Ab1130 a Microscopy-Based Assay to Monitor the Slc15a4/Tasl/Irf5 Activation Status in Rheumatic Diseases

Background: 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. Objectives: 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. Methods: 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. Results: 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. Conclusion: 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. REFERENCES: [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. Acknowledgements: 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. Disclosure of Interests: 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.