In vitro and in vivo study of the pathogenic role of PPARα in experimental periodontitis

Abstract Objective The purpose of this study is to investigate the pathogenic role of PPARα in periodontal antigen treated gingival cells in vitro and in experimental periodontitis in vivo . Methodology Gingival fibroblasts, gingival epithelial cells and splenocytes were isolated from C57BL/6J wild type (WT) mice and treated with fixed P. gingivalis at for 48 hours. The mRNA levels of PPARs, TNFα, IL-1β and IL-10 were detected by Real-time quantitative PCR. Silk ligatures after being soaked in the P.gingivalis suspension were tied around both maxillary second molars of WT mice or PPARα knock-out (KO) mice for two weeks. PPARα agonist fenofibrate and vehicle control were injected into the different side of the palatal gingiva on days 3, 6, and 9. At day 14, bone resorption and gingival mRNA expression levels of PPARs, TNFα, IL-1β and IL-10 were measured by micro-computed tomography and RT-qPCR respectively. Results P. gingivalis treatment downregulated the expression of PPARα, but not PPARβ or PPARγ, and increased the expression of TNF-α and IL-1β in Gingival fibroblasts, gingival epithelial cells and splenocytes from WT mice. Gingival mRNA levels of PPARα were significantly decreased in experimental periodontitis in WT mice. The bone loss of PPARα KO mice in experimental periodontitis was significantly higher than WT mice and was not reduced by fenofibrate treatment. Gingival TNFα protein expressions were significantly increased by P. gingivalis associated ligation and decreased by fenofibrate treatment in WT mice but not in PPARα KO mice. Conclusion This study suggests that PPARα plays an essential role in periodontitis.


Introduction
Periodontitis is a progressive inflammatory disease involved in the teeth surrounding tissues.
The inflammation can lead to alveolar osteolysis by increasing the osteoclast activity and inhibiting the osteoblast activity, 1,2 causing tooth loss and jaw bone deterioration in the periodontitis. 3 Besides local oral cavity pathology, chronic periodontitis is also associated with an increased risk of developing many systemic diseases. 4,5 Inflammation is usually driven by oral bacterial communities interacting with the host immune system and contributes to the inflammation in the process of periodontitis. 6 Among several main periodontal pathogens, Porphyromonas gingivalis (P.g), a gramnegative oral anaerobe, is one of the most prominent periodontal pathogens. 7 P.g mainly inhabits the subgingival sulcus of human oral cavity and is the main colonizer of dental plaque. The periodontitis is initiated by accumulating plaque, in which the virulence factors are released and induce the immune response. Although the exact mechanism is unknown, it has been hypothesized that the causative role of P.g in the periodontitis may be by synthesizing pathogenic factors by its virulence factors. 7 Lipopolysaccharide (LPS), a key component in the outer membrane of P.g, is a causative virulence factor of P.g. LPS plays a major pathogenic role that initiate and enhance the inflammation in the periodontitis. 8 LPS can function as a pathogen-associated molecular pattern to trigger or mediate the inflammatory response by binding to the Toll-like receptor and CD14. Studies have shown that this mechanism contributes to the inflammation and osteoclastogenesis in the periodontitis. 9 In the periodontitis, the inflammation initiates with the immune response of resident leukocytes to the bacterial biofilm and with the activation of the inflammation cytokines. 10 TNF-α 11 and IL-1β 12,13 are the most related pro-inflammatory cytokines and pathways, mediating the pathogenic process of periodontitis. TNF-α is the main connector to higher destructive periodontal disease and is the key pathogen to early inflammatory cytokines. The levels of TNF-α contribute to the onset of destructive periodontal diseases via several mechanisms. 14 IL-1β triggers many inflammatory reactions such as bone resorption and the production of tissue degrading proteinases. However, the exact mechanism that produces the TNF-α and IL-1β in periodontitis is still unclear.
Peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear hormonal transcription factor and regulates transcription of many genes involved in lipid metabolism, stress response, and inflammation. 15 PPARs belong to the phylogeny of the steroid receptor superfamily and are called nuclear hormone factors.
Its three subtypes include α, β/δ, and γ, and each one mediates many fatty acids (FAs)   Mouse gingival fibroblasts obtained from WT mice were cultured following a protocol previously described. 28 The gingival tissue was cut, minced and digested with collagenase I (2 mg/mL, Worthington Biochemical, Lakewood, NJ). After filtered by a nylon mesh filter and centrifuged for 10 min at 1,500 rpm, raw cells pellets were re-suspended and plated at a density of 10 5 cell/cm 2 . Cells were passaged at a

Statistical analysis
All the quantitative data were expressed as means ± SD. Unpaired Student's t test was performed to compare both groups of datasets in statistical analysis.
For multiple groups, differences were analyzed using the one-way analysis of variance (ANOVA) test followed by SNK-q multiple comparisons using GraphPad 6.0 software (La Jolla, CA). Statistical significance was set at p<0.05.

Results
P. gingivalis downregulated the expression of PPARα, but not PPARβ or PPARγ in vitro.  Figure   1 shows that all types of PPARs, including PPARα, PPARβ, and PPARγ, were expressed in these three types of cells. Then we tested if the expression of PPARs changed in periodontal conditions and if the P. gingivalis contributes to these changes. Primary cultured gingival fibroblasts, gingival epithelial cells, and splenocytes were exposed to P. gingivalis, a condition that mimics periodontitis. We quantified the transcriptional levels of PPARs of each group and compared to their controls. Figure 1A, 1D, and 1G show that exposure to P. gingivalis significantly decreased PPARα expression in all three cells. The reduction of PPARα in splenocytes was greater than in gingival cells. However, unlike the decreases of PPARα, the expression of PPARβ (Figure 1 B, 1E, and 1H) and PPAR gamma ( Figure 1C, 1F, and 1I) was not significantly affected in all three types of cells.
These results suggested that the PPARα, not PPARβ and PPAR gamma, is associated with the infection of periodontal pathogen.

P. gingivalis induced pro-inflammatory cytokines in vitro.
Since P. gingivalis infection is associated with a progressive increase of pro-inflammatory cytokines in periodontal diseases, we examined the effect of   This result agrees with the in vitro findings ( Figure   3D), which suggests that PPARα partly reverses TNF-α production while KO mice produce higher levels of TNF-α than WT.
Then we examined if PPARα has a similar TNFα regulating effect on immune cells under periodontitis conditions. The splenocytes of WT mice were exposed to LPS with/without the presence of fenofibrate. Figure   5C and 5D show that exposure to LPS increased TNFα, which was significantly reduced by fenofibrate.
We replaced fenofibrate by WY14643 in a parallel experiment, a synthesized PPARα agonist, to see treatment did not suppress this induction when PPARα was knock out ( Figure 5E, 5F). However, the induction levels of TNFα by LPS was significantly lower than PPARα knockout conditions when PPARα was re-expressed in KO cells and fenofibrate treatment reduced the induction of TNFα ( Figure 5E, 5F), similar to WT splenocytes ( Figure 5C, 5D). This result suggested that the reduction of TNF-α in periodontitis may depend on PPARα.
Figure 5-PPARα is essential to regulate the pro-inflammatory marker TNFα in ligature-induced experimental periodontitis. The same amount (50 µg) of gingival proteins from sites without ligation, ligation with vehicle treatment, ligation with fenofibrate treatment of WT and PPARα mice were used for Western blot analysis of TNFα (A), semiquantified by densitometry and normalized by β-actin levels (B) (mean±SD, n=4, *p<0.05, **p<0.01, N.S., no significance). Mouse splenocytes were separated from WT mice and treated with LPS (1µg/ ml), LPS(1µg/ml)+fenofibrate (50μM), LPS(1µg/ml)+WY14643(100μM), LPS(1µg/ml)+WY14643(100μM)+GW6471(10μM) for 48 hours. Total cell lysates were used for Western blot analysis of TNFα (C), semiquantified by densitometry and normalized by β-actin levels (D) (mean±SD, n=4, *p<0.05, **p<0.01). Mouse splenocytes were separated from PPARα mice and infected with adenovirus with control vector or PPARα vector for 24 hours and then treated with LPS (1µg/ml), LPS(1µg/ml)+fenofibrate (50μM) for 48 hours. Total cell lysates were used for Western blot analysis of TNFα (E), semiquantified by densitometry and normalized by β-actin levels (F) (mean±SD, n=4, **p<0.01, N.S., no significance) Our most essential finding is that PPARα plays a crucial role in periodontitis. We emphasize that PPARα, not PPARß or PPRAα, is affected by the P. gingivalis ligation. We evidence that the transcriptional level of genes antagonize the bone impairing mechanism of PPARγ and PPARβ mediation or if PPARα activation has a different bone regulating mechanism. Future study is needed to address that.
Our study has many limitations that must be Our study shows that PPARα is an essential transcription factor to regulate inflammation and bone loss in the P. gingivalis-associated ligature-induced experimental periodontitis. Activation of PPARα downregulated the expression and production of the pro-inflammatory cytokines, and inhibited the alveolar bone loss, which represents a new therapeutic target to treat periodontitis.