Interferons Are Pro-Inflammatory Cytokines in Sheared-Stressed Human Aortic Valve Endothelial Cells.

Calcific aortic valve disease (CAVD) is an athero-inflammatory process. Growing evidence supports the inflammation-driven calcification model, mediated by cytokines such as interferons (IFNs) and tumor necrosis factor (TNF)-α. Our goal was investigating IFNs' effects in human aortic valve endothelial cells (VEC) and the potential differences between aortic (aVEC) and ventricular (vVEC) side cells. The endothelial phenotype was analyzed by Western blot, qPCR, ELISA, monocyte adhesion, and migration assays. In mixed VEC populations, IFNs promoted the activation of signal transducers and activators of transcription-1 and nuclear factor-κB, and the subsequent up-regulation of pro-inflammatory molecules. Side-specific VEC were activated with IFN-γ and TNF-α in an orbital shaker flow system. TNF-α, but not IFN-γ, induced hypoxia-inducible factor (HIF)-1α stabilization or endothelial nitric oxide synthase downregulation. Additionally, IFN-γ inhibited TNF-α-induced migration of aVEC. Also, IFN-γ triggered cytokine secretion and adhesion molecule expression in aVEC and vVEC. Finally, aVEC were more prone to cytokine-mediated monocyte adhesion under multiaxial flow conditions as compared with uniaxial flow. In conclusion, IFNs promote inflammation and reduce TNF-α-mediated migration in human VEC. Moreover, monocyte adhesion was higher in inflamed aVEC sheared under multiaxial flow, which may be relevant to understanding the initial stages of CAVD.

Calcification Induced by Type I Interferon in Human Aortic Valve Interstitial Cells Is Larger in Males and Blunted by a Janus Kinase Inhibitor.

Objective- Calcific aortic valve disease is the most prevalent valvulopathy in Western countries. An unanticipated pathogenetic clue involving IFN (interferon) was disclosed by the finding of constitutive type I IFN activity associated with aortic valve calcification in children with the atypical Singleton-Merten syndrome. On this basis, the role of type I IFN on inflammation and calcification in human aortic valve interstitial cells (AVIC) was examined. Approach and Results- IFN-α was weakly proinflammatory but potentiated lipopolysaccharide-mediated activation of NF (nuclear factor)-κB and the ensuing induction of proinflammatory molecules in human AVIC. Stimulation with IFN-α and in combination with lipopolysaccharide promoted osteoblast-like differentiation characterized by increased osteoblastic gene expression, BMP (bone morphogenetic protein)-2 secretion, and ectopic phosphatase activity. Sex differences were observed. Likewise, IFN-α treatment of human AVICs in osteogenic medium resulted in increased formation of calcific nodules. Strikingly, IFN-α-mediated calcification was significantly higher in AVICs from males, and was blocked by tofacitinib, a JAK (Janus kinase) inhibitor, and by a BMP antagonist. A female-specific protective mechanism involving the activation of PI3K-Akt (protein kinase B) pathways and cell survival was disclosed. Females exhibited higher levels of BCL2 in valve cells and tissues and lower annexin V staining on cell stimulation. Conclusions- IFN-α acts as a proinflammatory and pro-osteogenic cytokine in AVICs, its effects being potentiated by lipopolysaccharide. Results also uncovered sex differences with lower responses in female AVICs and sex-specific mechanisms involving apoptosis. Data point to JAK/STAT (signal transducer and activator of transcription) system as a potential therapeutic target for calcific aortic valve disease.

Aging and amyloid ß oligomers enhance TLR4 expression, LPS-induced Ca2+ responses, and neuron cell death in cultured rat hippocampal neurons.

BACKGROUND: Toll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system recognizing diverse pathogen-derived and tissue damage-related ligands. It has been suggested that TLR signaling contributes to the pathogenesis of age-related, neurodegenerative diseases, including Alzheimer's disease (AD). AD is associated to oligomers of the amyloid ß peptide (Aßo) that cause intracellular Ca2+ dishomeostasis and neuron cell death in rat hippocampal neurons. Here we assessed the interplay between inflammation and Aßo in long-term cultures of rat hippocampal neurons, an in vitro model of neuron aging and/or senescence. METHODS: Ca2+ imaging and immunofluorescence against annexin V and TLR4 were applied in short- and long-term cultures of rat hippocampal neurons to test the effects of TLR4-agonist LPS and Aßo on cytosolic [Ca2+] and on apoptosis as well as on expression of TLR4. RESULTS: LPS increases cytosolic [Ca2+] and promotes apoptosis in rat hippocampal neurons in long-term culture considered aged and/or senescent neurons, but not in short-term cultured neurons considered young neurons. TLR4 antagonist CAY10614 prevents both effects. TLR4 expression in rat hippocampal neurons is significantly larger in aged hippocampal cultures. Treatment of aged hippocampal cultures with Aßo increases TLR4 expression and enhances LPS-induced Ca2+ responses and neuron cell death. CONCLUSIONS: Aging and amyloid ß oligomers, the neurotoxin involved in Alzheimer's disease, enhance TLR4 expression as well as LPS-induced Ca2+ responses and neuron cell death in rat hippocampal neurons aged in vitro.

The Flavone Luteolin Inhibits Liver X Receptor Activation.

Luteolin is a dietary flavonoid with medicinal properties including antioxidant, antimicrobial, anticancer, antiallergic, and anti-inflammatory. However, the effect of luteolin on liver X receptors (LXRs), oxysterol sensors that regulate cholesterol homeostasis, lipogenesis, and inflammation, has yet to be studied. To unveil the potential of luteolin as an LXRα/ß modulator, we investigated by real-time RT-PCR the expression of LXR-target genes, namely, sterol regulatory element binding protein 1c (SREBP-1c) in hepatocytes and ATP-binding cassette transporter (ABC)A1 in macrophages. The lipid content of hepatocytes was evaluated by Oil Red staining. The results demonstrated, for the first time, that luteolin abrogated the LXRα/ß agonist-induced LXRα/ß transcriptional activity and, consequently, inhibited SREBP-1c expression, lipid accumulation, and ABCA1 expression. Therefore, luteolin could abrogate hypertriglyceridemia associated with LXR activation, thus presenting putative therapeutic effects in diseases associated with deregulated lipid metabolism, such as hepatic steatosis, cardiovascular diseases, and diabetes.

Lipopolysaccharide and sphingosine-1-phosphate cooperate to induce inflammatory molecules and leukocyte adhesion in endothelial cells.

Given that TLRs and sphingosine-1-phosphate (S1P) are key players in inflammation, we explored the potential interplay between TLRs and S1P in the adhesion/inflammatory pathways in primary human endothelial cells. As determined by Western blot and flow cytometry, cells treated with LPS (a TLR4 ligand) and S1P showed significantly enhanced expression of adhesion molecules such as ICAM-1 and E-selectin compared with the effect of either ligand alone. Cell-type differences on E-selectin upregulation were observed. In contrast, no cooperation effect on ICAM-1 or E-selectin was observed with a TLR2/TLR1 ligand. Consistent with an increase in adhesion molecule expression, endothelial cell treatment with LPS plus S1P significantly enhanced adhesion of PBMCs under shear stress conditions compared with the effect of either ligand alone and exhibited comparable levels of cell adhesion strength as those after TNF-α treatment. Moreover, LPS and S1P cooperated to increase the expression of proinflammatory molecules such as IL-6, cyclooxygenase-2, and prostacyclin, as determined by ELISA and Western blot. The analysis of signaling pathways revealed the synergistic phosphorylation of ERK upon LPS plus S1P treatment of HUVEC and human aortic endothelial cells and cell-type differences on p38 and NF-κB activation. Moreover, pharmacological and small interfering RNA experiments disclosed the involvement of S1P(1/3) and NF-κB in the cooperation effect and that cell origin determines the S1P receptors and signaling routes involved. Sphingosine kinase activity induction upon LPS plus S1P treatment suggests S1P- Sphingosine kinase axis involvement. In summary, LPS and S1P cooperate to increase proinflammatory molecules in endothelial cells and, in turn, to augment leukocyte adhesion, thus exacerbating S1P-mediated proadhesive/proinflammatory properties.

[Lugol as a rare cause of anaphylactic reaction after colposcopy. Case report]. / Lugol como causa inusual de anafilaxia después de la colposcopia. Reporte de un caso clínico.

INTRODUCTION: Lugol is a solution composed of elemental iodine (5%) and potassium iodide (10%) together with distilled water, used during colposcopic assessment to identify possible cervical cell alterations. CASE REPORT: A 31-year-old female who presents an episode suggestive of anaphylaxis ninety minutes after a colposcopy exploration, successfully treated with intramuscular hydrocortisone and desclorfeniramine. During colposcopy Lugol solution and acetic acid was applied. Skin prick test (SPT) with Lugol solution was positive (papule 9x7 mm). Four control tests were negative. Intradermal tests (IDT) were positive with Lugol and elemental iodine, the last one turned-out irritant. It was ruled out possible cross-reactivity with other iodine preparations (Betadine®) and potassium iodide (Yoduk®). CONCLUSIONS: Our report demonstrates a rare case of allergy to Lugol solution with positive SPT and a clinical suggestive reaction, with tolerance to other iodine preparations and potassium iodide.

INTRODUCCIÓN: El lugol es una solución compuesta por yodo elemental (5%), yoduro de potasio (10%) y agua destilada, utilizada durante las colposcopias para detectar alteraciones celulares en el cérvix. REPORTE DE CASO: Paciente femenina de 31 años, que tuvo un evento de anafilaxia noventa minutos después de la colposcopia, tratada exitosamente con hidrocortisona por vía intramuscular y desclorfeniramina. Durante la colposcopia se aplicó lugol y ácido acético. La prueba de prick con lugol resultó positiva, con formación de una pápula de 9 x 7 mm, al igual que las pruebas intradérmicas para lugol y yodo elemental. Cuatro controles fueron negativos, excepto para yodo elemental, que resultó irritante en intradermorreacción. Se descartó reactividad cruzada con otras soluciones yodadas (Betadine®) y (Yoduk®). CONCLUSIONES: Reportamos un caso raro de alergia a lugol con prick positivo y una reacción clínica sugerente, con tolerancia a otras preparaciones yodadas y a yoduro de potasio.

[Lichenoid drug eruption with unfavorable evolution. Difficulties in diagnosis]. / Erupción liquenoide medicamentosa con evolución desfavorable. Dificultades en el diagnóstico.

We report the case of a woman who started with a lichenoid eruption, unfavorable evolution, for which a drug reaction was suspected. The final diagnosis was paraneoplastic pemphigus. Multidisciplinary care and evaluation by an Allergist is important in patients with severe skin reactions, suspected of drug reactions, due to the difficulty in establishing the diagnosis.

Se reporta el caso de una mujer que inició con erupción liquenoide, con evolución desfavorable, por lo que se sospechó una reacción medicamentosa. El diagnóstico final fue pénfigo paraneoplásico. Es importante la atención multidisciplinaria y la evaluación de un alergólogo en pacientes con reacciones cutáneas graves, por sospecha de reacciones farmacológicas, debido a la dificultad para establecer el diagnóstico.

Clinically used JAK inhibitor blunts dsRNA-induced inflammation and calcification in aortic valve interstitial cells.

Calcific aortic valve disease (CAVD) is the most prevalent valvulopathy worldwide. Growing evidence supports a role for viral and cell-derived double-stranded (ds)-RNA in cardiovascular pathophysiology. Poly(I:C), a dsRNA surrogate, has been shown to induce inflammation, type I interferon (IFN) responses, and osteogenesis through Toll-like receptor 3 in aortic valve interstitial cells (VIC). Here, we aimed to determine whether IFN signaling via Janus kinase (JAK)/Signal transducers and activators of transcription (STAT) mediates dsRNA-induced responses in primary human VIC. Western blot, ELISA, qPCR, calcification, flow cytometry, and enzymatic assays were performed to evaluate the mechanisms of dsRNA-induced inflammation and calcification. Poly(I:C) triggered a type I IFN response characterized by IFN-regulatory factors gene upregulation, IFN-ß secretion, and STAT1 activation. Additionally, Poly(I:C) promoted VIC inflammation via NF-κB and subsequent adhesion molecule expression, and cytokine secretion. Pretreatment with ruxolitinib, a clinically used JAK inhibitor, abrogated these responses. Moreover, Poly(I:C) promoted a pro-osteogenic phenotype and increased VIC calcification to a higher extent in cells from males. Inhibition of JAK with ruxolitinib or a type I IFN receptor blocking antibody blunted Poly(I:C)-induced calcification. Mechanistically, Poly(I:C) promoted VIC apoptosis in calcification medium, which was inhibited by ruxolitinib. Moreover, Poly(I:C) co-operated with IFN-γ to increase VIC calcification by synergistically activating extracellular signal-regulated kinases and hypoxia-inducible factor-1α pathways. In conclusion, JAK/STAT signaling mediates dsRNA-triggered inflammation, apoptosis, and calcification and may contribute to a positive autocrine loop in human VIC in the presence of IFN-γ. Blockade of dsRNA responses with JAK inhibitors may be a promising therapeutic avenue for CAVD.

Role of Toll Like Receptor 4 in Alzheimer's Disease.

Long-term evidence has confirmed the involvement of an inflammatory component in neurodegenerative disorders including Alzheimer's disease (AD). This view is supported, in part, by data suggesting that selected non-steroidal anti-inflammatory drugs (NSAIDs) provide protection. Additionally, molecular players of the innate immune system have recently been proposed to contribute to these diseases. Toll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system that recognize different pathogen-derived and tissue damage-related ligands. TLR4 mediated signaling has been reported to contribute to the pathogenesis of age-related neurodegenerative diseases, including AD. Although the pathophysiology of AD is not clear, soluble aggregates (oligomers) of the amyloid ß peptide (Aßo) have been proven to be key players in the pathology of AD. Among others, Aßo promote Ca2+ entry and mitochondrial Ca2+ overload leading to cell death in neurons. TLR4 has recently been found to be involved in AD but the mechanisms are unclear. Our group recently reported that lipopolysaccharide (LPS), a TLR4 receptor agonist, increases cytosolic Ca2+ concentration leading to apoptosis. Strikingly, this effect was only observed in long-term cultured primary neurons considered a model of aging neurons, but not in short-term cultured neurons resembling young neurons. These effects were significantly prevented by pharmacological blockade of TLR4 receptor signaling. Moreover, TLR4 expression in rat hippocampal neurons increased significantly in aged neurons in vitro. Therefore, molecular patterns associated with infection and/or brain cell damage may activate TLR4 and Ca2+ signaling, an effect exacerbated during neuronal aging. Here, we briefly review the data regarding the involvement of TLR4 in AD.

Selective attenuation of Toll-like receptor 2 signalling may explain the atheroprotective effect of sphingosine 1-phosphate.

AIMS: Vascular inflammation is a major atherogenic factor and Toll-like receptor (TLR) 2 ligands, including bacterial and serum lipoproteins, seem to be involved in atherogenesis. On this basis, we analysed the effect of lipoproteins and different lipid components on TLR2-dependent signalling. METHODS AND RESULTS: In TLR2-transfected human embryonic kidney 293 cells and human monocytes, oxidized low-density lipoproteins inhibited nuclear factor (NF)-kappaB-driven transcriptional activity and chemokine gene expression in response to TLR2 ligands. Sphingosine 1-phosphate (S1P) and oxidized palmitoyl-arachidonoyl-phosphatidylcholine, but not lipoprotein-carried lysophospholipids, inhibited TLR2 activation. Silencing experiments in TLR2-transfected 293 cells showed that the S1P-mediated attenuation effect is mediated by S1P receptors type 1 and type 2. To address the physiological significance of these findings, additional experiments were performed in human peripheral blood monocytes and monocyte-derived macrophages. In both cell types, S1P selectively attenuated TLR2 signalling, as NF-kappaB and extracellular signal-regulated kinase activation, but not c-Jun amino terminal kinase phosphorylation, were inhibited by physiologically relevant concentrations of S1P. Moreover, the attenuation of TLR2 signalling was partially reverted by pharmacological inhibition of phosphoinositide 3-kinase (PI3K) and Ras pathways. In addition, S1P inhibited the chemokine gene expression elicited by TLR2, but not by TLR4 ligands. CONCLUSION: These findings disclose a cross-talk mechanism between lipoprotein components and TLR in which engagement of S1P receptors exert selective attenuation of TLR2-dependent activation via PI3K and Ras signalling. A corollary to these data is that the negative cross-talk of S1P receptors and TLR2 signalling might be involved in the atheroprotective effects of S1P.

Lipopolysaccharide and interferon-γ team up to activate HIF-1α via STAT1 in normoxia and exhibit sex differences in human aortic valve interstitial cells.

In early stages of calcific aortic valve disease (CAVD), immune cells infiltrate into the valve leaflets and release cytokines such as interferon (IFN)-γ. IFN-γ has context-dependent direct effects, and also regulates other immune pathways. The purpose of this study was addressing the effects of IFN-γ on human aortic valve interstitial cells (AVICs), focusing on the pathogenic processes underlying CAVD. Strikingly, under normoxic conditions, IFN-γ induced hypoxia inducible factor (HIF)-1α expression, an effect strongly potentiated by the Toll-like receptor (TLR)-4 ligand lipopolysaccharide (LPS). Immunodetection studies confirmed the nuclear translocation of HIF-1α. Gene silencing showed that HIF-1α expression is dependent on signal transducer and activator of transcription (STAT)-1 expression. Consistent with HIF-1α induction, the secretion of the endothelial growth factor was detected by ELISA, and downregulation of the antiangiogenic factor chondromodulin-1 gene was observed by qPCR. Results also disclosed IFN-γ as a proinflammatory cytokine that cooperates with LPS to induce the expression of adhesion molecules, prostaglandin E2 and interleukins. Moreover, IFN-γ induced an osteogenic phenotype and promoted in vitro calcification that were markedly potentiated by LPS. Pharmacological experiments disclosed the involvement of Janus Kinases (JAK)/STATs as well as ERK/HIF-1α routes on the induction of calcification. Notably, IFN-γ receptor 1 expression, as well as ERK/HIF-1α activation, and the subsequent responses were more robust in male AVICs. This is the first report uncovering an immune and non-hypoxic activation of HIF-1α via STAT1 in AVIC. The aforementioned results and the sex-differential responses may be potentially relevant to better understand CAVD pathogenesis.

Toll-Like Receptors, Inflammation, and Calcific Aortic Valve Disease.

Inflammation, the primary response of innate immunity, is essential to initiate the calcification process underlying calcific aortic valve disease (CAVD), the most prevalent valvulopathy in Western countries. The pathogenesis of CAVD is multifactorial and includes inflammation, hemodynamic factors, fibrosis, and active calcification. In the development of CAVD, both innate and adaptive immune responses are activated, and accumulating evidences show the central role of inflammation in the initiation and propagation phases of the disease, being the function of Toll-like receptors (TLR) particularly relevant. These receptors act as sentinels of the innate immune system by recognizing pattern molecules from both pathogens and host-derived molecules released after tissue damage. TLR mediate inflammation via NF-κB routes within and beyond the immune system, and play a crucial role in the control of infection and the maintenance of tissue homeostasis. This review outlines the current notions about the association between TLR signaling and the ensuing development of inflammation and fibrocalcific remodeling in the pathogenesis of CAVD. Recent data provide new insights into the inflammatory and osteogenic responses underlying the disease and further support the hypothesis that inflammation plays a mechanistic role in the initiation and progression of CAVD. These findings make TLR signaling a potential target for therapeutic intervention in CAVD.

A conserved docking motif for CK1 binding controls the nuclear localization of NFAT1.

In resting cells, the NFAT1 transcription factor is kept inactive in the cytoplasm by phosphorylation on multiple serine residues. These phosphorylated residues are primarily contained within two types of serine-rich motifs, the SRR-1 and SP motifs, which are conserved within the NFAT family. Several different kinases have been proposed to regulate NFAT, but no single candidate displays the specificity required to fully phosphorylate both types of motifs; thus, the identity of the kinase that regulates NFAT activity remains unclear. Here we show that the NFAT1 serine motifs are regulated by distinct kinases that must coordinate to control NFAT1 activation. CK1 phosphorylates only the SRR-1 motif, the primary region required for NFAT1 nuclear import. CK1 exists with NFAT1 in a high-molecular-weight complex in resting T cells but dissociates upon activation. GSK3 does not phosphorylate the SRR-1 region but can target the NFAT1 SP-2 motif, and it synergizes with CK1 to regulate NFAT1 nuclear export. We identify a conserved docking site for CK1 in NFAT proteins and show that mutation of this site disrupts NFAT1-CK1 interaction and causes constitutive nuclear localization of NFAT1. The CK1 docking motif is present in proteins of the Wnt, Hedgehog, and circadian-rhythm pathways, which also integrate the activities of CK1 and GSK3.

Synergy between sphingosine 1-phosphate and lipopolysaccharide signaling promotes an inflammatory, angiogenic and osteogenic response in human aortic valve interstitial cells.

Given that the bioactive lipid sphingosine 1-phosphate is involved in cardiovascular pathophysiology, and since lipid accumulation and inflammation are hallmarks of calcific aortic stenosis, the role of sphingosine 1-phosphate on the pro-inflammatory/pro-osteogenic pathways in human interstitial cells from aortic and pulmonary valves was investigated. Real-time PCR showed sphingosine 1-phosphate receptor expression in aortic valve interstitial cells. Exposure of cells to sphingosine 1-phosphate induced pro-inflammatory responses characterized by interleukin-6, interleukin-8, and cyclooxygenase-2 up-regulations, as observed by ELISA and Western blot. Strikingly, cell treatment with sphingosine 1-phosphate plus lipopolysaccharide resulted in the synergistic induction of cyclooxygenase-2, and intercellular adhesion molecule 1, as well as the secretion of prostaglandin E2, the soluble form of the intercellular adhesion molecule 1, and the pro-angiogenic factor vascular endothelial growth factor-A. Remarkably, the synergistic effect was significantly higher in aortic valve interstitial cells from stenotic than control valves, and was drastically lower in cells from pulmonary valves, which rarely undergo stenosis. siRNA and pharmacological analysis revealed the involvement of sphingosine 1-phosphate receptors 1/3 and Toll-like receptor-4, and downstream signaling through p38/MAPK, protein kinase C, and NF-κB. As regards pro-osteogenic pathways, sphingosine 1-phosphate induced calcium deposition and the expression of the calcification markers bone morphogenetic protein-2 and alkaline phosphatase, and enhanced the effect of lipopolysaccharide, an effect that was partially blocked by inhibition of sphingosine 1-phosphate receptors 3/2 signaling. In conclusion, the interplay between sphingosine 1-phosphate receptors and Toll-like receptor 4 signaling leads to a cooperative up-regulation of inflammatory, angiogenic, and osteogenic pathways in aortic valve interstitial cells that seems relevant to the pathogenesis of aortic stenosis and may allow the inception of new therapeutic approaches.

Anti-inflammatory activity of Cymbopogon citratus leaves infusion via proteasome and nuclear factor-κB pathway inhibition: contribution of chlorogenic acid.

ETHNOPHARMACOLOGICAL RELEVANCE: Cymbopogon citratus (DC.) Stapf leaves infusion is used in traditional medicine for the treatment of inflammatory conditions, however little is known about their bioactive compounds. AIM OF THE STUDY: Investigate the compounds responsible for anti-inflammatory potential of Cymbopogon citratus (Cy) on cytokines production induced by lipopolysaccharide (LPS) in human and mouse macrophages, and the action mechanisms involved. MATERIALS AND METHODS: An essential oil-free infusion of Cy was prepared and polyphenol-rich fractions (PFs) were obtained from it by column chromatography. Chlorogenic acid (CGA) was identified, by HPLC/PDA/ESI-MS(n). The expression of cytokines, namely TNF-α and CCL5, was analyzed by real-time RT-PCR, on LPS-stimulated human macrophages. Activation of nuclear factor (NF)-κB, a master regulator of inflammation, was investigated by western blot and gene reporter assay. Proteasome activity was assessed using a fluorogenic peptide. RESULTS: Cymbopogon citratus extract and its polyphenols inhibited the cytokine production on human macrophages. This supports the anti-inflammatory activity of Cy polyphenols in physiologically relevant cells. Concerning the effect on the activation of NF-κB pathway, the results pointed to an inhibition of LPS-induced NF-κB activation by Cy and PFs. CGA was identified, by HPLC/PDA/ESI-MS(n), as the main phenolic acid of the Cy infusion, and it demonstrated to be, at least in part, responsible by that effect. Additionally, it was verified for the first time that Cy and PFs inhibited the proteasome activity, a complex that controls NF-κB activation, having CGA a strong contribution. CONCLUSIONS: The results evidenced, for the first time, the anti-inflammatory properties of Cymbopogon citratus through proteasome inhibition and, consequently NF-κB pathway and cytokine expression. Additionally, Cy polyphenols, in particular chlorogenic acid, were highlighted as bioactive compounds.

Viral and bacterial patterns induce TLR-mediated sustained inflammation and calcification in aortic valve interstitial cells.

BACKGROUND: Aortic stenosis shares some ethiopathological features with atherosclerosis and increasing evidence links Toll-like receptors (TLRs) to atherogenesis. METHODS: TLR-mediated inflammation and osteogenesis were investigated in human interstitial cells isolated from stenotic and non-stenotic aortic valves. TLR expression and signalling were evaluated by quantitative RT-PCR, flow cytometry, Western blot analysis, ELISA, and cytokine arrays. Osteogenesis was evaluated by measuring alkaline phosphatase activity. RESULTS: Interstitial cells from control valves express most TLRs, being TLR4 the most abundant, whereas cells from stenotic valves express higher TLR4 and TLR2 and lower TLR5 and TLR9 transcript levels. When pro-inflammatory pathways were analyzed, we observed that TLR4, TLR2 and TLR3 ligands induced an early activation of NF-κB and p38 MAPK activation in cells from control and stenotic valves. Strikingly, when TLRs sensing viral patterns were studied, a sustained TLR3-mediated activation of NF-κB, a κB-independent induction of catalytically active cyclooxigenase (COX)-2 and ICAM-1 expression, and induction of expression of several chemokines were observed. TLR4, but not TLR2, engagement produced a similar but NF-κB-dependent effect. Moreover, TLR3 and TLR4 agonists induced alkaline phosphatase expression and activity. CONCLUSIONS: Exposure of aortic valve interstitial cells to viral and Gram-negative bacteria molecular patterns induces distinct and long-term TLR-mediated pro-inflammatory and pro-osteogenic responses that might be relevant to the pathogenesis of degenerative aortic stenosis.

Cymbopogon citratus as source of new and safe anti-inflammatory drugs: bio-guided assay using lipopolysaccharide-stimulated macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Aqueous extracts of Cymbopogon citratus (Cy) leaves are used in traditional medicine for the treatment of inflammatory conditions, however, little is known about their mechanism of action. AIM OF THE STUDY: The aim of this study is to explore the anti-inflammatory properties of Cymbopogon citratus leaves and their polyphenol-rich fractions (PFs), as well its mechanism of action in murine macrophages. MATERIALS AND METHODS: A lipid- and essential oil-free infusion of Cy leaves was prepared (Cy extract) and fractionated by column chromatography. Anti-inflammatory properties of Cy extract (1.115 mg/ml) and its PFs, namely phenolic acids (530 µg/ml), flavonoids (97.5 µg/ml) and tannins (78 µg/ml), were investigated using lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophages as in vitro model. As inflammatory parameters, nitric oxide (NO) production was evaluated by Griess reaction, as well as effects on cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS) expression and on intracellular signaling pathways activation, which were analyzed by Western blot using specific antibodies. RESULTS: Cy extract inhibited iNOS expression, NO production and various LPS-induced pathways like p38 mitogen-activated protein kinase (MAPK), c-jun NH(2)-terminal kinase (JNK) 1/2 and the transcription nuclear factor (NF)-κB. The extracellular signal-regulated kinase (ERK) 1/2 and the phosphatidylinositol-3-kinase (PI3K)/Akt activation were not affected by Cy extract. Both phenolic acid- and tannin-rich fractions significantly inhibited NF-κB activation, iNOS expression and NO production but none of the PFs modulated MAPKs or PI3K/Akt activation. Neither Cy extract nor PFs affected LPS-induced COX-2 expression but LPS-induced PGE(2) production is inhibited by Cy extract and by phenolic acid-rich fraction. CONCLUSIONS: Our data provide evidence that support the usage of Cymbopogon citratus leaves extract in traditional medicine, and suggest that Cy, in particular its polyphenolic compounds, could constitute a natural source of a new and safe anti-inflammatory drug.

Differential roles of PI3-Kinase, MAPKs and NF-kappaB on the manipulation of dendritic cell T(h)1/T(h)2 cytokine/chemokine polarizing profile.

Dendritic cells (DC) are professional antigen-presenting cells with a unique capacity to initiate and modulate immune responses by their ability to prime naïve T-cells. Upon stimuli, DC experience several morphologic, phenotypic and functional changes in a process referred to as maturation. This process is crucial to the biological functions of DC since their maturation status confer them the ability to polarize distinct T-cell subsets. In this work we explored the relevance of PI3-Kinase, Mitogen-Activated Protein Kinases (MAPKs) and NF-kappaB on cytokines/chemokines and co-stimulatory molecules expression. As experimental model, we used a fetal skin-derived dendritic cell line (FSDC) induced to mature by treatment with lipopolysaccharide (LPS). Morphology and ultrastructure were analyzed by confocal and electron microscopies, respectively. Levels of phosphorylated proteins were evaluated by Western blot, production of cytokines/chemokines was analyzed by protein arrays and the expression of surface molecules was evaluated by flow cytometry. The effect of specific inhibitors of the studied signaling pathways on the transcription of cytokines/chemokines and co-stimulatory molecules was accessed by Quantitative Real-Time RT-PCR. The results showed that LPS induces significant morphological and ultrastructural changes in FSDC. Western blot analysis revealed that LPS challenge promotes an early and transient activation of NF-kappaB, ERK1/2, p38 MAPK, along with a more sustained PI3 kinase/AKT activation. The co-stimulatory CD40, CD80, CD86 and antigen-presenting MHC class I and II molecules were increased and among secreted molecules, interleukin IL-6, CCL5, G-CSF, CCL2, CXCL2 were strongly up-regulated. Using a pharmacological approach we observed that LPS-induced increase of these molecules was differentially regulated by the distinct signaling pathways. Moreover, the polarizing T(h)2 cytokines/chemokines induced by LPS in FSDC were found to be positively regulated by NF-kappaB and ERK and negatively modulated by p38 MAPK. Altogether these results suggest that the use of pharmacological inhibitors to manipulate DC maturation, namely the polarizing T(h)1/T(h)2 cytokine/chemokine profile, may be useful in the development of more specific immunotherapeutic protocols.