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IntroductionExosomes are major actors in the progression of cardiovascular diseases and potential associated-treatments. We showed previously that inactivation of the mouse P2Y4 nucleotide receptor induces a protection against myocardial infarction in the left anterior descending artery ligation model, characterized by smaller infarcts and reduced cardiac fibrosis and inflammation, compared to wild-type mice. This cardioprotection was associated with adiponectin and PD-L1 overexpression, regulatory leukocyte increase, and adipocyte beiging in the pericardial adipose tissue of P2Y4-null mice. We investigated here the contribution of exosome release in the cardioprotection observed in ischemic P2Y4-null mice.Methods and resultsInterestingly the reduction of cardiac fibrosis and T cell infiltration observed in P2Y4-null compared to wild-type ischemic heart was abolished after intraperitoneal injection of the exosome inhibitor GW4869 during myocardial infarction onset, as previously observed using an anti-PD-L1 blocking antibody. Additionally, GW4869 injection totally inhibited the increase in plasma PD-L1 level observed in P2Y4-null ischemic mice, as well as the higher T cell apoptosis in their pericardial adipose tissue, compared to wild-type mice. We observed increased expression of CDH13/T-cadherin, essential for adiponectin-driven exosome biogenesis, in P2Y4-null pericardial adipose tissue. Plasma exosomes were isolated from wild-type and P2Y4-null ischemic mice and characterized using nanoparticle tracking analysis and transmission electron microscopy experiments, as well as Western blot analysis of CD63 exosome marker and adiponectin expression. Our data support an increase in exosomes from adipocyte origin in the plasma of P2Y4-null ischemic mice. Flow cytometry experiments showed that P2Y4-null ischemic mice displayed an increased level of PD-L1+ plasma exosomes compared to wild-type ischemic mice. We finally demonstrated the capacity of total plasma exosomes from P2Y4-null ischemic mice to polarize macrophages into the anti-inflammatory M2c phenotype in vitro . M2c macrophages can inhibit T cell activation through PD-L1 regulation and play a central role in the resolution of cardiac inflammation to promote cardiac repair.DiscussionThese data support the role of the release of anti-inflammatory exosomes, and more particularly the exosomal form of PD-L1 and adiponectin, in P2Y4 loss-mediated cardioprotection. The study of regulators of cardioprotective exosomes could lead to the development of novel anti-inflammatory therapies to improve myocardial infarction outcome.

A better understanding of the immune function of pericardial adipose tissue is essential to adapt treatments after myocardial infarction. We showed previously that inactivation of mouse P2Y4 nucleotide receptor induces adiponectin overexpression and protection against myocardial infarction. We investigated here the inflammatory state of pericardial adipose tissue in ischemic P2Y4-deficient mice. We demonstrated that P2Y4-deficient mice displayed adipocyte beiging with increased PD-L1 expression and a higher number of regulatory leukocytes in their pericardial adipose tissue after left anterior descending artery ligation, compared to wild type mice. Effectively, a higher level of anti-inflammatory M2c macrophages and regulatory T cells was observed in pericardial adipose tissue of P2Y4 KO mice and correlated with reduced post-ischemic expansion of fat-associated lymphoid clusters. Interestingly, the anti-inflammatory effects observed in P2Y4 KO mice, were no more observed in P2Y4/adiponectin double KO ischemic mice. Finally, the reduction of T cell infiltration and cardiac fibrosis observed in P2Y4-deficient heart was lost after injection of anti-PD-L1 blocking antibody in ischemic mice. The present study defines P2Y4 as a regulator of PD-L1 and adiponectin, and as a potential target for anti-inflammatory therapies to improve myocardial infarction outcome. The combined effect of P2Y4 loss on adipocyte beiging and regulatory leukocyte increase highlights this nucleotide receptor as an important player in post-ischemic cardiac response.A better understanding of the immune function of pericardial adipose tissue is essential to adapt treatments after myocardial infarction. We showed previously that inactivation of mouse P2Y4 nucleotide receptor induces adiponectin overexpression and protection against myocardial infarction. We investigated here the inflammatory state of pericardial adipose tissue in ischemic P2Y4-deficient mice. We demonstrated that P2Y4-deficient mice displayed adipocyte beiging with increased PD-L1 expression and a higher number of regulatory leukocytes in their pericardial adipose tissue after left anterior descending artery ligation, compared to wild type mice. Effectively, a higher level of anti-inflammatory M2c macrophages and regulatory T cells was observed in pericardial adipose tissue of P2Y4 KO mice and correlated with reduced post-ischemic expansion of fat-associated lymphoid clusters. Interestingly, the anti-inflammatory effects observed in P2Y4 KO mice, were no more observed in P2Y4/adiponectin double KO ischemic mice. Finally, the reduction of T cell infiltration and cardiac fibrosis observed in P2Y4-deficient heart was lost after injection of anti-PD-L1 blocking antibody in ischemic mice. The present study defines P2Y4 as a regulator of PD-L1 and adiponectin, and as a potential target for anti-inflammatory therapies to improve myocardial infarction outcome. The combined effect of P2Y4 loss on adipocyte beiging and regulatory leukocyte increase highlights this nucleotide receptor as an important player in post-ischemic cardiac response.

Adipose tissue is a source of stem cells with a high potential of differentiation for cell-based regenerative therapies. We previously identified mouse P2Y 2, an ATP and UTP nucleotide receptor, as a regulator of adipogenic and endothelial differentiation of cardiac adipose-derived stem cells (cADSC). We investigated here the potential involvement of P2Y 2 receptor in the cardioprotective action of undifferentiated cADSC transplantation in mouse ischemic heart. Transplantation of cADSC was realized in the periphery of the infarcted zone of ischemic heart, 3 days after left anterior descending artery ligation. A strong reduction of collagen stained area was observed 14 days after cADSC injection, compared to PBS injection. Interestingly, loss of P2Y 2 expression totally inhibits the ability of transplanted cADSC to reduce cardiac fibrosis. A detailed gene ontology enrichment analysis was realized by comparing RNA-sequencing data obtained for UTP-treated wild type cASDC and UTP-treated P2Y 2 -null cASDC. We identified UTP target genes linked to extracellular matrix organization such as matrix metalloproteinases and various collagen types, UTP target genes related to macrophage chemotaxis and differentiation into pro-fibrotic foam cells, and a significant number of UTP target genes linked to angiogenesis regulation. More particularly, we showed that UTP regulated the secretion of CCL5, CXCL5, and CCL12 chemokines and serum amyloid apolipoprotein 3, in the supernatants of UTP-treated cADSC. Interestingly, CCL5 is reported as a key factor in post-infarction heart failure and in the reparative and angiogenic action of transplanted ADSC on ischemic tissue. We investigated then if a UTP-pretreatment of cADSC amplifies their effect on cardiac revascularization in mouse ischemic heart. Transplantation of cADSC was able to increase peri-infarct capillary density, 14 days after their injection. This beneficial effect on cardiac revascularization was enhanced by a UTP-pretreatment of cADSC before their transplantation, and not observed using P2Y 2 -null cADSC. Our data support that the efficacy of transplanted cADSC can be regulated by the release of inflammatory mediators such as extracellular nucleotides in the ischemic site. The present study highlights the P2Y 2 receptor as a regulator of cADSC cardioprotective action, and as a potential target for the therapeutic use of undifferentiated cADSC in post-ischemic cardiac ischemia.

Therapeutic angiogenesis is likely to require the administration of factors that complement each other. Activation of the receptor tyrosine kinase (RTK) Flk1 by vascular endothelial growth factor (VEGF) is crucial, but molecular interactions of other factors with VEGF and Flk1 have been studied to a limited extent. Here we report that placental growth factor (PGF, also known as PlGF) regulates inter- and intramolecular cross talk between the VEGF RTKs Flt1 and Flk1. Activation of Flt1 by PGF resulted in intermolecular transphosphorylation of Flk1, thereby amplifying VEGF-driven angiogenesis through Flk1. Even though VEGF and PGF both bind Flt1, PGF uniquely stimulated the phosphorylation of specific Flt1 tyrosine residues and the expression of distinct downstream target genes. Furthermore, the VEGF/PGF heterodimer activated intramolecular VEGF receptor cross talk through formation of Flk1/Flt1 heterodimers. The inter- and intramolecular VEGF receptor cross talk is likely to have therapeutic implications, as treatment with VEGF/PGF heterodimer or a combination of VEGF plus PGF increased ischemic myocardial angiogenesis in a mouse model that was refractory to VEGF alone.

Human P2Y 4 is a UTP receptor, while in mice it is activated by both ATP and UTP. P2Y 4 knockout (KO) in mice protects against myocardial infarction and is characterized by increased adiponectin secretion by adipocytes, and decreased cardiac inflammation and permeability under ischemic conditions. The relevance of these data has, however, not been explored to date in humans. In a population study comprising 50 patients with coronary artery disease (CAD) and 50 age-matched control individuals, we analyzed P2RY4 mutations and their potential association with CAD severity and fasting plasma parameters. Among the mutations identified, we focused our attention on a coding region polymorphism (rs3745601) that results in replacement of the asparagine at residue 178 with threonine (N178T) located in the second extracellular loop of the P2Y 4 receptor. The N178T variant is a loss-of-function mutation of the human P2Y 4 receptor and is encountered less frequently in coronary patients than in control individuals. In coronary patients, carriers of the N178T variant had significantly reduced jeopardy and Gensini cardiac severity scores, as well as lower resting heart rates and plasma levels of N-terminal pro-brain natriuretic peptide (NT-proBNP). Regarding fasting plasma parameters, the N178T variant was associated with a lower concentration of glucose. Accordingly, P2Y 4 KO mice had significantly improved glucose tolerance and insulin sensitivity compared with their WT littermate controls. The improvement of insulin sensitivity resulting from lack of the P2Y 4 receptor was no longer observed in the absence of adiponectin. The present study identifies a frequent loss-of-function P2Y 4 variant associated with less severe coronary artery atherosclerosis and lower fasting plasma glucose in coronary patients. The role of the P2Y 4 receptor in glucose homeostasis was confirmed in mouse. P2Y 4 antagonists could thus have therapeutic applications in the treatment of myocardial infarction and type 2 diabetes.

We aimed to study the role of the nucleotide receptor P2Y2R in the development of experimental autoimmune uveitis (EAU). EAU was induced in P2Y2+/+ and P2Y2-/- mice by immunization with IRBP peptide or by adoptive transfer of in vitro restimulated semi-purified IRBP-specific enriched T lymphocytes from spleens and lymph nodes isolated from native C57Bl/6 or P2Y2+/+ and P2Y2-/- immunized mice. Clinical and histological scores were used to grade disease severity. Splenocytes and lymph node cell phenotypes were analyzed using flow cytometry. Semi-purified lymphocytes and MACS-purified CD4+ T lymphocytes from P2Y2+/+ and P2Y2-/- immunized mice were tested for proliferation and cytokine secretion. Our data show that clinical and histological scores were significantly decreased in IRBP-immunized P2Y2-/- mice as in P2Y2-/- mice adoptively transfered with enriched T lymphocytes from C57Bl/6 IRBP-immunized mice. In parallel, naïve C57Bl/6 mice adoptively transferred with T lymphocytes from P2Y2-/- IRBP-immunized mice also showed significantly less disease. No differences in term of spleen and lymph node cell recruitment or phenotype appeared between P2Y2-/- and P2Y2+/+ immunized mice. However, once restimulated in vitro with IRBP, P2Y2-/- T cells proliferate less and secrete less cytokines than the P2Y2+/+ one. We further found that antigen-presenting cells of P2Y2-/- immunized mice were responsible for this proliferation defect. Together our data show that P2Y2-/- mice are less susceptible to mount an autoimmune response against IRBP. Those results are in accordance with the danger model, which makes a link between autoreactive lymphocyte activation, cell migration and the release of danger signals such as extracellular nucleotides.

ATP released in the early inflammatory processes acts as a danger signal by binding to purinergic receptors expressed on immune cells. A major contribution of the P2Y(2) receptor of ATP/UTP to dendritic cell function and Th2 lymphocyte recruitment during asthmatic airway inflammation was previously reported. We investigated here the involvement of P2Y(2) receptor in lung inflammation initiated by pneumonia virus of mice infection. We demonstrated that P2Y(2) (-/-) mice display a severe increase in morbidity and mortality rate in response to the virus. Lower survival of P2Y(2) (-/-) mice was not significantly correlated with excessive inflammation despite the higher level of neutrophil recruiters in their bronchoalveolar fluids. Interestingly, we observed reduced ATP level and lower numbers of dendritic cells, CD4(+) T cells and CD8(+) T cells in P2Y(2) (-/-) compared to P2Y(2) (+/+) infected lungs. Lower level of IL-12 and higher level of IL-6 in bronchoalveolar fluid support an inhibition of Th1 response in P2Y(2) (-/-) infected mice. Quantification of DC recruiter expression revealed comparable IP-10 and MIP-3α levels but a reduced BRAK level in P2Y(2) (-/-) compared to P2Y(2) (+/+) bronchoalveolar fluids. The increased morbidity and mortality of P2Y(2) (-/-) mice could be the consequence of a lower viral clearance leading to a more persistent viral load correlated with the observed higher viral titer. The decreased viral clearance could result from the defective Th1 response to PVM with a lack of DC and T cell infiltration. In conclusion, P2Y(2) receptor, previously described as a target in cystic fibrosis therapy and as a mediator of Th2 response in asthma, may also regulate Th1 response protecting mice against lung viral infection.

Exosomes are major actors in the progression of cardiovascular diseases and potential associated-treatments. We showed previously that inactivation of the mouse P2Y nucleotide receptor induces a protection against myocardial infarction in the left anterior descending artery ligation model, characterized by smaller infarcts and reduced cardiac fibrosis and inflammation, compared to wild-type mice. This cardioprotection was associated with adiponectin and PD-L1 overexpression, regulatory leukocyte increase, and adipocyte beiging in the pericardial adipose tissue of P2Y -null mice. We investigated here the contribution of exosome release in the cardioprotection observed in ischemic P2Y -null mice. Interestingly the reduction of cardiac fibrosis and T cell infiltration observed in P2Y -null compared to wild-type ischemic heart was abolished after intraperitoneal injection of the exosome inhibitor GW4869 during myocardial infarction onset, as previously observed using an anti-PD-L1 blocking antibody. Additionally, GW4869 injection totally inhibited the increase in plasma PD-L1 level observed in P2Y -null ischemic mice, as well as the higher T cell apoptosis in their pericardial adipose tissue, compared to wild-type mice. We observed increased expression of CDH13/T-cadherin, essential for adiponectin-driven exosome biogenesis, in P2Y -null pericardial adipose tissue. Plasma exosomes were isolated from wild-type and P2Y -null ischemic mice and characterized using nanoparticle tracking analysis and transmission electron microscopy experiments, as well as Western blot analysis of CD63 exosome marker and adiponectin expression. Our data support an increase in exosomes from adipocyte origin in the plasma of P2Y -null ischemic mice. Flow cytometry experiments showed that P2Y -null ischemic mice displayed an increased level of PD-L1 plasma exosomes compared to wild-type ischemic mice. We finally demonstrated the capacity of total plasma exosomes from P2Y -null ischemic mice to polarize macrophages into the anti-inflammatory M2c phenotype . M2c macrophages can inhibit T cell activation through PD-L1 regulation and play a central role in the resolution of cardiac inflammation to promote cardiac repair. These data support the role of the release of anti-inflammatory exosomes, and more particularly the exosomal form of PD-L1 and adiponectin, in P2Y loss-mediated cardioprotection. The study of regulators of cardioprotective exosomes could lead to the development of novel anti-inflammatory therapies to improve myocardial infarction outcome.

[This corrects the article DOI: 10.3389/fimmu.2022.1006934.].[This corrects the article DOI: 10.3389/fimmu.2022.1006934.].

Human P2Y is a UTP receptor, while in mice it is activated by both ATP and UTP. P2Y knockout (KO) in mice protects against myocardial infarction and is characterized by increased adiponectin secretion by adipocytes, and decreased cardiac inflammation and permeability under ischemic conditions. The relevance of these data has, however, not been explored to date in humans. In a population study comprising 50 patients with coronary artery disease (CAD) and 50 age-matched control individuals, we analyzed mutations and their potential association with CAD severity and fasting plasma parameters. Among the mutations identified, we focused our attention on a coding region polymorphism (rs3745601) that results in replacement of the asparagine at residue 178 with threonine (N178T) located in the second extracellular loop of the P2Y receptor. The N178T variant is a loss-of-function mutation of the human P2Y receptor and is encountered less frequently in coronary patients than in control individuals. In coronary patients, carriers of the N178T variant had significantly reduced jeopardy and Gensini cardiac severity scores, as well as lower resting heart rates and plasma levels of N-terminal pro-brain natriuretic peptide (NT-proBNP). Regarding fasting plasma parameters, the N178T variant was associated with a lower concentration of glucose. Accordingly, P2Y KO mice had significantly improved glucose tolerance and insulin sensitivity compared with their WT littermate controls. The improvement of insulin sensitivity resulting from lack of the P2Y receptor was no longer observed in the absence of adiponectin. The present study identifies a frequent loss-of-function P2Y variant associated with less severe coronary artery atherosclerosis and lower fasting plasma glucose in coronary patients. The role of the P2Y receptor in glucose homeostasis was confirmed in mouse. P2Y antagonists could thus have therapeutic applications in the treatment of myocardial infarction and type 2 diabetes.