NANO LETTERS

Nanosphere Induced Gene Expression in Human Dendritic Cells

Michiya Matsusaki,, Kristina Larsson,, Takani Akagi,| Malin Lindstedt, Mitsuru Akashi, and Carl A. K. Borrebaeck*,
Department of Molecular Chemistry, Graduate School of Engineering, Osaka UniVersity and CREST, Osaka, Japan, Department of Immunotechnology, Lund UniVersity, SE-220 07 Lund, Sweden, and Core Research for EVolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan
Received March 21, 2005

2005 Vol. 5, No. 11 2168-2173

ABSTRACT
The molecular mechanisms of nanosphere-induced mucosal immunization are important to decipher, since this can form the basis for novel approaches in, e.g., nasal vaccination. In this study, we have investigated the effect of nanospheres as antigen carriers on immature human dendritic cells. The results clearly indicate that tetanus toxoid immobilized nanospheres have a direct effect on human monocyte derived dendritic cells and induce a specific transcriptional profile involving genes crucial for phagocytosis and a protective immune response.

Polystyrene nanospheres (NS), coated with hydrophilic polymer chains containing functional groups,1 have recently proven useful as a carrier for biomolecules.2 Furthermore, intranasal immunization has been shown to induce a specific IgA response in the murine genital tract,3,4 using inactivated human immunodeficiency virus-1 (HIV-1) immobilized onto nanospheres (HIV-NS). In line with these results, intranasal immunization of macaques resulted in a specific anti-gp120 IgA and IgG genital tract response, using inactivated simian/ human immunodeficiency virus KU-2 capturing nanospheres (SHIV-NS).5 The molecular mechanisms behind these effects are largely unknown. Dendritic cells (DCs) are professional antigen presenting cells essential for the initiation of protective immune responses. DCs are present throughout the human body but are particularly localized at antigen-exposed sites, such as skin and mucosa. In these nonlymphoid tissues, DCs reside in an immature state lacking co-stimulatory membrane markers required for the initiation of T cell activation. Immature DCs can take up both particular and soluble antigens by either phagocytosis, receptor-mediated endocytosis, or macropinocytosis.6,7 The antigen uptake initiates maturation and migration of the DCs to secondary lymphoid tissue, where an antigen-specific activation of T cells is initiated. The ability of DCs to induce a primary response in T cells is in part a result of their up-regulation of
* Corresponding author: Department of Immunotechnology, P.O. Box 7031, Lund University, SE-220 07 Lund, Sweden; phone, +46 46 222 9613; fax, +46 46 222 42 00; e-mail, carl.borrebaeck@immun.lth.se. Osaka University and CREST. These authors have contributed equally to the study. Lund University. | Core Research for Evolutional Science and Technology (CREST). 10.1021/nl050541s CCC: $30.25 Published on Web 10/15/2005 2005 American Chemical Society

histocompatibility complex class II molecules (MHC class II) and costimulatory molecules, such as CD86 and CD80.8 In addition, the production of interleukin-12 (IL-12) by mature DCs is important for the induction of a type 1 T helper (Th1) response,9 which is considered to be beneficial for viral infections. Consequently, therapeutic vaccines for viral infections should be designed to deliver antigens to DCs, which then can orchestrate a protective immune response. In this study, the effects of antigen-coated and uncoated nanospheres on human dendritic cells have been evaluated to further our understanding of the mechanisms involved in mucosal immunization, using these nanospheres as vaccine carriers. To do this, we first immobilized tetanus toxoid on the surface of the polystyrene nanospheres, via their amino groups, and also confirmed the stability of the coupling by reanalysis after a week in storage. The synthetic preparation of nanospheres has been described previously.10 Briefly, tertbutyl methacrylate oligomers were prepared by the free radical polymerization of tert-butyl methacrylate with 2,2azobis(isobutyronitrile) (AIBN, initiator) and 2-mercaptoehtanol (chain transfer reagent). Poly(tert-butyl methacrylate) macro-monomers were synthesized by combining p-chloromethylstyrene with a hydroxyl group from the oligomers under alkaline conditions. After the hydrolysis of the macromonomers, poly(methacrylic acid)-corona polystyrenecore NS were prepared by the tree radical polymerization of macromonomers and styrene in water/ethanol (1/5 (v/v) with AIBN). The mean diameter of NS used in this study is 460 nm. Tetanus toxoid antigen (TT) was immobilized onto the surface of the nanospheres, using activation of carboxyl groups of the polystyrene nanospheres (10 mg) by water-

Figure 1. Phagocytosis of nanopsheres by immature MoDCs. The cells were analyzed after a 30 min incubation with FITC-NS at 37 C by FACScan (A) and after a 4 h incubation with FITC-NS by fluorescence microscopy (B). The FACS analysis was performed on a FACScan (BD Bioscience, San Jose, CA), using the FlowJo software (Star Inc., San Carlos, CA) for data analysis. Cells were stained for 10 min at 4 C, washed, and resuspended in PBS, containing 2% BSA (Sigma-Aldrich). Gates were set to exclude debris and nonviable cells. The following antibodies were used for FACS analysis: HLA-DR PE (BD Pharmingen, San Diego, CA) and CD86 FITC (DakoCytomation, Glostrup, Denmark). Appropriate isotype-matched, nonspecific antibody controls were used in all experiments to determine the level of background staining.

soluble carbodiimide (WSC) solution for 20 min. After centrifugation at 13 000 rpm, the nanospheres (1 mg/mL) were mixed with tetanus toxoid and kept at 4 C for 24 h. The nanospheres were washed three times with PBS, and the amount of immobilized tetanus toxoid was analyzed by use of a Micro BCA Protein Assay Reagent Kit (Pierce, Rockford, IL). The nanospheres used in the biological assays contained on average 40 ( 0.24 g of TT/mg of spheres, and this was reconfirmed a week after immobilization. DCs have to internalize the nanospheres in order to induce an immune response. Phagocytosis of nanospheres was demonstrated by adding FITC-conjugated nanospheres (FITCNS) to immature monocyte-derived dendritic cells (MoDC) at 37 C for 30 min. MoDCs were generated as described previously.11 Phagocytosis is temperature dependent and immature MoDCs and FITC-NS were incubated at 0 C for 30 min, as a control. MoDCs cultured at 37 C displayed significantly higher fluorescence intensity, compared to the control, demonstrating rapid phagocytosis of the nanospheres (Figure 1A). In addition, the uptake of FITC-NS after 4 h of incubation at 37 C was demonstrated by fluorescence microscopy (Figure 1B). The viability of the MoDCs was tested after nanosphere stimulation, since this is a crucial parameter in vaccinology as the cells have to remain viable long enough to induce an immune response. As the viability of MoDCs pulsed with NS, NS-TT, or TT alone for 3 days was almost identical to the medium control, it was clear that the nanospheres were not toxic to MoDCs (data not shown). To function, not only as a carrier but also as an adjuvant, the NS-TT should also affect the maturation process of MoDCs and induce markers known to be involved in T cell activation, such as, e.g., CD80/86 and HLA-DR. Consequently, we studied the effect of NS-TT on MoDC maturation and compared it to the effect of NS or tetanus toxoid alone. A maturational process was induced by NS-TT as upregulation of maturation markers, such as HLA-DR and CD86, was evident after 72 h (Figure 2B). Of note, MoDCs
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stimulated with NS alone showed expression levels, regarding maturation markers, similar to the medium control (Figure 2A,C). Furthermore, we also showed that TT alone had no effect on the maturation process, as compared to the medium control (Figure 2D). These results suggest that the vehicle, containing both polystyrene nanospheres and the tetanus toxoid antigen, not only is passively taken up by the dendritic cells but also affects the cellular maturation, since CD86 and HLA-DR are both markers involved in the induction of an immune response. To further characterize the effect of antigen-coated nanospheres on the dendritic cells, we performed global transcriptional analysis, using high-density microarrays. This analysis gives a transcriptional signature for each stimuli, resulting in a comprehensive comparative genomic view. Two different time points were selected for the transcriptional analysis, since it has been reported that the gene regulation involved in (i) phagocytosis, innate immune response, and apoptosis in human immature MoDCs reach a maximum after 4 h and (ii) transcription, T cell regulation, antigen presentation, and signaling reaches a maximum after 12 h.12 The quantitative differences in gene regulation are represented by Venn diagrams (Figure 3). For instance, we demonstrate that stimulation with TT alone induced a very marginal response in MoDCs. On the other hand, a large number of genes (100-175) were up-regulated >2-fold in NS-TT and NS-stimulated MoDCs, demonstrating that NS alone also had an effect on the gene regulation in MoDCs. However, the number of genes up-regulated by NS-TT were significantly higher compared to when NS alone was used, indicating that the combination of antigen and nanospheres had a synergistic effect on gene regulation. The pattern of up-regulated genes was similar for both time points, i.e., 4 and 12 h (Figure 3A,B). Furthermore, the level of downregulated genes was even more pronounced in the NS-TTpulsed dendritic cell, as compared to cells stimulated with either NS or TT alone (Figure 3C,D). In conclusion, the high
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Figure 3. Venn diagram representing the quantitative gene regulation in MoDCs stimulated with NS, NS-TT, or TT, respectively. The numbers of genes up-regulated >2-fold after 4 h (A) and 12 h (B) or down-regulated after 4 h (C) and 12 h (D) are given. Transcripts related to stimulation with NS are represented in red (up-regulated) and green (down-regulated), NS-TT in violet (up-regulated) and dark green (down-regulated), and TT in pink (up-regulated) and light green (down-regulated). Genes with intensity values lower than 50 were discarded.

Figure 2. Expression of maturational markers (CD86 and HLADR) on dendritic cells, challenged by (A) NS, (B) NS-TT, (C) medium (control), or (D) TT for 72 h. The expression was analyzed by flow cytometry.
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level of transcriptional activity in NS-TT-stimulated MoDCs demonstrate that antigen immobilized onto the surface of NS has a significantly more pronounced effect on the gene regulation in DCs, as compared to antigen alone. Phagocytosis and macropinocytosis are complex processes that involve changes in plasma membrane, cytoskeleton, vesicular traffic, signaling cascades, effector molecules, etc. and are essential for the host defense against infectious disease.12 When we performed a qualitative expression analysis, it was found that genes involved in processes associated with phagocytosis were up-regulated in both NS and NS-TT stimulated MoDCs (Figure 4A). Examples of genes, important for cytoskeletal rearrangement for efficient antigen uptake,13,14 are myosin IE (MYO1E), filamin B (FLNB), Cdc42 (CDC42SE1, CDC42, CDC42EP3), and Rho GTPase activating protein (ARHGAP26). Furthermore, upregulation of proteins involved in G-protein coupled receptor pathways (e.g., AKAP12), JAK-STAT cascade (e.g., JAK2, STAT2, PIAS1, SOCS3), and the p38/JNK MAP (mitogenactivated protein) kinase cascade (e.g., RAPGEF2, FGFR1) indicate activation of signaling cascades as a result of the phagocytosis. However, since immune response genes are central to the design of vaccine, we also made a focused analysis involving representative genes of this family (Figure 4B). It was evident that general inflammatory-associated genes, such as IL8,15 IL6R,16 IFNAR2,17 and IFNGR1,18 were up-regulated in both NS and NS-TT pulsed MoDCs. In addition, IL8 was also up-regulated by TT. Activation through IFN receptors (IFNAR2 and IFNGR1) induces the JAK-STAT cascade mentioned above, which results in transcriptional regulation and subsequently the outcome of the immune response.18 The regulation of the JAK-STAT pathway is important for controlling the immune response. Binding of cytokines to their receptors results in receptor aggregation, cross-phosphorylation, and activation of signaling molecules, such as JAKs and STATs. Phosphorylation of STATs allows for their dimerization and translocation to
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Figure 4. (A) Global heatmap of gene transcription, after 4 and 12 h stimulation by tetanus toxoid (TT), nanospheres (NS), or tetanus toxoid immobilized nanospheres (NS-TT). Totally 81 genes are selected. (B) Heatmap of immune response-associated genes, according to GeneOntology Consortium. Up-regulated genes (red) and down-regulated genes (green) were selected according to biological process, obtained from the Gene Ontology Consortium. RNA was isolated from NS, NS-TT, or TT-stimulated (4 and 12 h) MoDCs, as well as from unstimulated control MoDCs, derived from two different donors. Preparation of labeled cRNA, fragmentation, hybridization, and scanning of the Human Genome U133 Plus 2.0 arrays were performed according to the manufacturers protocol (Affymetrix Inc., Santa Clara, CA) as previously described.11 The fluorescence intensities were analyzed, using the GCOS Software 1.0 (Affymetrix Inc.). Further data normalization and analysis were performed, using the GeneSpring 7 software (Silicon Genetics, Redwood City, CA).

the nucleus, where they stimulate transcription of genes involved in the cytokine-induced response, resulting in gene products such as, e.g., SOCS19 and PIAS,20,21 involved in necessary negative feed-back loops. In this study, we detected transcriptional activation of the early genes in this signaling pathway (JAK2, STAT2), as well as up-regulation of the
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negative regulators SOCS3 and PIAS1 in both NS and NSTT stimulated MoDCs. This indicates that the JAK-STAT pathway is activated and regulated by a classical negativefeedback loop. Recognition through the Toll-like receptor-signaling pathway is also essential for the activation of the innate and
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subsequently the adaptive immune response22. Activation through Toll-like receptors (TLRs) activates the NFB pathway as well as MAP-kinase signaling pathways.23 Genes involved in these pathways were also found to be upregulated in MoDCs after stimulation by NS and NS-TT and the activation is indicated via TLR2 and/or TLR4, since PIK3R124 and NFB25 both are upregulated and is also followed by upregulation of the downstream gene CCL3.26 In addition, the MAP-kinase pathway is activated, which is confirmed by the expression of, e.g., FGFR1.27 Upregulation in both NS and NS-TT pulsed MoDCs was also observed for chemokine ligands, such as CCL3, CCL4, and CX3CL1, that are involved in lymphocyte trafficking.28 Interestingly, CD80, a gene involved in T cell activation, was somewhat more up-regulated in NS-TT stimulated MoDCs as compared to NS stimulated MoDCs. Other genes that were more highly expressed in NS-TT vs TT stimulated MoDC are, e.g., CD3729 (cell activation), ALCAM30 (cell adhesion), and IL4I131 (cytokine signaling). In addition, the IFN- inducible gene MDA532 was up-regulated in both NS and NS-TT after 4 h, but with a sustained expression after 12 h in only NS-TT stimulated MoDCs. Additional studies are now needed to validate our findings on both a protein as well as functional level. In summary, we report the first phenotypic and genotypic data explaining the in vivo effects of nanospheres as vaccine carriers for mucosal immunization. Nanospheres had a direct effect on human dendritic cells, inducing transcription of genes important for, e.g., phagocytosis as well as an immune response. Furthermore, the combination of antigen and nanospheres specifically induced transcription, involving genes important for protein transport, cell signaling, and immune response. Acknowledgment. We thank Dr. M. Baba for helpful discussions and Ann-Charlotte Olsson for expert technical assistance. This work was partially supported by the Swedish Foundation for Strategic Research (SSF-SIG), and by Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology agency (JST). References
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