Contribution of a Streptococcus mutans antigen expressed by a Salmonella vector vaccine in dendritic cell activation

Abstract

A Salmonella vector vaccine expressing the saliva-binding region (SBR) of the adhesin AgI/II of Streptococcus mutans has been shown to induce a mixed Th1/Th2 anti-SBR immune response in mice and to require Toll-like receptor 2 (TLR2), TLR4, and MyD88 signaling for the induction of mucosal anti-SBR antibody responses. Since dendritic cells (DC) are critical in innate and adaptive immunity, the present study assessed the role of SBR expression by the vector vaccine in DC activation. Bone marrow-derived DC from wild-type and TLR2, TLR4, and MyD88 knockout mice were stimulated with Salmonella vector BRD509, the SBR-expressing Salmonella vector vaccine BRD509(pSBRT7), or SBR protein, and the DC responses to different stimuli were compared by assessing costimulatory molecule expression, cytokine production, and signaling pathways. The DC response to both BRD509(pSBRT7) and BRD509 was dependent mainly on TLR4. BRD509(pSBRT7) and BRD509 induced upregulation of CD80, CD86, CD40, and major histocompatibility complex class II (MHC II) expression. Lower levels of interleukin-10 (IL-10) and IL-12p40 were produced by BRD509(pSBRT7)-stimulated DC than by BRD509-stimulated DC. Furthermore, BRD509(pSBRT7)-stimulated DC showed decreased p38 phosphorylation compared to that induced by DC stimulated with BRD509. However, BRD509(pSBRT7)-treated DC produced a higher level of IL-6 than BRD509-stimulated cells. The low IL-12p40 and high IL-6 cytokine profile expressed by BRD509(pSBRT7)-stimulated DC may represent a shift toward a Th2 response, as suggested by the increased expression in Jagged-1. These results provide novel evidence that a heterologous protein expressed by a Salmonella vector vaccine can differentially affect DC activation.

Fig. 1.

Upregulation of costimulatory molecules and MHC II on DC. DC derived from WT and TLR2, TLR4, and MyD88 KO mice were stimulated with BRD509 (MOI of 0.1), BRD509(pSBRT7) (MOI of 0.1), SBR (40 μg/ml), LPS (100 ng/ml), or Pam3CSK4 (300 ng/ml) for 24 h. Cells were stained with fluorescence-labeled antibodies against CD80, CD86, CD40, or MHC II, followed by FACS analysis. (A) Expression of CD80, CD86, CD40, and MHC II on DC from WT and TLR2, TLR4, and MyD88 KO mice following stimulation with BRD509, BRD509(pSBRT7), SBR, or unstimulated control. (B) Increased expression of MHC II on MyD88 KO DC compared to that on WT DC following different stimulation. Numbers represent the mean fluorescence intensity. Data are representative of three independent experiments.

Fig. 2.

Cytokine production by DC. DC derived from WT and TLR2, TLR4, and MyD88 KO mice were incubated with BRD509 (MOI of 0.1), BRD509(pSBRT7) (MOI of 0.1), SBR (40 μg/ml), LPS (100 ng/ml), or Pam3CSK4 (300 ng/ml) for 24 h. Culture supernatants were collected and assayed for IL-12p40, IL-10, TNF-α, and IL-6 by ELISA. Results are expressed as the means ± standard deviations (SD) of triplicate cultures from one of three independent experiments. (A) Cytokine production by WT DC. *, P < 0.05; **, P < 0.01. (B) Comparison of cytokine production by WT and TLR2, TLR4, and MyD88 KO DC. *, P < 0.05; †, P < 0.01 (compared with WT DC).

Fig. 3.

Cytokine production by BRD509- and BRD509(pSBRT7)-stimulated DC after 48 h of stimulation. DC from WT mice were stimulated with BRD509 (MOI of 0.1) or BRD509(pSBRT7) (MOI of 0.1). Forty-eight hours later, culture supernatants were collected and assayed for IL-12p40, IL-10, TNF-α, and IL-6 by ELISA. Results are expressed as the means ± SD of triplicate cultures from one of three independent experiments. **, P < 0.01 compared with the BRD509 group.

Fig. 4.

Stimulation of DC with BRD509(pSBRT7) results in decreased p38 phosphorylation. DC derived from WT and TLR2, TLR4, and MyD88 KO mice were stimulated with BRD509 (MOI of 0.1) or BRD509(pSBRT7) (MOI of 0.1) for 0, 10, 30, 60, and 120 min. Western blot analysis of p38, ERK1/2, and JNK phosphorylation (p-) in whole-cell lysates. Total p38 served as a loading control. Results are from WT (A) and TLR2 KO (B) DC. Data are representative of three independent experiments with similar results.

Fig. 5.

Inhibition of p38. DC derived from WT mice were pretreated with or without the selective p38 inhibitor (SB203580, 10 μM) for 1 h and then stimulated with BRD509 (MOI of 0.1), BRD509(pSBRT7) (MOI of 0.1), or SBR (40 μg/ml) for 24 h. The levels of IL-12p40, IL-10, TNF-α, and IL-6 in culture supernatants were determined by ELISA. Results are expressed as the means ± SD of triplicate cultures from one of three independent experiments. *, P < 0.05; **, P < 0.01 (compared to “no inhibitor” group).

Fig. 6.

Jagged-1 and Delta-1 expression in stimulated DC. DC derived from WT mice were incubated with BRD509 (MOI of 0.1), BRD509(pSBRT7) (MOI of 0.1), or SBR (40 μg/ml) for 12, 24, and 48 h. Cells were lysed, and whole-cell lysates were assessed for Jagged-1 (A) and Delta-1 (B) expression by Western blot analysis; β-actin served as a loading control (C). The number below each band represents the fold increase over baseline, quantitated by densitometry. Results are representative of three independent experiments with similar results.