Cytidine-phosphate-guanosine oligodeoxynucleotides in combination with CD40 ligand decrease periodontal inflammation and alveolar bone loss in a TLR9-independent manner

Abstract Local administration of toll-like receptor 9 (TLR9), agonist cytidine-phosphate-guanosine oligodeoxynucleotide (CpG ODNs), and CD40 ligand (CD40L) can decrease ligature-induced periodontal inflammation and bone loss in wild type (WT) mouse. Objective: This study aimed to explore whether such effect is dependent on TLR9 signaling. Material and Methods: Purified spleen B cells isolated from WT C57BL/6J mice and TLR9 knockout (KO) mice were cultured for 48 hours under the following conditions: CD40L, CpG+CD40L, CpG at low, medium and high doses. We determined B cell numbers using a hemocytometer at 24 h and 48 h. Percentages of CD1dhiCD5+ B cells were detected by flow cytometry. Interleukin-10 (IL-10) mRNA expression and protein secretion were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and by ELISA, respectively. The silk ligature was tied around the maxillary second molars for 14 days, during which the CpG+CD40L mixture or PBS was injected into palatal gingiva on days 3, 6, and 9. Results: For both WT and TLR9 KO mice, CpG significantly induced B cell proliferation, increased IL-10 mRNA expression and protein secretion of IL-10 but reduced CD1dhiCD5+ B cells population; local injection of CpG+CD40L mixture significantly decreased alveolar bone loss and the number of TRAP-positive cells adjacent to the alveolar bone surface, and significantly increased the gingival mRNA expression of IL-10 and decreased RANKL and IFN-γ mRNA expression. Conclusions: These results indicated that CpG plus CD40L decreased periodontal inflammation and alveolar bone loss in a TLR9-independent manner in ligature-induced experimental periodontitis.


Introduction
Periodontitis is characterized by inflammation and alveolar bone loss, which are closely related to bacteria-induced host immune responses. 3 Conventional therapies for periodontitis focus on clinical methods, such as removal of dental plaque and calculus, reduction of infection and periodontal surgery. Although these treatments are sufficient to treat periodontal diseases in many cases, the host immune response has been sustained in an imbalanced state. Recently, researchers have explored immune mechanisms and treatment strategies associated with periodontitis by looking for novel inflammatory pathways, 8 regulating host innate and adaptive immune responses, 9 and exploring immune mechanisms that inhibit alveolar bone loss. 29 Toll-like receptors (TLRs), a class of immune molecules closely related to periodontitis, consist of at least 13 proteins working as key pathogen recognition receptors (PRRs) in innate immunity system and respond to many kinds of microbial products and injuryinduced endogenous products. 13 Of these receptors, TLR9 is unique for its engagement in both microbial nucleic acids and danger-associated molecular patterns (DAMPs), and communication with other TLRs. 5 TLR9 can activate pro-and/or anti-inflammatory signaling pathways and mediate inflammatory responses in periodontitis pathogenesis. 8 In another study using oral gavage model of periodontitis, Kim, et al. 11 (2005) found that TLR9 Knockout (KO) mice show better resistance to periodontal inflammation and TLR9 can regulate TLR2-and TLR4-triggered inflammation by downstream signaling pathways.
TLR9 is not only activated by microbial nucleic acids, but also by synthetic cytidine-phosphateguanosine (CpG) DNA motifs. 11 At the molecular level, internalization of CpG activates TLR9 mediated sequence recruitment signals of MyD88, interleukin receptor associated kinase (IRAK) and TNFreceptor associated factor 6 (TRAF6), which activate downstream transcription factors (NF-κB and AP-1) causing the induction of proinflammatory cytokines. 9 Some studies have suggested that preventive therapy in mice using synthetic CpG can protect them against diverse infections. 21,22 Mouse B cell was one of the first definite cell types which has been proved to directly respond to immunostimulatory CpG-containing DNA sequences in vitro and in vivo. 12,17 Our recent study has also suggested that local administration of CpG

Bone morphometric analysis
The defleshed maxillae were bleached with 3% hydrogen peroxide for 8 hours and stained with 1% toluidine blue. The degree of alveolar bone loss was measured by Nikon microscope (SMZ745T, Nikon Instruments Inc). Images of the palatal alveolar bone of each maxilla were acquired, and the specific area presenting alveolar bone loss was measured by Image J software (NIH). The defined method of the area has been previously described. 16   but not in WT mice ( Figure 3D).

IL-10 mRNA expression and protein expression were increased by CpG stimulation in B cells from both WT and TLR9 KO mice
The levels of IL-10 mRNA and protein expression  Gingival IL-10 mRNA expression was increased and RANKL and IFN-γ mRNA expression was decreased after local administration of CpG+CD40L in WT mice and TLR9 KO mice.
The levels of gingival mRNA expression of IL-10, OPG, RANKL, IFN-γ, IL-1β and TNF-α were measured by qRT-PCR. In both WT and TLR9 KO mice, the mRNA level of gingival IL-10 expression was significantly increased and the mRNA levels of gingival RANKL and IFN-γ was significantly decreased CD40L can activate membrane-associated protein CD40 on B cells, inducing maturation of B cells into antibody-producing B cells. It is also crucial for activating regulatory B cells. 25 Our results showed that CpG alone induced activation of B10 function (IL-10 secretion), however, it also significantly reduced B10 frequency in cultured splenic B cells. In addition, CD40L alone induces B10 expansion but not IL-10 secretion.
Thus, the combination of CD40L with CpG will both induce B10 activation and maintain B10 population.
The synergy effects were shown in B cells from WT mice in vitro and ligation-induced periodontitis model in WT mice in vivo in our previous study. 29 Moreover, the effects of different concentration of CpG+CD40L on decreasing ligature induced periodontitis have been previously studied. 29 The previous experimental results showed that low concentration of CpG+CD40L (1 uM of CpG+0.1 ug/ml of CD40L) had a higher inhibitory effect on ligature induced periodontitis and alveolar bone loss than that of high concentration of

Conclusions
In conclusion, our study demonstrated that local administration of CpG+CD40L can decrease periodontal inflammation and alveolar bone loss in a TLR9-independent mechanism. The unknown mechanism remains to be investigated in the future, which is important to develop immunological interventions for treatments of periodontitis.