Budesonide promotes airway epithelial barrier integrity following double-stranded RNA challenge.

Airway epithelial barrier dysfunction is increasingly recognized as a key feature of asthma and other lung diseases. Respiratory viruses are responsible for a large fraction of asthma exacerbations, and are particularly potent at disrupting epithelial barrier function through pattern recognition receptor engagement leading to tight junction dysfunction. Although different mechanisms of barrier dysfunction have been described, relatively little is known about whether barrier integrity can be promoted to limit disease. Here, we tested three classes of drugs commonly prescribed to treat asthma for their ability to promote barrier function using a cell culture model of virus-induced airway epithelial barrier disruption. Specifically, we studied the corticosteroid budesonide, the long acting beta-agonist formoterol, and the leukotriene receptor antagonist montelukast for their ability to promote barrier integrity of a monolayer of human bronchial epithelial cells (16HBE) before exposure to the viral mimetic double-stranded RNA. Of the three, only budesonide treatment limited transepithelial electrical resistance and small molecule permeability (4 kDa FITC-dextran flux). Next, we used a mouse model of acute dsRNA challenge that induces transient epithelial barrier disruption in vivo, and studied the effects budesonide when administered prophylactically or therapeutically. We found that budesonide similarly protected against dsRNA-induced airway barrier disruption in the lung, independently of its effects on airway inflammation. Taken together, these data suggest that an under-appreciated effect of inhaled budesonide is to maintain or promote airway epithelial barrier integrity during respiratory viral infections.

An Influence of Modification with Phosphoryl Guanidine Combined with a 2'-O-Methyl or 2'-Fluoro Group on the Small-Interfering-RNA Effect.

Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2'-O-methyl (2'-OMe) or PG/2'-fluoro (2'-F); biophysical and biochemical properties were characterized for each duplex. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand.

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.

ILF3 contributes to the establishment of the antiviral type I interferon program.

Upon detection of viral infections, cells activate the expression of type I interferons (IFNs) and pro-inflammatory cytokines to control viral dissemination. As part of their antiviral response, cells also trigger the translational shutoff response which prevents translation of viral mRNAs and cellular mRNAs in a non-selective manner. Intriguingly, mRNAs encoding for antiviral factors bypass this translational shutoff, suggesting the presence of additional regulatory mechanisms enabling expression of the self-defence genes. Here, we identified the dsRNA binding protein ILF3 as an essential host factor required for efficient translation of the central antiviral cytokine, IFNB1, and a subset of interferon-stimulated genes. By combining polysome profiling and next-generation sequencing, ILF3 was also found to be necessary to establish the dsRNA-induced transcriptional and translational programs. We propose a central role for the host factor ILF3 in enhancing expression of the antiviral defence mRNAs in cellular conditions where cap-dependent translation is compromised.

Porcine deltacoronavirus nucleocapsid protein antagonizes IFN-ß production by impairing dsRNA and PACT binding to RIG-I.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in newborn piglets. Previous studies have suggested that PDCoV infection antagonizes RIG-I-like receptor (RLR)-mediated IFN-ß production to evade host innate immune defense, and PDCoV-encoded nonstructural protein nsp5 and accessory protein NS6 are associated with this process. However, whether the structural protein(s) of PDCoV also antagonize IFN-ß production remains unclear. In this study, we found that PDCoV nucleocapsid (N) protein, the most abundant viral structural protein, suppressed Sendai virus (SEV)-induced IFN-ß production and transcription factor IRF3 activation, but did not block IFN-ß production induced by overexpressing RIG-I/MDA5. Furthermore, study revealed that PDCoV N protein interacted with RIG-I and MDA5 in an in vitro overexpression system and evident interactions between N protein and RIG-I could be detected in the context of PDCoV infection, which interfered with the binding of dsRNA and protein activator of protein kinase R (PACT) to RIG-I. Together, our results demonstrate that PDCoV N protein is an IFN antagonist and utilizes diverse strategies to attenuate RIG-I recognition and activation.

Identification of Myricetin as an Ebola Virus VP35-Double-Stranded RNA Interaction Inhibitor through a Novel Fluorescence-Based Assay.

Ebola virus (EBOV) is a filovirus that causes a severe and rapidly progressing hemorrhagic syndrome; a recent epidemic illustrated the urgent need for novel therapeutic agents because no drugs have been approved for treatment of Ebola virus. A key contribution to the high lethality observed during EBOV outbreaks comes from viral evasion of the host antiviral innate immune response in which viral protein VP35 plays a crucial role, blocking interferon type I production, first by masking the viral double-stranded RNA (dsRNA) and preventing its detection by the pattern recognition receptor RIG-I. Aiming to identify inhibitors of the interaction of VP35 with the viral dsRNA, counteracting the VP35 viral innate immune evasion, we established a new methodology for high-yield recombinant VP35 (rVP35) expression and purification and a novel and robust fluorescence-based rVP35-RNA interaction assay ( Z' factor of 0.69). Taking advantage of such newly established methods, we screened a small library of Sardinian natural extracts, identifying Limonium morisianum as the most potent inhibitor extract. A bioguided fractionation led to the identification of myricetin as the component that can inhibit rVP35-dsRNA interaction with an IC50 value of 2.7 µM. Molecular docking studies showed that myricetin interacts with the highly conserved region of the VP35 RNA binding domain, laying the basis for further structural optimization of potent inhibitors of VP35-dsRNA interaction.

Antiviral activity of K22 against members of the order Nidovirales.

Recently, a novel antiviral compound (K22) that inhibits replication of a broad range of animal and human coronaviruses was reported to interfere with viral RNA synthesis by impairing double-membrane vesicle (DMV) formation (Lundin et al., 2014). Here we assessed potential antiviral activities of K22 against a range of viruses representing two (sub)families of the order Nidovirales, the Arteriviridae (porcine reproductive and respiratory syndrome virus [PRRSV], equine arteritis virus [EAV] and simian hemorrhagic fever virus [SHFV]), and the Torovirinae (equine torovirus [EToV] and White Bream virus [WBV]). Possible effects of K22 on nidovirus replication were studied in suitable cell lines. K22 concentrations significantly decreasing infectious titres of the viruses included in this study ranged from 25 to 50 µM. Reduction of double-stranded RNA intermediates of viral replication in nidovirus-infected cells treated with K22 confirmed the anti-viral potential of K22. Collectively, the data show that K22 has antiviral activity against diverse lineages of nidoviruses, suggesting that the inhibitor targets a critical and conserved step during nidovirus replication.

Design and Experimental Validation of Small Activating RNAs Targeting an Exogenous Promoter in Human Cells.

It is increasingly practical to co-opt many native cellular components into use as elements of synthetic biological systems. We present the design and experimental investigation of the first exogenous genetic construct to be successfully targeted by RNA activation, a phenomenon whereby small double-stranded RNAs increase gene expression from sequence-similar promoters by a mechanism thought to be related to that of RNA interference. Our selection of activating RNA candidates was informed by a custom-written computer program designed to choose target sites in the promoter of interest according to a set of empirical optimality criteria drawn from prior research. Activating RNA candidates were assessed for activity against two exogenously derived target promoters, with successful candidates being subjected to further rounds of validation as a precaution against potential off-target effects. A genetic platform was assembled that allowed activating RNA candidates to be simultaneously screened both for positive activity on the target reporter gene and for possible nonspecific effects on cell metabolism. Several candidate sequences were tested to appraise the utility of this platform, with the most successful achieving a moderate activation level with minimal off-target effects.

Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response.

Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.

Inhibition of antiviral innate immunity by birnavirus VP3 protein via blockage of viral double-stranded RNA binding to the host cytoplasmic RNA detector MDA5.

UNLABELLED: Chicken MDA5 (chMDA5), the sole known pattern recognition receptor for cytoplasmic viral RNA in chickens, initiates type I interferon (IFN) production. Infectious bursal disease virus (IBDV) evades host innate immunity, but the mechanism is unclear. We report here that IBDV inhibited antiviral innate immunity via the chMDA5-dependent signaling pathway. IBDV infection did not induce efficient type I interferon (IFN) production but antagonized the antiviral activity of beta interferon (IFN-ß) in DF-1 cells pretreated with IFN-α/ß. Dual-luciferase assays and inducible expression systems demonstrated that IBDV protein VP3 significantly inhibited IFN-ß expression stimulated by naked IBDV genomic double-stranded RNA (dsRNA). The VP3 protein competed strongly with chMDA5 to bind IBDV genomic dsRNA in vitro and in vivo, and VP3 from other birnaviruses also bound dsRNA. Site-directed mutagenesis confirmed that deletion of the VP3 dsRNA binding domain restored IFN-ß expression. Our data demonstrate that VP3 inhibits antiviral innate immunity by blocking binding of viral genomic dsRNA to MDA5. IMPORTANCE: MDA5, a known pattern recognition receptor and cytoplasmic viral RNA sensor, plays a critical role in host antiviral innate immunity. Many pathogens escape or inhibit the host antiviral immune response, but the mechanisms involved are unclear for most pathogens. We report here that birnaviruses inhibit host antiviral innate immunity via the MDA5-dependent signaling pathway. The antiviral innate immune system involving IFN-ß did not function effectively during birnavirus infection, and the viral protein VP3 significantly inhibited IFN-ß expression stimulated by naked viral genomic dsRNA. We also show that VP3 blocks MDA5 binding to viral genomic dsRNA in vitro and in vivo. Our data reveal that birnavirus-encoded viral protein VP3 is an inhibitor of the antiviral innate immune response and inhibits the antiviral innate immune response via the MDA5-dependent signaling pathway.

A fluorescence-based high-throughput screen to identify small compound inhibitors of the genotype 3a hepatitis C virus RNA polymerase.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) plays an essential role in the replication of HCV and is a key target for novel antiviral therapies. Several RdRp inhibitors are in clinical trials and have increased response rates when combined with current interferon-based therapies for genotype 1 (G1) HCV patients. These inhibitors, however, show poor efficacy against non-G1 genotypes, including G3a, which represents ~20% of HCV cases globally. Here, we used a commercially available fluorescent dye to characterize G3a HCV RdRp in vitro. RdRp activity was assessed via synthesis of double-stranded RNA from the single-stranded RNA poly(C) template. The assay was miniaturized to a 384-well microplate format and a pilot high-throughput screen was conducted using 10,208 "lead-like" compounds, randomly selected to identify inhibitors of HCV G3a RdRp. Of 150 compounds demonstrating greatest inhibition, 10 were confirmed using both fluorescent and radioactive assays. The top two inhibitors (HAC001 and HAC002) demonstrated specific activity, with an IC(50) of 12.7 µM and 1.0 µM, respectively. In conclusion, we describe simple, fluorescent-based high-throughput screening (HTS) for the identification of inhibitors of de novo RdRp activity, using HCV G3a RdRp as the target. The HTS system could be used against any positive-sense RNA virus that cannot be cultured.

Smoking decreases the response of human lung macrophages to double-stranded RNA by reducing TLR3 expression.

BACKGROUND: Cigarette smoking is associated with increased frequency and duration of viral respiratory infections, but the underlying mechanisms are incompletely defined. We investigated whether smoking reduces expression by human lung macrophages (Mø) of receptors for viral nucleic acids and, if so, the effect on CXCL10 production. METHODS: We collected alveolar macrophages (AMø) by bronchoalveolar lavage of radiographically-normal lungs of subjects undergoing bronchoscopies for solitary nodules (n = 16) and of volunteers who were current or former smokers (n = 7) or never-smokers (n = 13). We measured expression of mRNA transcripts for viral nucleic acid receptors by real-time PCR in those AMø and in the human Mø cell line THP-1 following phorbol myristate acetate/vitamin D3 differentiation and exposure to cigarette smoke extract, and determined TLR3 protein expression using flow cytometry and immunohistochemistry. We also used flow cytometry to examine TLR3 expression in total lung Mø from subjects undergoing clinically-indicated lung resections (n = 25). Of these, seven had normal FEV1 and FEV1/FVC ratio (three former smokers, four current smokers); the remaining 18 subjects (14 former smokers; four current smokers) had COPD of GOLD stages I-IV. We measured AMø production of CXCL10 in response to stimulation with the dsRNA analogue poly(I:C) using Luminex assay. RESULTS: Relative to AMø of never-smokers, AMø of smokers demonstrated reduced protein expression of TLR3 and decreased mRNA for TLR3 but not TLR7, TLR8, TLR9, RIG-I, MDA-5 or PKR. Identical changes in TLR3 gene expression were induced in differentiated THP-1 cells exposed to cigarette smoke-extract in vitro for 4 hours. Among total lung Mø, the percentage of TLR3-positive cells correlated inversely with active smoking but not with COPD diagnosis, FEV1% predicted, sex, age or pack-years. Compared to AMø of never-smokers, poly(I:C)-stimulated production of CXCL10 was significantly reduced in AMø of smokers. CONCLUSIONS: Active smoking, independent of COPD stage or smoking duration, reduces both the percent of human lung Mø expressing TLR3, and dsRNA-induced CXCL10 production, without altering other endosomal or cytoplasmic receptors for microbial nucleic acids. This effect provides one possible mechanism for increased frequency and duration of viral lower respiratory tract infections in smokers. TRIAL REGISTRATION: ClinicalTrials.gov NCT00281190, NCT00281203 and NCT00281229.

Effects of Brazilian green propolis on double-stranded RNA-mediated induction of interferon-inducible gene and inhibition of recruitment of polymorphonuclear cells.

BACKGROUND: Propolis is a bee product with various biological properties, including an antiviral activity when taken orally. However, its mechanisms at the cellular and molecular level are not well understood. RESULTS: We investigated the effect of propolis on antiviral signaling in A549 cells transfected with double-stranded RNA (dsRNA), a model for viral infection. Pretreatment of the cells with propolis inhibited poly I:C (synthetic dsRNA)-induced interferon (IFN)-ß expression. Propolis had no effect on the dsRNA-induced expression of RIG-I-like receptors (RLRs), which are known as intracellular viral RNA sensors. As to the effect on antiviral executor genes, propolis enhanced myxovirus resistance 1 (MX1) expression, whereas interferon-inducible gene 6-16 (G1P3) and 2'-5'-oligoadenylate synthetase (OAS) were unaffected. All of these genes belong to the IFN-inducible genes, suggesting that the effect of propolis on antiviral signaling is not necessarily mediated by the autocrine regulation by IFN-ß. Propolis pretreatment inhibited dsRNA-induced interleukin-8 (IL8) and CCL5 expression, and consequently lowered polymorphonuclear leukocyte (PMN) chemotactic activity in the cell-conditioned medium. CONCLUSION: Taken together, these results suggest that propolis may suppress excess inflammatory responses without affecting the innate immunity during viral infection.

Severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase.

Severe acute respiratory syndrome (SARS) is a highly contagious disease, caused by SARS coronavirus (SARS-CoV), for which there are no approved treatments. We report the discovery of a potent inhibitor of SARS-CoV that blocks replication by inhibiting the unwinding activity of the SARS-CoV helicase (nsp13). We used a Förster resonance energy transfer (FRET)-based helicase assay to screen the Maybridge Hitfinder chemical library. We identified and validated a compound (SSYA10-001) that specifically blocks the double-stranded RNA (dsRNA) and dsDNA unwinding activities of nsp13, with 50% inhibitory concentrations (IC(50)s) of 5.70 and 5.30 µM, respectively. This compound also has inhibitory activity (50% effective concentration [EC(50)] = 8.95 µM) in a SARS-CoV replicon assay, with low cytotoxicity (50% cytotoxic concentration [CC(50)] = >250 µM), suggesting that the helicase plays a still unidentified critical role in the SARS-CoV life cycle. Enzyme kinetic studies on the mechanism of nsp13 inhibition revealed that SSYA10-001 acts as a noncompetitive inhibitor of nsp13 with respect to nucleic acid and ATP substrates. Moreover, SSYA10-001 does not affect ATP hydrolysis or nsp13 binding to the nucleic acid substrate. SSYA10-001 did not inhibit hepatitis C virus (HCV) helicase, other bacterial and viral RNA-dependent RNA polymerases, or reverse transcriptase. These results suggest that SSYA10-001 specifically blocks nsp13 through a novel mechanism and is less likely to interfere with the functions of cellular enzymes that process nucleic acids or ATP. Hence, it is possible that SSYA10-001 inhibits unwinding by nsp13 by affecting conformational changes during the course of the reaction or translocation on the nucleic acid. SSYA10-001 will be a valuable tool for studying the specific role of nsp13 in the SARS-CoV life cycle, which could be a model for other nidoviruses and also a candidate for further development as a SARS antiviral target.

Recognition of double-stranded RNA by guanidine-modified peptide nucleic acids.

Double-helical RNA has become an attractive target for molecular recognition because many noncoding RNAs play important roles in the control of gene expression. Recently, we discovered that short peptide nucleic acids (PNA) bind strongly and sequence selectively to a homopurine tract of double-helical RNA via formation of a triple helix. Herein, we tested if the molecular recognition of RNA could be enhanced by α-guanidine modification of PNA. Our study was motivated by the discovery of Ly and co-workers that the guanidine modification greatly enhances the cellular delivery of PNA. Isothermal titration calorimetry showed that the guanidine-modified PNA (GPNA) had reduced affinity and sequence selectivity for triple-helical recognition of RNA. The data suggested that in contrast to unmodified PNA, which formed a 1:1 PNA-RNA triple helix, GPNA preferred a 2:1 GPNA-RNA triplex invasion complex. Nevertheless, promising results were obtained for recognition of biologically relevant double-helical RNA. Consistent with enhanced strand invasion ability, GPNA derived from d-arginine recognized the transactivation response element of HIV-1 with high affinity and sequence selectivity, presumably via Watson-Crick duplex formation. On the other hand, strong and sequence selective triple helices were formed by unmodified and nucelobase-modified PNA and the purine-rich strand of the bacterial A-site. These results suggest that appropriate chemical modifications of PNA may enhance molecular recognition of complex noncoding RNAs.

Small-molecule inhibitors of the TLR3/dsRNA complex.

The protein-RNA interface has been regarded as "undruggable" despite its importance in many biological processes. The toll-like receptor 3 (TLR3)/double-stranded RNA (dsRNA) complex provides an exciting target for a number of infectious diseases and cancers. We describe the development of a series of small-molecule probes that were shown to be competitive inhibitors of dsRNA binding to TLR3 with high affinity and specificity. In a multitude of assays, compound 4a was profiled as a potent antagonist to TLR3 signaling and also repressed the expression of downstream signaling pathways mediated by the TLR3/dsRNA complex, including TNF-α and IL-1ß.

dsRNA-induced expression of thymic stromal lymphopoietin (TSLP) in asthmatic epithelial cells is inhibited by a small airway relaxant.

RATIONALE: Thymic Stromal Lymphopoietin (TSLP) is considered a hub cytokine that activates dendritic cells and T-cells producing asthma-like Th2-inflammation. Viral stimuli, a major cause of asthma exacerbations, have been shown to induce overexpression of TSLP in asthmatic epithelium. Capsazepine has multiple effects and is of interest because it relaxes human small airways. Here we have explored effects of capsazepine on viral surrogate (dsRNA)-induced TSLP and other cytokines (TNF-alpha, IL-8) in human bronchial epithelial cells (HBEC) from healthy and asthmatic donors. METHODS: HBEC obtained from healthy and asthmatic subjects were grown and stimulated with dsRNA. Cells pre-treated with capsazepine (3-30 µM), dexamethasone (0.1-10 µM) or an IkappaB-kinase inhibitor (PS1145, 30 µM) were also exposed to dsRNA (10 µg/ml). Cells and supernatants were harvested for analyses of gene expression (RT-qPCR) and protein production (ELISA,Western blot). RESULTS: dsRNA-induced TSLP, TNF-alpha, and IL-8 in asthmatic and non-asthmatic HBEC. Dexamethasone attenuated gene expression and protein release whereas capsazepine dose-dependently, and similar to a non-relaxant NFkB inhibitor (PS1145), completely inhibited dsRNA-induced TSLP and TNF-alpha in both healthy and asthmatic HBEC. Capsazepine reduced dsRNA-induced IL-8 and it prevented dsRNA-induced loss of the NF-κB repressor protein IkBα. CONCLUSION: Additional to its human small airway relaxant effects we now demonstrate that capsazepine has potent anti-inflammatory effects on viral stimulus-induced cytokines in HBEC from healthy as well as asthmatic donors. Based on these data we suggest that exploration of structure-activity amongst the multifaceted capsazepinoids is warranted in search for compounds of therapeutic value in viral-induced, steroid-resistant asthma.

Cardiac glycosides are potent inhibitors of interferon-ß gene expression.

Here we report that bufalin and other cardiac glycoside inhibitors of the sodium-potassium ATPase (sodium pump) potently inhibit the induction of the interferon-ß (IFNß) gene by virus, double-stranded RNA or double-stranded DNA. Cardiac glycosides increase the intracellular sodium concentration, which appears to inhibit the ATPase activity of the RNA sensor RIG-I, an essential and early component in the IFNß activation pathway. This, in turn, prevents the activation of the critical transcription factors IRF3 and NFκB. Bufalin inhibition can be overcome by expressing a drug-resistant variant of the sodium pump and knocking down the pump by short hairpin RNA inhibits IFNß expression. Thus, bufalin acts exclusively through the sodium pump. We also show that bufalin inhibits tumor necrosis factor (TNF) signaling, at least in part by interfering with the nuclear translocation of NFκB. These findings suggest that bufalin could be used to treat inflammatory and autoimmune diseases in which IFN or TNF are hyperactivated.

High-throughput screening for TLR3-IFN regulatory factor 3 signaling pathway modulators identifies several antipsychotic drugs as TLR inhibitors.

TLR3 is one of the major innate immune sensors of dsRNA. The signal transduction pathway activated by TLR3, upon binding to dsRNA, leads to the activation of two major transcription factors: NF-kappaB and IFN regulatory factor (IRF) 3. In an effort to identify specific chemical modulators of TLR3-IRF3 signal transduction pathway, we developed a cell-based readout system. Using the IFN-stimulated gene 56 promoter-driven firefly luciferase gene stably integrated in a TLR3-expressing HEK293 cell line, we were able to generate a cell line where treatment with dsRNA resulted in a dose-dependent induction of luciferase activity. A screen of two pharmacologically active compound libraries using this system identified a number of TLR3-IRF3 signaling pathway modulators. Among them we focused on a subset of inhibitors and characterized their mode of action. Several antipsychotic drugs, such as sertraline, trifluoperazine, and fluphenazine, were found to be direct inhibitors of the innate immune signaling pathway. These inhibitors also showed the ability to inhibit IFN-stimulated gene 56 induction mediated by TLR4 and TLR7/8 pathways. Interestingly, they did not show significant effects on TLR3-, TLR7-, and TLR8-mediated NF-kappaB activation. Detailed analysis of the signaling pathway indicated that these drugs might be exerting their inhibitory effects on IRF3 via PI3K signaling pathway. The data presented in this study provide mechanistic explanation of possible anti-inflammatory roles of some antipsychotic drugs.

Involvement of phosphatidylinositol 3-kinase-mediated up-regulation of I kappa B alpha in anti-inflammatory effect of gemfibrozil in microglia.

The present study underlines the importance of PI3K in mediating the anti-inflammatory effect of gemfibrozil, a prescribed lipid-lowering drug for humans, in mouse microglia. Gemfibrozil inhibited LPS-induced expression of inducible NO synthase (iNOS) and proinflammatory cytokines in mouse BV-2 microglial cells and primary microglia. By overexpressing wild-type and dominant-negative constructs of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) in microglial cells and isolating primary microglia from PPAR-alpha-/- mice, we have demonstrated that gemfibrozil inhibits the activation of microglia independent of PPAR-alpha. Interestingly, gemfibrozil induced the activation of p85alpha-associated PI3K (p110beta but not p110alpha) and inhibition of that PI3K by either chemical inhibitors or dominant-negative mutants abrogated the inhibitory effect of gemfibrozil. Conversely, overexpression of the constitutively active mutant of p110 enhanced the inhibitory effect of gemfibrozil on LPS-induced expression of proinflammatory molecules. Similarly, gemfibrozil also inhibited fibrillar amyloid beta (Abeta)-, prion peptide (PrP)-, dsRNA (poly IC)-, HIV-1 Tat-, and 1-methyl-4-phenylpyridinium (MPP+)-, but not IFN-gamma-, induced microglial expression of iNOS. Inhibition of PI3K also abolished the inhibitory effect of gemfibrozil on Abeta-, PrP-, poly IC-, Tat-, and MPP+-induced microglial expression of iNOS. Involvement of NF-kappaB activation in LPS-, Abeta-, PrP-, poly IC-, Tat-, and MPP+-, but not IFN-gamma-, induced microglial expression of iNOS and stimulation of IkappaBalpha expression and inhibition of NF-kappaB activation by gemfibrozil via the PI3K pathway suggests that gemfibrozil inhibits the activation of NF-kappaB and the expression of proinflammatory molecules in microglia via PI3K-mediated up-regulation of IkappaBalpha.