JAK Inhibitors AG-490 and WHI-P154 Decrease IFN-γ-Induced iNOS Expression and NO Production in Macrophages

In inflammation, inducible nitric oxide synthase (iNOS) produces nitric oxide (NO), which modulates inflammatory processes. We investigated the effects of Janus kinase (JAK) inhibitors, AG-490 and WHI-P154, on iNOS expression and NO production in J774 murine macrophages stimulated with interferon-γ (IFN-γ). JAK inhibitors AG-490 and WHI-P154 decreased IFN-γ-induced nuclear levels of signal transducer and activator of transcription 1α (STAT1α). JAK inhibitors AG-490 and WHI-P154 decreased also iNOS protein and mRNA expression and NO production in a concentration-dependent manner. Neither of the JAK inhibitors affected the decay of iNOS mRNA when determined by actinomycin D assay. Our results suggest that the inhibition of JAK-STAT1-pathway by AG-490 or WHI-P154 leads to the attenuation of iNOS expression and NO production in IFN-γ-stimulated macrophages

Inhibition of p38 mitogen-activated protein kinase enhances c-Jun N-terminal kinase activity: Implication in inducible nitric oxide synthase expression

BACKGROUND: Nitric oxide (NO) is an inflammatory mediator, which acts as a cytotoxic agent and modulates immune responses and inflammation. p38 mitogen-activated protein kinase (MAPK) signal transduction pathway is activated by chemical and physical stress and regulates immune responses. Previous studies have shown that p38 MAPK pathway regulates NO production induced by inflammatory stimuli. The aim of the present study was to investigate the mechanisms involved in the regulation of inducible NO synthesis by p38 MAPK pathway. RESULTS: p38 MAPK inhibitors SB203580 and SB220025 stimulated lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression and NO production in J774.2 murine macrophages. Increased iNOS mRNA expression was associated with reduced degradation of iNOS mRNA. Treatment with SB220025 increased also LPS-induced c-Jun N-terminal kinase (JNK) activity. Interestingly, JNK inhibitor SP600125 reversed the effect of SB220025 on LPS-induced iNOS mRNA expression and NO production. CONCLUSION: The results suggest that inhibition of p38 MAPK by SB220025 results in increased JNK activity, which leads to stabilisation of iNOS mRNA, to enhanced iNOS expression and to increased NO production

Variants of beta-glucan polysaccharides downregulate autoimmune inflammation

Common infections and polysaccharides, from bacteria and yeasts, could trigger psoriasis and psoriatic arthritis (PsA), and possibly rheumatoid arthritis (RA). The objective of this study was to investigate the effects of beta-glucan polysaccharides in the effector phase of arthritis and as regulators of psoriasis and PsA-like symptoms in mice. Collagen antibody induced arthritis was studied as a model of RA and mannan-induced psoriasis (MIP) was used as model for psoriasis and PsA, using mice with a mutation of Ncf1 on the B10.Q genetic background, making them highly disease susceptible. The mice were exposed to three common variants: 1,6-beta-glucan, 1,3-beta-glucan and 1,3-1,6-beta-glucan. These beta-glucans down-regulated disease in mice if administered simultaneously, before or after mannan. Interestingly, the protection was macrophage mannose receptor (MMR/CD206) dependent with a more pronounced protection long-term than short-term. The number of resident peritoneal macrophages decreased after in vivo challenge with beta-glucan and mannan compared to mannan alone, whereas the numbers of infiltrating cells correspondingly increased, further indicating macrophages as key for beta-glucan mediated regulation. At the doses tested, beta-glucans could not induce arthritis, psoriasis or PsA in wild-type mice. However, beta-glucans could ameliorate the PsA-like symptoms representing a new unforeseen possibility to explore for future clinical treatment.beta-glucan exerted anti-inflammatory activities in a murine model of psoriasis and psoriatic arthritis is, at least in part, mediated via the activation of CD206 on macrophage

Superoxide Dismutase 3 Limits Collagen-Induced Arthritis in the Absence of Phagocyte Oxidative Burst

Extracellular superoxide dismutase (SOD3), an enzyme mediating dismutation of superoxide into hydrogen peroxide, has been shown to reduce inflammation by inhibiting macrophage migration into injured tissues. In inflamed tissues, superoxide is produced by the phagocytic NOX2 complex, which consists of the catalytic subunit NOX2 and several regulatory subunits (e.g., NCF1). To analyze whether SOD3 can regulate inflammation in the absence of functional NOX2 complex, we injected an adenoviral vector overexpressing SOD3 directly into the arthritic paws of Ncf1∗/∗ mice with collagen-induced arthritis. SOD3 reduced arthritis severity in both oxidative burst-deficient Ncf1∗/∗ mice and also in wild-type mice. The NOX2 complex independent anti-inflammatory effect of SOD3 was further characterized in peritonitis, and SOD3 was found to reduce macrophage infiltration independently of NOX2 complex functionality. We conclude that the SOD3-mediated anti-inflammatory effect on arthritis and peritonitis operates independently of NOX2 complex derived oxidative burst

The macrophage mannose receptor regulate mannan-induced psoriasis, psoriatic arthritis, and rheumatoid arthritis-like disease models

The injection of mannan into mice can result in the development of psoriasis (Ps) and psoriatic arthritis (PsA), whereas co-injection with antibodies toward collagen type II leads to a chronic rheumatoid-like arthritis. The critical event in all these diseases is mannan-mediated activation of macrophages, causing more severe disease if the macrophages are deficient in neutrophil cytosolic factor 1 (Ncf1), i.e., lack the capacity to make a reactive oxygen species (ROS) burst. In this study, we investigated the role of one of the receptors binding mannan; the macrophage mannose receptor (MR, CD206). MR is a C-type lectin present on myeloid cells and lymphatics. We found that mice deficient in MR expression had more severe mannan-induced Ps, PsA as well as rheumatoid-like arthritis. Interestingly, the MR-mediated protection was partly lost in Ncf1 mutated mice and was associated with an type 2 macrophage expansion. In conclusion, these results show that MR protects against a pathogenic inflammatory macrophage response induced by mannan and is associated with induction of ROS.</p

Dual Specificity Phosphatase 1 Regulates Human Inducible Nitric Oxide Synthase Expression by p38 MAP Kinase

The role of dual specificity phosphatase 1 (DUSP1) in inducible nitric oxide synthase (iNOS) expression in A549 human pulmonary epithelial cells, J774 mouse macrophages and primary mouse bone marrow-derived macrophages (BMMs) was investigated. iNOS expression was induced by a cytokine mixture (TNF, IFNγ and IL-1β) in A549 cells and by LPS in J774 cells, and it was inhibited by p38 MAPK inhibitors SB202190 and BIRB 796. Stimulation with cytokine mixture or LPS enhanced also DUSP1 expression. Down-regulation of DUSP1 by siRNA increased p38 MAPK phosphorylation and iNOS expression in A549 and J774 cells. In addition, LPS-induced iNOS expression was enhanced in BMMs from DUSP1(−/−) mice as compared to that in BMMs from wild-type mice. The results indicate that DUSP1 suppresses iNOS expression by limiting p38 MAPK activity in human and mouse cells. Compounds that enhance DUSP1 expression or modulate its function may be beneficial in diseases complicated with increased iNOS-mediated NO production

Pharmacological regulation of inducible nitric oxide synthase expression. Effects of Janus kinase inhibitors, orazipone and simendans

Uutta tietoa typpioksidista, tulehduksesta ja tulehduslääkkeistä Typpioksidi on elimistön signaalimolekyyli, joka säätelee mm. immunologista puolustusjärjestelmää. Tulehdussairauksissa, kuten astmassa, reumassa, nivelrikossa ja septisessä sokissa, typpioksidin tuotanto on lisääntynyt. Laboratoriokokeissa on osoitettu, että typpioksidin tuotannon estäminen lieventää tulehdusta. Tässä väitöskirjassa tutkittiin ja etsittiin uusia keinoja ja mekanismeja, joilla voidaan estää typpioksidin tuotantoa tulehduksessa. Tutkimuksessa havaittiin Janus kinaaseihin vaikuttavien yhdisteiden vähentävän typpioksidin tuottoa tulehdussoluissa. Typpioksidituotantoa vähensi myös oratsiponi, joka on anti-inflammatorisesti vaikuttava molekyyli. Oratsiponi esti typpioksidia tuottavan entsyymin, indusoituvan typpioksidisyntaasin, ilmentymistä. Myös levosimendaanin, jota käytetään vaikean sydämen vajaatoiminnan hoitoon, havaittiin estävän tehokkaasti typpioksidisyntaasigeenin aktivoitumista ja typpioksidin tuottoa. Tällä saattaa olla merkitystä levosimendaanin havaituissa positiivisissa vaikutuksissa sydämen vajaatoiminnan tai septisen sokin hoidossa. Väitöskirjatutkimuksessa havaittiin tutkituilla uusilla lääkekandidaateilla olevan tulehdusta vaimentavia ominaisuuksia ja saatiin selville ilmiön taustalla olevia molekulaarisia mekanismeja. Tutkimus lisää tietoa tulehdustautien syntymekanismeista ja tuloksia voidaan käyttää hyväksi kehitettäessä uusia tulehdusta vaimentavia lääkeaineita. Väitöskirja on tehty osana Tampereen yliopiston lääketieteellisen tiedekunnan laajempaa uusien tulehduslääkkeiden kehitysprojektia, jota johtaa professori Eeva Moilanen.Inflammation is a defence mechanism by which an organism reacts to pathogens and tissue damage. Activated inflammatory cells produce signalling molecules that modulate the inflammatory response and the progress of inflammation. The inflammatory response is usually beneficial and required for survival, but when inappropriately focused or regulated, or excessive or prolonged, the inflammatory reaction may be harmful to the host. This is the case in septic shock and in chronic inflammatory diseases like arthritis. One of the genes that is activated in inflammation is inducible nitric oxide synthase (iNOS). iNOS is an enzyme that catalyzes a reaction where nitric oxide is produced. Nitric oxide is a signalling molecule that modulates immune response and inflammation, regulates the tonus of the blood vessels and acts as a neurotransmitter. Even though nitric oxide has many positive effects, it has detrimental effects in chronic inflammation, where it is overproduced. Nitric oxide is involved in the pathophysiology of various inflammatory diseases including septic shock, rheumatoid arthritis, osteoarthritis and asthma. Compounds that inhibit iNOS expression or activity have been found to possess anti-inflammatory properties in various forms of experimentally-induced inflammation. In the present study, the pharmacological regulation of iNOS expression and nitric oxide production was investigated in activated macrophages. Janus kinase (JAK2 and JAK3) inhibitors, orazipone and simendans were found to inhibit iNOS expression and nitric oxide production. JAK2 inhibitor AG-490 and JAK3 inhibitor WHI-P154 decreased the activation of signal transducer and activator of transcription (STAT) 1, which may explain their inhibitory effects on iNOS expression. Orazipone inhibited the activation of NF-kappaB, one important transcription factor for iNOS expression. Orazipone also inhibited the activation of STAT1. The results suggest that the thiol modulating property of orazipone is involved in the mechanism by which orazipone inhibits iNOS expression. Simendans did not affect the activation or the DNA binding activities of STAT1 or NF-kappaB, but they inhibited NF-kappaB-dependent transcription. iNOS mRNA decay was not affected by simendans but they decreased iNOS mRNA expression by inhibiting iNOS promoter activity. In the present study, the pharmacological compounds used were found to have anti-inflammatory properties. The molecular mechanisms behind their effects were described. The information can be utilized in the development of novel anti-inflammatory drugs

Pharmacological regulation of inducible nitric oxide synthase expression. Effects of Janus kinase inhibitors, orazipone and simendans

Uutta tietoa typpioksidista, tulehduksesta ja tulehduslääkkeistä Typpioksidi on elimistön signaalimolekyyli, joka säätelee mm. immunologista puolustusjärjestelmää. Tulehdussairauksissa, kuten astmassa, reumassa, nivelrikossa ja septisessä sokissa, typpioksidin tuotanto on lisääntynyt. Laboratoriokokeissa on osoitettu, että typpioksidin tuotannon estäminen lieventää tulehdusta. Tässä väitöskirjassa tutkittiin ja etsittiin uusia keinoja ja mekanismeja, joilla voidaan estää typpioksidin tuotantoa tulehduksessa. Tutkimuksessa havaittiin Janus kinaaseihin vaikuttavien yhdisteiden vähentävän typpioksidin tuottoa tulehdussoluissa. Typpioksidituotantoa vähensi myös oratsiponi, joka on anti-inflammatorisesti vaikuttava molekyyli. Oratsiponi esti typpioksidia tuottavan entsyymin, indusoituvan typpioksidisyntaasin, ilmentymistä. Myös levosimendaanin, jota käytetään vaikean sydämen vajaatoiminnan hoitoon, havaittiin estävän tehokkaasti typpioksidisyntaasigeenin aktivoitumista ja typpioksidin tuottoa. Tällä saattaa olla merkitystä levosimendaanin havaituissa positiivisissa vaikutuksissa sydämen vajaatoiminnan tai septisen sokin hoidossa. Väitöskirjatutkimuksessa havaittiin tutkituilla uusilla lääkekandidaateilla olevan tulehdusta vaimentavia ominaisuuksia ja saatiin selville ilmiön taustalla olevia molekulaarisia mekanismeja. Tutkimus lisää tietoa tulehdustautien syntymekanismeista ja tuloksia voidaan käyttää hyväksi kehitettäessä uusia tulehdusta vaimentavia lääkeaineita. Väitöskirja on tehty osana Tampereen yliopiston lääketieteellisen tiedekunnan laajempaa uusien tulehduslääkkeiden kehitysprojektia, jota johtaa professori Eeva Moilanen.Inflammation is a defence mechanism by which an organism reacts to pathogens and tissue damage. Activated inflammatory cells produce signalling molecules that modulate the inflammatory response and the progress of inflammation. The inflammatory response is usually beneficial and required for survival, but when inappropriately focused or regulated, or excessive or prolonged, the inflammatory reaction may be harmful to the host. This is the case in septic shock and in chronic inflammatory diseases like arthritis. One of the genes that is activated in inflammation is inducible nitric oxide synthase (iNOS). iNOS is an enzyme that catalyzes a reaction where nitric oxide is produced. Nitric oxide is a signalling molecule that modulates immune response and inflammation, regulates the tonus of the blood vessels and acts as a neurotransmitter. Even though nitric oxide has many positive effects, it has detrimental effects in chronic inflammation, where it is overproduced. Nitric oxide is involved in the pathophysiology of various inflammatory diseases including septic shock, rheumatoid arthritis, osteoarthritis and asthma. Compounds that inhibit iNOS expression or activity have been found to possess anti-inflammatory properties in various forms of experimentally-induced inflammation. In the present study, the pharmacological regulation of iNOS expression and nitric oxide production was investigated in activated macrophages. Janus kinase (JAK2 and JAK3) inhibitors, orazipone and simendans were found to inhibit iNOS expression and nitric oxide production. JAK2 inhibitor AG-490 and JAK3 inhibitor WHI-P154 decreased the activation of signal transducer and activator of transcription (STAT) 1, which may explain their inhibitory effects on iNOS expression. Orazipone inhibited the activation of NF-kappaB, one important transcription factor for iNOS expression. Orazipone also inhibited the activation of STAT1. The results suggest that the thiol modulating property of orazipone is involved in the mechanism by which orazipone inhibits iNOS expression. Simendans did not affect the activation or the DNA binding activities of STAT1 or NF-kappaB, but they inhibited NF-kappaB-dependent transcription. iNOS mRNA decay was not affected by simendans but they decreased iNOS mRNA expression by inhibiting iNOS promoter activity. In the present study, the pharmacological compounds used were found to have anti-inflammatory properties. The molecular mechanisms behind their effects were described. The information can be utilized in the development of novel anti-inflammatory drugs