LAUSR

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. Description Decoding intracellular calcium signals in immune cells in live mice reveals their functional status during inflammation. Decoding Ca2+ signals The immune responses to infection and sterile inflammation are complex, involving the coordinated recruitment and activation of multiple cell types. To better understand the activation states of immune cells in the context of inflammation, Mehari et al. used various in vivo imaging techniques with transgenic Ca2+ reporter mice to measure the intensity and frequency of intracellular Ca2+ signals in macrophages and neutrophils as they responded to a laser-induced tissue injury. Analysis of these data revealed the kinetics of recruitment of tissue-resident macrophages and identified distinct patterns of intracellular Ca2+ signals in neutrophils that characterized the different stages of their recruitment from the blood to sites of injury. These findings suggest that in vivo monitoring of Ca2+ signals provides insights into the functional states of immune cells.