Background/Purpose: Interferon regulatory factor 5 (IRF5) is a key mediator of pathogen-induced immune responses that acts downstream of Toll-like receptors (TLRs), NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs). IRF5 polymorphisms leading to its elevated expression and activation have been detected in patients with autoimmune diseases; yet, the contribution of IRF5 to disease onset and/or severity remains to be fully elucidated. IRF5 typically remains inactive in the cytoplasm of a cell but upon stimulation by external signals, IRF5 undergoes post-translational modification(s), homo-dimerization, and nuclear translocation, where the dimeric protein induces transcription of antiviral and pro-inflammatory genes. Here, we report the evaluation of novel cell-penetrating peptides (CPPs) designed to disrupt IRF5 dimerization which is considered critical for nuclear translocation and function in immune cells.

Methods: We designed CPPs targeting IRF5 Helix 2 or Helix 5 regions based on a modelled structure of the IRF5 dimer. CPPs binding to the IRF5 monomer were measured in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Potencies of IRF5-CPPs were assessed in an IRF5 dimerization assay using recombinant biotin- and his-tagged IRF5 (222-467, S430D). Cell penetration was first tested in Hela cells with FITC-conjugated CPPs followed by confocal microscopy. The effects of IRF5-CPPs on nuclear localization and phosphorylation of IRF5 in CD14⁺ monocytes, CD123⁺BDCA2⁺ plasmacytoid dendritic cells (pDC) and CD19⁺ B cells were analyzed on an ImageStream Mark II cytometer following stimulation of peripheral blood mononuclear cells (PBMCs) with CpGA, R848 or SLE serum. Levels of inflammatory cytokines (IL-6, TNFα), IgG and IFNα in PBMCs were measured by AlphaLISA.

Results: Biochemical and imaging analyzes showed that IRF5-CPPs are cell permeable, non-cytotoxic at concentrations <50 µM, and directly bind to IRF5 (K_D = 0.5-0.93 µM). FRET assays revealed that IRF5-CPPs disrupt IRF5 homo-dimerization (IC₅₀ = 8.5-10.9 µM). Stimulation of PBMCs with TLR ligands revealed that IRF5-CPPs blocked pro-inflammatory cytokine production (IL-12, TNFα, and IL-6), IgG production in B cells, and IFNα production in pDCs. Inhibition of cytokine and IgG production from primary immune cells correlated with a significant, concentration-dependent reduction in the nuclear localization of phosphorylated IRF5. Similar findings were made in PBMCs derived from patients with systemic lupus erythematosus (SLE) or lupus nephritis (LN).

Conclusion: Rational design of novel cell-penetrating peptide inhibitors that target IRF5 dimerization not only provides new tools for the functional interrogation of IRF5 in healthy and disease-relevant cells, but also facilitates the development of new therapeutics to treat inflammatory and autoimmune disorders such as SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/assessing-interferon-regulatory-factor-5-irf5-function-in-human-primary-immune-cells-with-cell-penetrating-peptides/