AbstractDiets rich in fruits and vegetables (FV), which contain (poly)phenols, protect against age-related inflammation and chronic diseases. T-lymphocytes contribute to systemic cytokine production and are modulated by FV intake. Little is known about the relative potency of different (poly)phenols in modulating cytokine release by lymphocytes. We compared thirty-one (poly)phenols and six (poly)phenol mixtures for effects on pro-inflammatory cytokine release by Jurkat T-lymphocytes. Test compounds were incubated with Jurkat cells for 48 h at 1 and 30 µm, with or without phorbol ester treatment at 24 h to induce cytokine release. Three test compounds that reduced cytokine release were further incubated with primary lymphocytes at 0·2 and 1 µm for 24 h, with lipopolysaccharide added at 5 h. Cytokine release was measured, and generation of H2O2 by test compounds was determined to assess any potential correlations with cytokine release. A number of (poly)phenols significantly altered cytokine release from Jurkat cells (P<0·05), but H2O2 generation did not correlate with cytokine release. Resveratrol, isorhamnetin, curcumin, vanillic acid and specific (poly)phenol mixtures reduced pro-inflammatory cytokine release from T-lymphocytes, and there was evidence for interaction between (poly)phenols to further modulate cytokine release. The release of interferon-γ induced protein 10 by primary lymphocytes was significantly reduced following treatment with 1 µm isorhamnetin (P<0·05). These results suggest that (poly)phenols derived from onions, turmeric, red grapes, green tea and açai berries may help reduce the release of pro-inflammatory mediators in people at risk of chronic inflammation.

TNF-α is a key inflammatory mediator and is proposed to induce transcriptional responses via the mitochondrial generation of Reactive Oxygen Species (ROS). The aim of this study was to determine the effect of TNF-α on the production of myokines by skeletal muscle. Significant increases were seen in the release of IL-6, MCP-1/CCL2, RANTES/CCL5 and KC/CXCL1 and this release was inhibited by treatment with Brefeldin A, suggesting a golgi-mediated release of cytokines by muscle cells. An increase was also seen in superoxide in response to treatment with TNF-α, which was localised to the mitochondria and this was also associated with activation of NF-κB. The changes in superoxide, activation of NF-kB and release of myokines were attenuated following pre-treatment with SS-31 peptide indicating that the ability of TNF-α to induce myokine release may be mediated through mitochondrial superoxide, which is, at least in part, associated with activation of the redox sensitive transcription factor NF-kB.

TNF‐α is a key inflammatory mediator and is proposed to induce transcriptional responses via the generation of Reactive Oxygen Species (ROS). The aim of this study was to determine the role of mitochondrial superoxide generation in TNF‐α ‐ induced generation of myokines by skeletal muscle. Significant elevation in the release of IL‐6 (control untreated: 20.1pg/ml ± 2.3; TNF‐α treated: 73.8pg/ml ± 9.3), CCL2/MCP‐1 (980pg/ml ± 74.8; 31194pg/ml ± 5409), CCL5/RANTES (22.4pg/ml ± 2.09; 604pg/ml ± 93.4) and CXCL1/KC (236pg/ml ± 32.6; 3477pg/ml ± 383) and in superoxide, localised to the mitochondria were seen in response to TNF‐α treatment of muscle cells and this was associated with activation of NFκB. The TNF‐α mediated changes in superoxide, activation of NFκB and increased release of myokines were attenuated following pre‐treatment with the mitochondria targeted antioxidant, SS‐31 peptide (IL‐6, 11.9pg/ml ± 1.83; MCP‐1, 11322pg/ml ± 1124; RANTES, 252pg/ml ± 51.4; KC, 1166pg/ml ± 204) indicating that the ability of TNF‐α to induce myokine release is mediated through mitochondrial superoxide, which is, at least in part, associated with activation of the redox sensitive transcription factor NFκB. These novel findings provide evidence that targeting mitochondrial ROS could provide a potential for therapy for inflammatory myopathies. The authors thank the Medical Research Council and National Institute on Aging (grant AG‐020591) for their generous financial support and Dr M.B Reid (University of Kentucky Center for Muscle Biology) for the kind gift of the SS‐31 peptide.

Polyphenols and phenolic acids are abundant in fruits and vegetables. Twenty‐nine dietary phenolic compounds were screened for effects on cytokine release (interleukin 2 (IL2), interleukin 8 (IL8), and tumour necrosis factor α (TNFα)) by Jurkat T‐lymphocytes. Cells were treated with phenolic compounds for 48h, with or without stimulation with 25ng/ml phorbol myristate acetate and 5μg/ml phytohaemagglutinin (PMA/PHA) to induce cytokine release at the 24h time point.Polyphenols had more anti‐inflammatory effects than phenolic acids. In unstimulated cells, 1μM resveratrol decreased IL2 by 42±7% and IL8 by 32±8%, compared with vehicle controls (p<0.05). For PMA/PHA stimulated cells, 1μM isorhamnetin reduced IL2 by 50±4%, IL8 by 58±6% and TNFα by 63±7%, and curcumin reduced IL2 by 43±14%, IL8 by 30±7% and TNFα by 22±5 (p<0.05). Compounds that significantly modulated cytokine release were investigated for effects on glutathione redox status. Cells treated with 1µM epigallocatechin gallate (EGCG) or pyrogallol increased total and reduced glutathione compared with vehicle controls and reduced glutathione redox potentials from ‐211±3mV (controls) to ‐216±2mV (EGCG) and ‐221±2mV (pyrogallol; p<0.05). Polyphenols demonstrating anti‐inflammatory and redox modulating effects will be further investigated using proteomics to identify cellular pathways responsive to treatments with polyphenols.Grant Funding Source: Supported by BBSRC DRINC and Unilever

Quercetin and (‐)‐epigallocatechingallate (EGCG) are dietary polyphenols claimed to exert anti‐inflammatory effects. Jurkat T‐lymphocytes were incubated with 5μM quercetin or ECGC for 48h, with or without 25ng/ml phorbol myristoyl acetate and 5μg/ml phytohaemagglutinin (PMA/PHA) at 24h to induce cytokine release. Inflammatory cytokines interleukin 2 (IL2), 8 (IL8) and tumour necrosis factor α (TNFα) were measured. Unexpectedly, polyphenols had no significant anti‐inflammatory effects and EGCG increased IL8 release from stimulated cells by 174±18%. Polyphenols are digested to phenolic acids by the colonic microflora, thus we hypothesised anti‐inflammatory effects may be mediated by these metabolites. Quercetin and EGCG were fermented with normal gut bacteria (E. coli) or probiotic strain (L. rhamnosus) for 6h and phenolic acids were then isolated by solid phase extraction. Jurkat cells were incubated with the extracts for 48h with or without PMA/PHA stimulation at 24h.E. coli extract increased TNFα release from stimulated cells by 268±51% while L. rhamnosus extract reduced IL8 release from unstimulated cells by 34±2%. EGCG fermented with L. rhamnosus increased IL8 release from stimulated cells by 128±16%, consistent with effects of unfermented EGCG. The findings showed anaerobic bacterial fermentation did not modify polyphenol activity, but supported anti‐inflammatory effects of L. rhamnosus.Grant Funding Source: Supported by BBSRC DRINC

Altered circulating levels of inflammatory cytokines are a risk factor for the development of age related diseases. The objective of this study was to assess the effect of polyphenols or phenolic acids on the release of cytokines from a human CD4+ T‐cell line (Jurkat cells). Cells were treated with a range of polyphenols or phenolic acids at 1 μM or 30 μM. Cells were treated with phorbol 12‐myristate 13‐acetate (PMA) stimulation after 24 hours and cytokine release was determined 24 hours later. Four compounds were identified that resulted in a significant reduction in interleukin 2 (IL‐2) and tumour necrosis factor α (TNFα) release by PMA‐stimulated Jurkat cells. These molecules were dihydroferulic acid (−56.5% TNFα, p<0.001), feruloylglycine (− 49.9% TNFα, p<0.001), quercetin (−67.2% IL‐2, p<0.001; −91.8% TNFα, p<0.001), and 3‐O‐methylquercetin (−91.5% TNFα, p<0.001). A further 3 compounds, epigallocatechin gallate (−46.2% IL‐8, p<0.01), punicalagin (−43.1% IL‐8, p<0.05), and callistephin (−48.5% IL‐8, p<0.01), reduced the release of cytokines by non PMA‐stimulated Jurkat cells. In conclusion, we have identified 7 dietary polyphenols or phenolic acids that reduced the release of inflammatory cytokines by a human CD4+ T‐cell line. The authors thank the British Biological Sciences Research Council Diet and Health Research Industry Club (BBSRC DRINC) programme for financial support.

1. Investment in reproduction is anticipated to be costly and can decrease survival or future reproductive success. For males, substantial reproductive costs may be accrued when competing for mates, particularly when individuals need to invest heavily in the production of sexual signals to attract females. On a proximate level, increased male signalling effort can cause somatic damage because of oxidative stress, although this has been demonstrated only in species with visual sexual signals. 2. We tested whether reproductive effort (comprising reproduction, aggression and scent signalling) is associated with increased oxidative stress in male house mice (Mus musculus domesticus). Sexual signalling in this species involves the production and deposition of scent signals containing a high concentration of protein around a defended territory. Male reproductive investment was manipulated by housing males alone, with a female or with a female and in the vicinity of competitors. 3. Males breeding in the vicinity of competitors invested the most in olfactory signalling as well as having regular aggressive interactions with other males. These males tended to show greater oxidative damage to lipids in the gastrocnemius muscle but no other indication of increased oxidative stress. Instead, lipid oxidation was lower in the serum and liver of reproductive males compared with those housed alone. 4. Our results highlight that oxidative stress does not always occur simply as a function of increasing reproductive effort. The lack of a consistent increase in oxidative damage could be due to adaptive regulation of antioxidants and / or a consequence of the scent signalling system of house mice, which differs considerably from the visual signalling of birds previously examined in this context.

Investment in reproduction is costly and frequently decreases survival or future reproductive success. However, the proximate underlying causes for this are largely unknown. Oxidative stress has been suggested as a cost of reproduction and several studies have demonstrated changes in antioxidants with reproductive investment. Here, we test whether oxidative stress is a consequence of reproduction in female house mice ( Mus musculus domesticus ), which have extremely high energetic demands during reproduction, particularly through lactation. Assessing oxidative damage after a long period of reproductive investment, there was no evidence of increased oxidative stress, even when females were required to defend their breeding territory. Instead, in the liver, markers of oxidative damage (malonaldehyde, protein thiols and the proportion of glutathione in the oxidized form) indicated lower oxidative stress in reproducing females when compared with non-reproductive controls. Even during peak lactation, none of the markers of oxidative damage indicated higher oxidative stress than among non-reproductive females, although a positive correlation between protein oxidation and litter mass suggested that oxidative stress may increase with fecundity. Our results indicate that changes in redox status occur during reproduction in house mice, but suggest that females use mechanisms to cope with the consequences of increased energetic demands and limit oxidative stress.

Mice lacking Cu,Zn superoxide dismutase (SOD1) show accelerated, age-related loss of muscle mass. Lack of SOD1 may lead to increased superoxide, reduced nitric oxide (NO), and increased peroxynitrite, each of which could initiate muscle fiber loss. Single muscle fibers from flexor digitorum brevis of wild-type (WT) and Sod1(-/-) mice were loaded with NO-sensitive (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, DAF-FM) and superoxide-sensitive (dihydroethidium, DHE) probes. Gastrocnemius muscles were analyzed for SOD enzymes, nitric oxide synthases (NOS), and 3-nitrotyrosine (3-NT) content. A lack of SOD1 did not increase superoxide availability at rest because no increase in ethidium or 2-hydroxyethidium (2-HE) formation from DHE was seen in fibers from Sod1(-/-) mice compared with those from WT mice. Fibers from Sod1(-/-) mice had decreased NO availability (decreased DAF-FM fluorescence), increased 3-NT in muscle proteins indicating increased peroxynitrite formation and increased content of peroxiredoxin V (a peroxynitrite reductase), compared with WT mice. Muscle fibers from Sod1(-/-) mice showed substantially reduced generation of superoxide in response to contractions compared with fibers from WT mice. Inhibition of NOS did not affect DHE oxidation in fibers from WT or Sod1(-/-) mice at rest or during contractions, but transgenic mice overexpressing nNOS showed increased DAF-FM fluorescence and reduced DHE oxidation in resting muscle fibers. It is concluded that formation of peroxynitrite in muscle fibers is a major effect of lack of SOD1 in Sod1(-/-) mice and may contribute to fiber loss in this model, and that NO regulates superoxide availability and peroxynitrite formation in muscle.

During chronic inflammation muscle is exposed to inflammatory cytokines; a key player in this is TNF‐α. Muscle has protective mechanisms to adapt to stress, via further expression of cytokines and Heat shock proteins (HSPs), which can be released into the extracellular environment and then involved in apocrine or paracrine signalling pathways. C2C12 myotubes in vitro treated with TNF‐α (25ng/ml) showed significant up‐regulation of 7 pro‐inflammatory genes by qPCR array analysis. Significant release of cytokines (IL‐6, MCP‐1, KC, IP‐10 & RANTES) was seen after 3 & 6 hours. In addition, TNF‐α up‐regulated intracellular levels of HSP60 & 70, with specific release of HSP60 at 3 hours. Treatment of C2C12 myotubes with recombinant HSP60 and 70 induced significant release of MCP‐1 and RANTES at 3 hours. Adult and old C57Bl6 mice treated with TNF‐α (7.5μg/kg) i.v. were subject to microdialysis perfusion of the gastrocnemius muscle to measure direct cytokine release from whole muscle. Adult mice showed a significant release of IL‐1β & MCP‐1, 1 hour post TNF‐α; old mice showed elevated basal levels of IL‐1β & MCP‐1, with a blunted response to TNF‐α. Data show TNF‐α induces an immune and stress response in muscle in vitro and HSPs have a paracrine signalling effect inducing release of cytokines. Whole muscle in vivo releases cytokines in response to TNF‐α treatment which is blunted in old mice. Supported by University of Liverpool.

Oxidation of skeletal muscle proteins has been reported to occur following contractions, with ageing, and with a variety of disease states, but the nature of the oxidised proteins has not been identified. A proteomics approach was utilised to identify major proteins that contain carbonyls and/or 3-nitrotyrosine (3-NT) groups in the gastrocnemius (GTN) muscles of adult (5-11 months of age) and old (26-28 months of age) wild type (WT) mice and adult mice lacking copper, zinc superoxide dismutase (Sod1(-/-) mice), manganese superoxide dismutase (Sod2(+/-) mice) or glutathione peroxidase 1 (GPx1(-/-) mice). In quiescent GTN muscles of adult and old WT mice, protein carbonylation and/or formation of 3-NT occurred in several proteins involved in glycolysis, as well as creatine kinase and carbonic anhydrase III. Following contractions, the 3-NT intensity was increased in specific protein bands from GTN muscles of both adult and old WT mice. In quiescent GTN muscles from adult Sod1(-/-) , Sod2(+/-) or GPx1(-/-) mice compared with age-matched WT mice only carbonic anhydrase III showed a greater 3-NT content. We conclude that formation of 3-NT occurs readily in response to oxidative stress in carbonic anhydrase III and this may provide a sensitive measure of oxidative damage to muscle proteins.

Conjugated linoleic acids (CLA), derivatives of linoleic acid found in food products, inhibit chemically induced skin cancers in mice. However, their potential photoprotective properties remain unexplored. We examined whether CLA may modulate ultraviolet radiation (UVR)-induced secretion of interleukin (IL)-8 and prostaglandin E2 (PGE(2)), mediators implicated in UVR-induced inflammation and carcinogenesis, in human skin cells. Since tumour necrosis factor (TNF)-alpha is an early mediator of UVR effects, we also examined influence of CLA on TNF-alpha-induced mediator release. HaCaT keratinocytes were supplemented with CLA isomers cis-9-trans-11 (c9,t11-CLA; > or =90%), trans-10-cis-12 (t10,c12-CLA; > or =90%) or all trans-trans isomers (tt-CLA; 23.7%) in tetrahydrofuran/fetal calf serum (THF/FCS) or THF/FCS control. Supplementation of keratinocytes with c9,t11-CLA reduced Ultraviolet B(UVB)-induced IL-8 from 37 113 +/- 2903 pg/ng protein in control cells to 14 167 +/- 2063 pg/ng protein (P < 0.001). Similarly, t10,c12-CLA reduced UVB-induced IL-8 to 9786 +/- 1291.5 pg/ng protein (P < 0.001). Additionally, t10,c12-CLA and tt-CLA inhibited TNF-alpha-induced IL-8 from 11 669 +/- 1692 pg/ng protein in control cells to 5540 +/- 191 (P < 0.001) and 8082 +/- 1298 pg/ng (P < 0.01) protein, respectively. UVB-induced PGE(2) release was reduced by tt-CLA supplementation, from 4.8 +/- 1.2 to 1.6 +/- 0.8 pg/mg protein (P < 0.01), but increased by t10,c12-CLA to 8.8 +/- 1 pg/mg protein (P < 0.001). Influence of CLA on UVB-induced PGE(2) release was further explored in CCD922SK dermal fibroblasts. CLA isomers reduced UVB-induced PGE(2) in fibroblasts, reaching significance with c9,t11-CLA (98 +/- 5 falling to 0 pg/mg protein, P < 0.05). Hence, CLA isomers differentially modulate UVB effects on skin cells in vitro. CLA-containing foods have potential in photoprotection; the cutaneous effects of individual isomers warrant clinical study.

Background. Increased oxidative stress may play a role in morbidity and mortality of patients with renal failure. Most studies have examined serum markers of oxidation, but it is unclear whether oxidative stress is involved in skeletal muscle atrophy. Methods. This study examined markers of oxidative stress in the skeletal muscle of 10 haemodialysed patients and 10 control subjects. Biopsies from the quadriceps femoris were analysed for reduced and oxidized glutathione, protein thiols, malonaldehyde and heat shock proteins (HSP27, HSP60 and HSP70), superoxide dismutase and catalase activities. A novel microdialysis procedure was used to examine hydroxyl radical activity in the interstitial fluid of the tibialis anterior . Results. Patients had muscle atrophy with a reduced diameter of both type I and II fibres (by 15 and 20%, respectively). Muscle microdialysates contained 2,3- and 2,5-dihydroxybenzoates formed from salicylate indicating hydroxyl radical activity, with no differences between patients and control subjects. Muscle protein thiol and oxidized glutathione contents were unchanged in patients, but malonaldehyde content was reduced. In contrast, total muscle glutathione and heat shock protein contents were increased. Muscle superoxide dismutase activity was unchanged, but catalase activity was reduced in patients. Conclusions. The muscle of patients undergoing haemodialysis undergoes some adaptive responses in total glutathione content, heat shock protein content and catalase activity that are potentially related to chronic oxidative stress. However, there is no evidence of gross oxidation, nor any clear relationship between oxidative stress and muscle fibre atrophy, arguing against a direct role of oxidants in the degenerative processes.

Much data indicate that overt selenium deficiency induces a number of pathologies in animals and humans. The effects of chronic marginal undernutrition of this element are unclear, although it has been argued that such subjects will be at increased risk of developing various cancers. The dietary intake of selenium in the UK has fallen over the last 25 years, although no functional consequences of this have been recognized. Recent data demonstrate that restoration of selenium intakes in UK subjects induces biochemical effects with increased activity of selenium-dependent enzymes. Whether such biochemical changes are associated with functional changes is currently unclear and the subject of current investigation.