High content screening of CXCR2-dependent signalling pathways

Area of Science:

• Cellular and Molecular Pharmacology
• Immunology
• Biotechnology

Background:

• CXC-type chemokine receptor 2 (CXCR2) plays a crucial role in inflammatory responses.
• Understanding CXCR2 signaling pathways is vital for developing targeted therapeutics.
• Existing methods for analyzing CXCR2 signaling can be limited in throughput and scope.

Purpose of the Study:

• To develop and validate a high content screening (HCS) assay for quantifying CXCR2 signaling events.
• To investigate the coupling between CXCR2 internalization, ERK phosphorylation, and NFAT translocation.
• To enable pharmacological profiling of test compounds using multiplexed imaging.

Main Methods:

• Utilized CXCR2-transfected cells and stimulated with Gro-alpha or IL-8.
• Employed HCS with specific antibodies for CXCR2 and phosphorylated ERK1/2 (pERK).
• Quantified nuclear factor of activated T cells (NFAT) translocation using a red fluorescent protein (RFP) fusion construct.

Main Results:

• Stimulation induced CXCR2 internalization, pERK, and NFAT translocation.
• Receptor internalization was tightly coupled with ERK signaling.
• Multiplexed imaging demonstrated a strong correlation between CXCR2 internalization and NFAT translocation.
• Assay successfully profiled antagonistic test compounds regarding G-protein signaling and desensitization.

Conclusions:

• HCS and multiplexed imaging provide a robust platform for analyzing CXCR2 signaling.
• The developed assay enables comprehensive pharmacological characterization of compounds targeting CXCR2.
• This approach facilitates the discovery of novel therapeutics for inflammatory diseases.