Induction of Dendritic Cell Maturation and Activation by a Potential Adjuvant, 2-Hydroxypropyl-β-Cyclodextrin

2-Hydroxypropyl-β-cyclodextrin (HP-β-CD) is a chemically modified cyclic oligosaccharide produced from starch that is commonly used as an excipient. Although HP-β-CD has been suggested as a potential adjuvant for vaccines, its immunological properties and mechanism of action have yet to be characterized. In the present study, we investigated the maturation and activation of human dendritic cells (DCs) treated with HP-β-CD. We found that DCs stimulated with HP-β-CD exhibited a remarkable upregulation of costimulatory molecules, MHC proteins, and PD-L1/L2. In addition, the production of cytokines, such as TNF-α, IL-6, and IL-10, was modestly increased in DCs when treated with HP-β-CD. Furthermore, HP-β-CD-sensitized DCs markedly induced the proliferation and activation of autologous T lymphocytes. HP-β-CD also induced a lipid raft formation in DCs. In contrast, filipin, a lipid raft inhibitor, attenuated HP-β-CD-induced DC maturation, the cytokine expression, and the T lymphocyte-stimulating activities. To determine the in vivo relevance of the results, we investigated the adjuvanticity of HP-β-CD and the modulation of DCs in a mouse footpad immunization model. When mice were immunized with ovalbumin in the presence of HP-β-CD through a hind footpad, serum ovalbumin-specific antibodies were markedly elevated. Concomitantly, DC populations expressing CD11c and MHC class II were increased in the draining lymph nodes, and the expression of costimulatory molecules was upregulated. Collectively, our data suggest that HP-β-CD induces phenotypic and functional maturation of DCs mainly mediated through lipid raft formation, which might mediate the adjuvanticity of HP-β-CD.

effects of drugs by controlling compound release, increasing their stability, and regulating the metabolism of the incorporated molecules (4). Due to these physicochemical properties, cyclodextrins are commonly utilized as excipients of pharmaceutical agents, food products, and cosmetics. β-Cyclodextrin and some of its derivatives are widely used additives of commercial drugs because they are easy to produce, belong to generally recognized as safe (GRAS) materials for humans, and have improved solubility compared with the other cyclodextrins (4,5). 2-Hydroxypropyl-β-cyclodextrin (HP-β-CD) is a chemically modified derivative of β-cyclodextrin that exhibits an enhanced safety profile compared with its naturally occurring parent compound (4). HP-β-CD is used as an excipient for cardiac dysrhythmia, inflammation, and fungal disease medications (6). Furthermore, HP-β-CD has been proposed as a vaccine adjuvant because it markedly enhances humoral immune responses to an influenza vaccine without any adverse effects (7). However, the immunological properties and action mechanism of HP-β-CD need to be further characterized for the human use.
Dendritic cells (DCs) are professional antigen-presenting cells that bridge the innate and adaptive immunities. Immature DCs are characterized by high endocytic activity coincident with a low expression of costimulatory molecules and cytokines (8). When immature DCs meet microbial antigens or damage-associated molecular patterns, they begin the process of maturation (8,9). This process is accompanied by upregulation of (i) MHC associated with the antigen; (ii) costimulatory molecules including CD40, CD80, and CD86; and (iii) inflammatory cytokines such as IL-12, IL-6, and TNF-α (10). These phenotypic changes optimize conditions for T lymphocyte activation and differentiation (11,12). Since mature DCs potently stimulate adaptive immunity better than immature DCs, many vaccine adjuvants currently under development are designed to efficiently induce functional maturation and activation of DCs (13)(14)(15). In the present study, we investigated immunological function of HP-β-CD by determining its ability to mature and activate DCs leading to the induction of adaptive immunity.

Preparation of human Monocyte-Derived Dcs
Human peripheral blood samples donated by healthy adult male subjects (n = 15) were provided from the Korean Red Cross (Seoul, Korea) after obtaining informed consent. All experiments using human blood were conducted under the approval of the Institutional Review Board of Seoul National University. The peripheral blood was diluted in phosphate-buffered saline (PBS) and overlaid on the Ficoll-Paque PLUS, and peripheral blood mononuclear cells (PBMCs) were isolated by densitygradient centrifugation as previously described (16,17). PBMCs were washed with PBS three times to remove platelets and the remaining Ficoll. To isolate CD14 + monocytes, PBMCs were incubated with anti-human CD14 magnetic beads for 30 min at room temperature. The cells were separated on a magnetic field, and then CD14 + cells were enriched by positive selection. CD14 + monocytes were suspended at a concentration of 2 × 10 6 cells/ ml in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin solution. The isolated monocytes were differentiated into immature DCs in the presence of human recombinant GM-CSF (5 ng/ml) and IL-4 (10 ng/ml) for 6 days. Cell culture medium supplemented with GM-CSF and IL-4 was added every 3 days.

Preparation of hKec
Escherichia coli BL21 (DE3) strain obtained from Stratagene (La Jolla, CA, USA) was cultured in Luria Bertani broth at 37°C until reaching mid-log phase and then harvested by centrifugation. The harvested bacterial cells were resuspended in PBS and killed DC Maturation by HP-β-CD

analysis of Dc Phenotypes
Mouse BM-DCs (5 × 10 5 cells/ml) were stimulated with either HP-β-CD (0, 0.1, or 1 mg/ml) or LPS (100 ng/ml) in the presence of murine GM-CSF (10 ng/ml) for 24 h. Human monocytederived DCs (2.5 × 10 5 cells/ml) were stimulated with either HP-β-CD (0, 0.1, 0.3, or 1 mg/ml) or HKEC (1 × 10 7 CFU/ml) in the presence of GM-CSF (2.5 ng/ml) and IL-4 (5 ng/ml) for 24 h. The unstimulated or stimulated DCs were then stained with fluorochrome-conjugated monoclonal antibodies specific to CD80, CD83, CD86, MHC class II, PD-L1, or PD-L2 for 30 min on ice, and then the cells were washed once with PBS. The geometric mean fluorescence intensity (MFI) of each group of DCs was obtained by flow cytometric analysis. More than 8,500 events were acquired for each group, and cell debris and dead cells were gated out. Phenotypes of DCs were analyzed using flow cytometry with FACSCalibur (BD Biosciences) and FlowJo software (TreeStar, San Carlos, CA, USA).

analysis of lipid raft Formation
Dendritic cells (2.5 × 10 5 cells/ml) were stimulated with HP-β-CD (1 mg/ml) in the presence or absence of filipin (30 μg/ml) for 45 min. Cell staining was performed with the lipid raftlabeling kit according to the manufacturer's instructions. Briefly, unstimulated or HP-β-CD-stimulated DCs were washed once with ice-cold PBS and stained with Alexa Fluor ® 594-conjugated CTB conjugate for 10 min on ice. The DCs were washed once with ice-cold PBS and incubated with rabbit serum containing anti-CTB antibodies for 10 min on ice to crosslink lipid rafts on the surface of the DCs. Formation of lipid raft on the DCs was analyzed by confocal laser scanning microscopy (Carl Zeiss MicroImaging GmbH, Jena, Germany). Fluorescence intensity of DCs was analyzed by ZEN software, Lite Edition (Carl Zeiss, Oberkochen, Germany).

immunization with OVa Plus hP-β-cD in Mice
Seven-week-old male C57BL/6 mice were purchased from Orient Bio (Sungnam, Korea) and maintained in a specific pathogen-free animal facility. All experiments using animals were conducted under the approval of the Institutional Animal Care and Use Committee of Seoul National University. Care and treatment of the animals were carried out in accordance with the approved guidelines. Mice were anesthetized by intraperitoneal injection of Avertin (2,2,2-tribromoethanol and 2-methyl-2-butanol) and administered with 20 μg OVA with or without 3 mg HP-β-CD through a hind footpad. The mice were maintained for 24 h or 7 days and sacrificed to obtain the draining lymph nodes and the blood, respectively.

analysis of Dc activation in the Draining lymph nodes
Twenty-four hours after the immunization with OVA in the presence or absence of HP-β-CD, draining lymph nodes, including popliteal and inguinal lymph nodes, were harvested and dissociated into a single cell suspension on a cell strainer (BD Biosciences). To analyze DC populations in the lymph nodes, the cells were stained with fluorochrome-conjugated antibodies specific to CD11c, MHC class II, CD86, and CD80 at 4°C for 30 min. CD11c + MHC class II + cells in the lymph nodes and their phenotypic changes upon the administration of OVA in the presence or absence of HP-β-CD were analyzed by flow cytometry using FACSVerse (BD Biosciences).

Preparation of Mouse Bone
Marrow-Derived Dcs The statistical significance of differences between the experimental groups and the control group was analyzed using Student's t-test. P-values less than 0.05 were considered statistically significant. Results are presented as mean value ± SD or SEM.

hP-β-cD induces Maturation of human Monocyte-Derived Dcs
Maturation of DCs is an essential process for activating antigen-specific adaptive immunity and includes the upregulation of costimulatory molecules, MHC class I/II, and certain cytokines (12). Thus, we first examined the effects of HP-β-CD on the phenotypic maturation and cytokine production in human monocyte-derived DCs. Notably, HP-β-CD was not cytotoxic to DCs at concentrations up to 1 mg/ml ( Figure S1A in Supplementary Material). Stimulation with HP-β-CD remarkably augmented the expression of costimulatory molecules, such as CD80, CD83, and CD86 (Figures 1A,D), together with MHC class II (Figures 1B,E). Additionally, HP-β-CDtreated DCs exhibited increased expression of inhibitory molecules, such as PD-L1 and PD-L2 (Figures 1C,F). Moreover, HP-β-CD weakly increased the expression of proinflammatory cytokines, including TNF-α and IL-6, in a dose-dependent manner (Figures 1G,H). Furthermore, HP-β-CD treatment slightly increased IL-10 expression in DCs (Figure 1I), and IL-12p70 was not detected in the culture supernatant of HP-β-CD-treated DCs (data not shown). These results suggest that HP-β-CD upregulates the expression of maturation markers coincident with weak induction of cytokines in human monocyte-derived DCs.

hP-β-cD-sensitized Dcs elicit autologous T lymphocyte Proliferation and activation
To examine whether HP-β-CD potentiates the T lymphocyteactivating ability of DCs, unstimulated or HP-β-CD-stimulated DCs were cocultured with autologous T lymphocytes, and the extents of T lymphocyte proliferation and activation were analyzed. HP-β-CD-sensitized DCs significantly induced T lymphocyte proliferation and CD25 expression, an activation marker of T lymphocytes (Figures 2A,B). The enhancement of proliferation and CD25 expression was observed in CD4 + T lymphocytes but not in CD8 + T lymphocytes (Figures 2C,D). However, HP-β-CD did not directly enhance the proliferative activity or CD25 expression of the T lymphocytes, indicating that HP-β-CD requires the help of DCs to activate T lymphocytes ( Figures S2A,B in Supplementary Material). Therefore, the results suggest that HP-β-CD potentiates the ability of DCs to induce CD4 + cells.

hP-β-cD Triggers lipid raft Formation on the Dc Plasma Membrane
Lipid rafts are hydrophobic microstructures that play an important role as signal transduction platforms in many eukaryotic cells (20). Since HP-β-CD interacts with cellular cholesterol (21), an essential component of lipid rafts, we examined whether HP-β-CD induces lipid raft formation on the plasma membrane based on the hypothesis that lipid rafts are involved in the HP-β-CD-induced DC maturation. DCs were stimulated with HP-β-CD in the presence or absence of filipin, which disrupts a lipid raft formation, and fluorochrome-conjugated CTB was used to detect the formation of lipid rafts. As shown in Figure 3, HP-β-CD triggered lipid raft formation on the surface of DCs, whereas such effect was blocked by preexposure to the lipid raft inhibitor filipin.

inhibition of lipid raft Formation reduces hP-β-cD-induced Dc Maturation
Next, we further determined the role of lipid rafts in HP-β-CDmediated DC maturation by blocking the lipid raft formation using filipin. Of note, treatment with filipin and/or HP-β-CD was not cytotoxic to DCs ( Figure S1B in Supplementary Material). As shown in Figures 4A-C, treatment with filipin remarkably attenuated HP-β-CD-elicited expression of surface costimulatory molecules, PD-L1/L2, but not MHC class II. In addition, HP-β-CD-mediated induction of TNF-α, IL-6, and IL-10 in DCs was significantly reduced upon the inhibition of lipid raft ( Figure 4D). Furthermore, lipid raft inhibition in DCs stimulated with HP-β-CD abrogated their ability to activate T lymphocytes ( Figure 4E). These results suggest that HP-β-CD requires the formation of lipid rafts to trigger DC maturation and further to activate T lymphocytes.

hP-β-cD Potentiates humoral immune responses to coadministered antigens and Dc activation in the Draining lymph nodes in Mice
Next, we determined whether HP-β-CD has an adjuvanticity with a mouse footpad immunization model (22). To immunize mice, OVA with or without HP-β-CD was injected through a hind footpad, and titers of serum antibodies specific to OVA were determined. Coadministration with HP-β-CD and OVA efficiently increased OVA-specific total IgG in the blood ( Figure S3A in Supplementary Material). IgG1 was the major antibody subtype induced following immunization ( Figure S3B in Supplementary Material), and no IgG2a antibodies were detected (data not shown). As DCs are crucial in the mediation of naive T cell responses, we subsequently analyzed DC populations in the draining lymph nodes, including popliteal and inguinal lymph nodes upon administration of OVA with or without HP-β-CD. Mice administered with OVA with HP-β-CD showed an increase in the size and cell number of the draining lymph nodes (Figure 5A). OVA administration with HP-β-CD markedly augmented the number of CD11c + MHC class II + cells in the popliteal and inguinal lymph nodes (Figures 5B,C). In addition, the DCs showed an upregulation in the expression of costimulatory molecules, including CD80 and CD86 (Figures 5D,E). To determine whether DC activation in the draining lymph nodes of mice administered with OVA and HP-β-CD is directly attributed to stimulatory functions of HP-β-CD, we examined HP-β-CD effects on the maturation of BM-DCs generated in vitro. HP-β-CD markedly upregulated the expression of surface costimulatory molecules, such as CD80, CD83, and CD86, and MHC class II ( Figure 5F). In addition, HP-β-CD-treated BM-DCs modestly increased the production of proinflammatory cytokines including TNF-α and IL-6 in BM-DCs compared to LPS-stimulated BM-DCs ( Figure 5G). Collectively, these results suggest that HP-β-CD-induced DC maturation and activation in vivo that might be necessary for the adjuvanticity.

DiscUssiOn
Dendritic cells play a pivotal role in the induction of antigenspecific adaptive immune response by presenting the antigens to T cells and activating appropriate subtypes of T cells. HP-β-CD has long been utilized as a solubilizer and a delivery compound of hydrophobic drugs due to its physicochemical properties (6). However, recent studies have reported a novel beneficial effect of HP-β-CD on the immunogenicity of vaccines (7,23) as well as on the progression of incurable metabolic disorders (24) and viral infections (25). Although the previous findings have suggested that HP-β-CD could be a potential vaccine adjuvant (7,23), the effects of HP-β-CD on vaccine immunogenicity and DC properties have been poorly investigated. Here, we demonstrated that HP-β-CD has an adjuvanticity to OVA, and the maturation of DCs found in the draining lymph nodes in a mouse footpad immunization model. In vitro studies showed that HP-β-CD induces the maturation of DCs to induce the proliferation and activation of autologous T lymphocytes. Mechanism studies further showed that the lipid raft formation in DCs is essential for the HP-β-CD-induced DC maturation and its subsequent activation of autologous T cells. These results suggest that HP-β-CD is a promising vaccine adjuvant to potently induce the maturation and activation of DCs.
In the present study, we demonstrated that HP-β-CD has an adjuvanticity. Administration of HP-β-CD efficiently augmented the antigen-specific IgG in the blood. Notably, HP-β-CD predominantly induced IgG1 but not IgG2, indicating preferential enhancement of Th2 responses rather than Th1 responses. Consistent with our findings, intranasal or subcutaneous administration of HP-β-CD increased the immunogenicity of influenza vaccines (7,23). HP-β-CD seems to be appropriate as a mucosal adjuvant because it induces antigen-specific IgA and IgG in the airway mucosal tissues as well as in the blood (23).
Dendritic cell maturation is a prerequisite for the induction of adaptive immune response. Many previous studies have demonstrated that current vaccine adjuvants, such as aluminum salt (alum), cholera toxin (CT), and monophosphoryl lipid A (MPLA), can efficiently induce DC maturation (26)(27)(28). Here, we observed that HP-β-CD increased DCs in the draining lymph nodes as well as the upregulation of DC costimulatory molecules when coadministered with antigen. Additionally, HP-β-CD induced a marked increase in the expression of CD80, CD83, CD86, and MHC proteins on BM-DCs and human monocytederived DCs. Furthermore, stimulation with HP-β-CD resulted in weak but significant production of TNF-α, IL-6, and IL-10 in DCs. Therefore, like other adjuvants, HP-β-CD appears to be capable of stimulating DC maturation. However, in contrast to our DC Maturation by HP-β-CD Frontiers in Immunology | www.frontiersin.org October 2016 | Volume 7 | Article 435 observation, a previous study found that HP-β-CD did not induce a maturation marker of CD11c + DCs in the draining lymph node, even though it potentiated DC antigen uptake (7). Given the differences in a route of injection and doses of HP-β-CD, functional mode of DC activation in the tissues might be different.
Here, we found that HP-β-CD-sensitized DCs markedly induced the proliferation and activation of T lymphocytes, especially CD4 + cells, implying that HP-β-CD can potentially enhance Th-dependent immune responses. Notably, a study of mice immunized with an influenza vaccine suggested that HP-β-CD is a competent adjuvant capable of eliciting follicular Th cells and antibody production (7). Furthermore, other cyclodextrins, such as sulfolipo-cyclodextrin and dimethyl-βcyclodextrin, have been found to enhance antibody responses in a T lymphocyte-dependent manner (29,30). Given the fact that enhanced T cell responses have previously been observed with many existing adjuvants, including MPLA, saponin, and CT (31,32), this enhancement appeared to be mediated through DC activation. These adjuvants influence the types of adaptive immune responses that occur by modulating T lymphocyte differentiation. For instance, alum is an established potentiator of Th2-mediated humoral immunity (33). In addition, MPLA has been shown to preferentially induce Th1-skewed immune responses (34), whereas CT provokes Th1, Th17, and Th2 responses (35). Considering the previous finding that HP-β-CD administration markedly induces IL-13 and IL-5 production, HP-β-CD has been proposed to elicit Th2 responses (7).
In the present study, we observed that HP-β-CD-induced lipid raft formation and contributed to DC maturation. Specifically, filipin-mediated inhibition of lipid rafts abrogated HP-β-CDmediated phenotypic changes and functional activation of DCs. In line with this observation, many previous studies have demonstrated lipid raft involvement in the activation of various immune cells. Dispersion of lipid rafts has been shown to impair CD1a-mediated antigen presentation by DCs and subsequent activation of T lymphocytes (36). Furthermore, alum adjuvanticity has been proposed to have a critical relationship with the formation of signaling platforms via lipid sorting on DCs (26).
2-Hydroxypropyl-β-cyclodextrin can interact with cellular cholesterol (21); however, the precise effects of HP-β-CD on cholesterol seem to depend on its concentration. Low doses of HP-β-CD (below 1 mM) induce efflux or intermembrane transport of cholesterol, while high concentrations (10-100 mM) deplete the lipid molecules from the cell membrane (37,38). In the present study, DCs were treated with up to 1 mg/ml HP-β-CD, which is equivalent to 1.4 mM. This concentration is relatively low and is likely to mediate cholesterol accumulation and lipid raft formation on the cell membrane rather than to deplete cholesterol. In light of the observation that cholesterol accumulation in the membrane of human monocytes promotes the association of immuno-stimulatory receptor complexes, cholesterol loading in the plasma membrane is believed to be crucial for eliciting inflammatory immune responses (39). Since HP-β-CD can transport free cholesterol to the plasma membrane (40), HP-β-CD is likely to play a role in the formation of cholesterol-rich lipid rafts in DCs, thereby activating signals required for their phenotypic and functional maturation.
Recently, potential issues in relation to the efficacy and safety of vaccine adjuvant have been raised (41)(42)(43)(44). Thus, demand has increased for new adjuvants with minimal adverse effects and enhanced capacity to stimulate antigen-specific adaptive immune responses. HP-β-CD is GRAS in many Asian and European countries and is already widely utilized in various commercial products, including food and drugs. Our findings potentially inform the future application and improvement of vaccine adjuvants with HP-β-CD.
aUThOr cOnTriBUTiOns SH conceived the idea. SH and SK designed the experiments. SK and SH performed the experiments and/or interpreted the data. C-HY provided critical comments. All authors contributed to discussion of the results followed by writing and reviewing the manuscript.