Calcitriol inhibits osteoclastogenesis in an inflammatory environment by changing the proportion and function of T helper cell subsets (Th2/Th17)

Abstract Objectives Previously, we found that by regulating T helper (Th) cell polarization, calcitriol intervention inhibited lipopolysaccharide (LPS)‐induced alveolar bone loss in an animal periodontitis model, but the underlying cellular events remain unknown. Materials and methods In this study, mouse Th cells were incubated in an inflammatory environment in the presence of dendritic cells (DCs) and LPS. Then, the potential of the Th cells to undergo Th2/Th17 polarization, the RANKL expression of the polarized Th cells and the subsequent influences of the polarized Th cells on RAW264.7 cell osteoclastogenesis in response to calcitriol administration were assessed. Finally, the effects of calcitriol on antigen presentation by DCs during these cellular events were evaluated. Results In response to calcitriol administration, Th cells in an inflammatory environment exhibited an enhanced potential for Th2 polarization along with a decreased potential for Th17 polarization. In addition, RANKL expression in Th17‐polarized cells was largely inhibited. Furthermore, inflammation‐induced osteoclastogenesis in RAW264.7 cells was suppressed following coculture with calcitriol‐treated Th cells. During these cellular events, increased expression of Th2 promoters (such as OX‐40L and CCL17) and decreased expression of Th17 promoters (such as IL‐23 and IL‐6) were found in DCs. Conclusions Calcitriol can inhibit osteoclastogenesis in an inflammatory environment by changing the proportion and function of Th cell subsets. Our findings suggest that calcitriol may be an effective therapeutic agent for treating periodontitis.


| INTRODUC TI ON
Chronic periodontitis is characterized by local inflammation-induced bone loss due to the interaction between the host defence mechanism and plaque bacteria on the supporting tissues of the teeth. 1,2 During this inflammatory process, the host inflammatory response inevitably exerts an impact that induces systematic skeletal changes in alveolar bone. Specifically, the interplay between the immune system network and the receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of nuclear factor-κB (RANK)/osteoprotegerin (OPG) pathway has been recognized to play essential roles during the progressive damage of the bone until tooth loss. 1 In terms of the immune system network, T helper (Th) cells, the subsets of which are determined by immunity-associated antigen-presenting cells such as dendritic cells (DCs), can play both damaging and protective roles in bone loss by regulating the RANKL-OPG system. 3,4 Classically, Th cells can be divided into four types (Th1, Th2, Th17 and regulatory T cell (Treg)). Among these types, Th17 subsets with proinflammatory and pro-osteoclastogenic characteristics are reported to enhance leucocyte influx into periodontal tissues and intensify bone lesions through osteoclast precursor maturation and enhanced local RANKL levels. [5][6][7] In contrast, the Th2 phenotype and its characteristic cytokine, IL-4, can attenuate periodontal tissue destruction by inhibiting proinflammatory cytokine and RANKL expression. [7][8][9][10][11] Moreover, an increasing number of studies have also found that the functions of Th cells are closely related to RANKL-induced osteoclast differentiation. 8,12,13 In this context, researchers have attempted to attenuate inflammation-induced tissue destruction by modulating Th cells towards anti-inflammatory polarization. For example, promoting Th2 polarization and attenuating Th17 polarization by using bioactive immunomodulator agents, such as calcitriol, may be a practical strategy to inhibit tissue destruction. [14][15][16] Calcitriol has been confirmed to be an essential regulator in bone mineralization that functions by modulating the calcium and phosphate levels in the systemic circulation. 17,18 A deficiency in calcitriol can exacerbate osteopaenia and increase the risk of fracture in osteoporosis. 18,19 In addition to its widely known effects on bone homeostasis, calcitriol has also recently been shown to reverse the development of disease by modulating immune functions. 20 For example, calcitriol can exploit the antigen-presenting ability of DCs to regulate the polarization states of T cells, which contributes to inflammation resolution and prevents transplant rejection. 21,22 In our previously published study, we found that calcitriol supplementation could suppress periodontitis-induced bone destruction by promoting Th2 polarization and inhibiting Th17 polarization in an animal periodontitis model. 14 However, how calcitriol-induced Th cell polarization is associated with the activation of osteoclasts still requires further investigation. Therefore, the aim of the present study was to analyse the effects of calcitriol intervention on Th cell polarization and RANKL expression in an inflammatory environment in the presence of DCs and lipopolysaccharide (LPS). Then, the effect of Th cell polarization on RAW264.7 cell osteoclastogenesis was evaluated in vitro. To investigate whether calcitriol also influences the cellular behaviour of DCs, the biological effects of calcitriol on antigen presentation by DCs were assessed. Overall, our research was designed to uncover the underlying mechanism by which calcitriol affects LPSinduced osteoclastogenesis in vitro.

| Determination of the concentration of calcitriol used in cell cultures
First, a physiological saline solution was used to dissolve calcitriol (Roche Pharmaceuticals Ltd.). Then, we screened an appropriate concentration of calcitriol according to CCK8 assays. Briefly, both DCs and Th cells were seeded in triplicate in a 96-well plate at a density of 2 × 10 3 cells/well. After cell adhesion occurred, the cells were incubated with various concentrations of calcitriol (0, 1, 10 or 100 nmol/L). The CCK-8 assay was then performed on days 1, 2, 3, 4 and 5. For each test, 10 μL of CCK-8 solution was added to each test well, followed by an incubation at 37°C for 3 hours. 24 The optical absorbance (OD) value of the test well was then measured at 450 nm using a microplate reader (Infinite M200 Pro).

| Study design
Lipopolysaccharide, as a virulence factor from gram-negative bacteria, was used as a pathogenic agent to induce animal periodontitis in our previous research and other reports. 12,14 In this study, DCs in complete RPMI 1640 medium supplemented with 1 μg/mL LPS (from

| Effect of calcitriol administration on Th cell polarization in an inflammatory environment
The Th cells in the three groups (LPS group, LPS + DC group, LPS + DC + Cal group) were subjected to a 5-day incubation, and the polarization states of these Th cells were evaluated by flow cytometry. 8,25,26 Briefly, after immunostaining with an anti-CD4 PEconjugated antibody, Th cells were fixed with Intracellular Fixation & Permeabilization Buffer and then immunostained using an anti-IL-4 or anti-IL-17antibody to identify Th2 and Th17 cells, respectively.
Finally, the immunostained Th cells were detected and analysed with a Beckman Coulter Epics XL flow cytometer. In addition, the expression of Th2 polarization-related genes and proteins (GATA3, STAT5 and IL-4) 27,28 and Th17 polarization-related genes and proteins (STAT3, RORγT and IL-17) 16,[29][30][31] in the cultured Th cells was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis (for details, see Sections 2.8 and 2.9).

| Effect of calcitriol administration on RANKL expression in Th cells in an inflammatory environment
In parallel to the investigation of cell polarization, the gene and protein levels of RANKL in the Th cells (LPS group, LPS + DC group and LPS + DC + Cal group) following a 5-day incubation were determined by qRT-PCR and Western blot analysis (for details, see Sections 2.8 and 2.9). In addition, immunofluorescence and flow cytometry assays were performed to assess the proportion of IL-17 + /RANKL + cells. 13 Briefly, after Th cell smears were prepared, Th cells were incubated with primary antibodies against RANKL were determined by qRT-PCR and Western blot analysis (for details, see Sections 2.8 and 2.9). 33

| The effects of calcitriol administration on the antigen-presenting function of DCs
The antigen-presenting activity of DCs related to Th cell polarization was analysed using flow cytometry. 34 First, DCs were incubated in complete RPMI 1640 medium supplemented with 1 μg/ mL LPS in the absence (LPS group) or presence (LPS + Cal group) of 10 nmol/L calcitriol, while DCs cultured in only complete RPMI 1640 medium were designated the control (control group).
Following a 5-day incubation, the cells were harvested and incubated at 4°C for 1 hour with the following primary antibodies:

| The effects of calcitriol administration on the expression of Th2 or Th17 promoters in DCs
To clarify the role of DCs in calcitriol-mediated Th cell polarization,

| qRT-PCR
To determine the expression levels of related genes in cells,  Table 1.

| Western blot analysis
To determine the expression levels of related proteins in cells, total protein was extracted by using a lysis buffer (Sigma-Aldrich) and boiled for 10 minutes. Protein samples containing the same quantity (40 μg) were subjected to Western blot analysis according to protein concentrations measured by a BCA assay (Beyotime). Subsequently, the protein samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking in a 5% milk solution for 2 hours, the membranes were incubated with the following  . The data are shown as the mean ± SD; *P < .05 and **P < .01 represent significant differences between the indicated columns

| Statistical analysis
In this study, all experiments were performed in triplicate, and all values are expressed as the mean ± standard deviation (SD). Oneway analysis of variance (ANOVA) and Dunn's post hoc tests were applied to analyse comparisons among groups (no less than three groups). Statistical analysis was performed using SPSS software 17.0. Differences were considered statistically significant when P < .05.

| Calcitriol facilitated the polarization of Th cells towards Th2 phenotypes while inhibiting the polarization of Th cells towards Th17 phenotypes in an inflammatory environment
Flow cytometry analysis showed that the purities of CD4+ T cells and DCs isolated from mouse spleens with immunomagnetic beads were above 90% ( Figure S1A)

| Modulatory effects of calcitriol on the expression of Th2 promoters or Th17 promoters in DCs
To

| D ISCUSS I ON
Previously, we found that calcitriol could not only suppress LPSinduced bone loss but also regulate Th cell polarization in experimental periodontitis. 14 However, whether the immunomodulatory and RORγT has been found to be closely linked to alveolar bone loss induced by high IL-17 expression in mice with periodontitis. 16,29,30 Moreover, calcitriol treatment has also been reported to inhibit . The data are shown as the mean ± SD; *P < .05 and **P < .01 represent significant differences between the indicated columns useful measure to attenuate bone resorption in periodontitis by reducing NFATc1 and MMP-9.
As an immunological adjuvant, calcitriol has been found to di-  It has been reported that a high level of OX-40L in DCs can enhance Th2 polarization in vivo, which is considered an essential protective mechanism against tissue destruction in some disorders such as infectious, inflammatory and autoimmune diseases. 54 Additionally, both OX-40L and CCL17 in DCs can trigger Th2 polarization, which induces a low inflammatory response and plays a protective role in allergy under the effect of thymic stromal lymphopoietin. 35 Our data indicate that calcitriol has the crucial potential to block inflammatory reactions by upreg- In conclusion, the results reported in the current study support the conclusion that calcitriol can suppress inflammation-induced osteoclastogenesis in vitro by changing the proportion and function of Th cell subsets, which indicates that calcitriol may be a promising therapeutic agent for the treatment of chronic periodontitis.

ACK N OWLED G EM ENTS
We acknowledge our funding support from the National Natural

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.