewed previous studies on the two better known flavonoids, genistein and icariin, their structures, functions, action mechanisms, relative potency, and potential application in regulating bone remodeling and preventing bone loss. Genistein, an isoflavone abundant in soy, has dual functions on bone cells, able to inhibit bone resorption activity of osteoclasts and stimulate osteogenic differentiation and maturation of bone marrow stromal progenitor cells (BMSCs) and osteoblasts. Genistein is an estrogen receptor (ER)‐selective binding phytoestrogen, with a greater affinity to ERβ. Genistein inhibits tyrosine kinases and inhibits DNA topoisomerases I and II, and may act as an antioxidant. Genistein enhances osteoblastic differentiation and maturation by activation of ER, p38MAPK‐Runx2, and NO/cGMP pathways, and it inhibits osteoclast formation and bone resorption through inducing osteoclastogenic inhibitor osteoprotegerin (OPG) and blocking NF‐κB signaling. Icariin, a prenylated flavonol glycoside isolated from Epimedium herb, stimulates osteogenic differentiation of BMSCs and inhibits bone resorption activity of osteoclasts. Icariin, whose metabolites include icariside I, icariside II, icaritin, and desmethylicaritin, has no estrogenic activity. However, icariin is more potent than genistein in promoting osteogenic differentiation and maturation of osteoblasts. The existence of a prenyl group on C‐8 of icariin molecular structure has been suggested to be the reason why icariin is more potent than genistein in osteogenic activity. Thus, the prenylflavonoids may represent a class of flavonoids with a higher osteogenic activity. J. Cell. Physiol. 228: 513–521, 2013. © 2012 Wiley Periodicals, Inc.     

Liquid chromatography–tandem mass spectrometry analysis of metabolites in rats after administration of prenylflavonoids from Epimediums

The metabolites in rats after administration of icariside II, icariin, epimedin C and extracts of four Epimedium species were investigated. Feces, bile, plasma and urine samples were detected comprehensively using HPLC-ESI-MSⁿ method. The structures of metabolites were identified on the basis of their characteristic fragmentations in MSⁿ experiments. Totally, 54 metabolites were identified in these biosamples. Specific hydrolysis of 7-O glucosides in gut lumen and glucuronic acid conjugation in liver were considered as the main physiologic processes of prenylflavonoids. Icariside II and anhydroicaritin were the major intermediate products in forming of mono- and di-glucuronic acid conjugations in vivo. In general, this study revealed the possible metabolite profiles of prenylflavonoids in rats, and might aid the clinical use of different Epimedium species.     

Reactive oxygen species involved in prenylflavonoids, icariin and icaritin, initiating cardiac differentiation of mouse embryonic stem cells

The significant promoting effects of some prenylflavonoids on cardiac differentiation of mouse embryonic stem (ES) cells via reactive oxygen species (ROS) signaling pathway were investigated. The most effective differentiation was facilitated by icariin (ICA), followed by icaritin (ICT), while desmethylicaritin (DICT) displayed the weakest but still significant inducible effect. Contrarily, DICT demonstrated the strongest anti-oxidative activity while ICA displayed only little in vitro, which was well matched with the hydroxyl (OH) numbers and the positions in the molecular structures. Therefore, ROS signaling cascades were assumed to be involved in prenylflavonoids induced cardiomyogenesis. Treatment with ICA, intracellular ROS in embryoid bodies was rapidly elevated, which was abolished by the NADPH-oxidase inhibitor apocynin; elimination of intracellular ROS by vitamin E or pyrrolidine dithiocarbamate (PDTC) inhibited ICA induced cardiomyogenesis; ROS-sensitive extracellular-regulated kinase 1, 2 (ERK1, 2) and p38 activation were further observed, the cardiomyogenesis was significantly inhibited in the presence of ERK1, 2 or p38 inhibitor U0126 or SB203580, indicating the roles of NADPH-ROS-MAPKs signaling cascades in prenylflavonoids induced cardiac differentiation. There was no difference in Nox4 NADPH oxidase expression between ICA and ICT treatments, however, ROS concentration in EBs after ICT administration was lower than that after ICA treatment, followed by less activation of ERK1, 2, and p38. These results revealed that the significant promoting effects of prenylflavonoids on cardiac differentiation was at least partly via ROS signaling cascades, and the facilitating abilities preferentially based on the nature of prenylflavonoids themselves, but anti-oxidative activity determined by the OH numbers and the positions in the structures do influence the cardiomyogenesis in vitro.     

Biochemical characterization of a novel hyperthermophilic α-l-rhamnosidase from Thermotoga petrophila and its application in production of icaritin from epimedin C with a thermostable β-glucosidase

Epimedin C, a major flavonoid extracted from Herba Epimedii, is a precursor of minor flavonoid icaritin that is a desired drug candidate with remarkable anti-cancer activities. For enhancing the biotransformation efficiency of icaritin, a novel α-l-rhamnosidase gene was cloned from hyperthermophiles Thermotoga petrophila DSM 13995. TpeRha displayed optimal activity at a pH of 4.5 and a temperature of 90 °C. The K_m and K_cat of TpeRha for p-nitrophenyl-α-l-rhamnopyranoside were 2.99 mM and 651.73 s⁻¹, respectively. It displayed broad catalytic ability in cleavage of the outer and inner rhamnopyranosyl moieties on the C-3 carbon of epimedin C. Further, this enzyme was utilized to improve the efficiency of the co-conversion system in transforming epimedin C into icaritin, in combined with a thermostable β-glucosidase Tpebgl1. In addition, a transformation pathway (epimedin C -icariin - icariside I - icaritin) with a high efficiency for icaritin production was screened. After a two-stage transformation under optimized conditions (90 °C, pH 4.5, 80 U/mL of TpeRha and 1.2 U/mL of Tpebgl1), 1 g/L of epimedin C was transformed into 0.4337 g/L of icaritin within 150 min, with a corresponding molar conversion rate of 96.9 %. This is the first report of enzymatic transformation on preparing icaritin from epimedin C by using thermostable glycosidase.     

Icaritin and its glycosides enhance osteoblastic, but suppress osteoclastic, differentiation and activity in vitro

Icariin, a principal flavonoid glycoside in Herba Epimedii, is hypothesized to possess beneficial effects on bone mass. Icariin is metabolized to icariside II and then to icaritin in vivo. In the present study, we investigated the in vitro effects of icariin, icariside II and icaritin on both osteoblasts and osteoclasts. After treatment with these compounds at concentrations 10(-5)-10(-8) mol/l, osteoblasts were examined for proliferation, alkaline phosphatase activity, osteocalcin secretion and matrix mineralization, as well as expression levels of bone-related proteins. The formation of osteoclasts was assessed by counting the number of multinucleated TRAP-positive cells. The activity of isolated rat osteoclasts was evaluated by measuring pit area, actin rings and superoxide generation. Icariside II and icaritin increased the mRNA expression of ALP, OC, COL-1 and OPG, but suppressed that of RANKL. In addition, these compounds reduced the number of multinucleated TRAP-positive cells and the osteoclastic resorption area. Also decreases were observed in superoxide generation and actin ring formation that are required for osteoclast survival and bone resorption activity. These findings suggest that icaritin, which was more potent than icariin and icariside II, enhanced the differentiation and proliferation of osteoblasts, and facilitated matrix calcification; meanwhile it inhibited osteoclastic differentiation in both osteoblast-preosteoclast coculture and osteoclast progenitor cell culture, and reduced the motility and bone resorption activity of isolated osteoclasts.     

Avicularin suppresses cartilage extracellular matrix degradation and inflammation via TRAF6/MAPK activation

BackgroundOsteoarthritis (OA) is an intractable degenerative disease of the whole joint, which is characterized by synovitis inflammation, cartilage damage, and chronic pain. Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) performs an important role in OA.PurposeWe aim to investigate avicularin to protect cartilage extracellular matrix degradation (ECM) and suppresses inflammation both in rat and human chondrocytes.Methods5-Ethynyl-2′-deoxyuridine (EdU) staining, Quantitative real-time PCR, TRAF6 plasmid transfection, Western blot, Measurement of nitric oxide (NO), ROS detection and Immunofluorescence were utilized in vitro. micro-CT scanning, Safranin O-Fast Green, toluidine blue and immunohistochemistry staining were performed in vivo.ResultsIn vitro, avicularin attenuates the degradation of ECM and inflammation, which could inhibit the activation of TRAF6/MAPK pathway via targeting TRAF6. Increased MMP3 and MMP13 expressions and decreased Aggrecan and Collagen Ⅱ levels were observed in anterior cruciate ligament transection (ACLT) induced osteoarthritic rats. Interestingly, intra-articular injection of avicularin attenuates this phenomenon.ConclusionsTaken together, our results indicate that avicularin suppresses cartilage extracellular matrix degradation and inflammation via TRAF6/MAPK activation by targeting TRAF6. These observations identify TRAF6 as a relevant drug target, and avicularin may as a potential therapeutic agent in osteoarthritis.     

Increased Production of Endogenous Pyrogen and Lysozyme by Blood Monocytes in Sarcoidosis

Blood monocytes from patients with sarcoidosis were incubated in vitro, and secretion of endogenous pyrogen (EP), the protein which mediates fever, and lysozyme (L) were measured. After incubation with endotoxin, monocytes from 5 patients with sarcoidosis released twice as much EP as did monocytes from normal individuals (p < .001). Initial 24-hr secretion of L by monocytes from 6 of 11 additional patients with sarcoidosis exceeded the normal range of values for cells from 11 age- and sex-matched control individuals. Cells with initially augmented secretion rates continued to secrete increased amounts of L for 3 days. A correlation was noted between in vitro secretion of L by monocytes and serum levels of L in the same patient. These studies indicate that circulating mononuclear cells in some patients with sarcoidosis have an increased capacity to secrete EP and/or L prior to tissue localization.     

Effect of Vitamin D Therapy on Bone Turnover Markers in Postmenopausal Women with Osteoporosis and Osteopenia

ObjectiveTo (7) assess the rate of reduction in bone turnover with vitamin D and bisphosphonate therapies and (2) evaluate the clinical utility of bone-specific alkaline phosphatase (BSAP) in monitoring treatment response.MethodsWe retrospectively reviewed medical records of patients with newly diagnosed osteopenia and osteoporosis from 2002 to 2009 at Loyola University Medical Center. A cohort of postmenopausal women with hip or spine T-scores of less than -1, normal serum creatinine, and no prior vitamin D or bisphosphonate therapy was divided into vitamin D-deficient (n = 29) and vitamin D-sufficient (n = 13) groups. Vitamin D-deficient patients received high-dose vitamin D, whereas vitamin D-sufficient patients received orally administered bisphosphonates. BSAP levels at baseline and 1 year were compared.Resultsvitamin D therapy in the group with vitamin D deficiency led to a 26.7% decrease in BSAP (P < .01). Bisphosphonate therapy in the vitamin D-sufficient group led to a 32.7% decrease in BSAP (P = .01). The magnitude of BSAP change in the 2 study groups (6.74 ± 6.48 μg ∕ L and 8.72 ± 9.94 μgZL) did not differ significantly (P = .45).ConclusionThe results of this study suggest that correction of vitamin D deficiency in patients with osteopenia and osteoporosis can lead to a decrease in bone turnover as measured by BSAP and that the magnitude of this reduction is similar to that achieved with orally administered bisphosphonates. (Endocr Pract. 2011;17:873-879)     

Statistically designed enzymatic hydrolysis for optimized production of icariside II as a novel melanogenesis inhibitor

Three kinds of prenylated flavonols, icariside I, icariside II, and icaritin, were isolated from an icariin hydrolysate and their effects on melanogenesis evaluated based on mushroom tyrosinase inhibition and quantifying the melanin contents in melanocytes. Although none of the compounds had an effect on tyrosinase activity, icariside II and icaritin both effectively inhibited the melanin contents with an IC50 of 10.53 and 11.13 MM, respectively. Whereas icariside II was obtained from a reaction with beta-glucosidase and cellulase, the icariin was not completely converted into icariside II. Thus, for the high-purity production of icariside II, the reaction was optimized using the response surface methodology, where an enzyme concentration of 5.0 mg/ml, pH 7, 37.5 degrees C;, and 8 h reaction time were selected as the central conditions for the central composite design (CCD) for the enzymatic hydrolysis of icariin into icariside II using cellulase. Empirical models were developed to describe the relationships between the operating factors and the response (icariside II yield). A statistical analysis indicated that all four factors had a significant effect (p<0.01) on the icariside II production. The coefficient of determination (R2) was good for the model (0.9853), and the optimum production conditions for icariside II was an enzyme concentration of 7.5 mg/ml, pH 5, 50 degrees C, and 12 h reaction time. A good agreement between the predicted and experimental data under the designed optimal conditions confirmed the usefulness of the model. A laboratory pilot scale was also successful.     

miR-146a Enhances the Oncogenicity of Oral Carcinoma by Concomitant Targeting of the IRAK1, TRAF6 and NUMB Genes

MicroRNAs are short non-coding RNAs that regulate gene expression and are crucial to tumorigenesis. Oral squamous cell carcinoma (OSCC) is a prevalent malignancy worldwide. Up-regulation of miR-146 has been identified in OSCC tissues. However, the roles of miR-146 in carcinogenesis are controversial as it is suppressive in many other malignancies. The present study investigated the pathogenic implications of miR-146a in oral carcinogenesis. Microdissected OSCC exhibits higher levels of miR-146a expression than matched adjacent mucosal cells. The plasma miR-146a levels of patients are significantly higher than those of control subjects; these levels decrease drastically after tumor resection. miR-146a levels in tumors and in patients’ plasma can be used to classify OSCC and non-disease status (sensitivity: >0.72). Exogenous miR-146a expression is significantly increased in vitro oncogenic phenotypes as well as during xenograft tumorigenesis and OSCC metastasis. The plasma miR-146a levels of these mice parallel the xenograft tumor burdens of the mice. A miR-146a blocker abrogates the growth of xenograft tumors. miR-146a oncogenic activity is associated with down-regulation of IRAK1, TRAF6 and NUMB expression. Furthermore, miR-146a directly targets the 3′UTR of NUMB and a region within the NUMB coding sequence when suppressing NUMB expression. Exogenous NUMB expression attenuates OSCC oncogenicity. Double knockdown of IRAK1 and TRAF6, and of TRAF6 and NUMB, enhance the oncogenic phenotypes of OSCC cells. Oncogenic enhancement modulated by miR-146a expression is attenuated by exogenous IRAK1 or NUMB expression. This study shows that miR-146a expression contributes to oral carcinogenesis by targeting the IRAK1, TRAF6 and NUMB genes.     

Artesunate inhibits Sjögren's syndrome-like autoimmune responses and BAFF-induced B cell hyperactivation via TRAF6-mediated NF-κB signaling

BackgroundHyperactivation of B cells by activators has been demonstrated to play a central role in the pathogenesis of Sjögren's syndrome (SS). In this study, we found that artesunate (ART) can attenuate BAFF-induced B cell hyperactivation and SS-like symptoms in NOD/Ltj mice.PurposeTo determine the efficacy of ART in attenuating SS-like symptoms in vivo and explore the underlying mechanism in vitro.Study designART was intragastrically injected into SS-like NOD/Ltj mice. The cytokine hsBAFF was used to activate Raji and Daudi B cells to mimic B cell hyperactivation in vitro.MethodsThe efficacy of ART in inhibiting SS progression was studied in NOD/Ltj mice. Salivary flow rate, the number of lymphocytic infiltration foci, the level of autoantibodies and the extent of B cell infiltration were measured in the indicated groups. CCK-8 assays, flow cytometry-based EdU staining and Annexin V/PI staining were also used to detect the effect of ART on the survival and proliferation mechanism in BAFF-induced Raji and Daudi cells. Further studies determined that TRAF6 degradation is a potential mechanism by which ART determines B cell fate.ResultsTreatment with ART inhibited lymphocytic foci formation, B cell infiltration and autoantibody secretion in SS-like NOD/Ltj mice. In vitro assay results indicated that ART effectively inhibited BAFF-induced viability, survival and proliferation of neoplastic B cells. Mechanistically, ART targeted BAFF-activated NFκB by regulating the proteasome-mediated degradation of TRAF6 in Raji and Daudi cells.ConclusionART ameliorated murine SS-like symptoms and regulated TRAF6-NFκB signaling, thus determining survival and proliferation of B cells.     

NUMBL interacts with TRAF6 and promotes the degradation of TRAF6

Tumor necrosis factor-associated factor 6 (TRAF6) is an essential adaptor protein for IL-1R or TLR-mediated NF-κB signaling pathway activation. In previous work we have found NUMBL interacts with TAB2 and negatively regulates NF-κB signaling pathway. Here, we report that NUMBL directly binds to TRAF6 in vivo and in vitro. NUMBL down-regulates TRAF6 protein level and shortens its half-life. Furthermore, knockdown of NUMBL significantly increases endogenous TRAF6 protein level in the cultured cortical neurons. In vivo ubiquitination assays indicate that NUMBL promotes the assembly of K48-linked polyubiquitination chains on TRAF6, but has no significant effect on its K63-linked polyubiquitination. Our results collectively reveal that NUMBL interacts with TRAF6 and promotes the degradation of TRAF6 in vivo, leading to the inhibition of NF-κB signaling pathway.     

Xanthohumol and related prenylflavonoids from hops and beer: to your good health!

Xanthohumol (3'-[3,3-dimethyl allyl]-2',4',4-trihydroxy-6'-methoxychalcone) is the principal prenylated flavonoid of the female inflorescences of the hop plant ('hops'), an ingredient of beer. Human exposure to xanthohumol and related prenylflavonoids, such as 8-prenylnaringenin and isoxanthohumol, is primarily through beer consumption. Xanthohumol has been characterized a 'broad-spectrum' cancer chemopreventive agent in in vitro studies, while 8-prenylnaringenin enjoys fame as the most potent phytoestrogen known to date. These biological activities suggest that prenylflavonoids from hops have potential for application in cancer prevention programs and in prevention or treatment of (post-)menopausal 'hot flashes' and osteoporosis. Xanthohumol and 8-prenylnaringenin are metabolized into many flavonoid derivatives with modified 3,3-dimethyl allyl (prenyl) moieties. Xanthohumol is formed in lupulin glands by a specialized branch of flavonoid biosynthesis that involves prenylation and O-methylation of the polyketide intermediate chalconaringenin. Although a lupulin gland-specific chalcone synthase is known, the aromatic prenyltransferase and O-methyltransferase participating in xanthohumol have not been identified. The prenylflavonoid pathway is a possible target for breeding or biotechnological modification of hops with the aim of increasing xanthohumol levels for beer brewing and 8-prenylnaringenin levels for pharmaceutical production.     

Identification of TRAF6 as a ubiquitin ligase engaged in the ubiquitination of SopB,a virulence effector protein secreted by Salmonella typhimurium

The phosphoinositide phosphatase SopB is one of the effectors injected by Salmonellatyphimurium (S.typhimurium) that diversifies its function through a ubiquitin-dependent differential localization. However, it is unclear which E3 ubiquitin ligase is responsible for ubiquitination of SopB. Based on the E1-E2-E3 trio of enzymes responsible for the ubiquitin activation and translocation to substrate proteins, we constructed an in vitro assay of SopB ubiquitination. Using this assay, we purified an E3 ubiquitin ligase, TRAF6, from the Henle-407 S100 extraction that may be responsible for the ubiquitination of SopB. To investigate the functional correlation of TRAF6, we showed that recombinant TRAF6 specifically ubiquitinates SopB in a dose-dependent manner in vitro. Upon infection, the ubiquitination of SopB was absolutely blocked by TRAF6 deletion, as shown in Traf6^−/− mouse embryonic fibroblasts (MEFs) compared with Traf6^+/+ MEFs. However, the ectopic expression of TRAF6 in Traf6^−/− MEFs rescued the two species of ubiquitin-conjugated SopB, which strengthens the role of TRAF6 in SopB ubiquitination. The analysis of E2 revealed that UbcH5c and not other E2 conjugating enzymes are required for TRAF6-mediated SopB ubiquitination both in vitro and in vivo. In summary, these results suggest the relevance of UbcH5c/TRAF6 in SopB during S.typhimurium infection and thereby imply that S.typhimurium has evolved a mechanism of utilizing the host’s E3 ubiquitin ligase to modify and modulate the function of its effector protein in order to ensure pathogen and host cell survival.     

HIV-1 Nef induces proinflammatory state in macrophages through its acidic cluster domain: involvement of TNF alpha receptor associated factor 2

Background

HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNβ to activate STAT1, -2 and -3.

Methodology/Principal Findings

Searching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2.

Conclusions

Results reported here revealed TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNβ.     

Decrease of miR-146b-5p in monocytes during obesity is associated with loss of the anti-inflammatory but not insulin signaling action of adiponectin

Background

Low adiponectin, a well-recognized antidiabetic adipokine, has been associated with obesity-related inflammation, oxidative stress and insulin resistance. Globular adiponectin is an important regulator of the interleukin-1 receptor-associated kinase (IRAK)/NFκB pathway in monocytes of obese subjects. It protects against inflammation and oxidative stress by inducing IRAK3. microRNA (miR)-146b-5p inhibits NFκB-mediated inflammation by targeted repression of IRAK1 and TNF receptor-associated factor-6 (TRAF6). Therefore, we measured the expression of miR-146b-5p in monocytes of obese subjects. Because it was low we determined the involvement of this miR in the anti-inflammatory, antioxidative and insulin signaling action of globular adiponectin.

Methods

miR-146b-5p expression in monocytes of obese subjects was determined by qRT-PCR. The effect of miR-146b-5p silencing on molecular markers of inflammation, oxidative stress and insulin signaling and the association with globular adiponectin was assessed in human THP-1 monocytes.

Results

miR-146b-5p was downregulated in monocytes of obese persons. Low globular adiponectin decreased miR-146b-5p and IRAK3 in THP-1 monocytes, associated with increased mitochondrial reactive oxygen species (ROS). Intracellular ROS and insulin receptor substrate-1 (IRS1) protein were unchanged. Silencing of miR-146b-5p with an antisense inhibitor resulted in increased expression of IRAK1 and TRAF6 leading to more NFκB p65 DNA binding activity and TNFα. As a response IRAK3 and IRS1 protein increased. Mitochondrial and intracellular ROS production did not increase despite more inflammation. In addition, exposure of miR-146b-5p-depleted THP-1 monocytes to high levels of globular adiponectin resulted in an increased production of TNFα and intracellular ROS. Still, they did not lose their potential to increase IRAK3 and IRS1 protein and to decrease mitochondrial ROS.

Conclusion

miR-146b-5p, decreased in monocytes during obesity, is a major mediator of the anti-inflammatory action of globular adiponectin. It appears not to be involved in insulin signaling possibly by protective response of IRAK3 and lack of mitochondrial ROS production.