As the host’s first line of defense against pathogenic microorganisms (18, 23, 24), neutrophil homeostasis is key to periodontal health (1).

The periodontal lesion is initiated as acute inflammation characterized by increased numbers of neutrophils migrating into the gingival crevice through the junctional epithelium (1, 25) as a result of chemotaxis by plaque. They are activated by chemoattractants macrophage inflammatory protein-1α (MIP-1α), C-X-C motif ligand 8 (CXCL8) and constitutive higher reactive oxygen species (ROS) (26) and initiate phagocytosis with the assistance of antibodies and complement (27, 28), causing tissue damage (29) and excessive release of destructive molecules, which can be used to distinguish healthy and inflammatory periodontal tissues (30). In patients with periodontitis, recruitment, migration, and infiltration of neutrophils are increased in the early stage, while a significant reduction in phagocyte functions of neutrophils was observed in individuals with periodontitis (31). All of these changes are influenced by cytokines [e.g., granulocyte-colony stimulating factor (G-CSF)] (25), miRNAs [e.g., nod-like receptor 12 (NLRP12)] (32) and inflammasomes [e.g., nod-like receptor 12(NLRP12)] (33). Proinflammatory cytokines [e.g., tumor necrosis factor (TNF)-α and interleukin (IL)-8], neutrophil enzymes, eosinophil cationic protein (ECP), histidine decarboxylase, histamine and neutrophil elastase (NE) secreted by neutrophils are increased (34–37) and anti-inflammatory cytokines such as IL-10 are decreased (38). All of these changes are affected by bacteria (such as P. gingivalis) and bacterial products [such as leukocyte toxin (LtxA)] (36, 38).

Oxidative stress is considered to be an important component in various diseases (39). Polyphenols are now attracting attention as potential sources of agents that can inhibit, reverse, or delay the progression of diseases caused by oxidative stress and inflammatory processes. The highest concentration of active polyphenols has been found in the oral mucosa (40). Resveratrol, quercetin, and N-acetylcysteine (NAC) can reduce the production of ROS by neutrophils and upregulate the synthesis of the type 1 collagen gene, therefore contributing to the integrity of gingival tissues and prevention of periodontitis. Among the three, resveratrol has the best effect as an antioxidant that slows the progression of periodontitis. However, further studies using in vivo models are necessary to support the clinical use of antioxidants as a supplement to reduce oxidative stress and prevent periodontitis in humans (41).

Progress have been made in the treatment of periodontitis with vitamins targeting neutrophils. Clinical studies have shown that ascorbic acid (vitamin C) can reduce inflammation in patients with periodontitis possibly because it usually acts as a reducing agent and can be used to treat periodontitis by reducing the extracellular oxidants of neutrophils (42, 43). An L-ascorbic acid derivative, L-Ascorbic acid 2-phosphate magnesium salt (APM), is effective in decreasing cell damage through the suppression of H₂O₂-induced intracellular ROS and inhibited IL-8 production through the suppression of TNF-α-induced intracellular ROS. This suggests that local application of APM can help to prevent periodontal diseases (44). In addition, 1, 25 dihydroxivitamin D3 can promote neutrophil apoptosis in type 2 diabetic periodontitis through the P38/MAPK pathway, reducing periodontitis (45) ( Figure 2 ).

However, the approach of using small molecules based on the mechanism of oxidative stress using exogenous antioxidants such as vitamin C to treat other inflammation-related diseases has failed. Therefore, the prospects for these treatments are not very optimistic. Periodontitis may result from interference at the ROS level, and the future research focuses on disease-related ROS source-specific inhibition (46). In this regard, resveratrol has the greater advantage (44).

Monocytes, an important cellular defense system against pathogens, significantly increase in periodontitis tissues, especially intermediate monocytes (47). A significantly higher proportion of intermediate [Cluster of Differentiation (CD)14(+)CD16(+)] monocytes was observed in chronic periodontitis and they overexpressed human leukocyte antigen-DR (HLA-DR) and programmed cell death ligand 1 (PD-L1), indicating an activated inflammatory state (48). In addition, CD45RA(+) monocytes were increased in aggressive periodontitis (47). Depressed chemotaxis of monocyte results in increased periodontal destruction (49). There is also a reduction in the function of phagocytes of monocytes, suggesting a decrease in immune defenses in periodontitis (31). Pathogens stimulation of monocytes resulted in increased CD40 and CD54 expression, and enhanced the secretion of high levels of cytokines such as TNF-α, IL-1β, IL-6, IL-8, IL-17, IL-23, monocyte chemoattractant protein-1 (MCP-1) and interferon inducible protein-10 (50, 51).

Resveratrol can treat periodontitis by reducing P. gingivalis-mediated activation of the NF-κB signaling pathway. The effect on NF-κB activation likely results from the ability of resveratrol to act as a proliferator-activated receptor-γ (PPAR-γ) agonist. It can also attenuate triggering receptor expressed on myeloid cells-1(TREM-1) gene expression as well as soluble TREM-1 secretion in monocytes (52).

In addition, intracanal metformin for apical periodontitis has therapeutic efficacy. It can suppress lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and NO production by monocytes, therefore inhibiting LPS-enhanced chemokine (C-C motif) ligand 2 (CCL-2) synthesis (53). Both of these drugs can eventually reduce bone resorption and improve periodontitis.

Macrophages are central players in the destructive and reparative phases of periodontal disease (54). The behavior changes of macrophages are closely related to the pathogens. Pro-inflammatory macrophages increase and are activated in periodontitis (55). Increased proinflammatory responses, phagocytosis, and metabolic activity of macrophages in diseased periodontal tissue are mainly affected by various pathogenic bacteria such as Fecal coliforms and bacterial products such as LtxA (56, 57). Polarization is the main feature of macrophages in periodontitis that differentiates them from those in normal tissues (58). In periodontitis, macrophages tend to differentiate in the direction of M1, while M2 differentiation is inhibited significantly (48). Periodontitis is characterized by increased production of proinflammatory mediators and matrix-degrading enzymes by macrophages and increased osteoclastic activity (55). In addition, macrophages secrete increased proinflammatory cytokines such as TNF-α, interferon-γ (IFN-γ), IL-1α, IL-1β, IL-6, and IL-12; increased adhesion factors such as CXCL5 and CXCL1 (54, 59, 60); and increased inflammatory bodies such as NLRP3. In addition, the expression of other molecules such as toll-like receptors 2 (TLR2), TLR4, and nucleotide-binding oligomerization domain 2 (NOD2) is also increased (61), mainly by in vivo cytokines such as IL-17 and pathogenic bacteria such as Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) (62, 63).

There is much research on drugs targeting macrophages to treat periodontitis. Proanthocyanidins (PACN) and cranberry proanthocyanidins (PACs) are two of the most active substances with positive effects on both cell behavior and molecular expression. Because of a lower risk of development of resistance and side effects, PACN, a multicomponent plant-derived antibacterial substance, has become a promising alternative and adjunctive therapy candidate for the treatment of periodontitis. Pelargonium sidoides dendritic cell root extract (PSRE), as one of the most PACN-enriched plants, can inhibit IL-8 and prostaglandin E2 (PGE2) produced by LPS-induced fibroblasts and IL-6 by leukocytes, blocking the expression of CD80 and CD86 on the surface of macrophages and IL-1 and cyclooxygenase-2 (COX-2) in leukocytes. Thus, PACN could be an effective drug for periodontitis (64).

PACs can neutralize the cytolytic and proinflammatory responses in human macrophages treated with LtxA. It can protect macrophages against the cytotoxic effect of purified LtxA, inhibiting caspase-1 activation, and consequently decreasing the secretion of IL-1β and IL-18. Apart from the above therapeutic effects, highbush blueberry PACs can also inhibit the release of TNF-α, IL-6, CXCL8, matrix metalloproteinase-3 (MMP-3), MMP-9, and TREM-1 in a dose-dependent manner. PACs have been a potential candidate for the treatment and prevention of periodontal disease because of the combination of strong pathogen-selective antibacterial, anti-inflammatory, and gingival tissue protection properties (57, 65) ( Figure 3 ).

By summarizing the drugs studied in recent years, we found that most of the drugs can affect the polarization and infiltration behaviors of macrophages [such as CSINCpi-2 (66), Metformin (67), and CCL2 MPs (68)] and inhibit the production of a variety of pro-inflammatory cytokines by macrophages [such as PMX205 (69), CSINCpi-2 (66), 6-Shogaol (70)], a few drugs can promote the production of anti-inflammatory cytokines [e.g. PMX205 (69)]. In addition, they still have some other effects, such as promoting bone regeneration [Dioscin (71)], antibacterial (Perillyl alcohol [POH) (72)] and so on. The effects of other drugs targeting macrophages with positive effects either on cell behavior or molecular expression are shown in Table 1 .

T lymphocytes in tissues can maintain balanced in the gingival environment (83). The number of T cells is significantly higher and they play an important role in alveolar bone resorption (84–86). The differentiation of T cells is caused by ongoing microbial challenges and the ensuing inflammation (87). The activation of different T cell subtypes controls chronic inflammation through secretion of cytokines and regulation of osteoclast production, and they play an important role in determining whether the inflammatory lesion will lead to tissue-destructive periodontitis (83). Overall, Th1 and Th17 responses increase while Th2 and Treg responses decrease in periodontitis, which can independently or interactively increase the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio (88, 89). The early oral infection response is mainly attributed to pathogenic Th17 up regulation or protective Treg downregulation, and this imbalance determines the resorption of alveolar bone (90). Persistent oral P. gingivalis infection stimulates an initial IL-17A-based response changing into a later de novo Th1 response with only sporadic transdifferentiation of Th17 cells (87). As for the memory T cell subsets, a significant increase in the proportion of CD4(+)CD69(+) CD103(-) memory T cells was observed in periodontitis tissues compared with healthy gingiva (91). CD4(+) memory T cells from periodontitis tissues produced either IL-17 or IFN-γ whereas CD8(+) memory T cells produced only IFN-γ (91). In addition, during the development of periodontitis, the expression levels of IFN-γ (linked to Th cell polarization toward the Th1 cells), IL-17A, IL-17F, IL-1β, IL-6, IL-23 (linked to Th cell polarization toward the Th17 cells), TNF-α, RANKL, glucocorticoid-induced TNFR-related gene (GITR), T-bet, and GATA-3 are all highly increased (92–94). While less IL-4 (linked to Th cell polarization toward the Th2 cells), IL-10 (linked to Th cell polarization toward the Treg) and transforming growth factor-β (TGF-β) are detected in the patients with periodontitis (95). The more detail of changes of various T cell subtypes in periodontitis is summarized in Supplementary Table 1 .

Some drugs targeting T lymphocytes can regulate the differentiation of T cells to reduce inflammation, thereby reducing bone loss and improving periodontitis. Astragaloside IV (AsIV), as one of the active ingredients in the medicinal plant Astragalus membranaceus, can increase peripheral blood CD4(+)T cell percentages and the CD4(+)/CD8(+) T cell ratio, while the percentage of CD8(+) T cells can be significantly reduced, as well as TNF-α, IL-1β, IL-2, IgA, and IgG. The reduction in IgA and IgG may be because the drugs target CD4(+) T cells, reducing T cell-dependent antibody responses. By this mechanism, AsIV can slow the progress of periodontitis by suppressing inflammation (96).

Other drugs, for example, curcumin and calcitriol, can regulate the differentiation of Th cells, thus playing a therapeutic role. Both of them can inhibit the loss of alveolar bone by changing the proportion and function of Th cell subsets, which is manifested by the increase of Tregs and the decrease of Th17 cells. Calcitriol intervention can also increase Th2 polarization potential and decrease the Th1 promoter (97, 98). In addition, curcumin also exerts antibacterial and antioxidant effects (99, 100) ( Figure 4 ).

For periodontitis in patients with Parkinson’s disease, vitamin D can reduce peripheral blood CD3, CTL counts, proinflammatory cytokines in saliva, and autophagy-related proteins in whole peripheral blood mononuclear cells. Vitamin D had varied effects on reducing systemic inflammation and promoting the induction of autophagy-related proteins related to antibacterial function. This study has entered the clinical trial stage (101).

Imbalance in the inflammatory cytokine network is involved in the periodontal disease process. The interactions between the pathogens and host cells, including leukocytes, can lead to a cytokine cascade. Pro-inflammatory cytokines (such as IL-1 and TNF) lead to periodontitis while anti-inflammatory cytokines ameliorate the disease (102). At the site of periodontitis, the levels of proinflammatory cytokines such as TNF-α, IFN-γ, IL-1, IL-6, IL-12 and G-CSF are significantly increased (25, 54, 103) while anti-inflammatory cytokines are decreased (IL-4 and IL-10) (104). Cytokines and other molecules can be used to diagnose periodontitis, in which the combination of IL-6 and MMP-8 shows the best diagnostic performance (105).

Drugs that target cytokines have been mostly studied in vitro. They can inhibit inflammation and reduce bone resorption by inhibiting the production and action of pro-inflammatory factors. Among them, most drugs can inhibit the expression of TNF-α, such as trans-cinnamic aldehyde (106), kawa-205ME (107), β-carotene (108), calcitonin gene-related peptide (CGRP) (109), Platycarya strobilacea leaf extract (PLE) (110), and bismuth drugs (111). In addition, trans-cinnamic aldehyde (106), psoralen and angelicin (112), bismuth drugs (111), SIM-PPI (113), and benzydamine (114) can inhibit the expression of IL-1β. On the contrary, drugs that target cytokines have no effect on anti-inflammatory factors. This may be because some studies have shown that there is little change in anti-inflammatory cytokines in gingival crevicular fluid in patients with periodontitis (115). More detailed effects of these drugs are shown in Table 2 .

Oral probiotics are a relatively safe and effective adjunctive treatment for periodontitis. Their complementary use has the potential to improve disease indicators and reduce the need for antibiotics (142). In order to make better use of adjuvant therapy of probiotics, we believe that it is necessary to find more correct probiotics strains, gain more recognition from patients, and focus on developing more individualized treatment plans (143).

Probiotic-assisted routine treatment of periodontitis treatment (144) may have a positive impact on the immune prognosis of patients. It has been found that GCF/MMP-8 levels were significantly reduced in patients treated with scraping and root planning (SRP) and probiotics combined (145). Significantly reduced levels of pro-inflammatory cytokines IL-1ß and IL-8 were observed in patients with generalized chronic periodontitis treated with a probiotic buccal adjuvant containing Bifidobacterium animalis subsp. lactis (B. lactis) HN019 for SRP (146), while the levels of β-defensin-3, TLR4, CD57 and CD4 were significantly increased (147). Supplementation with probiotics containing Lactobacillus reuteri during periodontal therapy was associated with a significant decrease in MMP-8 levels and a significant increase in matrix metalloproteinase-tissue inhibitor (TIMP-1) levels. This suggests that lozenges reduce inflammatory markers in the short term (148). Using the intestinal symbiotic Akkermansia Muciniphila in an experimental periodontitis model induced by P. gingivalis, alveolar bone loss was improved. In vitro, bone marrow macrophages increased IL-10 and decreased IL-12, and expressions of connective integrity markers such as integrin -β1, e-cadherin, and ZO-1 in gingival epithelial cells were also increased. This proves that Akkermansia muciniphila can be considered as an adjunct to periodontal therapy (149).

As an independent means of treatment, the therapeutic effect of probiotics has also been positive. Gastric administration of Lactobacillus gasseri SBT2055 in mice enhanced immune regulation and prevented periodontitis through intestinal immune system. The expression and secretion of TNF-α and IL-6 decreased significantly. The mRNA and peptide products of β-defensin-14 were significantly increased in the distal mucosa and intestinal tract of mice (150).

In addition, probiotics have also been found to have a preventive effect. Prophylactic administration of a combination of omega-3 and probiotics reduced alveolar bone loss and improved serum IL-1β, IL-6, and IL-10 levels (151).

Existing meta-analyses have shown that probiotics have a positive therapeutic effect on periodontitis (152). Many probiotic strains have strong aggregation ability, strong adhesion ability to oral tissue, and high antagonistic activity against oral pathogens. And they were largely free of antibiotic resistance (153). However, this treatment does not seem to be a permanent solution, as most of these probiotics originate from the external oral microenvironment and may not succeed in permanently colonizing the oral cavity. For them to continue to play an active therapeutic role, we need to develop a more appropriate frequency of administration (154). Therefore, the extraction of probiotics from the mouth of healthy people may promote the permanent colonization of probiotics and may be a more ideal treatment method (155).

There have been many achievements in antibacterial treatment of periodontitis, but there is still no clinical treatment available. As for antibacterial therapy, the main method is to induce immunity to pathogens through vaccination.

P. gingivalis capsular defect mutant strains cause reduced loss of alveolar bone because of non-expression of RANKL and a decrease in Th1/Th17 cytokines, Th1/Th17 lymphocytes, and osteoclasts (156). Subcutaneously vaccination with formalin-killed P. gingivalis can result in upregulation of Tregs through the production of IL-10 and TGF-β, downregulation of Th17 cells and IL-17A production and inhibition of lymphocyte proliferation (157). P. gingivalis-specific inflammatory immune response can be protected by therapeutic vaccination with a chimera (KAS2-A1) (parenteral or intraoral administration) immunogen targeting the major virulence factors of the bacterium, the gingipain proteinases. In addition, this protection is characterized by an antigen-specific IgG1 isotype antibody and Th2 response, which produced an effective therapeutic intervention that protected against P. gingivalis-induced periodontitis (158).

To date, however, P. gingivalis vaccination has been studied only in animals, and no effective prophylactic human periodontal vaccine has been developed, with the reason for the failure of prophylactic human periodontal vaccines unknown (157). We consider patients with P. gingivalis-associated periodontitis have higher threshold levels of pathogens in the subgingival plaque and exhibit an inflammatory immune response. Therefore, therapeutic vaccination may exacerbate inflammation and bone resorption in these patients ( Figure 5 ).

PDLSCs in periodontitis tissues have impaired immune regulatory function due to changes in their inflammatory microenvironment, resulting in immune response imbalance and inflammation-related bone loss. PDLSCs provide new prospects and potential therapeutic cells for tooth regeneration and reconstruction of periodontal ligament tissue damaged by periodontal disease (172, 173).

PDLSCs possess low immunogenicity and marked immunosuppression via PGE2-induced T-cell anergy (174). PDLSCs can induce polarization of macrophages to the M2 phenotype, which contributes to enhanced periodontal regeneration after stem cell transplantation (175). In addition, PDLSCs significantly decrease the level of non-classical major histocompatibility complex glycoprotein CD1b on DCs, resulting in defective T cell proliferation, demonstrating their potential to be utilized in promising new stem cell therapies (176). The use of allogeneic PDLSCs in a miniature pig model led to a reduction in humoral immunity. This may be because PDLSCs inhibit B cell activation through intercellular contact, mainly mediated by programmed cell death protein 1 (PD-1) and PD-L1. In addition, PDLSCs can inhibit the proliferation, differentiation and migration of B cells. But interestingly, PDLSCs enhanced B cell viability by secreting IL-6 (177).

Approaches based on extracellular vesicles (EVs) appear to provide a new paradigm for cell-free therapies that overcomes many of the clinical limitations of current cell transplantation. As an ideal vector, EVs have been shown to display anti-inflammatory and immunosuppressive actions in different tissues and could represent a potent therapeutic tools against chronic inflammation during periodontitis (178).

EVs from PDLSCs grown on gelatin-coated alginate microcarriers may be used to target chronic inflammation during periodontitis in bioreactors. EVs permanently suppressed basal and LPS-induced activity of NF-κB in PDLSCs and partially suppressed inhibitory effect of anti-TLR4 blocking Ab, without affection to osteogenic mineralization (178). MicroRNA-155-5p in PDLSC-derived EVs can upregulate sirtuin-1 in CD4(+) T cells, thereby alleviating Th17/Treg imbalance (90). Furthermore, the conditioned medium of PDLSCs reduced mRNA levels of TNF-α in periodontal healing tissue (179).

GMSCs, a unique group of MSCs with the characteristic of inflammatory resistance, have been the focus of extensive research due to their easy accessibility, numerous distinct properties, including their homing to injury sites, their contribution to tissue regeneration and prominent immunomodulatory properties (160, 180).

It has been reported when transplanting GMSCs via the tail vein of mice, these cells were able to enter the site of periodontal injury (181). The delivery of GMSCs led to a significant decrease in TNF-α, IL-1β and IL-6 (M1 markers), a significant increase in IL-10 (M2 markers), thus inhibiting the activation of M1 macrophages (182, 183). GMSCs can also decrease the infiltration of DCs, MCs, CD8(+) T cells and Th17 cells, and increase the infiltration of Tregs (184). Hypoxic stimulation promoted the immunomodulatory properties of human GMSCs by enhancing the suppressive effects of human GMSCs on peripheral blood mononuclear cells (PBMCs) (185). In addition, GMSC-derived EVs can promote the conversion of M1 to M2 and reduce the proinflammatory cytokines produced by M1 macrophages (such as TNF-α, IL-1β and IL-12) (186).

However, many issues need to be resolved, such as costs, time-consuming culture procedures, insufficient stem cell sources, and other safety issues (187, 188).

In an induced rat model of periodontitis, SHED survived in periodontal tissue for about 7 days with minimal tissue diffusion. Then, the treatment of periodontitis with multi-dose SHED every 7 days can change the expression profile of cytokines in gingival crevicular fluid, with a reduction in the pro-inflammatory cytokines TNF-α, IFN-γ and IL-2, and an increase in the anti-inflammatory molecule IL-10. SHED can also promote the differentiation of macrophage M2 (189) and induce an immune regulatory phenotype in monocyte derived DC (moDCs) cells, thus increasing CD4(+)Foxp3(+)IL-10(+) T cells (190).

IFN-γ stimulated DFSCs by inducing immunomodulated effects of healthy donor PBMC, promoting the proliferation and differentiation of DFSCs, thereby inhibiting IL-4 and IFN-γ levels, increasing IL-10 levels, and increasing the number of CD4(+)FoxP3(+) cells (191). Dental follicle progenitor cells (DFPCs) can also sense and respond to LPS, resulting in the down-regulation of TLR4 mRNA expression and significantly increasing the migration of DFPCs. But IL-6 levels remained the same. Based on the role of DFPCs in the immune microenvironment of periodontitis, the potential of DFPCs as biological grafts for periodontal regeneration has been further confirmed (192).

Using BMMSCs in a rat model of periodontitis, significant reverse of alveolar bone lesion was observed after BMMSC transplantation. The expression of TNF-α, IFN-γ and IL-1β was down-regulated by BMMSC transplantation (193, 194). When combined with acetylsalicylic acid, the levels of TNF-α and IL-17 decreased, while the levels of IL-10 increased, and the inflammatory microenvironment was improved more (195). Injection of BMMSCs in a mouse model of periodontitis was also shown to reduce periodontitis inflammation (196). Meanwhile, BMMSCs-derived apoptotic extracellular vesicles (ApoEVs) could also regulate the polarization of macrophages (197).

DPSCs are capable of self-renewal and multidirectional differentiation, which provides a broad prospect for tooth regeneration. DPSCs have low immunogenicity and can inhibit lymphocyte proliferation and regulate cytokine production in vitro. DPSCs can inhibit T cell proliferation, B cell proliferation and mixed lymphocyte response. The number of Th17 cells in peripheral blood mononuclear cells co-cultured with DPSCs was significantly increased, while the number of Treg was significantly decreased. DPSCs significantly inhibited the secretion of TNF-α, IFN-γ, IL-2 and IL-17 and promoted IL-10 secretion without affecting IL-1β and IL-6 production (198, 199). These results have shed light on the therapeutic mechanism of DPSCs. In addition, DPSC-derived exosomes-incorporated chitosan hydrogel (DPSC-Exo/CS) can also facilitate macrophages to convert from a pro-inflammatory phenotype to an anti-inflammatory phenotype, thus ameliorating periodontal lesion (200).

The more detailed role of MSCs in the immune microenvironment of periodontitis is summarized in Table 3 .

Oral MSCs have the unique clinical advantage of availability in large numbers, controlling proliferation, migration and homing, multidirectional differentiation, and inflammatory responses. However, in order to convert laboratory periodontal regeneration methods to clinical application, the mechanisms of cell-based immunomodulatory and regeneration processes need to be understood (187).