Identifying Unique Pathogenic Macrophages in Systemic Sclerosis-ILD Using High Throughput Single-cell RNA-Seq
Identifying Unique Pathogenic Macrophages in Systemic Sclerosis-ILD Using High Throughput Single-cell RNA-Seq
Alveolar macrophages isolated from bronchoalveolar lavage (BAL) fluid from systemic sclerosis (SSc) patients with clinically significant lung fibrosis will be studied at baseline and at 6 months after enrollment to assess longitudinally the presence and persistence of an emergent, pro-fibrotic alveolar macrophage population, using single cell RNA-Seq technology to measure the individual transcriptome from each cell.
Using cutting-edge single cell RNA-Seq technology, we will identify in the BAL fluid of subjects of SSc-ILD emerging pathogenic cell populations in the lung that were previously unrecognized using standard RNA-Seq and microarray technologies, which lack the resolution to analyze transcriptomes of individual cells. Alveolar macrophages isolated from bronchoalveolar lavage (BAL) fluid from SSc patients with clinically significant lung fibrosis will be studied at baseline and at 6 months after enrollment to assess longitudinally the presence and persistence of an emergent, pro-fibrotic alveolar macrophage population.
Subjects with SSc-ILD will be recruited from the Scleroderma Program. We will recruit adults who fulfill 2013 American College of Rheumatology (ACR) SSc criteria and clinically relevant SSc-interstitial lung disease. Patients will undergo bronchoscopy with bronchoalveolar lavage at month 0 and then at month 6. Healthy control subjects will complete demographic and basic medical forms to ensure health. Pertinent clinical data will be downloaded from the Enterprise Data Warehouse, an electronic database in use at Northwestern that was designed to aggregate and store patient data from various medical systems, or through manual chart review, and entered into a RedCap database created specifically for this project.
During an elective bronchoscopy procedure in SSc and healthy control subjects the bronchoscope will be wedged into an affected lung segment guided by CT scanning. After wedging the bronchoscope, 120ml of sterile 0.09% normal saline will be instilled. All subsequent aliquots will be pooled. Up to 40-60 mL of BAL fluid will be obtained for analysis during each sampling. Alveolar macrophages will be sorted on a fluorescence-activated cell sorter (FACS) Aria III instrument. High-throughput single cell transcriptomic (Drop-seq) data will be processed on Northwestern high-performance computational cluster using Cell Ranger pipeline and post-processed using modified AltAnalyze pipeline.
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