469 - scRNAseq Reveals Sex-Dependent Responses to Hyperoxia in a Neonatal Mouse Model of Experimental BPD

Poster Number: 469
Publication Number: 469.432

Sheng Xia, Children's Mercy Hospitals and Clinics, Kansas City, MO, United States; Heather Menden, Childrens Mercy, Kansas City, MO, United States; Konner Winkley, Children's Mercy Hospitals and Clinics, Shawnee, KS, United States; Sherry Mabry, Children's Mercy, Kansas City, MO, United States; Venkatesh Sampath, Childrens Mercy Kansas City, Kansas City, KS, United States

Background: Lung injury induced by hyperoxia (HOX) results in alveolar simplification, a hallmark of bronchopulmonary dysplasia (BPD) in preterm infants. Strikingly, female preterm neonates are much less susceptible to BPD. The mechanisms by which hyperoxia induces less lung injury and BPD in females than in males have not been examined at single cell resolution. Literature suggests that lung capillary regeneration and decreased pro-inflammatory alveolar macrophage activation are key to lung recovery. Aero capillary EC (aCap) are specialized for air exchange and general capillary EC (gCAP) functions as a progenitor cell for EC recovery. Excessive activation of alveolar macrophages (AM) by HOX contributes to interruption of lung development. We posited that a lower AM response and a robust EC reparative capillary response in females during HOX lead to less BPD in female.

Objective: a) Compare male and female alterations in major lung cell populations after postnatal HOX exposure, and b) investigate male and female differences in transcriptional networks re-programmed by HOX in an experimental BPD model.

Design/Methods: C57BL6 mice were exposed to 85% oxygen (HOX) or ambient oxygen (RA) between P1-P14. Mouse lungs were digested to get single cell populations which underwent scRNA sequencing using the 10X genomics platforms. Quantitative reverse transcription PCR (qRT-PCR) and western blot were used to confirm scRNAseq data. Ingenuity Pathway Analysis (IPA) was applied to analyze and compare diseases and functions between female and male.

Results: Sequencing was done on 5000 cells/mouse in RA (4 males and 4 females) and HOX (4 males and 4 females). We identified 23 unsupervised lung cell clusters, which on supervised clustering yielded 4 major cell types (Fig. 1). Four clusters of endothelial cells (EC) were identified: Car4+ aCap and Plvap+ /VWF- gCap [capillary EC], VWF+ large vessel EC and Flt4+ lymphatic EC. scRNAseq indicated more Car4 expression in gCap capillary EC, with qRT-PCR and western blotting confirming more Car4 expression in female lung than in male lung after HOX treatment (Fig. 2). IPA analysis demonstrated that less immune response, movement, accumulation, and activation of AM in females than in males after HOX (Fig.3).Conclusion(s): Single cell survey in an HOX-model of BPD revealed less AM activation and more Car4 expression in gCap in females than males. Our results suggest that less AM response and more vigorous EC reparative response (more Car4+), and gCap differentiation to aCap in female lungs during HOX may lead to less BPD in females.
Figure 1 . Classification of cell types at P14 by scRNA-Seq in mouse lungs.22 lung cell clusters, grouping into 4 major cell types: Epithelial cells (Epi), Endothelial cells (EC), Stromal cells and immune cells. Room air (RA) vs Hyperoxia (HOX). Cell clusters are defined in Table.
Figure 2 More Car4 expression in lung EC in females vs. males after HOX treatment.A) scRNAseq data indicated that Car4 expression in gCap was 2-fold higher in females than in males in HOX. B) RNA was extracted from four groups of mouse whole lung after room air (RA) and hyperoxia (HOX) treatment. qRT-PCR was run to quantified Car4 mRNA expression. Room air male (RM), Room air female (RF), HOX male (HM) and HOX female (HF). # HF vs RF, n=4/group, p < 0.001. * HF vs HM, n=4/group, p < 0.01. C) cell lysate was extracted from mouse lung after RA and HOX treatment , and CAR4 protein expression was quantified by western blot. It indicated that CAR4 expression is higher in female than in male in both RA and HOX.YWHAZ as control. n=4/group.