Single cell autofluorescence imaging reveals immediate metabolic shifts of neutrophils with activation across biological systems
IntroductionNeutrophils are critical innate immune cells that heterogeneously respond to infection and inflammation by performing functions such as oxidative burst and NETosis, which require significant metabolic adaptation. Deeper insights into the single cell diversity of such metabolic changes wi...
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Frontiers Media S.A.
2025-08-01
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| author | Rupsa Datta Veronika Miskolci Gina M. Gallego-López Emily Britt Amani Gillette Aleksandr Kralovec Morgan A. Giese Morgan A. Giese Tongcheng Qian James Votava Wenxuan Zhao Jing Fan Jing Fan Anna Huttenlocher Anna Huttenlocher Melissa C. Skala Melissa C. Skala |
| author_facet | Rupsa Datta Veronika Miskolci Gina M. Gallego-López Emily Britt Amani Gillette Aleksandr Kralovec Morgan A. Giese Morgan A. Giese Tongcheng Qian James Votava Wenxuan Zhao Jing Fan Jing Fan Anna Huttenlocher Anna Huttenlocher Melissa C. Skala Melissa C. Skala |
| author_sort | Rupsa Datta |
| collection | DOAJ |
| description | IntroductionNeutrophils are critical innate immune cells that heterogeneously respond to infection and inflammation by performing functions such as oxidative burst and NETosis, which require significant metabolic adaptation. Deeper insights into the single cell diversity of such metabolic changes will help identify regulation of neutrophil functions in health and diseases. Due to their short lifespan and associated technical challenges, the early metabolic processes of neutrophil activation are not completely understood. New tools are needed to measure rapid changes in neutrophil metabolism on a single cell level.MethodsTo address this, we use optical metabolic imaging (OMI), which entails optical redox ratio and fluorescence lifetime imaging microscopy of intrinsic metabolic coenzymes NAD(P)H and FAD to assess the metabolic state of single neutrophils. Primary human neutrophils were imaged in vitro under a variety of activation conditions and metabolic pathway inhibitors, while metabolic and functional changes were confirmed with mass spectrometry, oxidative burst, and NETosis measurements.ResultsOur findings show rapid metabolic remodeling to a reduced redox state during activation. Additionally, heterogeneous metabolic response to pathogens (Pseudomonas aeruginosa and Toxoplasma gondii) was observed across neutrophils and human donors. Finally, consistent OMI changes with activation were confirmed between in vitro human and in vivo zebrafish larvae neutrophils. This study demonstrates the potential of OMI as a versatile tool for studying neutrophil metabolism and underscores its use across different biological systems, offering insights into neutrophil metabolic activity and function at a single cell level.ConclusionThis work addresses the critical need for advanced single-cell tools to monitor rapid and diverse metabolic changes in neutrophils, an underexplored area with significant implications for understanding immune responses and developing therapies for inflammatory diseases. Neutrophils, the body's first responders to infection and inflammation, undergo rapid metabolic changes upon activation. Using label-free optical metabolic imaging of intrinsic metabolic coenzymes NAD(P)H and FAD, we reveal distinct metabolic signatures in activated primary human neutrophils as well as neutrophils in live zebrafish larvae. Our findings highlight how pathogens and pharmacological stimuli heterogeneously rewire neutrophil metabolism within minutes, influencing immune responses. This noninvasive method offers insights into single-cell neutrophil metabolism immediately following activation, with implications for infection, inflammation, and immune disorders. |
| format | Article |
| id | doaj-art-6d5bdeaf417b4276a1d5000c8d80fdf7 |
| institution | DOAJ |
| issn | 1664-3224 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Immunology |
| spelling | doaj-art-6d5bdeaf417b4276a1d5000c8d80fdf72025-08-20T02:57:28ZengFrontiers Media S.A.Frontiers in Immunology1664-32242025-08-011610.3389/fimmu.2025.16179931617993Single cell autofluorescence imaging reveals immediate metabolic shifts of neutrophils with activation across biological systemsRupsa Datta0Veronika Miskolci1Gina M. Gallego-López2Emily Britt3Amani Gillette4Aleksandr Kralovec5Morgan A. Giese6Morgan A. Giese7Tongcheng Qian8James Votava9Wenxuan Zhao10Jing Fan11Jing Fan12Anna Huttenlocher13Anna Huttenlocher14Melissa C. Skala15Melissa C. Skala16Morgridge Institute for Research, Madison, WI, United StatesDepartment of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI, United StatesMorgridge Institute for Research, Madison, WI, United StatesMorgridge Institute for Research, Madison, WI, United StatesMorgridge Institute for Research, Madison, WI, United StatesMorgridge Institute for Research, Madison, WI, United StatesDepartment of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI, United StatesCellular and Molecular Biology Graduate Program, University of Wisconsin, Madison, WI, United StatesMorgridge Institute for Research, Madison, WI, United StatesMorgridge Institute for Research, Madison, WI, United StatesMorgridge Institute for Research, Madison, WI, United StatesMorgridge Institute for Research, Madison, WI, United StatesDepartment of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI, United StatesDepartment of Medical Microbiology and Immunology, University of Wisconsin, Madison, WI, United StatesDepartment of Pediatrics, University of Wisconsin, Madison, WI, United StatesMorgridge Institute for Research, Madison, WI, United StatesDepartment of Biomedical Engineering, University of Wisconsin, Madison, WI, United StatesIntroductionNeutrophils are critical innate immune cells that heterogeneously respond to infection and inflammation by performing functions such as oxidative burst and NETosis, which require significant metabolic adaptation. Deeper insights into the single cell diversity of such metabolic changes will help identify regulation of neutrophil functions in health and diseases. Due to their short lifespan and associated technical challenges, the early metabolic processes of neutrophil activation are not completely understood. New tools are needed to measure rapid changes in neutrophil metabolism on a single cell level.MethodsTo address this, we use optical metabolic imaging (OMI), which entails optical redox ratio and fluorescence lifetime imaging microscopy of intrinsic metabolic coenzymes NAD(P)H and FAD to assess the metabolic state of single neutrophils. Primary human neutrophils were imaged in vitro under a variety of activation conditions and metabolic pathway inhibitors, while metabolic and functional changes were confirmed with mass spectrometry, oxidative burst, and NETosis measurements.ResultsOur findings show rapid metabolic remodeling to a reduced redox state during activation. Additionally, heterogeneous metabolic response to pathogens (Pseudomonas aeruginosa and Toxoplasma gondii) was observed across neutrophils and human donors. Finally, consistent OMI changes with activation were confirmed between in vitro human and in vivo zebrafish larvae neutrophils. This study demonstrates the potential of OMI as a versatile tool for studying neutrophil metabolism and underscores its use across different biological systems, offering insights into neutrophil metabolic activity and function at a single cell level.ConclusionThis work addresses the critical need for advanced single-cell tools to monitor rapid and diverse metabolic changes in neutrophils, an underexplored area with significant implications for understanding immune responses and developing therapies for inflammatory diseases. Neutrophils, the body's first responders to infection and inflammation, undergo rapid metabolic changes upon activation. Using label-free optical metabolic imaging of intrinsic metabolic coenzymes NAD(P)H and FAD, we reveal distinct metabolic signatures in activated primary human neutrophils as well as neutrophils in live zebrafish larvae. Our findings highlight how pathogens and pharmacological stimuli heterogeneously rewire neutrophil metabolism within minutes, influencing immune responses. This noninvasive method offers insights into single-cell neutrophil metabolism immediately following activation, with implications for infection, inflammation, and immune disorders.https://www.frontiersin.org/articles/10.3389/fimmu.2025.1617993/fullneutrophillabel-freesingle-celloptical metabolic imagingmetabolismfluorescence lifetime imaging microscopy (FLIM) |
| spellingShingle | Rupsa Datta Veronika Miskolci Gina M. Gallego-López Emily Britt Amani Gillette Aleksandr Kralovec Morgan A. Giese Morgan A. Giese Tongcheng Qian James Votava Wenxuan Zhao Jing Fan Jing Fan Anna Huttenlocher Anna Huttenlocher Melissa C. Skala Melissa C. Skala Single cell autofluorescence imaging reveals immediate metabolic shifts of neutrophils with activation across biological systems Frontiers in Immunology neutrophil label-free single-cell optical metabolic imaging metabolism fluorescence lifetime imaging microscopy (FLIM) |
| title | Single cell autofluorescence imaging reveals immediate metabolic shifts of neutrophils with activation across biological systems |
| title_full | Single cell autofluorescence imaging reveals immediate metabolic shifts of neutrophils with activation across biological systems |
| title_fullStr | Single cell autofluorescence imaging reveals immediate metabolic shifts of neutrophils with activation across biological systems |
| title_full_unstemmed | Single cell autofluorescence imaging reveals immediate metabolic shifts of neutrophils with activation across biological systems |
| title_short | Single cell autofluorescence imaging reveals immediate metabolic shifts of neutrophils with activation across biological systems |
| title_sort | single cell autofluorescence imaging reveals immediate metabolic shifts of neutrophils with activation across biological systems |
| topic | neutrophil label-free single-cell optical metabolic imaging metabolism fluorescence lifetime imaging microscopy (FLIM) |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2025.1617993/full |
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