The main finding of the present study is that autophagy might be an important mechanism involved in chronic inflammatory diseases like periodontitis. Here, we have demonstrated, for the first time in patients with periodontitis, an enhancement of the autophagy phenomenon mediated by mitochondrial ROS in PBMCs. Also, our in vitro gingival fibroblast model showed how the periodontal etiological agent P. gingivalis LPS led to ROS-mediated autophagy. Periodontitis represents an example of how the organism responds to an insult. Here, some bacteria produce a local disease that may hasten the inflammatory systemic response, inducing and increasing autophagy . In this situation, cell metabolism is triggered to counteract the aggression. The key organelle for energy production and autophagic control in the cell, the mitochondria, is activated . In fact, it seems that mitochondrial 'health' should be fully considered when taking into account an organism's capacity to manage these pathological challenges. This may support the rising interest on the influence of mitochondria in inflammation-related diseases.
It is well known that the main source of cellular ROS is mitochondria. Moreover, it has been demonstrated that mitophagy/autophagy blockade leads to the accumulation of damaged ROS-generating mitochondria. This in turn activates the NLRP3 inflammasome which might explain the frequent association of mitochondrial damage with inflammatory diseases .
Recently, our group described that PBMCs from patients with periodontitis have a mitochondrial dysfunction characterized by lower CoQ10 levels and citrate synthase activity, together with high levels of ROS production . Also, we described that LPS-treated gingival fibroblasts raised oxidative stress and led to mitochondrial dysfunction in terms of lower protein expression, loss of mitochondrial mass and impaired membrane potential. These results agree with data from the present study in which the influence of periodontitis in modifying systemic defense mechanisms, plus other local effects at the gingival level, leads to enhanced ROS production. It has been reported that ROS production and oxidative stress are a common consequence of dysfunctional mitochondria and play important roles in the development of autophagy . We found increased expression of autophagy-related mRNA and proteins, demonstrating the activation of autophagy after ROS enhancement that occurred after mitochondrial dysfunction induced by P. gingivalis LPS. Moreover, lysosomal and autophagic markers (β-galactosidase, LC3 and LysoTracker staining) were higher in treated fibroblasts, indicating lysosomal proliferation. We confirmed these results by electron microscopy, which clearly showed the presence of laminar bodies and autophagosomes engulfing mitochondria.
Autophagy is a process by which cytosol and organelles are sequestered within double-membrane vesicles, delivering their contents for lysosome/vacuole degradation, followed by recycling of resulting materials . The induction of autophagy could be part of the cellular program leading to cell death, or it could reflect attempts by the cell to repair itself through the removal of damaged organelles. In this sense, autophagy might be induced to aid in removing damaged mitochondria. In the present study, we observed an important activation of autophagy-related mRNA and proteins after P. gingivalis LPS induction. Furthermore, we also confirmed by immunofluorescence that autophagosome markers such as LC3, co-localized with cytochrome c, a mitochondrial marker, and β-galactosidase, a typical lysosomal enzyme. These results agree with previous studies in which LPS-induced inflammation led to autophagy overexpression, both in cultured cardiomyocytes of adult rats  as well as in rat liver tissue .
To test if LPS treatment activated autophagy via the induction of ROS production, we cultured LPS-treated HGF with CoQ10, α-tocopherol, BHA and NAC, all of them very efficient antioxidants. It is worthwhile to underline that CoQ10 could act as a key molecule in this context for cell well-being, both for its antioxidant properties  and for its essential redox role in the mitochondrial respiratory chain . Results showed that all antioxidants significantly reduced acidic vacuoles induced by treatment with LPS. As stated before, we previously established the relationship between LPS treatment and HGF and ROS . In a recent investigation, a similar finding was also described in hepatic mitochondria from mice treated with a single dosage of LPS. The authors found that LPS administration affected mtDNA and eventually mitochondrial function, while the use of antioxidant treatments with Mn-Superoxide Dismutase, nitric oxide synthase inhibitors, superoxide or peroxynitrite scavengers prevented the above mentioned effects. Noteworthy, in our study, is that treatment with antioxidants also significantly decreased conversion of LC3-I to LC3-II, suggesting a reduction in autophagosome formation. CoQ10 and α-tocopherol, both lipophilic antioxidants, were more efficient in significantly attenuating ROS production, thus confirming the importance of ROS generated in the lipophilic environment of mitochondrial membranes. In the work by Choumar et al. , a role of superoxide anion (O2•¯), reacting with nitric oxide to form mtDNA and protein-damaging peroxynitrite, was pointed out. Recently, O2•¯ has been proposed as the major ROS regulating autophagy . These are new indications about the importance of proper preservation of structure and function of cell mitochondria. In this way, mitochondrial damage might lead to further enhanced ROS production, resulting in a downward spiral where mitochondrial viability is concerned. In turn, the accumulation of dysfunctional mitochondria is a very critical step because it is related to aging, cancer and neurodegenerative diseases .
Autophagy is like a double-edged sword, playing a role in cell survival as well as in cell death. It promotes cell death in some settings, but acts as a protective response in others. Thus, it is believed that selective mitochondrial autophagy (mitophagy) contributes to the maintenance of mitochondrial quality by eliminating damaged mitochondria or their excessive number , although little is known about this mechanism. It has been proposed that autophagy might act as an adaptive mechanism, defending organisms against the inflammatory process, and could be the background converging point with CVD. It may constitute an important physiological or pathophysiological response to cardiac stress, such as ischemia or pressure overload, which are frequently encountered in patients with coronary artery disease, hypertension, aortic valvular disease and congestive heart failure. The accumulation of autophagosomes has been noted in cardiac biopsy tissues of patients with these disorders, rodent models of these cardiac diseases, and isolated stressed cardiomyocytes . Inhibition of autophagy in the heart induces age-related cardiomyopathy in experimental animals . By contrast, induced autophagy in atherosclerosis plaque cells is a survival pathway in plaque stability and rupture . Consistent with what has been mentioned above, previous studies have supported the hypothesis that autophagy has a protective role in LPS-induced injury in cardiomyocytes . In agreement with this hypothesis on the protective role of autophagy, the present research demonstrates that disruption of autophagic processing by 3-MeA leads to cell death.
Given our results, we could hypothesize that mitochondrial dysfunction could represent a possible common functional derangement linking different inflammatory diseases such as periodontitis and CVD. In this sense, it could be a common event in all patients with periodontitis, namely a possible risk factor: in fact, mitochondria play an important role in proinflammatory signaling and ROS production that has also been shown to be an important activator of inflammasome-mediated inflammation . Autophagic turnover of cellular constituents, either general or specific for mitochondria (that is, mitophagy), eliminates dysfunctional or damaged mitochondria, thus counteracting degeneration, dampening inflammation and preventing unwarranted cell loss. To the best of our knowledge, this is the first time autophagy activation has been described in patients with periodontitis.