Ghodke, A. | bioRxiv Science

bioRxiv2024

The Immunoregulatory Architecture of the Adult Oral Cavity

The immunoregulatory architecture of human oral tissues remains poorly defined despite their central role as barrier interfaces. We present the first integrated single-cell and dual-platform spatial-proteotranscriptomic atlas of oral tissues, profiling >250,000 single-cell transcriptomes and >4 million spatially-resolved cells across 13 niches. Using our AI-enabled AstroSuite (TACIT, Constellation, STARComm, hist2omics), we defined tissue cellular neighborhoods (TCNs) and multicellular interaction modules (MCIMs) in health, revealing peri-epithelial fibroblast-centered hubs enriched for effector cytokines. We harmonized eight fibroblast subtypes (universal, immune, peri-epithelial, peri-vascular, peri-neural, APC-like, stress-responsive, and myofibroblasts) with stress-responsive subtypes partitioning between mucosae (Type I) and glands (Type II). Spatial multiomics mapped receptor-ligand circuits and showed mucosal stress-responsive fibroblasts as immunoregulatory hubs. In chronic periodontitis, niche-aware integration of healthy and diseased datasets revealed rewiring of fibroblast phenotypes and ligand::receptor networks into interdigitated inflammatory and reparative niches. Disease neighborhoods exhibited fragmentation, expansion of MHC-I, MHC-II, and PD-L1 fibroblasts, and predicted spatial engagement with T cells at ectopic lymphoid structures. Drug2Cell analysis highlighted druggable stromal::immune networks. Together, this proteotranscriptomic atlas positions fibroblasts as central architects of structural immunity in human oral tissues and establishes a scalable framework for precision targeting of stromal::immune ecosystems across other barrier organs in health and chronic disease.

cell biology↗

bioRxiv2024

Polybacterial Intracellular Macromolecules Shape Single-Cell Epikine Profiles in Upper Airway Mucosa

The upper airway, particularly the nasal and oral mucosal epithelium, serves as a primary barrier for microbial interactions throughout life. Specialized niches like the anterior nares and the tooth are especially susceptible to dysbiosis and chronic inflammatory diseases. To investigate host-microbial interactions in mucosal epithelial cell types, we reanalyzed our single-cell RNA sequencing atlas of human oral mucosa, identifying polybacterial signatures (20% Gram-positive, 80% Gram-negative) within both epithelial- and stromal-resident cells. This analysis revealed unique responses of bacterial-associated epithelia when compared to two inflammatory disease states of mucosa. Single-cell RNA sequencing, in situ hybridization, and immunohistochemistry detected numerous persistent macromolecules from Gram-positive and Gram-negative bacteria within human oral keratinocytes (HOKs), including bacterial rRNA, mRNA and glycolipids. Epithelial cells with higher concentrations of 16S rRNA and glycolipids exhibited enhanced receptor-ligand signaling in vivo. HOKs with a spectrum of polybacterial intracellular macromolecular (PIM) concentrations were challenged with purified exogenous lipopolysaccharide, resulting in the synergistic upregulation of select innate (CXCL8, TNFSF15) and adaptive (CXCL17, CCL28) epikines. Notably, endogenous lipoteichoic acid, rather than lipopolysaccharide, directly correlated with epikine expression in vitro and in vivo. Application of the Drug2Cell algorithm to health and inflammatory disease data suggested altered drug efficacy predictions based on PIM detection. Our findings demonstrate that PIMs persist within mucosal epithelial cells at variable concentrations, linearly driving single-cell effector cytokine expression and influencing drug responses, underscoring the importance of understanding host-microbe interactions and the implications of PIMs on cell behavior in health and disease at single-cell resolution. One-sentence summaryThis study reveals how persistent intracellular bacterial macromolecules in mucosal epithelial cells drive inflammatory signaling, offering new insights into microbial-host interactions and their potential impact on inflammatory disease treatment and drug efficacy.

cell biology↗