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Protocol for Evaluating In Vivo the Activation of the P2RX7 Immunomodulator
Serena Janho Dit Hreich1,2, Thierry Juhel3, Paul Hofman3,4,5,6
1Université Côte d'Azur, CNRS, INSERM, IRCAN, 28 avenue de Valombrose, 06108, Nice, France. serena.janho-dit-hreich@univ-cotedazur.fr.
Background:
P2RX7 is a purinergic receptor with pleiotropic activities that is activated by high levels of extracellular ATP that are found in inflamed tissues. P2RX7 has immunomodulatory and anti-tumor proprieties and is therefore a therapeutic target for various diseases. Several compounds are developed to either inhibit or enhance its activation. However, studying their effect on P2RX7's activities is limited to in vitro and ex vivo studies that require the use of unphysiological media that could affect its activation. Up to now, the only way to assess the activity of P2RX7 modulators on the receptor in vivo was in an indirect manner.
Results:
We successfully developed a protocol allowing the detection of P2RX7 activation in vivo in lungs of mice, by taking advantage of its unique macropore formation ability. The protocol is based on intranasal delivery of TO-PRO™-3, a non-permeant DNA intercalating dye, and fluorescence measurement by flow cytometry. We show that ATP enhances TO-PRO™-3 fluorescence mainly in lung immune cells of mice in a P2RX7-dependant manner.
Conclusions:
The described approach has allowed the successful analysis of P2RX7 activity directly in the lungs of WT and transgenic C57BL6 mice. The provided detailed guidelines and recommendations will support the use of this protocol to study the potency of pharmacologic or biologic compounds targeting P2RX7.
Insights
Researchers developed a new in vivo method to detect P2RX7 receptor activation in mouse lungs. This technique uses a fluorescent dye and flow cytometry to assess P2RX7 modulators, advancing drug development for inflammatory and tumor diseases.
Area of Science:
- Immunology
- Pharmacology
- Cell Biology
Background:
- P2RX7 receptor, activated by extracellular ATP in inflamed tissues, has immunomodulatory and anti-tumor properties.
- P2RX7 is a therapeutic target, but in vivo studies of its modulators are limited by unphysiological conditions.
- Current in vivo assessment of P2RX7 modulators is indirect, hindering drug development.
Purpose of the Study:
- To develop a direct in vivo method for detecting P2RX7 receptor activation in mouse lungs.
- To overcome limitations of in vitro/ex vivo studies for evaluating P2RX7 modulators.
Main Methods:
- Intranasal delivery of TO-PRO™-3, a DNA intercalating dye.
- Fluorescence measurement by flow cytometry to detect P2RX7-mediated macropore formation.
- Analysis in the lungs of wild-type and transgenic C57BL6 mice.
Main Results:
- Successfully established a protocol for in vivo P2RX7 activation detection in mouse lungs.
- Demonstrated that ATP enhances TO-PRO™-3 fluorescence in lung immune cells.
- Confirmed P2RX7-dependent fluorescence enhancement in vivo.
Conclusions:
- The developed protocol enables direct analysis of P2RX7 activity in mouse lungs.
- Provides guidelines for using this method to study P2RX7-targeting compounds.
- Facilitates in vivo evaluation of P2RX7 modulators for therapeutic applications.
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