Spatiotemporal atlas of pro-inflammatory NF-κB and anti-inflammatory STAT6 signaling using reporter mice during mRNA...

Immunization engages a highly coordinated series of innate and adaptive immune responses across multiple anatomical sites, a complexity that has contributed to the predominantly empirical nature of vaccine development. To address this limitation, we developed and validated a STAT6 reporter mouse that enables dynamic whole-body imaging and ex vivo characterization of STAT6-mediated anti-inflammatory signaling. We then integrated this model with an established NF-κB reporter system to dissect the immune activation triggered by two lipid nanoparticle (LNP)-formulated mRNA vaccines differing in RNA chemistry (unmodified versus N¹-methylpseudouridine-modified). The dual-reporter approach enabled the construction of a spatiotemporal atlas of vaccine-induced signaling, revealing distinct immune dynamics driven by RNA chemistry and identifying the liver as an early hub for both NF-κB and STAT6 activity following systemic administration. Correlation with serological data showed that early STAT6 activation followed by rapid signal resolution was associated with favorable humoral responses. These findings establish NF-κB and STAT6 reporter mice as rapid in vivo screening tools for early evaluation of vaccine immune potential and highlight their ability to inform more mechanistic and rational design of mRNA vaccine platforms, while emphasizing the role of the liver both as a primary LNP target and as an immunologically relevant organ.