Introduction

The oral mucosa must constantly respond to a diverse array of oral microbes, as well as to dietary and airborne antigens, making it one of the most active barrier tissues in the human body^{1, 2}. Consequently, oral barrier immunity acts as a crucial surveillance system, which must defend from pathogenic organisms, while ensuring sustained tolerance of commensal microbes and harmless antigens, without excessive inflammation³. Within this system, the junction between the gingiva and the tooth surface represents a weak link, wherein the initiation of localized inflammation often leads to chronic gingivitis and periodontitis. Once this gingival inflammation progresses, adjacent alveolar bone supporting the cervical area of the dentition is subjected to a localized, but often severe, osteoclastic bone resorption, resulting in tooth loss and disruption of the maxillofacial structure⁴. Globally, periodontitis ranks among the most significant contributors to poor health and decreased quality of life, imposing substantial economic and healthcare burdens, particularly in low and middle-income countries⁸. It is therefore acutely important to elucidate the molecular mechanisms underlying the initiation of gingival inflammation, in order to facilitate the development of effective preventive and therapeutic measures for this debilitating condition.

Critically, periodontitis is not only the most frequent cause of tooth loss in adults⁵, it is also associated with a number of severe non-communicable diseases, such as metabolic and cardiovascular diseases. A recent systematic review revealed that type 2 diabetes mellitus patients had significantly worse periodontal status with more tooth loss, and the presence of severe periodontitis increased the incidence of type 2 diabetes mellitus by 53%⁶. Chronic inflammatory conditions are also known to increase the risk of cardiovascular diseases, including valvular heart disease. Recently, a possible association has been recognized between periodontal inflammation—distinct from transient bacteremia due to dental procedures—and atherosclerotic cardiovascular disease⁷. Although a direct causal relationship between these non-communicative diseases and periodontitis has not been established, it is postulated that periodontitis triggers an inflammatory cascade that begins in oral barrier tissues and subsequently spreads throughout the body. Thus, elucidating the mechanisms underlying periodontitis development may further provide insight on the signaling pathways that induce and sustain systemic inflammation.

Clinical characterization of periodontitis patients and observations made during treatment have not yielded a comprehensive understanding of the mechanisms that promote periodontal disease. Rather, our current model for the pathological framework of periodontitis has been reconstructed from animal models, harvested immune cell analyses, and oral microbial associations^{4, 9}. Of particular importance has been the development of a ligature-induced periodontitis model in mice, combined with targeted gene mutations¹⁰. This model has shown that fully developed periodontal inflammation is closely associated with the abundant recruitment and excessive activation of neutrophils¹¹ and pathological induction of interleukin (IL)-17–secreting proinflammatory effector CD4⁺ T helper (Th)17 cells¹². Based on our current understanding of osteoimmunology, it was hypothesized that these pathological immune cells activate osteoclasts, leading to induction of periodontal alveolar bone resorption^{13 14}. However, the precise cascade of events leading to this pathological outcome has not been fully deciphered.

Investigation of the molecular mechanisms underlying chronic inflammation in immunological disorders is an intense area of research. Prior studies have shown that chemokines are involved in the trafficking and activation of inflammatory cells during both homeostasis and disease-associated inflammation¹⁵. These small proteins, which typically contain disulfide cysteine–cysteine (CC) and cysteine–X–cysteine (CXC) molecular signatures, act as ligands for cellular receptors that modulate inflammatory signaling pathways¹⁶. Such chemokine–receptor networks direct the migration of inflammatory cells, potentially amplifying the resulting tissue damage. However, in addition to immune cells, barrier tissues are also composed of epithelial cells and fibroblastic stromal cells, and increasing evidence suggests that stromal cells are involved in innate immune cell regulation^17–20. In particular, we and others have found that oral fibroblasts secrete cytokines and chemokines in response to microbial stimuli or a proinflammatory environment, suggesting a possible mechanism by which they may regulate immune cell function^{21, 22}. Therefore, a comprehensive elucidation of chemokine–receptor signaling networks will likely need to include all types of gingival cells.

In the present study, to better understand the pathways contributing to chronic oral inflammation, we determined the gingival single-cell transcriptomic atlas of mice with ligature-induced periodontitis and identified a novel subpopulation of fibroblasts activated to guide chronic inflammation (AG fibroblasts). Our findings suggest that chemokines and ligands derived from AG fibroblasts bind to and activate receptors involved in neutrophil and lymphocyte activation. We further show that Cd4^- innate lymphoid cells (ILCs) produce proinflammatory IL-17 cytokines in the inflamed gingiva, and provide evidence that type 3 ILCs (ILC3s) are responsible for cervical alveolar bone resorption in mice—a pathological feature consistent with the human periodontitis phenotype. Thus, the results from this study identify the AG fibroblast– neutrophil–ILC3 axis as a previously unrecognized mechanism contributing to pathological inflammation in periodontitis.

Results

Alterations in major cell type proportions during periodontitis development

Periodontal disease was induced in C57BL/6J mice by placing a ligature around the maxillary left second molar²³; gingival defect developed after Day 3 following ligature placement (Figure 1A and B). Three-dimensional (3D) reconstruction of micro-computed tomography (microCT) images (Figure 1C) further revealed a reduction in alveolar bone height starting from Day 3, which progressively increased on Day 7 (Figure 1D—figure supplement 1). Overall, the observed pattern of periodontal tissue degradation was consistent with that reported in previous studies^{24, 25}.

Figure 1.

Left-side palatal gingiva tissue was harvested from mice on Day 0 (i.e., healthy gingiva without ligature placement) and on Days 1, 4, and 7 after ligature placement, and gingival cells were dissociated for single-cell RNA sequencing (scRNA-seq)^{22, 26}. On Days 0 and 1, the major cell types identified included epithelial cells expressing cadherin 1 (Cdh1)²⁷ and type XVII collagen (Col17a1)²⁸, fibroblasts expressing type I collagen (Col1a1)²⁹ and lumican (Lum)³⁰, B cells expressing membrane spanning 4 domains A1 (Ms4a1)³¹ and cluster of differentiation 79A (Cd79a)³², T cells expressing epsilon subunit of T cell receptor complex (Cd3e)³³ and cluster of differentiation 5 (Cd5)³⁴, and myeloid cells expressing lysozyme 2 (Lyz2)³⁵ and integrin subunit alpha M (Itgam)³⁶ (Figure 1E and F—figure supplement 2). On Days 4 and 7, an additional endothelial cell fraction expressing selectin P (Selp)³⁷ and selectin E (Sele)³⁸ emerged (Figure 1E and F—figure supplement 2), suggesting increased inflammatory neovascularization with the progression of periodontal inflammation. The proportion of B cells was increased on Day 1, and the proportion of myeloid cells increased progressively from Day 4 to 7 (Figure 1F). In addition, the proportion of fibroblasts was increased on Day 7 (Figure 1F).

Myeloid cell composition and activity during periodontitis development

The infiltration of proinflammatory neutrophils into the gingiva has been extensively characterized in this mouse model of periodontitis^{11, 39}. Here, we found that macrophages expressing cluster of differentiation 86 (Cd86)⁴⁰ and integrin subunit alpha X (Itgax)⁴¹ were predominant on Day 0 in the myeloid cell fraction from healthy gingiva (Figure 2A and B— figure supplement 3). In contrast, the proportion of neutrophils expressing CXC motif chemokine receptor (Cxcr2)⁴² and G0/G1 switch gene 2 (G0s2)⁴³ increased after ligature placement and during periodontitis development from Day 1 to 7 (Figure 2A and B—figure supplement 3), suggesting continuous neutrophil infiltration into the early and established gingival lesion. Moreover, after ligature placement, gingival neutrophils upregulated the expression of triggering receptor expressed on myeloid cells 1 (Trem1), indicating that these cells are activated and participating in the amplification of inflammatory signals^{44, 45} (Figure 2C). Strikingly, the Trem1-expressing activated neutrophils also show upregulation of matrix metalloproteinase 9 (Mmp9; Figure 2C)—a protein associated with extracellular matrix degeneration within gingival tissue⁴⁶. We further detected expression of tumor necrosis factor alpha (Tnfa) and transforming growth factor beta 1 (Tgfb1) in both macrophages and neutrophils on Day 0 and after ligature placement (Figure 2D). Collectively, these myeloid cell behaviors are consistent with those reported in prior studies on periodontitis development, thus validating our scRNA-seq data.

Figure 2.

Myeloid cells are also known to stimulate other immune cells through the expression of CC motif chemokine ligands (CCLs) and CXC motif chemokine ligands (CXCLs), many of which are associated with periodontitis development⁴⁷. Our scRNA-seq analysis revealed that macrophages expressed Ccl2, Ccl9, Cxcl4, and Cxcl6, and neutrophils expressed Ccl3, Ccl4, Ccl6, Cxcl2, and Cxcl3 throughout periodontitis development (Figure 2E and F). These data suggest that chemokines and cytokines produced by macrophages and neutrophils in inflamed tissue may amplify and polarize the immune response toward chronic gingival inflammation.

Fibroblasts activated to guide leukocyte migration in periodontitis development

We previously identified two distinct subpopulations of gingival fibroblasts, differentiated by expression of type XIV collagen (Col14a1)²², and these were also detected in our current scRNA-seq data from Day 0 to 7 (Figure 3A). Gene ontogeny (GO) enrichment analysis of the Col14a1-expressing fibroblast subpopulation revealed expression of major gene clusters related to immune regulation, including “Regulation of leukocyte migration” (Figure 3B). This immune regulatory phenotype appears to be unique to Col14a1-expressing fibroblasts, which are therefore referred to as ‘fibroblasts activated to guide leukocyte migration’ or AG fibroblasts. The other subpopulation of Col1a1-expressing fibroblasts appeared to keep typical fibroblastic features and is thus referred to as KT fibroblasts (Figure 3C). An additional fibroblast subpopulation expressing smooth muscle aortic actin 2 (Acta2) was detected on Day 7, and these were identified as myofibroblasts⁴⁸ (MF) (Figure 3A). We found that the proportions of KT and AG fibroblasts were equal on Day 0 and on Days 1 and 4 after ligature placement (Figure 3E and F). However, on Day 7, the proportion of AG fibroblasts decreased, and the MF fraction emerged (Figure 3E and F).

Figure 3.

AG fibroblasts and immune surveillance in periodontitis development

To further characterize AG fibroblasts and their “Regulation of leukocyte migration” phenotype, we analyzed expression of CCL and CXCL chemokines within fibroblast subpopulations in our scRNA-seq dataset¹³. Results show that AG fibroblasts activated expression of Ccl8, Ccl11, Ccl19, Cxcl1, Cxcl11, and Cxcl12 immediately after ligature placement on Days 1 and 4 (Figure 4A). Chemokine expression levels then decreased on Day 7, suggesting that AG fibroblasts might initiate early leukocyte migration into gingival tissue. Given that activation of Toll-like receptors (TLR) is known to increase chemokine expression, we further assessed expression of TLRs in fibroblast subpopulations. We found that AG fibroblasts displayed temporal activation of Tlr2, Tlr3, and Tlr4 expression on Days 1 and 4 (Figure 4B). Similarly, the TLR downstream genes Myd88, Irak1, Map3k7, and Rela were also expressed on Days 1 and 4 (Figure 4B). These data suggest that AG fibroblasts may guide the establishment of an early inflammatory environment within the gingiva and thereby promote periodontitis pathogenesis.

Figure 4.

To validate the presence of AG fibroblasts, Day 1 gingiva and periodontal tissue histological sections were subjected to immunohistochemistry with antibodies against COL14A1 and CXCL12 (Figure 4C). We detected COL14A1- and CXCL12-positive AG fibroblasts localized near gingival epithelial cells in the connective tissue papillae and free gingiva, as well as in the cervical zone of the periodontal ligament (PDL) space (Figure 4C). We note that this localization pattern of AG fibroblasts appears to be highly suitable for early immune surveillance during periodontitis pathogenesis.

Role of AG fibroblasts in myeloid cell activation

The interaction between chemokine ligands and their receptors has been extensively studied. Here, to evaluate the interaction between chemokine ligands strongly expressed by AG fibroblasts and chemokine receptors in innate immune cells, we matched AG fibroblast-expressed chemokines to expression of their putative receptors in myeloid cells. Results show that AG fibroblasts may regulate macrophages via the expression Ccl8 and Ccl11, which encode chemokines that can interact with CC chemokine receptors (CCRs) encoded by Ccr2 and Ccr5 in macrophages. Similarly, gene expression signatures suggest that AG fibroblast-mediated neutrophil regulation may occur through CCL8–CCR1, CXCL1–CXCR2, and CXCL12–CXCR4 interactions (Figure 5A and B). Notably, all chemokine–receptor pairs are expressed throughout periodontitis development, although expression of factors mediating the interaction between AG fibroblasts and macrophages was decreased on Day 7.

Figure 5.

Intercellular communication may occur via other ligand–receptor interactions, which induce downstream target gene expression in recipient cells. Here, we performed NicheNet analysis to identify potential interactions between gingival fibroblasts and myeloid cells, revealing a trend toward increasing ligand–target interactions between fibroblasts and myeloid cells from Day 1 to Day 7 (Figure 5C and D). On Day 1, ligand expression was present primarily in AG fibroblasts (Figure 5E), whereas on Day 7, MF fibroblasts also exhibited prominent ligand expression (Figure 5F). Analysis of target gene expression revealed that both macrophages and neutrophils expressed receptors capable of interacting with AG fibroblast ligands on Day 1 (Figure 5G). In contrast, on Day 7, receptor expression was almost exclusively present in neutrophils, suggesting these cells are primarily targeted by MFs and AG fibroblasts at later stages of periodontitis development (Figure 5H).

T cells and ILCs in periodontitis development

We next analyzed expression of T cell-related genes in our scRNA-seq dataset and found that the Day 0 healthy gingiva exclusively contained Th cells expressing Cd4⁴⁹. However, after ligature placement, cytotoxic T (Tc) cells expressing Cd8⁴⁹ emerged, and on Day 7, Cd4^- Zbtb16⁺ ILCs and Cd4⁺Foxp3⁺ regulatory T (Treg) cells⁵⁰ were also detected (Figure 6A and B). The ILCs expressed Nfil3, a basic leucine zipper (bZIP) transcription factor required for ILC development⁵¹ (Figure 6C). However, to our surprise, expression of Rorγ, Il17a, and Il17f was only detected in ILCs (Figure 6C), indicating that this gingival subpopulation is predominantly composed of ILC3s.

Figure 6.

ILC3s and Th17 cells share similar regulatory functions. However, the role of ILC3s in osteoimmunology has not been fully deciphered. Macrophage-colony stimulating factor (M-CSF), encoded by Csf1, and receptor activator of nuclear factor kappa-Β ligand (RANKL), encoded by Tnfsf11, are known to play critical roles in osteoclast induction. We found that in gingival tissue, AG fibroblasts and neutrophils are the primary cellular sources of Csf1; ILC3s also express this gene, albeit at lower levels (Figure 6E). Expression of Tnfsf11 was detected in AG fibroblasts and ILC3s (Figure 6G). These observations suggest that ILC3s may function as inducers of inflammatory osteoimmune responses, similar to Th17 cells, leading to osteoclastogenesis in periodontitis downstream of AG fibroblast activation.

ILC3s are critical for cervical alveolar bone resorption in the mouse periodontitis model

We next examined the role of ILC3s in periodontitis pathogenesis by measuring ligature-induced gingival defects and alveolar bone resorption in Rag2^-/- mice, which lack functional B, Th, and Tc cells, and Rag2–IL-2 receptor common gamma (Il2rg) double-knockout mice (Rag2^-/-γc^-/-), lacking all lymphocytes, including ILCs. After ligature placement, gingival defects developed similarly in wild-type (WT), Rag2^-/-, and Rag2^-/-γc^-/- mice (Figure 7A and B). However, we found that alveolar bone loss was decreased in Rag2^-/- mice and nearly eliminated in Rag2^-/-γc^-/- mice (Figure 7C and D—figure supplement 4A). MicroCT image analysis further indicated a better perseveration of alveolar bone structure in Rag2^-/-γc^-/- mice, relative to the other groups (Figure 7C—figure supplement 4B). Histologically, osteoclastic resorption lacunae were observed on the alveolar bone surface at the cervical PDL and tooth apex PDL zones only in WT mice (Figure 7E and F). In addition, we detected a significant decrease in the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the cervical PDL zone of Rag2^-/-γc^-/- mice and in the apical PDL zone of both Rag2^-/- and Rag2^-/-γc^-/- mice (Figure 7G and H). Collectively, these data suggest that ILC3s, not Th17 cells, are responsible for cervical alveolar bone resorption in the mouse periodontitis model, a pathological phenotype consistent with human disease.

Figure 7.

The role of AG fibroblasts and neutrophils in ILC3 development in periodontitis

Based on our present data, we hypothesize that ILC3s within the gingival tissue play a pathological role in alveolar bone resorption in our mouse model. We therefore aimed to identify the cells that promote ILC3 development in mice. Similar to Th17 cells, ILC3 development was shown to be triggered by IL-6 and IL-23a^{52, 53}. A survey of our scRNA-seq data identified epithelial cells, fibroblasts, and myeloid cells as the source of Il6 in gingival tissue (Figure 8A). On Days 1 and 4 following ligature placement, AG fibroblasts primarily expressed Il6, whereas neutrophils became the predominant source on Day 7. We further found that gingival epithelial cells comprise at least four different subpopulations, plus an additional group displaying an epithelial–mesenchymal transition (EMT) phenotype on Day 7; Il6 was expressed by several of these epithelial subpopulations, including the EMT subgroup (Figure 8A—figure supplement 5A–D). Il23a was also detected in AG fibroblasts and myeloid cells (Figure 8B), with expression present in all epithelial cell subsets at various points in periodontitis development (Figure 8B— figure supplement 5D).

Figure 8.

Lastly, we evaluated potential interactions between chemokine ligands expressed by AG fibroblasts and neutrophils and chemokine receptors in innate immune cells. Our data suggest the presence of a chemokine–receptor association between ILC3s and both AG fibroblasts (Figure 8C) and neutrophils (Figure 8D), although interactions with other innate immune cells are also possible. NicheNet analysis further identified potential ligand–target gene associations between lymphocytes, including ILC3s, and both fibroblasts (Figure 8E) and myeloid cells (Figure 8F). Ligand expression was more prominent in AG fibroblasts than in other fibroblast subpopulations (Figure 8G) and elevated in neutrophils relative to macrophages (Figure 8H). Additionally, target gene expression was detected in ILC3s, although it was not specific to these cells (Figure 8I, J). Thus, in total, our data suggest a regulatory role for a newly identified AG fibroblast subpopulation in the gingiva, which appears to orchestrate chronic gingival inflammation, at least in the early stages, and to promote alveolar bone resorption via stimulation of neutrophils and ILC3s.

Discussion

In this study, we used scRNA-seq to generate a gingival cell transcriptomic atlas across the time course of periodontal inflammation development, leading to pathological alveolar bone resorption, in a mouse ligature-induced model of disease. Our results suggest the presence of a unique and previously uncharacterized subpopulation of gingival fibroblasts, referred to as AG fibroblasts, which show upregulated expression of TLRs and chemokines immediately after ligature placement (Figure 3 and 4). Previous studies have proposed that TLR-expressing gingival fibroblasts regulate innate immune responses in the oral barrier tissue^{54, 55}; however, the precise mechanisms underlying this fibroblast-mediated regulation remained poorly understood. Here, we found that not all gingival fibroblasts acquired an immune-sensing capability, but rather, AG fibroblasts represent a distinct fibroblast subpopulation, capable of upregulating TLRs and potentially responding to microbial and tissue damage signals. Consistent with this possibility, the temporal expression of TLRs was found to be associated with increased expression of CC and CXC chemokine ligands.

The signature gene of AG fibroblasts is Col14a1, which encodes a member of the Fibril-Associated Collagen with Interrupted Triple-helices (FACIT) superfamily⁵⁶. To our knowledge, our present findings represent the first observation of COL14A1 in the gingiva (Figure 4). However, in a previous study, we detected COL14A1-positive AG fibroblasts immediately under the gingival epithelium and in the cervical region of PDL, making them ideally situated to mediate immune surveillance for oral stimuli and microbial signals²². Given the strategic localization within the gingiva and the temporal expression of TLRs and chemokines after ligature placement, we hypothesize that AG fibroblasts play a previously unrecognized role in innate immune regulation during periodontitis development.

Neutrophils are well-established as crucial players in periodontitis progression. One study found that gingival neutrophils in chronic periodontitis patients are longer-lived than those from healthy patients⁵⁷. Moreover, it was shown that dysregulated neutrophils can induce destruction of periodontal connective tissue through the production of toxic molecules, such as reactive oxygen species, leading to initiation of bone resorption^{58, 59}. Critically, neutrophil function in periodontitis is dependent on trafficking to the affected gingiva, and accordingly, we found that the proportion of neutrophils increased after ligature placement (Figure 2). Neutrophils were previously reported to express a relatively limited number of chemokine receptors, including CXCR2, CXCR4, and CCR1^{60, 61}. Consistent with prior studies, we detected expression of these chemokine receptors in gingival neutrophils and linked them to ligands expressed by AG fibroblasts—CXCL1, CXCL12, and CCL8, respectively (Figure 5). In addition to chemokines, neutrophils can respond to various chemoattractants that modulate and finetune various cellular behaviors, such as migration direction, adhesion strength, and functional heterogeneity⁶². Here, we observed evidence for substantial intracellular communication between AG fibroblasts and neutrophils through ligand–target gene interactions (Figure 5). Although validation of each putative ligand–receptor interaction is beyond the scope of this study, our data suggest that AG fibroblasts may play an important role in neutrophil trafficking in both early and established periodontitis lesions.

A primary pathological consequence of periodontitis is uncontrolled alveolar bone resorption, leading to tooth loss, which is thought to be mediated by osteoimmune pathways involving IL-17–expressing Th17 cells⁶³. In contrast, analysis of our scRNA-seq revealed expression of Rorγ, Il17a, and Il17f not in CD4⁺ Th cells, but rather, in lymphocytes with ILC characteristics (Figure 6). ILCs share phenotypic and functional features with CD4⁺ T cells, although they lack antigen-specific T cell receptors⁵¹. In particular, the gingival ILCs detected in our study strongly expressed Rorγ, Il17a, and Il17f, but not Tbx21 and Gata3, indicating they are ILC3s, which are functionally similar to Th17 cells. Notably, recent clinical studies have reported the presence of ILCs in gingiva from human periodontitis patients^{64, 65} and leptin receptor–deficient mice⁶⁶. However, the specific role of these cells in alveolar bone loss had not been previously established.

In the present study, we explored the role of ILC3s in periodontitis-associated bone loss using Rag2^-/- and Rag2^-/-γc^-/- mice and observed differential patterns of alveolar bone resorption following ligature placement in these animals. Rag2^-/- mutation prevents V(D)J recombination required for generating immunoglobulin and T cell receptors, resulting in the production of functionally immature B and T cells, including Th17 cells. However, ILCs do not undergo genomic receptor rearrangements and, thus, are unaffected by Rag2^-/- mutation. In contrast, Rag2^-/-γc^-/- mice have the Rag2^-/- mutation in combination with the Il2rg^-/- mutation, which disables common γ chain cytokines (γc). Therefore, in addition to non-functional B and T cells, these animals also have defective γc-dependent ILCs. Here, we found that Rag2^-/- mice contained a reduced number of osteoclasts in the apical PDL area, indicative of reduced bone resorption in this region, whereas a significant loss of bone resorption was present in both the cervical and apical PDL areas of Rag2^-/-γc^-/- mice (Figure 7). Human periodontitis induces alveolar bone at the cervical PDL zone relative to the gingival legion. In contrast, apical alveolar bone resorption is observed in clinical cases of root canal infection (apical periodontitis). Therefore, our findings suggest that ILCs, which are differentially impacted by Rag2^-/-γc^-/- vs. Rag2^-/- mutation, may be involved in human periodontitis-like alveolar bone resorption near the site of gingival inflammation. It must be noted that Rag2^-/- and Rag2^-/-γc^-/- mice developed gingival defects similar to WT mice. However, this observation may be explained by results from our previous study, which suggest that early gingival degradation in the mouse model is mediated by cathepsin K (CTSK) secreted from gingival fibroblasts²², rather than by activated lymphocytes.

Differentiation of ILC3s and Th17 cells is mediated by similar environmental cues, with IL- 6 and IL-23a playing critical roles in both pathways^{67, 68}. Our present scRNA-seq data (Figure 8) suggest that AG fibroblasts and neutrophils are the primary cellular sources of Il6 during the early and later stages, respectively, of periodontitis development. In contrast, Il23a was found to be expressed by a variety of cell types, such as AG fibroblasts, myeloid cells, and multiple subsets of epithelial cells, including those with an EMT phenotype⁶⁹. Further, CC and CXC chemokine–receptor associations between ILC3s and both AG fibroblasts and neutrophils appeared to be non-specific. Therefore, our data suggest that rather than a specific trigger, the collective gingival environment, which includes AG fibroblasts, might contribute to ILC3 differentiation.

In conclusion, based on our present findings, we propose that a previously unrecognized AG fibroblast subpopulation in the gingiva can facilitate immune surveillance and participate in the pathological regulation of innate immune cells, such as proinflammatory neutrophils, within oral barrier tissue (Figure 9). Moreover, we hypothesize that ILC3s in the inflamed gingiva play a critical role in pathological alveolar bone resorption in the mouse model of periodontitis and, potentially, in human disease. Thus, the newly proposed AG fibroblast–neutrophil–ILC3 axis may hold valuable clues for unraveling the pathological mechanisms underlying periodontitis development. Moreover, these findings also provide a basis for investigation of new preventive and therapeutic strategies to contain oral barrier inflammation and potentially sever the link between periodontitis and debilitating non-communicative metabolic and cardiovascular diseases.

Figure 9.

Methods

Animal care

All protocols for animal experiments were reviewed and approved by the University of California Los Angeles (UCLA) Animal Research Committee (ARC# 2003-009) and followed the Public Health Service Policy for the Humane Care and Use of Laboratory Animals and the UCLA Animal Care and Use Training Manual guidelines. C57BL/6J WT, Rag2^-/-, and Rag2^-/-γc^-/- mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Animals had free access to regular rodent diet and water and were maintained in standard housing conditions with 12-h light/dark cycles in the Division of Laboratory Animal Medicine at UCLA.

Evaluation of gingival defect and alveolar bone resorption in a ligature-induced mouse model of periodontitis

A silk thread was gently tied around the left maxillary second molar of 8- to 12-week-old female WT, Rag2^-/-, and Rag2^-/-γc^-/- mice under general inhalation anesthesia with isoflurane (Henry Schein, Melville, NY, USA). On Days 1, 3, and 7 after ligature placement, WT mice were randomly chosen and euthanized by 100% CO₂ inhalation. WT mice without ligature placement were used as Day 0 pre-periodontitis control. Rag2^-/- and Rag2^-/-γc^-/- mice were euthanized on Day 7. The palatal tissue was digitally photographed, and maxillae were harvested and fixed in 10% buffered formalin (Thermo Fisher Scientific, Waltham, MA, USA). The gingival defect area was measured from digital photographs, using the ImageJ Java-based image-processing program (NIH, Bethesda, MD, USA), and normalized to the circumferential area of the maxillary first molar. Fixed maxillae were subjected to microCT imaging at an energy level of 60 kV and 166 µA, and 3D images were reconstructed from microCT scans (Skyscan 1275: Bruker, Billerica, MA, USA). Alveolar bone loss was assessed at three sites (mesiobuccal cusp, distobuccal cusp, and distal cusp) of the first molar, two sites (mesiobuccal cusp and distobuccal cusp) of the second molar, and one site (buccal cusp) of the third molar by measuring the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) on the buccal and palatal side of the alveolar bone. Total bone loss was calculated from the six-site total CEJ–ABC distance. The bone volume/total volume (BV/TV) ratio, bone surface, trabecular number (Tb.N), and trabecular thickness (Tb.Th) in the buccal side of alveolar bone of the second molar were determined using the proprietary analysis program (CTan: Bruker, Billerica, MA, USA). Statistical analysis to assess differences among multiple experimental groups was performed using two-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test; p < 0.05 was considered to be statistically significant.

Single cell dissociation from mouse maxillary gingiva

On Days 1, 4, and 7 after ligature placement, mice were euthanized by 100% CO₂ inhalation. And maxillary gingival tissues (n = 4 per group) were harvested from freshly isolated mouse maxillae.

Collagenase II treatment

The tissues were cut into 1-mm pieces and placed immediately into digestion buffer, containing 1-mg/ml collagenase II (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), 10-units/ml DNase I (Sigma–Aldrich, St. Louis, MO, USA), and 1% bovine serum albumin (BSA; Sigma–Aldrich) in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies). The tissues were incubated in digestion buffer for 20 min at 37 °C on a shaker at 150 rpm and then passed through a 70-µm cell strainer. The collected cells were pelleted at 1,500 rpm for 10 min at 4 °C and resuspended in phosphate-buffered saline (PBS; Life Technologies), supplemented with 0.04% BSA (Cell suspension A).

Trypsin treatment

Immediately following collagenase II treatment, tissues were incubated in 0.25% trypsin (Life Technologies) and 10-units/ml DNase I for 30 min at 37 °C on a 150-rpm shaker. Trypsin was neutralized with fetal bovine serum (Life Technologies), and the tissues were passed through a 70-µm cell strainer and washed with DMEM. The collected cells were then pelleted at 1,500 rpm for 10 min at 4 °C and resuspended in PBS with 0.04% BSA (Cell suspension B). Cell suspension A and Cell suspension B were combined in one tube. An equal number of combined cell suspensions A and B from four animals per group were combined for scRNA-seq analysis (10X Genomics, San Francisco, CA, USA).

Cell clustering and Identification

Cell Ranger was used to align reads, generate feature–barcode matrices, and perform clustering and gene expression analyses on the scRNA-seq data, and the output from this program was analyzed using the R-program Seurat (https://satijalab.org/seurat/). Cells with <2,400 genes detected or >1% mitochondrial gene expression were filtered out as low-quality cells. Individual gene counts for each cell were divided by the total gene counts for that cell and multiplied by a scale factor of 10,000; natural-log transformation was then applied to the counts. The FindVariableFeatures function was used to select 2,000 variable genes with default parameters, and the ScaleData function was used to scale and center the counts in the dataset. Principal component analysis and Uniform Manifold Approximation and Projection dimensional reduction were performed on variably expressed genes. The cluster markers were found using the FindAllMarkers function, and cell types were manually annotated based on the cluster markers. Cell types were assigned based on expression of cell marker genes, and gene expression within different cell types was displayed using dot plots and violin plots.

Histological analysis

Fixed maxillae were decalcified in 10% EDTA (Sigma–Aldrich) for 3 weeks and then embedded in paraffin. Histological cross-sections were stained with hematoxylin and eosin (HE) and evaluated on a light microscope. Adjacent paraffin sections (4 µm) were subjected to a heat-induced epitope retrieval procedure and then immunohistochemically stained with polyclonal antibodies to COL14A1 (#PA5-49916, Thermo Fisher Scientific) and CXCL12 (#PA5-30603, Thermo Fisher Scientific) at a 1:100 dilution, followed by secondary antibody application, diaminobenzidine staining, and methylene blue counterstaining.

Using maxillary cross-sections of WT, Rag2^-/-, and Rag2^-/-γc^-/- mice, osteoclasts were evaluated by TRAP staining using a commercially available kit (Acid Phosphatase TRAP kit, Sigma–Aldrich), according to the manufacturer’s instructions. TRAP-positive cells were counted in the 0.01-mm² area. Statistical analysis to assess the differences among multiple experimental groups was performed using two-way ANOVA with Tukey’s multiple-comparison test; p < 0.05 was considered to be statistically significant.

Functional annotation and pathway enrichment analysis

Annotation and visualization of GO terms were performed by Metascape (http://metascape.org/gp/index.html#/main/step1). The top-100 differentially expressed genes in each population were input and filtered with the term “immune”. Filtered genes were then input, and only “biological process” gene sets were retrieved from the GO database.

Ligand–target matrix prediction

NicheNet (v.1.0.0, https://github.com/saeyslab/nichenetr) was used to predict interactions between cell types. In brief, the integrated Seurat object containing each cell subpopulation was input into the NicheNet Seurat wrapper. Sender cells and receiver cells were determined, and interactions between active ligands expressed by sender cells and target receptors expressed by receiver cells were predicted based on information in signaling and ligand–receptor databases.

Acknowledgements

This study was supported by NIH grants R01DE022550, R44DE025524, and C06RR014529 and by SINTX Technologies.

Author contributions

I.N. conceptualized the project and obtained funding. T.K. and A.H. performed animal studies.

H.O. collected microCT data from the animal model. T.K. and A.G. performed scRNA-seq and data analysis. T.K. and I.N. drafted the manuscript. All authors gave final approval and agreed to be accountable for all aspects of the work.

Competing interests

I.N. is a consultant for FUJI FILM Corp and BioVinc LLC and received a research fund from SINTX Technologies, Inc. I.N. and A.H. received a research fund from Maruho Co. Ltd. The other authors declare no competing interests.

Data availability statement

We specify where the data supporting the results of this study can be found and provide hyperlinks to publicly archived datasets analyzed or generated during the study, where applicable. scRNA-seq data are found in NIH Gene Expression Omnibus (GSE228635).

Supplemental Information

Figure supplement 1.

Figure supplement 2.

Figure supplement 3.

Figure supplement 4.

Figure supplement 5.