Modulation of Gene Expression Related to Toll-Like Receptor Signaling in Dendritic Cells by Poly(γ-Glutamic Acid) Nanoparticles

Mice.Female C57BL/6 mice (H-2K^b; 6 to 8 weeks old) were purchased from Charles River (Yokohama, Japan). Experiments were carried out in accordance with the guidelines for animal experimentation of Kagoshima University.

Reagents.γ-PGA (average molecular weight = 380,000) was kindly provided by Meiji Seika (Tokyo, Japan). PAE, polymyxin B (PmB), and OVA were purchased from Sigma (St. Louis, MO).

Preparation of γ-PGA NPs.The synthetic procedures for γ-PGA NPs and OVA-NPs have been described in our previous reports (1, 36). The concentration of bacterial endotoxin in γ-PGA NPs was determined by a Limulus amoebocyte lysate assay (Seikagaku, Tokyo, Japan) and was found to be <10 endotoxin units/ml.

Preparation of DCs.Murine bone marrow-derived DCs were generated as previously described (26, 27, 36). Briefly, femurs and tibias were removed from mice and purified from the surrounding muscle tissues. Both ends of the bones were cut with scissors, and the marrow was flushed with phosphate-buffered saline (PBS), using a syringe with a 26-gauge needle. The suspension was passed through a 50-μm cell strainer. Bone marrow cells (2.5 × 10⁶ cells/10 ml) were suspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin G, 100 μg/ml streptomycin, 2-mercaptoethanol, and 20 ng/ml recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech, London, United Kingdom) and were placed into a petri dish. Fresh culture medium was added to the dish on day 3. On day 7, nonadherent or loosely adherent cells were harvested and used as immature DCs.

Oligonucleotide microarray.Immature DCs (1 × 10⁶ cells/ml) were incubated with either 300 μg/ml γ-PGA NPs, 1 μg/ml LPS, or 300 μg/ml unparticulate γ-PGA at 37°C. Six, 12, and 24 h after incubation, total RNA was extracted from the cells by use of an extraction kit (RNeasy; Qiagen, Hilden, Germany). The quality of the total RNA was examined by a model 2100 bioanalyzer (Agilent, Santa Clara, CA). Microarray experiments with the samples were carried out according to Agilent's protocol. Briefly, the total RNA (500 ng) was converted to cDNA with Moloney murine leukemia virus RT and the T7 promoter primer. The cDNA was transcribed and amplified with T7 RNA polymerase to produce a cRNA labeled with cyanine 3. The cyanine 3-labeled cRNA was purified with an RNeasy kit and examined for its concentration and labeling quality by use of a spectrophotometer. The cRNA was fragmented and hybridized to an Agilent whole-mouse-genome oligonucleotide microarray (4 × 44,000 slide format). After hybridization, the microarray was washed thoroughly and scanned with a microarray scanner (Agilent). The microarray scan data were processed with Future Extraction software (version 9.5.1; Agilent) according to the instructions in the manual. Cell culture and microarray experiments were repeated three times.

Data analysis.The expression level of each gene was analyzed by GeneSpring GX software (version 7.3.1; Agilent). Briefly, after importing the processed data into the software, they were normalized based on the default normalizing settings for one-color experiments, according to the GeneSpring 7.3 user's guide (Agilent). The normalized data were filtered on the basis of parameters in certain specific columns of the original data files to remove the control and other inappropriate spots.

Real-time RT-PCR.DCs (1 × 10⁶ cells/ml) were incubated with either 300 μg/ml γ-PGA NPs, 1 μg/ml LPS, 5 μg/ml zymosan (InvivoGen, San Diego, CA), 1 μg/ml class B CpG (InvivoGen), 1 μg/ml poly(I:C) (InvivoGen), or PBS at 37°C. Six hours after incubation, total RNA was extracted from the cells with an RNeasy kit. The quality of the total RNA was examined by a model 2100 bioanalyzer. Real-time RT-PCR analysis of the samples was carried out using a mouse Toll-like receptor (TLR) signaling pathway PCR array (SuperArray; SuperArray Biosciences, Frederick, MD), which includes primer pairs specific to 84 genes related to TLR-mediated signaling pathways, according to the manufacturer's protocol. Briefly, cDNA was synthesized from the total RNA (1 μg) by use of an RT² first-strand kit (SuperArray Biosciences) and was subjected to real-time PCR with an RT² real-time PCR SYBR green/ROX kit (SuperArray Biosciences) on an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA). The PCR consisted of 1 cycle of 95°C for 10 min and 50 cycles of 95°C for 15 s and 60°C for 1 min. The expression levels of target genes were normalized to those in PBS-treated DCs (control).

Effect of PmB on DC activation.DCs (1 × 10⁶ cells/ml) were incubated with either 100 μg/ml γ-PGA NPs or 1 μg/ml LPS in the absence or presence of 10 μg/ml PmB. Twenty-four hours after incubation, the cells and culture supernatants were collected. The TNF-α level in the culture supernatants was determined by use of a sandwich enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, Carlsbad, CA). For phenotypic analysis, the cells were washed with PBS and stained with a phycoerythrin (PE)-conjugated anti-CD40 monoclonal antibody (MAb; BD Biosciences, San Jose, CA) or a PE-conjugated anti-CD80 MAb (BD Biosciences) for 30 min at 4°C. After being washed, the cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences).

Analysis of spleen cells.Mice (3 to 5 mice in each group) were immunized subcutaneously with either PBS (100 μl), OVA alone (10 μg of OVA in 100 μl of PBS), CFA + OVA (10 μg of OVA in 100 μl of CFA), alum + OVA (10 μg of OVA in 100 μl of alum), or OVA-NPs (10 μg of γ-PGA NPs carrying 10 μg of OVA in 100 μl of PBS) on day 0. On day 10, spleen cells were collected by use of lympholyte-M (Cedarlane, Burlington, Ontario, Canada) and were analyzed by pentamer staining, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay. For pentamer staining, the cells were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-CD8 MAb (Proimmune, Oxford, United Kingdom) and an allophycocyanin-conjugated pentameric H-2K^b complex (BD Biosciences) with the OVA_257-264 cytotoxic T-lymphocyte (CTL) epitope. After being washed with PBS containing 0.1% sodium azide and 0.1% bovine serum albumin, the cells were fixed with PBS containing 2.5% formaldehyde (Wako, Osaka, Japan), washed with PBS, and analyzed by flow cytometry. For intracellular cytokine staining, the cells were incubated with BD GolgiPlug (BD Biosciences) and either OVA_257-264 peptide (10 μg/ml) or medium alone for 10 h. After being washed, the cells were incubated with a purified anti-mouse CD16/32 MAb (BD Biosciences) followed by staining with a PE-conjugated anti-CD8 MAb (BD Biosciences). The cells were permeabilized with Cytofix/Cytoperm Plus (BD Biosciences) and stained with an FITC-conjugated anti-IFN-γ MAb, an FITC-conjugated anti-IL-2 MAb, or an FITC-conjugated anti-TNF-α MAb (all from BD Biosciences) for 30 min at 4°C. The cells were washed with PBS and subjected to flow cytometric analysis. For ELISPOT assay, the cells were cultured with either medium alone, the OVA_257-264 peptide (10 μg/ml), or the control peptide (10 μg/ml) in an ELISPOT plate (BD Biosciences). After incubation for 24 h at 37°C, the plate was washed, and IFN-γ-producing cells were measured by ELISPOT assay according to the manufacturer's protocol.

Microarray data accession numbers.The microarray data are available at the Gene Expression Omnibus (GEO). The accession numbers are GSE15087 and GSE15089, which contain the data on comparative gene expression modulated by γ-PGA NPs, LPS, and unparticulate γ-PGA and the data on the time course of gene expression modulated by γ-PGA NPs, respectively.

Gene expression in DCs after treatment with γ-PGA NPs.DC maturation involves coordinated regulation of several genes. To identify the genes modulated during the maturation process of DCs, comprehensive gene expression in bone marrow-derived DCs was examined by use of a whole-mouse-genome oligonucleotide microarray after treatment with either γ-PGA NPs, LPS, or unparticulate γ-PGA and was compared to that in DCs treated with PBS (control). Among the genes that could be analyzed (41,267 genes), the expression of 8,006 and 13,282 genes was found to be modulated in DCs, with statistical significance (P < 0.05), after treatment with γ-PGA NPs and LPS, respectively (data not shown). However, the expression of only 20 genes was significantly upregulated or downregulated in DCs after treatment with unparticulate γ-PGA. The correlation coefficient for the relative expression levels of analyzed genes was 0.67 between γ-PGA NP-treated DCs and LPS-treated DCs (Fig. 1A). However, no such positive correlation was observed (r = −0.0026) between γ-PGA NP-treated DCs and unparticulate γ-PGA-treated DCs (Fig. 1B).

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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.

The expression levels of the genes related to immune responses are summarized in Table 1. Among the genes for cytokines and cytokine receptors, 8 genes were upregulated >100-fold 6 h after treatment with γ-PGA NPs. These gene products included IL-6, CSF2 (GM-CSF), IL-12β, IL-1α, CSF3 (granulocyte colony-stimulating factor [G-CSF]), IL-1 family member 6, IL-23α, and inhibin β. In addition, 14 genes were upregulated >10-fold. A similar result was observed in DCs treated with LPS. The expression of some chemokine and chemokine receptor genes was also highly upregulated by treatment with γ-PGA NPs or LPS (Table 1). An interesting finding is that the genes related to Th1 cytokines, such as IL-12β, IL-23α, IL-12α, and IFN-γ, were equally or even more upregulated in γ-PGA NP-treated DCs than in LPS-treated DCs, whereas those related to Th2 cytokines, such as IL-10, IL-33, and IL-19, were less upregulated in γ-PGA NP-treated DCs than in LPS-treated DCs. Except for a few genes, the expression of most chemokine and chemokine receptor genes was enhanced in both γ-PGA NP-treated DCs and LPS-treated DCs (Table 1). In particular, the expression of the genes related to the neutrophil-attracting chemokines CXCL1, CXCL2, and CXCL5 was remarkably enhanced in γ-PGA NP-treated DCs. It has been demonstrated that CCR7 is upregulated during DC maturation and does play an important role in leading DCs to lymph nodes (16). In fact, the CCR7 gene was upregulated 5.5- and 11.4-fold in γ-PGA NP-treated DCs and LPS-treated DCs, respectively. On the other hand, the CCR2 gene was expressed only in immature DCs (Table 1). The expression of the genes related to costimulatory molecules (CD40, CD80, and CD86) and adhesion molecule (CD54) was also upregulated in γ-PGA NP-treated DCs as well as LPS-treated DCs.

γ-PGA NPs modulate the expression of TLR signaling-related genes.Since the microarray analysis revealed that like LPS, γ-PGA NPs could modulate gene expression in DCs, the effect of γ-PGA NPs on the expression of TLR signaling-related genes was examined by quantitative real-time RT-PCR and compared to that of LPS, CpG, zymosan, and poly(I:C). Consistent with the results of microarray analysis, γ-PGA NPs and LPS upregulated the gene expression of IL-1α, IL-1β, IL-6, IL-10, IL-12α, lymphotoxin A (LTA), TNF-α, and CXCL10 (Fig. 2A). The correlation coefficient between γ-PGA NP-treated DCs and LPS-treated DCs was high (r = 0.920). A high correlation coefficient (r = 0.918) for gene expression was also obtained between γ-PGA NP-treated DCs and CpG-treated DCs (Fig. 2B). In addition, significant increases in cyclooxygenase-2 (COX-2) gene expression were observed in DCs treated with γ-PGA NPs, LPS, CpG, and zymosan. The correlation coefficient for gene expression between γ-PGA NP-treated DCs and zymosan-treated DCs was 0.708 (Fig. 2C), while there was no correlation (r = 0.394) between γ-PGA NP-treated DCs and poly(I:C)-treated DCs (Fig. 2D).

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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.

γ-PGA NP-induced DC maturation is not due to LPS contamination.Although γ-PGA is a bacterial product, γ-PGA NPs contain LPS at a concentration far below the threshold that activates cytokine production and costimulatory molecule expression in DCs (33, 35). To confirm that LPS contamination is indeed not responsible for the activation of DCs by γ-PGA NPs, the cells were treated with γ-PGA NPs or LPS after preincubation with PmB and analyzed for protein expression by ELISA and flow cytometry. TNF-α production from DCs was enhanced by treatment with both LPS and γ-PGA NPs (Fig. 3A). However, such enhancement was significantly reduced by preincubation of LPS with PmB but not by preincubation of γ-PGA NPs with PmB. A similar result was obtained for the surface expression of costimulatory molecules, where CD40 and CD86 expression was strongly upregulated by treatment with both LPS and γ-PGA NPs (Fig. 3B). Again, the upregulation was annihilated by preincubation of LPS with PmB but not by preincubation of γ-PGA NPs with PmB. These results indicate that the induction of DC maturation by γ-PGA NPs is due not to LPS contamination but to their own effect on DCs.

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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.

γ-PGA NPs are more effective than conventional adjuvants.To determine the comparative effects of γ-PGA NPs and conventional adjuvants in inducing antigen-specific cellular immune responses, mice were injected subcutaneously with either PBS, OVA, OVA-NPs, OVA + CFA, or OVA + alum. When the antigen-specific T-cell response was examined by flow cytometry, a marked increase of OVA antigen-specific CD8⁺ T cells was observed for the cells obtained from the OVA-NP-immunized mice, whereas little increase of the antigen-specific CD8⁺ T cells was identified for the cells from mice immunized with OVA alone, OVA + CFA, or OVA + alum (Fig. 4A). Since the cytokine production from immune cells is an important marker of their activation, the intracellular cytokine assay is able to detect the state of activation in individual cells. When the production of intracellular cytokines in the antigen-specific CD8⁺ T cells was examined, marked increases of IFN-γ, TNF-α, and IL-2 production were observed for the cells obtained from the OVA-NP-immunized mice. Again, little increases of such cytokine production were observed for the cells from mice immunized with OVA alone, OVA + CFA, or OVA + alum (Fig. 4B). Furthermore, the most induction of IFN-γ-producing cells was achieved by immunization with OVA-NPs, as determined by ELISPOT assay (Fig. 4C). These results suggest that γ-PGA NPs are a more efficient adjuvant than CFA and alum in inducing cellular immune responses.

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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.