605 - Single cell transcriptomic analysis of neonatal murine lungs after hyperoxia inducedacute lung injury in the immediate post injury and sequela stages

Poster Number: 605
Publication Number: 605.1217

Background: Oxygen supplementation therapy or Hyperoxia (HO) disrupts lung development
leading to bronchopulmonary dysplasia (BPD). In this study, single cell RNA sequencing
(scRNA-seq) was performed to understand the molecular and cellular dynamics during post-
hyperoxia and recovery phases.

Objective: To investigate cell-type specific transcriptional changes of lungs immediate post-
hyperoxic injury and during recovery.

Design/Methods: Neonatal mice were exposed to normoxia (NO) or HO (95% O 2 ) from PN1 to PN 7 and
the lungs were digested enzymatically (collagenase and neutral protease) in gentle MACS™
Octo Dissociator. The cell suspension was then multiplexed and processed by 10x genomics
(chromium). Raw sequencing reads from the gene expression libraries were processed and
demultiplexing expression data with MULTI-seq barcode libraries was performed. Data quality
control, integration, and clustering followed by differential expression analysis and gene set
enrichment analysis (GSEA) was done. The expression of markers involved in fibrosis,
inflammation and angiogenesis in the top 50 differentially expressed genes were studied.

Results: The genes involved in fibrosis (Ccnd1, Acta2, Serpine, Col18A), TGF downstream
(Acta2, Tgfbr2, Serpine, Cdkn1a), TP53 (Serpine, Bax, Cdkn1a) and the inflammation (Lcn2)
were looked at. There is significant upregulation of many of these genes on postnatal day 7 (PN
7). The inflammatory marker Saa3 and Lcn2 was significantly upregulated in the Col 13a1+, Col
14a1+ and Cap/Art clusters in HO. Bax was increasingly expressed by many clusters including
AT1 and AT2 clusters. Cdkn1a was upregulated in all the cell clusters studied. Tgfbr2 was
accentuated in the Col13a1+ fibroblast cluster. TGF and TP53 signaling is known to inhibit
AT2- AT1 transition as well. In addition to the dysregulation of Reactive oxygen Species (ROS),
inflammatory and apoptosis pathways, significant deregulation of the genes involved in
glycolytic, oxidative phosphorylation and fatty acid metabolism was also established in different
cell clusters including AT2 cells. Gene signatures for epithelial-mesenchymal transition were
also seen dysregulated on PN7. The upregulated genes related to fibrosis and inflammation
were not differently regulated on PN28. However, certain genes involved in inflammation,
angiogenesis and epithelial-mesenchymal transition were found to be dysregulated.

Conclusion(s): Our study shows distinct cellular and transcriptomic changes in post-hyperoxic
neonatal lung injury and reparative phases.