Sepsis is a complex clinical condition resulting from a serious bloodstream infection. With mortality rates as high as 50%, improved treatments are needed. Regulatory T cells (Tregs), a subset of T lymphocytes, promote the resolution of inflammation. Septic patients have elevated levels of circulating Tregs. Platelets influence the proliferation and activation of Tregs in vitro. However, modulating platelet‐Tregs interaction during sepsis may restraing Treg proliferation, leading to the restoration of immunologic homeostasis. P2Y12 is a purinergic receptor present on platelets and T lymphocytes. Blocking P2Y12 improves the outcome of sepsis. We investigated whether blocking P2Y12 alters platelet–Treg interaction in vivo. We used the murine model of sepsis, cecal ligation, and puncture (CLP) and we blocked P2Y12 using the P2Y12 antagonist, clopidogrel. Twenty‐four hours after surgery, we measured Treg population sizes in the spleens of the Sham, CLP, and CLP + clopidogrel groups. We investigated the effect of blocking P2Y12 in vitro using cocultures of human platelets and T cells with or without anti‐CD3/CD28. P2Y12 was blocked using AR‐C69931MX. Treg population sizes were reduced in the septic mice treated with clopidogrel compared with untreated septic mice. Aggregation of platelets and CD4+ T cells was reduced in treated CLP mice compared with untreated CLP mice. P2Y12 antagonism changes how platelets influence T cells in vitro, depending on T‐cell activation. In conclusion, blockade of the P2Y12 signaling pathway restrains Treg proliferation in vivo and in vitro. Targeting platelets to control Treg proliferation and activity may be a promising strategy for treating sepsis.

Purinergic signaling plays a complex role in inflammation. Nucleotides released by T lymphocytes, endothelial cells, and platelets during inflammation induce cellular responses by binding to receptors that regulate intracellular signaling pathways. Previous studies have found that purinergic signaling can have both proinflammatory and anti-inflammatory effects, but the roles of specific pathways in specific cell types are poorly understood. We investigated the role of the P2Y 12 signaling pathway in the activation of T lymphocytes in vitro. We isolated peripheral blood mononuclear cells (PBMCs) from healthy donors and pretreated them with ADP (a P2Y 12 agonist), AR-C69931MX (a P2Y 12 antagonist), or both. We then stimulated PBMC using phytohemagglutinin (PHA) or anti-CD3/CD28 antibodies. We found that ADP affects T cell responses in term of cell activity and receptor expression through both P2Y 12-dependent and P2Y 12-independent pathways and other responses (cytokine secretion) primarily through P2Y 12-independent pathways. The ADP-mediated effect changed over time and was stimulus-specific.

Sepsis, a complex clinical syndrome resulting from a serious infection, is a major healthcare problem associated with high mortality. Sex-related differences in the immune response to sepsis have been proposed but the mechanism is still unknown. Purinergic signaling is a sex-specific regulatory mechanism in immune cell physiology. Our studies have shown that blocking the ADP-receptor P2Y12 but not P2Y1 receptor was protective in male mice during sepsis, but not female. We now hypothesize that there are sex-related differences in modulating P2Y12 or P2Y1 signaling pathways during sepsis. Male and female wild-type (WT), P2Y12 knock-out (KO), and P2Y1 KO mice underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. The P2Y12 antagonist ticagrelor or the P2Y1 antagonist MRS2279 were administered intra-peritoneally after surgery to septic male and female mice. Blood, lungs and kidneys were collected 24 hours post-surgery. Sepsis-induced changes in platelet activation, secretion and platelet interaction with immune cells were measured by flow cytometry. Neutrophil infiltration in the lung and kidney was determined by a myeloperoxidase (MPO) colorimetric assay kit. Sepsis-induced platelet activation, secretion and aggregate formation were reduced in male CLP P2Y12 KO and in female CLP P2Y1 KO mice compared with their CLP WT counterpart. Sepsis-induced MPO activity was reduced in male CLP P2Y12 KO and CLP P2Y1 KO female mice. CLP males treated with ticagrelor or MRS2279 showed a decrease in sepsis-induced MPO levels in lung and kidneys, aggregate formation, and platelet activation as compared to untreated male CLP mice. There were no differences in platelet activation, aggregate formation, and neutrophil infiltration in lung and kidney between female CLP mice and female CLP mice treated with ticagrelor or MRS2279. In human T lymphocytes, blocking P2Y1 or P2Y12 alters cell growth and secretion in vitro in a sex-dependent manner, supporting the data obtained in mice. In conclusion, targeting purinergic signaling represents a promising therapy for sepsis but drug targeting purinergic signaling is sex-specific and needs to be investigated to determine sex-related targeted therapies in sepsis.

Platelets are well known for their roles in hemostasis and thrombosis, and are increasingly recognized for their abilities to interact with white blood cells during inflammatory diseases, via secreted soluble factors as well as cell–cell contact. This interaction has been investigated in animal models and patient samples and has shown to be implicated in patient outcomes in several diseases. Platelet‐leukocyte co‐cultures are widely used to study platelet‐leukocyte interactions ex vivo. However, there is a paucity with regard to the systematic characterization of cell activation and functional behaviors of platelets and leukocytes in these co‐cultures. Hence we aimed to characterize a model of platelet‐leukocyte co‐culture ex vivo. Human peripheral blood mononuclear cell (PBMC) and platelets were isolated and co‐cultured for 5 days at 37 °C in the presence or absence of anti‐CD3/CD28 antibodies or PHA. We evaluated PF‐4 secretion and p‐selectin expression in platelets as markers of platelet activation. Lymphocyte activation was assessed by cell proliferation and cell population phenotyping, in addition to platelet‐lymphocyte aggregation. Platelet secretion and p‐selectin expression is maintained throughout the co‐culture, indicating that platelets were viable and reactive over the 5 days. Similarly PBMCs were viable and maintained proliferative capacity. Finally, dynamic heterotypic conjugation between platelets and T lymphocytes was also observed throughout co‐culture (with a peak at days 3 and 4) upon T lymphocyte activation. In conclusion, this in vitro model can successfully mimic the in vivo interaction between platelets and T lymphocytes, and can be used to confirm and/or support in vivo results.

Sepsis is a complex clinical syndrome resulting from a serious bloodstream infection. With hospital mortality rates of affected patients reportedly as high as 50%, improved methods for treating sepsis are urgently needed. To begin development of new pharmacologic therapies, we investigated the effect of an antiplatelet treatment on the proliferation of regulatory T cells (Tregs) in a murine model of sepsis. Tregs are a subset of T lymphocytes that downregulate the immune response and promote the resolution of inflammation. Septic patients have elevated levels of circulating Tregs, and this increased prevalence is associated with increased patient mortality. Platelets, which regulate inflammation through cell-cell interactions and through secretion of inflammatory mediators,have been shown to alter the proliferation and activation of Tregs in vitro. However, the influence of platelets on Tregs in vivohas not been fully investigated. We propose that suppression of platelet functions during sepsis may restrain Treg proliferation, leading to the restoration of immunological homeostasis. To study the influence of platelets on Treg proliferation in vivo, we blocked the P2Y12signaling pathway and measured the resulting population sizes of Tregs in septic mice. P2Y12is a Giprotein-coupled purinergic receptor present on platelet surfaces. Stimulation of P2Y12by ADP leads to platelet aggregation and potentiation of platelet secretion. To block the P2Y12signaling pathway, we used the P2Y12antagonist clopidogrel. To induce sepsis in mice, we used cecal ligation and puncture (CLP). Clopidogrelwas administered orallywith a loading dose (30 mg/kg in PBS) one day before surgery and a maintenance dose (10 mg/kg in PBS) two hours prior to surgery. The nonseptic mice in the negative control group (sham) were treated with PBS only. Twenty-four hours after surgery, we isolated cells from the spleens of the mice in each treatment group (sham, CLP, and CLP with clopidogrel) and measured Treg population sizes by incubating the cells with anti-CD4, anti-CD25,and anti-Foxp3 antibodies. Tregs were identified by their positive staining for CD4, CD25, and Foxp3. We found that Tregpopulation sizes were reduced in the septic mice treated with clopidogrel compared with those in the untreated septic mice (Figure 1A).Additionally, we used flow cytometry (forward and side light scattering) to investigatewhether P2Y12antagonism altered the aggregation of platelets and CD4+T cells in whole blood.Platelets and CD4+T cells wereidentified by their positive staining with PE-anti CD41 and FITC-anti CD4, respectively. Events that were double positive for FITC and PE were identified as aggregates and reported as a percentage of gated CD4+T cells.We found that aggregation of platelets and CD4+T cells was reduced in the septic mice treated with clopidogrel (15 ±5 %) compared with that in the untreated septic mice (38 ±6 %) (n= 3, p<0.05 treated CLP vs. untreated CLP). We investigated the effect of blocking the P2Y12signaling pathway in vitrousing co-cultures of human platelets and T cells. Human platelets and T cells were isolated from healthy donors and cultured in the presence or absence of anti-CD3/CD28 (5 μg/mLeach) antibodies for 5 days at 37°C in a humidified atmosphere containing 5% CO2. To block the P2Y12signaling pathway in vitro, we used AR-C69931MX (100 nM). We measured Treg population sizes using flow cytometry as described above. We found that Treg population sizes increased when resting T cells were exposed to platelets, AR-C, or both (Figure 1B). In contrast, we found that Treg population sizes decreased when CD3/CD28-stimulated T cells were exposed to a combination of platelets and AR-C (Figure 1B). Our data indicate that blockade of the P2Y12signaling pathway changes how platelets influence T cells in vitro, depending on whether the T cells have been activated. In conclusion, blockade of the P2Y12signaling pathway restrains Treg proliferation in vivoand in vitro. Our study indicates that targeting platelets to control Treg proliferation and activity may be a promising strategy for treating sepsis. Disclosures No relevant conflicts of interest to declare.