| BackgroundsHeatstroke(HS)is a life-threatening disease.Liver is a common feature of severe HS.About 31.9%of HS patients have liver damage.The mortality of severe HS-liver failure(ALF)was>70%.However,the mechanism of HS liver damage is still poorly understood.The present view is that the primary physicochemical damage of hepatocytes initiates a secondary unbalanced inflammatory reaction of immune cells(such as macrophages)and aggravates the damage.Exosome is a novel mechanism of intercellular communication.Studies have shown that in non-alcoholic steatohepatitis and alcoholic hepatitis,the number of exosomes secreted by hepatocytes increases,which contains unique cargos and has a pro-inflammatory effect that promotes the polarization of M1 phenotype of macrophages and further aggravates liver inflammation.AimsTo investigate whether hepatic exosomes released after heat stress can be taken up by macrophages and mediates its polarization to the pro-inflammatory M1 phenotype and mediates liver damage by promoting macrophage inflammatory phenotype in vivo.In addition,the target protein of interest in HS hepatocyte exosomes was screened by protein profiling to verify its role in mediating the proinflammatory function of exosomes.Explore the mechanism of target protein enrichment in donor cells and the action mechanism in recipient cells.Finally,the serum exosomes as early diagnosis and prognostic evaluation marker in heatstroke liver injury were validated in clinical studies.Methods1.Exosomes in HepG2 hepatocyte supernatant,animal and heatstroke patients serum were separated by ultra-high-speed gradient centrifugation.The morphology and diameter of exosomes were identified by transmission electron microscopy and NTA,and the expression of characteristic surface markers CD9,CD63,CD81,Tsg-101 and endoplasmic reticulum marker gp96 was detected by Western-blot.2.The uptake of hepatocyte exosomes fluorescently labeled with PKH67 by macrophages was verified by direct absorption experiments.3.Macrophage M1/M2 phenotype surface marker expression was analysed by flow cytometry.4.mRNA level of M1/M2 related cytokines in macrophages was determined by PCR.5.ELISA method was used to determine the level of histone H3 in HS hepatocyte exosome,plasma exosomes,cell supernatant and plasma,and the level of macrophages M1/M2 related cytokines in cell supernatant and plasma.6.Exosome reinfusion mouse model was constructed by injecting exosomes(40 ug)through tail vein.7.PKH-67-labeled exosomes were injected through tail vein,and frozen sections were taken from each organ to observe the ingestion of exosomes.Observe the co-localization of exosomes and F4/80-labeled macrophages in the liver.8.Liver damage was detected by HE/MPO/F4/80 staining of liver tissue.9.Detect the levels of ALT,AST and LDH in the supernatant and plasma by commercial Kit.10.iTRAQ protein profiling to analyze differences in hepatic exosomal protein expression profile after HS.11.Nuclear translocation of histone in hepatocytes,colocalization of PKH-67-labeled exosomes with TLR4,EEA1,Rab5 and Rab7 in hepatocytes,and with EEA1,Rab5,Rab7 and Lampl in macrophages was observed by confocal microscopy.12.Western-blot analysis of hepatocyte cytoplasm Alix,Tsg101,histone H3 and nuclear histone H3 expression.13.Histone H3 overexpressing cell line was constructed by lentivirus.14.siRNA knockdown of histone H3,TLR4 and caspase-3 in hepatocyte,clathrin and TLR9 in macrophage and TLR9 in vivo.15.Full capase inhibitor was used to inhibit hepatocyte apoptosis.Nacrostatin was used to inhibit necrotic apoptosis.TLR9 agonist(ODN1688),TLR9 inhibitor(ODN2088),negative agonist(CpG-ODN)was used to treat macrophages in vitro and i.p.in vivo.The exosome inhibitor GW4869 was used to treat macrophages in vitro and injected through the tail vein in vivo.16.In vitro THP-1 cells were added with PMA to stimulate its differentiation into macrophages.17.Intraperitoneal injection of chlorinated phospholipids to remove macrophages.18.HS hepatocyte model:Hepatocytes were heat-shocked for 1h at a 43?,60%humidity incubator,then recooled for 0,3,6,and 9 hours,respectively;HS mouse model:C57 mice were placed in a 42.5?,60%humidity incubator for 1h,and then recooled for 0,3,6,9,1 2,and 24 hours,respectively.19.The data were expressed as the number(%),meanąSD,or median(25th-75th percentiles).Means of two groups were compared using Student's t test.The correlations were analysed by Pearson's correlation test.p<0.05 was considered significantly significant.All tests were performed using the GraphPad software version 7.0.Results1.Heat shock induces the release of a large number of exosomes from hepatocytes,which are taken up by macrophages by clathrin mediated endocytic pathway and induce the polarization of macrophages into M1 phenotype.1.1 Hepatocyte exosomes isolated by ultra-high-speed gradient centrifugation are consistent with the typical characteristics of exosomes by morphology,diameter distribution and surface marker expression detection.1.2 The number of exosomes secreted by hepatocytes increased after heat shock and was positively correlated with the degree of hepatocyte injury.1.3 Hepatocyte exosomes can be taken up by macrophages via clathrin-mediated endocytic pathway in vitro,and exosomes re-infused in vivo can also be taken up by liver in situ macrophages.1.4 HS-hepatic exosomes can activate the polarization of macrophages to the M1 phenotype in vitro and reinfusion of HS hepatocyte exosomes in vivo can also induce liver damage and liver inflammation through Ml phenotype polarization of macrophages.2.The exosomes released by hepatocytes after HS are enriched in histone H3,and HS induces nuclear translocation of histone in hepatocytes and assembles into exosomes by binding to the TLR4 receptor on the endosomal system.2.1 Through iTRAQ-based protein profiling analysis,it was found that HS significantly changed the protein expression in exosomes released by hepatocytes.Histone H3 is one of the top 10 high expressed protein and has strong disease correlation,as a result was chosen as the target protein.2.2 The enrichment of histone H3 in hepatocyte-derived exosomes after HS was further verificated and the level was positively correlated with the degree of hepatocyte injury.2.3 Histones in hepatocyte exosomes are derived from the nucleus.After HS,histones are first translocated from the nucleus to the cytoplasm,and then released to the extracellular without being degraded.This process is independent of cell death.2.4 Cytoplasmic histones are transported into the MVB by binding to the TLR4 receptor on the endosomal system and subsequently assembled into the exosomes and released.2.5 Histone H3 is located inside the exosomes.2.6 After HS,the elevation of histone H3 level in hepatocyte exosome was earlier than that in the supernatant.The elevation of serum exosomal H3 in the mouse HS model was also earlier than serum free H3.3.After HS,hepatocytes transfer exosomal histone H3 to macrophages and activate them to polarized into M1 phenotype via TLR9 receptor.3.1 HS hepatocyte exosomes can transfer histone H3 to macrophages,and histone H3 in HS hepatocytes exosome can also be taken up by the liver in vivo.3.2 The transfer of histones between hepatocytes and macrophages is a direct way through horizontal protein delivery,rather than by means of exosomes that transmit H3 mRNA or activate H3 mRNA transcription in macrophages.3.3 HS hepatocyte exosomes promote macrophage polarizaiton to M1 phenotype in vitro and induce liver damage and inflammation in vivo mainly through its cargo histone H3.3.4 HS hepatocyte exosome histone H3 mediates the polarizaiton of macrophages to M1 phenotype and induces liver injury and inflammation through TLR9 receptor in vitro and in vivo.4.Serum exosomes of patients with severe heatstroke and liver injury high express histone H3,and its level is related to the severity of disease and has a prognostic value.4.1 The incidence liver injury(ALI)in severe heatstroke is as high as 45.45%,although most of them are mild ALI,the incidence of severe ALI is not low.The mortality of severe ALI is significantly increased,the length of hospital stay is prolonged and the incidence of combined organ damage increased.4.2 Serum exosomal histone H3 level was higher in patients with liver injury than without liver injury,with severe liver injury than mild liver injury and non-surviors than surviors.It was increased in non-surviors by time while decreased in survivors.The changes of the serum free histone are not as significant as exosomal histones.4.3 The level of histone H3 in serum exosomes was significantly positively correlated with liver injury score(MELD),disease severity score(APACHE II)and organ failure score(SOFA).4.4 The area under the ROC curve for serum exosomal H3 levels to diagnose liver injury or severe liver injury and identify non-survivors was high,similar to the traditional indicator ALT/AST/LDH and higher than free histone H3 in plasma.Conclusion1.HS induces the release of a large number of exosomes from hepatocytes,which are taken up macrophages by the clathrin-mediated endocytic pathway and promote the polarization of macrophages to the M1 phenotype mainly through the enriched cargo histone H3.2.HS induces the translocation of histone from nucleus into the cytoplasm in hepatocytes,and enters MVB through the TLR4 receptor on the endosomal system and transports it to the exosomes,which is an actively enrichment process independent of cell death.3.Histones in HS hepatocyte exosomes acted through TLR9 in vitro and in vivo to promote macrophage transformation to M1 phenotype and induce liver damage and inflammation.4.Serum exosomes of patients with severe HS are enriched in histone H3,which is a potential prognostic marker for liver injury. |
PDF Full Text Request