During periodontal inflammation, bacteria residing in the biofilm and certain bacterial components are recognized via pattern recognition receptors (PRRs). Toll-like receptors, present on host cells, detect molecules broadly shared by microorganisms; these molecules, such as lipopolysaccharides, lipoproteins, and lipoteichoic acids, are described using the term “pathogen-associated molecular patterns” (PAMPs) (41). Recognition of PAMPs, via binding to these PRRs, has been shown to induce the secretion of chemoattractant proteins, namely, chemokines, which in turn attract PMNs at the site of infection. Extravasated PMNs enter the gingival tissues and subsequently enter the crevice through the junctional epithelium. Their extravasation is mediated by the combined action of high- and low-affinity adhesive interactions and implicates crucial morphological changes in both PMNs and endothelial cells (42). Tissue-derived cytokines enhance endothelial adhesion molecule expression, whereas tissue-derived chemokines induce changes on leukocyte integrins, enabling them to acquire a high-affinity state (43). The initial contact, namely, “tethering” with the vascular wall and adhering to endothelial cells, is mediated by selectins and their ligands. L-selectin is expressed by leukocytes, whereas P- and E-selectins are expressed on activated endothelial cells and platelets (Figure 2). Tethering slows the speed of circulating PMNs and favors subsequent interactions mediated by integrins, which permit the firm adherence of leukocytes on endothelial cells. Via interactions with their endothelial counter-receptors, β2-integrins, as well as lymphocyte function-associated antigen 1, play essential roles in regulating the firm adhesion of PMNs to the endothelium (44). The abovementioned events halt the traveling PMNs on the vascular wall and enable their diapedesis into the infected tissue (45, 46).

In this way, PMNs transmigrate in large numbers into tissues and neutralize pathogens by engulfing them via phagocytosis, release of bactericidal proteins stored in their intracellular granules and production of ROS (47). To avoid PMN-induced tissue damage, it has been proposed that the presence of PMNs in the tissues is regulated via a “feedback loop.” Recruited PMNs undergo apoptosis and are consequently removed by macrophages. It has been described that phagocytosis of microbial or damaged tissue cells by PMNs stimulates their apoptosis, an essential step in resolution of inflammation. Incubation with RvE1 for 24 h has been demonstrated to facilitate phagocytosis-induced PMN apoptosis (33).

Extravasated apoptotic PMNs are cleared via phagocytosis by tissue macrophages. This action hampers the expression of interleukin (IL)-23, which consequently suppresses the generation of IL-17 and granulocyte colony stimulating factor and, thus, downregulates granulopoiesis in the bone marrow (44). Patients with periodontitis exhibit a higher number of PMNs and longer lived PMNs in gingival tissues, as well as reduced PMN chemotactic accuracy, compared with healthy individuals (48, 49). Fredman et al. (50) also reported elevated expression of integrins on the circulating PMNs and monocytes of localized aggressive periodontitis (LAgP) patients, indicating that the resting cells within the whole blood of these patients circulate in a primed state.

It has been suggested that the persistent recruitment of PMNs in inflamed periodontal tissues might be partially due to the inability to control the subgingival microbial challenge. There is evidence that RvE1 can, to a certain extent, counter-regulate this stage in the inflammatory reaction. Administration of RvE1 reduced the infiltration of leukocytes, primarily PMNs, by 50% in peritoneal exudates of mice with zymosan-induced peritonitis (51). This can be possibly explained by the effect of RvE1 on the expression of adhesion molecules on leukocytes. In fact, RvE1 reduces the L-selectin and integrin CD18 expression in peripheral blood PMNs and monocytes, thus impairing binding to their endothelial counterreceptors. In the same direction, administration of RvE1 resulted in 40% downregulation in leukocyte rolling, as confirmed via intravital microscopy (52). Additionally, in vivo experiments in rats with ligature-induced periodontitis showed that local treatment with RvE1 solution significantly suppressed inflammatory cell infiltration and downregulated expression of inflammation-associated genes in the gingival tissues (17).

Apart from their well-described functions in thrombosis and hemostasis, platelets have also been implicated in inflammation. Increasing evidence supports that platelets, including their interactions with inflammatory cells, play a crucial role in atheromatic plaque initiation, instability and rupture (53). The thrombogenic capacity of platelets depends on their ability to aggregate, a process mediated by selectins and integrins (54). Platelets from periodontitis patients demonstrate increased activation in comparison to platelets from healthy controls. In this activation process, P-selectin mediates coaggregation and inflammation by regulating cell–cell interactions between platelets, leukocytes and endothelial cells. In fact, periodontitis patients demonstrated increased plasma levels of sP-Selectin, as well as elevated binding of platelet glycoprotein IIb–IIIa complex, a recognized measure of platelet aggregation (55). Moreover, platelets from periodontitis patients exhibited increased sensitivity to activation by oral bacteria and formed increased numbers of platelet–monocyte complexes compared to healthy controls (56). Incubation of platelet-rich plasma with RvE1 blocked adenosine diphosphate (ADP)-induced platelet aggregation in healthy donors, demonstrating that RvE1 acts selectively and inhibits platelet aggregation (52). The molecular pathway behind this inhibitory action was further elucidated by Fredman et al. (54), who demonstrated that, via stereoselective binding to ChemR23 on platelets, RvE1 reduces P-selectin surface mobilization and induces platelet–actin polymerization. Platelet responses to ADP require the activation of two G-protein-coupled receptors, namely, P2Y1 and P2Y12 (57). In general, the intracellular pathway involves the inhibition of adenyl cyclase (via activation of P2Y12) and increase in intracellular Ca²⁺ (via activation of P2Y1). Of note, RvE1 did not alter Ca²⁺ mobilization, indicating that RvE1 exerts its counterregulatory effect by P2Y12-ADP signaling via ChemR23 (54).

Peripheral blood PMNs from chronic periodontitis patients exhibit excess ROS production following stimulation with bacterial stimuli (stimulated hyperreactivity), as well as in the absence of exogenous stimulation (intrinsic hyperactivity), when compared to age-/sex- and smoking-matched healthy controls (58, 59). As a result, their prolonged presence in the tissues, in combination with the release of the abovementioned molecules and tissue-degrading enzymes, can result in collateral tissue damage (60). After incubation with RvE1, superoxide generation was blocked >90% in PMNs from patients with LAgP. When incubated with increasing concentrations of RvE1 and subsequently challenged with N-formyl-methionyl-leucyl-phenylalanine, PMNs from LAgP patients and controls responded with an 80% reduction in superoxide generation (16), providing an indication that this lipid mediator counteracts the collateral tissue damage induced by PMNs in periodontal inflammation (Figure 2).

At a later stage in the inflammatory reaction, leukocytes of the monocyte/macrophage lineage are recruited to the inflammatory milieu. PMNs that have already been recruited appear to mediate this switch by releasing soluble factors that stimulate monocyte recruitment (61). Monocyte-derived macrophages replace the PMNs as the predominant leukocytes and perform an impressive repertoire of functions involving elimination of bacterial stimuli, recruitment of other cells to the site of infection, clearance of the excess PMNs, generation of cytokines/chemokines and activation of the lymphocyte-mediated adaptive immune response. In the presence of RvE1, the production of TNF-α and IL-8 by monocytes in a whole blood culture model was significantly reduced, whereas RvE1 did not trigger the generation of other cytokines, such as IL-10, interferon-γ, and IL-1β (52). Consistent with their functional role, macrophages acquire different phenotypes. In fact, they can modify their metabolic setup and polarize from a healing/growth-promoting setting (M2 phenotype) to a killing/inhibitory function (M1 phenotype) (62). Although it has been previously documented in mice that macrophages can convert to an M2 phenotype at the later stages of an M1 response, more recent evidence failed to confirm these findings both in mouse and in human macrophages (63, 64).

As long as the bacterial influx in periodontitis is successfully eradicated, the acute inflammatory process will result either in complete resolution with healing and return of the tissue to its preinflammatory state or elimination of the infection with fibrosis. An alternative outcome includes failure to clear the infection followed by establishment of a chronic inflammatory lesion (65). The above-described pleiotropic functions of macrophages clearly illustrate their central role in regulating the resolution of inflammation and return to homeostasis. The reduction of PMN influx is a critical prerequisite for resolution of inflammation. As previously mentioned, after performing their actions at the inflamed site, PMNs are programmed to undergo apoptosis and are cleared by macrophages (Figure 2), a process called “efferocytosis.” Ingestion of apoptotic PMNs induces changes in the macrophage phenotype and guides the cell toward a resolution phase phenotype (M2 phenotype). This “proresolving” macrophage secretes reparative mediators and anti-inflammatory cytokines, such as IL-10, transforming growth factor-β, and vascular endothelial growth factor, promoting tissue regeneration and return to homeostasis (66). Apoptotic PMNs also secrete mediators, which inhibit leukocyte infiltration. Additionally, since the migration of PMNs would persist in the presence of chemokines, macrophage-specific Metalloproteinase 12 is produced, resulting in the degradation of chemokines with a consequent reduction in leukocyte recruitment (67). As discussed above, incubation with RvE1 reduced L-selectin and CD18 expression in peripheral blood monocytes, indicating that the tissue homing of monocytes is hampered (52). It might thus be plausible to hypothesize that apart from circulating monocytes, M2 macrophages might also originate from tissue-resident or already-recruited macrophages (62, 68).

Isolated macrophages from patients with LAgP exhibit impaired phagocytosis compared to those isolated from healthy controls. After 15 min incubation with 1 nM of RvE1, the phagocytic capacity of LAgP macrophages was enhanced to a level that was similar to that of healthy controls, indicating a possible rescue of the phagocytic defect (50). In addition, when RvE1 was introduced into the peritoneum of rats with zymosan-induced peritonitis, a new subset of proresolving macrophages was identified. These macrophages secreted significantly lower levels of TNF-α, IL-1β, and IL-10 upon stimulation with LPS, compared to other macrophage subpopulations, indicating that RvE1 may reduce the production of proinflammatory cytokines by macrophages ex vivo (69).

Failure to resolve inflammation within gingival tissue results in the expansion of the inflammatory infiltrate adjacent to alveolar bone. Bone resorption and formation are regulated by the interplay between the receptor activator of nuclear factor-kappa B ligand (RANKL) expressed by OBs, the RANKL receptor (RANK) on OC precursor cells and the soluble decoy receptor osteoprotegerin (OPG) (70). In addition to its expression on OBs, RANKL is also expressed by other cell types, including T- and B-lymphocytes. In the case of unresolved periodontal inflammation, the consequent production of cytokines, chemokines and other mediators stimulates the increased expression of RANKL by these cells. When the expression of RANKL outweighs that of OPG, it allows RANKL to bind RANK on OC precursors and induce the differentiation of pre-OCs to mature OCs, thereby favoring inflammation-induced bone resorption (71). Multiple clinical studies have confirmed that in periodontitis patients, the RANKL/OPG ratio in gingival tissues and gingival crevicular fluid is higher than in healthy controls (72).

Evidence provided by Herrera et al. has shown that nanomolar doses of RvE1 inhibit RANKL-induced OC growth and differentiation, thus downregulating bone resorption in vitro (73). This action is mainly mediated through binding of RvE1 to the BLT1 receptor expressed on OCs. Interestingly, in contrast to RvE1, addition of similar doses of EPA to OC cultures increased OC formation, implying that not EPA but one of its metabolic derivatives (RvE1) acts directly on these cells to exert the abovementioned actions. In addition, via binding to ChemR23 expressed on OBs, RvE1 increases the generation of OPG by OBs and limits bone resorption in vitro (74). The contribution of this lipid mediator to promoting bone formation and inhibiting bone resorption was also confirmed in vivo in a rabbit ligature-induced periodontitis model (16). Prevention of Porphyromonas gingivalis-induced periodontitis in rats by local application of RvE1 solution was investigated in a 6-week experiment. The results showed that topical treatment with RvE1 prevents and resolves ligature-induced periodontitis with regeneration of soft and hard periodontal tissues to their predisease condition. DNA checkerboard analysis was also performed to investigate changes at the level of periodontal microbiota through the course of the experiment. In the presence of P. gingivalis, other bacteria in the biofilm increased as well. After the ligature, P. gingivalis application stopped, P. gingivalis disappeared from the biofilm, and the resident microflora returned to its normal levels in the RvE1 treated group. However, in the placebo group, the inflammation-induced periodontal breakdown progressed, and the microflora including P. gingivalis, Actinobacillus actinomycetemcomitans, and Fusobacterium nucleatum became more complex (16, 19). Apart from the possible regenerative potential of RvE1, such a finding also confirms the ecological plaque hypothesis proposed by Marsh; according to this hypothesis, the microenvironment conditions, and more specifically the inflammation level, triggers and determines the composition of the microflora (13). The results of the ligature-induced periodontitis study were recently confirmed in a rat model (17).

In addition to RvE1, other proresolving mediators, mainly lipoxins, were employed in several studies of periodontal inflammation. Lipoxins are generated by individual LOs (5-LO, 12-LO, and 15-LO) via trans-cellular biosynthesis, act within a local microenvironment and are rapidly enzymatically inactivated (75). A significant contribution to the understanding of their proresolving actions originates from several ex vivo and in vivo animal studies. Lipoxins were shown to counter PMN responses during inflammation, to prevent periodontal bone loss and to promote regeneration in the presence of existing periodontitis (22, 76). Activated PMNs isolated from the peripheral blood of localized juvenile periodontitis patients but not from healthy controls appeared to produce lipoxin A4 (LXA4). The same lipid compound was detected in the gingival crevicular fluid of localized juvenile periodontitis patients, suggesting a possible immunomodulatory role within the local inflammatory milieu of the periodontium (77). Higher levels of lipoxins produced by transgenic rabbits overexpressing 15-LO have been associated with protection against bone resorption when the animals were subjected to ligature-induced periodontitis with P. gingivalis. In contrast, non-transgenic rabbits exhibited significant soft- and hard-tissue destruction. Along these lines, when the rabbits were treated with lipoxin stable analogs and subjected to ligature-induced periodontitis, they exhibited markedly reduced bone loss and local inflammation (78). Similarly, ATL lipoxin analogs inhibited recruitment of leukocytes stimulated by P. gingivalis in a different in vivo model (77).

In addition to their direct proresolving effect of reducing leukocyte infiltration, SPMs have also been associated with indirect roles. Lipoxins promoted the endogenous biosynthesis of other SPMs, such as resolvins, and vice versa, thus amplifying the resolution signaling (76, 79). In ex vivo studies of LAgP, the administration of another SPM, Maresin 1, enhanced bacterial killing and restored the phagocytic defect of LAgP phagocytes (80).

The application of benzo-LXA4, combined with surgical treatment of osseous defects in domesticated miniature pigs, resulted in the formation of new alveolar bone, new connective tissue attachment and new cementum, providing a promising field for research on periodontal regeneration (76). Another link between SPMs and tissue regeneration was provided for the first time by research conducted on human periodontal ligament stem cells (hPDLSCs). HPDLSCs have been characterized as important players in tissue regeneration, and recent evidence has confirmed their ability to biosynthesize SPMs, including resolvin D1, D2, D5, and D6, protectin D1, maresins, and LXB4, thereby uncovering new properties of these cells (81). Along this line, LXA4 induced the proliferation, migration, and wound-healing capacity of hPDLSCs acting through ALX/FPR2 (81).

A large number of studies conducted during the last two decades have focused on the benefits of dietary PUFAs in the treatment of inflammatory conditions. The results from short-term studies providing individuals with ω-3 fatty acid supplements demonstrated reductions in IL-1β and TNF-α synthesis in cell cultures. In one of the studies, the reduction was more dramatic in older compared to younger women (82, 83).

Regarding periodontal inflammation, a pilot study in which PUFA supplementation was provided as a monotherapy in the treatment of experimental gingivitis for 21 days failed to demonstrate a significant benefit of the dietary supplementation (84). Similar findings were reported for a clinical trial in which subjects with chronic periodontitis received oral hygiene instructions in conjunction with 3 g of PUFAs daily (test group) or oral hygiene instructions combined with placebo for 28 weeks (control group). No significant improvements were demonstrated with respect to clinical outcomes when PUFAs were provided as a monotherapy for chronic periodontitis (85). However, when combined with non-surgical treatment of periodontitis [scaling and root planning (SRP), dietary supplementation with ω-3 fatty acids significantly reduced the gingival index, probing depth, and clinical attachment gain compared to SRP combined with placebo (86)].

Based on the potential synergy of action reported for ω-3 PUFAs and aspirin, El-Sharkawy et al. evaluated the combination of low-dose aspirin together with PUFA supplementation and SRP. The test group received dietary supplementation of fish oil (900 mg of EPA + DHA) and 81 mg of aspirin in combination with SRP, whereas the control group was treated with SRP and a placebo. Significant improvements in clinical periodontal parameters, with reductions in the mean probing depth and in the number of residual deep pockets, were demonstrated in the test group (87). In another randomized controlled trial, Naqvi et al. compared the effect of DHA supplementation and low-dose aspirin (test group) with the daily consumption of placebo (control group) in patients with moderate periodontitis who did not receive any SRP for 3 months. The authors concluded that the test group presented with significantly decreased mean pocket depth and gingival index, in comparison to the control group (88). Finally, Elkhouli et al. (89) evaluated the additional benefit of a 6-month daily PUFA supplementation and low-dose aspirin in conjunction with regenerative therapy in class II furcation defects. At 6 months, the test group presented a significantly greater reduction in mean probing pocket depth (0.9 mm) and gain in clinical attachment level (0.6 mm) compared to the control group (89). Apart from the improvement in clinical parameters, the experimental protocol achieved a host modulatory effect reflected by a significant reduction in the levels of proinflammatory markers, such as IL-1b, in the gingival crevicular fluid (88, 89). In the absence of a third group, which should have used ω-3 fatty acids as a monotherapy, the positive results cannot be attributed to PUFA alone, since long-term aspirin use in patients with periodontitis was found to be associated with significantly lower mean values of periodontal attachment loss compared to periodontal patients who did not receive this medication (90).

Although these findings open a promising pharmacologic avenue for an adjunct to periodontal treatment, the limited number of subjects, the variability in the dose of PUFAs within studies, the short-term follow-up and the non-investigated possible side effects are few of the limitations of the abovementioned studies. One possible side effect of ω-3 fatty acids was reported in the late 1970s by Dyerberg and Bang, who demonstrated that the Greenlandic Inuits had significantly prolonged bleeding times compared with healthy Danes. The authors proposed that ω-3 fatty acids affect platelet function and aggregation, a finding later confirmed by other studies (91). However, according to later studies, such prolongation of bleeding time did not exceed normal limits and did not result in clinically significant bleeding in patients using high doses of ω-3 fatty acids alone or in combination with antiplatelet or anticoagulant therapies (92, 93).