Mari Hämäläinen

Anti-Inflammatory Effects of Flavonoids: Genistein, Kaempferol, Quercetin, and Daidzein Inhibit STAT-1 and NF-κB Activations, Whereas Flavone, Isorhamnetin, Naringenin, and Pelargonidin Inhibit only NF-κB Activation along with Their Inhibitory Effect on iNOS Expression and NO Production in Activated Macrophages

In inflammation, bacterial products and proinflammatory cytokines induce the formation of large amounts of nitric oxide (NO) by inducible nitric oxide synthase (iNOS), and compounds that inhibit NO production have anti-inflammatory effects. In the present study, we systematically investigated the effects of 36 naturally occurring flavonoids and related compounds on NO production in macrophages exposed to an inflammatory stimulus (lipopolysaccharide, LPS), and evaluated the mechanisms of action of the effective compounds. Flavone, the isoflavones daidzein and genistein, the flavonols isorhamnetin, kaempferol and quercetin, the flavanone naringenin, and the anthocyanin pelargonidin inhibited iNOS protein and mRNA expression and also NO production in a dose-dependent manner. All eight active compounds inhibited the activation of nuclear factor-κB (NF-κB), which is a significant transcription factor for iNOS. Genistein, kaempferol, quercetin, and daidzein also inhibited the activation of the signal transducer and activator of transcription 1 (STAT-1), another important transcription factor for iNOS. The present study characterises the effects and mechanisms of naturally occurring phenolic compounds on iNOS expression and NO production in activated macrophages. The results partially explain the pharmacological efficacy of flavonoids as anti-inflammatory compounds.

Breast milk fatty acids, eicosanoids, and cytokines in mothers with and without allergic disease.

Allergic disease (AD), including atopic eczema, asthma, allergic rhinitis, and food allergy, is characterized by an imbalance between cytokines produced by distinct T-helper cell subtypes. Whether this imbalance can be transferred from mother to breast milk remains to be established. The objective was to investigate the concentrations and interactions of nutritional and inflammatory factors in breast milk. Breast milk samples were collected from mothers with AD (n = 43) and without AD (n = 51). The concentrations of transforming growth factor (TGF)-β2, tumor necrosis factor-α, IL-4, IL-10, prostaglandin E2, and cysteinyl leukotrienes were measured by immunoassays and fatty acid composition by gas chromatography. Mothers with AD had a lower concentration of TGF-β2 in breast milk [median (interquartile range), 420 (278–701) ng/L] compared with those without AD [539 (378–1108) ng/L;p = 0.003], whereas other cytokines, prostaglandin E2, and cysteinyl leukotriene concentrations or fatty acid composition were not significantly different between the groups. The breast milk inflammatory factors and fatty acid composition were shown to be related. A positive association was observed between TGF-β2 and the proportion of polyunsaturated fatty acids (p = 0.038) and a negative association between TGF-β2 and the proportion of saturated fatty acids (p = 0.029) in breast milk. The reduced TGF-β2 concentration in the breast milk of mothers with AD may interfere with the development of the mucosal immune system of the breast-fed infant. The observed associations between nutritional and inflammatory factors in breast milk suggest that it may be possible to influence the immunologic properties of breast milk by dietary intervention of the mother.

Effects of Flavonoids on Prostaglandin E2 Production and on COX-2 and mPGES-1 Expressions in Activated Macrophages

Prostaglandin E2 (PGE2) has a central role in inflammation and both cyclooxygenase-2 (COX-2) and prostaglandin E synthases are critical enzymes in its synthesis. In inflammation, bacterial products and cytokines enhance the expression of COX-2 and inducible microsomal prostaglandin E synthase-1 (mPGES-1) which are functionally coupled to result in increased PGE2 formation in macrophages and tissue cells. In the present study, we systematically investigated the effects of 26 naturally occurring flavonoids on PGE2 production and on COX-2 and mPGES-1 expression in activated macrophages. Twelve flavonoids, i.e., flavone, luteolin-7-glucoside, kaempferol, isorhamnetin, morin, quercetin, naringenin, taxifolin, pelargonidin, daidzein, genistein, and genistin effectively inhibited lipopolysaccharide (LPS)-induced PGE2 production. Four flavonoids (flavone, isorhamnetin, daidzein, and genistein) inhibited significantly LPS-induced COX-2 expression, while mPGES-1 expression was downregulated by kaempferol and isorhamnetin. The present study characterizes the effects of flavonoids on PGE2 production and on COX-2 and mPGES-1 expression in activated macrophages. The results add to our knowledge of the anti-inflammatory actions of flavonoids and introduce kaempferol and isorhamnetin as compounds capable of downregulating the expression of mPGES-1.

Calcineurin inhibitors, cyclosporin A and tacrolimus inhibit expression of inducible nitric oxide synthase in colon epithelial and macrophage cell lines

Nitric oxide (NO) production is increased in inflammatory bowel disease and selective inducible nitric oxide synthase (iNOS) inhibitors have proved to be anti-inflammatory in experimentally induced colitis. The aim of the present study was to test if drugs used in the treatment of inflammatory bowel disease effect on NO production in colon epithelial and macrophage cell lines. We tested the effects of cyclosporin A, tacrolimus (FK-506), methotrexate, sulfasalazine, 5-aminosalicylic acid and two novel TNF-alpha antagonists etanercept and infliximab on endotoxin-induced NO production in human T84 colon epithelial cells and in murine J774 macrophages. Cyclosporin A and FK-506 inhibited iNOS expression, and subsequent NO production, in a dose-dependent manner at therapeutically achievable drug concentrations in both cell lines. The effect was most pronounced when cyclosporin A was given 1 h prior to 4 h after endotoxin, and declined thereafter, indicating that cyclosporin A does not inhibit iNOS activity. Neither cyclosporin A nor FK-506 altered the activation of nuclear factor-kappaB (NF-kappaB) that is a critical transcription factor for iNOS. Sulfasalazine inhibited NO production slightly only when given at high (100 microM) drug concentrations. Methotrexate, 5-aminosalicylic acid and TNF-alpha antagonists infliximab and etanercept were practically ineffective. Two inhibitors of phosphatase calcineurin, cyclosporin A and FK-506, inhibited iNOS expression and NO production in human T84 colon epithelial cells and in murine J774 macrophages by an NF-kappaB independent manner. These findings are implicated in the anti-inflammatory action of these compounds.

Effects of levo- and dextrosimendan on NF-κB-mediated transcription, iNOS expression and NO production in response to inflammatory stimuli

Levosimendan is used in the treatment of decompensated heart failure. It increases the contractility of the myocardium by sensitizing troponin C to calcium. In addition, levosimendan has been reported to have beneficial effects in experimental models of septic shock. Because heart failure and sepsis have been associated with excessive nitric oxide (NO) production through inducible NOS (iNOS), we investigated the effects of the simendans on NO production and iNOS expression and on generation of pro‐inflammatory cytokines.

Breast Milk Fatty Acids May Link Innate and Adaptive Immune Regulation: Analysis of Soluble CD14, Prostaglandin E2, and Fatty Acids

In addition to its role in sensing intraluminal microbial antigens, soluble (s)CD14 may regulate immune responses by its lesser known function as a lipid carrier with possible influences in the production of fatty acid-derived eicosanoids. We investigated the interrelations of fatty acids, prostaglandin E2 (PGE2), and sCD14 and their role in infant atopic eczema during the first year of life. Serum and breast milk samples from mothers and serum samples from their infants were collected at infants age 3 mo and analyzed for sCD14 and PGE2 concentrations and for fatty acid compositions. The main correlation of sCD14 was with arachidonic acid (20:4n-6) (AA). Dihomo-γ-linolenic acid (20:3n-6) (DHGLA) and the ratio of n-6 to n-3 fatty acids correlated positively and docosahexaenoic acid (22:6n-3) (DHA) and sum of n-3 fatty acid negatively with PGE2 in mothers serum and linoleic acid (LA) negatively with PGE2 in breast milk. Soluble CD14 tended to be higher and LA, total polyunsaturated fatty acid (PUFA), and sum of n-6 fatty acids were lower in breast milk received by infants with atopic eczema compared with those without. These results suggest that fatty acids contribute to the regulation of innate and adaptive immune responses and link intraluminal exposures, mothers diet, and microbes.

Cytokine and adipokine alterations in patients with schizophrenia treated with clozapine.

Metabolic syndrome is associated with both schizophrenia and antipsychotic medication, especially clozapine, with alterations in inflammatory cytokines and adipokines. However, the data in this field is heterogeneous and the sample sizes of the patients are limited. In this study we assessed the serum levels of cytokines/adipokines IL-6, IL-1Ra, hs-CRP and adiponectin, and components of metabolic syndrome in 190 patients with treatment resistant schizophrenia treated with clozapine. Substantial metabolic comorbidity was found in this patient group; overweight/obesity, smoking, hypertriglyceridemia, low HDL-cholesterol, high HOMA-IR, low adiponectin levels, elevated hs-CRP levels and elevated IL-1Ra levels. Elevated IL-1Ra levels are associated with insulin resistance, obesity and hypertriglyceridemia. Low adiponectin levels were associated with hypertriglyceridemia, low HDL cholesterol and high glucose, and in male patients also with obesity and high IL-1Ra levels. After controlling for confounding factors age and smoking, levels of IL-1Ra and hs-CRP associated with obesity, and the levels of IL-6 associated with obesity in female patients. We conclude that there are partly gender dependent cytokine and adipokine alterations in patients with schizophrenia on clozapine treatment associated with metabolic comorbidity. The genetic background of these cytokine alterations needs to be further investigated.

Attenuation of TNF production and experimentally induced inflammation by PDE4 inhibitor rolipram is mediated by MAPK phosphatase‐1

3′,5′‐Cyclic nucleotide PDE4 is expressed in several inflammatory and immune cells, and PDE4 catalyses the hydrolysis of cAMP to 5′AMP, down‐regulating cAMP signalling in cells. MAPK phosphatase‐1 (MKP‐1) is an endogenous p38 MAPK signalling suppressor and limits inflammatory gene expression and inflammation. In the present study, we investigated the effect of a PDE4 inhibitor rolipram on MKP‐1 expression and whether MKP‐1 is involved in the anti‐inflammatory effects of rolipram.

Monosodium iodoacetate-induced inflammation and joint pain are reduced in TRPA1 deficient mice – potential role of TRPA1 in osteoarthritis

OBJECTIVES Intra-articularly injected monosodium iodoacetate (MIA) induces joint pathology mimicking osteoarthritis (OA) and it is a widely used experimental model of OA. MIA induces acute inflammation, cartilage degradation and joint pain. Transient Receptor Potential Ankyrin 1 (TRPA1) is an ion channel known to mediate nociception and neurogenic inflammation. Here, we tested the hypothesis that TRPA1 would be involved in the development of MIA-induced acute inflammation, cartilage changes and joint pain. METHODS The effects of pharmacological blockade (by TCS 5861528) and genetic depletion of TRPA1 were studied in MIA-induced acute paw inflammation. Cartilage changes (histological scoring) and joint pain (weight-bearing test) in MIA-induced experimental OA were compared between wild type and TRPA1 deficient mice. The effects of MIA were also studied in primary human OA chondrocytes and in mouse cartilage. RESULTS MIA evoked acute inflammation, degenerative cartilage changes and joint pain in wild type mice. Interestingly, these responses were attenuated in TRPA1 deficient animals. MIA-induced paw inflammation was associated with increased tissue levels of substance P; and the inflammatory edema was reduced by pretreatment with catalase, with the TRPA1 antagonist TCS 5861528 and with the neurokinin 1 receptor antagonist L703,606. In chondrocytes, MIA enhanced interleukin-1 induced cyclooxygenase-2 (COX-2) expression, an effect that was blunted by pharmacological inhibition and genetic depletion of TRPA1. CONCLUSIONS TRPA1 was found to mediate acute inflammation and the development of degenerative cartilage changes and joint pain in MIA-induced experimental OA in the mouse. The results reveal TRPA1 as a potential mediator and drug target in OA.

YKL-40 as a Novel Factor Associated with Inflammation and Catabolic Mechanisms in Osteoarthritic Joints

YKL-40 is associated with tissue injury and inflammation, and consequently to diseases in which these mechanisms lead to tissue degradation, for example, asthma and rheumatoid arthritis. The purpose of the present study was to investigate if YKL-40 is also a significant factor in osteoarthritis (OA) by assessing associations of YKL-40 with mediators related to the pathogenesis of OA: cartilage destructing matrix metalloproteinases (MMPs) and proinflammatory cytokines interleukin-6 (IL-6) and interleukin-17 (IL-17). Cartilage, synovial fluid (SF), and plasma samples were obtained from 100 OA patients undergoing total knee replacement surgery. SF levels of YKL-40 (1027.9 ± 78.3 ng/mL) were considerably higher than plasma levels (67.2 ± 4.5 ng/mL) and correlated with YKL-40 released from cartilage samples obtained from the same patients (r = 0.37, P = 0.010), indicating that YKL-40 is produced by OA cartilage. Interestingly, YKL-40 concentrations in OA SF correlated positively with MMP-1 (r = 0.36, P = 0.014), MMP-3 (r = 0.46, P = 0.001), IL-6 (r = 0.57, P < 0.001), and IL-17 (r = 0.52, P = 0.010) levels. Moreover, IL-6 and IL-17 enhanced YKL-40 production in human primary chondrocyte cultures. The present study introduces YKL-40 as a cartilage-derived factor associated with mediators of inflammation and cartilage destruction involved in the pathogenesis of OA.