Periodontitis and peri-implantitis are the inflammatory diseases that are initiated by the bacteria (1). Recent investigations into the regulation of the inflammation in the periodontitis have revealed that the chronic inflammatory diseases such as periodontitis are characterized by an imbalance in the proinflammatory and proresolution mediators and can be characterized by a failure of the resolution pathways in the late stages of the acute inflammatory response (2, 3). Resolution of the inflammation is driven by a group of specialized proresolving mediators (SPMs) comprising the lipoxins, resolvins, protectins, and maresins that are derived from the arachidonic acid or omega-3 polyunsaturated fatty acids (PUFAs) (4). Resolution of the inflammation is an active process in which SPMs interact with their respective receptors on the cells to halt the neutrophil influx, skew monocytes/macrophages toward the M2-like phenotype with increased phagocytosis, and to limit the adaptive immune activation (5). These actions lead to a well-orchestrated quick return to the immune homeostasis. Importantly, administration of exogenous SPMs in pathological inflammatory lesions breaks the cycle of the chronicity and facilitates the return to the physiological form and function (5).

The repertoire of SPM biosynthesis is mainly the innate immune cells such as neutrophils, macrophages, and macrophage precursor monocytes (4). In addition, the resident stromal cells can produce SPMs under certain conditions and express SPM receptors. (3). The synthetic pathways of SPMs rely on the lipoxygenase enzymes that are processed by the various cell types (6). In humans, 5-lipoxygenase is the main enzyme for the leukotriene production (7), which creates a proinflammatory environment, whereas 12-lipoxygenase and 15-lipoxygenase are the main enzymes that drive SPM biosynthesis to promote the resolution phase (8). It is also important to note that SPMs act in an autocrine and/or paracrine fashion; SPMs further activate the lipoxygenase enzymes and accelerate SPM biosynthesis (9). Thus, regulation of the lipoxygenase enzymes in the disease is critical to understand the synthetic mechanisms of SPMs that fail in the chronic inflammatory diseases.

Resolution of the inflammation promotes the tissue regeneration, which emphasizes the role that the control of the inflammation plays in wound healing. Hasturk et al. provided the first evidence that the local application of resolvin E1 (RvE1) promoted the periodontal regeneration in a rabbit periodontitis model (10). Wang et al. later showed that Maresin1 (MaR1) accelerated the extraction socket regeneration by skewing the macrophages toward a proresolution M2-like phenotype (11). Cianci et al. has shown that the human periodontal ligament stem cells (hPDLSCs) release the abundant SPMs including lipoxin A4 (LXA4) that regulates the regenerative function of hPDLSCs (12). A recent study from our group has further demonstrated that RvE1 and MaR1 improve hPDLSCs regenerative properties by enhancing the proliferation, migration, and regulating molecular machinery to overcome the negative impact of the inflammation (13). In addition to producing SPMs and reacting to SPMs, the immunomodulatory properties of PDLSCs are also reflected in their rich synthesis of the immune markers and the impact on the immune cells. Enhancing the understanding of the immunomodulatory role of PDLSCs will provide valuable insights by further improving the periodontal regeneration.

In this study, we investigated PDLSCs from a large animal—the Yorkshire pig—to establish a large animal model to optimize translational value in regeneration related studies. The immunomodulatory properties of SPMs on pig PDLSCs (pPDLSCs) were examined to investigate the regulation of the lipoxygenase enzymes in pPDLSCs and to characterize the interaction between pPDLSCs and immune cells.

In this study, we investigate the two major characteristics of PDLSCs: the stem cell function and immunomodulation. PDLSCs resemble other MSCs in their multipotency to differentiate into the fibroblasts, osteoblasts, and cementoblasts, confirming prior observations (22). Thus, PDLSCs are critical to restoring the periodontal tissues lost due to the disease. Importantly, PDLSCs play an important immunomodulatory role defined by the PDLSCs response to the inflammation. This immunomodulatory response of PDLSCs is manifested by the immune responsive actions and the influence of inflammation on the properties of the stem cells.

Periodontal ligament stem cells are conveniently harvested from the deciduous or permanent teeth, providing a reliable and reproducible cell source for the study of the periodontal regeneration. In this study, we used PDLSCs isolated from the deciduous teeth of the Yorkshire pig. We chose the porcine model, since they share the physiological and anatomical similarities to the humans in their teeth and tooth-supporting structures; pigs have been widely used in the periodontal regeneration studies (23–25). Indeed, in this study, pPDLSCs exhibited similar MSC markers to human PDLSCs (Supplementary Figure 1) as previously reported (13). Thus, the Yorkshire pig provides an excellent large animal model to guide the ongoing regeneration research leading up to human clinical trials.

Periodontal regeneration occurs in an inflammatory environment and PDLSCs sense the inflammation cues and produce the inflammatory cytokines (26). We demonstrate that there are synergistic actions of IL-1β and IL-17 that regulate PTGS2 and IL-6 gene expression by pPDLSCs (Figure 2). Individual actions of IL-1β or IL-17 on MSCs including PDLSCs have been well documented. Carrero et al. reported that in response to IL-1β challenge, bone marrow MSCs exhibited the major changes in the immune phenotype with increased production of the multiple inflammatory cytokines including cytokines critical for the recruitment of the leukocytes (27). IL-1β appears to have dual functions on PDLSCs. Mao et al. showed that the lower doses of IL-1β enhanced the osteogenic properties of PDLSCs, while the higher doses of IL-1β inhibited the osteogenic properties of PDLSCs (28). A recent study support this observation showing that IL-1β and tumor necrosis factor-α (TNF-α) together inhibit hPDLSCs proliferation, migration, and inhibit osteogenesis and cementogenesis (13).

Interleukin-17 is mainly released by T-helper 17 (Th17) cells during the inflammation with contribution from the other resident cells (29). Opinions on the impact of IL-17 on MSCs are divergent. In some studies, IL-17 was shown to cooperate with the other inflammatory cytokines to promote the immunosuppressive functions of MSCs (30, 31). In other studies, IL-17 enhanced proinflammatory cytokine production by MSCs that positively influenced osteogenesis (32). There are few reports of the direct impact of IL-17 on PDLSCs. Dordević et al. reported that IL-17 negatively regulated osteogenesis by PDLSCs and inhibited PDLSCs proliferation and migration (33). IL-1β and IL-17 together inhibit the proliferation of PDLSCs, agreeing with the negative regulatory role of IL-17. At the same time, the synergistic actions of IL-1β and IL-17 suggest that the stem cell niche in PDL tissues is profoundly affected by the inflammation; thus, understanding how to predictably regulate the local inflammatory environment will be important for the periodontal regeneration.

In the time-course experiments, it is interesting to note that PTSG2 and IL-6 were significantly more upregulated at 24 h and 48 h (Figures 3A,B). This mRNA induction pattern was specific to PTGS2 and IL-6, as the transcription of CCL8 peaked at an earlier time point and was almost depleted at 48 h (Supplementary Figure 3). It is possible that after a long exposure to the inflammatory cues, PDLSCs partially lose stemness properties and behave like a resident periodontal ligament fibroblast and are, thus, more immune sensitive. Fibroblasts are known to be the major source of PGE2 in the periodontal tissues (34). In Figure 3C, we also show activation of the corresponding NF-κB and MAPK pathways. Both the pathways are the immune response pathways downstream of IL-1β or IL-17; their activation plays an important role in defining the stem cell fate and in shaping the stem cell immune properties (27, 28, 35). The immunomodulatory role of MaR1 in lowering the proinflammatory cytokine production of PDLSCs was evident (Figure 4). However, we found inconsistency between the mRNA and protein data for the detection of IL-6. The levels of PGE2 were an order of greater magnitude. The level of PGE2 was hundreds of nanograms (~200 ng/ml), but ~2 ng/ml for IL-6 and ~20 pg/ml for CCL8. This suggests that the immunomodulatory properties of PDLSCs have specific patterns and some mediators are more dominant in the case of PGE2. Ern et al. demonstrated that PGE2 inhibits the osteogenic differentiation of PDLSCs (36). MaR1-hampered PGE2 production may be biologically important for two reasons: to alleviate the osteogenesis inhibition by PGE2 or change the dynamics of the crosstalk between PDLSCs and immune cells.

An additional objective of this study was to investigate the regulations of the lipoxygenase enzymes that mediate the production of SPMs including LOX5 and its activating protein FLAP, LOX12, and LOX15. These lipoxygenases catalyze the oxygenation of PUFAs to form SPM precursors (6). The SPM family includes lipoxins derived from arachidonic acids; E-series resolvins derived from eicosapentaenoic acid (EPA); and D-series resolvins, protectins, and maresins derived from docosahexaenoic acid (DHA) (37). LOX15 is predominantly expressed in the leukocytes and resident epithelium and LOX12 is predominantly expressed in the platelets (6, 38). In addition to participating in SPM biosynthesis, LOX5 and its activating protein FLAP induce the inflammation catalyzing the synthesis of the leukotrienes derived from arachidonic acid (39). In this study, we found that neither LOX5 nor FLAP expression was affected by SPM challenge of pPDLSCs (Supplementary Figure 2). However, under the same conditions, LOX12 and LOX15 were significantly elevated (Figure 1). These findings suggest that SPMs shift PDLSCs toward the resolution of the inflammation by activating LOX12 and LOX15, while maintaining baseline levels of LOX5.

The crosstalk between PDLSCs and immune cells is an important component of PDLSCs immune-modulatory properties. Overall, MSCs have the ability to recruit the leukocytes to the local tissues for the immune regulation (27). PDLSCs promote the polarization of the monocytes/macrophages toward a resolution phenotype (M2-like phenotype) to enhance the regeneration (40). Due to the limited markers to detect the conventional M1-like (proinflammation) or M2-like (resolution of inflammation) phenotype macrophages in the pigs, we utilized CD14, CD16, and CD163 to differentiate the phenotype macrophages. In humans, CD14⁺CD16⁻ monocytes (classical) contribute more to proinflammation and CD14⁺CD16⁺ monocytes (non-classical) are viewed as anti-inflammatory (21). CD163, a conventional M2 marker, has divergent expression patterns within CD14⁺CD16⁻ or CD14⁺CD16⁺ cells in different diseases (41). We demonstrate the feasibility of using CD14⁺CD16⁺/CD14⁺CD16⁻ ratio to differentiate between the proinflammation and resolution of inflammation phenotype macrophages (Figure 5). CM from pPDLSCs treated with the cytokines (IL-1β and IL-17) drives monocytes/macrophages toward a proinflammatory phenotype compared to the control pPDLSC CM; MaR1 skews the system toward the resolution of inflammation (Figure 6). CM was collected 48 h after pPDLSCs were challenged with the cytokines and/or SPMs. By this time, pPDLSCs already exhibit the major immune gene regulatory changes and secrete many proteins to influence the expression of CD14/CD16/CD163 markers. It is not within the scope of this project, but worthwhile to further investigate what specific molecules contribute to the immune marker changes within the CM. MaR1 has already been shown to skew macrophages toward a proresolution M2-like phenotype (11). Because in PMA-differentiated PBMCs, MaR1 demonstrates the direct actions on promoting the resolution of inflammation, we conclude that MaR1 skews the inflammation toward the resolution through the direct and indirect mechanisms via pPDLSCs.

Specialized proresolving mediators have shown promise in the preclinical studies for the promotion of the periodontal regeneration (10). The principle that control of the inflammation is critical to the regeneration appears to be universal to all the tissues and evolutionarily conserved. Recently, the Serhan group explored the function of SPM/protein conjugates in planarian regeneration further linking the resolution of the inflammation and regeneration (42). Likewise, Norling et al. clearly demonstrate the role of SPMs in the wound healing and regeneration (43).

In conclusion, it is becoming clear that PDLSCs function cannot be separated in situ from the local inflammatory environment. PDLSCs function is negatively impacted by the curtailed local inflammation and regeneration. The role of SPMs in PDLSCs function is critical to their function mechanistically operating by the regulation of the mediator and the cellular surrounding environment including cytokine production and function and the modulation of the phenotype macrophages. This is further evidenced by the previously described production of SPMs by PDLSCs (12) and the response of PDLSCs to exogenously provided SPMs. Thus, continued elucidation of the actions and mechanisms of SPMs in stem cell-mediated periodontal and peri-implant regeneration will likely lead to improved regeneration outcomes in the future human therapies.

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author/s.

NY, AR, and AD contributed to the design of the study, performing experiments, and writing of the manuscript. TVD contributed to the design of the study, discussions, writing, and critical review of the manuscript.

This study is supported by the NIDCR R01DE025020 (TVD) and NIDCR T90DE026110 (AR).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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