Differential methanogenic archaea-induced TLR8-dependent signaling engages gene classes regulated by NF-κB p65 and STAT1/2

Archaea are prevalent in the human gut but lack canonical bacterial cell wall-associated immune ligands. Time-resolved RNA-seq of human PBMCs showed that Methanosphaera stadtmanae and Methanobrevibacter smithii induced a shared immune program but with distinct kinetics: M. stadtmanae triggered an earlier and stronger response, whereas M. smithii acted more gradually. Conserved early-upregulated genes separated into Class I genes, preferentially induced by M. stadtmanae, and Class II genes, similarly induced by both. M. stadtmanae showed greater early uptake, while responses to both archaea were broadly TLR8-dependent. ChIP-qPCR linked the stronger Class I gene activation induced by M. stadtmanae to greater NF-κB p65 binding at Class I promoters, whereas STAT1/2 binding at Class II promoters occurred with both stimuli. Dose-response analyses with archaeal RNA inputs supported distinct activation thresholds, with Class II genes at lower input and Class I genes requiring higher input. Together, these findings support an input-tuned TLR8-dependent mechanism shaping differential immune programs towards archaea.