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bioRxiv · 10.1101/2025.08.07.669054

Sex-Specific Metabolic Programming in Human Neutrophil Subsets

Yennemadi, A. S.·Murphy, F. K.·Keane, J.·Leisching, G.
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Abstract

BackgroundSex differences in immune responses are well-documented, with females exhibiting more robust immunity against infections but higher susceptibility to autoimmune diseases, while males often demonstrate more severe inflammatory pathology. Neutrophils which are key players in the innate immune response, display sex-based functional differences, but whether these extend to metabolic programming, particularly in low-density neutrophils (LDNs), remains unknown. MethodsWe isolated LDNs and normal-density neutrophils (NDNs) from healthy human donors using density gradient centrifugation and negative selection. Cellular metabolism was assessed via Seahorse XF analysis (measuring oxygen consumption rate, OCR, and extracellular acidification rate, ECAR), alongside flow cytometry for maturity markers (CD16hi/lo). ResultsMale LDNs exhibited significantly higher basal OCR and ATP production than female LDNs, while no sex differences were observed in NDNs. Strikingly, male LDNs had higher OCR and glycolysis than their matched NDNs, whereas female NDNs were more oxidative than their LDNs. These metabolic differences were independent of neutrophil maturity, as CD16hi frequencies did not differ between subsets or sexes. ConclusionsOur study reveals, for the first time, subset-specific sexual dimorphism in neutrophil metabolism whereby male LDNs adopt a hypermetabolic (oxidative/glycolytic) phenotype, while female NDNs retain higher oxidative capacity. This reprogramming occurs independently of developmental stage, suggesting sex hormones or epigenetic regulation may drive these differences. These findings provide a metabolic basis for sex-biased immune responses and highlight the need for sex-stratified approaches in neutrophil-targeted therapies. Plain English SummaryMen and women fight infections differently, and our study could reveal why--their immune cells produce energy in distinct ways. We examined two types of neutrophils (infection-fighting cells), normal cells (NDNs) and low-density cells (LDNs) found in inflammation. Using advanced metabolic measurements, we discovered that mens LDNs use more oxygen and generate more energy than womens. Surprisingly, while mens LDNs were more active than their normal neutrophils, women showed the opposite--their normal neutrophils were more energetic than their LDNs. These differences werent due to cell maturity, suggesting biological distinctions between sexes. This may help explain why men often have worse outcomes in diseases like sepsis (where oxygen-driven inflammation dominates), while women are more prone to autoimmune diseases like lupus. Our findings could lead to better sex-specific treatments by dampening overactive immune responses in men or adjusting metabolism in women to prevent autoimmune flares. This research highlights why medical studies must consider sex differences, as one-size-fits-all treatments may miss key biological variations. HighlightsO_LICD16hi frequencies show no sex differences, however female (but not male) NDNs contain significantly more mature CD15+ CD16hi cells than their LDNs, revealing female-specific maturation differences between subsets. C_LIO_LIFirst report of sex-specific metabolic differences in LDNs where males exhibit significantly higher basal respiration and ATP production than female LDNs. C_LIO_LIMale LDNs show higher OCR than matched NDNs, while female NDNs are more oxidative than their LDNs. C_LI

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Yennemadi, A. S., Murphy, F. K., Keane, J., Leisching, G.. 2025-08-10. Sex-Specific Metabolic Programming in Human Neutrophil Subsets. https://doi.org/10.1101/2025.08.07.669054

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