Abstract
Lipid nanoparticle (LNP)-mediated mRNA delivery has emerged as a powerful platform for both immunostimulatory and immunomodulatory applications. However, the influence of local immunometabolic cues on the intracellular fate and translational efficiency of mRNA-LNPs remains poorly understood. In this study, we investigated how interferon-gamma (IFN-γ), a potent inducer of indoleamine 2,3-dioxygenase 1 (IDO1), and tryptophan (Trp) deprivation independently and combinatorially affect mRNA-LNP function in DC2.4 dendritic cells. These two cues are canonical drivers of immunoregulatory microenvironments, particularly those that favor tolerogenic dendritic cell programming and the induction of regulatory T cells. Using dual-reporter mRNA constructs and high-resolution confocal imaging, we show that IFN-γ stimulation reduces total cellular mRNA uptake and lysosomal accumulation without affecting translation efficiency and endosomal escape efficiency. Whereas Trp deprivation also reduces the overall cellular uptake of mRNA-LNPs, it also significantly impairs protein synthesis from mRNA-LNPs and modestly reduces endosomal escape, despite having minimal impact on lysosomal mRNA levels. Spatial compartmentalization analysis revealed that IFN-γ and Trp limitation disrupt distinct steps in the delivery-translation cascade, acting independently but additively to suppress the ultimate protein translation from mRNA-LNPs in DC2.4 dendritic cells. These findings highlight the importance of considering local metabolic and cytokine contexts when deploying mRNA-LNPs for immunological applications. Our work provides mechanistic insights into how immunoregulatory environments impair the delivery and translation of mRNA-LNPs, suggesting strategies to tune delivery outcomes for tolerogenic purposes.