cJun-assisted loading of RelA DNA-binding promotes synergistic lung epithelial antimicrobial responses

Lower respiratory tract infections remain a leading cause of death worldwide, highlighting the need for host-directed therapies. We previously identified an inhaled immunostimulatory combination of Pam2CSK4 (Pam2) and CpG oligodeoxynucleotide ODN M362 (ODN) that synergistically protects against bacterial, viral, and fungal pneumonias by reprogramming lung epithelial defenses. Using RNA-seq, dual transcription factor ChIP-seq, and reverse phase protein array, we define the transcriptional basis of this synergy. Cooperative regulation by the AP-1 component cJun and the NF-κB subunit RelA is central to the Pam2+ODN response. cJun occupancy distinguishes synergistic genes, whereas RelA occupancy aligns with expression directionality. Promoter-proximal cJun occupancy facilitates enhanced RelA loading, enabling rapid transcriptional reprogramming that strengthens epithelial survival pathways and amplifies microbicidal effector programs. This AP-1-assisted NF-κB cooperation establishes inducible resistance in lung epithelium and provides a mechanistic foundation for host-directed therapies against respiratory infection.