Alteration Of TLR Pathway In Innate Immune Cells And Bacterial 16SrRNA In Patients With ACLF And Optimization Of Prognositication Model

Backgrounds and objectivesAcute-on-chronic liver failure is critical entity caused by acute deterioration of chronic liver disease,usually accompanied by multi-organ failure and high short-term mortality.Intense hepatic inflammation "spills" to circulation resulting in systemic inflammation,which plays a critical role in the occurrence and progression of ACLF.Meanwhile,the alteration of bacterial DNA burden and microbiome composition in peripheral circulation may occur,and the dysbiosis of microbiota may excerbate ACLF.This study is aimed to investigate the derangement of peripheral circulatory immunity and alteration of circulating 16SrRNA at this setting.The study would deepen our understanding of the pathogenesis of ACLF.Besides,an optimization of existing prognostic model using a panel of inflammatory surrogates to accurately predict the prognosis of ACLF patients and provide a basis for clinical decision-making for management.Methods:1.50 HB-ACLF patients,23 healthy controls(HCs)and 25 patients with compensated liver cirrhosis(C-LC)were enrolled from August 2017 to May 2018 and followed up for 28 days.Plasma were collected.Chemokines/cytokines were detected by cytometric bead array system(CBA).The plasma DNA was extracted and bacterial DNA was measured by amplifying V3-V4 section of 16S rRNA.And microbial composition was analyzed by 16S rDNA pyrosequencing.2.A total of 27 patients,including 9 with compensated cirrhosis,9 with de-compensated cirrhosis and 9 with ACLF,were enrolled in the study from August 2017 to October 2017.9 healthy controls were enrolled in parallel.Peripheral immune cells were isolated,RNA was extracted and alterations in TLR signaling pathways were evaluated using an RT2 ProfilerTM PCR Array.Differentially expressed genes were identified and TLR-signaling maps were drawn.3.151 Patients with HB-ACLF were prospectively enrolled from six hospitals between August 2017 and March 2018.Their plasma was detected for sCD163,Neutrophil gelatinase-associated lipocalin(NGAL)and copeptin by enzyme-linked immunosorbent assay(ELISA).The association between these biomarkers and mortality was analyzed.And the performance of MELD,APASL-ACLF Research Consortium score(AARC-ACLF score)and the CLIF Consortium ACLF score(CLIF-C ACLFs)with or without biomarkers were compared.Results:1.The circulating bacterial DNA was significantly increased in HB-ACLF patients compared to that in the control groups,whereas the overall 16SrRNA diversity was significantly decreased.HB-ACLF patients were enriched with 16SrRNA of Moraxellaceae,Sulfurovum,Comamonas,and Burkholderiaceae but were depleted in Actinobacteria,Deinococcus-Thermus,Alphaproteobacteria,Xanthomonadaceae and Enterobacteriaceae in circulation compared to controls.Network analysis revealed a direct positive correlation between Burkholderiaceae DNA and chemokine IP-10 in HB-ACLF patients.The relative abundance of Prevotellaceae DNA independently predicted 28-day mortality.Inferred functional metagenomics predicted an enrichment of bacteria with genes related to methane,alanine,aspartate,glutamate,pyrimidine,purine and energy metabolism.2.Using health controls as reference,TLR-signaling genes were more extensively depressed in Polymorphonuclear(PMN)and peripheral blood mononuclear cell(PBMC)of ACLF patients than those in patients with cirrhosis.TLR3,IFNG,IL1B,CXCL8,TBK1,PTGS2 and LTA were down-regulated but TLR9,FADD,IRAKI,CD 14,HPSA1A,IL10,ELK1 were up-regulated in neutrophils of ACLF.In PBMCs,TLR3,TLR9,TLR10,SIGIRR,IFNG,CCL2,CXCL10,PTGS2,IFNB1,LTA,IL2,IL12A and IRAK2 were down-regulated,IRAK1,CD14,IL10 were up-regulated.3.151 patients were enrolled.Advanced ACLF patients had significantly higher levels than early ACLF individuals of plasma biomarkers sCD163(P=0.001),NGAL(P=0.006)and copeptin(P=0.049).34 deaths occurred during the 28-day follow-up(22.5%).sCD163 and NGAL showed a strong independent association with 28-day mortality.Scoring systems incorporating sCD163 and NGAL had better discrimination and calibration,as measured by AUROCs,the Akaike information criteria(AIC),integrated discrimination improvement(IDI)and net reclassification improvement(NRI).Conclusions1.HB-ACLF patients display increased circulating microbial DNA burden,reduced 16SrRNA diversity,implying altered microbiome composition and a shift in microbiome functionality.The alteration in circulating microbial DNA is associated with systemic inflammation(SI)and clinical outcome in HB-ACLF.2.An immunosuppressive transcriptional signature of the TLR signaling pathway is observed in PBMCs and neutrophils of ACLF patients.3.sCD163 and NGAL are independently associated with short-term mortality in hepatitis B-associated ACLF.Use of a combination of sCD163 and NGAL improves prognostication.