[Bulk RNA-seq]: Characterization of transcriptional and chromatin accessibility landscape of human bronchial epithelial cells treated with synergistic immunostimulatory ligands.

Lower respiratory tract infections remain a leading cause of death worldwide. The increasing prevalence of antibiotic-resistant respiratory pathogens has been a key driver of efforts to develop host-directed therapies that protect patients against pneumonia. Our laboratory has previously reported discovery of an immunostimulatory therapeutic comprised of a synthetic diacetylated lipopeptide, Pam2CSK4 (Pam2), and a class C CpG oligodeoxynucleotide, ODN M362 (ODN). When delivered by inhalation, this dyad protects synergistically against pneumonias caused by Gram-positive and -negative bacteria, seasonal and pandemic viruses, and multiple fungi. Protection is achieved by manipulating the immune defense response from lung epithelial cells. Here, we dissect the lung epithelial transcriptional response to explain the observed synergistic signaling, using RNA-seq and dual transcription factor ChIP-seq. We find cooperation of RelA and cJun following Pam2+ODN treatment that synergistically confers broad inducible resistance against respiratory pathogens. Specifically, transcription factor binding site (TFBS) analysis revealed that cJun occupancy at promoter regions of Pam2+ODN-induced genes facilitated increased RelA occupancy during differential expression, demonstrating cJUN-assisted loading of RelA as a key mechanism of the synergistic signaling events. Increased or decreased RelA occupancy at promoter sites dictated gene up- or down-regulation, respectively, while increased or decreased cJun occupancy at promoter sites directed synergistic or antagonistic gene expression patterns. Together, RelA and cJun work at promoter regions to control directionality and magnitude of gene expression after Pam2+ODN stimulation, rapidly reprograming the lung epithelial transcriptome to combat respiratory infections.