Study On The Correlation Of Monocyte And Macrophage Apoptosis With The Prognosis Of Sepsis At The Early Stage And Its Regulatory Mechanism

BackgroundSepsis is a common disease with high mortality rate in the department of emergency.At the early stage of sepsis,monocytes and macrophages are activated to release inflammatory factors,recruiting neutrophils to form extracellular traps(NETs)to capture and kill pathogens.When inflammation is hyperactive,the death of immune cells and the release of damage-associated molecular patterns(DAMPs)lead to multiple organ dysfunction syndrome(MODS),which is an important cause of death in sepsis at the early stage.A large number of immune cell death also leads to persistent immunosuppression,and secondary infection is the main cause of death in sepsis at the middle and late stages.In recent years,treatment of cytokine storms has reduced early sepsis mortality,but effective methods to intervene immune cell death in sepsis are still lacking in clinic.Part Ⅰ Analysis of clinical features and prognostic risk factors in early sepsis ObjectiveA single disease database of sepsis was established to analyze the differences of clinical characteristics of the patients in different prognosis groups and to screen the independent risk factors of poor prognosis,focusing on the correlation between the immune cell amounts and the prognosis of patients,so as to provide scientific basis for the accurate assessment of sepsis status and prognosis at the early stage.MethodsEthical approval was obtained,inclusion and exclusion criteria were established,and clinical data of enrolled subjects were collected(including physiological indicators,infection sites,test results,disease scores,and 90-day prognosis).The levels of inflammatory cytokines,chemokines,apoptosis and pyroptosis-related molecules in plasma were detected by Luminex and ELISA.The early clinical characteristics of the patients in different prognosis groups were compared.The independent risk factors of death were analyzed by Logistic and COX regression,and the predictive efficacy of clinical indicators was evaluated by receiver operating characteristic(ROC)curve.Results(1)Compared with the survival group,peripheral monocyte count and percentage were significantly reduced in the death group at the early stage,but the heart rate,respiratory rate,plasma ALT,AST,and lactate contents,and SOFA scores were significantly increased.(2)Logistic and COX regression analysis showed that decreased monocyte percentage and increased blood lactic acid levels were independent risk factors for poor prognosis of sepsis at the early stage.ROC curve indicated that the proportion of monocytes had the best predictive efficacy.(3)The plasma levels of inflammatory factor TNF-,chemokine CCL2,pro-apoptotic molecule BAX,and pyroptosis molecule GSDMD were increased in sepsis,especially in the death group.Anti-apoptotic molecule BCL2 was significantly decreased.ConclusionDecreased proportion of monocytes and increased lactic acid are the independent risk factors for the death of patients with sepsis at the early stage,and the plasma levels of apoptosis and pyroptosis-related molecules are significantly increased in the septic death group compared to those in the survival and healthy groups.Part Ⅱ High-throughput sequencing of PBMCs reveals the death pattern of immune cells in sepsis at the early stageObjectiveBased on high-throughput sequencing of PBMCs,the main death patterns of different types of immune cells in sepsis at the early stage were analyzed in different prognosis groups,and the relationship between immune cell death and prognosis was investigated.MethodsTranscriptome sequencing was conducted on PBMCs of patients in healthy,survival,and death groups,and the differential genes between groups were screened and functionally enriched,focusing on the changes of signaling pathways related to immune cell death.qRT-PCR and Western blotting were used to verify the expression of differential genes at the transcriptional and translational levels.The morphology and structure of immune cells in different prognosis groups were observed by electron microscopy.The percentage of apoptosis and necrosis of immune cells was analyzed by flow cytometry,and the differences between groups were compared.Single-cell RNA sequencing reveals the maj or cell death pattern of different types of immune cells in early sepsis,focusing on macrophages and neutrophils involved in the innate immunity.Results(1)Transcriptome sequencing showed that the differentially expressed genes in PBMCs of septic patients were significantly enriched in the apoptotic signaling pathways compared with those of the healthy group.The apoptotic molecules(BID,CASP4/5)were up-regulated,and the anti-apoptotic molecule BCL2 was down-regulated in sepsis,especially in the death group.(2)Compared with the survival group,the proportion of apoptosis and necrosis of immune cells in the death group was significantly increased,with obvious nuclear shrinkage and margination of heterochromatin,and significantly increased intracellular apoptosis molecules(BAX,Caspase3)and decreased anti-apoptotic molecule BCL2.(3)Single-cell RNA sequencing showed that the differential genes of immune cells in the early sepsis were enriched in different death-related signaling pathways.Neutrophils were the most significant in necrosis,necrotic apoptosis,and apoptosis signaling pathways.Monocytes were most prominent in apoptosis,autophagy,and ferroptosis.ConclusionAt the early stage of sepsis,the apoptotic molecules in peripheral immune cells were upregulated,the apoptotic signaling pathways were significantly activated,the apoptotic ratio was increased with typical apoptotic morphological changes observed,especially in the death group.Single-cell RNA sequencing revealed that apoptosis was most pronounced in monocytes during sepsis.Part III Correlation between liver macrophage apoptosis and the severity of sepsisObjectiveTo explore the apoptosis of liver macrophages(Kupffer cells)in sepsis and its correlation with the severity of the disease through cellular and animal experiments.MethodsMurine macrophage RAW264.7 cells were treated with LPS(200 ng/ml)to analyze the apoptosis of macrophages in sepsis in vitro.The percentage of apoptotic cells was analyzed by flow cytometry.Morphological and structural changes of the cells were analyzed by transmission electron microscopy.qRT-PCR and Western blotting were used to detect the expression of apoptotic genes in the cells at the transcriptional and translational levels.Male C57BL/6 mice were divided into sham-operated group and sepsis group(mild and severe).The mice in the sepsis group were treated with cecal ligation and puncture(CLP).After CLP,the general state of the mice was observed and the disease score was recorded.Twenty-four hours later,the samples were collected and the plasma levels of inflammatory factors and apoptotic molecules were detected by Luminex and ELISA.Liver and lung tissue sections were stained with hematoxylin-eosin(HE)to analyze the pathological changes and scores,and the apoptosis rate of Kupffer cells was analyzed by immunofluorescence staining(Tunel,F4/80).qRT-PCR and western blotting were used to detect the expression of apoptotic genes in liver at the transcriptional and translational levels.Results(1)The apoptosis rate of RAW264.7 cells was significantly increased after LPS treatment.The heterochromatin in the nucleus was marginated and the perinuclear space was widened.Cellular apoptosis molecules BAX and Caspase3 were significantly upregulated,while anti-apoptotic BCL2 was downregulated after LPS treatment.(2)After CLP,the hair of mice was fluffy,the activity of mice was reduced,and the disease score was significantly increased.Inflammatory infiltration and cellular apoptosis in the liver and lung tissues significantly increased following CLP.The expression of pro-apoptotic genes(BAX,Caspase3)in the liver cells significantly increased,while anti-apoptotic gene BCL2 decreased.(3)Compared with the mild group,plasma levels of inflammatory molecules(TNF-and IL-6),apoptotic molecule(BAX),and liver injury marker(ALT and AST)in the severe group were increased,and the apoptosis rate of Kupffer cells was also significantly elevated.ConclusionApoptosis of liver macrophages(Kupffer cells)significantly increased in sepsis and is related to the severity of the disease.Part Ⅳ The regulatory mechanism of ADAR1 improving the prognosis of sepsis by inhibiting macrophage apoptosisObjectiveBased on the sequencing results and the previous research on ADAR1,the regulatory mechanism of macrophage apoptosis was further explored,to find new therapeutic intervention targets for sepsis.MethodsThe binding sites were predicted and RNP-IP experiments were performed to analyze the direct binding effects of ADAR1 on miR-122 and its A-to-I editing effects.Dual-luciferase assay was performed to explore the targeting relationship between miR-122 and BCL2A1.RAW 264.7 cells were transfected with ADAR1 adenovirus and ADAR1-specific si-RNA(si-ADAR1)to specifically overexpress and knockdown ADAR1,respectivley.Cells were transfected with miR-122 mimic and inhibitor to overexpress and inhibit miR-122.The percentage of apoptotic cells was analyzed by flow cytometry.The morphological and structural changes of cells in different treatment groups were observed by electron microscopy.The cellular expression of apoptosis molecules was analyzed by immunofluorescence staining,and the transcriptional and translational expressions of ADAR1,miR-122,BAX,and BCL2Ala were detected by qRT-PCR and Western blotting,respectively.Finally,the mechanism of ADAR1/miR-122/BCL2A1 regulating macrophage apoptosis and affecting the prognosis of septic mice was verified by animal experiments.The mice in the treatment group(CLP+OE-ADAR1)were injected with ADAR1 adenovirus through the tail vein after CLP.Results(1)After ADAR1 knockdown,the expression of pro-apoptotic molecules BAX and Caspase3 was upregulated,while BCL2Ala was downregulated in RAW264.7 cells.The apoptosis ratio was significantly increased with obviously apoptotic morphology.Overexpression of ADAR1 showed opposite changes.(2)RNP-IP confirmed that ADAR1 directly bound to and edited miR-122.The dual-luciferase report assay suggested that miR-122 targeted BCL2A1 to regulate its expression.Overexpression of miR-122 resulted in a significant decrease of BCL2Ala and an increased proportion of apoptotic cells,and inhibition of miR-122 led to increased BCL2Ala.(3)ADAR1 could reduce Kupffer cell apoptosis,alleviate liver injury,and improve the survival rate of CLP mice through regulating miR-122/BCL2A1 pathway.ConclusionADAR1 can inhibit Kupffer cell apoptosis,alleviate liver injury,and improve the survival rate of sepsis through miR-122/BCL2A1 signaling pathway.To sum up,clinical investigation found that the reduction of monocytes at the early stage of sepsis was a risk factor for poor prognosis,and high-throughput sequencing showed that monocyte apoptosis increased in sepsis,especially in the septic death group.Animal and cellular experiments verified that macrophage(Kupffer cell)apoptosis increased at the early stage of sepsis,which was related to the severity of the disease.Besides,ADAR1 could inhibit Kupffer cell over-apoptosis through miR-122/BCL2A1 signaling pathway to relieve liver injury and reduce the mortality of sepsis.In conclusion,we confirm that apoptosis of mononuclear macrophages at the early stage of sepsis is associated with the prognosis of sepsis,and find novel therapeutic intervention targets for sepsis.