Systemic Analysis Of The Immune Features And Identification Of Diagnostic And Prognostic Immunologic Biomarkers In Elderly Septic Patients

By 2050,the number of persons above 65-year old is estimated to be higher than that of teenagers and young adults worldwide.Meanwhile,the number of age-related diseases has steadily increased,with infection being one of the leading causes of death among the elderly.Sepsis,a dysregulated systemic inflammatory and immune response to infection,is also one of the main causes of death in clinical intensive care units.Even though researchers have made efforts on exploring pathogenesis of sepsis,the complexity of disease still brings many difficulties in clinical diagnosis and treatment,due to the diversity of pathogenesis and the complexity of immune response.Sepsis develops faster and causes higher mortality in the elderly compared to the younger patients.Although hundreds of diagnostic or prognostic biomarkers related to sepsis have been found,they are mainly derived from the younger patients,thus making it more difficult to be applied in the elderly septic patients whose immune system is quite different.Furthermore,lack of appropriate cohort analysis and unreliable biomarkers have posed the difficulty in the diagnosis and treatment of the elderly septic patients.To overcome these problems,we have performed a comprehensive study on the immune features of the elderly septic patients by using flow cytometry,multiplex cytokines detection and RNA sequence?RNA-seq?.1.Global study on immune cells of elderly sepsis102 cases of elderly septic patients?Sep-Pt?,30 cases of elderly pneumonia as infectious controls?Inf-Ctrl?and 31 cases of elderly healthy donors?HD?were recruited from Shanghai Dongfang Hospital.We used flow cytometry to determine the immune cells in the peripheral blood of the elderly septic patients on day1,day3and day7 and others on one time point equivalent to day1.We found that on day1,neutrophils,monocytes and antibody-secreting cells?ASCs?were increased in the Sep-Pt group compared to the groups of Inf-Ctrl and HD.By contrast,eosinophils,basophils,dendritic cells,natural killer cells and T cells were reduced in the Sep-Pt group.Next the diagnostic value of different immune cell pararmeters was evaluated,and“basophils”was found to be the best with the area under the receiver operating characteristic?AUROC?of 0.945,which was more sensitive than that of procalcitonin?PCT?,a commonly used diagnostic pararmeterc for sepsis in clinics.We also assessed the prognostic value of immune cells for early?3-day?and 28-day mortality of septic patients,and found day1 CD62L⁺CD16^-subset of neutrophil?Neu?combined with sequential organ failure assessment?SOFA?score was the best predictor of early mortality.In addition,day1 CD28⁺PD1⁺percentage of regulatory T cells?Treg?,day3 CD28⁺PD1⁺%CD4⁺Treg combined with lactate and SOFA score,day3 eosinophils?Eos?percentage of CD45 combined with lactate and SOFA score,as well as day7 Eos%CD45 combined with lactate were the best predictive values for28-day mortality when evaluated at the respective time points.2.Global study on plasma cytokines of elderly sepsisWe used multiplex assay and ELISA to analyze plasma cytokines of 97 cases of Sep-Pt,30 cases of Inf-Ctrl and 31 cases of HD.We found that plasma cytokines were increased significantly in Sep-Pt group,especially in those who did not survive.For diagnosis,day1 hepatocyte growth factor?HGF?,interleukin?IL?1R?and IL6 showed the best results,with AUROC over 0.8.In addition,macrophage inflammatory protein1?,IL8 combined with acute physiology and chronic health evaluation?APACHE?II score on day1 had the best predictive value for early mortality.Moreover,day1stromal cell-derived factor 1?combined with stem cell factor?SCF?and TNF?,day3SCF combined with and SOFA score,and day7 IL8 combined with APACHE II score were the best predictors for 28-day mortality prognosis when evaluated at the respective time points.3.Comparisons of the sepsis and septic shock in the elderlyAccording to the degree of severity,sepsis can be divided into normal sepsis and septic shock.In our research cohort,58 patients were classified as normal sepsis and44 patients as septic shock.From day1,day3 to day7,plasma cytokines such as monocyte chemotactic protein1,HGF,IL1R?,IL8 and IL10 were significantly increased in septic shock.In the analysis of early mortality of normal sepsis group,we found day1 IL8 had the best predictive value,followed by HGF,both were more sensitive than routine clinical indicators.In the analysis of 28-day mortality prediction in normal sepsis,we found day1 CD28⁺PD1⁺%CD4⁺Treg combined with SOFA score had the best predictive value.On day3,lactate combined with CD56^loNK%PBMC?peripheral blood mononuclear cell?,CD28⁺PD1⁺%CD4⁺Treg combined and IL27,had the best prediction values when total immune cells and T cells were considered respectively.On day7,CD56^loNK%PBMC combined with SOFA score,and CD27⁺CD45RA^-%CD4⁺Treg combined with PCT had the best predictive values when total immune cells and T cells were considered respectively.In the early mortality analysis of the septic shock group,we found IL8 combined with APACHE II score had the best predictive value.For 28-day mortality,day1 antibody secreting cells?ASCs?combined with PCT was the best predictor,while day3 SOFA score combined with PBMC,CD4⁺T helper cell and interferon?inducible protein10?IP10?had the best predictive values when total immune cells and T cells were considered respectively.day7 Eos%CD45 combined with Lactate,IL8 combined with APACHE II score had the best predictive values when total immune cells and cytokines were considered respectively.4.Study on the molecular and functional features of ASCs in elderly septic shock From the results above,ASCs was found to increase significantly in elderly septic patients.Therefore,we performed RNA-seq and ASCs-T cells co-culture to study gene expression and immunomodulation pattern of septic ASCs.The RNA-seq results showed that compared to ASCs from healthy donors,the immune-related pathways in septic ASCs were down-regulated,while the metabolism-related pathways were significantly enhanced.Besides,septic ASCs could promote the proliferation and differentiation of T cells,and were positively correlated with the amount of T follicular helper cells in peripheral blood.Kinetic analysis showed that immunoglobulin A in septic shock survivors tended to increase,but remained unchanged or even decreased in those who died,and IgA⁺%ASCs significantly decreased in patients who died of septic shock.In conclusion,in this study we have characterized the feature of immune disorders in elderly septic patients and identified a series of diagnostic and prognostic biomarkers,which could provide important clues for future studies of elderly sepsis.