The θ‐defensin retrocyclin 101 inhibits TLR4‐ and TLR2‐dependent signaling and protects mice against influenza infection

Despite widespread use of annual influenza vaccines, seasonal influenza‐associated deaths number in the thousands each year, in part because of exacerbating bacterial superinfections. Therefore, discovering additional therapeutic options would be a valuable aid to public health. Recently, TLR4 inhibition has emerged as a possible mechanism for protection against influenza‐associated lethality and acute lung injury. Based on recent data showing that rhesus macaque θ‐defensins could inhibit TLR4‐dependent gene expression, we tested the hypothesis that a novel θ‐defensin, retrocyclin (RC)‐101, could disrupt TLR4‐dependent signaling and protect against viral infection. In this study, RC‐101, a variant of the humanized θ‐defensin RC‐1, blocked TLR4‐mediated gene expression in mouse and human macrophages in response to LPS, targeting both MyD88‐ and TRIF‐dependent pathways. In a cell‐free assay, RC‐101 neutralized the biologic activity of LPS at doses ranging from 0.5 to 50 EU/ml, consistent with data showing that RC‐101 binds biotinylated LPS. The action of RC‐101 was not limited to the TLR4 pathway because RC‐101 treatment of macrophages also inhibited gene expression in response to a TLR2 agonist, Pam3CSK4, but failed to bind that biotinylated agonist. Mouse macrophages infected in vitro with mouse‐adapted A/PR/8/34 influenza A virus (PR8) also produced lower levels of proinflammatory cytokine gene products in a TLR4‐independent fashion when treated with RC‐101. Finally, RC‐101 decreased both the lethality and clinical severity associated with PR8 infection in mice. Cumulatively, our data demonstrate that RC‐101 exhibits therapeutic potential for the mitigation of influenza‐related morbidity and mortality, potentially acting through TLR‐dependent and TLR‐independent mechanisms.