TIPE2 regulates periodontal inflammation by inhibiting NF-κB p65 phosphorylation

Abstract The roles and molecular mechanisms of tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) in periodontitis remain largely unknown. Objective This study aimed to determine the expression of TIPE2 and NF-κB p65 in rat Porphyromonas gingivalis-induced periodontics in vivo. Methodology Periodontal inflammation and alveolar bone resorption were analyzed using western blotting, micro-computed tomography, TRAP staining, immunohistochemistry, and immunofluorescence. THP-1 monocytes were stimulated using 1 μg/ml Pg. lipopolysaccharide (Pg.LPS) to determine the expression of TIPE2 in vitro. TIPE2 mRNA was suppressed by siRNA transfection, and the transfection efficiency was proven using western blotting and real-time PCR. The NF-κB pathway was activated by treating the cells with 1 μg/ml Pg.LPS to explore related mechanisms. Results The expression of both TIPE2 and NF-κB p65 was increased in the gingival tissues of rat periodontitis compared with normal tissues. Positive expression of TIPE2 was distributed in inflammatory infiltrating cells and osteoclasts in the marginal lacunae of the alveolar bone. However, strong positive expression of TIPE2 in THP-1 was downregulated after Pg.LPS stimulation. TIPE2 levels negatively correlated with TNF-α and IL-1β. Decreased TIPE2 in THP-1 further promoted NF-κB p65 phosphorylation. Mechanistically, TIPE2 knockdown upregulated NF-κB signaling pathway activity. Conclusions Taken together, these findings demonstrate that TIPE2 knockdown aggravates periodontal inflammatory infiltration via NF-κB pathway. Interventions aimed at increasing TIPE2 may help in the therapeutic applications for periodontitis.


Introduction
Periodontitis is one of the most common human inflammatory diseases, which is closely related to both bacterial infections and host immunity. 1,2 A dysregulated immune response can lead to aggravated periodontal inflammation and bone resorption by allowing the excessive recruitment of pro-inflammatory cells to the periodontal tissues. 3 Perturbed immune cells, including monocytes/macrophages in periodontitisaffected sites, play a critical role in the pathogenesis of periodontitis. 4,5 Studies have shown that both negative regulation of inflammatory cells and interventions aimed at increasing anti-inflammatory cells can assist in therapeutic applications for periodontitis. 6 Therefore, it is of interest to explore effective molecular targets for immune cell regulation.
Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2), a member of the tumor necrosis factor-alphainduced protein-8 family, is a novel negative regulator of innate and adaptive immune responses, and its selective immune expression maintains immune homeostasis and prevents hyperresponsiveness.
TIPE2 is preferentially expressed in lymphoid tissues, and its deletion leads to lethal Toll-like receptor and T cell receptor activation, multiorgan inflammation, splenomegaly, and premature death. 7 TIPE2 inhibits M1 macrophage-related neutrophilic inflammation in asthma 8 and accelerates IL-4-induced immunomodulatory M2 differentiation. 9 Decreased TIPE2 in peripheral blood mononuclear cells is associated with the disease progression of chronic hepatitis, 10 asthma, 11 systemic lupus erythematosus, 12 and osteoporosis. 13 TIPE2 protects cardiomyocytes from ischemia-reperfusion-induced apoptosis by decreasing cell autophagy via the mTORC1 signaling pathway. 14 Reduced TIPE2 expression is inversely associated with proinflammatory cytokines and positively correlated with bone mineral density. 13 TIPE2 has also been shown to promote host resistance to Pseudomonas aeruginosa and inhibit NF-κB signaling and inflammatory infiltration in keratitis. 15 Although studies have highlighted that TIPE2 is an important regulator of the immune response in various diseases, the roles and molecular mechanisms of TIPE2 in periodontitis remain largely unknown.
In this study, we investigated the expression of TIPE2 in rat periodontitis induced by Porphyromonas gingivalis in vivo. Additionally, we stimulated THP-1 monocytes with 1 μg/ml of Pg. lipopolysaccharide (Pg.

LPS) to determine the expression of TIPE2 in vitro.
Targeting TIPE2 in the immune response may prove to be beneficial in facilitating the healing process of periodontitis.

Methodology Animals
A total of 18 cage-reared male Wistar rats (6-week-old, weight: 260-280 g) were provided with adequate normal hard food and water under a suitable laboratory feeding environment (12-h light/12-h dark cycle, 22-24°C, and 45% relative humidity). Gingival tissues from both sides of the maxillary second molars were taken from six rats for western blotting two weeks after ligation, whereas gingival tissues from six normal rats were used as controls (n=6).
Additionally, six left maxillary bones from six rats were collected four weeks after ligation for micro-computed tomography (CT), whereas six right maxillary bones were taken for TRAP staining, immunohistochemistry, and immunofluorescence.

Results
Increased TIPE2 in gingival tissues of rat periodontitis According to western blot results, the expression of NF-kB p65 and p38 was significantly increased in the gingival tissues, suggesting that NF-kB and p38 pathways were activated and that gingival inflammation occurred. The expression of TIPE2 in the inflammatory gingival tissues was also significantly higher than that in normal gingival tissues, suggesting that TIPE2 participated in gingival inflammation (Figure 2).

Histochemical localization of TIPE2 in rat periodontitis
Significant alveolar bone resorption of the maxillary second molar was observed at four weeks after ligation (Figure 3a

Expression of TIPE2 in LPS-stimulated THP-1 cells in vitro
Strong positive expression of TIPE2 was detected in THP-1 cells using immunofluorescence (Figure 6a).

Decreased TIPE2 promoted NF-κB p65 phosphorylation
The knockdown effect was verified using western blotting and real-time PCR. siRNA transfection suppressed TIPE2 mRNA and protein levels in the THP-1 cells (Figure 7a and b).  showed that the expression of NF-kB p65 and p38 was significantly increased, suggesting that the classical inflammatory NF-kB and p38 signaling pathways were activated, and gingival inflammation occurred. The expression of TIPE2 in the inflammatory gingival tissues was also significantly higher than that in the normal gingival tissues, suggesting that TIPE2 participates in periodontal inflammation. *p<0.05 In this study, strong positive expressions of TIPE2 in THP-1 cells were decreased after Pg.LPS stimulation. It has been reported that decreased TIPE2 in peripheral blood mononuclear cells is associated with the progression of various diseases. [10][11][12][13] We assumed that the decreased TIPE2 in the monocyte/macrophage lineage could aggravate the process of periodontitis.
To further explore the molecular mechanisms of TIPE2 in periodontitis, TIPE2 mRNA was suppressed by siRNA transfection. The results of western blotting showed that NF-κB p65 was increased at 20 min in the interference group. This result suggested that