Interleukin-36 (IL-36) Ligands Require Processing for Full Agonist (IL-36α, IL-36β, and IL-36γ) or Antagonist (IL-36Ra) Activity

Abstract IL-36α, IL-36β and IL-36γ (formerly IL-1F6, F8 and F9) are IL-1 family members that signal through the IL-1 receptor family members IL-1Rrp2 (IL-1RL2) and IL-1RAcP. IL-36Ra (formerly IL-1F5) has been reported to antagonize IL-36γ. However, our previous attempts to demonstrate IL-36Ra antagonism were unsuccessful. Here we demonstrate that IL-36Ra antagonist activity is dependent upon removal of its amino-terminal methionine. IL-36Ra starting at valine 2 is fully active and capable of inhibiting not only IL-36γ but also IL-36α and IL-36β. Valine 2 of IL-36Ra lies 9 amino acids upstream of an aliphatic-X-Asp motif conserved in all IL-1 family members. In further experiments, we show that truncation of IL-36α, IL-36β and IL-36γ to this same point increases their specific activity approximately 1000-10,000 fold (from EC50≥1µg/ml to EC50≤1ng/ml). Inhibition of truncated IL-36β activity requires approximately 100-1000 fold excess of IL-36Ra, similar to the ratio required for IL-1Ra to inhibit IL-1β. Chimeric receptor experiments demonstrate that the extracellular but not the cytoplasmic domain of IL-1Rrp2 or IL-1R1 is required for inhibition by their respective natural antagonists. IL-36Ra binds to IL-1Rrp2 and pretreatment of IL-1Rrp2 expressing cells with IL-36Ra prevents an IL-36β-mediated co-immunoprecipitation of IL-1Rrp2 with IL-1RAcP. Taken together these results suggest the mechanism of IL-36Ra antagonism is analogous to the mechanism of IL-1Ra, such that IL-36Ra binds to IL-1Rrp2 and prevents IL-1RAcP recruitment and the formation of a functional signaling complex. In addition, truncation of IL-36α, IL-36β and IL-36γ dramatically enhances their activity suggesting post-translational processing is required for full activity.