Formation And Regulation Of Neutrophil Extracellular Traps In Patients With Acute-on-chronic Liver Failure

BackgroundPatients with liver failure or end-stage liver disease are more susceptible to infection, causing tissue damage and organ failure. It’s reported that patients with advanced cirrhosis suffered neutrophil dysfunction, especially in those who were complicated with infection. Neutrophil dysfunction could also be observed in patients with acute liver failure, meanwhile neutrophil function indexes phagocytosis and oxidative burst could predict the severities and outcomes of liver failure.Neutrophils are the most abundant white blood cells in peripheral blood, act as first-line defenders, quickly gather around the sites of infection and inflammation, kill the pathogens through phagocytosis, degranulation and neutrophil extracellular traps (NETs).NETosis is a special form of cell death, differs from the two classic forms of cell death (necrosis and apoptosis), occur particularly after being stimulated with some special stimuli like phorbol ester (PMA), some sort of bacteria and fungi. Neutrophils underwent NETosis released their DNA and proteins to the extracellular spaces to form a web-like structure, known as NETs. Most mechanisms known involed in the NETosis were based on the PMA-induced NADPH oxidase-dependent NETs formation, required activation of Raf-MEK-ERK signaling pathway, mainly manifested as autophagy and superoxide species (ROS) generation. Blocking one of the pathways mentioned above will witch NETs formation to apoptosis eventually. Important steps involved in NADPH oxidase-dependant NETs formation including neutrophil elastase-nuclear translocation, histone H3 citrullination, calcium flux and histone H1 and H4 degradation. Pathogens are fixed to the NETs formed DNA web-like structure, limilating infection, then killed by the neutrophil-related proteins and other imunne moleculars and cells. Meanwhile, some imunne moleculars were degraded by NETs helped limiting inflammation. In addition, NETs also helped to promote thrombosis and tumor metastasis.Chronic liver disease, particularly in the end stage, is characterized by clinical bleeding and decreased levels of most procoagulant factors, with the notable decrease of platelet counts. Platelets involve in thrombosis, tissue reconstruction, material transportation and immune response. It has already been reported that platelets expressed Toll-like receptor (TLR) 1-9. After bacterial lipopolysaccharide (LPS) bound to TLR4 expressed upon platelets, platelets were activated, after which the platelets would release a variety of bioactive substances into the blood, including a variety of phagocytosis and biosynthesis related proteins, small non-protein molecules and some hydrolases. In addition, activated platelets also promote the activation of antigen-presenting cells and NETs formation.AimTo observe the formation and regulation of neutrophil extracellular traps in patients with acute-on-chronic liver failure.MethodAll the study subjects were divided into three groups including chronic hepatitis group (CHB), cirrhosis group (CIR) and acute-on-chronic liver failure group (ACLF) according to the relative guidelines of Chinese Society of Hepatology recommended and collected all the relevant clinical information and results of blood routine test, liver function laboratory test, coagulation function laboratory test and patients’ prognosis. And then we determined the concentration of plasma NETs-related markers myeloperoxidase (MPO) and polymorphonuclear elastase (PMNE) using enzyme-linked Immunosorbent (ELISA) kits. We compared the NETs formation in patients with different stages of liver diseases and the ability to form NETs of isolated neutrophils from these patients to form NETs, as well as to explore the plasma NETs-related markers’s relation with these patients’outcomes.We successfully modeled the healthy control group, chronic liver injury group and LPS caused liver injury group mouse model based on previous reported study. Then we stained liver biopsy sections with HE, Fraser-Lendrum and immunofluorescence staining, after which we observed the formation of NETs under microscopy in assessment of NETs formation in endotoxemia. And we recorded neutrophil-platelet aggregates percentage and neutrophil-platelet aggregates PAC-1 positive percentage in patients with CHB and ACLF before or after LPS stimulation under flow cytometry. After observed the influences of 5-HT on NADPH-dependent and NADPH oxidase-independent NETs by quantified NETs formation using fluorescence plate-reader and observed under microscopy, we compared the plasma serotonin (5-HT) concentration between the patients with chronic hepatitis B and liver failure before and after adenosine diphosphate (ADP) stimulation. Then we stimulated the isolated-neutrophils from patients with different stages of liver disease with PMA or ionomycin after which we quantified NETs formation using fluorescence plate-reader and observed under microscopy to compare the ability to form NETs.We found the possible 5-HT receptor (HTR) involved through activation or inhibition of HTR. At last, we studied the role of 5-HT in several important mechanisms involved in the NETs formation through fluorescence quantitation, as well as western blot and immunofluorescence staining.ResultFormation of neutrophil extracellular traps in patients with acute-on-chronic liver failureCell free MPO and PMNE considered to be NETs-related markers, we compared the plasma MPO and PMNE levels to evaluate the formaiton of NETs in patients with chronic hepatitis B, cirrhosis or acute-on-chronic liver failure. We found plasma MPO levels (log10 ng/ml) and PMNE levels (log10 pg/ml) of patients with acute-on-chronic liver failure were not statistically significant compared with the patients with chronic hepatitis B or cirrhosis (MPO:CHB vs. ACLF:3.194[2.85-4.23] vs.3.219[2.70-3.89], P=0.3800; CIR vs. ACLF:3.145[2.45-3.93] vs. 3.219[2.70-3.89], P=0.6347。 PMNE:CHB vs. ACLF:5.02[4.34-5.74] vs. 5.029[4.44-6.16], P=0.7488; CIR vs.ACLF:4.921 [4.4-6.06] vs.5.029[4.44-6.16], P=0.0767).We divided all the patients into survival or death group according to the outcomes in day 30. We found plasma MPO levels (log10 ng/ml) were not statistically significant between two groups (3.154[2.45-3.93] vs.3.245[2.71-3.89], P=0.2535), but the plasma PMNE levels (log10 pg/ml) were lower in the survival group with no statistic significance (4.941 [4.4-6.16] vs.5.044[4.58-5.61], P=0.0565).We further divided the patients with cirrhosis or acute-on-chronic liver failure (classified as end-stage liver disease in this assay) while alanine aminotransferase (ALT)≥200IU/L into survival or death group according to the outcomes in day 30. It turned out that the plasma PMNE levels were lower in the survival group than the death group (4.941 [4.4-6.16] vs.5.044[4.58-5.61], P=0.0205), and the plasma PMNE levels could predict the prognosis of these patients in day 30 to some extent (AUC=0.7243, P=0.0216). Additionally, the levels of plasma PMNE of these patients correlated with total bilirubin (TBIL) levels positively (Rs=0.3912, P=0.0167), but had no correlation with other liver function indicators such as ALT, aspartate aminotransferase (AST) and albumin (ALB) (P>0.05). We also found that the plasma PMNE levels of these patients had positive correlation with prothrombin time (PT, Rs=0.3707, P=0.0167), thrombin time (TT, Rs=0.4002, P=0.0386), prothrombin normalized ratio (PT-INR, Rs=0.3718, P=0.0235) and activated partial thromboplastin time (APTT, Rs=0.2715, P=0.0055), but had a negative correlation with prothrombin activity (PTA, Rs=-0.3312, P=0.0452). And the levels of plasma PMNE of these patients had no correlation with fibrinogen (FIB, Rs=0.0145, P=0.8955).Regulation of neutrophil extracellular traps in patients with acute-on-chronic liver failureAs the most well accepted theory of pathophysiologic mechanisms of acute-on-chronic liver failure, "PIRO" concept consider infection is one of the most important precipitating events. So we built a liver injury mouse model caused by endotoxemia by intraperitoneal injection of LPS. As the Fraser-Lendrum staining of acute-on-chronic liver injury mouse model liver biopsy sections showed, the liver structure were severely damaged, causing no definite recognition of blood clots in the liver. But frozen liver sections of acute liver injury mouse model with PMNE immunofluorescence staining showed that NETs formed in the periportal vein.LPS-stimulated platelets induced neutrophils to form NETs, which is one of the most common mechanisms of infection-induced NETs formation. We stimulated the whole blood from chronic hepatitis B and acute-on-chronic liver failure patients with LPS. However, the results of flow cytometry tests showed that neutrophil-platelet aggregates percentage (P=0.8426) and PAC-1 positive percentage among neutrophil-platelet aggregates (P=0.9730) were not significant different between the two groups. And the percentage of neutrophil-platelet aggregates (CHB:5.334[4.34-7.35]% Neutrophils vs.11.81 [5.8-20.3]% Neutrophils, P=0.0285 ACLF:5.66[3.63-11.58]% Neutrophils vs.13.1 [4.51-23.1]% Neutrophils, P=0.0222) and positive percentage of PAC-1 among neutrophil-platelet aggregates (CHB:2.272[1.16-4.19]% Neu-Plt aggregates vs.5.574[1.84-10.2]% Neu-Plt aggregates, P=0.0266 ACLF: 2.229[0.615-6.58]% Neu-Plt aggregates vs.5.334[1.88-11.2]% Neu-Plt aggregates, P=0.0292) increased in patients with chronic hepatitis B and acute-on-chronic liver failure after LPS stimulation.Activated platelets released various bioactive substances, which include serotonin (5-HT), one of the most abundant substances in platelet. We found that by adding 100μM 5-HT, PMA-induced NADPH oxidase-dependent NETs formation was inhibited (P=0.0002), with concentration-dependent manner.5-HT could not suppress ionomycin-induced NADPH oxidase-independent neutrophil death, but 100μM 5-HT significantly reduced ionomycin-induced spread NETs.95% of serotonin stored in platelets. We found plasma levels of 5-HT (ng/ml) were higher in patients with chronic hepatitis B than in those with liver failure, before (330.9[128.8-546.7] vs.165.8[28.73-610.6], P=0.0289) or after (330.9[128.8-546.7] vs.389[160.6-546.7], P=0.0391) ADP stimulation of whole blood. And the increased percentage higher in patients with acute-on-chronic liver failure with no statistic significance (124.4[0.615-6.58] % Control vs.99.71 [89.53-107.4] % Control, P=0.0718).5-HT (ng/ml) had no increase in acute-on-chronic liver failure patients (165.8[28.73-610.6] vs.156.2[30.84-546.7], P=0.5625).But after 180 minutes of PMA stimulation, NETs formations were significantly less in isolated neutrophils from patients with acute-on-chronic liver failure than those with chronic hepatitis B (144[100.4-178.9]% Control vs.315[178-428]% Control, P=0.0043), with no statistic significance with cirrhosis patients (144[100.4-178.9]% Control vs.271[131.1-620.4]% Control, P=0.1320). These findings were consistent with the results observed under microscopy. But there were no statistically significant between three groups stimulated NETs formation with ionomycin (CHB vs. ACLF:238.3[144-369.6]% Control vs.185[131.5-313]% Control, P=0.1797 CIR vs.ACLF:191.7[119.3-434]% Control vs.185[131.5-313]% Control, P=0.5714).Regulation of serotonin on the NETs formationWe found 5-HTR2b agonist significantly inhibited NETs formation (P=0.0108) under PMA stimulation, and 5-HTR2b antagonist obviously deprived such inhibition (P=0.0177) consistently, however 5-HTR2b mRNA was undetectable with sensitive PCR. And Among all the important parts of NADPH oxidase-dependent NETs formation, we found that 5-HT coexisted with decreased ROS levels (60 min: P=0.0054; 120 min:P<0.0001; 180 min:P<0.0001) and inhibited PMNE-nuclear translocation, with exception of histone H3 citrulliation.5-HT seemed unable to suppress autophagy from preliminary experitment results.DiscussionsFormation of neutrophil extracellular traps in patients with acute-on-chronic liver failureThe presence of various damage-associated molecular patterns (DAMPs) in liver failure patients, recognized as a danger signal that could activate the innate immune response, which include NETs form in the sites of inflammation. Theoretically, formation of NETs should increased in liver failure patients, but our results shown no increase in overall acute-on-chronic liver failure patients compared with chronic hepatitis B or cirrhosis patients, with a increase in advanced acute-on-chronic liver failure patients. We considered these related to some of the acute-on-chronic liver failure patients entered the stable or recovering phaze, in which the anti-flammatory response enhanced, leading to lower or lack of NETs formation.Regulation of neutrophil extracellular traps in patients with acute-on-chronic liver failureMouse model and recent reports support the idea of NETs formation causing by liver injury. But our results showed that there were no diffence of neutrophil-platelet aggregates percentage between the chronic hepatits B or acute-on-chronic liver failure patients, which imples there may be some substance from platelets help to promote NETs formation.Activated platelets released various bioactive substances, which include serotonin (5-HT), one of the most abundant substances in platelet. Unexpectedly, we found that PMA-induced NADPH oxidase-dependent and ionomycin-induced NADPH oxidase-independent NETs formation was inhibited by serotonin.As 95% of serotonin stored in platelets, we found that plasma concentration of 5-HT and amount of 5-HT released after ADP stimulation in patients with chronic hepatitis B were higher than acute-on-chronic liver failure patients.Serotonin suppressed NETs formation, with lower level of platelet and lower level of serotonin in plasma and platelet, may lead to increased formation of NETs in advanced acute-on-chronic liver failure patients. But our results showed that after 180 minutes of PMA stimulation, NETs formation of isolated neutrophils from patients with acute-on-chronic liver failure was significantly less than patients with chronic hepatitis B, which indicated the impaired capacity of NETs formation in patients with acute-on-chronic liver failure. These results consistent with early reports.These implies that the regulation of NETs is complicated, and further study need to be done.Regulation of serotonin on the NETs formationAfter further investigation, we found the inhibition of 5-HT in NETs formation involved HTR2b. But with no mRNA expression of HTR2b, we thought the possible reason may lie on the inactive transcription of neutrophil. Next step, we will try to detect the protein of HTR2b on neutrophils.ROS generation, autophagy, PMNE-nuclear translocation, histone H3 citrullination, calcium flux and histone HI and H4 degradation are the important parts involved in NADPH oxidase-dependent NETs formation, but we were only certained that 5-HT affected the generation of ROS and PMNE translocation, but not the histone H3 citrullination. The rest need more investigation.In summary, we hypothesized that in the sinusoidal, activated platelets bound to neutrophils, enabled the ROS generation and PMNE translocation in neutrophil, causing NETs formation. Platelets aggregated and adhered to NETs. Meanwhile activated platelets released a variety of bioactive substances, increased the level of serotonin. The high concentrations of serotonin in this limited space stimulated HTR2b of the subsequent accumulated neutrophils, with inhibition of ROS generation and PMNE translocation, led to the inhibition of NETs formation.