Autophagy in periodontitis patients and gingival fibroblasts: unraveling the link between chronic diseases and inflammation

Abstract

Background: Periodontitis, the most prevalent chronic inflammatory disease, has been related to cardiovascular diseases. Autophagy provides a mechanism for the turnover of cellular organelles and proteins through a lysosome-dependent degradation pathway. The aim of this research was to study the role of autophagy in peripheral blood mononuclear cells from patients with periodontitis and gingival fibroblasts treated with a lipopolysaccharide of Porphyromonas gingivalis. Autophagy-dependent mechanisms have been proposed in the pathogenesis of inflammatory disorders and in other diseases related to periodontitis, such as cardiovascular disease and diabetes. Thus it is important to study the role of autophagy in the pathophysiology of periodontitis.

Methods: Peripheral blood mononuclear cells from patients with periodontitis (n = 38) and without periodontitis (n = 20) were used to study autophagy. To investigate the mechanism of autophagy, we evaluated the influence of a lipopolysaccharide from P. gingivalis in human gingival fibroblasts, and autophagy was monitored morphologically and biochemically. Autophagosomes were observed by immunofluorescence and electron microscopy.

Results: We found increased levels of autophagy gene expression and high levels of mitochondrial reactive oxygen species production in peripheral blood mononuclear cells from patients with periodontitis compared with controls. A significantly positive correlation between both was observed. In human gingival fibroblasts treated with lipopolysaccharide from P. gingivalis, there was an increase of protein and transcript of autophagy-related protein 12 (ATG12) and microtubule-associated protein 1 light chain 3 alpha LC3. A reduction of mitochondrial reactive oxygen species induced a decrease in autophagy whereas inhibition of autophagy in infected cells increased apoptosis, showing the protective role of autophagy.

Conclusion: Results from the present study suggest that autophagy is an important and shared mechanism in other conditions related to inflammation or alterations of the immune system, such as periodontitis.

Figure 1

Reactive oxygen species production and autophagy in periodontitis patients. (A) ROS production was analyzed in PBMCs from patients with and without periodontitis by flow cytometry as described in Methods. *P < 0.001 periodontitis versus no periodontitis. (B). Expression of LC3 transcripts in PBMCs from patients with and without periodontitis assessed by real-time PCR as described in Methods.*P <0.001 periodontitis versus no periodontitis. (C) Correlation between ROS levels and MAP-LC3 mRNA levels in PBMCs from patients with periodontitis. Data represent the mean ± SD of three separate experiments. non perio: participants without periodontitis; PBMC: peripheral blood mononuclear cells; perio: patients with periodontitis; ROS: reactive oxygen species; SD: standard deviation.

Figure 2

Autophagy in human gingival fibroblasts treated with lipopolysaccharide (10 μg/mL). (A) mRNA levels of ATG12, and LC3 in control and LPS-treated fibroblasts. Statistical significance: *control versus LPS-treated HGF (P < 0.01) (B) Protein expression of Atg12 and LC3. Protein levels were determined by densitometric analysis of three different western blots and normalized to GADPH signal.*P < 0.01, between control and LPS-treated fibroblasts. (C) Representative images of autophagic markers (LC3, β-galactosidase) in control and LPS-treated fibroblasts that were visualized by immunofluorescence and light microscopy respectively, as described in Methods. Bar = 25 μm. Data represent the mean ± SD of three separate experiments. CTL: control; IOD: integrated optical intensity; LPS: lipopolysaccharide.

Figure 3

Ultrastructure of lipopolysaccharide-treated human gingival fibroblasts (10 μg/mL). Control fibroblasts show mitochondria with typical ultrastructure. Laminar bodies and autophagosome with mitochondria were present in LPS-treated fibroblasts (black arrow); Bar = 500 nm. LPS: lipopolysaccharide.

Figure 4

Action of antioxidants on human gingival fibroblast-related autophagy and mitochondrial co-localization of lysosomal markers of autophagy. (A) Quantification of acidic vacuoles in control and LPS-treated fibroblasts by Lysotracker staining and flow cytometry analysis after antioxidant treatment.*P < 0.01 between control and LPS-treated fibroblasts.**P < 0.01 between LPS-treated fibroblasts and LPS + antioxidants. (B) Quantification of mitochondrial ROS in control and LPS-treated fibroblasts by MitoSOX staining and flow cytometry analysis after antioxidant treatment.*P < 0.01 between control and LPS-treated fibroblasts.**P < 0.01 between LPS-treated fibroblasts and LPS + antioxidants. (C) Protein expression of LC3 in HGF treated with LPS 10 μg/mL and antioxidants performed by western blotting as described in Methods. **P < 0.001 between LPS-treated fibroblasts and LPS + antioxidants. (D) Mitochondrial-induced ROS degraded by autophagy. Mitochondrial ROS production was localized by Mitosox Red staining. Cells were then harvested, fixed and immunostained with LC3 (autophagosome marker) and examined in a fluorescence microscope as described in Methods. Data represent the mean ± SD of three separate experiments. α-toc: α-tocopherol; BHA: butylated hydroxyanisole; CTL: control; CoQ₁₀: coenzyme Q₁₀; HGF: human gingival fibroblasts; LPS: lipopolysaccharide; NAC: N-acetylcysteine.

Figure 5

Indices of cell viability and apoptosis in control and lipopolysaccharide-treated fibroblasts after autophagy arrest by 3-methyl adenine (20 mM). Results are expressed as mean ± SD of three independent experiments. *P < 0.01 between control and LPS-treated fibroblasts. CTL: control: LPS: lipopolysaccharide.