Macrophages are involved in a remarkably diverse array of homeostatic processes of vital importance to the host. In addition to their critical role in immunity , macrophages are also widely recognized as ubiquitous mediators of cellular turnover and maintenance of extracellular matrix homeostasis [13–18]. However, beyond their essentiality in immunity and tissue homeostasis, the macrophage has also been implicated in the evolution of periodontal pathological processes including periodontal disease and DIGO [11, 19, 20, 31, 32]. This investigation posited that macrophage-derived expression of proinflammatory cytokines, MMPs and/or TIMP expression is blunted upon exposure to PHT and/or HPPH hindering the ability of these cells to contribute to the fibroblast-mediated degradation of exuberant ECM proteins seen in DIGO. Since plaque-induced gingival inflammation exacerbates the manifestations of PHT-induced GO , we exposed macrophage cultures to purified LPS from the periodontal pathogen, A. actinomycetemcomitans (Aa) and examined protein levels of MMPs, TIMPs and proinflammatory cytokines in conditioned media. Aa can be isolated from plaque samples of patients with GO  while Aa LPS, a TLR4 agonist, strongly induces MMP and pro-inflammatory cytokine expression [28–30, 35].
We exposed macrophage cultures to 2 different concentrations of PHT and HPPH. And while PHT plasma levels of 10-20 μg/mL are necessary to effectively maintain effective seizure control [22–24], disturbances in plasma as well as gingival concentrations of PHT are likely associated with DIGO. Indeed, Güncü et al  compared PHT levels in plasma and gingival crevicular fluid (GCF), a serum exudate, from subjects who demonstrated gingival overgrowth (responders) vs. those who did not (non-responders). Although PHT was detected in all of the GCF and plasma samples, the mean concentration of PHT was significantly greater in GCF compared to plasma (294.99 ± 430.15 μg/mL vs. 16.09 ± 4.21 μg/mL, respectively). Further, the concentration of plasma PHT was significantly higher in responders compared to non-responders (16.09 ± 4.21 μg/mL vs. 9.93 ± 4.56, respectively).
MMP-1 is recognized as an important mediator of connective tissue remodeling reported to be present at high concentrations in inflamed gingiva . In the present study, supernatant MMP levels did not demonstrate any significant differences in response to PHT and HPPH alone at either dose compared to untreated macrophage cultures although we noted a trend for higher levels of MMP-1 and MMP-3. This finding was attributed to donor-specific variations in responses to these agents and serve to highlight clinical observations that approximately 50% of patients taking PHT develop GO [1, 2]. This notion is supported by the finding that fibroblasts derived from subjects with cyclosporine-A (CSA)-induced gingival overgrowth produce significantly lower levels of MMP-1 than fibroblasts derived from subjects without overgrowth . In the present study, supernatant levels of several MMPs were significantly decreased relative to LPS-only cultures in a dose-dependent manner suggesting that PHT and HPPH may mitigate the macrophage's ability to degrade ECM proteins by limiting its natural response to produce metalloproteinases. Such a dose response is consistent with other studies which have demonstrated, not only a similar effect on MMP-1 and MMP-3 at the protein and mRNA level [39–43], but also that a threshold of serum concentration of CSA helps to govern this mechanism [44–49].
MMP activity is counteracted by the actions of TIMPs. Here we report that exposure of macrophages to LPS was associated with an increase in TIMP-1 levels while exposure to high concentration (50 μg/mL) of PHT and HPPH, on the other hand, significantly reduced TIMP-1 levels. This finding is in agreement with in-vitro and in-vivo studies which report a relative reduction in MMP-1 and MMP-8/TIMP-1 in gingival fibroblasts and in serum and GCF concentration in CSA-associated gingival overgrowth subjects [50, 51]. This reflects more a decrease in MMP production rather than an increase in TIMP. In fact, this corresponds with our findings in that supernatant levels of TIMP-1 in samples treated with both LPS and high doses of PHT or HPPH were not significantly different relative to untreated controls (Fig. 2). The net effect on ECM metabolism is based on the relative ratios of MMP and TIMP. When MMP levels decrease and/or TIMP levels increase, the turnover of ECM diminishes, potentially leading to an exuberant accumulation of these proteins. In this study, elevated levels of PHT in LPS-stimulated macrophages were associated with decreases in both MMP and TIMP levels. Therefore the decrease in TIMP-1 levels was counteracted by decreases in MMP levels. As a result, the macrophage's synergistic relationship with the fibroblast would be compromised leading to DIGO. Indeed, monocytes (macrophage precursors) can stimulate fibroblasts to produce MMP-1 by cell-cell interactions while conditioned media from monocytes is capable of inducing MMP-1 production in fibroblasts . How PHT and HPPH impact monocyte/macrophage-fibroblast interactions and MMP production requires further study.
PHT is known to affect Na+ as well as Ca2+ metabolism , (e.g., Ca2+ channels) and it is likely that this will impact MMP/TIMP and cytokine levels . Indeed, Na+ channels have been linked to activation of macrophages and microglia  and accumulating evidence indicates that sodium channel blockers can contribute to modulation of immune functions . PHT has been reported to ameliorate the inflammatory response associated with experimental autoimmune encephalomyelitis in mice , modulate intracellular signaling cascades to TLR ligands  and significantly reduce LPS-induced phagocytosis in-vitro . Here we report a dose-dependent inhibition of macrophage function by way of suppressed supernatant levels of MMP-1, MMP-3, MMP-9, TIMP-1 and TNF-α by PHT in human macrophages challenged with LPS. PHT has been reported to inhibit both activation of T-type calcium channels and RANKL-induced expression of c- fos protein in bone marrow-derived macrophages implying that calcium signals play a role in c- fos expression . PHT was also shown to inhibit NFATc1 signaling in these cells. Further, in atrial myocytes, pharmacological inhibition of NFAT with 11R-VIVIT almost completely blunted the stretch-induced up-regulation of active-MMP-2/-9 . Kiode et al  suggested that PHT may inhibit NFATc1 signals through suppression of c- fos expression. Since c- fos/AP-1 regulates the expression of numerous inflammatory cytokines and MMPs/TIMPs via promoter AP-1 binding motif [59, 60], suppression of c- fos may provide a possible mechanism whereby MMPs/TIMPs and possibly cytokine levels are inhibited.
In contrast to PHT we report a dose-dependent inhibition of MMP-9 and TIMP-1 by HPPH in cultures challenged with LPS. These discrepancies may be attributed to differences in the interactions of these drugs with target molecules. Kobayashi et al  reported that PHT and 5-(4-methylphenyl)-5-phenylhydantoin, which contain a phenyl or methylphenyl group at both R2- and R3-positions activated the ligand binding domain of human pregane X receptor (hPXR), a member of the nuclear receptor family of ligand-activated transcriptional factors, whereas 5-(4-hydroxyphenyl)-5-phenylhydantoin did not. Alternatively, it is possible that higher concentrations of HPPH may be required to achieve results similar to that observed with PHT as evident by the trend for blunting of MMP-1 and MMP3 at higher doses of HPPH (Fig. 1A, C). Nevertheless, these findings serve to highlight the impact of PHT and HPPH, on the macrophage's ability to contribute to ECM turnover and underscore the importance of Na+ and Ca2+ channels in activated macrophages.
An interesting finding of our study was the suppression of TNF-α but not IL-6 by PHT. IL-6 enhances proliferation of fibroblasts and exerts a positive effect on collagen and glycosaminoglycan synthesis [62, 63]. At high levels, TNF-α has been reported to inhibit collagen synthesis  and increase MMP synthesis in gingival fibroblasts [65–67], which contributes to gingival breakdown. Conversely, at low levels (< 10 ng/ml) TNF-α stimulates cellular proliferation, induces production of ECM and inhibits phagocytosis of collagen by gingival fibroblasts [68, 69]. Since TNF-α enhances MMP-1  and MMP-9  expression, the blunting of TNF-α levels observed in the present study may have contributed to the decrease in supernatant levels of MMP-1 and MMP-9. In microglial cells, blockade of sodium channels with PHT significantly reduced LPS-induced secretion of IL-1α, IL-1β, and TNF-α, but not IL-6 or IL-10 suggesting that sodium channels participate in the process of cytokine release . In agreement, we noted specific modulation of LPS-induced TNF-α but not IL-6 in the presence of high concentrations of PHT (50 μg/ml). Black et al  also demonstrated that tetrodotoxin, a sodium channel blocker, inhibited secretion of IL-1α, IL-1β, and TNF-α secretion but to a lesser degree than PHT, in spite of similar inhibitory actions on sodium channels. This difference was likely due to the effects on Ca2+ metabolism by PHT. It was also interesting to note that HPPH had no effect on TNF-α levels. As discussed above this may be due to differences in the interactions of HPPH with target molecules or that higher dose of HPPH is required for inhibition of TNF-α.
In macrophages, increased TNF-α production in response to LPS challenge is associated with a transient increase in intracellular calcium [72, 73] so that intracellular calcium may participate as a second messenger in TLR4-dependent signaling [72, 74]. Insight into a possible mechanism linking intracellular calcium and cytokine levels was recently demonstrated using RAW macrophages . Using a pharmacological approach, Yamashiro et al  examined the role of transient receptor potential vanilloin 4 (TRPV2), a calcium permeable channel, in LPS-induced calcium mobilization and induction of cytokines. They reported that LPS-induced IL-6 production was due at least in part by calcium mobilization solely from intracellular sources and partly by entry of extracellular calcium through TRPV2. Further, they reported that in addition to calcium mobilization through the IP3-receptor, TRPV2-mediated intracellular calcium mobilization involved NFκB-dependent TNF-α and IL-6 expression, while extracellular calcium entry is involved in NFκB-independent IL-6 production. Collectively, these findings may provide insights into how PHT and HPPH modulate cytokine and possibly MMP/TIMP levels. Future studies will be necessary to evaluate the impact of these agents on intracellular and extracellular calcium levels in macrophages prior to LPS challenge and their correlation to cytokine and MMP/TIMP production.