320 - Hyperoxia increases alveolar macrophage senescence both in culture and in lung tissue: mechanisms and impact on lung injury.

Publication Number: 320.5387

Katy Hegarty, The Warren Alpert Medical School of Brown University, Providence, RI, United States; Hongwei Yao, Providence VA Medical Center, Providence, RI, United States; Bethany G. McGonnigal, --None--Brown University, North Dartmouth, MA, United States; Ivy Lin, Brown University, Providence, RI, United States; Alper Uzun, The Warren Alpert Medical School of Brown University, Providence, RI, United States; Joselynn Wallace, The Warren Alpert Medical School of Brown University, Cranston, RI, United States; Phyllis A.. Dennery, The Warren Alpert Medical School of Brown university, Providence, RI, United States

Background: We previously reported a predominant increase in lung macrophage senescence on postnatal day (pnd) 7 in mice exposed to hyperoxia (95% O2) for 3 days from birth, and that hyperoxia leads to increased lung glycolysis.

Objective: To understand what regulates hyperoxia-induced macrophage senescence and to understand the effect of macrophage senescence on neonatal lung injury.

Design/Methods: Cultured mouse alveolar macrophages (MH-S) were exposed to normoxia (21% O2/5% CO2) or hyperoxia (95% O2/5% CO2) for 4 or 24 h. After 4 h, cells were treated with a selective 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 inhibitor (3PO) or vehicle. Glycolysis stress tests were performed and p21 gene expression and loss of nuclear lamin-b, markers of senescence, were evaluated as was γH2AX protein content, a marker of DNA damage. To understand what proteins senescent macrophages release through their senescence associated secretory phenotype (SASP), hyperoxia exposed MH-S cells were tagged with DDAOG, a marker of senescence, sorted using FACS and plated for 24 hours. The conditioned media from the DDAOG+ macrophages as well as that from DDAOG- non-senescent hyperoxia exposed and air exposed controls were analyzed by proteinomics. In addition, the recovered SASP was intranasally injected into naïve newborn mice and lungs were collected at 10 days for histology. Mean linear intercept (MLI) and radial alveolar count (RAC) were assessed in lungs slices as was vessel density using von Willebrandt factor staining..

Results: Further evaluation of the senescent macrophage clusters detected by ScRNASeq (Yao et al, Nat Comm 2023) demonstrated that senescent macrophages are predominantly Ear2 high and interstitial macrophages ( Figure 1), both serve in homeostasis and repair. In hyperoxia, γH2AX, p21 levels and lamin b1 exclusion were significantly increased in MH-S. Glycolysis was also significantly increased after 4 h, and 24 h. Incubation with 3PO significantly reduced glycolysis and markers of senescence (Figure 2). In vivo, mice treated with the SASP from DDAOG+ senescent macrophages had higher MLI and reduced vessel density compared to that of air controls and, mice administered MH-S conditioned media from non-senescent ( Figure 3) macrophages.

Conclusion(s): These data suggest that increased glycolysis leads to macrophage senescence in vitro and that senescent macrophages secrete SASP factors that play a role in lung injury in vivo. Further evaluation of these factors may reveal druggable targets to prevent or ameliorate lung injury.

Heterogeneity of alveolar macrophages with hyperoxic exposure
Upper panel: diversity of alveolar macrophages from neonatal lungs; lower panel: abundance of senescent Ear2 high and interstitial macrophages recovered from the lungs of neonatal mice exposed to hyperoxia. Three groups are compared: senescent, oxygen exposed non-senescent air air exposed.

Regulation of senescence by glycolysis
MHS cells were exposed to air or hypoxia for 4 or 24 hours. Glycolysis was measured using the Seahorse analyzer ( left panel). Effect of inhibition of glycolysis with 3PO on p21 (marker of senescence) gene expression using qPCR . *** p< 0.001 vs air exposed; ** p<0.01 vs air exposed. ††: p< 0.05 vs vehicle control

Effect of macrophage SASP on lung morphology and vessel abundance
Panel A representative image of lung histology with H and E staining after intranasal injection of conditioned media from senescent macrophages. Mean linear intercept and radial alveolar counts are shown in B. * p< 0.05 vs air. Panel C: representative image of endothelial cell density after intranasal injection of conditioned media using immunohistochemical detection of vonWillebrand factor ( green). DAPI is shown in blue. Arrowheads show blood vessels.. Quantitation is shown in panel D. ** p<0.01 vs air