C67-04 Host-directed Immunotherapy of Lung Epithelium With Pam2+odn Promotes Pathogen-agnostic Antiviral Responses by Reprogramming Nf-kb and Ap-1

Abstract Rationale Host-directed immunotherapy can ameliorate the global incidence and mortality burden of pneumonia caused by both known and emerging pathogens. We have reported an immunostimulatory dyad of pattern recognition receptor ligands, Pam2 CSK4 and ODN M362 ("Pam2+ODN"), that synergistically reduce pathogen burden and promote host survival against respiratory infections. Here, we combine high-content analysis of bulk and single-cell models of mouse and human lung epithelium to understand the anti-infectious transcriptional and mechanistic responses after stimulation with Pam2+ODN. Methods We modeled multi-Omics host responses conserved between mouse (GSE26864, GSE289984) and human (GSE299678, GSE299702, GSE299552) lung epithelium after Pam2+ODN stimulation to discover differential transcriptional and epigenetic changes related to lung protection against respiratory pathogens. NGS reads were aligned to GRCh38 for feature (gene/peaks) calling, and differential analysis and functional enrichment was done in R, QIAGEN IPA and HOMER. We evaluated transcriptional responses in vitro (HBEC3-KT, MLE-15) and in vivo (C57BL/6J) in the presence or absence of Pam2+ODN and/or transcription factor inhibitors (NF-kB and/or AP-1). We tested antiviral protection by challenging our models with Influenza A and MHV coronavirus at multiple MOIs and time points. We included high-throughput imaging flow cytometry, fluorescent imaging, qPCR, and co-immunoprecipitation of host targets: RelA and pathogen Influenza A (M1, NS1) or MHV coronavirus (N) to associate transcriptional dynamics, gene expression changes and pathogen burden reduction outcomes with antiviral and antimicrobial protection. Results Both mouse and human lung epithelium showed differential expression of genes downstream of NF-kB and AP-1 transcription factor families. In vitro we detected increased co-expression and nuclear colocalization of RelA and cJun in MLE-15 and HBEC3-KT cells using immunofluorescence and imaging flow cytometry, these dual transcription factor complexes also colocalized with H3K27Ac accessible genomic sites after 24 hours of Pam2+ODN stimulation. Also, after 24-hour infection, (e.g. Influenza A), we detected activation of p-S536 RelA and p-S73 cJun only in the Pam2+ODN treated cells when compared to sham treated cells. RNA-seq and ChIP-seq revealed increased RelA binding at promoter sites of Pam2+ODN-regulated genes associated with cJun bindings sites, suggesting a regulatory mechanism of transcriptional activity at the host lung epithelium. Additional to pathogen burden reduction, we observed reversal of MHV coronavirus-induced genes, and colocalization of Influenza A proteins with RelA in Pam2+ODN-stimulated lung epithelial cells. Conclusion Pam2+ODN activates NF-kB and AP-1 families in lung epithelium by cooperation of RelA and cJun that lead to pathogen-agnostic antiviral responses. This abstract is funded by: National Heart, Lung, and Blood Institute R35 HL144805