The function of epithelial tissues is the protection of the organism from chemical, microbial, and physical challenges which is indispensable for viability (10). Keratinocytes form a barrier through tight junctions, adherens junctions, and gap junctions. Bacteria, in turn, can disrupt the epithelial barrier by inducing leukocytes to produce proteolytic enzymes that degrade inter-epithelial junctions, inflammatory cytokines that downregulate the expression of adhesion molecules and keratinocyte apoptosis that disrupts a continuous barrier (10–12). Bacteria can penetrate epithelial cells and reach the basal layer within 24 hours (13). The severity of periodontitis is positively correlated with the extent of epithelial tissue damage. A reduction of epithelial cells is found in moderate or severe periodontitis (14). Bacterial invasion of the oral epithelium causes increased ROS production, which can lead to mitochondrial damage and accumulation and the production of pro-inflammatory factors (15). P. gingivalis can modulate gingival keratinocytes to enhance mRNA levels of inflammatory factors and stimulate apoptosis (16) and degrade the proteins that form intercellular adhesions (17, 18). Other invasive bacteria include A. actinomycetemcomitans, T. denticola, and F. alocis (19). The use of protease inhibitors such as leupeptin has been shown to partially mitigate the loss of barrier function induced by P. gingivalis, implicating the involvement of microbial proteolytic enzymes in disrupting the epithelial barrier (16).

In addition to providing a physical barrier to microorganisms as part of the host immune defense, the gingival epithelium also expresses a variety of pattern recognition receptors (PRRs) that enable it to recognize microbiota-associated molecular patterns (MAMPs) and respond by secreting cytokines, chemokines and antibacterial peptides. Keratinocytes in the epithelial barrier play a key role in the initiation of the host immune response.

Single-cell RNA sequencing analysis has identified gingival epithelial subpopulations that contribute to inflammatory signatures, antimicrobial defense and neutrophil recruitment in periodontitis (14, 20). scRNA-seq analysis of gingiva indicates an overall reduction in epithelial cells in subjects with periodontitis (14, 20, 21). Caetano et al. identified ten subpopulations of gingival epithelial cells in periodontitis (14). These subpopulations comprised two basal cell clusters, three epithelial clusters expressing high levels of cell cycle genes, one cluster expressing genes associated with extracellular matrix organization and angiogenesis, and four distinct gingival epithelial subpopulations with transcriptomes linked to immune regulation. The latter express transcripts that encode factors that stimulate B-lymphocyte receptor signaling and neutrophil-mediated immunity. Although the overall population of epithelial cells decreased, there was an increase in immune-related epithelial subpopulations (14). Williams et al. identified three keratinocyte subpopulations including a basal cell cluster, a cluster enriched in genes involved in cornification, and a cluster with a gene expression profile consistent with inflammatory responses (20). The gene expression profiles of these cells indicated a shift towards an inflammatory state, with upregulated pathways related to antimicrobial responses and cytokine biosynthesis in subjects with periodontitis (20). Thus, there is an increase in gingival epithelial subpopulations with pro-inflammatory gene signatures with periodontitis and an overall reduction of non-inflammatory epithelial cells. A distinctive junctional epithelial population was characterized by elevated expression levels of serum amyloid A-proteins (SAA) (21). These proteins were found to trigger the secretion of inflammatory cytokines through interaction with the TLR2 pathway in human gingival fibroblasts.

Stromal cells in the gingiva consist of mesenchymal stem cells, pericytes and fibroblasts and contribute to tissue integrity, immune regulation, and repair processes. They express several receptors needed to recognize microbes and produce cytokines and chemokines in response. Approximately 30 years ago Yu and Graves suggested that gingival fibroblasts through the production of CCL2 could play an important role in recruiting monocytes and macrophages to inflamed gingiva (22). scRNA-seq data supports the concept that fibroblasts are an important part of the host response. Williams et al. characterized five distinct fibroblast subclusters (20). Among these clusters, two exhibited a transcriptome profile linked to matrix synthesis and tissue remodeling, while the remaining three featured gene signatures associated with immune functions, including leukocyte proliferation, granulocyte migration, and complement activation. Notably, individuals with periodontitis showed a general decrease in fibroblast subpopulations but displayed a specific increase in inflammatory fibroblast subpopulations in parallel with findings with epithelial cells ( Figure 1 ). There was an upregulation of genes associated with neutrophil recruitment such as CXCL1 and CXCL8. Caetano et al. identified five distinct fibroblast populations, one pericyte population, and one myofibroblast subpopulation (14). Among the fibroblasts, three subpopulations exhibited enrichment of genes associated with extracellular matrix production, while two other fibroblast subpopulations displayed an inflammatory profile. In the context of periodontitis, a marked reduction was observed in the myofibroblast and pericyte subpopulations, accompanied by an increase in inflammatory fibroblasts, while the other subpopulations remained unchanged. Consequently, both investigations identified a decline in fibroblast numbers, accompanied by an expansion of pro-inflammatory stromal cells in subjects with periodontitis. This data suggests a unique restructuring of the epithelial and stromal compartments in periodontitis, with a specific emphasis on facilitating neutrophil recruitment.

Unconventional T-cells include natural killer T (NKT) cells, γδ T-cells and mucosal-associated invariant T (MAIT) cells that express CD3. They are more prevalent in gingiva from subjects with periodontitis (59). NK T-cells are a specialized subset of T-cells with αβ T-cell receptors (TCRs) and NK cell receptors on their surface (59, 60). Gram-negative bacteria possess glycosphingolipids that can activate NK T-cells via antigen presenting cells. NK T-cells can enhance RANKL production, osteoclastogenesis, and alveolar bone loss in mice following oral P. gingivalis inoculation and in other models (61, 62).

γδ T-cells express T-cell receptors (TCR) consisting of the gamma and delta chains, with a limited diversity, and do not express CD4 or CD8 (63). These cells are stimulated by a variety of signals, such as direct antigen binding to TCR, stimulation of toll-like receptors or cytokine stimulation. They are found in the epithelium or in the connective tissue adjacent to the epithelium and make up the majority of T-cells in epithelial tissues (64). γδ T-cells are elevated in inflamed human gingiva (64), and are increased to a greater extent than αβ T-cells (64). γδ T-cells stimulate the recruitment of macrophages and neutrophils and produce IL-17A and IFNγ (65). In mice, they are the principal source of IL17A. In the oral inoculation model of periodontitis, γδ T-cells have distinct pathogenic functions, and their reduction significantly reduces loss of alveolar bone. However, this linkage does not exist in the ligature model, pointing out an important difference in the two primary murine models of periodontitis (65). scRNA-seq data reveals an approximately 20% decrease in γδ T-cells among all cell types in individuals with periodontitis compared to healthy individuals (20), contrasting with a 30% increase among immune cells observed in a mouse model of periodontitis compared to the healthy state (29) ( Figure 1 ). Using different reference populations could be a potential reason for the contrasting findings and species differences (64). Another difference may be due to the fact that most human studies represent inflamed tissue that may not exhibit current disease activity, a significant limitation in most human studies, whereas the disease activity is typically progressing in murine models (66).

MAIT have a restricted T-cell receptor (TCR) response (67). Notably, TCR-independent mechanisms such IL-18 signaling can activate MAIT cells. They differ in how they react to various microorganisms, and this diversity may help them discriminate between dangerous pathogens and beneficial commensal species. Germ-free mice have fewer MAIT cells and MAIT cell populations increase during infection, suggesting a protective function against microbial challenge (67). Emerging evidence indicates that MAIT cells may contribute to the development of periodontitis by producing proinflammatory cytokines like IL-17 and TNF when activated by pathogenic microorganisms in the oral cavity. Further research is required to comprehensively elucidate the precise role of MAIT cells in periodontitis (68). A scRNA-seq study reported a significant decline in MAIT cells in subjects with periodontitis (20) The scRNAseq data revealed an upregulation of nucleotide oligomerization domain (NOD)−like receptor signaling pathways, apoptosis, IL-17 signaling, and TNF signaling in MAIT cells from periodontitis subjects (69).

Lymphoid cells that lack T-cell receptors but are of lymphocyte lineage include innate lymphoid cells (ILC) with three subtypes (ILC1, ILC2, and ILC3). Rather than reacting to antigen, these cells react directly to signals of stress and danger. They possess pattern recognition receptors (TLR2, TLR4, TLR9, NLRP3, RAGE, P2X7 and P2Y2, etc) on their cell surface that respond to danger-associated molecular patterns (LPS, S100 proteins, AGEs, ATP, ROS, etc). ILCs are primarily tissue-resident cells and are classified according to the cytokines they generate (70). ILC1 cells express similar cytokines to Th1 cells such as IFNγ, ILC2 cells produce cytokines similar to Th2 cells such as IL-4, IL-5, IL-9, and IL-13 and ILC3 cells produce IL17A, similar to Th17 cells. According to scRNA-seq data, a mouse model of periodontitis exhibits a reduction of over 30% in ILC cells among immune cells in animals with induced periodontitis (29). ILC1 was the predominant subset, comprising over 60% of ILCs in mice and humans. In humans, the percent ILCs were not significantly altered in the gingiva of subjects with periodontitis. Notably, a small proportion of ILC1 cells expressed RANKL and and ILC3 produced IL17A suggesting they could participate in bone resorption (71). The plasticity, differentiation, tissue-specific migration and accumulation of ILC subpopulations may be an important modulator of the local immune response (72).

NK cells are lymphocytes belonging to the innate immune system (60). NK cells are cytolytic, killing viral or bacterial-infected, or malignant cells, and can exert pro-inflammatory effects. Through the release of granzymes and perforin, NK cells directly destroy their targets. NK cells produce cytokines like IFN, TNF, IL-5, IL-13, and GM-CSF that upregulate activity in other cells, particularly macrophages and contribute to the control of infections (60, 73) ( Figure 1 ). NK cells have a role in senescent cell clearance. They are stimulated by bacteria through toll-like receptors (TLRs) and cytokines produced by cells such as dendritic cells (73).

NK cells tend to have proinflammatory influences in periodontitis (60). This is manifested through cytokine production, cytotoxic effects, and dendritic-cell crosstalk. Moreover, increased numbers of NK cells in patients wtih periodontitis and decreased numbers after periodontal therapy have been observed (60). Additionally, NK cells are correlated to the regulation of T-cell proliferation and suppression of B-cells in periodontitis (60). Overall, these findings suggest that NK cells play a role in the pathogenesis of periodontitis, particularly through their proinflammatory influences (60).

Naive CD4+ T-cells are capable of differentiating into a variety of functional and phenotypical T helper (Th) cell subsets, Th1, Th2, Th17, Treg and Tfh cells (67, 74). Th1 cells are pro-inflammatory and produce IL-1β and IFN-γ to promote inflammation and are associated with tissue damage in periodontitis. Th2 cells play a key role in the production of antibodies. Interleukin-4 (IL-4) and other Th2-cell-derived cytokines are anti-inflammatory and are considered to reduce bone loss. However, antibodies produced by Th2 responses activate complement and could potentially be pro-inflammatory. Th17 cells promote inflammation through IL-17 production. Th17 cells are increased in human periodontitis; reducing Th17 cell numbers reduces alveolar bone resorption in experimental periodontitis (75). In humans, Th17 cells are the principal source of IL17A. IL-17 stimulates osteoblast-lineage cells to secrete RANKL and GM-CSF to enhance osteoclast formation and bone resorption (76). IL-17A also stimulates fibroblasts, epithelial cells, and endothelial cells to produce RANKL, MMP, PGE2, and chemokines to promote the progression of periodontitis (77). IL-17A can affect immune cells such as macrophages, neutrophils, dendritic cells, and B-cells. Regulatory T-cells (Treg) stimulate immunosuppression and resolution of inflammation through production of TGF-b, cytotoxic T lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), programmed cell death protein 1 (PD1), T-cell immune receptors with Ig and ITIM structural domains (TIGIT), and T-cell immunoglobulin and mucin-containing structural domain 3 (Tim-3) (78). The percentage of these cells increases in the later stages of periodontitis to reduce disease progression and reestablish homeostasis (79). Interestingly, increased RANKL promotes the induction of Tregs and increases formation of M2 macrophages, thus facilitating the resolution of inflammation (79). T follicular helper (Tfh) cells play an important role in the regulation of humoral immunity and germinal center responses, and in periodontitis, may promote local B-cell activation, and maintain a long-term humoral immune response (80). Tfh cells in older individuals may contribute to increased inflammation in periodontitis (74).

Th22 cells are a subpopulation of T-helper cells that produce IL-22 and TNF, which have been linked to the pathogenesis of periodontitis by increasing inflammation (81) and the number of Th17 cells in periodontal lesions (82). Oral inoculation of bacteria in mice stimulates the production of IL-22 through increased numbers of IL-22-expressing CD4+ T-cells in periodontitis-affected tissues (83). This increase is associated with higher levels of RANKL and alveolar bone resorption.

scRNA-seq analysis indicates that T-cells constitute the largest lymphocyte population, followed by B-cells and plasma cells (20) ( Figure 1 ). In human samples with periodontitis there is an overall expansion of T-cells (20, 21). Various studies have identified distinct subclusters of T-cells within single-cell RNA sequencing datasets from both healthy and diseased conditions. These subclusters include CD4+, MAIT, CD8+, γδ T-cells, Treg, TH17, and NK T-cells, which are consistently observed across different studies, albeit with varying proportions in periodontitis (20, 21, 29). Human studies indicate an approximately 25% decrease in CD4⁺ T-cells in human subjects (20) and a similar reduction of over 30% in mouse periodontitis models (30).

CD8+ T-cells kill virally or bacterially infected cells. CD8+ T-cells are fewer in number than CD4+ T-cells in periodontitis lesions (84). CD8+ cytotoxic T lymphocytes produce TNF, IFN-γ and kill cells through expression of Fas ligands, pore-forming proteins (perforins) and proteases (granzyme) (85). CD8+ regulatory T lymphocytes (CD8+ Tregs) produce CTLA4, TGF-b and IL-10 to resolve inflammation. Systemic administration of CTLA-4 reduces alveolar bone resorption in experimental periodontitis (86). Like pro-inflammatory CD4+ T-cells, the pro-inflammatory CD8+ cytotoxic T lymphocytes likely promote periodontitis whereas the pro-resolving CD8+ Tregs help prevent or reduce it. In human gingival tissue, scRNA-seq studies indicate a small ~10% increase in CD8+ T-cells in subjects with periodontitis compared to non-periodontitis subjects (20, 21). Interestingly, periodontitis was associated with an increase in expression of CCL4, CCL4L2, and CCL3L3 in both CD8 T-cells and NK cells. Elevated levels of the CCR5 ligands in cytotoxic CD8+ T-cells underscores their potential role in recruiting inflammatory cells during periodontitis (21).

B-cells are part of the humoral component of the adaptive immune system and are specialized in producing antibodies. B-cells can also present antigens and enhance inflammation through cytokine production, opsonization, and complement fixation mediated by the antibodies they produce. B-cells and plasma cells are increased in periodontitis (87). Hub genes are located at critical nodes in biological processes such as chronic inflammation. Interestingly, a recent study pointed to B-cells as expressing a high number of hub genes that are correlated with inflammation in periodontitis (88). B-cells produce RANKL to promote bone loss (79). Evidence that B-cells contribute to periodontitis was shown in a ligature-induced murine model in which there was significantly less bone loss in B-cell deficient mice (89). On the other hand, B-cells can potentially reduce periodontitis by limiting bacterial invasion. In support of the latter, reduced dendritic cell activation of B-cells increases periodontitis in an oral inoculation murine model (55). B regulatory cells (Bregs) can reduce inflammation and limit excessive inflammatory responses similar to Tregs. Bregs produce anti-inflammatory cytokines such as IL-10, and inhibit alveolar bone resorption (90, 91). Plasma cells that produce anti-inflammatory cytokines IL-35 and IL-37 also inhibit alveolar bone loss (92). Taken together, evidences suggests that B lymphocytes have a dual role in modulating the progression of periodontitis and can both promote and inhibit alveolar bone resorption depending on the specific conditions.

scRNA-seq studies observed an overall increase in the proportion of B-cells in human subjects with periodontitis compared to healthy controls (21, 50). Three distinct B-cell populations were consistently detected including memory B-cells, IgG-producing plasma B-cells, and follicular B-cells (20). Caetano et al. (14) reported a distinct increase in memory B-cells in moderate periodontitis compared to healthy individuals. The increase is backed up by several publications using alternative approaches showing there is a significant increase in plasma cells in periodontitis compared to healthy individuals (14, 20, 21, 50) ( Figure 1 ).