Expression of Toll-Like Receptor 2 by Dendritic Cells Is Essential for the DnaJ-ΔA146Ply-Mediated Th1 Immune Response against Streptococcus pneumoniae

Abstract

The fusion protein DnaJ-ΔA146Ply could induce cross-protective immunity against pneumococcal infection via mucosal and subcutaneous immunization in mice in the absence of additional adjuvants. DnaJ and Ply are both Toll-like receptor 4 (TLR4) but not TLR2 ligands. However, we found that TLR2^-/- mice immunized subcutaneously with DnaJ-ΔA146Ply showed significantly lower survival rates and higher bacterial loads in nasal washes than did wild-type (WT) mice after being challenged with pneumococcal strain D39 or 19F. The gamma interferon (IFN-γ) level in splenocytes decreased in TLR2^-/- mice, indicating that Th1 immunity elicited by DnaJ-ΔA146Ply was impaired in these mice. We explored the mechanism of protective immunity conferred by DnaJ-ΔA146Ply and the role of TLR2 in this process. DnaJ-ΔA146Ply effectively promoted dendritic cell (DC) maturation via TLR4 but not the TLR2 signaling pathway. In a DnaJ-ΔA146Ply-treated DC and naive CD4⁺ T cell coculture system, the deficiency of TLR2 in DCs resulted in a significant decline of IFN-γ production and Th1 subset differentiation. The same effect was observed in adoptive-transfer experiments. In addition, TLR2^-/- DCs showed remarkably lower levels of the Th1-polarizing cytokine IL-12p70 than did WT DCs, suggesting that TLR2 was indispensable for DnaJ-ΔA146Ply-induced IL-12 production and Th1 proliferation. Thus, our findings illustrate that dendritic cell expression of TLR2 is essential for optimal Th1 immune response against pneumococci in mice immunized subcutaneously with DnaJ-ΔA146Ply.

Keywords: DnaJ-ΔA146Ply; Th1 immunity; Toll-like receptor 2; dendritic cells; protein vaccine.

FIG 1

Expression and purification of recombinant protein DnaJ-ΔA146Ply. (A) Purified DnaJ-ΔA146Ply (lane 2) was analyzed by SDS-PAGE and then stained with Coomassie brilliant blue. Lane 1, molecular mass markers. (B) BMDCs were stimulated with 5 μg/ml of DnaJ-ΔA146Ply, DnaJ-ΔA146Ply digested with protease K or pretreated with polymyxin B, and LPS (100 ng/ml) for 24 h. The quantities of TNF-α and IL-6 in the culture medium were measured by ELISA. All data are expressed as means ± SDs (n = 3), and statistical significance (***, P < 0.001; ns, not significant) is indicated for treatments compared to the controls.

FIG 2

Deficiency of TLR2 impairs the protection against pneumococcal infection induced by DnaJ-ΔA146Ply. WT and TLR2^−/− mice were immunized subcutaneously with 18 μg of DnaJ-ΔA146Ply or PBS as a vehicle control three times at 14-day intervals. The antibody titers of anti-DnaJ IgG (A) and subtypes IgG1, IgG2a, IgG2b, and IgG3 (B) in serum were measured 1 week after the last immunization. Mice were challenged with D39 (1,000 CFU) or 19F (1 × 10⁸ CFU) 2 weeks after the last immunization, and the survival rates were monitored (n = 10) (C) and bacterial loads in nasal wash were determined 72 h postinfection (n = 4 to 6) (D). (E) Histopathology of hematoxylin and eosin-stained lung sections from WT and TLR2^−/− mice immunized with DnaJ-ΔA146Pl 72 h postinfection. *, P < 0.05 based on Mann-Whitney U or log rank test.

FIG 3

TLR2^−/− mice exhibit deficient Th1 immune responses elicited by DnaJ-ΔA146Ply. Seven days after the last immunization, suspensions of splenocytes (1 × 10⁵/well) were cultured and exposed to 5 μg/ml of DnaJ-ΔA146Ply for 72 h at 37°C. Levels of IFN-γ (A), IL-17A (B), and IL-4 (C) in culture medium were determined by ELISA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

DnaJ-ΔA146Ply induces phenotypic and functional maturation of DCs. (A) Immature BMDCs (1 × 10⁶/well) were treated with 5 μg/ml of DnaJ-ΔA146Ply or 100 ng/ml of LPS for 24 h and then stained with FITC-conjugated anti-CD11c and phycoerythrin (PE)-conjugated anti-CD40, -CD86, and MHC-II. The percentage of positive cells was analyzed by flow cytometry. Immature BMDCs (1 × 10⁵/well) were stimulated with LPS or 1, 5, or 10 μg/ml of DnaJ-ΔA146Ply for 24 h. Levels of TNF-α (B), IL-6 (C), IL-1β (D), IL-23 (E), and IL-12p70 (F) in culture medium were assayed by ELISA. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (for LPS or DnaJ-ΔA146Ply groups compared with medium control).

FIG 5

DnaJ-ΔA146Ply induces DC maturation via TLR4 but not TLR2. BMDCs from WT, TLR2^−/−, and TLR4^−/− mice were stimulated with 100 ng/ml of LPS, 10 μg/ml of PGN, or 5 μg/ml of DnaJ-ΔA146Ply for 24 h. (A) Surface costimulatory molecules CD40, CD86, and MHC-II were analyzed by flow cytometry. (B to E) Cytokines TNF-α and IL-6 in supernatants were measured with ELISA. (F) Fluorescence intensity of the anti-His antibody bound to DnaJ-ΔA146Ply-treated DCs. DCs derived from WT, TLR2^−/−, and TLR4^−/− mice were treated with DnaJ-ΔA146Ply for 30 min, fixed, and stained with DAPI and anti-His antibody. The data are shown as means ± SDs. ***, P < 0.001.

FIG 6

MAPK, NF-κB, and PI3K-Akt pathways are involved in DC maturation mediated by DnaJ-ΔA146Ply. (A) BMDCs from WT, TLR2^−/−, and TLR4^−/− mice were treated with 5 μg/ml of DnaJ-ΔA146Ply for the indicated times and lysed in lysis buffer. The phosphorylation of corresponding molecules was analyzed by Western blotting. (B and C) BMDCs from WT mice were pretreated with specific inhibitors of p38 (SB203580), ERK1/2 (U0126), Jun N-terminal protein kinase (JNK) (SP600125), PI3K (LY294002), and NF-κB (BAY11-7082) for 1 h prior to stimulation with DnaJ-ΔA146Ply. Cytokines TNF-α and IL-6 in supernatants were quantified with ELISA kits. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 7

Absence of TLR2 in DCs impairs Th1 polarization in a DC and CD4⁺ T cell coculture system. Immature BMDCs from WT and TLR2^−/− mice were treated with 5 μg/ml of DnaJ-ΔA146Ply or 100 ng/ml of LPS for 24 h and then cocultured with purified naive CD4⁺ T cells sorted from spleens of WT and TLR2^−/− mice, respectively. (A to C) After 72 h, IFN-γ, IL-17A, and IL-4 levels in culture medium were assayed by ELISA. (D to F) Intracellular cytokines IFN-γ, IL-17A, and IL-4 were analyzed by flow cytometry. *, P < 0.05 for IFN-γ level in WT CD4⁺ T cells cocultured with WT BMDCs compared to TLR2^−/− BMDCs.

FIG 8

Adoptive transfer of DnaJ-ΔA146Ply-treated TLR2^−/− DCs results in deficient Th1 immune responses, and TLR2 regulates IL-12p70 production. (A to C) Immature BMDCs from WT and TLR2^−/− mice were treated with 5 μg/ml of DnaJ-ΔA146Ply for 24 h and then adoptively transferred intraperitoneally into naive mice at days 0, 14, and 28. One week after the last transfer, splenocytes of mice subjected to transfer were isolated and levels of IFN-γ, IL-17A, and IL-4 were measured. (D) BMDCs from WT and TLR2^−/− mice were treated with 5 μg/ml of DnaJ-ΔA146Ply, 100 ng/ml of LPS, or 10 μg/ml of PGN. After 24 h, the IL-12p70 in supernatants was quantified using ELISA. *, P < 0.05 for mice subjected to adoptive transfer with WT BMDCs compared to TLR2^−/− BMDCs. ***, P < 0.001 for IL-12 concentration in WT BMDCs compared to TLR2^−/− BMDCs in DnaJ-ΔA146Ply treatment groups.