LL37 and Cationic Peptides Enhance TLR3 Signaling by Viral Double-stranded RNAs

Abstract

BackgroundToll-like Receptor 3 (TLR3) detects viral dsRNA during viral infection. However, most natural viral dsRNAs are poor activators of TLR3 in cell-based systems, leading us to hypothesize that TLR3 needs additional factors to be activated by viral dsRNAs. The anti-microbial peptide LL37 is the only known human member of the cathelicidin family of anti-microbial peptides. LL37 complexes with bacterial lipopolysaccharide (LPS) to prevent activation of TLR4, binds to ssDNA to modulate TLR9 and ssRNA to modulate TLR7 and 8. It synergizes with TLR2/1, TLR3 and TLR5 agonists to increase IL8 and IL6 production. This work seeks to determine whether LL37 enhances viral dsRNA recognition by TLR3.Methodology/Principal FindingsUsing a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3. The presence of LL37 also increased the cytokine response to rhinovirus infection in BEAS2B cells and in activated human peripheral blood mononuclear cells. Confocal microscopy determined that LL37 could co-localize with TLR3. Electron microscopy showed that LL37 and poly(I:C) individually formed globular structures, but a complex of the two formed filamentous structures. To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling. This is the first demonstration that LL37 and other RNA-binding peptides with cell penetrating motifs can activate TLR3 signaling and facilitate the recognition of viral ligands.Conclusions/SignificanceLL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA.

The Human Antimicrobial Peptide LL-37, but Not the Mouse Ortholog, mCRAMP, Can Stimulate Signaling by Poly(I:C) through a FPRL1-dependent Pathway

“…Poly(I:C) and LPS were from Invivogen. Reovirus (RV) dsRNA S4 were prepared as described in Lai et al (9). Antimicrobial peptides with or without covalently attached fluorophores were custom-synthesized to greater than 95% purity by AnaSpec Inc.…”

“…BEAS-2B cells were cultured in BEGM media with its supplements (Lonza Inc.) (9,17). NK-92MI cells were propagated in ␣-minimum essential medium with final concentrations of 2 mM L-glutamine, 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 M folic acid, 12.5% horse serum, and 12.5% FBS.…”

“…LL-37 binding to lipopolysaccharides (LPS) can down-regulate signaling by Toll-like receptor 4 (TLR4), 2 binding to ssRNA can up-regulate TLR7 and TLR8 signaling, and interaction with flagellin can activate TLR5 signaling (3,7,8). Most relevant to this work, LL-37 binds doublestranded (ds) RNA, including poly(I:C) to enhance TLR3 signaling above the level observed with poly(I:C) alone (7,9,10). How LL-37 functions in concert with dsRNA to affect TLR3 signaling remains to be better understood.…”

Non-coding Double-stranded RNA and Antimicrobial Peptide LL-37 Induce Growth Factor Expression from Keratinocytes and Endothelial Cells

“…LL-37 has been shown to bind dsRNA and traffic it to endosomes where it releases the dsRNA in a pH-dependent manner (44). Additionally, structural changes of LL-37⅐ligand complex were shown by electron microscopy illustrating that LL-37 and poly(I:C) individually formed globular structures, but a complex of the two formed filamentous structures (23). Thus, the LL-37 data presented here correlate with previous evidence supporting the role of LL-37 in facilitating the response to nucleic acids.…”

“…Through its charge and amphipathic nature, LL-37 promiscuously binds multiple DAMP molecules such as single-stranded DNA (18), single-stranded RNA (19), dsRNA (20), and bacterial polysaccharides (21,22). LL-37 is also an immunogenic effector molecule can that alter the signal response of TLRs to a LL-37⅐DAMP complex such as enhancing dsRNA sensing through TLR3 (23) or inhibiting LPS sensing through TLR4 (24).…”