Christine E. Loscher

Anti-inflammatory effects of EPA and DHA are dependent upon time and dose-response elements associated with LPS stimulation in THP-1-derived macrophages

The long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) of fish oil, eicosapentanoic (EPA) and docosahexanoic (DHA) acids are considered cardioprotective. Inflammation elicited by macrophages is increasingly recognised in the aetiology of metabolic syndrome. This study investigated the differential anti-inflammatory potential of EPA and DHA through cytokine production and nuclear factor (NF)-kappaB signalling in a human macrophage model. We investigated the dependency of LC n-3 PUFA immune-modulation on concentration and duration of lipopolysaccharide (LPS) activation. Interleukin (IL)-1beta, IL-6 and tumor necrosis factor-alpha secretion from EPA, DHA and control cells were differentially limited by LPS concentration. In all cases, there was no benefit in activating cells with >0.1 microg/ml LPS. LC n-3 PUFA decreased proinflammatory cytokines production, an effect modulated by LPS concentration. Expression of the transcription factor NF-kappaB p65 was significantly reduced in the nucleus and retained in the cytoplasm of EPA- and/or DHA-treated macrophages during 5-h activation with 0.1 microg/ml LPS. Nuclear binding of p65 was significantly reduced in EPA- and DHA-treated cells at 2-h LPS activation. Over the time course, expression of nuclear IkappaBalpha was significantly reduced, cytoplasmic NF-kappaB p50 significantly increased and cytoplasmic cleaving enzyme IkappaB inhibitor complex significantly reduced in LC n-3 PUFA-treated cells. EPA and DHA down-regulated the production of proinflammatory cytokines associated with the aetiology of metabolic syndrome, NF-kappaB transcriptional activity and upstream cytoplasmic signalling events. Immune responses are dynamic, and the present study suggests a nutrient sensitive window of LPS activation at which EPA and DHA are strongly anti-inflammatory.

Antidiabetic Effects of cis-9, trans-11–Conjugated Linoleic Acid May Be Mediated via Anti-Inflammatory Effects in White Adipose Tissue

Adipose tissue may be the source of insulin desensitizing proinflammatory molecules that predispose to insulin resistance. This study investigated whether dietary fatty acids could attenuate the proinflammatory insulin-resistant state in obese adipose tissue. The potential antidiabetic effect of cis-9, trans-11–conjugated linoleic acid (c9,t11-CLA) was determined, focusing on the molecular markers of insulin sensitivity and inflammation in adipose tissue of ob/ob C57BL-6 mice. Feeding a c9,t11-CLA–enriched diet reduced fasting glucose (P < 0.05), insulin (P < 0.05), and triacylglycerol concentrations (P < 0.01) and increased adipose tissue plasma membrane GLUT4 (P < 0.05) and insulin receptor (P < 0.05) expression compared with the control linoleic acid–enriched diet. Interestingly, after the c9,t11-CLA diet, adipose tissue macrophage infiltration was less, with marked downregulation of several inflammatory markers in adipose tissue, including reduced tumor necrosis factor-α and CD68 mRNA (P < 0.05), nuclear factor-κB (NF-κB) p65 expression (P < 0.01), NF-κB DNA binding (P < 0.01), and NF-κB p65, p50, c-Rel, p52, and RelB transcriptional activity (P < 0.01). To define whether these observations were direct effects of the nutrient intervention, complimentary cell culture studies showed that c9,t11-CLA inhibited tumor necrosis factor-α–induced downregulation of insulin receptor substrate 1 and GLUT4 mRNA expression and promoted insulin-stimulated glucose transport in 3T3-L1 adipocytes compared with linoleic acid. This study suggests that altering fatty acid composition may attenuate the proinflammatory state in adipose tissue that predisposes to obesity-induced insulin resistance.

Conjugated Linoleic Acid Suppresses NF-κB Activation and IL-12 Production in Dendritic Cells through ERK-Mediated IL-10 Induction

Polyunsaturated fatty acids (PUFA) have been shown to modulate immune responses and have therapeutic effects in inflammatory disorders. However, the influence of PUFA on dendritic cells (DC), key cells of the innate immune system in shaping adaptive immune responses, has not yet been defined. In this study, we examine the effects of the cis-9, trans-11 isomer of conjugated linoleic acid (c9, t11-CLA), a dietary PUFA found in meat and dairy products, on murine DC activation. Treatment of DC with c9, t11-CLA suppressed LPS-induced IL-12, enhanced IL-10R expression, and enhanced IL-10 production at the transcriptional and protein level. The suppression of IL-12 by c9, t11-CLA was found to be IL-10 dependent. We investigated the involvement of the MAPK, ERK, and the transcription factor, NF-κB, in this IL-10-mediated effect. c9, t11-CLA enhanced ERK activation after LPS stimulation, and inhibition of ERK resulted in abrogation of IL-10 and recovery of IL-12 production. c9, t11-CLA decreased NF-κB:DNA binding after LPS stimulation, which was concomitant with delayed translocation of NF-κBp65 into the nucleus and an increase in IκBα. These effects were reversed by addition of a neutralizing anti-IL-10 Ab. Our findings demonstrate that c9, t11-CLA suppresses IL-12 production by LPS-stimulated DC by ERK mediated IL-10-induction. Furthermore, these IL-10-mediated effects are dependent on inhibition of NF-κB activation. This is the first study to demonstrate that c9, t11-CLA can enhance transcription and production of the anti-inflammatory cytokine IL-10, while inhibiting the Th1-promoting cytokine IL-12, and may explain certain of its immunosuppressive properties.

A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins.

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4−/− and Myd88−/−, but not TRIF−/− mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.

Divergent mechanisms of cis9, trans11-and trans10, cis12-conjugated linoleic acid affecting insulin resistance and inflammation in apolipoprotein E knockout mice: a proteomics approach

Conjugated linoleic acids (CLA) affect atherogenesis, but mechanisms are not well understood. We explored how two isomers of CLA, cis9, trans11‐CLA and trans10, cis12‐CLA, affected lipid and glucose metabolism, as well as hepatic protein expression, in apolipoprotein E knockout mice. After 12 wk of intervention, plasma triglyceride, NEFA, and glucose concentrations were significantly higher in the trans10, cis12‐CLA group, whereas plasma triglyceride, NEFA, glucose, and insulin concentrations were significantly lower in the cis9, trans 11‐CLA group, compared with control mice consuming linoleic acid. Proteomics identified significant up‐ or down‐regulation of 113 liver cytosolic proteins by either CLA isomer. Principal component analysis revealed that the treatment effect of cis9, trans11‐CLA was mainly explained by the up‐regulation of different posttranslational forms of heat shock protein 70 kD. In contrast, the treatment effect of trans10, cis12‐CLA was mainly explained by up‐regulation of key enzymes in the gluconeogenic, β‐oxidation, and ketogenesic pathways. Correlation analysis again emphasized the divergent effects of both CLA isomers on different pathways, but also revealed a linkage between insulin resistance and increased levels of hepatic serotransferrin. Thus, our systems biology approach provided novel insights into the mechanisms by which individual CLA isomers differentially affect pathways related to atherogenesis, such as insulin resistance and inflammation. Baukje De Roos, Garry Rucklidge, Martin Reid, Karen Ross, Gary Duncan, Maria A. Navarro, Jose M. Arbones‐Mainar, Mario A. Guzman‐Garcia, Jesus Osada, John Browne, Christine E. Loscher, Helen M. Roche Divergent mechanisms of cis9, trans11‐ and trans10, cis12‐conjugated linoleic acid affecting insulin resistance and inflammation in apolipoprotein E knockout mice: a proteomics approach. FASEB J. 19, 1–21 (2005)

IL-1F5 mediates anti-inflammatory activity in the brain through induction of IL-4 following interaction with SIGIRR/TIR8.

Similarity in structure and sequence homology has led to the identification of new members of the interleukin‐1 (IL‐1) ligand and receptor superfamilies. IL‐1F6, IL‐1F8 and IL‐1F9 have been shown to signal through IL‐1R‐related protein 2 and IL‐1 receptor accessory protein leading to activation of NFκB, while IL‐1F7 and IL‐1F10 interact with the IL‐18 receptor and the soluble IL‐1 receptor type I respectively. In contrast, identification of a biological role for IL‐1F5 has remained elusive, with conflicting data relating to its possible ability to antagonize IL‐1F9‐stimulated activation of NFκB in Jurkat cells transfected with IL‐1R‐related protein 2. In this study, we set out to investigate a possible role for IL‐1F5 in the brain and report that it antagonizes the inflammatory effects of IL‐1β and lipopolysaccharide (LPS) in vivo and in vitro including the inhibitory effect on long‐term potentiation (LTP) in rat hippocampus. We demonstrate that IL‐1F5 induces IL‐4 mRNA and protein expression in glia in vitro and enhances hippocampal expression of IL‐4 following intracerebroventricular (i.c.v.) injection. The inhibitory effect of IL‐1F5 on LPS‐induced IL‐1β is attenuated in cells from IL‐4‐defective (IL−4−/− mice). Our findings suggest that IL‐1F5 mediates anti‐inflammatory effects through its ability to induce IL‐4 production and that this is a consequence of its interaction with the orphan receptor, single Ig IL‐1R‐related molecule (SIGIRR)/TIR8, as the effects were not observed in SIGIRR−/− mice. In contrast to its effects in brain tissue, IL‐1F5 did not attenuate LPS‐induced changes, or up‐regulated IL‐4 in macrophages or dendritic cells, suggesting that the effect is confined to the brain.

Whole-Cell but Not Acellular Pertussis Vaccines Induce Convulsive Activity in Mice: Evidence of a Role for Toxin-Induced Interleukin-1β in a New Murine Model for Analysis of Neuronal Side Effects of Vaccination

ABSTRACT Immunization with the whole-cell pertussis vaccine (Pw), while effective at preventing whooping cough in infants, has been associated with local, systemic, and neuronal reactions, including fevers and convulsions in children. In contrast, the new acellular pertussis vaccines (Pa) have a considerably improved safety profile. The lack of an appropriate animal model has restricted investigations into the mechanisms by which neurological reactions are induced by vaccination. Here we describe a novel murine model wherein seizure-like behavioral changes are induced following parenteral administration of Pw. The proinflammatory cytokine interleukin-β (IL-1β), production of which has been associated with many neurodegenerative conditions, was significantly increased in the hippocampus and hypothalamus of vaccinated animals. Accompanying this change was a decrease in release of the inhibitory neurotransmitters γ-aminobutyric acid and adenosine in the hippocampus. Seizure-like behavioral changes were significantly reduced following inhibition of IL-1β production by the administration of an inhibitor of IL-1β-converting enzyme and were almost completely abrogated in IL-1 receptor type I knockout mice. These results suggest a causal relationship between IL-1β induction and convulsive behavior following Pw vaccination. Significantly, Pa neither increased IL-1β nor induced behavioral changes in mice, but did induce the anti-inflammatory cytokine IL-10. In contrast, administration of active pertussis toxin and lipopolysaccharide, residual in Pw but absent from Pa, also induced convulsive activity. Our findings provide the first direct evidence of an immunological basis for pertussis vaccine reactogenicity and suggest that active bacterial toxins are responsible for the neurologic disturbances observed in children immunized with Pw.

Omega-3 fatty acids attenuate dendritic cell function via NF-κB independent of PPARγ☆

Long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) have been shown to modulate the immune response and have therapeutic effects in inflammatory disorders. PUFA are also peroxisome proliferators-activator receptor-gamma (PPARγ) ligands; a family of ligand-activated transcription factors, which when activated antagonise the pro-inflammatory capability of nuclear factor κB (NF-κB). PPARγ plays a role in dendritic cell (DC) maturation and n-3 PUFA have been shown to affect DC maturation by decreasing activation of NF-κB. While n-3 PUFA can function as PPAR ligands, it is not known whether the NF-κB-mediated immunomodulatory properties of n-3 PUFA are PPARγ-dependent. In this study we examined whether the immunomodulatory effects of n-3 PUFA on DC activation were mediated through activation of PPARγ. Treatment of murine bone marrow derived DCs with docosahexaenoic acid (DHA; 25 μM) and eicosapentaenoic acid (EPA; 25 μM) attenuated LPS-induced DC maturation. This was characterised by suppression of IL-12 production and expression of CD40, CD80, CD86 and MHC II and enhanced production of IL-10 and expression of IL-10R. This was coincident with enhanced PPARγ expression, suppressed NF-κB activity and increased the physical interaction and cellular colocalization between NF-κB with PPARγ. To understand the functional implication of the physical association of PPARγ with NF-κB, we determined whether the specific PPARγ inhibitor, GW9662 could abolish the anti-inflammatory effect of n-3 PUFA Inhibiting PPARγ did not impede the NF-κB-mediated anti-inflammatory cytokine profile induced by EPA and DHA alone. Thus n-3 PUFA activate PPARγ and interact with NF-κB in DC. However, the anti-inflammatory effects of EPA and DHA on DCs are independent of PPARγ.

Proinflammatory Responses in the Murine Brain after Intranasal Delivery of Cholera Toxin: Implications for the Use of AB Toxins as Adjuvants in Intranasal Vaccines

Intranasal delivery of vaccines provides an attractive alternative to parenteral delivery, but it requires appropriate mucosal adjuvants. Cholera toxin (CT) is a powerful mucosal adjuvant, but it can undergo retrograde transport to the brain via the olfactory system after intranasal delivery. We demonstrate that intranasal delivery of CT increases the expression of interleukin-1 beta , cyclooxygenase-2, and chemokine messenger RNA in the murine hypothalamus, whereas parenterally delivered CT has little effect. Our findings suggest that CT can induce proinflammatory mediators in the brain when it is administered intranasally but not parenterally, and they raise concerns about the use of AB toxins as adjuvants in intranasal vaccines.

Interleukin-1 receptor antagonist exerts agonist activity in the hippocampus independent of the interleukin-1 type I receptor

Interleukin-1 receptor antagonist (IL-1ra) selectively and competitively inhibits the actions of IL-1 at its receptors and has not been reported to have agonist activity. This study demonstrates that stimulation of synaptosomes with IL-1ra in vitro, mimicked the effects of IL-1 beta by decreasing glutamate release and increasing JNK phosphorylation. These effects of IL-1ra, but not IL-1 beta, were maintained in IL-1 type I receptor (IL-1RI) defective mice. IL-1 beta blocked these IL-1ra-induced effects suggesting that it may also act independently of IL-1RI in some circumstances. Furthermore, IL-1ra mimicked the inhibitory effect of IL-1 beta on long-term potentiation (LTP) in the hippocampus. These data, taken together with our findings that IL-1ra binds to hippocampal synaptosomes in the absence of IL-1RI, provide evidence that IL-1ra exerts agonist activity in the hippocampus independent of IL-1RI.