TLR4 recognizes ionizable lipid nanoparticles to induce NF-kB and IRF responses 2951

Description Lipid nanoparticles (LNPs) are essential in messenger RNA (mRNA) vaccines, aiding in mRNA delivery. Their role as adjuvants and the mechanisms by which they stimulate an innate immune response are unclear. To address these questions, we studied three ionizable LNP formulations to evaluate their ability to and mechanisms by which they stimulate innate signaling. Using the THP-1 monocyte cell line to model innate activation, we assessed the Interferon Regulatory Factor (IRF) and Nuclear Factor-kappa Beta (NF-?B) responses to empty LNPs from the mRNA-1273 and BNT162b2 vaccines, and a third LNP compositionally similar to that of BNT162b2. Signaling was detectable at 48 hours, though the magnitude varied. Importantly, the addition of mRNA to the LNP did not increase the activation of THP-1. Using TLR4, MyD88, or TRIF-deficient THP reporters, we identified TLR4-MyD88 as the predominant signaling axis for the three LNPs. Notably, removal of the ionizable lipid component abrogated signaling, highlighting its critical role. Our findings demonstrate that three LNPs with ionizable lipid components activate the TLR4-MyD88 pathway, leading to NF-?B and IRF responses in THP-1 cells. The ionizable lipid is responsible for this activation, and the presence of mRNA did not provide additional stimulation. Overall, these findings underscore the role of ionizable LNPs in triggering the innate immune response and highlight their role as adjuvants that can be optimized for vaccine design. Funding Sources Supported by federal funds from the Defense Health Program (HU00012020067, HU00012020094) and the Immunization Healthcare Branch (HU00012120104).