Spatiotemporal atlas of pro-inflammatory (NF-kB) and anti-inflammatory (STAT6) signalling using reporter mice during mRNA vaccination

Abstract

The immunization process unfolds through a precisely orchestrated sequence of innate and adaptive events across distinct anatomical sites. Although many mechanisms underlying vaccination are well described, most vaccines have been developed empirically, partly due to the lack of tools enabling rapid, organ-specific analysis of immune activation. To address this gap, we developed and validated a novel STAT6 reporter mouse enabling dynamic in vivo whole-body imaging and ex vivo analysis of STAT6-mediated anti-inflammatory signalling, and combined it with an established NF-{kappa}B reporter model to dissect immune activation induced by two LNP-encapsulated mRNA vaccines encoding the same antigen but differing in RNA chemistry (unmodified versus N{superscript 1}-methyl-pseudouridine (m{superscript 1}{Psi})-modified). This dual-reporter system enabled the creation of a spatiotemporal atlas of vaccine-induced signalling, revealing chemistry-dependent immune dynamics and identifying the liver as a predominant early hub for both NF-{kappa}B and STAT6 activity following systemic administration. Integration with antibody measurements demonstrated that early STAT6 activation followed by rapid signal resolution--rather than prolonged NF-{kappa}B-mediated inflammation--correlated with robust humoral responses, suggesting that monitoring NF-{kappa}B and STAT6 dynamics could provide predictive insight into vaccine immunogenicity. Together, these findings establish NF-{kappa}B and STAT6 reporter mice as rapid in vivo screening tools for the early assessment of vaccine immunogenicity and performance. By enabling dynamic, organ-resolved immune profiling, this approach paves the way for more rational, mechanism-driven design of mRNA vaccines and underscores the importance of further investigating the effects of vaccines on the liver, both as a primary LNP target and as an immunologically tolerogenic organ.