Resolution of inflammation in periodontitis: a review

Abstract

Inflammation is a physiological response to an injury or infection. It is supposed to be self-limiting, stopping when the situation recovers to normal to protect the tissue. This self-limiting action is called “resolution of inflammation”. Currently, periodontitis is thought to be the result of failed resolution of inflammation; specifically, it is the result of excessive inflammation that leads to gingival recession and alveolar bone loss. In this review, we will focus on the processes of resolution of inflammation in periodontitis, which may be a therapeutic target of periodontitis.

Introduction

Inflammation is assumed to occur to protect tissue that has been infected or injured. There are different results after the onset of an inflammatory response: ideally, the pathogenic stimuli are eliminated, and the tissues return to homeostasis, which is the optimal response; however, if the resolution process fails, the inflammation becomes uncontrolled and chronic [1]. Recently, it has become clear that the inflammatory process includes key checkpoints that regulate its onset, maintenance, and resolution [2,3].

Periodontitis is globally prevalent and is the major cause of tooth loss in adults [4]. Over the last four decades, there is no evidence that the prevalence of periodontitis has changed over time [5]. Periodontitis is an inflammation induced by bacterial pathogens. It is characterized by gingival recession and alveolar bone loss [6]. Until now, the conventional therapy to periodontitis is scaling and root planning [7]. Nowadays it is recognized that the bacterial biofilm-induced body response is the essential cause of periodontal destruction during the pathogenesis of the disease [8]. It is now thought that periodontitis is the result of uncontrolled and unresolved inflammation [9,10]. Therefore, we should more deeply examine the molecular and cellular mechanisms of inflammation resolution and target them to treat periodontitis. The resolution of inflammation in periodontitis should be an active process involving a series of cells and processes. Inflammation-induced recruitment of neutrophils should change to apoptosis in a timely manner. Pro-inflammation macrophages should be reprogrammed to become pro-resolution macrophages. Lymphangiogenesis and lymphatic function play a key role in the balance of inflammation in periodontitis. Regarding the adaptive immune responses, Treg cells are important for inhibiting excessive inflammation. More importantly, clinical trials show that pro-resolution mediators are effective for resolving periodontitis [11].

Neutrophil apoptosis

Neutrophils constitute 40-60% of the white blood cell population and form the body’s first line of defense against invading pathogens, such as bacteria. They are the first leukocytes to enter the inflammatory site, and they can eliminate pathogens via multiple mechanisms, such as proteolytic enzymes, antimicrobial proteins and reactive oxygen species [12]. Under normal conditions, the longevity of neutrophils is short, lasting approximately a few hours. During inflammation, the longevity of neutrophils increases because of their activation, which ensures that they participate in the inflammatory responses [13]. Neutrophils have been shown to be present in the inflamed periodontium, and chemotaxis disorders in neutrophils will increase the severity of periodontitis [14,15].

However, the excessive presence of neutrophils will prolong the process of inflammation by continuing to produce antigens and secrete inflammatory cytokines and will damage the tissue by secreting protein enzymes and toxic oxygen [16]. Therefore, the timely apoptosis and phagocytosis of neutrophils is extremely important for resolving inflammation and restoring tissue. The morphology of apoptotic neutrophils is infrequently detected during the resolution stage [17]. Neutrophil apoptosis is mediated by various extrinsic and intrinsic factors; for example, cell-to-cell Fas/Fasl contact can induce apoptosis, and reactive oxygen species (ROS) from the NAPDH oxidase also limits the survival of neutrophils. Furthermore, some environmental circumstances, such as hypoxia, interfere with neutrophil longevity [3,18,19]. Apoptotic neutrophils are removed via efferocytosis, through which they are found and eaten by macrophages via specific signals. Lysophos-phatidylcholine (LPC), sphingosine 1-phosphate (S1P), fractalkine (CX3CL1) help macrophages find apoptotic neutrophils. Phosphatidylserine (PS) is the signal that apoptotic neutrophils express to indicate that they should be eaten [3,20]. Neutrophil apoptosis helps to resolve inflammation not only by decreasing the number of existing neutrophils but also by expressing signals that block the recruitment of more neutrophils and influence the function of other immune cells, such as annexin A1 and lactoferrin. The subsequent efferocytosis inhibits the secondary necrosis of neutrophils, which would otherwise release toxic granules and oxygen species and harm the tissue [19].

The study of apoptosis in periodontitis began a decade ago, but few later studies have been conducted. Gamonal and colleagues [21] demonstrated that the cytokines TNFα and GM-CSF were elevated in gingival crevicular fluid. In a later ex vivo gingival tissue biopsy culture, 100 ug/ml GM-CSF was added to the medium for 2 and 4 days. Apoptosis was measured using the terminal TdT-mediated Dutp-biotin nick-end labelling (TUNEL) technique, and Bax expression was observed by immunohistochemistry. The results showed that apoptosis was reduced in the GM-CSF treated gingiva. This research implied a novel mechanism of periodontitis in which GM-CSF-induced delays in neutrophil apoptosis contributed to the failed resolution of periodontitis. The results of a recent study were consistent with this conclusion [22]. Svetislav Zaric and colleagues found that P. gingivalis LPS could only induce partial immune tolerance in the human monocytic cell line THP-1 because although the secretion of TNFα was decreased, the production of IL-8 remained high after repeated LPS challenges. Furthermore, the incubation of neutrophils with the supernatant of monocytes with impaired immune tolerance displayed a high migration index and increased apoptosis. This result reinforced the belief that the delayed apoptosis of neutrophils prolongs the inflammation that characterizes periodontitis. Another study positively confirmed the importance of neutrophil apoptosis in periodontitis [24]. Isolated neutrophils were induced to apoptosis in vitro which was confirmed with Annexin V positivity and the loss of CD16 expression. Then, isolated monocytes were cultured with apoptotic neutrophils or fresh neutrophils or alone. All three groups were stimulated with P. gingivalis LPS. The ELISA results showed that IL-10 levels were increased and IL-1β levels were inhibited in the apoptotic neutrophil-treated group compared with the other groups. This study’s results demonstrated a typical mechanism through which neutrophil apoptosis helps resolve inflammation by producing a signal to stimulate monocytes to produce pro-resolution cytokines and suppress pro-inflammatory cytokines. This suggests that neutrophil apoptosis may be a potential target for resolving periodontitis. Currently, there are several methods for modulating neutrophil apoptosis, such as the use of pharmacological inhibitors of cyclin-dependent kinases to drive neutrophil apoptosis [24,25]. Targeting apoptosis-associated pathways is another means of promoting resolution. Pharmacological inhibitors of ERK1/2, which is involved in the MAPK signal pathway, suppress the production of neutrophil survival factors and indirectly increase the apoptosis of neutrophils, which helps to resolve inflammation [26].

Macrophage reprogramming

Macrophages exist in nearly all tissues in the human body. Under normal or inflammatory situations, the circulating peripheral blood mononuclear cells recruit into the tissues and differentiate into macrophages [27]. There are many specific types of macrophages in the corresponding tissues: osteoclasts in the bone, microglial cells in the central nervous system, Kupffer cells in the liver and others [28]. In contrast with the replenishment function exhibited by recruited monocytes, macrophages may represent a self-renewal feature similar to that of stem cell and may proliferate in the tissue once they are activated by a stimulus [29]. Regarding the periodontium, after stimulation by P. gingivalis, monocytes and macrophages are recruited to the periodontium, which results in periodontitis [6].

Because of their cellular differentiation, widespread tissue distribution and responsiveness to various endogenous and exogenous stimuli, macrophages have marked phenotypic heterogeneity [30]. The classically activated macrophages (AAM or M1) activated by T helper 1(Th1)-cytokine interferon-r represent the most common and best known type. The concept of alternatively activated macrophages (CAM or M2), which are stimulated by Th2-type cytokines, has gained attention in the past decades [31-34]. In 1992 [34], Simon Gordon and colleagues identified the alternatively activated macrophages stimulated by IL-4, which is significantly different from the macrophages classically activated by IFN-r. The alternatively activated macrophages have a higher level of macrophage mannose receptor and secrete lower amounts of inflammatory cytokines. In 1994 [35], Gordon reported another type of alternatively activating macrophage that was stimulated by IL-13. The classically activated macrophages have features that are quite different from those of the alternatively activated macrophages. In terms of cell morphology, M2 cells are elongated, while M1 cells are round and pancake-like [36]. In terms of function, M1 cells are pro-inflammatory, while M2 cells are pro-resolution and pro-healing [37]. because of their different functions, each cell type dominates different stages in the cascade of inflammation. From the onset to the peak of inflammation or injury, M1 cells are dominant. Then, as the process turns to the resolution of inflammation and the recovery of tissue, M1 cells engage in the phagocytosis of apoptotic cells and transfer the process to the M2 cells [2,30]. The M1 cells’ surface markers are CD68 and major histocompatibility complex-II, while the M2 cells’ surface markers are CD163 and CD206. M1 cells secrete more pro-inflammatory cytokines, including TNF-α, iNOS and other, whereas M2 cells secrete more immunoregulative cytokines, such as Arginase-1 and the pro-healing cytokines IL-10 and TGF-β [38,39]. Furthermore, considerable in vitro and in vivo evidence indicates that M2 cells interact with stem cells or stem cell-like cells and support their proliferation and differentiation, while the M1 cells do not. Conversely, stem cell transplantation into a microenvironment will promote the transfer of M1 cells to M2 cells and have a pro-healing role [40,41].

We have always known that macrophages are amplified in periodontitis. However, little is known about the different macrophages involved in the process of periodontitis. Mariely Navarrete and colleagues used IFN-r, IL-6, IL-4 and FXIII-A as indirect markers of the classical and alternative macrophages to show that both of the cell types exist in the inflamed periodontium [42]. More recently [43], Roselind S. Lam and colleagues demonstrated that the ratio of M1/M2 was increased in a mouse model of periodontitis compared with the control group. Additionally, the M1-related cytokines and chemokines TNF-α and MIP are increased in the collected supernatants of peritoneal macrophages. Furthermore, the depletion of macrophages by clodronate liposomes significantly reduces macrophage infiltration and prevents P. gingivalis-induced bone resorption in mice. The results were consistent with another recent research that the M1-type markers outnumber the M2-type markers in both periodontium and serum in periodontitis mice compared to control mice [44]. Another in vitro study using co-culture of oral commensals and pathogens with macrophages suggested that P. gingivalis, as an oral pathogen, may program macrophages to M1 and result in a pro-inflammatory environment [45]. These results prove the pro-inflammation nature of M1 in periodontitis and suggest that M2 would dominate M1 in the resolution of inflammation in periodontitis. The same group also tested the different responses of M1 and M2 to P. gingivalis stimulation [46]. M1 and M2 cells were induced from murine bone marrow macrophages and then incubated with a low or high dose of P. gingivalis. In M1 cells, the high dose of P. gingivalis LPS significantly increased the expression of CD40, CD86, inducible nitric oxide synthase (iNOS) and nitric oxide (NO). As to M2 cells, P. gingivalis LPS lightly increased the expression of CD206 and YM-1 but did enhance arginase expression. The in vitro research implied that the various macrophages have different functions in the process of periodontitis. In the research by Qunzhou Zhang and colleagues [47], the mucosa of normal and periodontitis patients were collected and compared with that of patients with bisphosphonate-related osteonecrosis of the jaws. The results showed that the proportion of iNOS+ M1 cells was significantly higher than that of the normal group, but the proportion of CD206+ M2 cells was significantly lower than that of the normal group. The above-mentioned studies suggest that it is worthwhile to attempt to rescue periodontitis using an M2-based tissue engineer. Kara L. Spiller and colleagues confirmed that tissue engineering scaffolds primed with an M2-inducer showed a high capacity for vascularization, which is consistent with the pro-healing activity of M2 cells [48]. Anusuya Das and colleagues used nanofibers composed of an S1P receptor agonist to treat mandibular defects; they obtained better regeneration than when normal nanofibers were used because of the M2 cell recruitment associated with the S1P receptor agonist nanofibers [49]. In the attempt to improve the osseointegration of a nanostructured titanium surface of an oral implant, Qianli Ma demonstrated that mediation by macrophage polarization plays a key role. Titanium surfaces that can recruit more M2 cells will achieve better implant osseointegration [50].

Lymphangiogenesis and lymphatic function

The lymphatic system participates in the regulation of the microenvironment by returning proteins, cells, molecules and fluid to the circulating blood. Furthermore, it removes dead or mutant cells, microbes and foreign matter under pathogenic conditions [51,52]. During the embryonic period, the formation of LVs is initiated from a subset of venous endothelial cells of the cardinal vein that express Prox-1. In adulthood, lymphangiogenesis will occur only under certain conditions, such as tissue injury, inflammation and tumors [53]. Lymphangi-ogensis and lymphatic function play a complex role in inflammation which is accompanied by vascular responses, migration and the activation of leukocytes. To relieve the inflammation, pathogens, debris and excessive inflammatory cells must be removed; thus, the need for appropriate lymphatic drainage arises under inflammatory conditions [54]. Lymphangiogensis is a context-dependent reaction that alters the process of inflammation and tissue repair. The most studied signaling pathway of inflammatory lymphangiogenesis is the vascular endothelial growth factor (VEGF)-C and VEGF receptor 3 pathway [55]. The VEGF-A and VEGF receptor 2 pathway is also known to contribute to inflammatory lymphangiogenesis. T cell-derived IFN-r is a vital anti-lymphangiogenic cytokine. During the development of inflammation, pro-lymphangiogenic factors overweigh anti-lymphatic factors, which results in an outburst of lymphatic vessels. When the inflammation process turns to resolution, the anti-lymphatic factors dominate, and the excessive lymphatic network regresses (54). The markers of mature LECs are Prox-1, VEGF-3, LYVE-1 and podoplanin. The lymph travels through different sizes of channels from the inflamed tissue to the blood circulation: lymph capillaries, collecting vessel, lymph nodes, lymphatic trunks and lymphatic ducts [56]. During the initiation of inflammation, lymphatic vessels recruit inflammatory cells from the circulatory system to the inflamed tissue and pass the antigen-loaded immune cells into the lymph nodes. The lymphatic vessels recruit CCR7+ dendritic cells and T and B cells by secreting CCL21 (also known as secondary lymphoid chemokine, SLC) [57]. Furthermore, the LECs themselves interact with immune cells and contribute to the peripheral tissue’s inflammatory response. During the resolution of inflammation, the lymphatic vessels help drain away the apoptotic leukocytes, hostile debris, and fluid. Afterward, the additional lymphatic vessels should fade away and recover to normal levels. If they do not, the excess lymphatic vessels will cause unwanted inflammation by transporting antigens into the lymph nodes, and the immune response will continue. Thus, inflammation could be partially mediated by targeting lymphangiogenesis [54,57].

To date, little attention is paid to the role of lymphatic vessels in oral tissue. Lymphatic vessels in the gingiva of mice were first described using lymphatic vessel endothelial hyaluronan receptor (LYVE)-1 as a specific marker [58,59]. Under normal conditions, gingival lymphatic vessels are located in the lamina propria. In normal condition, lymphangiogenesis was recently observed after infection with P. gingivalis [57,60,61]. When the inflammation process began, both the number and the size of the lymphatic vessels increased. The number of VEGF-C+/CD45+ immune cells increased significantly, while there was no change in the VEGF-D/CD45+ cells [60]. This implies that VEGF-C might be mainly responsible for inflammatory lymphangiogenesis in periodontitis. The trapping of VEGF-C and D by soluble VEGFR-3 results in genetically modified K14-VEGFR3-Ig mice that are deficient in dermal and gingival lymphatic vessels [62]. However, the regression of inflammation is specific to the lymphatics because the blood vessel network does not seem to be influenced in this model. New mice models offer more opportunities to investigate lymphangiogenesis and its role in periodontal diseases. Lilian E. Mkonyi and colleagues observed that K14-VEGFR3-Ig mice developed more severe bone resorption than WT mice in a model of periodontitis induced by P. gingivalis [63]. The histological examination of the periodontal tissue revealed higher numbers of F4/80+ macrophages in the defects, and increased levels of the pro-inflammatory cytokines IFN-r, IL-1β, RANKL and M-CSF confirmed the presence of more serious inflammation. These cytokines are essential to the activation of osteoclasts. Furthermore, the production of antibody IgG to P. gingivalis was impaired, which implied an impaired humoral immune response. This may be the result of damaged gingival drainage following the reduced capacity of antigen-presenting cells to travel to the lymph nodes. The above-mentioned studies show that effective lymphangiogenesis is necessary for the resolution of inflammation. However, there is insufficient research regarding the role of excessive lymphangiogenesis in the uncontrolled inflammation of periodontitis, and translational research regarding lymphangiogenesis in human periodontitis is lacking. VEGF-C can significantly increase lymphangiogenesis, and anti-lymphatic treatment with anti-VEGFR-3 antibody (mF4-31C1) can inhibit additional lymphangiogenesis [54]. However, attention must be paid to the complexity and different contexts of lymphatic biology during treatment. Lymphatic target therapy could be a double-edged sword. Investigations of the lymphatic biology of the gingiva during inflammation will surely open a new window onto the pathogenesis of periodontitis. Translational research is worth conducting, as are therapeutic trials of lymphangiogenesis modulation in periodontitis.

Immune-suppressive Treg cells

Tregs are a subset of T lymphocytes that express CD4, CD25 and the transcription factor Foxp3 and have an immune suppression feature. They are important for ensuring immune homeostasis to prevent T cell-induced immune responses from damaging the host [64]. Treg cells include natural Tregs (nTregs) and inducible Tregs (iTregs). The two types of Tregs differ in terms of development and function. nTregs reside in the thymus and constitute 5-10% of the naïve CD4+ T cells pool in normal individuals. iTregs are driven by naïve T cells primed by IL-10 and TGF-β in the peripheral tissues. nTregs play a role in immune homeostasis via cell contact, while iTreg cells mediate the immune response by secreting suppressive cytokines [65]. Treg cells limit chronic inflammation by suppressing and regulating the T helper cells. In addition, Treg cells can exert immune-regulatory functions independent of inhibiting Th cells [66]. In Rag1-/- mice, which lack T and B cells, the transfer of Treg cells can eliminate lung inflammation by inducing alveolar neutrophil apoptosis and macrophage efferocytosis, thus decreasing the level of pro-inflammatory cytokines. Furthermore, Tregs play a role in bone biology. Emerging evidence shows the inhibition of Treg cells during osteoclastogenesis. Treg cells can inhibit osteoclastogenesis and the formation of osteoclasts via the secretion of IL-10, TGF-β and IL-4. Cell-to-cell contact via cytotoxic T lymphocyte antigen 4 (CTLA4) also plays a role [67]. In a pilot study including 7 subjects with healthy gingiva and 8 with chronic periodontitis, a higher proportion of CD4+CD25+Foxp3+CD45RB+ Treg in the peripheral blood samples of chronic periodontitis subjects compared to that of healthy ones [68].

Because of the immune modulation potential of Tregs, they are considered a promising immunotherapy source for several immune-related diseases [69]. The transfer of autologous Treg cells is relatively easy and generally includes a protocol of isolation, enrichment and re-infusion. Other strategies that aim to induce Treg cells in vivo, such as the injection of IL-2, have also been studied. A third method for Treg therapy is the conversion of antigen-specific conventional T cells to Treg cells, although this process is characterized by many uncertainties [70]. It is clear that Treg cells are present in periodontitis [71]; furthermore, they are observed during later stages of the disease. R. Kobayashi and colleagues demonstrated that IL-10+CD4+ T cells are increased in the inflamed gingiva, as are CD4+CD25+Foxp3+ T cells during the late period of P. gingivalis infection [72]. Garlet GP and colleagues [73] demonstrated progressive alveolar bone loss 15 days post-infection in a model of A. actinomycetemcomitans-induced periodontitis, while CD4+Foxp3+ Treg cells were only observed 30 days pi. The levels of IL-10 and CTLA-4 were positively correlated with the number of Treg cells. However, the Treg cells were decreased in the anti-GITR group, the severity of alveolar bone loss increased 30 days pi, and the infiltration of inflammatory cells was increased. However, periodontal infection control was not affected because the level of A. actinomycetemcomitans in the periodontal tissue and the level of serum CRP were not influenced. The researchers concluded that the protective mechanism of Treg cells in periodontitis was related to the secretion of CTLA-4, TGF-β, and IL-10. In another study [74], C-C motif chemokine ligand 22 (CCL22) was made into a degradable polymer, poly lactic-co-glycolic acid (PLGA). CCL22-releasing microparticles were injected into the local gingiva of mice in a mouse periodontitis model. The measurements of the area between the cementoenamel junction and the alveolar bone crest were decreased 10 days pi in the CCL22 group. The mechanism for this decrease was the recruitment of Treg cells because CCL22 is the ligand of CCR4, which is highly expressed in Treg cells. The mice that received CCL22-releasing microparticles expressed higher levels of the Treg cells markers. The levels of the pro-inflammatory cytokines were down-regulated. The transgenic CCR4ko mice showed significant impairment in Treg cells migration, which resulted in increased alveolar bone loss and higher pro-inflammatory cytokine levels. Another ex vivo study [75] showed that CD4+CD25+ Treg cells could affect the antimicrobial response and inhibit reactive oxygen and pro-inflammatory cytokines. Furthermore, Tregs can reverse the survival of neutrophils and promote their apoptosis. These results provide direct evidence of the pro-resolution feature of Treg cells and show the important roles of Treg cells in the resolution of periodontitis. The recruitment or enrichment of Treg cells may be a promising treatment for periodontitis.

Induction of pro-resolution mediators

At present, the most attractive pro-resolution mediators are a number of lipid mediators derived from polyunsaturated fatty acids (PUFAs). These include lipoxin (LX), resolvin (Rv)-D, resolvin-E, protectin, maresin [76-78]. Lipoxins are derived from the omega-6 polyunsaturated fatty acid arachidonic acid. LXs are produced quickly and work in a paracrine or autocrine manner [79]. LipoxinA4 (LXA4) works by binding to its high-affinity receptor formyl-peptide receptor2/lipoxin A4 receptor (ALX/FPR2), which belongs to the formyl peptide receptor superfamily [80]. In addition, recent data demonstrated that LXA4 shares GPR32 with another pro-resolution mediator, Resolvin D1 [81]. The most frequently recognized mechanism involved in the inflammation resolution ability of LXs is their ability to prevent the recruitment of neutrophils [82]. However, LXs can also upregulate the phagocytosis of apoptotic neutrophils by macrophages, which might stimulate the alternative activation of macrophages [83,84]. In the research by Souza et al. [85], 15-epi-lipoxin A4 accompanied by an annexin-1-derived peptide can prevent TNF-α production and alleviate inflammation in a manner similar to that of IL-10, another inflammation modulating cytokine. Another study [86] demonstrated that LXA4 induced the expression of suppressor of cytokine signaling (SOCS)-2 via AhR and LXAR in dendritic cells and served to down-regulate inflammation. In the recent study by Emma Borgeson and colleagues [87], LXA4 had a protective role in P. gingivalis-induced inflammation; by inhibiting the Rho GTPase-signal pathway and decreasing CD11b/CD18, LXA4 prevented P. gingivalis-induced cell activation. Furthermore, LXA4 also decreased the aggregation and ROS in the whole blood, which is important for the prevention of some forms of general periodontitis-related inflammation, such as atherosclerosis. T. E. Van Dyke’s group demonstrated [88] the pro-resolution feature of a novel lipoxin analogue (benzo-lipoxin A4, bLXA4) in a miniature pig model of periodontitis. To imitate the endogenous relief provided by lipoxin, they used bLXA4-contaning nano-pro-resolving medicines (NPRM) to treat periodontal defects. The results showed a dramatic regeneration of hard and soft tissues, and the histological results confirmed the decreased infiltration of inflammatory cells.

Resolvins and protectins are derived from omega-3 polyunsaturated fatty acids. There are two forms of resolvins: D and E [89]. ChemR23 is the first receptor identified for RvE1. RvD1 binds to two GPCRs: ALX and GPR32 [90]. Similar to LXA4, RvE1 and RvD1 can stop PMN infiltration and promote macrophages’ phagocytosis of apoptotic neutrophils [91,92]. RvD1 was proven to contribute to the alternative activation of macrophages [93], and RvE1 could induce the apoptosis of activated CD4+ T cells, which is an important step in the resolution of inflammation [93]. The role of Rv in the resolution of periodontitis has been confirmed by several studies. Hatice Hasturk et al. [94] applied 4 µg of RvE1 per tooth in a model of ligature- and P. gingivalis-induced periodontitis in rabbits. The treatment was administered three times per week for 6-week period. Quantitative morphologic study showed that the RvE1 application regenerated periodontitis-related bone loss to pre-disease levels, and the clinical periodontitis parameters were all improved. Histological evaluation showed a significant elimination of inflammatory cells in the RvE1 group. Hard tissue slices demonstrated a recovery of the periodontal ligament, cementum, and alveolar bone. The TRAP-stained sections confirmed a low number of osteoclasts in the RvE1 group, and the RvE1 group showed much lower levels of serum CRP and IL-1β. This series of data showed the full range of periodontitis resolution and regeneration induced by RvE1. Another study showed a similar result, and a binding experiment showed that RvE1 specifically binds to neutrophils [95]. Li Gao et al. [96] found that alveolar bone loss was decreased in ligature-induced periodontitis, and leukocytes were cleared in the peritoneal cavity of transgenic mice that overexpressed the RvE1 receptor chemR23. In vitro bone cultures exposed to RvE1 showed increased expression of osteoprotegerin (OPG). In another in vitro culture, osteoclasts were limited in the presence of RvE1, and resorption pit formation was decreased [97]. Similarly, RvD1 down-regulated P. gingivalis-induced inflammation by eliminating pro-inflammatory cytokines such as IL-6 and monocyte chemotactic protein (MCP)-1 in human gingival fibroblasts; furthermore, RvD1 had a positive effect on cell survival [98]. In addition to local application, omega-3 polyunsaturated fatty acids have also been administered via the diet [99]. In a parallel-design, double-blind clinical trial, eighty patients with chronic periodontitis were assigned to two treatment groups, one that underwent conventional scaling and root planing with a placebo and another that underwent scaling and root planing with the addition of dietary fish oil (900 mg eicosapentaenoic acid and docosahexaenoic acid) and 81 mg aspirin. The results showed that probing depth was decreased in the omega-3 group compared with the conventional group, and the salivary RANKL and MMP-8 concentrations were reduced. In a more recent study, administration of Maresin 1 at 1 nM enhanced macrophage phagocytosis and bacterial killing of periodontal pathogens [100].

Several other molecules have the potential to mediate the resolution of inflammation. H₂O₂ has been shown to resolve inflammation by clearing neutrophils by both pharmacological and genetic means. In transgenic gp91phox-/- mice, the recruitment of neutrophils was not affected, but clearance was delayed. A low dose of H₂O₂ at the peak of inflammation induced the apoptosis of neutrophils [101]. Low concentrations of H₂O₂ have been shown to promote the proliferation of periodontal ligament fibroblasts and increase their osteoblastic differentiation [102]. However, a low concentration of H₂O₂ should be used to resolve periodontitis because high concentrations of H₂O₂ have been shown to be pro-inflammatory by inducing IL-8 [103]. TNFRs have four cysteine-rich domains (CDR). CDR1 does not mediate the function of TNF-α immediately, but it plays a role in prompting TNFRs to form polymers; thus, it is called a preligand-binding assembly domain (PLAD) [104]. TNFR1-PLAD can bind to TNFR1 and block the combination of sTNFα and TNFR1. Additionally, TNFR1-PLAD can inhibit the nuclear movement of NF-κb [105]. TNFR1-PLAD has a potential application for the resolution of periodontitis, which we are exploring in our current research.

Discussion

The identification of inflammatory resolution as an active process has improved our understanding of the cascade of inflammation. Inflammation is necessary for the body to fight pathogenic invaders and keep the tissue intact; however, once the threat is past, the process is supposed to move to the resolution stage [106]. Failure of the resolution of inflammation leads to prolonged and chronic inflammation [107]. During the resolution stage, a series of processes occur and interact with each other. First, the pro-inflammatory chemokines cease production and become depleted, which leads to the blockage of neutrophil infiltration. Apoptosis is then induced in the existing neutrophils. Neutrophil apoptosis is central to the resolution of inflammation and influences the features of other immune cells. Macrophages engage in efferocytosis of the apoptotic neutrophils and transfer to alternatively activated macrophages. AAM is characterized by pro-resolution and pro-healing cells. Excessive immune cells and pathogens are drained to the circulatory system through the lymphatic system. However, lymphogenesis is supposed to be blocked in a timely manner to avoid the uncontrolled production of antigens. In the adoptive immune system, Treg cells can limit the other Th cells and produce some rehabilitative cytokines. Furthermore, many autologous lipids and cytokines have the potentials to resolve the inflammation.

Periodontitis is an excessive inflammation caused by microbial biofilm and the host immune response [108]. It is thought that periodontitis is a result of the failed resolution of inflammation in the periodontium. Any mistake in inflammation resolution process described above may lead to the prolonged inflammation and tissue destruction of periodontitis. Preprogrammed neutrophil apoptosis is delayed, which gives neutrophils an opportunity to promote excessive inflammation and destroy the tissue. The dominant macrophages in periodontitis are classically activated macrophages that do not successfully transition to alternatively activated macrophages. Lymphangio-genesis is indispensable to the resolution of periodontitis; without it, the periodontitis will become more serious. Regulatory T cells are shown to help heal periodontitis and eliminate inflammation. Many pro-resolution lipids are used to cure periodontitis and exhibit considerable potential for resolution based on their observed tissue regeneration and decreased inflammation.

Despite our progress in the area of resolution, additional work must be performed; in particular, we need more in-depth research into the various processes involved in the resolution of periodontitis and the specific methods that can influence them in an ideal direction. Future studies should examine how we can specifically induce neutrophil apoptosis without influencing other types of cells, whether there any methods that can help M1 transition to M2, and whether M2-based tissue engineering can help resolve periodontitis. Lymphangiogenesis is a complex biological process, and translational research is needed in this new field. Further trial modulations of lymphangiogenesis should be performed. Recruiting Treg cells to the inflammation site seems to offer potential for resolving periodontitis. At present, many lipids and cytokines have demonstrated usefulness for resolving periodontitis, and the field is still active.

Disclosure of conflict of interest

None.