Ionizable lipid nanoparticles of mRNA vaccines elicit NF-κB and IRF responses through toll-like receptor 4

Ionizable lipid nanoparticles (LNP) that have enabled the success of messenger RNA (mRNA) vaccines have been shown to be immunostimulatory in the absence of mRNA. However, the mechanisms through which they activate innate immune cells is incompletely understood. Using a monocyte cell line, we compared the ability of three LNP formulations to activate transcription factors Nuclear Factor-kappa B (NF-κB) and Interferon Regulatory Factor (IRF). Comparison of signaling in knockout cell lines illustrated a role for Toll-like receptor (TLR) 4 in initiation of this signaling cascade and the contribution of the ionizable lipid component. Activation induced by empty LNPs was similar to that induced by LNPs containing mRNA, indicating that LNPs may provide the majority of innate stimulation for the mRNA vaccine platform. Our findings demonstrate that ionizable lipids within LNPs signal through TLR4 to activate NF-κB and IRF, identifying a mechanism for innate activation that can be optimized for adjuvant design.