Single-Cell and Spatially Resolved Interactomics of Tooth-Associated Keratinocytes: Exploring the Role of Structural Immunity in Periodontitis

Background

Periodontitis is a chronic inflammatory disease that affects the periodontal tissues and impacts billions of people globally, potentially resulting in tooth loss. It is primarily caused by polybacterial dysbiosis in the subgingival biofilm and is associated with systemic diseases such as cardiovascular diseases, type 2 diabetes, Alzheimer’s disease, and inflammatory bowel disease. Despite this, personalized precision treatments for periodontitis remain challenging due to limited understanding of the cellular populations in the periodontal tissue and their functional roles in health and disease. Specifically, although the heterogeneity of tooth-associated epithelial keratinocytes has been recognized for decades, functional annotations remain incomplete.

In recent years, the concept of “structural immunity,” which highlights the immune-regulatory roles of non-hematopoietic cells (e.g., fibroblasts, epithelial cells), has gained traction. Keratinocytes, in particular, may not only passively permit microbial infection through barrier breakdown but may also actively regulate immune responses through stem cell reprogramming and immune memory. Thus, this study aims to investigate the structural immune properties of keratinocytes in periodontitis, understand how their differentiation states are altered, and explore their interactions with other immune and microbial systems through single-cell and spatially resolved approaches.

Paper Information

This study was a collaboration among researchers from prominent international institutions, including Quinn T. Easter, Bruno Fernandes Matuck, Germán Beldorati Stark, and others, with Kevin M. Byrd as the corresponding author. The paper was published in Nature Communications (2024) with DOI: 10.1038/s41467-024-49037-y.

Research Workflow Details

The study primarily focused on constructing an integrated single-cell transcriptomic map and spatial interactomic analysis system to investigate keratinocyte subpopulations related to periodontitis. The workflow consists of the following key parts:

Step 1: Construction of Single-Cell Transcriptomic Atlas

The researchers integrated four published single-cell RNA sequencing (scRNAseq) datasets of human periodontal tissue, covering 34 samples (including health, gingivitis, and periodontitis states) and totaling 105,918 cells. The data was annotated and analyzed using the open-access tool cellenics, identifying 17 major cell types and refining 5 new gingival keratinocyte subpopulations. Particular attention was given to sulcular keratinocytes (SK) and junctional keratinocytes (JK), which were classified into stem/progenitor cells and their differentiated progeny. This classification was validated through markers such as keratin 19 (KRT19).

Step 2: Spatial Transcriptomics Validation

Using multiplexed in situ hybridization (MISH) techniques, the researchers validated the spatial heterogeneity of keratinocytes in periodontal tissues. Specific markers such as CXCL14, NEAT1, and FDCSP were used to locate SK and JK keratinocytes around the teeth. This provided insights into their distribution under healthy and diseased conditions, as well as their differentiation trajectories and inflammation-associated phenotypes.

Step 3: Cell-Cell Communication Analysis

Leveraging CellPhoneDB technology, the team analyzed the expression of various chemokines (e.g., CXCL1, CXCL8, IL-1B) to predict cell-cell communication pathways from SK/JK to innate and adaptive immune cells. The findings revealed that JK keratinocytes preferentially interact with macrophages and neutrophils, while SK keratinocytes primarily communicate with T cells and B cells.

Step 4: Single-Cell Metagenomics and Host-Microbe Interactomics

Single-cell metagenomics identified significant bacterial burdens, particularly periodontal pathogens such as Porphyromonas gingivalis, in SK and JK keratinocytes. The presence of bacterial signals was confirmed with 16S rRNA probes, shedding light on the cell-specific distribution of bacterial loads. For the first time, 3D imaging revealed the co-localization of multiple bacterial species within keratinocyte stem cells and their progeny.

Step 5: Multiplexed Immunofluorescence and Immune Composition Analysis

Through multiplexed immunofluorescence (MIF) staining, the team unveiled changes in the microenvironments surrounding SK and JK cells in periodontitis. They observed an immunosuppressive microenvironment near JK cells characterized by PD-L1+ expression, while SK regions preferentially recruited adaptive immune cells (e.g., T cells) and formed tertiary lymphoid structures.

Key Findings and Conclusions

1. Data-Driven Evidence: SK and JK keratinocytes exhibit disrupted differentiation trajectories and heightened expressions of inflammatory mediators in periodontitis. This includes multiple chemokines and IL-1 family members (e.g., IL-1A, IL-1B, IL-36G), which may exacerbate chronic inflammation by recruiting immune cells.

2. Bacterial Signal Associations: For the first time, spatially resolved analyses revealed a direct correlation between polybacterial burden (Polybacterial Burden) and the inflammatory phenotypes of SK/JK keratinocytes. These bacterial signals may exist in the form of outer membrane vesicles (OMVs).

3. Immune-Structural Interactions: JK keratinocytes predominantly signal innate immune cells (e.g., macrophages, neutrophils), while SK keratinocytes preferentially engage with adaptive immune cells (e.g., T cells, B cells). This functional division in immune communication likely plays a critical role in regulating health and disease states.

Significance and Highlights of the Study

This study represents a comprehensive annotation of the heterogeneity and immune-interacting properties of SK and JK keratinocytes in periodontitis using single-cell and multimodal spatial omics technologies. The introduction of the concept of “keratokines” (chemokines secreted by keratinocytes) underscores the idea that these keratinocytes are central players in the “every cell is an immune cell” framework of structural immunity.

In addition, the findings on polybacterial interactions and their impact on the host–microbial symbiosis system offer new perspectives for cross-species and cross-system disease regulation. This study lays a solid foundation for early diagnosis, intervention, and personalized treatment strategies for periodontal diseases.