AB1130 A MICROSCOPY-BASED ASSAY TO MONITOR THE SLC15A4/TASL/IRF5 ACTIVATION STATUS IN RHEUMATIC DISEASES
Authors: L. Heinz, F. Kartnig, D. Sieghart, D. Mrak, D. Aletaha
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> Compelling evidence from human genetics and mouse experimentation implicates an important role for the transcription factor IRF5 as a driver in multiple autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and systemic sclerosis (SSc). Importantly, elevated nuclear IRF5 levels have been reported in monocytes, B cells and plasmacytoid dendritic cells from SLE patients. IRF5 activation occurs upon engagement of endosomal Toll-like receptors (TLR7/8/9) via the recently identified SLC15A4/TASL adapter complex [1], which might therefore serve as an attractive target for novel therapeutic approaches [2]. The quantitative assessment of the IRF5 activation state in relevant immune cell subsets is however technically challenging. <h3>Objectives:</h3> The goal of this study is to establish a high-content microscopy-based nuclear translocation assay that allows for the simultaneous and cell type-specific assessment of IRF5 and NF-κB activation status in peripheral blood mononuclear cell (PBMC) subsets. The assay will serve as a tool to profile (I) pathway-specific innate immune stimuli, (II) small molecule inhibitors and (III) IRF5 activation status in different rheumatic disease entities. <h3>Methods:</h3> Plasmacytoid dendritic cell (pDC) lines or primary human PBMCs from patients and healthy controls were pre-treated and activated with reference stimuli and thereafter immuno-labelled with IRF5- and NF-κB/p65-specific fluorescent antibodies. In addition, PBMCs were pre-stained for immune cell subset-specific surface markers and confocal microscopy was used for image acquisition. A computational image analysis pipeline was developed and allowed for the cell compartment-specific quantification of transcription factor activation as well as immune cell identification based on surface markers. <h3>Results:</h3> Kinetic analysis of IRF5 and NF-κB nuclear translocation in pDCs upon endosomal TLR stimulation revealed distinct temporal dynamics and CRISPR/Cas9-mediated knockout of SLC15A4 or TASL in pDC lines confirmed the specific requirement of the SLC15A4/TASL adapter complex for IRF5, but not NF-κB activation. Differential nuclear translocation of IRF5 and NF-κB upon stimulation was confirmed in relevant immune cell subsets, including monocytes, B cells and T cells. Profiling of a comprehensive set of innate immune stimuli including Pam3CSK4, poly(I:C), LPS, R848, CpG-ODN, iE-DAP, MDP and cGAMP in human PBMC immune cell subsets allowed to determine a cell-type-specific response pattern and confirmed a central role for endosomal TLRs in IRF5 activation. <h3>Conclusion:</h3> The established assay serves as a powerful approach for the specific and quantitative assessment of IRF5 activation in an immune cell subset-specific context in single cell resolution. Therefore, the method represents an important novel tool to mechanistically characterize RMDs regarding the involvement of the SLC15A4/TASL/IRF5 pathway. <h3>REFERENCES:</h3> [1] Heinz LX, Lee J, Kapoor U, et al. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7–9. Nature. 2020;581:316–22. [2] Boeszoermenyi A, Bernaleau L, Chen X, et al. A conformation-locking inhibitor of SLC15A4 with TASL proteostatic anti-inflammatory activity. Nat Commun. 2023;14:6626. <h3>Acknowledgements:</h3> We thank Brigitte Meyer, Carl-Walter Steiner and Birgit Niederreiter for technical support. This work was supported by a grant (RMG2235) from the European Alliance of Associations for Rheumatology (EULAR). The content is solely the responsibility of the authors and does not necessarily represent the official views of EULAR. <h3>Disclosure of Interests:</h3> Leonhard Heinz: None declared, Felix Kartnig: None declared, Daniela Sieghart: None declared, Daniel Mrak: None declared, Daniel Aletaha received grants, speaker fees, or consultancy fees from Abbvie, Gilead, Galapagos, Eli Lilly, Janssen, Merck, Novartis, Pfizer, Sandoz, and Sanofi.