| Background and PurposeSepsis is a clinical syndrome of life-threatening organ dysfunction caused by the host’s maladjusted response to infection.Sepsis can induce multiple organ failure,which is the most common cause of death in the intensive care units(ICU).Sepsis triggers a complex body immune response that changes over time,accompanied by the development of pro-inflammatory and anti-inflammatory mechanisms.Therefore,most septic patients rapidly show severe immunosuppression,which is closely related to poor prognosis.In the 2020 Chinese expert consensus on the diagnosis and treatment of sepsis immunosuppression and a review of sepsis immunity published in Military Medical Research,they are all pointed out that the body immune dysfunction is involved in the pathophysiological process of sepsis.Lymphocyte(LY)can be an important indicator of acquired immune dysfunction in patients with sepsis.Patients with low lymphocyte had higher mortality and risk of developing severe disease than those with regular lymphocyte.Studies have shown that the gut microbiome is actively involved in shaping and maintaining the normal acquired immune system of the gut mucosal.Phenotypic differentiation of specific lymphocyte lineages in the gut mucosal immune system depends on the unique components of the microbiome,but the specific mechanisms remain unclear.The gut microbiota contributes to host physiology by producing a myriad of metabolites that act within the host as signaling molecules and substrates for metabolic reactions.Considering the interaction between gut microbiota and host metabolism,we hypothesize that septic patients with persistent lymphopenia(PL)had specific microflora patterns and metabolic characteristics.We performed a prospective study to explore the molecular pattern of microbiota and metabolic interactions in septic patients with persistent lymphopenia for evaluating potential functional associations among gut microbiota,metabolome,and host in the disease cohort,which may provide new clues for further exploration of pathogenesis.MethodsIn the prospective study,blood,fecal samples and clinical data were collected from patients with sepsis from the department of critical medicine of the First Affiliated Hospital of Dalian Medical University.Patients were divided into persistent lymphopenia group(≥3d)and non-persistent lymphopenia(NPL)group(<3d)according to lymphocyte.Clinical immune cells,clinical prognosis,gut microbiota and metabolite characteristics of the two groups were compared.Lymphocyte subgroup analysis and Th typing analysis were performed on blood samples.In order to identify differential gut microbiota and metabolites,we used the 16S r RNA gene amplicons sequencing and untargeted metabolomics analysis to test the fecal samples.Spearman rank correlation analysis was conducted on differential gut microbiota and metabolites,differential gut microbiota and clinical parameters,and differential gut metabolites and clinical parameters.Results(1)A total of 36 patients were enrolled in this study and divided into the persistent lymphopenia group with 21 patients and the non-persistent lymphopenia group with 15patients.The mean age of the patients was 66.0±14.7 years,including 25 males(69.4%)and 11 females(30.6%).(2)The 28d mortality[38.1%(8/21)vs 0%(0/15),P<0.05]and 90d mortality[47.6%(10/21)vs 6.7%(1/15),P<0.05]were higher in the persistent lymphopenia group than in the non-persistent lymphopenia group.Hospital mortality[14.3%(3/21)vs0%(0/15),P>0.05],total length of stay[16.0(8.0～22.8)d vs 15.0(9.5～21.5)d,P>0.05],length of stay in ICU(16.4±12.5d vs 14.9±8.0d,P>0.05),multiple organ failure[33.3%(7/21)vs 20.0%(3/15),P>0.05]and mechanical ventilation[71.4%(15/21)vs93.3%(14/15),P>0.05]had no significant difference.(3)Comparing immune cells between the two groups,the persistent lymphopenia group showed that CD3~+T/LY[59.4(44.1～69.1)%vs 70.9(66.0～75.7)%,P<0.05],CD3~+T cells(289.6±173.5cells/μl vs 961.8±532.7 cells/μl,P<0.05),CD3~+CD4~+T cells[145.0(86.0～193.0)cells/μl vs 495.0(309.5～946.5)cells/μl,P<0.05],CD3~+CD8~+T cells(110.2±96.3cells/μl vs 249.8±101.4cells/μl,P<0.05),Th1 cells[54.0(26.8～87.8)cells/μl vs 135.0(55.0～234.0)cells/μl,P<0.05],Th2 cells[88.5(40.0～125.8)cells/μl vs137.0(85.5～224.5)cells/μl,P<0.05],CD45RA~+Treg cells[1.0(0.0～1.0)cells/μl vs1.0(1.0～2.5)cells/μl,P<0.05]were significantly lower than those in the non-persistent lymphopenia group.(4)16S r RNA results:Staphylococcus,Peptostreptococcus,Bulleidia,Leuconostoc were different gut microbiota at the genus level in the feces of patients with the persistent lymphopenia group.Bacteroides,Parabacteroides,Butyricimonas,Mycobacterium,Isobaculum were different gut microbiota at the genus level in the non-persistent lymphopenia group.(5)The correlation analysis between immune cells and different microbiota found that CD3~+T cells were positively correlated with Bacteroides,Butyricimonas and Isobaculum,but negatively with Leuconostoc and Staphylococcus.CD3~+CD4~+T cells were positively correlated with Bacteroides,Butyricimonas and Isobaculum,and negatively with Leuconostoc and Staphylococcus.CD3~+CD8~+T cells were positively correlated with Isobaculum and negatively with Staphylococcus.Th1 cells were positively correlated with Bacteroides and negatively with Staphylococcus.Treg cells were positively correlated with Butyricimonas.CD45RA~+Treg cells were negatively correlated with Leuconostoc.CD45RA~+Treg/Total Treg were were positively correlated with Mycobacterium and negatively with Leuconostoc.Th1/Th17 cells and(Th1/Th17)/CD3~+CD4~+T were positively correlated with Butyricimonas and Isobaculum.Th1/CD3~+CD4~+T and Th1/Th2 were negatively correlated with Staphylococcus and Bulleidia.Th17 cells were positively correlated with Butyricimonas.Treg/CD3~+CD4~+T were negatively correlated with Leuconostoc.CD3~+CD4~+T/LY were positively correlated with Bacteroides.(6)Metabolomics analysis results:A total of 9 differential metabolites were obtained by unidimensional analysis,which were 7-DHCA,α-Ketoisovaleric acid,α-MCA,β-MCA,HCA,LCA-3S,CA,UCA and citramalic acid.α-Ketoisovaleric acid was increased and 7-DHCA,α-MCA,β-MCA,HCA,LCA-3S,CA,UCA,and citramalic acid were decreased in the persistent lymphopenia group compared with the non-persistent lymphopenia group.Correlation analysis between different metabolites and different microbiota found that 7-DHCA was positively correlated with Bacteroides,Parabacteroides and Isobaculum,and negatively with Staphylococcus.UCA was positively correlated with Parabacteroides,and negatively with Staphylococcus.α-MCA was positively correlated with Bacteroides,and Isobaculum.α-Ketoisovaleric acid was positively correlated with Leuconostoc.Citramalic acid was positively correlated with Bacteroides,Parabacteroides and Butyricimonas,and negatively with Staphylococcus.The correlation between immune cells and differential metabolites found that CD3~+T/LY was positively correlated withβ-MCA.CD3~+T cells were positively correlated withβ-MCA.CD3~+CD4~+T cells were positively correlated withβ-MCA and HCA.Th1 cells were positively correlated with HCA and citramalic acid.CD45RA~+Treg/Total Treg was negatively correlated withα-Ketoisovaleric acid.90 mortality was negatively correlated with 7-DHCA,CA andβ-MCA.Conclusion(1)Compared with the non-persistent lymphopenia group,the 28d mortality and 90d mortality of septic patients in the persistent lymphopenia group were higher,while the CD3~+T cells,CD3~+CD4~+T cells,CD3~+CD8~+T cells,Th1 cells,Th2 cells and CD45RA~+Treg cells of septic patients in the persistent lymphopenia group were significantly lower.We hypothesize that immune dysfunction in septic patients with the persistent lymphopenia group may be one of the main causes of death.(2)16S r RNA results showed that there were fewer distinct species of gut microbiota in the persistent lymphopenia group than in the non-persistent lymphopenia group.(3)Non-targeted metabolomics results found that bile acid metabolites and fatty acid metabolites were significantly reduced in septic patients with the persistent lymphopenia group,while organic acid metabolites were significantly increased.(4)The host receptors of septic patients were interacted with the microbiome.Bile acids may be involved in a variety of inflammatory pathways in the gut regulatory immune cells. |
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