Inserting “OFF-to-ON” BODIPY tags into cytokines: A fluorogenic interleukin IL-33 for real-time imaging of immune cells

Reese, Abigail E., de Moliner, Fabio, Mendive-Tapia, Lorena, Benson, Sam, Kuru, Erkin, Bridge, Thomas, Richards, Josh, Rittichier, Jonathan, Kitamura, Takanori, Sachdeva, Amit, McSorley, Henry J. and Vendrell, Marc (2024) Inserting “OFF-to-ON” BODIPY tags into cytokines: A fluorogenic interleukin IL-33 for real-time imaging of immune cells. ACS Central Science, 10 (1). 143–154. ISSN 2374-7951

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Abstract

The essential functions that cytokine/immune cell interactions play in tissue homeostasis and during disease have prompted the molecular design of targeted fluorophores to monitor their activity in real time. Whereas activatable probes for imaging immune-related enzymes are common, many immunological functions are mediated by binding events between cytokines and their cognate receptors that are hard to monitor by live-cell imaging. A prime example is interleukin-33 (IL-33), a key cytokine in innate and adaptive immunity, whose interaction with the ST2 cell-surface receptor results in downstream signaling and activation of NF-κB and AP-1 pathways. In the present work, we have designed a chemical platform to site-specifically introduce OFF-to-ON BODIPY fluorophores into full cytokine proteins and generate the first nativelike fluorescent analogues of IL-33. Among different incorporation strategies, chemical aminoacylation followed by bioorthogonal derivatization led to the best labeling results. Importantly, the BODIPY-labeled IL-33 derivatives─unlike IL-33-GFP constructs─exhibited ST2-specific binding and downstream bioactivity profiles comparable to those of the wild-type interleukin. Real-time fluorescence microscopy assays under no wash conditions confirmed the internalization of IL-33 through ST2 receptors and its intracellular trafficking through the endosomal pathway. We envision that the modularity and versatility of our BODIPY labeling platform will facilitate the synthesis of minimally tagged fluorogenic cytokines as the next generation of imaging reagents for real-time visualization of signaling events in live immune cells.

Item Type:	Article
Additional Information:	Acknowledgments: L.M.-T. acknowledges funding from the Wellcome Trust Institutional Strategic Support Fund (ISSF) at the University of Edinburgh. A.S. acknowledges funding from the University of East Anglia. H.J.M. acknowledges funding from a Wellcome Investigator award (221914/Z/20/Z). M.V. acknowledges funds from EPSRC (EP/W015706/1) and an ERC Consolidator Grant (DYNAFLUORS, 771443). The authors thank Flow Cytometry, Confocal Advanced Light Microscopy, and SIRCAMS Mass Spectrometry facilities at the University of Edinburgh for the technical support. For open access, the authors applied a CC-BY public copyright license to any Author Accepted Manuscript version arising from this submission. The authors acknowledge BioRender.com for assistance with figure creation.
Faculty \ School:	Faculty of Science > School of Chemistry
UEA Research Groups:	Faculty of Science > Research Groups > Chemistry of Life Processes
Depositing User:	LivePure Connector
Date Deposited:	10 Jan 2024 01:39
Last Modified:	27 Jan 2024 03:14
URI:	https://ueaeprints.uea.ac.uk/id/eprint/94141
DOI:	10.1021/acscentsci.3c01125

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