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Published on: February 18, 2020
Intravital calcium imaging in myeloid leukocytes identifies calcium frequency spectra as indicators of functional
Fitsumbirhan T Mehari1,2, Meike Miller1,2, Robert Pick3
1Medizinische Klinik und Poliklinik I, University Hospital, LMU Munich, 81377 Munich, Germany.
Insights
Analyzing calcium (Ca2+) signaling in myeloid cells reveals new ways to assess immune cell activation during inflammation. Frequency spectra of Ca2+ signals offer insights into leukocyte behavior in vivo.
Area of Science:
- Immunology
- Cell Biology
- Biophysics
Background:
- Assessing leukocyte activation in vivo typically relies on indirect measures like cell shape and migration.
- A deeper understanding of the spatiotemporal dynamics of leukocyte activation during inflammation is needed.
- Novel parameters are required to dissect these complex cellular processes.
Purpose of the Study:
- To establish intracellular calcium (Ca2+) signal analysis as a tool for assessing myeloid leukocyte activation in vivo.
- To investigate the spatiotemporal patterns of Ca2+ signaling in macrophages and neutrophils during inflammation.
- To explore the role of Ca2+ channels and G protein-coupled receptors in modulating these Ca2+ signals.
Main Methods:
- Intravital microscopy using Ca2+ reporter mouse strains to visualize leukocyte Ca2+ signals in vivo.
- Two-photon microscopy to observe Ca2+ transients in tissue-resident macrophages during sterile inflammation.
- High-resolution intravital spinning disk confocal microscopy to analyze neutrophil Ca2+ signaling patterns during migration.
- Bioinformatic analysis of Ca2+ signal frequency spectra to characterize activation patterns.
Main Results:
- Tissue-resident macrophages exhibited distinct spatiotemporal Ca2+ transients in response to sterile inflammation.
- Intracellular Ca2+ signaling patterns of neutrophils during migration were identified in vivo.
- These Ca2+ signaling patterns were modulated by the Ca2+ channel Orai1 and Gαi-coupled GPCRs.
- Analysis of Ca2+ signal frequency spectra provided insights into leukocyte Ca2+ oscillations.
Conclusions:
- Intravital Ca2+ imaging combined with bioinformatic analysis offers a novel method to assess leukocyte activation in vivo.
- Ca2+ frequency spectra represent an additional dimension for evaluating leukocyte activation and migration during inflammatory responses.
- This approach enhances the understanding of immune cell dynamics in the context of inflammation.
Abstract:
The assessment of leukocyte activation in vivo is mainly based on surrogate parameters, such as cell shape changes and migration patterns. Consequently, additional parameters are required to dissect the complex spatiotemporal activation of leukocytes during inflammation. Here, we showed that intravital microscopy of myeloid leukocyte Ca2+ signals with Ca2+ reporter mouse strains combined with bioinformatic signal analysis provided a tool to assess their activation in vivo. We demonstrated by two-photon microscopy that tissue-resident macrophages reacted to sterile inflammation in the cremaster muscle with Ca2+ transients in a distinct spatiotemporal pattern. Moreover, through high-resolution, intravital spinning disk confocal microscopy, we identified the intracellular Ca2+ signaling patterns of neutrophils during the migration cascade in vivo. These patterns were modulated by the Ca2+ channel Orai1 and Gαi-coupled GPCRs, whose effects were evident through analysis of the range of frequencies of the Ca2+ signal (frequency spectra), which provided insights into the complex patterns of leukocyte Ca2+ oscillations. Together, these findings establish Ca2+ frequency spectra as an additional dimension to assess leukocyte activation and migration during inflammation in vivo.
