Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context.

Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gαq, Gα11, Gα12, and Gα13 or ß-arrestins, we showed that Gαq and Gα11 are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of ß-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or ß-arrestin.

IL-18 is required for the TH1-adaptation of TREG cells and the selective suppression of TH17 responses in acute and chronic infections.

Interleukin (IL)-18, a member of the IL-1 family of alarmins, is abundantly released in the lungs following influenza A (IAV) infections yet its role in orchestrating the local adaptive immune response remains ill defined. Through genetic disruption of the IL-18 receptor, we demonstrate that IL-18 not only promotes pulmonary TH1 responses but also influences regulatory T cells (TREG) function in the infected lungs. As the response unfolds, TREG cells accumulating in the lungs express Helios, T-bet, CXCR3, and IL-18R1 and produce interferon γ in the presence of IL-12. During IAV, IL-18R1 is required for TREG cells to control TH17, but not TH1, responses and promote a return to lung homeostasis, revealing a novel mechanism of selective suppression. Moreover, this observation was not limited to the lungs, as skin-localized TREG cells require an IL-18 signal to specifically suppress IL-17A production by TH17 and γδ T cells in a model of chronic cutaneous Leishmania major infection. Overall, these results uncover how IL-18 orchestrates the tissue adaptation of TREG cells to selectively favor TH1 over TH17 responses during TH1-driven immune responses and provide a novel perspective into how IL-18 dictates the immune response during viral and parasitic infections.