P2Y₁₂activation in the spleen during sepsis

Introduction: Sepsis is a complex clinical syndrome that results from dysregulation of the body’s response to invading pathogens. With mortality rates at 50%, improving methods for treating sepsis is critical. The spleen is the immune organ where the immune response initiates, and it is a key participant in immune cell maturation and platelet production. Indeed, patients undergoing splenectomy have a high risk of developing post-operative sepsis, hence, targeting the spleen can be a strategy for modulating the dysregulated immune response in sepsis. The ADP-receptors P2Y 12 play a sex-specific role in sepsis, and it is expressed in platelets and T lymphocytes. We have shown that blocking P2Y 12 reduced sepsis-induced spleen damage and reduced splenic regulatory T cells in male mice. Now we aim to investigate whether blocking P2Y 12 can restore spleen functions in both sexes. We hypothesize that P2Y 12 activation contributes to spleen damage and cellular composition. Methods: Males and Female C57BL6/6J mice underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. P2Y 12 antagonist, ticagrelor (30 mg/kg) was administered intraperitoneally after surgery. Twenty-four hours post-surgery, the spleen was harvested. To assess total cell counts in the spleen, we measured leukocyte, neutrophil, and platelet counts using Hemavet® Multispecies Hematology System. We analyzed the cell population (T and B lymphocytes, monocytes, and platelets) using flow cytometry. We assessed spleen damage by measuring Myeloperoxidase (MPO) content with ELISA and by evaluating the disruption of different organ compartments using histological classification. P2Y 12 protein levels were analyzed using Western blotting. Single-cell splenocytes were obtained from the spleen and activated with LPS for 48 hours. Cell migration with or without P2Y 12 blockage was measured using a transwell system. Results: Interestingly, sepsis significantly decreased splenic lymphocyte, neutrophil, and platelet count compared to their sham. Flow cytometry data showed a lower percentage of T and B lymphocytes, monocytes, and platelets in the spleen upon sepsis. Blocking P2Y 12 increased immune cell content (lymphocyte, neutrophil, and platelet) in the spleen compared with the untreated mice. It also prevented spleen tissue damage by decreasing MPO levels and restoring the percentage of white pulp compared to untreated CLP. The results were comparable between the sexes. Interestingly, protein levels of P2Y 12 were increased in septic male mice but not in females. Blocking P2Y 12 diminished protein P2Y 12 in the spleen of male mice but not female. Blocking P2Y 12 increased splenocyte migration in vitro in cells isolated from male mice but not females. Conclusions: Blocking P2Y 12 reduced sepsis-induced disorganization in the spleen and restored immune cell content in both sexes. In males, but not females, the effects could be due to decreased P2Y 12 protein levels and increased migration. This work was supported by the National Institute of Health, grant AI156627-01 to EL. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.