Clinical Study Of Adult Community-acquired Pneumonia And Changes Of TLR2 And Cytokines After Infection With Mycoplasma Pneumoniae

Part I Clinical study of adult community-acquired pneumoniaBackground Community-acquired pneumonia（CAP）has high hospitalization rate and high mortality in the world.At present,most of the etiological studiesof CAP focus in children and are systematic.There are few studies on the etiology of adult CAPin China and abroad.Most researches of adult CAP focus on the incidence in different age groups,but lack systematic study.In the early years,a large-scale multi-center etiological study of adult CAP was conducted under the leadership Prof.You-ningLiu in 2006 in China.In recent years,Prof.BinCao and Prof.Yi Shi have jointly led the nation-wide multi-center clinical research on adult CAP in China,base on which the 2016 Chinese Diagnosis and Treatment Guidelines for Adult CAPwere revised to guide thestandardizedclinical treatment of adult CAP.At present,worldwide studies showed variances in the pathogen constitution of adult CAP in different regions.In addition,due to the differences in drug use tendencyand empiric treatment of adult CAP was different in different regions,the resistance of M.pneumoniae to macrolide drugs is also different.It is of great social value to carry out clinical research in order to understand the etiological composition and drug resistance of adult CAP in local region,so as to guide the empiric treatment of adult CAP.This study mainly investigated the etiological composition,drug resistance and clinical characteristics of adult CAP caused bydifferent pathogens in the city of Yantai,and provided basis for empiric treatment of local adult CAP.Objectives 1.To clarify the pathogen constitution of adult CAP and the resistance of M.pneumoniae to macrolides in Yantai.2.Drug resistance of Mycoplasma pneumoniae to macrolides.3.To understand the clinical characteristics of CAP caused by various pathogens.4.To analyze the irrational use of antibiotics in the empiric treatment of adult CAP in Yantai.5.Clinical symptoms and laboratory tests of CAP caused by different pathogens were compared between groups.6.To evaluate the sensitivity and specificity of clinical diagnosis of viral infection compared with viral nucleic acid.7.To analyze the clinical characteristics of severe community-acquired pneumonia8.30-day mortality and 90-day readmission.Methods This was a prospective study that enrolled 239 adult CAP patients admitted to the Department of Respiratory Medicine of Yantai Yuhuangding hospital from December 2015 to December 2017.Patients’ general information,underlying diseases,vital signs,clinical symptoms and physical signs were collected.Patients received routine blood test,CRP,ESR,blood gas analysis,biochemical tests and chest CT.Theirnasopharyngeal swabs were taken for the nucleic acid examination of respiratory tract viruses and M.pneumoniae.Samples of respiratory tract were collected for bacterial culture,pathogenidentification and drug sensitivity test.The score of pneumonia severity index（PSI）was calculated and graded.The clinical data,laboratory test results and empiric treatment protocols of CAP caused by different pathogenic stains were analyzed,and the clinical symptoms and laboratory tests of CAP caused by different pathogens were compared between groups.The mechanism of drug resistance of M.pneumoniae to macrolides was also analyzed.Results 1.Pathogen constitution of adult CAP in Yantai:virus accounted for 33.1%,M.pneumoniae accounted for 21.3%,and bacteria accounted for 5.4%of all enrolled cases.2.Viral pneumonia mainly occurred in winter and spring,with an average patient age of 49.5±20.7years old,average body temperature of 37.5±1.2℃,average leucocyte count of 7.9 ± 3.7 × 109/L,average duration of fever of 1.9 ± 1.8 days.Pulmonary imaging findings:27 cases（34.2%）with single lung involvement,52 cases（65.8%）with double lung involvement,most of which showed ground-glass exudation.In viral pneumonia,influenza virus accounted for 53.2%,adenovirus accounted for 17.7%,parainfluenza virus accounted for 10.1%,and rhinovirus accounted for 8.8%of the cases.3.Mycoplasmalpneumonia mainly occurred in autumn and winter,with average patient age of 36.4 ± 16.8 years old,average body temperature of 38.5 ± 1.3℃,average leucocyte count of 7.7 ± 2.9 × 109/L,and average duration of fever of 3.3±2.1 days.4.Drug resistance of M.pneumoniae:the resistance rate of M.pneumoniae to macrolides was 100%.The irrational use of antibiotics accounted for 25.2%of the cases.5-Compared with patients with viral pneumonia,the body temperature andduration of fever of patients with mycoplasmal pneumonia were significantly higher（P<0.05）.6.The mean age of 12 patients with SCAP was 68.7± 17.6 years（18,82）.Eight patients（75%）had underlying disease,and the average NLR of neutrophil count to lymphocyte count（24.2±28.8）was much higher than that of patients with common pneumonia（1.43±12.4）.7.30-day mortality and 90-day readmission were respectively 2.5% and 9.6%.Conclusions 1.The pathogen constitution of adult CAP in Yantai wasmainly viral pneumonia,followed by mycoplasmal pneumonia.2.The bacterial detection rate is low,but it is still dominated by Streptococcus pneumoniae。3.influenza virus was the main virus,followed by adenovirus,parainfluenza virusand rhinovirus.4.In adult CAP in Yantai,the resistance of M.pneumoniae to macrolide reached 100%,whereas the drug sensitivity to quinolones and tetracyclines remained good.For patients with clinically highly suspected M.pneumoniae infection,empiric use of respiratory quinolones or tetracyclineswas recommended.5.Comparingmycoplasmal pneumonia and viral pneumonia,we found although the clinical symptoms（fever,duration of fever,etc.）of mycoplasmal pneumonia were significantly heavier than those of viral pneumonia,the proportion of PSI grade Ⅲand Ⅳ viral pneumonia was significantly higher than that of mycoplasmal pneumonia.6.In the empiric use of antibiotics foradult CAPin Yantai,the irrational use of antibiotics was serious,therefore it is necessary to improve the management of antibiotics in this region.7.The mean age of SCAP,the proportion of complicated underlying diseases and the proportion of NLR were higher than those of ordinary pneumonia.8.The 30-day fatality rate and 90-day readmission rate were not high.Part II Changes of TLR2 and cytokines after infection with M.pneumoniaeBackground M pneumoniae not only causes respiratory tract infection,but also affects other organs,including the liver,heart,kidney and central nervous system,and even causes life-threatening infection.In recent years,there are many studies on the pathogenesis of M. pneumoniae.Previous studies have found that the pathogenicity of M. is mainly related to its adhesion and toxin secretion abilities.In recent years,as research further develops,it has been found that M. pneumoniae is associated with immune response.The membrane lipoproteins（LAMP-s）of M.pneumoniae have been identified as immune factors,which can combine with TOLL-like receptor （TLR） to promote the release of inflammatory factors and participate in the pathological process.TOLL receptors are the most studied pattern recognition receptors（PRRS）.At present,several studies compared the serum level of TLR2 and cytokines between patientsinfected with M.pneumoniae and healthy people,in order to understand the changes of various inflammatory factors after M. pneumoniae infection.Some studies were carried out in mouse models.The levels of TLR2 and inflammatory factors were measured in the alveolar lavage fluid or serum of infected animals.However,few studies have been carried out in cells,and the conclusions were inconsistent.In this study,the human monocyte cell line THP-1 was stimulated with different concentrations of M. pneumoniaemembrane lipoproteins.We measured the expression level of TLR2,detected the changes of IL-6 and IL-10 level in the supernatant of THP-1 cells before and after TLR2 interference,and analyzed whether the expression IL-6 and IL-10 were correlated with the induction concentration of membrane lipoprotein,in order to understand the mechanism ofliost immune response caused by M.pneumoniae infection from the cellular level and to provide basis for further study of M.pneumoniae.Objectives l.To determine the relationship between the expression level of TLR2 in THP-1cells and the stimulation concentration of exogenous membrane lipoprotein.2.THP-1 cells were stimulated with different concentrations of exogenous membrane lipoprotein before and after TLR2 knockdown,and IL-10 level in the culture supernatant was determined.IL-10 and IL-6 levels in the culture supernatant were measured to determine their correlation with TLR2 after M pneumoniae infection.3.the change of cytokine IL-6,IL-10Methods1.The protein level of TLR2 in THP-1 cells was determined by Western blot.2.After THP-1 cells were stimulated with different concentrations of exogenous membrane lipoproteins,the mRNA expression of TLR2 was detected by qPCR.3.After THP-1 was stimulated with different concentrations of exogenous membrane lipoprotein,the expression levels of IL-6 and IL-10 in cell culture supernatant were detected by ELISA.4.After interfered with TLR2 expression,the THP-1 cells were stimulated again withdifferent concentrations of exogenous LAMP-s.The levels of secreted IL-6 and IL-10 in the supematantof THP-1 cells were measured by ELISA.Results 1.ExogenousLAMP-s protein promoted the expression of TLR2 in THP-1 cells.2.The expression of TLR2 mRNA in THP-1 cells treated with 2μg/mL and 4μg/mL LAMP-s protein increased by 1.46 and 2.57 folds,respectively（P< 0.05）.2.Fluorescence microscopy showed a 70% infection rate of THP-1 cells by lentiviras.Compared with the untreated KD group,TLR2 mRNA expression both increased after treated with 2 μg/mL and 4μg/mL LAMP-s protein,but the TLR2 expression did not rise significantly with increased stimulation concentration.3.Changes of IL-6 level after infection with M.pneumoniae membrane lipoprotein After THP-1 cells were treated with 2 μg/mL or 4 μg/mL LAMP-s proteins,the levels of IL-6 in the supernatant of THP-1 cells were 1.50 pg/mL and 1.54 pg/mL,respectively.The concentration of IL-6 in the supernatant of the untreated control group was 1,18 pg/ml.The concentration of IL-6 in the supernatant of THP-1 cells significantly increased after LAMP-s stimulation（P< 0.05）.After interference of TLR2 gene expression in THP-1 cells,the concentration of IL-6 in the supernatant of THP-1 cells was 0.38 pg/mL.After treated with 2μg/mL and 4μg/mL LAMP-s proteins,the concentrations of IL-6 in the supernatant of THP-1 cells were 0.64 pg/mL and 0.65 pg/mL,respectively（P< 0.05）.4.Changes of IL-10 after infection with M pneumoniae membrane lipoprotein After THP-1 cells were treatment with 2μg/mL and 4μg/mL LAMP-s proteins,the concentrations of IL-10 in the supernatant of THP-1 cells were 8.01 pg/mL and5.27 pg/mL,respectively.The concentration of IL-10 in the supernatant of control group was 5.97 pg/mL,the difference was statistically significant（P< 0.05）.The concentration of IL-10 in the supernatant of THP-1 cells was 5.61 pg/mL after interference of the TLR2 and there was no significant difference in the content of IL-10 in the cell supernatant from the control group without interference with TLR2.After treatment with 2 μg/mL and 4μg/mL LAMP-s protein,the concentration of IL-10 in the supernatant of THP-1 cells increased to 8.34 pg/mL and 8.65 pg/mL,respectively（P<0.05）.The concentration of IL-10 in the supernatant of THP-1 cells was3.20 pg/tnL after interference of the TLR4 which was not significantly different from that of the control group.After treatment with 2μg/mL and 4jag/mLLAMP-s protein,the concentration of IL-10 in the supernatant of THP-1 cells increased to 4.94 pg/mL and pg/mL,respectively（P< 0.05）.Conclusions1.TLR2 was expressed in THP-1,and the expression level raised with the increase of exogenous LAMP-s concentration.2.After interfering with TLR2 expression in THP-1 cells,the mRNA expression level of TLR2 in THP-1 cells stimulated with exogenous LAMP-swas higher than that in the control group,TLR2 was involved in the host immune response caused by mycoplasma pneumoniae infection.The expression of IL-6 correlated with TLR2.The expression of IL-6 was up-regulated with the increase of LAMP-s concentration.3.The expression of IL-10 was not related to TLR25 but increased with the increase of LAMP-s concentration.The expression of IL-10 was related to TLR4.