The Mechanism Of Stored Red Blood Cell-transfusion Aggravating Bacterial Infection-induced Liver Injury

Stored red blood cells transfusion,which is an independent risk factor leading to death,can induce post-traumatic inflammation,enhance infection,cause sepsis,lead to infectious complications.But the specific mechanism is not clear.Therefore,to solve the clinical problem of stored of RBCs transfusion inducing post-traumatic inflammation and enhancing the infection,we must strengthen the pathogenesis study.The vital organs of the body,especially the liver,play an important role in the occurrence and development of sepsis.At the same time,the liver is also the main organ in the iron circulation and the removal of senile RBCs.According to the regulation of liver activity in sepsis,erythrocyte clearance and the theory of "stored RBCs inducing post-traumatic inflammation and enhancing infection",we hypothesize that stored RBCs transfusion enhances bacterial infection and causes sepsis,which may be associated with increased liver injury caused by the stored RBCs.In order to verify the above hypothesis,this study will be based on visual monitoring technology to monitor the stored RBCs aggravating sepsis and causing liver injury process,exploring its mechanism.Although Kupffer cells are closely related to sepsis-induced liver injury and the relationship between stored RBCs and macrophages has also been demonstrated,the effect of stored RBCs transfusion on Kupffer cells after bacterial infection and its effect on liver.The mechanism of intracellular signaling pathways has not been reported so far,especially in the transfusion of stored RBCs during sepsis in the liver after Kupffer cell polarization and activation.The mechanism is still unclear.Therefore,this study will further explore the stored RBCs transfusion aggravating bacterial infection and promoting liver injury and Kuffer cell-mediated immune response,and analyze its molecular mechanism of liver injury.The purpose of this study is to understand the stored RBCs on liver damage regulation and its impact on bacterial infection,to provide a scientific basis of safe blood transfusion for traumatic patients.It is of great significance to improve the safety and effectiveness of clinical transfusion.The results show that: 1.Transfusions of stored RBCs enhanced P.aeruginosa-induced liver injury in vivoFirst,in vitro imaging was performed to confirm that the luciferase activity correlated positively with the number of bacterial CFUs.Next,a sepsis mouse model was induced by tail-vein injection of P.aeruginosa,a typical gram-negative bacterium.To confirm the infection,the mice were analyzed by in vivo imaging.As shown,bioluminescence readings correlated with bacteria CFUs numbers,which were determined by conventional enumeration,demonstrating that bioluminescence can be applied to monitor changes in bacterial survival.To investigate the effect of stored-RBCs transfusions on P.aeruginosa-induced sepsis,WT mice were injected intravenously with P.aeruginosa together with fresh or stored RBCs.The data indicated that transfusing stored RBCs could exacerbate sepsis and decrease survival in P.aeruginosa-infected mice.The data demonstrated that P.aeruginosa injection induced the highest luciferase signal in the hepatic region.The serum levels of ALT and AST in mice treated with fresh RBCs increased,but were significantly reduced compared to those of mice treated with stored RBCs.Histological examination of liver sections demonstrated concordant results.These results indicated that transfusions with stored RBCs enhanced P.aeruginosa-induced liver injury in vivo,and liver injury exacerbated the severity of sepsis.2.Increased levels of M1-polarized Kupffer cells were responsible for the enhanced proinflammatory responses mediated by transfused stored RBCsTo determine the proinflammatory effects of transfusing stored RBCs,the levels of the proinflammatory cytokines IL-6,IL-1?,and tumor necrosis factor-?(TNF-?)were monitored in P.aeruginosa-infected mice.When P.aeruginosa-infected mice were transfused with stored RBCs,the release of TNF-?,IL-6,and IL-1? was significantly higher than the levels in mice transfused with fresh RBCs at 2 h post-infection,indicating an important involvement of TNF-?,IL-6,and IL-1? in stored-RBCs transfusion-associated liver injury.These results indicated that stored-RBCs transfusions could amplify systemic inflammatory responses to bacterial infection during sepsis and that excessive inflammatory responses resulted in liver injury.To investigate the cellular origins of inflammatory cytokines,We incubated LPS with ex vivo-generated,BMDMs after incubation with stored RBCs or fresh RBCs.The IL-6 and TNF-? levels in the supernatant of the stored-RBCs-treated group were significantly higher than those observed in the fresh-RBCs-treated group,suggesting that incubation with stored RBCs leads to markedly enhanced macrophage activation.As determined by flow cytometry,among F4/80+ macrophages,M1-specific markers were more highly expressed by cells treated with stored RBCs,compared to those treated with fresh RBCs,whereas M2-specific markers showed no difference.M1-polarized macrophages played an important role in the enhanced proinflammatory responses mediated by transfused stored RBCs.To determine the effects of transfusing stored RBCs on Kupffer cells in the mouse liver,Kupffer cells marks of phenotypes in the mouse liver were assessed by quantitative PCR post-transfusion.The result indicated that the injection of stored RBCs led to dramatic Kupffer cell fluctuations in the livers of mice infected with P.aeruginosa.Tissue samples from the stored-RBCs-treated group had higher i NOS,TNF-?,and MCP1 m RNA levels,which were measured to quantify M1 subpopulations,compared to those in the fresh-RBCs-treated group.The data revealed,for the first time,that the in vivo transfusion of stored RBCs could significantly enhance the polarization of liver-resident Kupffer cells towards an M1 phenotype,which is responsible for the enhanced proinflammatory response mediated by transfused,stored RBCs.3.Stored RBCs induced hepatic apoptosis by disturbing the dynamic balance between the NF-?B and JNK pathwaysTo further explore the mechanism of stored RBCs transfusion enhancing bacterial infection-induced liver injury,we studied The effect of stored RBCs transfusion on NF-?B and JNK signaling pathways in sepsis mice hepatocytes by the early visualized mouse model of the liver NF-?B and caspase-3.A patt B-NF-?B-Fluc reporter was integrated in mouse hepatocytes by hydrodynamic gene delivery,so that the dynamic activation of NF-?B could be monitored by BLI.The data indicated that bacterial infection upregulated mouse liver NF-?B activation,while the NF-?B activation could be inhibited by the transfusion of stored RBCs.EMSA analysis documented concordant results,as evidenced by reduced signaling at 8 h post-transfusion.The reporter-expression cassette att B-ANLuc(DEVD)BCLuc integrated permanently into the mouse liver chromosome,and caspase-3 activity could be evaluated under true physiological conditions by BLI.The data indicated that the JNK and caspase pathways were enhanced by the transfusion of stored RBCs.Western blot analysis showed a significant reduction in phosphorylation of the p65 protein in liver samples from the fresh-RBCs group compared with liver samples from the stored-RBCs group.The results demonstrates that NF-?B activation,which is important for sustaining hepatocyte survival,was inhibited following the transfusion of stored RBCs in P.aeruginosa-infected mice,whereas the JNK-mediated hepatocyte apoptosis pathway was enhanced.Stored RBCs therefore induce hepatic injury by disturbing the dynamic balance between the NF-?B and JNK pathways.In summary,We showed that transfusions with stored RBCs enhanced sepsis-induced liver injury in vivo,and liver injury exacerbated the severity of sepsis and decreased survival in P.aeruginosa-infected mice.Stored-RBCs transfusions enhanced the production of proinflammatory cytokines such as TNF-?,IL-6,and IL-1?,which play important roles in sepsis-associated liver injury in P.aeruginosa-infected mice.Further study showed that the enhanced inflammation observed was associated with increased activation of M1-polarized Kupffer cells.Moreover,the M1-polarized Kupffer cells and secreted proinflammatory cytokines exerted their effects on hepatocytes through enhanced Jun N-terminal kinase activation and inhibited NF-?B activation,demonstrating that transfusion with stored RBCs disrupted the balance between cell survival and cell death in the liver.Understanding the mechanisms whereby stored RBCs might contribute to these complications will likely be helpful in providing guidance towards making transfusions safer.