B10 Cells Alleviate Periodontal Bone Loss in Experimental Periodontitis

Abstract

B10 cells can regulate inflammatory responses in innate immunity. Toll-like receptors (TLRs) play an important role in B cell-mediated immune responses in periodontal disease. This study aimed to determine the effects of TLR-activated B10 cells on periodontal bone loss in experimental periodontitis. Spleen B cells isolated from C57BL/6J mice were cultured with Porphyromonas gingivalis lipopolysaccharide (LPS) and cytosine-phospho-guanine (CpG) oligodeoxynucleotides for 48 h. B10-enriched CD1d^hi CD5⁺ B cells were sorted by flow cytometry and were adoptively transferred to recipient mice through tail vein injection. At the same time, P. gingivalis-soaked ligatures were placed subgingivally around the maxillary second molars and remained there for 2 weeks before the mice were euthanized. Interleukin-10 (IL-10) production and the percentage of CD1d^hi CD5⁺ B cells were significantly increased with treatment with P. gingivalis LPS plus CpG compared to those in mice treated with P. gingivalis LPS or CpG alone. Mice with CD1d^hi CD5⁺ B cell transfer demonstrated reduced periodontal bone loss compared to the no-transfer group and the group with CD1d^lo CD5^- B cell transfer. Gingival IL-10 mRNA expression was significantly increased, whereas expressions of receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG), tumor necrosis factor alpha (TNF-α), and IL-1β were significantly inhibited in the CD1d^hi CD5⁺ B cell transfer group. The percentages of CD19⁺ IL-10⁺ cells, CD19⁺ CD1d^hi CD5⁺ cells, and P. gingivalis-binding CD19⁺ cells were significantly higher in recovered mononuclear cells from gingival tissues of the CD1d^hi CD5⁺ B cell transfer group than in tissues of the no-transfer group and the CD1d^lo CD5^- B cell transfer group. This study indicated that the adoptive transfer of B10 cells alleviated periodontal inflammation and bone loss in experimental periodontitis in mice.

Keywords: B cell; CpG; LPS; Toll-like receptor; lipopolysaccharide; periodontitis.

FIG 1

Changes in IL-10 mRNA and protein levels in mouse splenocyte B cells after LPS, CpG, and LPS+CpG treatment. Splenocyte B cells were separated from nonimmunized C57BL/6J mice and cultured with P. gingivalis LPS (10 μg/ml), CpG (10 μM), and P. gingivalis LPS (10 μg/ml) plus CpG (10 μM) for 48 h. (a) IL-10 mRNA expression levels in cell lysates of the control, LPS, CpG, and LPS+CpG groups were determined by real-time PCR in duplicates. (b) Secreted IL-10 protein levels in the supernatants of the same groups as the ones mentioned above were measured in duplicates by using an enzyme-linked immunosorbent assay kit (means ± standard errors; n = 6) (*, P < 0.05; **, P < 0.01).

FIG 2

B10 cell expansion in mouse splenocyte B cells after LPS, CpG, and LPS+CpG treatments and IL-10 mRNA expression levels in the CD1d^hi CD5⁺ B cell subset. Splenocyte B cells were separated from nonimmunized C57BL/6J mice and cultured with P. gingivalis LPS (10 μg/ml), CpG (10 μM), and P. gingivalis LPS (10 μg/ml) plus CpG (10 μM) for 48 h. (a) IL-10-expressing B cells (CD19⁺ IL-10⁺ B cells) in control and treatment groups were detected by using flow cytometry in duplicates. (b) The percentages of CD19⁺ IL-10⁺ B cells in control and treatment groups were quantified and analyzed by using FlowJo software (means ± standard deviations; n = 5) (*, P < 0.05). (c) IL-10 mRNA expression levels in cell lysates of the untreated total B cell group, the LPS+CpG-treated total B cell group, the CD1d^lo CD5⁻ B cell subset from the LPS+CpG-treated group, and the CD1d^hi CD5⁺ B cell subset from the LPS+CpG-treated group were determined by real-time PCR (means ± standard errors; n = 5) (*, P < 0.05; **, P < 0.01).

FIG 3

Adoptive transfer of CD1d^hi CD5⁺ B cells inhibits P. gingivalis-associated ligature-induced periodontal bone loss. P. gingivalis-soaked ligatures were placed subgingivally around the maxillary second molars of C57BL/6 mice on day 1 and were retained for 2 weeks to establish an experimental periodontitis model. CD1d^hi CD5⁺ B cells and CD1d^lo CD5⁻ B cells were sorted from splenic B cells separated from P. gingivalis-immunized donor mice by flow cytometry and then transferred (1 × 10⁶ cells in 100 μl PBS per mouse) into recipient mice through the tail vein on day 2; the same amount of PBS were injected into the control group without ligation and the group with ligation and no transfer. (a) Maxillae were collected on day 14, and the buccal and palatal alveolar bone resorption areas around maxillary second molars were measured. (b) Data are presented as bone resorption area per square millimeter at a magnification of ×30 (means ± standard errors; n = 6) (**, P < 0.01; NS, no significant difference).

FIG 4

Adoptive transfer of CD1d^hi CD5⁺ B cells suppresses gingival expressions of RANKL/OPG, TNF-α, and IL-1β but increases IL-10 expression. Gingival tissues from mice of the no-transfer control group, the CD1d^lo CD5⁻ B cell transfer group, and the CD1d^hi CD5⁺ B cell transfer group were isolated under a surgical microscope at day 14 after ligation. The gingival mRNA expression levels of RANKL/OPG (a), IL-10 (b), TNF-α (c), and IL-1β (d) were detected and analyzed by RT-qPCR, as indicated, in duplicates, and the relative levels were normalized to the GAPDH level (means ± standard errors; n = 6) (*, P < 0.05; **, P < 0.01).

FIG 5

Adoptive transfer of CD1d^hi CD5⁺ B cells increases the numbers of IL-10-expressing B cells, CD1d^hi CD5⁺ B cells, and P. gingivalis-binding B cells in gingival tissue. Gingival tissues from mice of the no-transfer control group, the CD1d^lo CD5⁻ B cell transfer group, and the CD1d^hi CD5⁺ B cell transfer group were isolated under a surgical microscope at day 14 after ligation. Mononuclear cells were recovered from gingival tissues of each group (total number of cells ranging from 1.3 × 10² to 2.7 × 10³ cells per tissue). The percentages of CD19⁺ IL-10⁺ cells (a and d), CD19⁺ CD1d^hi CD5⁺ cells (b and e), and P. gingivalis-binding CD19⁺ cells (c and f) in these mononuclear cells were measured by flow cytometry in duplicates and were analyzed by using FlowJo software (means ± standard errors; n = 6) (**, P < 0.01).