344 - From Protector to Perpetrator: Is there a role for hyperoxia-Induced Macrophage Senescence in Neonatal Lung Disease?

Publication Number: 1328.344

Olivia Spielman, Brown University, Providence, RI, United States; Bethany McGonnigal, Brown University, Providence, RI, United States; Sorel Yimga Ouonkap, Brown University, Providence, RI, United States; Phyllis À.. Dennery, Brown University, Providence, RI, United States

Background: Exposure to hyperoxia and ventilation in neonates leads to alveolar and vascular simplification, an impetus for bronchopulmonary dysplasia (BPD). Our publicly available scRNAseq data from mouse lung samples revealed a significant increase in senescence at pnd(postnatal day)7 after 3 days of hyperoxic exposure and recovery in room air, particularly among macrophages. We hypothesized that in addition to disseminating the Senescence Associated Secretory Phenotype (SASP) to propagate damage, senescence prevents macrophages from completing essential defensive functions such as phagocytosis and migration to the site of injury thus jeopardizing the newborn lung.

Objective: To evaluate the impact of hyperoxia induced senescence on macrophage functions including secretion of SASP, phagocytosis, and migration, in cultured alveolar macrophages (MH-S).

Design/Methods: MH-S were grouped into hyperoxia (95% O2/5% CO2 x 24 h) and air-exposed control (5% CO2/air x 24 h). Senescence was evaluated in cell lysates by measuring p21 gene expression with PCR, lamin B1 exclusion with immunohistochemistry, and DNA damage with Western analysis of γH2AX immunoreactive protein levels. The conditioned media from air and hyperoxia-exposed senescent MH-S was assessed by proteomics. Phagocytic potential was evaluated through Western analysis for the Transgelin-2 protein. Functional Phagocytosis Assays using FITC beads and FACS were performed and further analyzed through staining with senescence marker DDAOG to identify overlay between FITC and DDAOG positive MHS. Scratch Wound Healing Assays to assess migratory potential were visualized through microscopy and analyzed with Studio R.

Results: Hyperoxia altered healthy MHS to the senescent phenotype, characterized by increased p21 gene expression, γH2AX levels, and Lamin B1 exclusion. Comparing hyperoxia-exposed MH-S to controls, we identified decreased Transgelin-2 protein levels under hyperoxic exposure, and through Phagocytosis Assays, a significant reduction in phagocytic ability (75% vs 100% n=6). Further reduction in phagocytic activity was observed in the sorted senescent cells. Scratch assays revealed a lower migratory ability of hyperoxia-exposed MH-S vs. controls.

Conclusion(s): In hyperoxia, macrophages are significantly less able to perform their essential functions while increasing their ability to propagate injury via the SASP. We speculate that hyperoxia-induced senescent macrophages may impede proper lung repair in neonates, thus contributing to BPD. Therapeutic approaches targeting senescent lung macrophages may be important in mitigating neonatal hyperoxic lung injury.