Crystallinity of Double-Stranded RNA-Antimicrobial Peptide Complexes Modulates Toll-Like Receptor 3-Mediated Inflammation

Abstract

Double-stranded RNA (dsRNA) induces production of pro-inflammatory cytokines in normal human epidermal keratinocytes (NHEK) by specific binding to endosomal Toll-like receptor-3 (TLR3). Recently, it has been shown that hyperactivation of TLR3 in psoriatic keratinocytes by dsRNA can occur in the presence of human antimicrobial peptide (AMP) LL37. Here, we combine synchrotron X-ray scattering, microscopy, computer simulations, and measurements of NHEK cytokine production to elucidate a previously unanticipated form of specific molecular pattern recognition. LL37 and similar ⍺-helical AMPs can form proinflammatory nanocrystalline complexes with dsRNA that are recognized by TLR3 differently than dsRNA alone. dsRNA complexes that activate IL-6 production in NHEK and those that do not are both able to enter cells and colocalize with TLR3. However, the crystallinity of these AMP-dsRNA complexes, specifically the geometric spacing between parallel dsRNA and the repeat number of ordered dsRNA, strongly influence the level of TLR3 activation. Crystalline complexes that present dsRNA at a spacing that matches with the steric size of TLR3 can recruit and engage multiple TLR3 receptors, driving receptor clustering and immune amplification, whereas crystalline complexes that exhibit poor steric matching do not. Reverse-transcription quantitative PCR (RTqPCR) of IL-6 during siRNA knockdown of TLR3 confirms that cytokine production is due to TLR3: High levels of IL-6 transcription are observed for sterically-matched complexes without TLR3 knockdown, whereas such activity is abrogated with TLR3 knockdown.

Cathelicidin promotes inflammation by enabling binding of self-RNA to cell surface scavenger receptors

“…This structure is consistent with close contact and charge compensation between the cationic peptides and anionic dsRNA, with a spatial periodicity of 3.76 nm and 3.70 nm between adjacent dsRNA within the complex (respective nanocrystal sizes of L = 9.0 nm and 16.6 nm). Interestingly, the structure of the LL34(I20P)-dsRNA complex is consistent with the capacity to enable dsRNA to be recognized by TLR3 31 . Therefore, we concluded that it was unlikely that the loss of immunological activity of LL34(I20P) was due to disruption of binding and structured complex formation with dsRNA or interference with TLR3 recognition.…”

Functional Reciprocity of Amyloids and Antimicrobial Peptides: Rethinking the Role of Supramolecular Assembly in Host Defense, Immune Activation, and Inflammation

“…Cognate to LL37-DNA complexes, we found that LL37-dsRNA complexes formed nanocrystalline structures with well-defined inter-dsRNA spacings, and that complexes that maximally activated TLR3 had spacings perfectly matched with the steric size of TLR3. A mathematical model and computer simulation of TLR3 binding to spatially periodic AMP-dsRNA complexes recapitulated the experimental data and showed that both the inter-dsRNA spacing and the number of repeat units within the complexes were primary determinants of immune activation (56). This validated the idea that innate immune receptors like TLR9 and TLR3 can recognize both single ligands, as well as the crystallinity of spatially periodic, geometrically patterned ligands templated by molecular chaperones like AMPs.…”

Psoriasis and Antimicrobial Peptides

“…We found that the complex formed by LL-37 and dsRNA binds to scavenger receptors and causes uptake of the complex into the intracellular space [98]. We also demonstrated that LL-37 and similar α-helical AMPs can form pro-inflammatory nanocrystalline complexes with dsRNA that are recognized by TLR3 differently from dsRNA alone [101]. Self-nucleic acids (DNA, RNA) are DAMPs released from damaged cells and extracellular matrix to activate the innate immune system.…”