The Mechanism Of Akkermansia Muciniphila And Bile Acids In SFTSV-infection

Background and objective:Severe fever with thrombocytopenia syndrome（SFTS）is a new acute tick-borne infectious disease caused by severe fever with thrombocytopenia syndrome virus.The main clinical symptoms are acute fever,gastrointestinal symptoms,myalgia,thrombocytopenia and leukopenia.Severe cases are accompanied by unconsciousness,skin petechiae,gastrointestinal and pulmonary hemorrhage,etc.,while inflammatory cytokines,encephalitis,myocarditis and multiple organ failure may even occur in fatal cases.SFTS is mainly transmitted through tick bites,and there are few reports of interpersonal dissemination due to contacts with blood or body fluids of patients.The population is generally susceptible,and the high level of mortality in severe cases.In recent years,its incidence rate has been rising,the epidemic areas have been spreading,and there are no effective special therapeutic drugs and vaccines.Therefore,SFTS has become one of the most harmful tick-borne diseases in China,affecting global public health safety.The composition,metabolites,colony structure and abundance of intestinal symbiotic bacteria are related to the physiological and pathological process of the host.So intestinal symbiotic bacteria are called the"microbial organs"of the human body.Studies have confirmed that intestinal symbiotic bacteria are involved in the occurrence and development of obesity,cancer and inflammatory bowel disease,and play an important role in anti-pathogen infection.The change of bile acids is related to intestinal symbiotic bacteria,which can adjust the content of bile acid and the composition of bile acids components,and effects the host.Similarly,the change of bile acids composition will reshape the structure of intestinal symbiotic bacteria,prevent the growth of some bacteria,and maintain the homeostasis of intestinal.The relationship among intestinal symbiotic bacteria,bile acids and host are profound and complicated.This study intends to explore the interaction of them by integrating the technologies of microbiome,metabolomics,and transcriptome,and combining with SFTS cohort study as well as in vitro and in vivo experiments,in order to provide important references for clinical treatment,drug development and disease prognosis of SFTS.Method:The clinical cohort study included 455 donors,including 233 SFTS surviving patients,27 SFTS deceased patients,176 non-SFTS fever patients and 19 healthy volunteers.According to the Guidelines for Prevention and Treatment of Severe Fever with Thrombocytopenia Syndrome（2010 edition）issued by the Ministry of Health of China,patients with SFTS were diagnosed.Experimental research part:1)High-throughput amplicon sequencing technology of 16S r RNA gene was utilized to identify and analyze the microbial composition in fecal samples;2)Liquid chromatography-tandem mass spectrometry（LC-MS/MS）was adopted to proceed the quasi-targeted metabolomics detection of serum and detect the bile acid content in mouse serum;3)Real-time quantitative PCR was used to detect the gene expression levels of inflammatory factors IL-1B and IL-6 under different treatment conditions;4)Using ELISA to detect the contents of IL-1βand IL-6 mature protein in serum and cell culture supernatant under different treatment conditions;5)Western blot was applied to detect the expression levels of related proteins including inflammatory factors,cell membrane and intracellular receptors,NF-κB signaling pathway and NLRP3 inflammatory pathway.6)The main signal pathway that changed after GCDCA treatment in SFTSV-infected THP-1 were identified by RNA-seq;7)Gene expressions of TLR8 and My D88 in THP-1 cells were knocked down by lentivirus vector,and the knock-out efficiency was detected by Western blot;8)Small interfering RNA（si RNA）was utilized to knock down the expressions of TGR5 and FXR in THP-1 cells,and the knock-out efficiency was detected by Western blotAnimal research part:1)Establish a half-death model of mice infected with SFTSV by intraperitoneal injecting anti-IFNAR1 Ig G.2)To establish the TGR5 receptor deletion mouse model,TGR5^gfp/gfp mice were injected with Tamoxifen intraperitoneally to induce the expression of cre enzyme.3)Abx mice were subjected to gavage with1010 colony-forming units of A.muciniphila at 6 d post Abx oral administration.After48 h of colonization,faecal samples were collected to determine the efficiency of colonization.Results:1.Fecal microbiome analysis of SFTS patients.The fecal microbial composition and relative abundance of 260 patients hospitalized with SFTS,176 non-SFTSV febrile patients and 19 healthy controls were identified by high-throughput amplicon sequencing technology of full-length 16S r RNA gene.Both the phylum and genus level,there are significant differences in the intestinal symbiotic bacteria composition of SFTS patients,and only A.muciniphila exhibits significant differences in the surviving and deceased patients of SFTS.This suggests that A.muciniphila a may be an important intestinal symbiotic bacterium help for recovery.In different courses,the relative abundance of A.muciniphila was increased in surviving patients during hospitalization,which was significantly higher than patients with fever without SFTSV infection.There is also a significant difference between the early stage and the middle stage of the disease course,which indicated the specific protective effects of A.muciniphila in SFTS.There is no significant correlation between the viral load in serum and the relative abundance of A.muciniphila.The contents of inflammatory factors IL-1βand IL-6 in serum of SFTS patients were negatively correlated with the relative abundance of A.muciniphila.It is suggested that there may be a correlation between A.muciniphila and inflammation in patients with SFTS.2.The function of bile acids in SFTS patientsBy the quasi-targeted metabolomic sequencing,there were 69 differential metabolites between surviving SFTS patients and non-SFTS patients,and 153differential metabolites between surviving SFTS patients and deceased patients.Among these differential metabolites,the composition of bile acids changed significantly,and there were significant differences in conjugated primary bile acids and secondary bile acids between surviving SFTS patients and deceased patients.GCDCA,which were significantly increased between surviving SFTS patients and non-SFTS patients,and between SFTS survivors and dead patients,negatively correlated inflammatory cytokines IL-1βand IL-6 in serum.There were significant differences in inflammatory cytokines in different levels of GCDCA groups.Speculated that GCDCA may be an important conjugated primary bile acid with anti-inflammatory function.In order to verify the speculation,detected the expression of IL-1B significantly decreased in peripheral mononuclear blood cells（PBMCs）from SFTS patients,which treated with GCDCA.In SFTSV-infected PBMCs from healthy controls,it was found that GCDCA treatment could significantly decrease the expression of IL-1B and IL-6,and the content of mature IL-1βand IL-6 proteins in cell supernatant.All these data indicate that GCDCA inhibit the inflammation.3.Inflammation inhibited by conjugated bile acids GCDCA through TGR5-NF-κB signal axis.To further explore the mechanism of GCDCA,THP-1 cells were selected as the cell model.After GCDCA pretreatment,it was found that the gene expression of IL-1B and IL-6 in SFTSV-infected cells significantly decreased,and the intracellular contents of IL-1βprotein precursor,IL-1βand IL-6 mature proteins expressions in supernatants also reduced significantly,which was consistent with the results in PBMCs.Transcriptome data analysis found that a large number of genes are up-regulated in biological processes,such as cytokine activation,chemokine-mediated signal pathway,inflammatory response,immune response and NF-κB activation signal pathway.After GCDCA treatment,the number of up-regulated genes in these biological processes decreased.Western blot showed that GCDCA treatment decreased the protein contents of P50 subunit of NF-κB and the phosphorylated-P65,which inhibited the transcription of inflammatory cytokines and reduced inflammation.Knock down the bile acid receptor TGR5,the inhibitory effect of GCDCA on inflammation disappeared,indicating that GCDCA works through the TGR5-NF-κB signal axis.CDCA is predominantly conjugated to glycine（which would form TCDCA）and rarely conjugated to taurine（to form GCDCA）in mice,so use the TCDCA in mouse model.The significant difference in the survival rate between the TCDCA-treated and untreated Abx WT mic was completely lost in the TCDCA-treated and untreated Abx TGR5^-/-mice.A.muciniphila also can increase the survival rate in SFTSV-infected mice through improve content of TCDCA by its metabolite harmaline.However,the protective effect of A.muciniphila colonization also required TGR-5 signaling,because the fatality rate was not restored in TGR5^-/-mice after A.muciniphila reconstitution.Together,these data indicate that A.muciniphila-driven TCDCA confers protection against SFTSV systemic infection via TGR5 signal in vivo.Conclusions:In this study,we described an A.muciniphila–BA–TGR5 axis that limits host NF-κB-mediated immunopathogenic responses resulting from infections of SFTSV.Critically ill and deceased patients with SFTS showed a significantly lower abundance of fecal A.muciniphila.Conjugated bile acids can protect host and reduce mortality by inhibiting systemic inflammation mediated by NF-κB through TGR5 receptor.In summary,these results suggesting a potential role for A.muciniphila as a microbial biomarker in predicting the outcome of systemic SFTSV infection,and provide an important reference for clinical individualized treatments.