Effects of 1,25-dihydroxyvitamin D3 on experimental periodontitis and AhR/NF-κB/NLRP3 inflammasome pathway in a mouse model

Abstract Vitamin D has been known to have important regulatory functions in inflammation and immune response and shows inhibitory effects on experimental periodontitis in animal models. However, the potential mechanism has yet to be clarified. Recent studies have highlighted Aryl hydrocarbon receptor (AhR) and its downstream signaling as a crucial regulator of immune homeostasis and inflammatory regulation. Objective: This study aimed to clarify the effect of 1,25-dihydroxyvitamin D3 (VD3) on experimental periodontitis and AhR/nuclear factor-κB (NF-κB)/NLR pyrin domain-containing 3 (NLRP3) inflammasome pathway in the gingival epithelium in a murine model. Methodology: We induced periodontitis in male C57BL/6 wild-type mice by oral inoculation of Porphyromonas gingivalis (P. gingivalis), and subsequently gave intraperitoneal VD3 injection to the mice every other day for 8 weeks. Afterwards, we examined the alveolar bone using scanning electron microscopy (SEM) and detected the gingival epithelial protein using western blot analysis and immunohistochemical staining. Results: SEM images demonstrated that alveolar bone loss was reduced in the periodontitis mouse model after VD3 supplementation. Western blot analyses and immunohistochemical staining of the gingival epithelium showed that the expression of vitamin D receptor, AhR and its downstream cytochrome P450 1A1 were enhanced upon VD3 application. Additionally, VD3 decreased NF-κB p65 phosphorylation, and NLRP3, apoptosis-associated speck-like protein, caspase-1, interleukin-1β (IL-1β) and IL-6 protein expression. Conclusions: These results implicate the alleviation of periodontitis and the alteration of AhR/NF-κB/NLRP3 inflammasome pathway by VD3 in the mouse model. The attenuation of this periodontal disease may correlate with the regulation of AhR/NF-κB/NLRP3 inflammasome pathway by VD3.


Introduction
Periodontitis is a bacterium-induced chronic immunoinflammatory disease that leads to loss of gingival tissue and bone support of the dentition. 1 Porphyromonas gingivalis (P. gingivalis) has been reported to be important in periodontitis and it can release virulence factors, such as lipopolysaccharide, inducing host immune response and periodontal tissue damage. 2 After being exposed to lipopolysaccharide from P. gingivalis, gingival epithelial cells produced a variety of inflammatory mediators, including interleukin-1β (IL-1β) and interleukin-6 (IL-6), exerting various immunomodulatory functions in periodontal tissues. 3,4 Thus, regulating the inflammatory response in gingival epithelia may become a potential strategy for periodontitis treatment.
Recently, vitamin D 3 has emerged as a crucial regulator of the immune system, and it has immunomodulatory properties in different inflammatory diseases, including inflammatory bowel disease and oral lichen planus. 5,6 Active form of vitamin D 3 , 1,25-dihydroxyvitamin D 3 (VD3), is reported to alleviate excessive inflammatory response in many epithelial cells, including intestinal epithelial cells and keratinocytes. 6,7 Although in clinical research, the beneficial effect of VD3 on periodontitis still remains to be confirmed; 8,9 reduced alveolar bone loss has been observed in murine experimental periodontitis after supplementation with the stable form of VD3. 10,11 These findings suggest that VD3 may have potential protective effect on periodontitis, and this effect might be linked to its immunomodulatory functions on the oral epithelium.
Aryl hydrocarbon receptor (AhR) is a ligandactivated transcription factor, and its immunoregulatory function has been highlighted recently. 12 AhR activation has been shown to improve immune homeostasis in epithelial cells, and stimulation of oral commensal bacteria can enhance its activation in oral epithelial cells. 13,14 NLR pyrin domain-containing 3 (NLRP3) is a pattern recognition receptor with a key role in host defense against pathogens. 15 NLRP3 assembles a multi-protein complex (inflammasome), which consists of NLRP3, apoptosis-associated speck-like protein (ASC) and caspase-1. 16 Current research on macrophages has shown that the NLRP3 inflammasome has a crosstalk with AhR and nuclear factor-κB (NF-κB), a pivotal regulator of inflammation-related gene transcription. 17 other day from 11 wk of age, and they were injected the last time 1 day before sacrifice, at wk 19. VD3 was dissolved in sterile corn oil (VD3 dose: 2.5 μg/kg body weight), and sterile corn oil was used as vehicle (the mice in N and P groups were given only corn oil). All was accepted as significant.

VD3 reduces alveolar bone loss in experimental periodontitis
Quantitative analysis of alveolar bone loss revealed that both P. gingivalis-infected groups (V and P groups) had more bone loss than the normal control group. However, V group showed obviously decreased bone loss after VD3 administration for 8 wks when compared to P group ( Figure 1). This result indicates the attenuated bone loss after VD3 treatment.

VD3 increases VDR expression in gingival epithelia
A s s h o w n by w e s t e r n b l o t a n a l y s e s a n d immunohistochemical staining, VDR protein levels in the gingival epithelium were markedly increased in V group compared with other two groups (N and P groups). Moreover, no significant difference in VDR expression was observed between N and P groups  VD3 inhibits IL-1β and IL-6 production in gingival epithelia Western blot analyses showed amplified expression of IL-1β and IL-6 in P group compared with N group.
Furthermore, attenuated expression of these cytokines was found in V group compared with P group (Figure 4).
Similarly, immunohistochemical staining revealed that the expression of both cytokines was greater in mice with periodontitis than in their normal counterparts (P vs. N, and V vs. N), while the expression was reduced  "Note: Protein expression of VDR, AhR, CYP1A1, p-p65, NLRP3, ASC, caspase-1, IL-1β and IL-6 in the gingival epithelium of the mouse model was examined using immunohistochemical staining. Staining intensity was quantified, and shown as mean optical density. After 1,25-dihydroxyvitamin D3 treatment, the expression of VDR, AhR and CYP1A1 was enhanced in mice with periodontitis, while the expression of p-p65, NLRP3, ASC, caspase-1, IL-1β and IL-6 was inhibited. Data are presented as the mean ± SD (n = 10). VDR: P<0.05 for P vs. V mice, and N vs. V mice. AhR, CYP1A1, p-p65, NLRP3, ASC, caspase-1, IL-1β and IL-6: P<0.05 for N vs. P mice, P vs. V mice, and N vs. V mice. N, normal control; P, Porphyromonas gingivalis infection; V, Porphyromonas gingivalis infection with 1,25-dihydroxyvitamin D3 treatment.   Table 1).

Discussion
Recently, VD3 has been considered an important regulator of immune response in different inflammatory diseases, such as type 1 diabetes and inflammatory bowel disease. 5,21 In clinical research, vitamin D supplementation was reported to decrease the  which processes pro-IL-1β into its mature form IL-1β. 17,40 In immune cells such as macrophages, through the interaction with NF-κB, activated AhR signaling can inhibit NLRP3 inflammasome formation and subsequent secretion of IL-1β, and other proinflammatory cytokines, improves inflammatory response. 17 In clinical research, the VD3 effect on periodontal disease still remains controversial. Several studies on adults showed that lower serum 25-hydroxyvitamin D 3 levels were significantly associated with periodontitis. 41,42 However, some investigators demonstrated that serum vitamin D levels or vitamin D supplementation did not seem to be related to periodontal status. 8  Thus, in the study on periodontitis, especially on its inflammatory regulation, we should consider the genetic differences between different animal species, and between experimental animals and humans.
This study showed the VD3 effect on experimental periodontitis in a mouse model, but there were some limitations. First, the mouse strain we used was more susceptible to human oral bacterium P. gingivalis and tended to exhibit severe periodontitis, compared with some other mouse strains and animal species. Second, we did not compare the in vivo VD3 concentrations before and after VD3 treatment to show the its supplementation levels. Third, we did not examine some inflammatory markers closely related to periodontitis, such as periodontal inflammatory infiltrate in mice. To further elucidate the precise effect and mechanisms of VD3 on periodontitis, in future experiments we can choose different animals to induce periodontitis, such as different mouse strains with AhR knockdown or rat ligature models, to mimic the complex human host response to microbial challenge and treatment.
Moreover, we can detect more parameters, as periodontal VD3 concentrations and inflammatory infiltrate in periodontal tissues, to confirm the VD3 status and periodontal inflammation.

Conclusions
We observed that VD3 attenuated P. gingivalis-