Abstract Introduction/Rationale Immune cell populations, in particular monocytes and macrophages, have been associated with fibrosis in Interstitial Lung Disease (ILD). However, the mechanisms by which they contribute to fibrosis progression are not completely understood as most studies have analyzed tissues collected from end-stage disease. We aimed to characterize immune cells in lung tissue from patients with early ILD using cryobiopsy samples. We hypothesized that we would identify pro-fibrotic macrophage populations in the alveolar and interstitial spaces, and that these cells would co-localize with other cells in fibrotic niches. Methods We utilized spatial transcriptomics to distinguish cell populations in tissue from individuals without lung disease (n=2), early ILD (n=6), and end-stage ILD (n=1). Early ILD tissues were collected using cryobiopsies from patients undergoing bronchoscopy for diagnosis of ILD. Importantly, 5/6 participants had preserved lung function (pFVC ≥ 70%) at the time of cryobiopsy sampling. End-stage ILD tissue was from a participant who underwent lung transplantation. Normal tissue was collected from organ donors without known lung disease. Spatial transcriptomics was performed using the 10x VisiumHD platform which allowed for transcriptome-wide expression coverage at single-cell resolution. Pixels were grouped into cells using bin2cell and then cell types were annotated using cellTypist. Cells identified as myeloid and lymphoid were extracted and unsupervised hierarchical clustering was performed using Seurat to identify cell subpopulations. Network analysis using CellChat and CellNest was also performed to identify putative cell communication networks and transcriptional niches. Results Cryobiopsies were from participants primarily with autoimmune disease (Figure 1). Four out of 6 participants have gone on to develop a progressive pulmonary fibrosis phenotype. An estimated 391,362 cells were captured, of which 66,138 were lymphoid and 27,831 were myeloid. Diseased samples had an expansion of lymphocytes, in particular B-cells. Myeloid cells were clustered into subpopulations that included mature alveolar macrophages, proliferating macrophages, and interstitial macrophages. Macrophages expressing putative pro-fibrotic genes including CHIT1 and SPP1 were also identified. Cell localization in tissue confirmed predicted cell identities and revealed clusters of lymphocytes and plasmablasts in the interstitium. Conclusions This study utilized clinically indicated cryobiopsies to characterize cell populations present during the early stages of ILD. We identified lymphoid and myeloid subpopulations within these tissues and characterized their relative abundance and localization. Further work will focus on co-localization of these cells with fibroblasts and using additional computational approaches to define cell trajectories and transcriptional niches to compare early disease, end-stage disease, and non-diseased tissues. This abstract is funded by: This work was supported by the Parker B. Francis Foundation (SEH) and the National Institutes of Health: NHLBI K23HL175120 (SEH), NHLBI R01HL16925 (EDM), NHLBI R01HL149676 (CM)and NIAID U19AI167891 (CM)
Holton et al. (Fri,) studied this question.
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