IntroductionThe epithelium serves as a sophisticated and dynamic interface that acts not only as a physical barrier but also as an active orchestrator of tissue homeostasis, immune surveillance, and host defense.1,2 Following microbial challenge, the preservation of epithelial integrity and its communication with underlying connective tissue are critical to the pathogenesis of chronic inflammatory disorders.3,4,5 In the intestinal epithelium, for instance, dysregulation of conserved pathways such as Wnt/β-catenin and Notch impairs crypt regeneration and Paneth cell function, compromising barrier integrity and promoting bacterial translocation in inflammatory bowel disease.6,7 Similarly, in skin, defects in keratinocyte differentiation and filaggrin expression, together with altered transforming growth factor-β (TGF-β) signaling, sustain the chronic inflammation seen in atopic dermatitis.8,9 Across tissues, the mechanisms driving epithelial dyshomeostasis during chronic inflammation are highly diverse, shaped by organ-specific microenvironments and functional demands.10 This contextual regulation underscores the need to study epithelial behavior within its native tissue framework to uncover both shared and unique principles of inflammatory dysregulation.In the oral cavity, disruption of the gingival epithelial barrier can trigger periodontitis, a highly prevalent chronic inflammatory disease linked to systemic conditions including diabetes, cardiovascular disease, and adverse pregnancy outcomes.11,12,13,14,15 Periodontal pathogens and their virulence factors disrupt intercellular junctions such as E-cadherin and occludin, facilitating bacterial invasion.16,17,18 Activation of epithelial Toll-like receptors (e.g., TLR2, TLR4, TLR5) by microbial components engages mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling, leading to epithelial apoptosis and barrier breakdown.19,20 Subsequent immune activation within the underlying connective tissue fuels a self-sustaining inflammatory cascade that promotes osteoclastogenesis and alveolar bone loss.21,22,23 Fibroblasts further contribute to pathological remodeling via reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) production, degrading the extracellular matrix and amplifying local inflammation.24,25 Despite these insights, a spatially resolved understanding of epithelial-connective tissue crosstalk in periodontitis remains limited.The application of single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) in periodontium research has become a cutting-edge focus, revealing unprecedented cellular heterogeneity and intricate cell-cell communication networks. A comprehensive single-cell atlas of gingival tissues has been established, revealing the heterogeneity of immune cells across disease states, the differentiation potential of pericytes, and the transition of fibroblasts to a C3-secretory proinflammatory phenotype, as extensively documented in recent studies.26,27,28 In addition, Quinn et al. integrated single-cell datasets to characterize keratinocyte heterogeneity, identifying a distinct differentiation state and immunomodulatory function of KRT19+ keratinocytes in periodontitis.29 These cells notably upregulate the expression of genes encoding multiple effector cytokines, including CXCL1, CXCL8, IL1A, and IL1B. Furthermore, by coupling spatial proteomics, the authors revealed the spatial heterogeneity of the peri-epithelial immune microenvironment, demonstrating differently enriched clustering of innate and adaptive immune cells around keratinocyte subclusters. Through integrated scRNA-seq and ST analyzes, Zhang et al. revealed that cigarette smoke exposure compromises epithelial barrier integrity and disrupts fibroblast-epithelial cell crosstalk in gingival tissues.30 Despite these insights, the mechanistic roles of specific epithelial subclusters, their differentiation dynamics, and the direct in situ impact of the local microbial community on epithelial remodeling remain incompletely characterized.To address these gaps, in the present study, we integrated scRNA-seq, ST, and metagenomic sequencing of paired gingival tissues and subgingival plaque from healthy individuals and patients with periodontitis to investigate host-microbe interactions in situ. Briefly, we summarize here the conceptual framework that is further integrated in Fig. 8. We define inflammatory epithelial remodeling in periodontitis and identify Epithelium-3, located near the junctional epithelium, as a spatially restricted inflammatory epithelial region. Within this region, we further identified an SAA1+ inflammatory epithelial subcluster. The following therefore proceeds from spatial epithelial identification to microbial-triggered epithelial reprogramming, and finally to downstream connective tissue and immune microenvironment remodeling.ResultsSpatial transcriptomics integrated with scRNA-seq reveals the organizational landscape of the human gingival epitheliumTo characterize periodontitis gingival epithelium features, we established ST (10× Genomics Visium) and scRNA-seq (10× Genomics Chromium) methods. Limited healthy gingival biopsy size allowed paired scRNA-seq for only a subset of controls. Spatial transcriptomics was performed on 4 healthy controls and 6 periodontitis patients, while scRNA-seq was conducted on a partially overlapping cohort of 2 healthy controls and 6 periodontitis patients, and the scRNA-seq reference was mapped onto the ST sections to reconstruct gingival tissue organization (Fig. 1a, b). A total of 3520 Visium spatial spots were obtained from ST sections. Damaged and unrecognizable spots were discarded. The epithelium region was first annotated based on the morphology of hematoxylin and eosin (H&E) staining of each tissue section (Fig. S1a, b) and further confirmed by the expression of known epithelial marker genes (KRT5, KRT76, and CSTB) (Fig. 1c, and Fig. S1c). The gingival epithelium could be clustered into 4 epithelium regions (Fig. 1d). In terms of histological anatomy, Epithelium-2 predominantly corresponded to the stratum spinosum of the sulcular epithelium, whereas Epithelium-1 aligned with both the stratum granulosum and stratum corneum layers. Of particular clinical significance, Epithelium-3 occupied the basal layer of the junctional epithelium. In contrast, Epithelium-4 maintained its characteristic location in the basal layer of the oral gingival epithelium, serving as an internal reference for epithelial homeostasis. Gene Set Variation Analysis (GSVA) revealed that the functions of all epithelium regions were consistent with the corresponding anatomical levels (Fig. S1d).Fig. 1Full size imageSpatial transcriptomics (ST) and scRNA-seq landscape in the gingival epithelium. a The gingival tissues were collected from 4 healthy controls and 6 periodontitis patients, and the gingival epithelium region. ST was performed on gingival tissues from 4 healthy controls and 6 periodontitis patients. scRNA-seq was performed on 2 healthy controls and 6 periodontitis patients. Because healthy gingival biopsies were limited in size and tissue allocation prioritized ST to preserve spatial information, paired ST and scRNA-seq data were available for only a subpopulation of healthy controls. b Schematic drawing of the combined scRNA-seq and ST. c The Epithelium in the slice of ST was recognized by marker genes (KRT76, KRT5, CSTB). d A dot plot showing the 4 major subclusters in different colors in the health group (n = 4) and periodontitis group (n = 6). e Uniform manifold approximation and projection (UMAP) of epithelial cell subclusters. The black circle indicates SAA1+EpiA total of 37,122 cells were subsequently captured from the raw scRNA-seq data. Six cell clusters were identified: the epithelial cell cluster (KRT5), the endothelial cell cluster (PLVAP), the B and plasma cell cluster (CD79A), the natural killer (NK) cell (XCL1) and T-cell cluster (CD3D), the fibroblast cluster (COL3A1), and the myeloid cell cluster (LYZ) (Fig. S2a, b). The epithelial cells were then clustered into 6 distinct subclusters (Fig. 1e) according to their marker genes (Figs. S2c and S3d): COL17A1+Epi, KRT6C+Epi, IGLC2+Epi, VIM+Epi, SAA1+Epi, and SPRR1B+Epi. According to Gene Ontology (GO) functional analysis (Data S1), COL17A1+Epi and KRT6C+Epi have differentiation functions, while IGLC2+Epi plays a role in signal transduction. SAA1+Epi showed significant inflammatory features. VIM+Epi represents the epithelial-mesenchymal transformation (EMT) epithelial group, and SPRR1B+Epi is a group of mature keratinocytes.SAA1+Epi epithelial subcluster is responsible for the pathogenesis of periodontitisIn terms of the landscape of the human gingival epithelium, Epithelium-3, which is the primary interface for subgingival plaque interactions, exhibited high expression of the genes MMP13, CXCL6, SAA2, and PTHLH, suggesting its potential role in gingival inflammation (Fig. S3a). Moreover, the proportion of Epithelium-3 in patients with periodontal disease significantly increased (Fig. S3b), indicating its function in periodontitis. Gene set enrichment analysis (GSEA) analysis revealed that Epithelium-3 was responsive to immune activity, especially bacteria and lipopolysaccharide (LPS) (Fig. 2a), which confirmed the special role of Epithelium-3 in bacterial defense. Epithelium-3 significantly upregulated pathways related to epithelial cell immune activation and proinflammatory immune responses (Fig. S1d), indicating that Epithelium-3 functions as both a microbial sentinel and an inflammatory orchestrator. Epithelium-3 directly converts plaque challenge into sustained tissue destruction, driving the transition from microbial colonization to chronic periodontitis.Fig. 2Full size imageSAA1+Epi performs the functions of the Epithelium-3. a Dot plot of functions enriched in the Epithelium-3 by using GSEA analysis with Bayesian Testing, P