Modulation of gene expression related to Toll-like receptor signaling in dendritic cells by poly(gamma-glutamic acid) nanoparticles

Abstract

Poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs) have previously been reported as an efficient antigen delivery system with adjuvant activity. In this study, the gene expression in murine bone marrow-derived dendritic cells (DCs) treated with gamma-PGA NPs was examined by oligonucleotide microarray analysis and compared with that in cells treated with other adjuvants. The gene expression of proinflammatory chemokines, cytokines, and costimulatory molecules was upregulated considerably in DCs treated with gamma-PGA NPs. The upregulation pattern was similar to that in DCs treated with lipopolysaccharide (LPS) but not to that in DCs treated with unparticulate gamma-PGA. The activation of DCs by gamma-PGA NPs was confirmed by real-time reverse transcriptase PCR (RT-PCR) analysis of genes related to Toll-like receptor (TLR) signaling. The effect of gamma-PGA NPs on DCs was not annihilated by treatment with polymyxin B, an inhibitor of LPS. Furthermore, the immunization of mice with gamma-PGA NPs carrying ovalbumin (OVA) as an antigen significantly induced antigen-specific CD8(+) T cells and antigen-specific production of interleukin-2, tumor necrosis factor alpha, and gamma interferon from the cells. Such activities of gamma-PGA NPs were more potent than those obtained with immunization with OVA plus aluminum hydroxide or OVA plus complete Freund's adjuvant. These results suggest that gamma-PGA NPs induce a CD8(+) T-cell response by activating innate immunity in a fashion different from that of LPS. Thus, gamma-PGA NPs may be an attractive candidate to be developed further as a vaccine adjuvant.

FIG. 1.

Gene expression in DCs treated with γ-PGA NPs versus LPS (A) and γ-PGA NPs versus unparticulate γ-PGA (B). Murine bone marrow-derived DCs (1 × 10⁶ cells/ml) were treated with either 300 μg/ml γ-PGA NPs, 1 μg/ml LPS, or PBS for 6 h. Total RNA was extracted from the treated DCs, purified, and subjected to oligonucleotide microarray analysis. The expression level of each gene was normalized to that of the control (PBS treatment). The expression profiles are shown by scatter plots. The genes upregulated >5-fold were plotted.

FIG. 2.

Gene expression related to TLR signaling in DCs treated with γ-PGA NPs versus LPS (A), γ-PGA NPs versus CpG (B), γ-PGA NPs versus zymosan (C), and γ-PGA NPs versus poly(I:C) (D). DCs (1 × 10⁶ cells/ml) were treated with either 300 μg/ml γ-PGA NPs, 1 μg/ml LPS, 1 μg/ml CpG, 5 μg/ml zymosan, 1 μg/ml poly(I:C), or PBS for 6 h. Total RNA was extracted from the treated DCs, purified, and subjected to real-time RT-PCR analysis. The expression level of each gene was normalized to that of the control (PBS treatment). The genes upregulated >2-fold are indicated by their product names.

FIG. 3.

Effect of PmB on cytokine production and costimulatory molecule expression induced by γ-PGA NPs and LPS. DCs were incubated with either γ-PGA NPs or LPS in the absence or presence of 10 μg/ml PmB. (A) Culture supernatants were collected and their TNF-α level determined by ELISA. (B) The cells were stained with a PE-conjugated anti-CD40 MAb or a PE-conjugated anti-CD86 MAb and analyzed by flow cytometry. The number in each histogram indicates the mean fluorescence intensity.

FIG. 4.

Antigen-specific T-cell responses induced by OVA-NPs, CFA, and alum. Mice were immunized subcutaneously once with either PBS, OVA, OVA-NPs, OVA + CFA, or OVA + alum. T cells were obtained from the spleen. (A) The antigen (OVA)-specific CD8⁺ T cells were analyzed by pentamer staining. (B) IL-2, TNF-α, and IFN-γ production of the antigen-specific CD8⁺ T cells was measured by intracellular cytokine staining. (C) Spleen cells from the immunized mice were restimulated with the control or antigen peptide. The number of IFN-γ-producing cells was determined by ELISPOT assay. SFU, spot-forming units. All results represent the means for three separate experiments.