Background:Sepsis is often accompanied by lactic acidemia and acute lung injury(ALI).Clinical studies have established that high serum lactate levels are associated with increased mortality rates in septic patients.We...Background:Sepsis is often accompanied by lactic acidemia and acute lung injury(ALI).Clinical studies have established that high serum lactate levels are associated with increased mortality rates in septic patients.We further observed a significant correlation between the levels of cold-inducible RNA-binding protein(CIRP)in plasma and bronchoalveolar lavage fluid(BALF),as well as lactate levels,and the severity of post-sepsis ALI.The underlying mechanism,however,remains elusive.Methods:C57BL/6 wild type(WT),Casp8^(-/-),Ripk3^(-/-),and Zbp1^(-/-)mice were subjected to the cecal ligation and puncture(CLP)sepsis model.In this model,we measured intra-macrophage CIRP lactylation and the subsequent release of CIRP.We also tracked the internalization of extracellular CIRP(eCIRP)in pulmonary vascular endothelial cells(PVECs)and its interaction with Z-DNA binding protein 1(ZBP1).Furthermore,we monitored changes in ZBP1 levels in PVECs and the consequent activation of cell death pathways.Results:In the current study,we demonstrate that lactate,accumulating during sepsis,promotes the lactylation of CIRP in macrophages,leading to the release of CIRP.Once eCIRP is internalized by PVEC through a Toll-like receptor 4(TLR4)-mediated endocytosis pathway,it competitively binds to ZBP1 and effectively blocks the interaction between ZBP1 and tripartite motif containing 32(TRIM32),an E3 ubiquitin ligase targeting ZBP1 for proteasomal degradation.This interference mechanism stabilizes ZBP1,thereby enhancing ZBP1-receptor-interacting protein kinase 3(RIPK3)-dependent PVEC PANoptosis,a form of cell death involving the simultaneous activation of multiple cell death pathways,thereby exacerbating ALI.Conclusions:These findings unveil a novel pathway by which lactic acidemia promotes macrophage-derived eCIRP release,which,in turn,mediates ZBP1-dependent PVEC PANoptosis in sepsis-induced ALI.This finding offers new insights into the molecular mechanisms driving sepsis-related pulmonary complications and provides potential new therapeutic strategies.展开更多
In sepsis, macrophage bacterial phagocytosis is impaired, but the mechanism is not well elucidated. Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern that causes inflamm...In sepsis, macrophage bacterial phagocytosis is impaired, but the mechanism is not well elucidated. Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern that causes inflammation. However, whether eCIRP regulates macrophage bacterial phagocytosis is unknown. Here, we reported that the bacterial loads in the blood and peritoneal fluid were decreased in CIRP^(−/−) mice and anti-eCIRP Ab-treated mice after sepsis. Increased eCIRP levels were correlated with decreased bacterial clearance in septic mice. CIRP−/− mice showed a marked increase in survival after sepsis. Recombinant murine CIRP (rmCIRP) significantly decreased the phagocytosis of bacteria by macrophages in vivo and in vitro. rmCIRP decreased the protein expression of actin-binding proteins, ARP2, and p-cofilin in macrophages. rmCIRP significantly downregulated the protein expression of βPIX, a Rac1 activator. We further demonstrated that STAT3 and βPIX formed a complex following rmCIRP treatment, preventing βPIX from activating Rac1. We also found that eCIRP-induced STAT3 phosphorylation was required for eCIRP’s action in actin remodeling. Inhibition of STAT3 phosphorylation prevented the formation of the STAT3-βPIX complex, restoring ARP2 and p-cofilin expression and membrane protrusion in rmCIRP-treated macrophages. The STAT3 inhibitor stattic rescued the macrophage phagocytic dysfunction induced by rmCIRP. Thus, we identified a novel mechanism of macrophage phagocytic dysfunction caused by eCIRP, which provides a new therapeutic target to ameliorate sepsis.展开更多
基金supported by grants from the USA VA(1I01BX004838 and IK6BX006297)the Shenzhen Science and Technology Program(JCYJ20230807142311024).
文摘Background:Sepsis is often accompanied by lactic acidemia and acute lung injury(ALI).Clinical studies have established that high serum lactate levels are associated with increased mortality rates in septic patients.We further observed a significant correlation between the levels of cold-inducible RNA-binding protein(CIRP)in plasma and bronchoalveolar lavage fluid(BALF),as well as lactate levels,and the severity of post-sepsis ALI.The underlying mechanism,however,remains elusive.Methods:C57BL/6 wild type(WT),Casp8^(-/-),Ripk3^(-/-),and Zbp1^(-/-)mice were subjected to the cecal ligation and puncture(CLP)sepsis model.In this model,we measured intra-macrophage CIRP lactylation and the subsequent release of CIRP.We also tracked the internalization of extracellular CIRP(eCIRP)in pulmonary vascular endothelial cells(PVECs)and its interaction with Z-DNA binding protein 1(ZBP1).Furthermore,we monitored changes in ZBP1 levels in PVECs and the consequent activation of cell death pathways.Results:In the current study,we demonstrate that lactate,accumulating during sepsis,promotes the lactylation of CIRP in macrophages,leading to the release of CIRP.Once eCIRP is internalized by PVEC through a Toll-like receptor 4(TLR4)-mediated endocytosis pathway,it competitively binds to ZBP1 and effectively blocks the interaction between ZBP1 and tripartite motif containing 32(TRIM32),an E3 ubiquitin ligase targeting ZBP1 for proteasomal degradation.This interference mechanism stabilizes ZBP1,thereby enhancing ZBP1-receptor-interacting protein kinase 3(RIPK3)-dependent PVEC PANoptosis,a form of cell death involving the simultaneous activation of multiple cell death pathways,thereby exacerbating ALI.Conclusions:These findings unveil a novel pathway by which lactic acidemia promotes macrophage-derived eCIRP release,which,in turn,mediates ZBP1-dependent PVEC PANoptosis in sepsis-induced ALI.This finding offers new insights into the molecular mechanisms driving sepsis-related pulmonary complications and provides potential new therapeutic strategies.
基金supported by the US National Institutes of Health(NIH)grants R35GM118337,R01HL076179,R01AA028947,U01AI133655 and U01AI170018MA is supported by the US NIH grants R01GM129633 and U01AI170018.
文摘In sepsis, macrophage bacterial phagocytosis is impaired, but the mechanism is not well elucidated. Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern that causes inflammation. However, whether eCIRP regulates macrophage bacterial phagocytosis is unknown. Here, we reported that the bacterial loads in the blood and peritoneal fluid were decreased in CIRP^(−/−) mice and anti-eCIRP Ab-treated mice after sepsis. Increased eCIRP levels were correlated with decreased bacterial clearance in septic mice. CIRP−/− mice showed a marked increase in survival after sepsis. Recombinant murine CIRP (rmCIRP) significantly decreased the phagocytosis of bacteria by macrophages in vivo and in vitro. rmCIRP decreased the protein expression of actin-binding proteins, ARP2, and p-cofilin in macrophages. rmCIRP significantly downregulated the protein expression of βPIX, a Rac1 activator. We further demonstrated that STAT3 and βPIX formed a complex following rmCIRP treatment, preventing βPIX from activating Rac1. We also found that eCIRP-induced STAT3 phosphorylation was required for eCIRP’s action in actin remodeling. Inhibition of STAT3 phosphorylation prevented the formation of the STAT3-βPIX complex, restoring ARP2 and p-cofilin expression and membrane protrusion in rmCIRP-treated macrophages. The STAT3 inhibitor stattic rescued the macrophage phagocytic dysfunction induced by rmCIRP. Thus, we identified a novel mechanism of macrophage phagocytic dysfunction caused by eCIRP, which provides a new therapeutic target to ameliorate sepsis.
