The Role of Gingival Fibroblasts in the Pathogenesis of Periodontitis

Abstract

Gingival fibroblasts (GFs) are essential components of the periodontium, which are responsible for the maintenance of tissue structure and integrity. However, the physiological role of GFs is not restricted to the production and remodeling of the extracellular matrix. GFs also act as sentinel cells that modulate the immune response to oral pathogens invading the gingival tissue. As an important “nonclassical” component of the innate immune system, GFs respond to bacteria and damage-related signals by producing cytokines, chemokines, and other inflammatory mediators. Although the activation of GFs supports the elimination of invading bacteria and the resolution of inflammation, their uncontrolled or excessive activation may promote inflammation and bone destruction. This occurs in periodontitis, a chronic inflammatory disease of the periodontium initiated and sustained by dysbiosis. In the inflamed gingival tissue, GFs acquire imprinted proinflammatory phenotypes that promote the growth of inflammophilic pathogens, stimulate osteoclastogenesis, and contribute to the chronicity of inflammation. In this review, we discuss the biological functions of GFs in healthy and inflamed gingival tissue, highlighting recent studies that provide insight into their role in the pathogenesis of periodontal diseases. We also draw parallels with the recently discovered fibroblast populations identified in other tissues and their roles in health and disease. This knowledge should be used in future studies to discover more about the role of GFs in periodontal diseases, especially chronic periodontitis, and to identify therapeutic strategies targeting their pathological interactions with oral pathogens and the immune system.

Fibroblasts are essential components of the stroma responsible for the maintenance of tissue structure and integrity. The main function of fibroblasts is the production and remodeling of the extracellular matrix (ECM), but it is now clear that they also have the capacity to respond to pathogens and damage-related signals by producing cytokines, chemokines, and other inflammatory mediators. By modulating host responses to pathogenic stimuli, fibroblasts act as sentinel cells representing an important “nonclassical” component of the innate immune system (Davidson et al. 2021). However, like immune cells, their uncontrolled activation leads to chronic inflammation and tissue damage caused by the excessive recruitment of leukocytes, the secretion of proteolytic enzymes such as matrix metalloproteinases (MMPs) and cathepsins, and the induction of osteoclastogenesis (Davidson et al. 2021).

The development of “omics” technologies, particularly single-cell transcriptomics, has led to rapid progress in fibroblast biology, allowing the characterization of fibroblast heterogeneity in different tissues and the discovery of their unique roles in health and disease (Davidson et al. 2021). Recent landmark studies have described the contributions of fibroblasts to organ-specific immune responses, characterized specific synovial fibroblast subsets that drive pathogenesis in rheumatoid arthritis (Croft et al. 2019), and identified a skin fibroblast population that is necessary for the development of inflammatory lesions in atopic dermatitis (Ko et al. 2022). A similar diversity has also been observed in cancer-associated fibroblast (CAF) populations, which exhibit tumor-restraining or tumor-promoting activities as well as context-dependent plasticity (Chen et al. 2021). Together with studies showing that the model of “innate immune memory” driven predominantly by epigenetic mechanisms is also operational in specific fibroblast populations (Crowley et al. 2018), this work has accelerated the development of therapeutic strategies targeting the destructive aspects of fibroblast activation in chronic inflammatory diseases.

Gingival fibroblasts (GFs) are among the most abundant cells in gingival tissue, and their interaction with oral pathogens has been studied for several decades. Early reports of significant decreases in the numbers of GFs during periodontitis were followed by in vivo studies identifying the molecular mechanisms underlying GF apoptosis (Zappa et al. 1992; Graves et al. 2001). These observations indicate that excessive GF apoptosis may limit the ability to repair gingival tissue damage caused by oral pathogens. Subsequent in vitro experiments have provided strong evidence that GFs not only passively respond to oral bacteria penetrating the epithelial barrier or cytokines that are present in inflamed gingival tissue but may also adopt imprinted proinflammatory phenotypes that promote the growth of inflammophilic pathogens and contribute to the chronicity of inflammation.

The recently published single-cell atlases of the human oral barrier tissues not only revealed a previously unappreciated heterogeneity of stromal cell populations (Caetano et al. 2021; Huang et al. 2021) but also confirmed the ability of GFs to drive inflammatory processes in periodontitis. In a seminal study comparing healthy and periodontitis-affected oral mucosa, Williams et al. (2021) identified the specific hyperresponsiveness of GFs toward neutrophil recruitment. Apart from GFs, periodontal ligament fibroblasts represent an important stromal cell population of the periodontium, and their involvement in the pathological processes in periodontitis has been thoroughly summarized elsewhere (Jönsson et al. 2011). In this review, we discuss recent insights into the functions of GFs in health and disease, highlighting their contribution to chronic inflammation and gingival tissue damage in periodontal disease. We also draw parallels with fibroblast populations in other diseases that could help to determine how GFs promote the pathogenesis of periodontitis and identify therapeutic strategies to target GF interactions with oral pathogens and the immune system.

Inflammatory Activation of GFs by Oral Pathogens and Their Virulence Factors

Complex host–pathogen interactions occur during the development and progression of periodontitis, with immune cells, epithelial cells, and also GFs playing important roles. The immunohistochemical analyses of the inflamed gingival tissue from patients with periodontitis suggested that GFs may be an important source of the monocyte chemokine C-C motif chemokine ligand-2 (CCL2) (also known as monocyte chemoattractant protein 1) (Yu and Graves 1995). The recent single-cell transcriptomic analysis of oral mucosa from patients with periodontitis and healthy controls confirmed and extended those early observations. GFs have been identified as the main cell population expressing inflammatory chemokines, particularly C-X-C motif chemokine ligand 1 (CXCL1), CXCL2, CXCL12, CCL2, and CCL19, that are responsible for the recruitment of neutrophils and lymphocytes to inflamed gingival tissue (Williams et al. 2021). In vitro studies demonstrated that GFs also produce a plethora of other inflammatory mediators that are involved in the pathogenesis of periodontitis, including cytokines (interleukin [IL]–6 and IL-1β), MMPs (MMP1, MMP3, MMP8, and MMP9), and prostaglandin E2 (PGE2) (Yucel-Lindberg and Båge 2013). This essential immune function of GFs relies on their ability to recognize and respond potently to a variety of pathogens and danger signals. GFs express multiple pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) 1 to 9, nucleotide-binding oligomerization domain-containing protein 1–2 (NOD1–2), and protease-activated receptor 1 (PAR-1) (Palm et al. 2015), allowing them to engage distinct signaling pathways and activate unique transcriptional programs in response to different pathogens (Fig. 1). The sentinel role of GFs is supported by studies demonstrating the elevated expression of several TLRs in GFs isolated from patients with periodontitis compared to healthy controls (Wang et al. 2003).

Figure 1.

Pattern recognition receptors involved in the gingival fibroblast (GF) inflammatory response to oral pathogens and their virulence factors. GFs express pattern recognition receptors that enable them to recognize and distinguish a variety of pathogens and modulate the host immune response accordingly. Toll-like receptor (TLR) 2, which recognizes both types of Porphyromonas gingivalis fimbriae (FimA and Mfa1) and lipoteichoic acid (LTA) from Filifactor alocis, plays a central role in pathogen recognition by GFs. TLR4 responds to lipopolysaccharide (LPS) from various oral Gram-negative bacteria. Protease-activated receptor 1 (PAR-1) is activated by proteinase cleavage, for example, by gingipains from P. gingivalis. Nucleotide-binding oligomerization domain-containing protein (NOD) 1 and NOD-2 are intracellular receptors activated by meso-diaminopimelic acid (meso-DAP) present in peptidoglycans from Gram-negative oral bacteria, and muramyl dipeptide (MDP), a cell wall component in all bacteria, respectively. Although these receptors activate distinct signaling cascades, they all converge on nuclear factor (NF)–κB activation, which promotes the transcription of inflammatory genes. Created with BioRender.com.

GFs express multiple PRRs, but many reports indicate that their responses to pathogens rely predominantly on TLR2. Gene silencing experiments have shown that TLR2 is required to induce the production of IL-6 and IL-8 following infection with Porphyromonas gingivalis, whereas PAR-1 is involved only in the production of IL-6. In contrast, the knockdown of TLR4 had no effect on the induction of cytokine production by P. gingivalis in GFs (Palm et al. 2015). Similarly, the inflammatory activation of GFs stimulated with P. gingivalis lipopolysaccharide (LPS) or synthetic TLR ligands was mediated by TLR2 rather than TLR4 (Morandini et al. 2013), although TLR2 activation by P. gingivalis LPS is caused by contaminating lipoproteins (Nativel et al. 2017). The recent analysis of a commercially available GF cell line immortalized with human telomerase (hTERT) provided additional evidence that TLR2 is necessary for P. gingivalis to activate GFs (Lagosz-Cwik et al. 2021). The immortalized cells express significantly lower levels of TLR2 than primary GFs, probably due to TLR2 promoter hypermethylation, and they fail to upregulate inflammatory mediators in response to P. gingivalis infection. Notably, the hTERT-immortalized GFs express TLR4 at levels comparable to primary cells and retain a partial ability to respond to Fusobacterium nucleatum (Lagosz-Cwik et al. 2021). This observation shows that, despite the predominant role of TLR2 in GF activation, other PRRs are involved in responses to different species of oral bacteria.

The effects of individual virulence factors on GF activation have been studied in most detail for P. gingivalis LPS (Tsai et al. 2014; Brinson et al. 2016; Herath et al. 2016). However, the number of in vitro studies examining GF responses to other virulence factors has recently increased (Table 1). It is important to note that GFs can distinguish between even minor structural variants of certain virulence factors, such as the acylation of LPS or the citrullination of fimbriae (Herath et al. 2016; Wielento et al. 2022).

Table 1.

Effects of Virulence Factors from Oral Pathogens on GFs: In Vitro Studies.

Virulence Factor	Bacterium	Biological Effects	Activated Receptors and Signaling Pathway(s)	References
LPS	Porphyromonas gingivalis	IL-8 and PGE2 production; synergistic with IL-1β to increase Myd88 expression	MEK/ERK pathway; activation of ERK, JNK, p38, and NF-κB pathways; TLR4–NF-κB signaling	(Tsai et al. 2014; Brinson et al. 2016)
LPS1690 (penta-acylated)	Porphyromonas gingivalis	Proinflammatory effect: production of IL-6, IL-8, cyclophilin, annexins, iNOS, galectin, cathepsins, and heat shock proteins; upregulation of MnSOD expression		(Herath et al. 2016)
LPS1435/1449 (tetra-acylated)	Porphyromonas gingivalis	Anti-inflammatory effect: upregulation of annexins A2 and A6		(Herath et al. 2016)
Citrullinated FimA fimbriae	Porphyromonas gingivalis (ATCC 33277)	Increased expression of COX-2, mPGES-1, IL-6, and IL-8	PGE2 synthesis pathway, p38 and NF-κB pathways, TLR2-dependent signaling	(Wielento et al. 2022)
Mfa1 fimbriae	Porphyromonas gingivalis (ATCC 33277)	Increased expression of CXCL1, CXCL3, ICAM1, and selectin E (in mouse GFs)	TLR4-dependent (mainly) and TLR2-dependent signaling	(Takayanagi et al. 2020)
Lipooligosaccharide	Treponema denticola	Increased secretion of IL-6, IL-8, CCL2, NO, PGE2, and MMP3	Fos, MAP kinases, and NF-κB signaling	(Tanabe et al. 2008)
Bacterial DNA	Aggregatibacter actinomycetemcomitans	IL-6, IL-1β, and TNF production		(Soto-Barreras et al. 2017)
Lipoteichoic acid	Filifactor alocis	Increased expression of TNF, IL-6, IL-8, and MMP2	NF-κB, ERK, JNK, and p38 pathways	(Yoo and Lee 2022)
TD92 protein	Treponema denticola	Activation of the caspase 4/cathepsin G complex		(Jun et al. 2018)

COX-2, cyclooxygenase 2; ERK, extracellular signal-regulated kinase; GF, gingival fibroblast; IL, interleukin; iNOS, inducible nitric oxide synthase; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAP, mitogen-activated protein; MMP, matrix metalloproteinase; mPGES-1, microsomal prostaglandin E synthase 1; NF-κB, nuclear factor–κB; NO, nitric oxide; PGE2, prostaglandin E2; TLR, Toll-like receptor; TNF, tumor necrosis factor.

Modulation of GF Activation by the Environment of the Oral Mucosa

The inflammatory activation of GFs and their responses to pathogens are also dynamically modulated by the local microenvironment of the oral mucosa, such as inflammatory cytokines present in the affected gingival tissue. Several cytokines, chemokines, MMPs, and components of the PGE2 synthesis pathway are upregulated in GFs exposed to tumor necrosis factor (TNF) or IL-1β (Båge et al. 2010; Maksylewicz et al. 2019). Furthermore, the involvement of nuclear factor–κB (NF-κB) and mitogen-activated protein (MAP) kinases in this process has been thoroughly characterized (Båge et al. 2010). Interestingly, the cytokines interferon-γ (IFN-γ) and IL-4 skew polarized GFs into functionally distinct subtypes that differ in their potential to activate immune cells and effect tissue repair (Ha et al. 2022), as well as their responses to oral pathogens (Jang et al. 2021). Although these in vitro phenotypes represent the extremes of the polarization spectrum and are unlikely to exist in vivo, single-cell analysis of the healthy oral mucosa identified transcriptionally distinct subtypes of oral fibroblasts that are characterized by gene signatures associated either with inflammatory responses or with collagen synthesis and matrix remodeling (Williams et al. 2021).

The local inflammatory environment also affects the sensitivity of GFs to oral pathogens and vice versa. IL-1β and P. gingivalis LPS synergistically induce the production of inflammatory cytokines by upregulating the adapter molecule MyD88 (Brinson et al. 2016). In contrast, the TNF-induced production of IL-8 by GFs is inhibited by P. gingivalis, and the underlying mechanism is dependent on proteolytic activity (Palm et al. 2013). GFs also respond potently to damage signals released from necrotic cells. Exposure to necrotic cell extracts not only stimulates GFs to produce IL-6 and IL-8 but also increases their sensitivity to bacterial virulence factors by upregulating TLR2 (Mori et al. 2015).

Inflammatory Phenotype of GFs from Patients with Periodontitis

The ability of GFs to act as the “innate immune memory” of the oral mucosa is another key aspect of their involvement in host immune responses. Cultured GFs isolated from patients with periodontitis are characterized by sustained inflammatory activation, unlike cells isolated from healthy individuals (Wang et al. 2003; Baek et al. 2013; Liu et al. 2021). GFs from patients with periodontitis also generate reactive oxygen species in larger amounts than healthy controls, which is probably driven by the elevated expression of mitochondrial p53 (Liu et al. 2021). The mechanisms underlying the imprinted inflammatory features of GFs from patients with periodontitis are not yet clear. In other diseases, such features are often driven by epigenetic mechanisms. Accordingly, the stimulation of GFs with IL-1β or PGE2 causes specific changes in DNA methylation at the promoters of inflammatory genes (Seutter et al. 2020).

There are conflicting reports concerning the responsiveness of GFs from healthy donors and patients with periodontitis to bacterial challenge. Some studies demonstrated that GFs isolated from patients with periodontitis became more responsive to stimulation with P. gingivalis LPS (Kang et al. 2016) or infection with live bacteria (Baek et al. 2013), whereas others showed a decrease in cytokine production compared to healthy controls (Fitzsimmons et al. 2018). Further research is needed to resolve these apparent discrepancies, but it is important to note that GFs do not develop tolerance to stimulation with P. gingivalis LPS (Ara et al. 2009).

Invasion of GFs by Oral Bacteria

The invasion of gingival tissues by periodontopathogens plays a key role in the development and progression of periodontitis. Internalized bacteria not only evade the host immune response but also penetrate host cells to facilitate systemic dissemination (Sakanaka et al. 2016). There is evidence that oral bacteria can invade GFs, although neither the mechanism nor the biological implications of this process are as thoroughly characterized as in gingival epithelial cells or macrophages. The analysis of pathogenic bacteria invading GFs in vitro demonstrated the low invasive potential of P. gingivalis, Treponema denticola, and Tannerella forsythia (Jang et al. 2017). In contrast, GF infection with F. nucleatum and Campylobacter gracilis results in high intracellular bacterial loads (Dabija-Wolter et al. 2009; Jang et al. 2017). This process could be driven in part by intracellular replication, as in the case of F. nucleatum (Dabija-Wolter et al. 2009).

The ability of P. gingivalis to invade GFs increased in a dose-dependent manner when cocultured or pretreated with F. nucleatum, suggesting that the latter induces the expression of molecules necessary for the internalization of P. gingivalis (Jang et al. 2017). Similarly, the invasion of GFs by P. gingivalis is facilitated by Candida albicans, which is frequently isolated from periodontal pockets. This process does not require bacterial adhesion to the cell surface and is possibly driven by the activation (mediated by dectin 1 and TLRs) of lipid rafts required for bacterial entry (Tamai et al. 2011). These data indicate that GFs may not be the primary cellular reservoir of key oral pathogens given the low efficiency of invasion, although this can change dynamically depending on the surrounding microbial community as well as the strain-dependent and environment-dependent expression of bacterial virulence factors. Additional studies using 3-dimensional gingival tissue models and more complex oral biofilms are required to assess the true contribution of GF invasion by oral pathogens to the systemic dissemination of bacteria and evasion of the immune response.

New Insights into GF Pathobiology in Periodontitis

The involvement of GFs in periodontitis is not limited to the production of inflammatory mediators and interactions with oral pathogens and their virulence factors. GFs participate in many other biological processes, such as alveolar bone remodeling, ECM turnover, and inflammatory cell death (Fig. 2). The contribution of GFs to these processes in healthy individuals promotes tissue homeostasis, but it may perpetuate chronic inflammation and exacerbate tissue damage during periodontitis.

Figure 2.

The many roles of gingival fibroblasts (GFs) in gingival tissue from patients with periodontitis and healthy controls. Under physiological conditions, GFs maintain connective tissue integrity by balancing the production of tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs). GFs also protect against alveolar bone resorption by releasing osteoprotegerin (OPG) and/or interleukin (IL)–4, which bind to receptor activator of NF-κB (RANK) on the surface of (pre)osteoclasts. In response to pathogens, GFs produce cytokines and chemokines. In healthy gingiva, this leads to the recruitment of neutrophils and the elimination of bacteria. In periodontal disease, GFs may exacerbate inflammation and tissue damage. The inflammatory environment and dysbiotic microflora promote GF activation and cytokine production. This leads to the infiltration of excessive numbers of immune cells to the inflamed gingival tissue. In addition, pathogenic bacteria and their virulence factors induce pyroptosis, a type of inflammatory cell death. GFs thus produce IL-1β, which further contributes to the chronicity of inflammation. In addition, oral pathogens and proinflammatory mediators stimulate GFs to produce elevated levels of MMPs and receptor activator of NF-κB ligand (RANKL), which cause extracellular matrix (ECM) degradation and the stimulation of osteoclastogenesis, respectively. Together, these processes contribute to the destruction of tooth-supporting tissue, alveolar bone resorption, and eventual tooth loss in periodontitis. Created with BioRender.com.

Regulation of Osteoclastogenesis and Osteogenesis by GFs

Alveolar bone resorption, which is one of the hallmarks of periodontitis, is another key aspect of gingival tissue homeostasis regulated by GFs. Bone remodeling relies on the equilibrium between osteogenesis and osteoclastogenesis, which are driven by osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL), respectively. In healthy individuals, GFs release OPG and IL-4 to suppress osteoclast differentiation, highlighting their bone-protective role in the absence of pathogenic bacteria or chronic inflammation (Ujiie et al. 2016). In contrast, the RANKL/OPG ratio in GFs has been shown to shift in favor of osteoclastogenesis in the presence of oral pathogens. P. gingivalis induces RANKL expression and downregulates the OPG gene in GFs to increase the RANKL/OPG ratio, which promotes osteoclast formation (Belibasakis et al. 2007). Similarly, supragingival and subgingival biofilms differentially affect the RANKL/OPG ratio in GFs. Subgingival biofilms are highly virulent, significantly increasing the osteoclastogenic potential of GFs compared to supragingival biofilms (Belibasakis et al. 2011).

Functional studies have not yet provided clear evidence that GFs promote osteoclastogenesis under inflammatory conditions. Conditioned media from GFs infected with P. gingivalis increase the number of osteoclasts in the absence of other stimuli, but they suppress osteoclast formation mediated by macrophage colony-stimulating factor (M-CSF) and RANKL (Scheres et al. 2011). Similarly, the chronic exposure of GFs to TLR2 or TLR4 agonists suppresses osteoclast formation when GFs are cocultured with peripheral blood mononuclear cells (Karlis et al. 2020).

GFs may also affect alveolar bone homeostasis by modulating osteogenesis. GFs release antagonists of bone morphogenetic proteins, such as Gremlin-1, which suppress osteoblast differentiation (Ghuman et al. 2019). However, GFs display a degree of plasticity that allows their transdifferentiation into functional osteoblasts (Cho et al. 2017). In vitro, this is achieved by treating GFs with a DNA methyltransferase inhibitor, which causes the demethylation and reexpression of osteogenic lineage genes (Cho et al. 2017). It is unclear whether GF-to-osteoblast transdifferentiation occurs in the gingival tissue under physiological conditions, but it may be possible in the future to treat periodontitis-related bone loss by targeting the epigenetic changes in GFs.

ECM Turnover by GFs

GFs actively regulate ECM turnover in gingival tissue by modulating the levels and/or activity of MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs) (Yucel-Lindberg and Båge 2013). A hepatocyte growth factor (HGF)–neutralizing antibody was recently shown to suppress collagen degradation by GFs, revealing an important role for GF-derived HGF in the regulation of ECM turnover. Elevated levels of HGF correlate with disease severity in patients with periodontal disease, so these observations indicate the therapeutic potential of HGF-targeting drugs (Yamaguchi et al. 2021). Regulation of the ECM is also fine-tuned by interactions between GFs and other cell types. The coculture of GFs with the monocytic cell line U937 significantly increased the production of MMP2 by GFs, and this was dependent on the upregulation of the glycoprotein CD147 (Lai et al. 2020).

GF Interactions with Immune Cells

GFs modulate the host immune responses not only through the pathogen-induced release of inflammatory mediators but also by direct contact with immune cells and/or by secretion of factors that affect immune cell functions. In the coculture of monocytes and GFs, IL-6 released by GFs enhanced MMP1 production by monocytes (Sundararaj et al. 2009). In contrast, P. gingivalis–induced TNF production by the differentiated THP-1 macrophages was diminished in the presence of oral fibroblast populations, including GFs, periodontal ligament fibroblasts, and fibroblasts derived from peri-implant mucosal tissue (Tzach-Nahman et al. 2017). This effect was partly mediated by IL-6 and IL-10 secreted by fibroblasts and required cell–cell contact (Tzach-Nahman et al. 2017). GFs have also been shown to support the survival and retention of lymphocytes and promote CD4⁺ and CD8⁺ T-cell proliferation (Moonen et al. 2018). Interestingly, stromal–immune cell cross-talk is bidirectional as coculture with lymphocytes significantly increases messenger RNA (mRNA) expression of IL6, IL1A, and IL1B in GFs (Murakami et al. 1999).

Inflammatory Cell Death of GFs

Increased levels of pyropotosis, a type of programmed inflammatory cell death, significantly contribute to the chronicity of inflammation in periodontitis (Xu et al. 2022). Recent studies have shown that GFs undergo pyroptosis when exposed to oral pathogens or their virulence factors. For example, P. gingivalis LPS synergistically induces the activation of NLRP3 inflammasomes in GFs cultured under hypoxic conditions. This is associated with the induction of IL-1β and gasdermin D expression, which promote pyroptosis and exacerbate the GF inflammatory response (Yang et al. 2021). Similarly, interaction between the T. denticola protein TD92 and α5β1 integrin activates cathepsin G, which in turn activates caspase 4. The resulting complex drives pyroptosis and IL-1β secretion (Jun et al. 2018). GFs undergoing inflammatory cell death are therefore a potential source of IL-1β in the inflamed gingival tissue.

Conclusions and Future Perspectives

GFs are tissue-resident sentinel cells that maintain gingival tissue homeostasis and modulate the immune response to invading oral pathogens. However, inappropriate or excessive GF activation promotes inflammation and bone destruction in periodontitis, significantly contributing to the persistence of the disease. Although the mechanistic basis and functional consequences of GF interactions with pathogenic bacteria have been thoroughly characterized, our current understanding of GF biology relies predominantly on GF bulk cultures that originate from collagenase-digested gingival tissue specimens. This experimental system generates a heterogeneous population of cells that cannot reveal differences between the GF subtypes present in gingival tissue. Future research should therefore focus on state-of-the-art approaches to unravel the functional specialization of different GF subpopulations residing in the gingival tissue of patients with periodontitis and healthy controls. This research can be guided by the recent discovery of distinct fibroblast populations that play specific roles in the pathogenesis of rheumatoid arthritis and atopic dermatitis. In rheumatoid arthritis, immune effector fibroblasts expressing fibroblast activation protein α (FAPα) and thymus cell antigen 1 (THY1) drive inflammation, whereas FAPα⁺THY1⁻ fibroblasts selectively mediate bone and cartilage damage (Croft et al. 2019). In atopic dermatitis, a subpopulation of fibroblasts expressing paired-related homeobox 1 (Prx1) maintains skin homeostasis, and the disruption of NF-κB signaling in this population is sufficient to induce inflammatory lesions (Ko et al. 2022). It is unclear whether similarly diverse GF functional phenotypes regulate distinct aspects of pathophysiology in the inflamed gingival tissue that characterizes periodontal disease.

Evidence from studies of fibroblasts in other tissues indicates that their anatomical diversity, immune potential, and memory of previous inflammatory or infectious events may be driven by epigenetic modifications, such as DNA methylation and histone modifications (Crowley et al. 2018). In this context, epigenetic regulatory mechanisms in GFs have received little attention. Dysregulated DNA methylation profiles have been reported in total gingival tissue from patients with periodontitis, but this type of analysis has not been extended to isolated GF populations (Jurdziński et al. 2020). Therefore, novel epigenomic and transcriptomic methods that provide a comprehensive characterization of epigenetic landscapes should be applied to GFs. This will not only improve our understanding of the molecular mechanisms underlying the imprinted pathological phenotypes of GFs in periodontal disease but may also facilitate the identification of novel targets for host modulation therapy. In this regard, the anti-inflammatory potential of drugs targeting histone modifications has been demonstrated in GFs from patients with periodontal disease (Maksylewicz et al. 2019; Lagosz et al. 2020).

Although our understanding of GF functional heterogeneity is still limited, the publication of a human mucosal cell atlas was an important milestone in the field that identified the unique functionality of oral fibroblasts in the immune system (Williams et al. 2021). By combining this atlas with increasingly sophisticated experimental approaches, we will undoubtedly accelerate research focusing on stromal–immune interactions in healthy and inflamed gingival tissue, paving the way for the therapeutic targeting of pathogenic GF populations.

Author Contributions

A. Wielento, K.B. Lagosz-Cwik, contributed to design, data analysis and interpretation, drafted the manuscript; J. Potempa, contributed to data conception and design, critically revised the manuscript; A.M. Grabiec, contributed to conception and design, data analysis and interpretation, drafted the manuscript. All authors critically reviewed the manuscript and approved the final draft.