Abstract
The transcription factor JUN is highly expressed in pulmonary fibrosis. Its induction in mice drives lung fibrosis, which is abrogated by administration of anti-CD47. Here, we use high-dimensional mass cytometry to profile protein expression and secretome of cells from patients with pulmonary fibrosis. We show that JUN is activated in fibrotic fibroblasts that expressed increased CD47 and PD-L1. Using ATAC-seq and ChIP-seq, we found that activation of JUN rendered promoters and enhancers of CD47 and PD-L1 accessible. We further detect increased IL-6 that amplified JUN-mediated CD47 enhancer activity and protein expression. Using an in vivo mouse model of fibrosis, we found two distinct mechanisms by which blocking IL-6, CD47 and PD-L1 reversed fibrosis, by increasing phagocytosis of profibrotic fibroblasts and by eliminating suppressive effects on adaptive immunity. Our results identify specific immune mechanisms that promote fibrosis and suggest a therapeutic approach that could be used alongside conventional anti-fibrotics for pulmonary fibrosis.
Highlights
The transcription factor JUN is highly expressed in pulmonary fibrosis
For profiling with mass cytometry, single-cell suspensions of 14 representative lung samples, 11 fibrotic and 3 normal, were stained with a panel of 41 metal-conjugated antibodies (Supplementary Data 1) including 3 antibodies (CD45, CD31 and CK7) that allowed for manual gating of four distinct cell lineages: CD45+ leukocytes, CK7+ epithelial cells, CD31+ endothelial cells and CD45−CK7−CD31− fibroblasts (Fig. 1b, gating strategy in Supplementary Fig. 7 and live cells counts in Supplementary Table 2)
We detected that the frequency of fibroblasts was 5-fold higher in fibrotic lungs (15% in normal lungs compared to 80% in fibrotic lungs), and leukocytes were 3-fold lower (60% normal compared to 20% in fibrotic lung)
Summary
PD-L1 and CD47 are upregulated in fibrotic fibroblasts. To systematically profile the pathophysiology of human pulmonary fibrosis, we applied an -omics approach combining multiparameter single-cell mass cytometry and genome-wide chromatin accessibility assays together with a multiplexed Luminex secretome analysis as outlined in (Fig. 1a). The JUN enrichment observed in these two cases correlated with an increase in chromatin accessibility (detected by ATAC-seq) in lung-fibroblast cells compared to normal This is interesting as these changes are only present in our primary lung fibroblasts but not in any of the other previously published data on JUN ChIP-seq performed on cancer-cell lines such as A549, MCF-7, H1-hESC, HepG2 or K562. To demonstrate the physiological relevance of these findings, we compared our ATAC-seq data with published gene expression profiling from fibrotic and normal lungs[45] and found an overlap of 70 genes between the two datasets; among the most significant were genes encoding the pro-fibrotic epithelial–mesenchymal transition pathway, indicating that the JUN pathway could be a driver of fibrotic progression in pulmonary fibrosis (Supplementary Fig. 4c).
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