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Objective Platelets modulate hemostasis and immune responses via interactions with immune cells, through secretion of immune-modulators and cell-cell interactions. The P2Y12 receptor mediates ADP-induced aggregation and secretion in platelets. Approach using a mouse model of intra-abdominal sepsis and acute lung injury, we investigated the role of the P2Y12 receptor in neutrophil migration and lung inflammation in P2Y12 null mice and in mice pre-treated with the P2Y12 antagonist clopidogrel. Results our data show a decrease in circulating white blood cells and a decrease in platelet activation and platelet-leukocyte interactions in treated mice compared to untreated. Additionally, lung injury and platelet sequestration were diminished in clopidogrel-treated mice compared to their untreated septic littermates. Similar results were observed in P2Y12 null mice: platelet activation and platelet-leukocyte aggregates were decreased in septic P2Y12 null mice compared to wild-type. P2Y12 null mice were refractory to lung injury compared to wild-type. Lastly, to evaluate P2Y12 independent effects of clopidogrel, we pre-treated P2Y12 null mice. Interestingly, the number of circulating neutrophils was reduced in treated septic P2Y12 null mice, suggesting neutrophils as a target for clopidogrel pleiotropic effects. No difference was observed in P2Y1 null mice during sepsis, indicating that the P2Y12 receptor is responsible for the effects. Conclusions P2Y12 null mice are refractory to sepsis-induced lung injury, suggesting a key role for activated platelets and the P2Y12 receptor during sepsis.

Aims/hypothesis: To investigate the phenotypic effects of common polymorphisms on adipose tissue metabolism and cardiovascular risk factors, we set out to establish a biobank with the unique feature of allowing a prospective recruit-by-genotype approach. The first use of this biobank investigates the effects of the peroxisome proliferatoractivated receptor-γ (PPARγ) Pro12Ala polymorphism on integrative tissue-specific physiology. We hypothesised that Ala12 allele carriers demonstrate greater adipose tissue metabolic flexibility and insulin sensitivity. Materials and methods: From a comprehensive population register, subjects were recruited into a biobank, which was genotyped for the Pro12Ala polymorphism. Twelve healthy male Ala12 carriers and 12 matched Pro12 homozygotes underwent detailed physiological phenotyping using stable isotope techniques, and measurements of blood flow and arteriovenous differences in adipose tissue and muscle in response to a mixed meal containing [1,1,[1][2][3][4][5][6][7][8][9][10][11][12][13]

BackgroundNeuroinflammation often develops in sepsis leading to activation of cerebral endothelium, increased permeability of the blood-brain barrier (BBB), and neutrophil infiltration. We have identified protein kinase C-delta (PKCδ) as a critical regulator of the inflammatory response and demonstrated that pharmacologic inhibition of PKCδ by a peptide inhibitor (PKCδ-i) protected endothelial cells, decreased sepsis-mediated neutrophil influx into the lung, and prevented tissue damage. The objective of this study was to elucidate the regulation and relative contribution of PKCδ in the control of individual steps in neuroinflammation during sepsis.MethodsThe role of PKCδ in mediating human brain microvascular endothelial (HBMVEC) permeability, junctional protein expression, and leukocyte adhesion and migration was investigated in vitro using our novel BBB on-a-chip (B3C) microfluidic assay and in vivo in a rat model of sepsis induced by cecal ligation and puncture (CLP). HBMVEC were cultured under flow in the vascular channels of B3C. Confocal imaging and staining were used to confirm tight junction and lumen formation. Confluent HBMVEC were pretreated with TNF-α (10 U/ml) for 4 h in the absence or presence of PKCδ-i (5 μM) to quantify neutrophil adhesion and migration in the B3C. Permeability was measured using a 40-kDa fluorescent dextran in vitro and Evans blue dye in vivo.ResultsDuring sepsis, PKCδ is activated in the rat brain resulting in membrane translocation, a step that is attenuated by treatment with PKCδ-i. Similarly, TNF-α-mediated activation of PKCδ and its translocation in HBMVEC are attenuated by PKCδ-i in vitro. PKCδ inhibition significantly reduced TNF-α-mediated hyperpermeability and TEER decrease in vitro in activated HBMVEC and rat brain in vivo 24 h after CLP induced sepsis. TNF-α-treated HBMVEC showed interrupted tight junction expression, whereas continuous expression of tight junction protein was observed in non-treated or PKCδ-i-treated cells. PKCδ inhibition also reduced TNF-α-mediated neutrophil adhesion and migration across HBMVEC in B3C. Interestingly, while PKCδ inhibition decreased the number of adherent neutrophils to baseline (no-treatment group), it significantly reduced the number of migrated neutrophils below the baseline, suggesting a critical role of PKCδ in regulating neutrophil transmigration.ConclusionsThe BBB on-a-chip (B3C) in vitro assay is suitable for the study of BBB function as well as screening of novel therapeutics in real-time. PKCδ activation is a key signaling event that alters the structural and functional integrity of BBB leading to vascular damage and inflammation-induced tissue damage. PKCδ-TAT peptide inhibitor has therapeutic potential for the prevention or reduction of cerebrovascular injury in sepsis-induced vascular damage.

Thienopyridines are a class of antiplatelet drugs that are metabolized in the liver to several metabolites, of which only one active metabolite can irreversibly antagonize the platelet P2Y12 receptor. Possible effects of these drugs and the role of activated platelets in inflammatory responses have also been investigated in a variety of animal models, demonstrating that thienopyridines could alter inflammation. However, it is not clear whether it is caused only by the P2Y12 antagonism or whether off-target effects of other metabolites also intervene. To address this question, we investigated P2Y12 KO mice during a LPS-induced model of systemic inflammation, and we treated these KO mice with a thienopyridine drug (clopidogrel). Contrary to the reported effects of clopidogrel, numbers of circulating WBCs and plasma levels of cytokines were increased in LPS-exposed KO mice compared with WT in this inflammation model. Moreover, both spleen and bone marrow show an increase in cell content, suggesting a role for P2Y12 in regulation of bone marrow and spleen cellular composition. Finally, the injury was more severe in the lungs of KO mice compared with WT. Interestingly, clopidogrel treatments also exerted protective effects in KO mice, suggesting off-target effects for this drug. In conclusion, the P2Y12 receptor plays an important role during LPS-induced inflammation, and this signaling pathway may be involved in regulating cell content in spleen and bone marrow during LPS systemic inflammation. Furthermore, clopidogrel may have effects that are independent of P2Y12 receptor blockade.

Platelets play an important role not only during thrombosis, but also in modulating immune responses through their interaction with immune cells and by releasing inflammatory mediators upon activation. The P2Y12 receptor is a Gi-coupled receptor that not only regulates ADP-induced aggregation but that can also dramatically potentiate secretion, when platelets are activated by other stimuli. Considering the importance of P2Y12 receptor in platelet function, a class of anti-platelet drugs, thienopyridines, have been designed and successfully used to prevent thrombosis. This review will focus on the role of activated platelets in inflammation and the effects that P2Y12 antagonism exerts on the inflammatory process. A change in platelet functions was noted in patients treated with thienopyridines during inflammatory conditions, suggesting that platelets may modulate the inflammatory response. Further experiments in a variety of animal models of diseases, such as sepsis, rheumatoid arthritis, myocardial infarction, pancreatitis and pulmonary inflammation have also demonstrated that activated platelets influence the inflammatory state. Platelets can secrete inflammatory modulators in a P2Y12–dependent manner, and, as a result, directly alter the inflammatory response. P2Y12 receptor may also be expressed in other cells of the immune system, indicating that thienopyridines could directly influence the immune system rather than only through platelets. Overall the results obtained to date strongly support the notion that activated platelets significantly contribute to the inflammatory process and that antagonizing P2Y12 receptor can influence the immune response.

Graphical abstract

The increased risk of thromboembolism and higher incidence of cardiovascular disorders are among the most common causes of morbidity in patients suffering from autoimmune diseases. In this study we tested the hypothesis that IL-17A, a key pro-inflammatory cytokine involved in the development of autoimmune diseases, exerts pro-aggregant effects on both human and mouse platelets. Human or murine platelets were incubated with IL-17A for 2 min at 37°C prior the addition of the stimuli. Aggregation was monitored in a light transmission aggregometer measuring changes in turbidity with continuous observation over a 5-min interval after the addition of the stimuli. IL-17RA, CD42b and CD62P expression as well as fibrinogen bindings were measured by FACS while Erk-2 phosphorylation was analyzed by western blot using phospho-specific antibodies. Pre-incubation with IL-17A increased ADP-, but not collagen-induced platelet aggregation and accelerated CD62P expression and exposure of fibrinogen binding sites. These effects were associated with a faster kinetic of ADP-induced Erk-2 phosphorylation and were lost in platelets deficient in the IL-17 receptor. Together these results unveil a novel aspect of the inflammatory nature of IL-17A suggesting, at the same time, that therapeutic strategies targeting this cytokine might provide further benefit for the treatment of autoimmune diseases by reducing the risk of cardiovascular-related pathologies.