Differential methanogenic archaea-induced TLR8-dependent signaling is governed by NF-κB p65- and STAT1/2-controlled gene classes

Prevalent in the human gut yet lacking canonical cell wall-derived host-recognition motifs found in bacteria, human-associated archaea Methanosphaera stadtmanae and Methanobrevibacter smithii show immune activity and disease links but remain largely underexplored. Time-resolved RNA-seq in human PBMCs identified an immune program shared with bacterial or viral stimuli across both archaeal species, yet their kinetics diverge, with M. stadtmanae inducing earlier and stronger activation and M. smithii eliciting more gradual responses. Among conserved early-upregulated genes, two classes emerged: Class I is preferentially induced by M. stadtmanae , whereas Class II is similarly induced by both species. To investigate the origin of the Class I/II phenotype, we measured uptake and applied TLR8 inhibition, finding greater early uptake with M. stadtmanae and broad TLR8 dependence across readouts. Mechanistically, ChIP-qPCR demonstrated that M. stadtmanae , but not M. smithii , drives NF-κB p65 binding at Class I promoters, while STAT1/2 binding at Class II promoters occurs with both stimuli. Dose-response analyses with RNA inputs established distinct activation thresholds, with Class II at low dose (via STAT1/2) and Class I only at higher dose (via p65). Together, these findings define an input-tuned logic in which archaeal inputs gate TLR8-governed immune programs. More broadly, variation in archaeal inputs may tune immune tone and help explain divergent disease links, supporting dose-aware TLR8 agonist/antagonist modulation for diagnosis, therapy, and adjuvant design.