A106-02 Sex as a Biological Variable and Immunometabolism: Focus on Alveolar Macrophages

Abstract Rationale Our previous findings show that AMs exhibit sexual dimorphism in the developing murine and human lung, possibly contributing to sex-biased outcomes in pulmonary disorders like bronchopulmonary dysplasia (BPD), a chronic lung disease affecting preterm infants. Alveolar macrophages (AMs) are important for maintaining lung homeostasis and coordinating immune responses during respiratory distress. Their function is tightly linked to cellular metabolism, particularly the balance between oxidative phosphorylation and glycolysis. These insights underscore the need to investigate how biological sex influences AM metabolic states and mitochondrial function to uncover sex-specific mechanisms driving disease vulnerability. Methods AMJ2-C11 and MH-S (female and male immortalized mouse AM cell lines, respectively) were cultured at room air and hyperoxia (85% FiO2, 48 hrs). Cellular bioenergetics at baseline and after hyperoxia were assessed using a Seahorse XFe analyzer with the Mito-Stress and Glyco-Stress assays, measuring oxygen consumption rate (OCR) and Extracellular acidification rate (ECAR) as proxies for mitochondrial and glycolytic function, respectively (Figure 1A, B). Next, male and female AMs were treated with LPS and IFN-gamma (100ng/ml;48 hours; M1polarization) and were sequenced to measure changes in the transcriptome. Findings were also validated in neonatal AMs isolated from PND 7 mice. (n = 3-4 replicates/group). Results AMJ2 (female AMs) cells showed higher oxygen consumption than MH-S (male AMs) cells, with significant declines in maximal OCR and Spare Respiratory Capacity (SRC) after hyperoxia exposure (Fig. 1C, E-G; top: baseline, bottom: room air vs hyperoxia). AMJ2 cells exhibited higher glycolytic function at baseline with no significant decline after hyperoxia. MH-S cells showed no change in glycolytic function (Fig. 1D, H-J; top: baseline, bottom: room air vs hyperoxia). The sex-specific transcriptomic changes (Fig 2) in gene expression and biological pathways upon exposure to pro-inflammatory stimuli included pathways related to T-cell activation and angiogenesis in females (positively enriched) and pathways related to metabolism in male AMs (negatively enriched). Similar metabolic findings were observed in primary neonatal AMs. Conclusion Male and female AMs exhibit distinct shifts in their immunometabolic phenotypes during adaptation to oxidative stress and pro-inflammatory stimuli. Identifying the mechanisms underlying these differences may elucidate the reasons behind sex-specific differences in disease pathophysiology. This abstract is funded by: NIH