Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
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 (KD = 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.
To cite this abstract in AMA style:Banga J, Srinivasan D, Sun CC, Milletti F, Huang KS, Hamilton S, Hoffman AF, Qin YG, Panicker S, Lu G, Li D, Qian H, Bolin DR, Liang L, Wartchow C, Fotouhi N, DeMartino JA, Tan SL, Chen G, Barnes BJ. Assessing Interferon Regulatory Factor 5 (IRF5) Function in Human Primary Immune Cells with Cell-Penetrating Peptides [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/assessing-interferon-regulatory-factor-5-irf5-function-in-human-primary-immune-cells-with-cell-penetrating-peptides/. Accessed September 20, 2021.
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