| BACKGROUND:Hypersensitivity pneumonitis(HP)was one kind of interstitial lung disease(ILD).Guideline of adult HP diagnosis published in 2020 recommended that HP classified as non-fibrotic(NFHP)and fibrotic(FHP)on the basis of the predominant presence or absence of radiological and/or histopathological fibrosis.FHP was characterized with impaired lung function and poor prognosis.Up to now,factors associated with the development of FHP were unclear,prognostic biomarkers were limited and the mechanism of HP need to be further explored.Bronchoalveolar lavage fluid(BALF)was accessible specimen,which could reflect lung pathological changes during the development of HP.Single-cell transcriptome sequencing could be used to explore the heterogeneity of BALF cells from HP patients.Comparison of cell types and differential expression genes could assist to understand the development of FHP.OBJECTIVE:This research contained three parts:in the first part,we summarized clinical characteristics and prognosis of FHP patients and explore factors associated with the presence of fibrosis;the second part was conducted to assess prognostic potential of serum CYFRA 21-1 applied as a biomarker to predict adverse outcome in FHP patients;in the third part,we aimed to contrast the single cell atlas of BALF cells from HP patients and investigated the heterogeneity of BALF cells from FHP patients with single-cell transcriptome sequencing.Part Ⅰ:Clinical characteristics and factors associated with the development of fibrotic hypersensitivity pneumonitisMETHODS:Patients enrolled in this part derived from one prospective cohort study,who were screened from January 2017 to August 2021.The diagnosis of HP was determined through ILD multidisciplinary team discussion.Comparison of clinical features between NFHP and FHP were made.Survival between NFHP group and FHP group was compared using Kaplan-Meier analysis.Logistic regression analysis was performed to explore factors associated with the presence of fibrosis.RESULTS:A total of 202 patients with HP were enrolled,including 87(43.1%)NFHP patients and 115(56.9%)FHP patients.Patients with FHP were older and more frequently presented with dyspnea,crackles,and digital clubbing than patients with NFHP.Also,FHP patients have higher serum level of several tumor markers,sever impairment of lung function and lower percentage of BALF eosinophils than NFHP patients.BALF lymphocytosis was present in both groups,but it was less pronounced in the FHP group than NFHP group.12 patients suffered from adverse outcomes and all of these patients were FHP.Univariate and multivariate regression analyses revealed that age≥65 years,percentage of lymphocytes in BALF<20%,and percentage of eosinophils in BALF≥1.75%were factors associated with the presence of fibrosis in HP.CONCLUSIONS:This study summarized the differences of clinical features and prognosis of FHP patients.Level of several serum tumor markers in FHP patients were higher than NFHP patients.Age≥65 years,percentage of lymphocytes in BALF<20%,and percentage of eosinophils in BALF≥1.75%were factors associated with the presence of fibrosis in HP.Part Ⅱ:serum CYFRA 21-1 could predict adverse outcomes in fibrotic hypersensitivity pneumonitis:A cohort studyMETHODS:Patients enrolled in this part derived from one prospective cohort study,who were screened from January 2015 to July 2021.Patients were classified as NFHP and FHP on the basis of radiology and histopathology.Level of serum CYFRA 21-1 was compared between NFHP and FHP.Immumohistochemical staining were performed on histopathologic sections of the FHP lung transplant patients and donors.Correlation analysis was performed to between serum CYFRA 21-1 and indicators of FHP disease severity.Cox regression analysis and Kaplan-Meier plot were used to explore the relationship between serum CYFRA 21-1 and death/lung transplantation.Area under the curve(AUC)on receiver operating characteristic curve was applied to estimate performance of predicting FHP outcome.RESULTS:Compared with NFHP group,serum CYFRA 21-1 level was significantly increased in FHP group.Expression level of CYFRA 21-1 in FHP patients were also verified in the lung tissue and it was expressed mainly in pulmonary epithelial cell.86 FHP patients were enrolled in this part and 24 died or performed lung transplantation.Serum CYFRA 21-1 was associated with FHP disease severity indicators including:FVC%predicted,TLC%predicted,DLCO%predicted,6-minute walking distance,and fibrosis scores on chest CT.In the COX regression analysis,after adjusted for age,gender,smoking status,FVC%predicted,DLCO%predicted,fibrosis scores,traction bronchiectasis and mosaic perfusion,serum CYFRA 21-1 was still an independent risk factor for death/lung transplantation(HR=1.36,95%CI 1.10-1.68,P=0.005).Serum CYFRA 21-1 showed great performance in assessing the probability of death/lung transplantation(AUC=0.70).And death/lung transplantation was more frequently in the higher serum CYFRA 21-1 group(≥3.75ng/ml)than in the lower serum CYFRA 21-1 group(<3.75ng/ml)(log-rank test,P=0.001).In addition,there were 30 patients being eligible for evaluating whether developing progression.Serum CYFRA 21-1 level was significantly higher in FHP progression group than non-progression group.The AUC of serum CYFFA 21-1 and the development of FHP progression was 0.64 while AUC of the combination of serum CYFRA 21-1 and age,FVC%pred and DLCO%pred was 0.89.Also,risk of FHP progression was higher with the increasing serum CYFRA 21-1.CONCLUSIONS:The expression level of CYFRA 21-1 in serum and lung tissue wassignificantly increased in FHP patients.Serum CYFRA 21-1 concentration was correlated with FHP disease severity.Increasing serum CYFRA 21-1 was an independent risk factor for the death/lung transplantation.And the higher serum CYFRA 21-1,the higher risk of progression.Therefore,serum CYFRA 21-1 had the potential to be one biomarker to assess adverse outcome in FHP patients.Part Ⅲ:The heterogeneity of BALF cells from fibrotic hypersensitivity pneumonitisMETHODS:In this part,BALF cells from healthy controls,NFHP and FHP patients were collected to performed single cell transcriptome sequencing.Cell types annotation,subpopulation analysis and differential gene enrichment analysis were conducted.RESULTS:BALF specimens from 2 health controls,2 NFHP and 3 FHP patients were collected and 61102 cells were obtained after sequencing and screening.And identified cells after annotation included macrophages,mDC cells,pDC cells,T cells,B cells,NK cells and epithelial cells.In BALF of NFHP patients,lymphocytosis were pronounced while in FHP patients,macrophages were dominant.Macrophages were classified as 6 subclusters.In the FHP patients,subclusters of macrophages were characterized with highly expression of CCL18,PLAC8,HIST1H4 and IL32.Differential genes expressed in macrophages from FHP group were mostly related with regulation of defense reaction.T cells were classified into 10 subclusters and B cells were classified into 3 subclusters.Both T cells and B cells were mainly derived from NFHP patients and they accounted for only minor portions of FHP BALF cells.In FHP patients,subclusters of T cells were mainly FTL+CD4+T cells and B cells were mainly memory B cells.CONCLUSIONS:macrophages accounted for the majority of BALF cells in FHP patients while T cells and B cells were the major cells in NFHP patients’ BALF.In FHP,subclusters of macrophages characterized with highly expression of CCL18,PLAC8,HIST1H4 and IL32 while subclusters of T cells were mainly FTL+CD4+T cells.These cells may play important roles in the development of FHP but further experiments were needed to verify their function. |
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