Disease Specific Signatures Identified by RNA‐seq of Sorted Lung Cellular Populations
Authors: Reyfman Paul A , Morales‐Nebreda Louisa , Walter James M , McQuattie‐Pimentel Alexandra , Chen Ching‐I , Anekalla Kishore R , Abdala‐Valencia Hiam , Yacoub Tyrone J , Antalek Matthew , Chi Monica , Chiu Stephen F , Gonzalez Francisco , Homan Philip J , Soberanes Saul , Bharat Ankit , DeCamp Malcolm M , Bhorade Sangeeta M , Bartom Elizabeth T , Morimoto Richard I , Balch William , Sznajder Jacob I , Chandel Navdeep S , Ridge Karen M , Bagheri Neda , Amaral Luis AN , Budinger GR Scott , Perlman Harris , Winter Deborah , Misharin Alexander V 
Published: June 2021

Rationale

Fibrotic interstitial lung disease (ILD) is among the top indications for patients undergoing lung transplantation worldwide. While our group and others have demonstrated a role for monocyte‐derived alveolar macrophages in the development of lung fibrosis in murine models, it is unknown if human alveolar macrophages play a similar role in the development of fibrotic ILD. We hypothesized that transcriptional profiling of AMs isolated from lung transplant donors and recipients as well as from murine alveolar macrophages in a bleomycin‐induced lung fibrosis model would demonstrate homology of involved genes.

Methods

Fluorescence‐activated cell sorting (FACS) was used to isolate AMs (CD45+ CD169+ HLA‐DR+) from samples of 18 donor lungs and 22 explanted lungs from patients at the time of lung transplantation and to isolate separately tissue‐resident and monocyte‐derived AMs from mice at fourteen and nineteen days following the administration of intratracheal bleomycin. RNA extraction from sorted cells was performed with poly(A) enrichment, followed by single‐end RNA‐seq. Exploratory data analysis using principle component analysis (PCA) and K‐mean clustering were performed. Differentially expressed genes were estimated between donors and patients with interstitial lung disease, and between murine tissue‐resident and monocyte‐derived AMs during bleomycin‐induced fibrosis.

Results

The median age of the 22 lung transplant recipients was 56 (IQR 13–64). Among recipients, there were eleven patients with the diagnosis of fibrotic ILD (five with scleroderma‐associated ILD, three with myositis‐ILD, two with idiopathic pulmonary fibrosis, and one with mixed connective tissue disease ILD). PCA of sorted populations showed grouping by diagnosis. 61 genes were identified that were differentially expressed (adjusted p < 0.05) in AMs between donors and patients with fibrotic ILD, and that were also homologs of genes highly expressed in murine monocyte‐derived AMs relative to tissue‐resident AMs during bleomycin‐induced lung fibrosis. Hierarchical clustering of samples according to expression of these homologous genes showed just one ILD sample clustering among donors, and of 61 total differentially expressed homologous genes, 51 were upregulated in AMs from patients with fibrotic ILD and only 10 were downregulated.

Conclusions

Transcriptional profiling of AMs collected at the time of lung transplantation identifies homology between human fibrotic interstitial lung disease and a murine model of bleomycin‐induced lung fibrosis. By focusing on key lung cellular populations such as alveolar macrophages, future transcriptional studies of lung disease in humans may identify new pathways and new potential targets for therapy for patients with lung disease.

Support or Funding Information

5T32HL076139‐13, AG049665, HL071643, ES013995, The Veterans Administration, DOD W81XWH‐15‐1‐0215