Study On The Correlation Between Circulating Histones And The Pathogenesis Of Multiple Organ Dysfunction Syndrome In Acute Critical Illnesses

Background:Multiple organ dysfunction syndrome(MODS)is characterized by dysfunction of two or more organs in acutely and critically ill patients who require medical interventions to maintain homeostasis in ICUs.Affected patients have high mortality rates of around 30�100%,depending on the number of dysfunctional organs.Therefore,MODS has become the leading cause of morbidity and mortality in current ICU practice.MODS is mediated by harmful simultaneous effects on multiple organs rather than a chain reaction from one organ to another.However,the identification of common mediators remains undefined.Coagulation activation,microcirculatory failure,and oxygen deprivation as well as bacterial toxins have been proposed as potential mediators but have not been fully proven.Histones are well-conserved proteins essential in DNA packaging and gene regulation.During cell damage,chromatin will be cleaved and then histones are going to be extracellularly released.Nucleosomes/histones are rapidly cleared by the liver and are rarely detected in the blood,unless extensive tissue damage or cell death occurs during a very short period,for example,in acute critical illnesses.Objectives:Extracellular histones can damage a single organ.This study will elaborate whether circulating histones mediate the occurrence and development of MODS in critical illnesses via the secondary hit,such as trauma,acute pancreatitis,sepsis,etc.Meanwhile,the sources of circulating histones will be preliminary discussed in critically ill mice.Methods:Generally,we aligned clinical studies,in vitro experiments,and animal models of trauma,acute pancreatitis and sepsis to elucidate the role of circulating histone in the occurrence and development of MODS.To explore the sources of circulating histones,sepsis models with wide-type(WT)C57BL/6j mice,WT BALB/C and BALB/C nude mice were used.Splenectomy was performed in those mice to evaluate the contribution of lymphocyte death to circulating histones.Pan-Caspases inhibitor Z-VAD-FMK was applied to observe if cell apoptosis is a major source of circulating histones.Meanwhile,B-and T-cell lines after steroid treatment were employed to explore if extracellular histone levels would increase during apoptosis.1.Characteristics and clinical significance of circulating histones in patients with acute critical illnessesPlasma histones of 420 ICU patients and 10 healthy donors were detected by western blotting(WB),and then the concentrations were calculated by certain conversion methods.Functional parameters of liver and kidney as well as the cardiac troponin T and oxygenation index of the patients were collected;SOFA scores at 48h-72h after admission were gathered in time.The correlation between circulating histone concentrations and those clinical data above was analyzed.2.Cytotoxic effect of extracellular histones on cells derived from various organsFirstly,the human endothelial cell line EA.hy926 was treated with healthy human sera after adding different doses of calf thymus histones or sera from critically ill patients with different concentrations of histones;anti-histone single chain variable fragments(ahsc Fv)and Heparin were used as extracellular histone antagonists.And then the mouse myocardial cells(HL-1),the human primary bronchial-alveolar epithelial cells(HSAEp C),human immortalized hepatocytes(THLE-3)and human primary renal cortical epithelial cells(HRCE)were treated with critically ill patient sera with high levels of circulating histones;flow cytometry was used to detect the PI positive rate of various cells at the end of the above treatment to assay the cytotoxic effect of extracellular histones.3.The role of circulating histones in the pathogenesis of MODS in mouse models of acute critical illnessesWide type(WT)C57BL/6j mice were used in this part of study.The free-fall strike of heavy objects and different strike times were used to establish mouse models of closed with three different severities.Acute pancreatitis models with different pancreas injury severities were induced by intraperitoneal injection of 4 times or 12 times of cerulenin,or by retrograde injection of bile acid salt into the mouse common bile duct.For CLP models,75%length of cecum was ligated,and then two or four fecal overflow puncture holes were made to establish the mouse models of less severe and severe sepsis;ahsc Fv and heparin were intravenously or intramuscular injected to treat the mice with various severities of diseases.Plasma histones of the mice above was detected by WB;plasma ALT,BUN and c Tn I were tested to assay the liver,renal functions as well as the myocardial damage of the mice with various injuries.The results of lung injury indexes were evaluated by tissue injury score under microscope;the histopathological characteristics of multiple organs were observed after H&E staining or anti-activated caspase-3 immunohistochemistry.The correlation between the levels of circulating histones and parameters of damaged multiple organ functions was statistically analyzed.The effect of anti-histone treatment on survival rates within72h of mice in severe CLP model was also evaluated.4.A preliminary study on the sources of circulating histones in critically ill miceWT C57BL/6j mice,WT BALB/c mice and BALB/c nude mice were used in this part of study.Splenectomy was performed in both WT C57BL/6j mice and BALB/c nude mice prior sepsis model generation.To avoid additional trauma from surgical procedures,we generated sepsis in mice by intraperitoneal injection of E.coli K-12instead of CLP operations.Since nude mice are immune compromised,we used LPS injection to induce sepsis.To rule out the possibility that high levels of circulating histones caused lymphocyte apoptosis via its cytotoxicity instead of the outcome of apoptosis,high-dose of calf thymus histones were injected into the C57BL/6j mice via the tail vein to reach similar levels detected in severe sepsis.To block apoptosis in vivo,pan-Caspases inhibitor,Z-VAD-FMK,was injected subcutaneously.Survival rate evaluation of WT BALB/c,BALB/c nude and BALB/c nude mice after splenectomy was conducted within 40h after LPS injection.It is generally believed that necrosis but not apoptosis release histones.To address this controversy,Daudi cells(a B lymphocyte cell line)and Jurkat cells(a T lymphocyte cell line)were treated with hydrocortisone to induce apoptosis only;FACS and WB were used to detect the apoptosis induction and the subsequently released extracellular histone H3 in the culture media.Results:1.Characteristics and clinical significance of circulating histone in patients with acute critical illnessesIn general,the concentrations of circulating histones in ICU patients were significantly higher than that in healthy donors.According to the etiology of patients into the overall stratification,sepsis,severe trauma and severe pancreatitis patients were admitted to the hospital with high levels of circulating histones,but there was no significant difference between the concentrations of circulating histone in those three diseases.Spearman rank correlation tests showed that there were positive correlations between circulating histone levels and the organ functional parameters,including ALT,BUN,Pa O₂/Fi O₂,and troponin T(c Tn T).Patients with MODS on admission or who died within 28 days of hospitalization had higher levels of circulating histones at the time of admission;in addition,circulating histone concentrations at admission can not only predict the occurrence of MODS between 48h-72h after hospitalization but also are helpful to predict the 28-day mortality.2.Cytotoxic effects of extracellular histones on cells derived from various organsAfter being added with exogenous histones,sera from healthy donors became to have a dose-dependent toxic effect on the human endothelial cell line EA.hy926;after treatment of EA.hy926 cells with a series of patient sera with histone levels over30?g/ml,the survival rate was also significantly reduced.Ahsc Fv or Heparin can block the cytotoxicity of extracellular histones.After treatment with healthy human sera containing 50?g/ml calf thymus histones or patient sera with histone levels exceeding50?g/ml,cells from different organs(mouse cardiomyocytes,human hepatocytes,human renal cortical epithelial cells and Human bronchial-alveolar epithelial cells)all showed reduced viability at the same time.3.Role of circulating histones in the pathogenesis of MODS in mice with acute critical illnessesPlasma histone concentration-time curves showed that the peak time of circulating histone concentrations of mice with moderate trauma,less severe CLP and TCL-pancreatitis were at 8h,16h and 16h after modeling respectively;circulating histone concentrations at the peak time points increased with the severities of the disease.After correlation analysis,it was found that circulating histone concentrations were individually and positively correlated with ALT,BUN,elevated troponin I(c Tn I),lung tissue injury scores(LIS);anti-histone reagents such as ahsc Fv and non-anticoagulant heparin can significantly decreased the values of ALT,BUN,c Tn I,LIS in mouse models of severe trauma,severe CLP.No matter when first anti-histone treatment were started,the 72-hour survival rate of severe CLP model mice was significantly improved in comparison with model groups.Anti-activated Caspase-3 immunohistochemical staining of the tissue sections from various organs revealed that there was excessive cell apoptosis in the thymuses and spleens of mice with severe trauma,severe CLP or TCL induced pancreatitis.4.A preliminary study on the source of circulating histones in critically ill miceIntraperitoneal injection of an appropriate amount of E.coli K-12 can successfully induce sepsis in healthy C57BL/6j mice,and the circulating histone concentration was at a high level(322.8�100.6?g/ml)10 hours later.The value continued to increase and the peak time was at 16h after bacterial injection(478.2�166.4?g/ml);the level of circulating histones was still high(424.8�154.1?g/ml)at 24h.Near-infrared spectroscopy of multiple organs showed that the apoptosis signal from thymuses and spleens increased significantly from 4 hours after bacteria injection,and the signal of apoptotic thymuses continued to be upregulated until 24h;the peak value of spleen apoptosis signal happened 8h after bacterial injection.Being confirmed by H&E staining and anti-activated caspase-3 immunohistochemistry of multiple organ tissues and 24 hours after the intraperitoneal injection of E.coli,thymocytes and splenocytes of C57BL/6j mice were both seriously lost,and there were large apoptosis areas in both of them;there was no apparent apoptosis in the heart,liver,kidney or any other important organs.After statistical analysis,it was found that the number of apoptotic cells in spleens of septic mice had a moderate positive correlation with the concentration of circulating histone protein(r=0.78;P<0.001).Calf thymus histones were injected into the C57BL/6j mice via the tail vein to reach the similar levels detected in severe sepsis,but no extensive lymphocyte apoptosis was observed in IHC staining at different time points.Those data suggest that apoptosis in the spleen and thymus are the sources of circulating histones but not the consequences.Subcutaneous injection of the pan-caspases inhibitor Z-VAD-FMK effectively blocked the thymic and splenic cell apoptosis,and the concentrations of circulating histones also decreased accordingly.Therefore,it suggests that apoptosis promotes the increase of circulating histone concentrations.Compared with C57BL/6j mice after intraperitoneal injection of E.coli,thymus deletion(BALB/c node mice)or thymus deletion combined with spleen removal(BALB/c node mice+splenectomy)effectively reduced the circulating histone levels of septic mice induced by intraperitoneal injection of high dose of LPS;Meanwhile,the 40h-survival rate of those mice was also significantly improved.After steroid treatment,Daudi and Jurkat cells cultured in vitro all demonstrated different levels of apoptosis depending on the dose of hydrocortisone(FACS data);western blotting also confirmed that there were no obvious histone H3bands in the culture media without steroid treatment,whereas high levels of extracellular histone H3 appeared in the supernatants of those apoptotic cells and the concentrations depended on the degrees of cell apoptosis.Conclusions:1.High levels of circulating histones on admission in patients with severe trauma,severe pancreatitis and sepsis are closely related to MODS development and progression,and even to the poor prognosis.2.Sera from healthy donors spiked with histones or critically ill patient sera with high levels of circulating histones show cytotoxicity which is not cell-type specific.3.Circulating histones demonstrated their direct cytotoxicity via secondary hit to mediate the occurrence and development of MODS in a variety of primary acute critical illnesses such as severe trauma,severe pancreatitis,and sepsis.Anti-histone therapies can effectively protect multiple organ functions in those mice,and significantly prolong the survival in mouse model of severe CLP.4.Spleens and thymuses are important sources of high-level circulating histones in septic mice,and the underlying mechanism mainly depends on the extensive apoptosis of thymocytes and splenocytes.