TLR9 Signaling Is Required for the Porphyromonas gingivalis-Induced Activation of IL-10-Expressing B Cells

Abstract

Immune cell pattern-recognition receptors such as Toll-like receptors (TLRs) play important roles in the regulation of host responses to periodontal pathogens. Our previous studies have demonstrated that immune regulatory B cells were activated by TLRs and alleviated periodontitis inflammation and bone loss. The purpose of this study is to determine the role of TLR9 signaling in the activation and IL-10 production of the primed-immune B cells in vitro. Wild-type (WT) and TLR9 knockout (TLR9KO) mice (C57BL/6 background, n = 5) were pre-immunized intraperitoneally with 1 × 10⁸ formalin-fixed P. gingivalis and boosted once with 1 × 10⁷ formalin-fixed P. gingivalis. Isolated splenocytes and purified B cells from each mouse were cultured with 1 × 10⁸ formalin-fixed P. gingivalis for 48 h. Immunocytochemistry was performed to detect CD45⁺ IL-10⁺ cells. Levels of IL-10 expression and secretion in splenocytes and B cells were detected using qRT-PCR and ELISA, respectively. After stimulation with fixed P. gingivalis, the percentage of CD45⁺ IL-10⁺ B cells and the level of IL-10 expression were significantly increased (p < 0.01) in splenocytes and purified B cells isolated from WT mice. However, these changes were not observed in splenocytes and purified B cells from TLR9KO mice when the cells were treated with fixed P. gingivalis. The percentage of CD45⁺ IL-10⁺ B cells was significantly reduced in splenocytes and purified B cells from TLR9KO mice compared to those from WT mice when challenged with P. gingivalis. IL-10 expression in B cells from TLR9KO mice was significantly decreased compared to those from WT mice at both the mRNA and protein levels. Additionally, P. gingivalis-induced up-regulation of TNF-α mRNA expressions were consistently observed in B cells from both WT and TLR9KO mice. P. gingivalis-induced B10 activation and IL-10 production during adaptive responses by primed B cells requires TLR9 signaling and can be achieved independent of T-cell help.

Keywords: B10 cells; P. gingivalis; TLR-9; adaptive immune response; periodontal disease.

Figure 1

Under P. gingivalis stimulation for 24 h, the CD45⁺IL-10⁺ cells and IL-10 mRNA level of splenocytes and B cells isolated from pre-immunized wild-type mice were calculated. (A) The representative ICC images of CD45⁺IL-10⁺ cells in each group. CD45+ cells were labeled with rat anti-mouse CD45 antibody conjugated with emerald anti-rat antibody (blue) and IL-10+ cells were labeled with goat anti-mouse IL-10 antibody conjugated with permanent red anti-goat antibody (red). (B,C) The percentage of CD45⁺IL-10⁺ cells among splenocytes (B) and B cells (C) were calculated based on cell immunocytochemistry staining. (D,E) The IL-10 mRNA expression in splenocytes (D) and B cells (E) were analyzed by quantitative RT-PCR. The data were shown as mean ± SD; significance calculated by unpaired t-test was indicated as ** p < 0.01, *** p < 0.001, **** p < 0.0001 (n = 5).

Figure 2

Under P. gingivalis stimulation for 24 h, the CD45⁺IL-10⁺ cells, IL-10 mRNA level and secreted IL-10 protein levels of splenocytes and B cells isolated from pre-immunized TLR9 knockout mice were calculated. (A) The representative ICC images of CD45⁺IL-10⁺ cells in each group. CD45 cells were labeled with rat anti-mouse CD45 antibody conjugated with emerald anti-rat antibody (blue) and IL-10 were labeled with goat anti-mouse IL-10 antibody conjugated with permanent red anti-goat antibody (red). (B,E) The percentage of CD45⁺IL-10⁺ cells among splenocytes (B) and B cells (E) were calculated based on cell immunocytochemistry staining. (C,F) The IL-10 mRNA expression in splenocytes (C) and B cells (F) was analyzed by quantitative RT-PCR. (D,G) The secreted IL-10 protein levels in the supernatants were measured by a mouse IL-10 enzyme-linked ELISA kit using a 1:10 assay diluent in PBST. The data were shown as mean ± SD; significance calculated by unpaired t-test was indicated as ** p < 0.01, **** p < 0.0001, and ns (no significant difference) as p > 0.05 (n = 5).

Figure 3

Under P. gingivalis stimulation for 24 h, the CD45⁺IL-10⁺ cells of splenocytes and B cells, and secreted IL-10 protein levels of B cells isolated from both pre-immunized wild-type and TLR9 knockout mice were calculated. (A,B) The percentage of CD45⁺IL-10⁺ cells among splenocytes (A) and B cells (B) were calculated based on cell immunocytochemistry staining. (C) The secreted IL-10 protein levels in the supernatants were measured by a mouse IL-10 enzyme-linked ELISA kit using a 1:10 assay diluent in PBST. The data were shown as mean ± SD; significance calculated by unpaired t-test was indicated as * p < 0.05, **** p < 0.0001 (n = 5).

Figure 4

Under P. gingivalis stimulation for 24 h, the IL-10 mRNA level of splenocytes and B cells isolated from both wild-type and TLR9 knockout pre-immunized mice were calculated. (A) Splenocytes from wild-type mice; (B) B cells from wild-type mice; (C) Splenocytes from TLR9 knockout mice; (D) B cells from TLR9 knockout mice. The data were shown as mean ± SD; significance calculated by unpaired t-test was indicated as * p < 0.05, ** p < 0.01, and ns (no significant difference) as p > 0.05 (n = 5).