G(alpha)s-to-G(alpha)i switch of G protein-coupled receptor signalling in rheumatoid arthritis synovial fibroblasts

In dieser Arbeit wurde die Bedeutung einer Gαs-zu-Gαi-Signalumschaltung am β2-AR in synovialen Fibroblasten von RA-Patienten untersucht. Verglichen wurden die Signalwege in synovialen Fibroblasten von RA-Patienten mit Fibroblasten aus den inguinalen Lymphknoten von gesunden Kontrollen. Es konnte gezeigt werden, dass die Voraussetzungen für eine Gαs-zu-Gαi-Umschaltung durch gemeinsames Binden von β-Arrestin und PDE4 an den β2-AR gegeben waren. Außerdem konnten sowohl Gαs- als auch Gαi-Proteine nachgewiesen werden. In vielen Fällen konnte eine Umschaltung der Signalweiterleitung des β-AR detektiert werden, die durch RA oder die Simulation einer Entzündung durch Hypoxie oder/und IL-1β auch bei Gesunden ausgelöst wurde. Bei der Stimulation des β-AR, der typischerweise antiinflammatorische Signalwege über Gαs induziert, konnten daraufhin proinflammatorische Effekte detektiert werden. Diese unterschieden sich stark von den cAMP-vermittelten Effekten der stimulierten Adenylatzyklase, an die das Signal ohne die Umschaltung üblicherweise weitergeleitet worden wäre. Die beschriebene Umschaltung konnte oftmals durch Hemmung des Gαi durch Pertussis-Toxin oder Hemmung der PKA durch H89 rückgängig gemacht werden, sodass die Wirkung des stimulierten β-AR der Wirkung der aktivierten Adenylatzyklase entsprach. Diese Wirkung der Gαi- und PKA-Inhibitoren entspricht der Hypothese einer Gαs-zu-Gαi-Umschaltung, wie sie bereits zuvor auch schon in anderen Arbeiten beobachtet wurde. Außerdem konnte in dieser Arbeit gezeigt werden, dass die GRK2/3 und GRK2/5 ebenfalls großen Einfluss auf die Umschaltung haben. Zusammenfassend lässt sich sagen, dass es bei den synovialen Fibroblasten von RA-Patienten zu einer Gαs-zu-Gαi-Umschaltung kommt, die proinflammatorische Effekte mit sich bringt. Durch die Simulation einer Entzündung durch Hypoxie oder/und IL-1β konnte diese Umschaltung teilweise auch bei Fibroblasten aus inguinalen Lymphknoten von Gesunden provoziert werden

Differential inflammation-mediated function of prokineticin 2 in the synovial fibroblasts of patients with rheumatoid arthritis compared with osteoarthritis

Prokineticin 2 (PK2) is a secreted protein involved in several pathological and physiological processes, including the regulation of inflammation, sickness behaviors, and circadian rhythms. Recently, it was reported that PK2 is associated with the pathogenesis of collagen-induced arthritis in mice. However, the role of PK2 in the pathogenesis of rheumatoid arthritis (RA) or osteoarthritis (OA) remains unknown. In this study, we collected synovial tissue, plasma, synovial fluid, and synovial fibroblasts (SF) from RA and OA patients to analyze the function of PK2 using immunohistochemistry, enzyme-linked immunosorbent assays, and tissue superfusion studies. PK2 and its receptors prokineticin receptor (PKR) 1 and 2 were expressed in RA and OA synovial tissues. PKR1 expression was downregulated in RA synovial tissue compared with OA synovial tissue. The PK2 concentration was higher in RA synovial fluid than in OA synovial fluid but similar between RA and OA plasma. PK2 suppressed the production of IL-6 from TNFα-prestimulated OA-SF, and this effect was attenuated in TNFα-prestimulated RA-SF. This phenomenon was accompanied by the upregulation of PKR1 in OA-SF. This study provides a new model to explain some aspects underlying the chronicity of inflammation in RA

Acute exposure to air pollution particulate matter aggravates experimental myocardial infarction in mice by potentiating cytokine secretion from lung macriphages

Clinical, but not experimental evidence has suggested that air pollution particulate matter (PM) aggravates myocardial infarction (MI). Here, we aimed to describe mechanisms and consequences of PM exposure in an experimental model of MI. C57BL/6J mice were challenged with a PM surrogate (Residual Oil Fly Ash, ROFA) by intranasal installation before MI was induced by permanent ligation of the left anterior descending coronary artery. Histological analysis of the myocardium 7 days after MI demonstrated an increase in infarct area and enhanced inflammatory cell recruitment in ROFA-exposed mice. Mechanistically, ROFA exposure increased levels of the circulating pro-inflammatory cytokines TNF-α, IL-6, and MCP-1, activated myeloid and endothelial cells, and enhanced leukocyte recruitment to the peritoneal cavity and the vascular endothelium. Notably, these effects on endothelial cells and circulating leukocytes could be reversed by neutralizing anti-TNF-α treatment. We identified alveolar macrophages as the primary source of elevated cytokine production after PM exposure. Accordingly, in vivo depletion of alveolar macrophages by intranasal clodronate attenuated inflammation and cell recruitment to infarcted tissue of ROFA-exposed mice. Taken together, our data demonstrate that exposure to environmental PM induces the release of inflammatory cytokines from alveolar macrophages which directly worsens the course of MI in mice. These findings uncover a novel link between air pollution PM exposure and inflammatory pathways, highlighting the importance of environmental factors in cardiovascular disease.Fil: Marchini, Timoteo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Wolf, Dennis. University Of Freiburg; AlemaniaFil: Anto Michel, Nathaly. University Of Freiburg; AlemaniaFil: Mauler, Maximilian. University Of Freiburg; AlemaniaFil: Dufner, Bianca. University Of Freiburg; AlemaniaFil: Hoppe, Natalie. University Of Freiburg; AlemaniaFil: Beckert, Jessica. University Of Freiburg; AlemaniaFil: Jäekel, Markus. University Of Freiburg; AlemaniaFil: Magnani, Natalia Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Duerschmied, Daniel. University Of Freiburg; AlemaniaFil: Tasat, Deborah Ruth. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; ArgentinaFil: Alvarez, Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Reinöhl, Jochen. University Of Freiburg; AlemaniaFil: von zur Muhlen, Constantin. University Of Freiburg; AlemaniaFil: Idzko, Marco. University Of Freiburg; AlemaniaFil: Bode, Christoph. University Of Freiburg; AlemaniaFil: Hilgendorf, Ingo. University Of Freiburg; AlemaniaFil: Evelson, Pablo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Zirlik, Andreas. University Of Freiburg; Alemani

Extracellular ATP Induces Vascular Inflammation and Atherosclerosis via Purinergic Receptor y 2 in Mice

Objective - A solid body of evidence supports a role of extracellular ATP and its P2 receptors in innate and adaptive immunity. It promotes inflammation as a danger signal in various chronic inflammatory diseases. Thus, we hypothesize contribution of extracellular ATP and its receptor P2Y 2 in vascular inflammation and atherosclerosis. Approach and Results - Extracellular ATP induced leukocyte rolling, adhesion, and migration in vivo as assessed by intravital microscopy and in sterile peritonitis. To test the role of extracellular ATP in atherosclerosis, ATP or saline as control was injected intraperitoneally 3× a week in low-density lipoprotein receptor -/- mice consuming high cholesterol diet. Atherosclerosis significantly increased after 16 weeks in ATP-treated mice (n=13; control group, 0.26 mm2; ATP group, 0.33 mm2; P=0.01). To gain into the role of ATP-receptor P2Y 2 in ATP-induced leukocyte recruitment, ATP was administered systemically in P2Y 2 -deficient or P2Y 2 -competent mice. In P2Y 2 -deficient mice, the ATP-induced leukocyte adhesion was significantly reduced as assessed by intravital microscopy. P2Y 2 expression in atherosclerosis was measured by real-time polymerase chain reaction and immunohistochemistry and demonstrates an increased expression mainly caused by influx of P2Y 2 -expressing macrophages. To investigate the functional role of P2Y 2 in atherogenesis, P2Y 2 -deficient low-density lipoprotein receptor -/- mice consumed high cholesterol diet. After 16 weeks, P2Y 2 -deficient mice showed significantly reduced atherosclerotic lesions with decreased macrophages compared with P2Y 2 -competent mice (n=11; aortic arch: control group, 0.25 mm 2; P2Y 2 -deficient, 0.14 mm2; P=0.04). Mechanistically, atherosclerotic lesions from P2Y 2 -deficient mice expressed less vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 RNA. Conclusions - We show that extracellular ATP induces vascular inflammation and atherosclerosis via activation of P2Y 2.Fil: Stachon, Peter. Albert Ludwigs University of Freiburg; AlemaniaFil: Geis, Serjosha. Albert Ludwigs University of Freiburg; AlemaniaFil: Peikert, Alexander. Albert Ludwigs University of Freiburg; AlemaniaFil: Heidenreich, Adrian. Albert Ludwigs University of Freiburg; AlemaniaFil: Anto Michel, Nathaly. Albert Ludwigs University of Freiburg; AlemaniaFil: Üenal, Fatih. Albert Ludwigs University of Freiburg; AlemaniaFil: Hoppe, Natalie. Albert Ludwigs University of Freiburg; AlemaniaFil: Dufner, Bianca. Albert Ludwigs University of Freiburg; AlemaniaFil: Schulte, Lisa. Albert Ludwigs University of Freiburg; AlemaniaFil: Marchini, Timoteo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Cicko, Sanja. Albert Ludwigs University of Freiburg; AlemaniaFil: Korcan Ayata, Cemil. Albert Ludwigs University of Freiburg; AlemaniaFil: Zech, Andreas. Albert Ludwigs University of Freiburg; AlemaniaFil: Wolf, Dennis. Albert Ludwigs University of Freiburg; AlemaniaFil: Hilgendorf, Ingo. Albert Ludwigs University of Freiburg; AlemaniaFil: Willecke, Florian. Albert Ludwigs University of Freiburg; AlemaniaFil: Reinöhl, Jochen. Albert Ludwigs University of Freiburg; AlemaniaFil: von zur Muhlen, Constantin. Albert Ludwigs University of Freiburg; AlemaniaFil: Bode, Christoph. Albert Ludwigs University of Freiburg; AlemaniaFil: Idzko, Marco. Albert Ludwigs University of Freiburg; AlemaniaFil: Zirlik, Andreas. Albert Ludwigs University of Freiburg; Alemani

CD40L Deficiency Attenuates Diet-Induced Adipose Tissue Inflammation by Impairing Immune Cell Accumulation and Production of Pathogenic IgG-Antibodies

BACKGROUND: Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L--an established marker and mediator of cardiovascular disease--induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo. METHODOLOGY/PRINCIPAL FINDINGS: WT or CD40L(-/-) mice consumed a high fat diet (HFD) for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L(-/-) mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L(-/-) mice. However, CD40L(-/-) mice consuming HFD were not protected from the onset of diet-induced obesity (DIO), insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L(-/-) mice consuming a low fat diet (LFD) showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels. CONCLUSION: We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease

Proinflammatory α-Adrenergic Neuronal Regulation of Splenic IFN-γ, IL-6, and TGF-β of Mice from Day 15 onwards in Arthritis

Introduction: In arthritic mice, a sympathetic influence is proinflammatory from the time point of immunization until the onset of disease (days 0-32), but reasons are unknown. Disruption of the major anti-inflammatory pathway through G(alpha s)-coupled receptors probably play a role. For example, noradrenaline cannot operate via anti-inflammatory beta(2)-adrenoceptors but through proinflammatory alpha(1/2)-ad-renoceptors. This might happen, first, through a loss of sympathetic nerve fibers in inflamed tissue with low neurotransmitter levels (noradrenaline only binds to high-affinity alpha-adrenoceptors) and, second, through an alteration in G-protein receptor coupling with a predominance of alpha-adrenergic signaling. We hypothesized that both mechanisms play a role in the course of collagen type II-induced arthritis (CIA) in the spleen in mice. Methods: In CIA mice, nerve fiber density in the spleen was quantified by immunohistochemistry techniques. The functional impact of sympathetic nerve fibers in the spleen was studied by a micro-superfusion technique of spleen slices with a focus on the secretion of IFN-gamma and IL-6 (proinflammatory) and TGF-beta (anti-inflammatory). Results: During CIA, sympathetic nerve fibers get increasingly lost from day14 until day 55 after immunization. The influence of electrically released noradrenaline diminishes in the course of arthritis. At all investigated time points (days 14, 32, and 55), only proinflammatory neuronal alpha-adrenergic effects on cytokine secretion were demonstrated (i.e., stimulation of IFN-gamma and IL-6 and inhibition of TGF-beta). Conclusion: Sympathetic nerve fibers are rapidly lost in the spleen, and only proinflammatory alpha-adrenergic neuronal regulation of cytokine secretion takes place throughout the course of arthritis. These results support a predominance of a proinflammatory alpha-adrenergic sympathetic influence in arthritis

A thyroid hormone network exists in synovial fibroblasts of rheumatoid arthritis and osteoarthritis patients

While patients with rheumatoid arthritis (RA) sometimes demonstrate thyroidal illness, the role of thyroid hormones in inflamed synovial tissue is unknown. This is relevant because thyroid hormones stimulate immunity, and local cells can regulate thyroid hormone levels by deiodinases (DIO). The study followed the hypothesis that elements of a thyroid hormone network exist in synovial tissue. In 12 patients with RA and 32 with osteoarthritis (OA), we used serum, synovial fluid, synovial tissue, and synovial fibroblasts (SF) in order to characterize the local thyroid hormone network using ELISAs, immunohistochemistry, imaging methods, tissue superfusion studies, cell-based ELISAs, flow cytometry, and whole genome expression profiling. Serum/synovial fluid thyroid hormone levels were similar in RA and OA (inclusion criteria: no thyroidal illness). The degradation product termed reverse triiodothyronine (reverse T3) was much lower in serum compared to synovial fluid indicating biodegradation of thyroid hormones in the synovia I environment. Superfusion experiments with synovial tissue also demonstrated biodegradation, particularly in RA. Cellular membrane transporters of thyroid hormones, DIOs, and thyroid hormone receptors were present in tissue and SF. Density of cells positive for degrading DIOs were higher in RA than OA. TNF increased protein expression of degrading DIOs in RASF and OASF. Gene expression studies of RASF revealed insignificant gene regulation by bioactive T3. RA and OA synovial tissue/SF show a local thyroid hormone network. Thyroid hormones undergo strong biodegradation in synovium. While bioactive T3 does not influence SF gene expression, SF seem to have a relay function for thyroid hormones

Adjustment of Micro- and Macroporosity of ß-TCP Scaffolds Using Solid-Stabilized Foams as Bone Replacement

To enable rapid osteointegration in bioceramic implants and to give them osteoinductive properties, scaffolds with defined micro- and macroporosity are required. Pores or pore networks promote the integration of cells into the implant, facilitating the supply of nutrients and the removal of metabolic products. In this paper, scaffolds are created from ß-tricalciumphosphate (ß-TCP) and in a novel way, where both the micro- and macroporosity are adjusted simultaneously by the addition of pore-forming polymer particles. The particles used are 10–40 wt%, spherical polymer particles of polymethylmethacrylate (PMMA) (Ø = 5 µm) and alternatively polymethylsilsesquioxane (PMSQ) (Ø = 2 µm), added in the course of ß-TCP slurry preparation. The arrangement of hydrophobic polymer particles at the interface of air bubbles was incorporated during slurry preparation and foaming of the slurry. The foam structures remain after sintering and lead to the formation of macro-porosity in the scaffolds. Furthermore, decomposition of the polymer particles during thermal debindering results in the formation of an additional network of interconnecting micropores in the stabilizing structures. It is possible to adjust the porosity easily and quickly in a range of 1.2–140 μm with a relatively low organic fraction. The structures thus prepared showed no cytotoxicity nor negative effects on the biocompatibility