578 - Decreased Glycolysis Reduces Senescence in Cultured Lung Macrophages Exposed to Hyperoxia and Increased Glycolysis is Associated with Hyperoxia-Induced Lung Senescence In Vivo.

Poster Number: 578
Publication Number: 578.1254

Background: Bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants, is characterized by alveolar and vascular simplification. BPD is often associated with hyperoxia exposure and mechanical ventilation. We previously reported a transient increase in lung macrophage senescence on postnatal day (pnd) 7 in mice exposed to hyperoxia (95% O2) for 3 days as neonates, a murine model of BPD. Our previous findings demonstrated that hyperoxia leads to dysregulated metabolism, such as increased glycolysis.

Objective: Whether this dysregulated metabolism causes neonatal hyperoxia-induced senescence in macrophages is unknown. We hypothesized that reducing glycolysis inhibits hyperoxia-induced senescence in macrophages.

Design/Methods: Mouse alveolar macrophage (MH-S) cells were exposed to normoxia (21% O2 /5% CO2 ) or hyperoxia (95% O2 /5% CO2 ) at 37 ⁰ C for 4 h or 24 h. After 4 h of exposure, MH-S cells were treated with a selective 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 inhibitor (3PO, 10 µM dissolved in 0.02% DMSO) or vehicle (0.02% DMSO) for 20 h. A glycolysis stress test was performed using a XF24 Seahorse Analyzer. qRT-PCR was performed to measure p21 gene expression, a marker of senescence. Western blot was used to evaluate loss of lamin b and γH2AX, markers of senescence and DNA damage, respectively. C57BL/6J mice ( < 12 h old) were exposed to normoxia (21% O 2 ) or hyperoxia (95% O 2 ) for 3 days. Lung tissues were harvested on pnd3 and pnd7 to measure lactate levels.

Results: γH2AX levels were significantly increased in MH-S cells exposed to hyperoxia for 24 h compared to the normoxia group. There were no changes in γH2AX levels between normoxia and hyperoxia groups after 4 h of exposure. Hyperoxic exposure increased p21 gene expression at 24 h but not at 4 h in MH-S cells. Lamin b1 levels were not changed regardless of exposure durations. Glycolysis was significantly increased in MH-S cells exposed to hyperoxia for 4 h, and further augmented at 24 h compared to normoxia controls. 3PO incubation significantly reduced basal glycolysis and p21 gene expression in MH-S cells exposed to hyperoxia. In mice exposed to hyperoxia, whole lung lactate levels were significantly higher on both pnd3 and pnd7 as compared to the normoxic group, whereas macrophage senescence was only increased on pnd7.

Conclusion(s): Suppressing glycolysis reduces senescence in hyperoxia-exposed cultured macrophages. Furthermore, macrophage senescence and increased lung glycolysis occur in a mouse model of BPD. These data implicate increased glycolysis as an underlying mechanism of macrophage senescence in BPD.