Endothelial P2Y2 and P2X7 receptors colocalizes with Caveolin-1 boosting Monocyte Adhesion in Intestinal Schistosomiasis

Intravascular schistosomiasis promotes endothelial cell damage and ATP release. Recently we showed a positive cooperation between endothelial P2Y₂ and P2X7 receptors (P2Y₂R/P2X7R) triggering an increased monocyte adhesion during schistosomiasis (Front. Immunol. 2024;14:1328897). However, the role of Cav-1 to endothelial P2Y₂R/P2X7R crosstalk and intestinal inflammation remains unknown. Primary cultures of mesenteric endothelial cells were obtained from control and S. mansoni-infected mice (50-70 d.p.i) (CEUA UFRJ A07/26-124-22). Confluent cells were incubated with vehicle (basal), UTP (100 μM, 5h) and ATP (500 µM, 10 min) in the presence or absence of siRNA Cav-1 transfection (SI02731813; 1 pmol) for 48h. Western blotting was used to analyze co-immunoprecipitation. Cells were imaged (Zeiss Celldiscover 7, LSM 900) and analyzed by ImageJ software. Functional data was performed by co-culture assay.  Data were expressed as mean and SEM. P2Y₂R and P2X7R are coimmunoprecipitated at same levels in both groups (n=4). The colocalization of Cav-1 and P2X7R or P2Y₂R were higher in the infected than in the control group (311.5% ± 31.5 compared to 100.1% ± 13.5 and 312.6% ± 38.4 compared to 100.0% ± 14.8, respectively, p < 0.001, n=4). Moreover, in the infected group, the P2Y₂R/P2X7R coactivation increased the P2X7/Cav-1 colocalization compared to basal levels (from 100.1% ± 12.6 to 213.2% ± 21.0, p < 0.001, n=4) without altering protein expression (p > 0.05), suggesting a dynamic redistribution of P2Y₂R/P2X7R into Cav-1 microdomains. Cav-1 knockdown inhibited P2Y₂R/P2X7R-induced monocyte adhesion (from 11.2 ± 1.2 to 5.4 ± 0.3 cells/field; p < 0.001, n = 4). Our data unveiled an endothelial P2Y₂R/P2X7R basal complex with Cav-1-dependent crosstalk during schistosomiasis, which may be an important pathway favoring mesenteric inflammation. Support: CNPq, INCT "Sinalização Purinérgica: Desafios para a Saúde do Século XXI", CNPq 409156/2024-8, CAPES, FAPERJ, FIOCRUZ (RJ).