A Study On The Clinical Importance Of AECOPD Based On Induced Sputum Inflammatory Cellular Classification

Background: Chronic pulmonary obstructive disease (COPD) is characterized asan incompletely reversible airflow obstruction, and has a nature of heterogeneity.Hence, it is very important to assess the pathophysiology of airway of COPD. Inducedsputum is a non-invasive method and a useful tool to evaluate inflammatory cells andmediators in the airway lumen in the setting of acute exacerbation of COPD. However,the inhalation of hypertonic saline solution to induce sputum may cause abronchoconstrictive response, so it is crucial to evaluate the success and safety ofsputum induction. Furthermore, the patients with COPD are classified into fourphenotypes based on the sputum inflammatory cellular profile, to investigate thedifference in clinical characteristics, laboratory test, mediators expression, effect oftreatment and diagnosis among subgroups. This inflammatory phenotypeclassification is not only useful for the management of AECOPD patients, but alsovaluable for investigating the pathogenesis of AECOPD.Objectives: The aims of this study are to assess the safety and efficacy of sputuminduction in adults with AECOPD, to establish a modified protocol to ensure thesafety of sputum induction in AECOPD, and to investigate the sputum cellularprofiles to classify patients with AECOPD to provide new insight in the mechanismsof AECOPD and individual therapy.Methods:1. According to the modified protocol on sputum induction,83AECOPDsubjects and26healthy controls underwent a sputum induction, processing and cellcounting. The outcome measures included fall in lung function during induction andsuccess of sputum induction, and determines which variables were independentlyassociated with a fall in FEV1as percentage from baseline.2. Found out the cutoff values of the95th percentile of sputum neutrophils andeosinophils from healthy controls, respectively. 3. Their demographic and clinical characteristics were recorded, and their lungfunction,chest CT,6mintues walk test, BODE scores, CCQ scores and clinical routinetest were examined. The serums of all subjects were collected.4. The phenotypes of sputum inflammatory cells were characterised, and theconcentrations of sputum and serum amyloid-A (SAA), C-reactive protein (CRP),interleukin-6(IL-6), and matrix metalloproteinase-9(MMP-9) were measured. Basedon the sputum inflammatory cell profiles, individual patients were categorized intoone of the four subgroups with inflammatory eosinophilic, neutrophilic,paucigranulocytic, and mixed granulocytic AECOPD. The comparison was performedin clinical characteristics, lung function, BODE scores, length of hospital stay, levelsof inflammatory mediators and prognosis.5. The AECOPD patients were reevaluated within12-14months after dischargein stable phase, including sputum induction, processing, cell counting, lung function,6minutes walk test, BODE scores and routine test. The serums were collected again.6. The analysis was performed by comparing between the changes of thephenotypes, clinical characteristics, lung function, BODE scores and the expression ofinflammatory mediators from AECOPD to stable phase.Results:1. The assessment of safety: according to the modified protocol, the sputuminduction to AECOPD is safe and efficacy. Adults hospitalized with AECOPD hadmoderate to very severe airflow obstruction. Sputum induction was successful in over80%of subjects. The percentage decrease in FEV1from baseline by GOLD categorywas median1.2(IQR,0.5-3.3)[GOLD II],2.3(1.3-3.2)[GOLD III],5.2(3.3-8.6)[GOLDIV], and1.4(0.5-3.2)[control], respectively. A fall in FEV1of>20%occurred in onlyone subject with AECOPD who was in GOLD category III. The decrease inpercentage of FEV1from baseline was greatest in the second stage of induction, andcorrelated with that of the final stage (r=0.589; P=0.01). The fall in FEV1duringinduction increased with GOLD category (P <0.05).2. Predictor of fall in lung function: in the multivariate linear regression model,increased age (r=0.25, P=0.04) and decreased post SABA FEV1/predicted (r=-0.31,P=0.01) were associated with a greater fall in FEV1as percentage from baseline.There were weaker correlations with the fall in FEV1and some medicines used beforeSI, such as long acting β2-agonists (r=-0.15, P=0.06) and theophylline (r=-0.21, P=0.08).3. AECOPD phenotypes and GOLD categories: all of the AECOPD patients werestratified, according to the number of neutrophils (>61%) and eosinophils (>2.5%) inthe sputum samples, which were the cutoff values of the95th percentile of healthycontrols, respectively. Individual patients were classified into the eosinophilic COPD(EO) with sputum eosinophils>2.5%of total cells, the neutrophilic COPD (NE) withneutrophils>61%, the paucigranulocytic COPD (PA) with eosinophils≤2.5%andneutrophils≤61%, and the mixed granulocytic COPD (MC) with eosinophils>2.5%and neutrophils>61%. There were10(12%) eosinophilic,36(43%) neutrophilic,5(6%) mixed granulocytic, and32(39%) paucigranulocytic AECOPD patients. Inpaucigranulocytic AECOPD, there were15patients for GOLDⅡ,16for Ⅲ,1for Ⅳ,respectively; in neutrophilic AECOPD,4for Ⅱ,26for Ⅲ,6for Ⅳ, respectively; ineosinophilic AECOPD,7for Ⅱ,3for Ⅲ, respectively; in mixed granulocyticAECOPD,1for Ⅲ,4for Ⅳ,respectively.4. Clinical characteristics and expression of inflammatory mediators: the patientswith mixed granulocytic or neutrophilic AECOPD had a higher BODE score, moresputum inflammatory cells, lower lung function, and longer hospital stay,accompanied by higher concentrations of sputum MMP-9, IL-6and CRP, and serumSAA, IL-6and CRP. Notably,83%of patients with neutrophilic AECOPD displayedevidence of bacterial infection and many of them responded poorly to standardtherapies.5. The stability of phenotypes and prognosis: we followed up61out83AECOPD patients for about14months. These patients with stable COPD remained inthe same group, except for two patients from the EO to PA, two patients from the PAto NE group, and1patient from the NE to PA group. The kappa statistic (95%confidence interval) was0.87(0.76-0.98)(P <0.01), indicating substantial agreementin classifications between the visits. In addition, patients with mixed granulocytic orneutrophilic stable COPD remained at lower lung functions, higher BODE scores,inflammatory cell counts, serum and sputum inflammatory mediators and higherlevels of inflammation. These result indicated there were higher local and systemicinflammatory mediators, lung function damage and poor prognosis. Conclusion:1. Sputum induction can be safely and successfully performed in patients withmoderate-to-very severe COPD who experience an exacerbation by using thismodified induction protocol. The early decrease in FEV1can be used to predict themaximum fall. A multivariate linear regression model showed increased age anddecreased post SABA FEV1/predicted were associated with a greater fall in FEV1aspercentage from baseline.2. According to sputum inflammatory cell-based classification, differentphenotypes of patients with AECOPD exhibited various properties of phathophy-siology. In the four phenotypes, there were significant difference in clinical symptoms,BODE scores, GOLD categories, lung function, inflammatory cell counts in bloodand sputum, pathogens, expressions of inflammatory mediators, responses to standardtherapy and prognosis. The classification based on sputum inflammatory cell countssuggests each type of inflammatory cell, such macrophages, neutrophils andeosinophils, plays different roles, and lead to pathophysiological changes throughexpressing diverse inflammatory mediators.3. The classification based on sputum inflammatory cell counts displaysheterogeneous inflammation, and substantial agreement in classifications during longterm visits. These results indicate that each phenotype experiences its ownphathophysiological process, and cause different digrees and patterns of damagementin the function and parenchyma. The classification will lay a foundation for thepathophysiology of AECOPD, and provide the scientific evidence to the individualtherapy.