Tiina Leppänen

Bilberries reduce low-grade inflammation in individuals with features of metabolic syndrome

SCOPE Low-grade inflammation is a hallmark of cardiometabolic risk. Bilberries (Vaccinium myrtillus) are rich in polyphenols with potential anti-inflammatory properties. We studied the impact of bilberries on inflammation and gene expression profile in peripheral blood mononuclear cells in subjects with metabolic syndrome. METHODS AND RESULTS In randomized, controlled dietary intervention, the participants consumed either a diet rich in bilberries (n = 15) or a control diet (n = 12). The bilberry group consumed daily an equivalent dose of 400 g fresh bilberries, while the control group maintained their habitual diet. No differences were found between the groups in body weight, glucose, or lipid metabolism, but bilberry supplementation tended to decrease serum high-sensitivity C-reactive protein, IL-6, IL-12, and LPS concentrations. An inflammation score was significantly different between the groups (p = 0.024). In transcriptomics analyses (three participants with improved oral glucose tolerance test in the bilberry group), Toll-like receptor signaling, cytoplasmic ribosomal proteins, and B-cell receptor signaling pathways were differently regulated. QPCR analyses (n = 13 and 11 in the bilberry and control groups, respectively) showed decreased expression of MMD and CCR2 transcripts associated with monocyte and macrophage function associated genes. CONCLUSION Regular bilberry consumption may reduce low-grade inflammation indicating decreased cardiometabolic risk in the long term.

Lingonberry (Vaccinium vitis-idaea) and European Cranberry (Vaccinium microcarpon) Proanthocyanidins: Isolation, Identification, and Bioactivities

European, small-fruited cranberries (Vaccinium microcarpon) and lingonberries (Vaccinium vitis-idaea) were characterized for their phenolic compounds and tested for antioxidant, antimicrobial, antiadhesive, and antiinflammatory effects. The main phenolic compounds in both lingonberries and cranberries were proanthocyanidins comprising 63-71% of the total phenolic content, but anthocyanins, hydroxycinnamic acids, hydroxybenzoic acids, and flavonols were also found. Proanthocyanidins are polymeric phenolic compounds consisting mainly of catechin, epicatechin, gallocatechin, and epigallocatechin units. In the present study, proanthocyanidins were divided into three groups: dimers and trimers, oligomers (mDP 4-10), and polymers (mDP > 10). Catechin, epicatechin, A-type dimers and trimers were found to be the terminal units of isolated proanthocyanidin fractions. Inhibitions of lipid oxidation in liposomes were over 70% and in emulsions over 85%, and in most cases the oligomeric or polymeric fraction was the most effective. Polymeric proanthocyanidin extracts of lingonberries and cranberries were strongly antimicrobial against Staphylococcus aureus, whereas they had no effect on other bacterial strains such as Salmonella enterica sv. Typhimurium, Lactobacillus rhamnosus and Escherichia coli. Polymeric fraction of cranberries and oligomeric fractions of both lingonberries and cranberries showed an inhibitory effect on hemagglutination of E. coli, which expresses the M hemagglutin. Cranberry phenolic extract inhibited LPS-induced NO production in a dose-dependent manner, but it had no major effect on iNOS of COX-2 expression. At a concentration of 100 μg/mL cranberry phenolic extract inhibited LPS-induced IL-6, IL-1β and TNF-α production. Lingonberry phenolics had no significant effect on IL-1β production but inhibited IL-6 and TNF-α production at a concentration of 100 μg/mL similarly to cranberry phenolic extract. In conclusion the phenolics, notably proanthocyanidins (oligomers and polymers), in both lingonberries and cranberries exert multiple bioactivities that may be exploited in food development.

TRPA1 Contributes to the Acute Inflammatory Response and Mediates Carrageenan-Induced Paw Edema in the Mouse

Transient receptor potential ankyrin 1 (TRPA1) is an ion channel involved in thermosensation and nociception. TRPA1 is activated by exogenous irritants and also by oxidants formed in inflammatory reactions. However, our understanding of its role in inflammation is limited. Here, we tested the hypothesis that TRPA1 is involved in acute inflammatory edema. The TRPA1 agonist allyl isothiocyanate (AITC) induced inflammatory edema when injected intraplantarly to mice, mimicking the classical response to carrageenan. Interestingly, the TRPA1 antagonist HC-030031 and the cyclo-oxygenase (COX) inhibitor ibuprofen inhibited not only AITC but also carrageenan-induced edema. TRPA1-deficient mice displayed attenuated responses to carrageenan and AITC. Furthermore, AITC enhanced COX-2 expression in HEK293 cells transfected with human TRPA1, a response that was reversed by HC-030031. This study demonstrates a hitherto unknown role of TRPA1 in carrageenan-induced inflammatory edema. The results also strongly suggest that TRPA1 contributes, in a COX-dependent manner, to the development of acute inflammation.

Effects of ellagitannin‐rich berries on blood lipids, gut microbiota, and urolithin production in human subjects with symptoms of metabolic syndrome

Ellagitannins are polyphenols abundant in strawberries, raspberries, and cloudberries. The effects of a mixture of these berries were studied in a randomized controlled trial with subjects having symptoms of metabolic syndrome. The study focused on serum lipid profiles, gut microbiota, and ellagitannin metabolites. The results indicate that bioavailability of ellagitannins appears to be dependent on the composition of gut microbiota.

PPARα agonists inhibit nitric oxide production by enhancing iNOS degradation in LPS-treated macrophages

Nitric oxide (NO) production through the inducible nitric oxide synthase (iNOS) pathway is increased in response to pro‐inflammatory cytokines and bacterial products. In inflammation, NO has pro‐inflammatory and regulatory effects. Peroxisome proliferator‐activated receptors (PPARs), members of the nuclear steroid receptor superfamily, regulate not only metabolic but also inflammatory processes. The aim of the present study was to investigate the role of PPARα in the regulation of NO production and iNOS expression in activated macrophages.

Protein Kinase C and its Inhibitors in the Regulation of Inflammation: Inducible Nitric Oxide Synthase as an Example

Protein kinase C (PKC) is a family of ten isoenzymes that play a crucial role in cellular signal transduction. Studies with PKC knockout animals have revealed that many of the isoenzymes are involved in cell growth, proliferation and differentiation. Several PKC isoenzymes have also been shown to be important mediators in inflammation and immunity, particularly in lymphocyte responses. However, less is known about the role of PKC in the regulation of the expression of inflammatory genes. In inflammatory processes, nitric oxide is primarily produced by inducible nitric oxide synthase (iNOS) in inflammatory cells, such as macrophages. In innate immunity, nitric oxide functions as an effector molecule towards the infectious organisms. Increased levels of nitric oxide are also produced by inflammatory and tissue cells in inflammatory diseases, such as asthma and arthritis. In this MiniReview, the role of PKC isoenzymes in the pathogenesis and as a potential drug target in inflammation will be discussed presenting iNOS as an example of an inflammatory gene regulated by the pleiotropic PKC signalling pathway.

The expression of interleukin-12 is increased by MAP kinase phosphatase-1 through a mechanism related to interferon regulatory factor 1.

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a nuclear tyrosine/threonine phosphatase that inhibits p38 mitogen-activated protein kinase (MAPK) activity. We and others have shown that MKP-1 deficiency leads to excessive activation of innate immunity and inflammatory gene expression. Surprisingly, the present study shows that MKP-1 is a positive regulator of IL-12 expression in macrophages suggesting a stimulatory effect on Th1 type immune response. In the present study, we found that LPS-induced expression of IL-12p40 was lower in primary mouse peritoneal macrophages (PMs) and bone marrow-derived macrophages from MKP-1 deficient mice than in cells from wild-type mice whereas TNF expression was enhanced as expected. Correspondingly, the inhibition of p38 MAPK by pharmacologic inhibitors BIRB 796 and SB 202190 enhanced LPS-induced IL-12p40 production. Silencing of interferon regulatory factor 1 (IRF1) by siRNA inhibited the expression of IL-12p40 in J774 macrophages, showing that IRF1 is an important factor regulating IL-12p40 expression. BIRB 796 enhanced LPS-induced expression of IRF1 in J774 macrophages and in PMs from wild-type mice, and IRF1 expression was reduced in PMs from MKP-1 deficient mice. In conclusions, our results show that MKP-1 increases and p38 MAPK decreases the expression of IL-12 by enhancing the expression of IRF1. MKP-1, through regulation of IRF1 and IL-12, therefore may be an important factor supporting the development of Th1 type of immune response and anti-microbial defense.

Pinosylvin and Monomethylpinosylvin, Constituents of an Extract from the Knot of Pinus sylvestris, Reduce Inflammatory Gene Expression and Inflammatory Responses in Vivo

Scots pine (Pinus sylvestris) is known to be rich in phenolic compounds, which may have anti-inflammatory properties. The present study investigated the anti-inflammatory effects of a knot extract from P. sylvestris and two stilbenes, pinosylvin and monomethylpinosylvin, isolated from the extract. Inflammation is characterized by increased release of pro-inflammatory and regulatory mediators including nitric oxide (NO) produced by the inducible nitric oxide synthase (iNOS) pathway. The knot extract (EC50 values of 3 and 3 μg/mL) as well as two of its constituents, pinosylvin (EC50 values of 13 and 15 μM) and monomethylpinosylvin (EC50 values of 8 and 12 μM), reduced NO production and iNOS expression in activated macrophages. They also inhibited the production of inflammatory cytokines IL-6 and MCP-1. More importantly, pinosylvin and monomethylpinosylvin exerted a clear anti-inflammatory effect (80% inhibition at the dose of 100 mg/kg) in the standard in vivo model, carrageenan-induced paw inflammation in the mouse, with the effect being comparable to that of a known iNOS inhibitor L-NIL. The results reveal that the Scots pine stilbenes pinosylvin and monomethylpinosylvin are potential anti-inflammatory compounds.

Running a Marathon Induces Changes in Adipokine Levels and in Markers of Cartilage Degradation – Novel Role for Resistin

Running a marathon causes strenuous joint loading and increased energy expenditure. Adipokines regulate energy metabolism, but recent studies have indicated that they also exert a role in cartilage degradation in arthritis. Our aim was to investigate the effects of running a marathon on the levels of adipokines and indices of cartilage metabolism. Blood samples were obtained from 46 male marathoners before and after a marathon run. We measured levels of matrix metalloproteinase-3 (MMP-3), cartilage oligomeric protein (COMP) and chitinase 3-like protein 1 (YKL-40) as biomarkers of cartilage turnover and/or damage and plasma concentrations of adipokines adiponectin, leptin and resistin. Mean marathon time was 3∶30∶46±0∶02∶46 (h:min:sec). The exertion more than doubled MMP-3 levels and this change correlated negatively with the marathon time (r = –0.448, p = 0.002). YKL-40 levels increased by 56% and the effect on COMP release was variable. Running a marathon increased the levels of resistin and adiponectin, while leptin levels remained unchanged. The marathon-induced changes in resistin levels were positively associated with the changes in MMP-3 (r = 0.382, p = 0.009) and YKL-40 (r = 0.588, p<0.001) and the pre-marathon resistin levels correlated positively with the marathon induced change in YKL-40 (r = 0.386, p = 0.008). The present results show the impact of running a marathon, and possible load frequency, on cartilage metabolism: the faster the marathon was run, the greater was the increase in MMP-3 levels. Further, the results introduce pro-inflammatory adipocytokine resistin as a novel factor, which enhances during marathon race and associates with markers of cartilage degradation.

Betulin Derivatives Effectively Suppress Inflammation in Vitro and in Vivo.

Betulin is a pharmacologically active triterpenoid found in the bark of the birch tree (Betula sp. L.). Betulin and betulinic acid are structurally related to anti-inflammatory steroids, but little is known about their potential anti-inflammatory properties. In the present study, the inflammatory gene expression and the anti-inflammatory properties of betulin, betulinic acid, and 16 semisynthetic betulin derivatives were investigated. Betulin derivatives 3, 4, and 5 selectively inhibited the expression of the inducible nitric oxide synthase (iNOS) in a post-transcriptional manner. They also inhibited nitric oxide (NO) production but had no effect on the other inflammatory factors studied. More interestingly, a new anti-inflammatory betulin derivative 9 with a wide-spectrum anti-inflammatory activity was discovered. Compound 9 was found to suppress the expression of cytokines interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1), as well as that of prostaglandin synthase-2 (COX-2) in addition to iNOS. The in vivo anti-inflammatory effect of compound 9 was indicated via significant suppression of the carrageenan-induced paw inflammation in mice. The results show, for the first time, that the pyrazole-fused betulin derivative (9) and related compounds have anti-inflammatory properties that could be utilized in drug development.