Sophia, A Ali
Dr. Hydar Ali
| Sophia, A Ali | Dr. Hydar Ali |
Sophia, A Ali, Dr. Hydar Ali
Basic & Translational Sciences, University of Pennsylvania, School of Dental Medicine
Introduction
Mast cells (MCs) are innate immune cells that upon stimulation release important mediators of inflammation. Activation of MCs via G Protein-Coupled Receptor X2 (MRGPRX2) is associated with host defenses and inflammatory conditions. Adaptor proteins (β-arrestins) are involved in the desensitization and internalization of G-coupled protein receptors. β-Arrestin isoforms 1 and 2 may also be involved in the regulation of MRGPRX2-mediated MC activation, but their distinct functions are unknown. Characterizing roles of β-Arrestins can further the understanding of MC regulation. The mouse ortholog of MRGPRX2 (MrgprB2) binds the same ligands but is regulated differently. The ligands used are neuropeptide Substance P (SP), pro-adrenomedullin peptide (PAMP), and rocuronium. SP is known to correlate with inflammation and pain, PAMP has a role in histamine-independent itch, and rocuronium is a drug that causes degranulation in MRGPRX2-expressing MCs. The research question is: Do β-arrestins regulate human MRGPRX2 mast cell function and response?
Methods
The first goal of this study is to “humanize” bone marrow derived mouse MCs (BMMCs) with MRGPRX2. This was done by collecting bone marrow cells from wild type C57BL/6, β-Arrestin1-/-, and β-Arrestin2-/- mice and transducing them with retrovirus particles encoding human MRGPRX2. Cells were differentiated into BMMCs in culture medium containing IL-3 and Stem Cell Factor. After four weeks, cell surface MRGPRX2 expression was determined by flow cytometry using phycoerythrin-conjugated receptor-specific antibody. The second goal of the study is to determine the role of β-arrestin1 and β-arrestin2 on MRGPRX2-mediated MC activation. Functional assays will be conducted with each ligand (calcium mobilization, receptor internalization, and degranulation). For comparison, the degranulation assay will be conducted with peritoneal mouse MCs that express MrgprB2 (wild type, β-Arrestin1-/-, and β-Arrestin2-/-).
Results
At this time, bone marrow cells were collected from the mice, transduced with retrovirus encoding MRGPRX2, and differentiated into MCs. However, while all β-Arrestin1-/-, and β-Arrestin2-/- BMMCs expressed MRGPRX2 at similar levels, only ~50% of wild-type cells expressed MRGPRX2. Flow cytometry will be used to purify the cells that express MRGPRX2 and these cells will be used for functional studies.
Conclusion
This study is ongoing, but it is expected that functional assays will reveal differences between each group.