Polybacterial intracellular macromolecules shape single-cell inflammatory profiles in upper airway epithelia

Mucosal epithelial cells of the upper airways are continuously exposed to microbes throughout life. Specialized niches such as the anterior nares and the tooth are especially susceptible to dysbiosis and chronic inflammatory diseases. Here, we reanalyzed our v1-Human Periodontal Atlas, identifying polybacterial signatures (20% Gram-positive; 80% Gram-negative) and distinct responses of bacterial-associated epithelia. Fluorescence microscopy detected numerous persistent polybacterial intracellular macromolecules (PIMs) within human oral keratinocytes (HOKs), including bacterial rRNA, mRNA, and glycolipids. PIM levels directly correlated with enhanced receptor-ligand signaling in vivo. Inflammatory "keratokines" targeting immune cells were synergistically upregulated in lipopolysaccharide-challenged HOKs, while endogenous lipoteichoic acid (LTA) correlated with CXCL1/8 expression in vitro and in vivo. Application of Drug2Cell suggested altered drug efficacy predictions based on PIM detection-agnostic of disease state. CXCL1/8 expression again correlated with LTA in epithelial cells of the nasal cavity, oropharynx, and trachea. Thus, PIMs shape epithelial single-cell profiles across upper airway mucosae.