The Associations Of Serum S100A9 With The Severity And Prognosis In Patients With Community-acquired Pneumonia And Its Potential Mechanism

Background and ObjectiveCommunity-acquired pneumonia（CAP）,infected by pathogenic microorganism in the community,is one of common infectious diseases.Due to the increasing aged population in China,the continuous variation of pathogens,the rise of antibiotic resistance,the elevated population with chronic diseases or immunity activity decline,high treatment cost,and a poor prognosis and high mortality rate,CAP has become an important disease that threatens public health.Not only that,the clinical characteristics are atypical,CAP are always with many complications and an unsatisfactory effect of experiential treatments.Many improvements of diagnostic techniques and treatment methods have been obtained.But there are still many difficult of diagnose and evaluation for CAP patients.CAP severity scores have been widely to use to assess the severity of CAP patients.However,because of too many clinical markers and longer detection time,these restricts the application of CAP severity scores in the clinical practice.In recent years,many serum biomarkers are found and used for disease diagnosis and classification.More and more serum biomarkers have been found and used to evaluate the severity and prognosis in CAP patients.Serum biomarkers have exerted important roles in the CAP diagnosis.S100 Calcium Binding Protein A9（S100A9）,a small Ca²⁺binding protein recognized as an alarmin,is released by stressed cells:an endogenous danger signal,which promotes and exacerbates the inflammatory response.S100 protein family is widely expressed and acts as both intracellular and extracellular signal molecules.S100A9 and S100A8 form a heterodimer molecule binding calcium and zinc,which is mainly expressed in innate immune cells,especially in neutrophils,monocytes and macrophages.It has been found that S100A9 is elevated in many diseases.Previous studies by our research group found that S100A8/S100A9 is elevated in patients with COPD and is positively correlated with inflammatory cytokines,indicating that S100A9can be used as a biomarker for disease diagnosis.A large number of data indicate that S100A9 may play an important role in lung diseases.However,the role of S100A9protein in CAP patients is still unknown.Therefore,the main purpose of this study is to analyze the correlation between serum S100A9 and the severity and prognosis of CAP patients based on a prospective cohort study.In addition,the role of pulmonary S100A9elevation and its potential mechanism were explored in mice of CAP model.MethodsFrom May 1,2018 to June 30,2020,220 CAP patients were recruited from the Department of Respiratory and Critical Care Medicine of the Second Affiliated Hospital of Anhui Medical University.Demographic and clinical data were collected from the electronic medical record system.All subjects must meet the CAP diagnostic criteria.Inclusion criteria:（1）All subjects were diagnosed as CAP;（2）All subjects were over 18 years old;（3）All subjects volunteered to sign a written notice of consent.Exclusion criteria include:severe impairment of immune function;pregnant woman;Patients with other lung diseases,such as severe complications,chronic obstructive pulmonary disease,lung cancer and asthma;The hospital stay is less than 5 days.The healthy control subjects were from the physical examination center.The severity of pneumonia was scored by CAP,including Pneumonia Severity Index（PSI）,CURB-65,CRB-65,CURXO and SMART-COP.The whole blood was collected and centrifuged at six o’clock in the morning,and the serum was collected and the levels of inflammatory cytokines and serum S100A9 were measured by enzyme-linked immunosorbent assay（ELISA）.The mouse model of pneumonia was established by lipopolysaccharide（LPS）intratracheal instillation.All mice were sacrificed at 4 h and 24 h after LPS exposure.Lung specimens and serum samples were collected.The mouse model of pneumonia and lung injury were evaluated.The protein expression of S100A9 was detected in lung tissues by western blotting,and the level of serum S100A9 was measured via ELISA.Pulmonary NF-κB signaling was determined through western blotting and immunohistochemistry（IHC）.The levels of pro-inflammatory cytokines and chemokines m RNAs were estimated by RT-PCR.In order to explore the role of S100A9 rise on acute LPS exposure-induced mouse model of pneumonia,S100A9 sh RNA was used to knock-down S100A9 m RNA in lungs.The inhibitory effects of S100A9 sh RNA pretreatment on acute LPS exposure-induced mouse model of pneumonia progression and lung injury were analyzed.In addition,the antagonistic effects of S100A9 sh RNA pretreatment on acute LPS exposure-activated pulmonary NF-κB signaling and inflammatory cytokines secretion were estimated.ResultsCompared with control subjects,the level of serum S100A9 was increased in CAP patients at admission.The gradual increase of serum S100A9 was parallel to the severity score of CAP.Based on the CRB-65 score,the serum S100A9 level of CAP patients with≥3 scores were significantly higher than that of other grades.Based on SMART-COP score,the serum S100A9 level of CAP patients with 7-8 scores was significantly higher than that of other grades.According to the CURXO score,the serum S100A9 level of severe CAP patients was significantly higher than that of mild CAP patients.Based on the CURB-65 score,the serum S100A9 level is the lowest at0-1 and the highest at 3-5.Similarly,according to the PSI score,the level of serum S100A9 in grade III is higher than that in grade II and I,and the level of serum S100A9is the highest in grade IV.Correlation analysis found that serum S100A9 and CAP severity score（CURB-65,CRB-65,PSI,CURXO and SMART-COP）,blood routine parameters（WBC,NLR and MON）and inflammatory cytokines（TNF-α,IL-1βand CRP）.In addition,logistic regression analysis showed that there was a positive correlation between serum S100A9and CAP severity score.The serum S100A9 levels of living and dead patients were analyzed.A total of 8 CAP patients died during the observation period.The serum S100A9 level of dead patients was significantly higher than that of surviving CAP patients.The serum S100A9 level of patients with hospital stay>14 days was higher than that of patients with hospital stay<8 days and 8-14 days.Logistic regression was used to analyze the relationship between serum S100A9 level and the prognosis of CAP patients.The results showed that the serum S100A9 level was positively correlated with the length of hospital stay and the risk of death.In order to control the confounding factors,multiple logistic regression analysis was conducted.The results showed that serum S100A9 was positively correlated with the risk of death.The diagnostic prediction ability of serum S1009A level was analyzed by ROC curve AUC.The AUC of serum S100A9 level to CAP was 0.788（95%CI:0.699,0.878）,and the sensitivity and specificity of serum S100A9 level to predict CAP were73.5%and 82.5%,respectively.We also analyzed the predictive ability of serum S1009A level on disease severity in CAP patients.The optimal critical value of serum S100A9 level is 213.6 pg/m L,the sensitivity is 76.0%,and the specificity is 82.0%.Moreover,animal experiments indicated that acute LPS exposure obviously induced lung injury,elevated lung weight,lung coefficient,and the ratio wet weight to dry weight,caused inflammatory cell infiltration and destruction of alveolar wall,at 4 h and 24 h after LPS injection.These results hinted that acute lung exposure successfully established mouse model of pneumonia.In addition,acute LPS exposure elevated the expression levels of S100A9 in mice lungs and serum samples,activated pulmonary NF-κB signaling,promoted NF-κB p65 and NF-κB p50 nuclear translocation,and upregulated m RNA levels of pro-inflammatory cytokines（Tnf-α,Il-6）and chemokines（Mcp-1,Mip-2）in lung tissues.As expected,S100A9 sh RNA significantly alleviated LPS-induced mouse model of pneumonia and lung injury,inhibited NF-κB signaling activation and inflammatory cytokines secretion.ConclusionBased on a prospective cohort study and animal experiments,this study analyzed the correlation between serum S100A9 and severity and prognosis of CAP patients at admission,and explored the potential mechanism and role of S100A9 elevation in CAP.Serum S100A9 is positively correlated with the severity of CAP.At the time of admission,S100A9 with high serum increases the risks of death and hospitalization of CAP patients.S100A9 elevation activates pulmonary NF-κB signaling,promotes downstream target genes expression and inflammatory cytokines secretion,induces mouse model of pneumonia and lung injury,indicating that S100A9 may play a certain role in the pathophysiology of CAP.Serum S100A9 can be regarded as a diagnostic biomarker and can be used for the clinical management of CAP in future clinical practice.