Platelets HMGB1 Participate In Host Defense And Neutrophils Recruitment During Sepsis

Part 1 Platelets participate in host defense during CLPObjective Platelets were recognized to play an important role in hemostasis. More and more evidence indicated that platelets play an important role in inflammation and infection. Sepsis is the leading cause of death in critical ill patients. However, the role of platelet on host responses to polymicrobial intra-abdominal sepsis remains unknown.MethodsWild type mice were subjected to cecal ligation and puncture model. Anti-a-GpIbaantibody was injected by intra-peritoneum injection2 hours before CLP. Mice blood was collected 2 hours after antibody injection and 18hours after CLP and blood were analyzed by cell counting machine. Peritoneal lavage fluid, blood and lung were collected 18 hours after CLP, and applied to 5% sheep blood agar platelets for bacterial culture. Colony forming units were counted after overnight incubation in 37℃ incubator. Plasma IL-6 level was measured to detect the systemic inflammatory response.Results The anti-a-GpIbaantibodyinjected mice showed significantly reduced circulating platelets compared to control mice. The platelets depletion mice had significantly increased bacterial load in peritoneal lavage fluid, blood and lung homogenates. Consistently, the systemic inflammatory response was increased compared to control mice, which was associated with bacterial load.ConclusionsAnti-a-GpIbaantibodycan significantly reduced platelets counts in circulation. Platelets play an important role in host defense.Part 2 platelets TLR4 knock out doesnot affect host defense but can attenuates inflammatory response during endotoxemiaObjectivePlatelets have TLR4 expression on their surface. Toll like receptor 4 (TLR4) is a pathogen associated molecule pattern. TLR4 has cell specific functions, but the function of platelets TLR4 in sepsis and endotoxemia remains unknown.Methods WT, TLR4 KO, PF4 TLR4 KO mice were subjected to cecal ligation and puncture model and LPS injection (10mg/Kg, ip). Blood was collected 6h and 18h after LPS injection. IL-6, IL-1β and HMGB1 levels were measure to detect the systemic inflammatory response. And platelets and white blood cells were counted by cell counting machine.18 hours after CLP, peritoneal lavage fluid, blood and lung were collected 18 hours after CLP, and cultured for bacterial culture. Colony forming units were counted after overnight incubation in 37℃ incubator. Plasma were collected to detected IL-6, IL-1β level. NETs were measured by detecting MPO-DNA complex level by ELISA.ResultsPF4 TLR4 KO mice showed significantly reduced IL-6, IL-1β and HMGB1 level compared to TLR4 flox mice during endotoxemia. The PF4 TLR4 KO mice showed higher platelet and white blood cell counting. The bacterial load in peritoneal lavage fluid, blood and lung showed no difference among each strain. Consistently, the systemic inflammatory response was around the same level with control mice.Conclusions Platelet TLR4 participate in inflammatory response but not host defense and NETs formation.Part 3 Platelets HMGB1 participate in host defense and neutrophils recruitment during sepsisObjectivePlatelets are recognized to play an important role in inflammation and this may occur through platelet-neutrophil (PMN) interactions. Platelets are known to express a number of inflammatory mediators, including HMGB1. Furthermore, HMGB1 have well-established roles in the pathogenesis of sepsis but neither the sources of HMGB1 nor the importance of platelet HMGB1 has been addressed.Methods WT, HMGB1 flox, HMGB1 PF4 mice were subjected to cecal ligation and puncture.18 hours after CLP, peritoneal lavage fluid, blood and lung were collected 18 hours after CLP, and applied to 5% sheep blood agar plates for bacterial culture. Colony forming units were counted after overnight incubation in 37℃ incubator. Plasma were collected to detected IL-6, IL-1β level. NETs were measured by detecting MPO-DNA complex level by ELISA. We isolated platelets from WT, PF4-HMGB1KO, and HMGB1 flox mice and stimulated these cells with collagen and thrombin. We next co-cultured resting and activated platelets with PMNs.ResultsThe bacterial load in the peritoneum, blood, and lung were significantly higher in the PF4-HMGB1KO mice than the controls. The increased bacterial counts in the HMGB 1-platlet specific knockouts were associated with higher plasma IL6 levels at 18 hrs. We found that P-selectin expression in the PF4-HMGB1KO platelets was lower compared to platelets from wild type or HMGB1 flox after stimulation. The platelet-PMN interaction was significantly lower in the activated HMGB1 knockout platelets than WT platelets. The PMN co-cultured with activated WT platelets also produced more reactive oxygen species than those cultured with the PF4-HMGB1KO platelets.ConclusionsThese data show that HMGB1 can participate in the host defense to intra-abdominal polymicrobial sepsis by regulating PMN recruitment and possibly through direct platelet-PMN interaction.