The Pro-inflammatory Effect And Mechanism Of Candida Isolated From The Lower Respiratory Tract On Chronic Obstructive Pulmonary Disease

Objective Through a retrospective study on the isolation of Candida from respiratory secretions of patients with acute exacerbation of chronic obstructive pulmonary disease(AECOPD),the clinical characteristics and risk factors of acute exacerbation in AECOPD patients with positive and negative isolation were compared and analyzed.To study the phagocytosis of monocyte-derived macrophages(MDMs)and local and systemic inflammatory response in AECOPD with positive Candida isolation in the lower respiratory tract.Then an animal model was established to further investigate the effects of Candida albicans airway colonization on pseudomonas aeruginosa pneumonia,alveolar macrophages(AMs)and lung Th17 in chronic emphysema rats.Methods 1.A retrospective analysis of the etiological test results and clinical data of 225 patients with AECOPD who were hospitalized in the First Affiliated Hospital of Guangxi Medical University from January 1,2018 to December 31,2019.The patients were divided into Candida-positive group and Candida-negative group for comparative analysis.2.The clinical data,induced sputum and peripheral blood of 75 patients with AECOPD hospitalized in the First Affiliated Hospital of Guangxi Medical University from January 1,2020 to December 31,2020 were collected and divided into Candida-positive group(n=32)and Candida-negative group(n=43)according to the detection results of Candidaalbicans in the lower respiratory tract,and the induced sputum and peripheral blood of 30 healthy subjects with normal lung function and non-smoking were collected during the same period.The contents of IL-17,IFN-γIL-1βand IL-6 in serum and sputum supernatant were detected by Luminex kit.The proportion of helper T lymphocyte 1(Th1)and helper T lymphocyte 17(Th17)in peripheral blood of each group was detected by flow cytometry,and the phagocytic function of MDMs in each group was compared by flow cytometry combined with fluorescence microscope.3.(1)Wistar rats were randomly divided to air exposure control group(NC)and cigarette smoke exposure and elastase group(CS+PPE).The CS+PPE group was exposed to cigarette smoke for 4 weeks,followed by an airway drip of elastase,while the NC group was exposed to air alone.The lung was stained with pathological HE and the mean interlining interval(MLI)was calculated.(2)The rat airway was infused with Candida albicans(10~6CFU)to establish colonization model.Rats were divided into NC group(NC C.albicans colonization)and CS+PPE group(CS+PPE C.albicans colonization).The lung homogenate colony number and the rate of weight change were observed at 24 hour,72 hour,7 day,14 day and21 day.The levels of IL-17 and IL-6 in supernatant of BALF supernatant were detected by ELISA.(3)Rats were divided into NC group,CS+PPE group,and CS+PPE C.Albicans colonization group.The Th17 differentiation and AMs activation and phenotype in pulmonary alveolar lavage fluid(BALF)were detected by Flow Cytometry after candida albicans solution was dropped into the airway for 72 hours.The phagocytosis of AMs was observed by flow cytometry and fluorescence microscope.(4)Pseudomonas aeruginosa(10~5CFU)was drip-infused into the airway of CS+PPE C.albicans colonization group and CS+PPE non-albicans colonization group.The incidence of pneumonia was compared by general view of lung and colony count of homogenate culture.Statistical methods such as independent sample T test,Mann-Whitney test,Chi-square test,Survival curve drawing by Kaplan-meier method and Binary Logistic regression analysis were used to analyze the data.P<0.05 indicated that the difference was significant.Results 1.(1)A total of 88 AECOPD patients(39.1%)were positive for Candida in lower respiratory tract secretions,and the species composition ratio was:Candida albicans(87.5%),Candida glabrata(10.2%),Candida tropicalis(6.8%),Candida parapsilosis(2.3%),Candida krusei(1.1%).(2)There was no significant difference between the two groups in gender,age,course of disease,BMI,complications and comorbidities.The smoking index and CAT score in the Candida-positive group were higher than those in the Candida-negative group,and the proportion of m MRC-4 and GOLD-4 patients in the Candida-positive group was higher,and the differences were statistically significant.In terms of acute exacerbations,although there was no significant difference between the two groups in the number of AEs in the past 1 year,the proportion of the Candida-positive group requiring hospitalization for AEs in the past 1 year was significantly higher than that in the Candida-negative group.(3)The absolute value of lymphocytes,CD8~+Tlymphocyte count,FEV1%pred,and FEV1 levels in the Candida-positive group were significantly lower than those in the Candida-negative group,and the results were statistically different.There is no significant difference in other aspects.(4)Logistic regression analysis indicated that Candida and m MRC-4 were not only independent risk factors for acute exacerbations in COPD patients in the past year,but also independent risk factors for acute exacerbations requiring hospitalization in the past year.(5)Survival analysis showed that 20 patients(14.6%)died in the candida-negative group and 36 patients(40.9%)died in the candida-positive group.There was significant difference in survival rate between two groups.2.(1)There was no significant difference in age,sex,BMI,course of disease,days of hospitalization and complications among the three groups.The clinical manifestations,laboratory tests and pulmonary function of the two groups were consistent with the results of the first part of the study.(2)Compared with the Candida-negative group,the Candida-positive group had a higher proportion of patients with a history of ICU admission,hospitalization for acute exacerbation,and the interval between the recurrence of acute exacerbation was significantly shorter.The differences were statistically significant.(3)The levels of IL-17,IFN-γ,IL-1βand IL-8 in the serum and sputum supernatant of AECOPD patients were higher than those of the healthy control group,and the levels of IL-17 in serum and sputum supernatant and IL-1βand IL-6 in sputum supernatant in Candida positive group were higher than those in Candida negative group.(4)The proportion of Th1 and Th17 cells in the peripheral blood of COPD patients was significantly higher than that of the healthy controls,and the difference was statistically significant.The proportion of Th17 cells in the Candida-positive group was higher than that in Candida-negative group,but there was no difference in Th1 proportion between the two groups.(5)The phagocytosis rate of MDMs in COPD patients was significantly lower than that in healthy controls,and that in Candida-positive group was significantly lower than that in Candida-negative group.The differences were statistically significant.3.(1)After exposure to cigarette smoke exposure combined with elastase,pulmonary emphysema-like changes were observed under the microscope in the CS+PPE group,with the alveolar cavity enlargement,alveolar rupture and fusion,and inflammatory cell infiltration.The MLI of CS+PPE group was significantly higher than that of NC group,and the results were statistically significant.(2)There were no difference in activity status,fecal appearance,and rate of weight change among NC group,CS+PPE group,NC C.albicans colonization group and CS+PPE C.albicans colonization group.Candida albicans was gradually removed,and the removal efficiency of CS+PPE C.albicans colonization group was lower than that of NC C.albicans colonization group.IL-17 and IL-6 in the supernatant of BALF in NC C.albicans colonization group and CS+PPE C.albicans colonization were higher than those in NC group and CS+PPE group,respectively.The results were statistically different.(3)Compared with CS+PPE group,the phagocytosis of AMs was lower,the proportion of CD86~+and CD163~+cells in AMs was increased,and the levels of Th17 and IL-17 in BALF were significantly increased in CS+PPE C.albicans colonization group.The results were statistically different.(4)The incidence of pseudomonas aeruginosa pneumonia in CS+PPE C.albicans colonization group was significantly higher than that in CS+PPE group.The results were statistically different.Conclusion Candida was detected in lower respiratory secretions of more than1/3 of hospitalized patients with AECOPD,mainly Candida albicans.Candida isolated from lower respiratory may be related to the severity of symptoms,airflow limitation,prognosis and recurrent acute exacerbation in patients with COPD.Candida and m MRC Grade-4 was an independent risk factor for exacerbations in COPD patients.The impaired phagocytic function of MDMs was more severe in AECOPD patients with positive Candida isolated in the lower respiratory tract.Candida in airway can affect the local and systemic immune responses associated with Th17 cells in COPD patients.Candida albicans airway colonization promoted a stronger Th17 inflammatory response in the lung in CS+PPE rats.Although the number of AMs activation increased and the phagocytosis decreased significantly.Candida albicans airway colonization increased the risk of pseudomonas aeruginosa pneumonia in CS+PPE rats.