Mechanisms of Enhanced Antigen Delivery to Murine Dendritic Cells by the Cationic Liposomes

There is an increased demand for vaccines to prevent and/or treat illness and mortality caused by the infectious diseases. We have recently established that liposomes composed of cationic lipids act as adjuvant for nasal vaccine formulation. However, the molecular mechanism(s) behind the adjuvant effect remain unrevealed. To this end, we have studied the enhancement of antigen uptake by murine dendritic cell line, DC2.4 cells, by the cationic liposomes and the specific pathways involved in the process. We have observed that the uptake of ovalbumin (OVA) into DC2.4 cells is greatly increased when co-cultured with the cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] (DC-chol). However, this enhancement was blocked by pretreatment of DC2.4 cells with chlorpromazine and methyl-β-cyclodextrin, indicating the involvement of clathrinand caveolin-independent lipid raft-dependent endocytic pathways in the process. Our results implied, at least in part, that enhanced uptake of antigens induced by the cationic liposomes could be a possible mechanism for the induction of immune responses. Although further studies are needed to understand the precise mechanisms behind the adjuvant effects of DOTAP/DC-chol liposome, this approach is quite useful for the development of vaccine system to combat various diseases.


Introduction
The demand for vaccines is greatly increased, as vaccination seems to be the only How to cite this paper: Takahashi, S., Tada, R., Negishi, Y. and Aramaki, Y. (2017) Mechanisms of Enhanced Antigen approach in preventing and/or treating illness and mortality associated with infectious diseases [1] [2].Despite that the traditional vaccine systems, such as live-attenuated and inactivated vaccines, are quite effective, their clinical usage for deadly pathogens have still been challenging because of the safety concerns and risks for infections [3] [4] [5].Recently, there has been an emphasis on new generation subunit vaccines due to its safety features compared to both live-attenuated and inactivated vaccines [6] [7].However, for these subunit vaccines, an adjuvant component, which potentiates host immunity, is crucial for the effective induction of antigen-specific immune responses as the antigenic property of proteins are inherently poor when administered to the host [8].
While numerous adjuvants have been explored experimentally, their translation to clinical usage in humans is extremely slow.Thus a clinical need for the development of novel adjuvants arises.Adjuvants generally exhibit their immune-enhancing activities to the antigens through the following mechanisms: 1) the depot effect by which releases antigen from adjuvant carriers at the site of injection gradually, 2) activation of host innate immune responses and 3) increased antigen uptake by antigen-presenting cells (APCs) and in turn its presentation to T cells [9].Dendritic cells (DCs) are one of the most favored targets for vaccine delivery because of their primary role in the initiation of acquired immune responses via antigen internalization followed by antigen processing and presentation to lymphocytes, especially to T cells via the major histocompatibility complex (MHC) molecules [10] [11].Nanoparticles such as liposomes and gold nanoparticles are now considered to be not only efficient drug carriers but also potent adjuvants useful for vaccine formulations.For example, a nanogel composed of cholesteryl-group-bearing pullulan acts as an adjuvant by promoting antigen delivery to dendritic cells [12] [13].Similarly, a well-known carrier for nucleic acid delivery, polyethyleneimine (PEI), is reported to be a potent adjuvant as the release of host double-stranded DNA (dsDNA) into the extracellular milieu triggers Ifr3-dependent innate immune signaling [14].Cationic liposome is recognized as a powerful tool for vaccine development as well as for nonviral gene delivery system [15] [16] [17] [18].We recently demonstrated that the cationic liposomes composed of 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] (DC-chol), termed as DOTAP/DC-chol liposomes, are served as a potent adjuvant.The detailed molecular mechanism(s) behind the adjuvant effects of the cationic liposomes are not completely elucidated.We have previously reported that cationic liposome promotes antigen uptake into the DCs located at nasal-associated lymphoid tissues (NALTs) in vivo [19] [20].Although, how the cationic liposome increases antigen uptake by DCs remains unclear till date.In general, the difference in the uptake mechanism(s) of particulate and soluble forms of antigenic proteins by DCs might be the reason behind this increase in uptake efficiency of antigens tagged to a delivery system [21] [22].Understanding this process might be crucial for optimization of liposomal design, including its features like particle size and lipid composition, for inducing maximum immune responses.
The goal of this study is to understand the molecular mechanisms involved in the increase in antigen uptake into DCs by the cationic liposomes.Here, we investigated the role of various endocytic pathways on enhanced ovalbumin (OVA) uptake into murine dendritic cell lines, DC2.4,mediated by the cationic liposomes by perturbing various endocytic pathways using specific chemical inhibitors.

Preparation of Cationic Liposomes
Liposomes were prepared as described earlier [19].Briefly, 10 μmol of total lipids (DOTAP:DC-chol at 1:1 mol ratios) were evaporated to dryness in a glass tube and desiccated for at least 1 h in vacuo.The resultant lipid films were hydrated by the addition of 250 μl of phosphate-buffered saline (PBS; Wako Pure Chemical Industries, Tokyo Japan)) and then vortexed for 5 min at room temperature (RT).The prepared multilamellar vesicles were extruded 10 times by passing through a 100 µm-pore polycarbonate membrane (Advantec, Tokyo, Japan) and then sterilized by filtration through 0.45 μm filter membranes (Iwaki, Tokyo, Japan).The particle size and ζ-potential of the liposomes were measured by Nicomp 380 ZLS (Particle Sizing Systems; Port Richey, USA).The liposomes used in this study had a particle size of 149.3 ± 5.5 nm with a ζ-potential of 8.9 ± 2.0 mV.The fluorescent-labeled cationic liposomes (DiI liposomes) were prepared in the same way by combining 0.2% DiI with 10 µmol of DOTAP:DC-chol 1:1 mixture.The DiI liposomes prepared here had a particle size of 143.2 ± 5.0 nm with a ζ-potential of 9.8 ± 2.0 mV.
Briefly, 5 mg of FITC in 0.4 mL of DMSO was added drop-wise to 4 mL of OVA (50 mg/mL) in 0.1 M sodium carbonate buffer (pH 9.0) with gentle agitation, in dark.After 8 h incubation at 4˚C, the reaction was quenched by the addition of ammonium chloride to a final concentration of 50 mM and incubated further for 2 h to obtain the crude FITC-OVA.The conjugate was then purified by gel filtration chromatography using Toyopearl HW-55F (Tosoh Corporation, Tokyo, Japan) resin.The protein concentration of purified FITC-OVA preparation was determined by the BCA Protein Assay Kit (Wako Pure Chemical Industries).

In Vitro Cytotoxic Assay
The cytotoxic effects of the liposomes on DC2. as the percentage (%) with respect to the untreated control group.

Flow Cytometry
DC 2.4 cells were cultured in RPMI 1640 medium as described in section 2.5.
The cells were plated on a 24-well flat bottom plate at 5 × 104 cells/well density.

Statistical Analysis
Statistical differences in cytotoxicity assay and internalization assay were assessed using one-way ANOVA with Bonferroni's post-hoc test and unpaired t-test with Welch's correction, respectively.

Toxicity of the Cationic Liposomes on Murine Dendritic Cell Line, DC2.4
It has been reported previously that the cationic liposomes generally show cytotoxicity against various types of cells [24] [25] [26] [27].Hence, we have studied the cellular toxicity of cationic liposomes on DC2.4 cells in vitro to determine the optimal concentration of cationic liposomes to be used for further experiments.The viability of cells treated with various concentrations of cationic liposomes was assessed using an MTT assay.The cationic liposomes show a significant cellular toxicity on DC2.4 cells evidenced by a reduction in living cells (~50%) at concentrations above 200 nmol/mL.Whereas the lower concentrations of cationic liposomes (below 100 nmol/mL) did not show any cytotoxicity against DC2.4 cells in vitro (Figure 1).Therefore, we have used the cationic liposomes at 100 nmol/mL concentration for further experiments in this study.

Effect of the Cationic Liposomes on OVA Uptake by DC2.4 Cells
We evaluated whether the cationic liposome promotes OVA uptake by DC2.4 cells in vitro.The cationic liposomes markedly increased the uptake of OVA by DC2.4 cells compared to an OVA alone-treated group as evidenced by an increase in MFI (39.6 ± 12.8 and 14.3 ± 2.5, respectively) by flow cytometry analysis (Figure 2(a)).Furthermore, confocal microscopic analysis also supported the increased uptake of OVA when combined with the cationic liposomes (Figure 2(b)).These results revealed that cationic liposomes enhance the cellular uptake of OVA in murine dendritic cells, thereby acting as a carrier for antigen delivery.

Mechanism of Enhanced Uptake of OVA by the Cationic Liposomes
It is well-known that most endocytic pathways are energy-dependent and hence are inhibited at low temperatures (e.g.4˚C) [28] [29].Therefore, we have examined the temperature-dependency of OVA internalization in DC2.4 cells to understand the possible mechanisms of enhancement in uptake by the cationic liposomes.Figure 3 shows that OVA internalization is increased by the cationic liposomes at 4˚C (MFI 26.7) which is otherwise inhibited (MFI 11.1), suggesting the involvement of energy-dependent endocytic pathways in the process.Various endocytic pathways, including phagocytosis, clathrin-mediated endocytosis (CME), lipid raft-mediated endocytosis, and macropinocytosis, are orchestrated to internalize a variety of molecules into the cell compartments [30] [31].To identify the precise mechanism(s) of endocytosis mediating the increased uptake of OVA by the cationic liposomes, the effect of perturbation of endocytic pathways by various chemical inhibitors (Table 1) were examined (Figure 4).The the cationic liposomes.However, genistein, which inhibits caveolae-mediated (lipid raft-mediated) endocytic pathway, did not affect the uptake of OVA, implying that the endocytic pathway involved here might be clathrin-mediated and caveolae-independent lipid raft-mediated pathways, which is reported to be cholesterol-dependent previously [32] [33].Further, we have studied the  endocytic pathways involved in the uptake of FITC-OVA and the cationic liposomes separately in the absence of each other.In the case of antigenic OVA, the uptake of protein was inhibited by the treatment of chlorpromazine (Figure 5), conforming the involvement of CME, as reported previously [34] [35], On the other hand, the pre-treatment of chlorpromazine, methyl-β-cyclodextrin, and cytochalasin D clearly suppressed cationic liposome uptake by DC2.4 cells (35.3%, 24.1% and 23.3%, respectively), indicating the implication of concerted mechanisms using clathrin-, cholesterol-, and actin-dependent pathways for the uptake of the cationic liposomes (Figure 6).Taken together, OVA uptake increased by the addition of the cationic liposomes may be mediated by clathrinand caveolae-independent lipid raft-mediated endocytic pathways.

Complex Formation Is Crucial for the Uptake of OVA Enhanced by the Cationic Liposomes
OVA is a typical acidic protein (pI = 4.7) it and possesses a negative charge.
Therefore, possible interaction of OVA with the cationic liposomes is via an electrostatic force and it is hypothesized that DC2.4 cells might engulf OVA as a complex with cationic liposomes.To verify this hypothesis, we have analyzed localization of OVA and the cationic liposomes when co-cultured with the cells.
As shown in Figure 7, confocal imaging revealed that OVA (green) and liposomes (red) were largely co-localized in the cytosolic compartment of DC2.4 cells represented in yellow, implying that DC2.4 cells might uptake OVA as a complex with the cationic liposomes.To validate our hypothesis further, we compared OVA uptake in presence of cationic liposomes and after pre-treatment with cationic liposomes.In the latter case, the cells were treated with cationic liposomes first and OVA was added after washing out these liposomes.Figure 8 shows that increased uptake of OVA by liposomes could not be observed if DC2.4 cells are incubated separately with OVA and liposomes, indicating that the co-existence of OVA and liposomes is required for the enhancement in uptake of OVA mediated by the liposomes.Cumulatively, OVA interacted with the cationic liposomes maybe via electrostatic force and the uptake by DC2.4 cells is via both clathrin-and cholesterol-dependent endocytic pathways.

Discussion
We have reported that the cationic liposome composed of DOTAP and DC-chol acts as a potent adjuvant in mice.However, the underlying mechanism(s) were mostly unknown.In this study, we have investigated the pathways by which OVA is taken up by murine DCs cell line, DC2.4 cells, when co-cultured with the cationic liposomes, to evaluate whether specific endocytic pathways are contributing to antigenic delivery to APCs by the cationic liposomes.Our data herein show the following findings: 1) addition of cationic liposomes together   Recently there has been an increase in demand of safe and effective adjuvant which can enhance the immune response to the antigenic protein [36].Generally, adjuvants exhibit their immune-enhancing activities against antigenic protein through the following mechanisms: 1) the depot effect that gradually releases antigen from adjuvant carriers at the site of administration, 2) activation of host innate immune responses, and 3) increased antigen uptake by APCs and presentation toward T cells.However, research suggested that the depot effect will be  [39].In the present study, we focused on whether the cationic liposomes, a potent adjuvant we have reported previously, act as an antigen delivery system into murine dendritic cells, DC2.4 cells.Numerous studies have reported that for example, a nanogel consisting of a cholesteryl-groupbearing pullulan induced antigen-specific antibody response by delivering antigens to DCs, implying adjuvant-free vaccination [12] [39].These facts demonstrate that the antigen delivery to APCs is an important step for the induction of antigen-specific immune responses towards the antigen.
Since antigen internalization by APCs is essential for the effective antigen presentation onto MHC class II molecule, the delivery of antigens is a rational strategy for the initiation of adjuvant activities.We herein explored antigen delivery capacities of the cationic liposome to DC2.4 cells.We demonstrated that OVA in the presence of the cationic liposomes increased the cellular uptake to DC2.4 cells (Figure 2, Figure 7 and Figure 8).Perturbation studies using the chemical inhibitors for endocytic pathways indicated that the molecular mechanism(s) of enhanced uptake of OVA to DC2.4 cells rely on clathrin-and caveolae-independent lipid raft-mediated endocytic pathways (Figures 3-6).It is known that the interaction of nanoparticles like liposomes with cell membrane plays a significant role in the endocytic processes.These interactions depend upon hydrophobicity, size, and shape [40].Recent evidences have shown that the cellular internalization of nanoparticles is highly influenced by the nature of their surface.For instance, cationic nanoparticles are readily taken up by cells compared to anionic nanoparticles [41].Additionally, endocytic pathways involved in the process of internalization of nanoparticles by various cells are also influenced by nature of the surface of nanoparticles.Amphiphilic nanoparticles are preferentially taken up via lipid raft/caveolin-mediated process whereas, small polymeric particles are shown to enter the cell via clathrin-and caveolin-independent endocytosis [42] [43].

Conclusions
This study indicated that cellular uptake of antigens may be important for the adjuvant effects of the cationic liposome.Additionally, since the cationic liposomes exert cellular toxicity at higher concentrations both in vitro and in vivo, the use of these liposomes may pose a danger by releasing DAMPs at the site of administration.We are currently testing this possibility to understand the molecular mechanism(s) behind the adjuvant effects of cationic liposomes.This information may lead to development of efficient vaccine systems using liposomal formulations.
4 cells were examined by an MTT assay.Briefly, DC2.4 cells were maintained in RPMI 1640 medium (Wako Pure Chemical Industries) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Biowest, Nuaillé, France), 100 μg/mL of streptomycin sulfate salt (Merck), and 100 U/mL of penicillin G potassium salt (Merck).The cells were cultured in a 96-well flat-bottom plate (Thermo Fisher Scientific) at a cell density of 5 × 10 4 cells/well in 0.1 mL culture media containing the liposomes (0 -400 nmol/mL) for 48 h at 37˚C in a humidified atmosphere containing 5% CO 2 and 95% air.After incubation, the cells were centrifuged and washed twice with fresh RPMI 1640 medium and treated with 0.5 mg/mL of MTT (dissolved in PBS and filter sterilized by a 0.22 μm PES membrane) for 2 h at 37˚C.The intracellular formazan crystals formed by the reduction of MTT by viable cells were dissolved in 0.01 M HCl solution containing 10% sodium dodecyl sulfate (SDS) to form a purple color.The optical density was measured at 570 nm using 650 nm as reference.The intensity of the color in each well is directly proportional to the number of viable cells.The cell viability in liposome treated DCs was calculated

Figure 1 .
Figure 1.Cytotoxicity of the cationic liposomes on DC2.4 cells.DC2.4 cells were cultured for 48 h in the presence or absence of various concentrations of the cationic liposome.The cell viabilities were then assessed by the MTT assay.The cell viability (%) was calculated on the basis of the ratio to the untreated group.The values are expressed as the mean ± SD of triplicate cultures from three independent experiments.Significance was evaluated by a one-way ANOVA with Boferroni's post-hoc test: *p < 0.05, N.S.: not significant.

Figure 2 .
Figure 2. Antigen uptake by DC2.4 cells following co-culture with the cationic liposomes.(A) DC2.4 cells were treated with vehicle (black), FITC-OVA (50 μg/mL) (orange), or FITC-OVA (50 μg/mL) in combination with cationic liposomes (20 nmol/mL) (blue) for 1 h.FITC-OVA uptake was then analyzed using flow cytometry as mean fluorescence intensities of the cells.The values are expressed as the mean ± SEM of duplicate cultures from three independent experiments.Significance was evaluated by an unpaired t-test with Welch's correction: *p < 0.05, and (b).Images from confocal microscopy showing FITC-OVA uptake.FITC-OVA = green; DAPI = blue.

Figure 3 .
Figure 3.Effect of temperature on enhanced uptake of FITC-OVA by the cationic liposomes.DC2.4 cells were treated with vehicle (black) or FITC-OVA (50 μg/mL) in combination with cationic liposomes (20 nmol/mL) for 1 h at 4˚C (orange) or 37˚C (blue).(a) FITC-OVA uptake was then analyzed using flow cytometry as mean fluorescence intensities of the cells.The values are expressed as the mean ± SEM of duplicate cultures from three independent experiments.Significance was evaluated by an unpaired t-test with Welch's correction: *p < 0.05; (b) Confocal microscopy images for FITC-OVA uptake at different temperatures.FITC-OVA = green; DAPI = blue.

Figure 4 .
Figure 4. Effect of various chemical inhibitors of endocytic pathways on enhancement FITC-OVA uptake by the cationic liposomes.DC2.4 cells were pre-treated with various chemical inhibitors of endocytic pathways for 30 min at 37˚C, and then treated with vehicle (black), FITC-OVA (50 μg/mL) (orange), or FITC-OVA (50 μg/mL) in combination with cationic liposomes (20 nmol/mL) (blue) for 1 h at 37˚C.FITC-OVA uptake was further analyzed using flow cytometry as mean fluorescence intensities of the cells.The values are expressed as the mean ± SEM of duplicate cultures from three independent experiments.Significance was evaluated by an unpaired t-test with Welch's correction: *p < 0.05, N.S.: not significant.

Figure 5 .
Figure 5. Mechanisms of uptake of FITC-OVA by DC2.4 cells.DC2.4 cells were pre-treated with various chemical inhibitors for endocytic pathways for 30 min at 37˚C, and then treated with vehicle or FITC-OVA (50 μg/mL) for 1 h at 37˚C.FITC-OVA uptake was further analyzed using flow cytometry as mean fluorescence intensities of the cells.The values are expressed as the mean ± SEM of duplicate cultures from three independent experiments.Significance was evaluated by an unpaired t-test with Welch's correction: *p < 0.05, N.S.: not significant.

Figure 6 .
Figure 6.Mechanisms of uptake of cationic liposome by DC2.4 cells.DC2.4 cells were pre-treated with various chemical inhibitors for endocytic pathways for 30 min at 37˚C, and then treated with vehicle (black) or the cationic liposomes (20 nmol/mL) for 1 h at 37˚C.The uptake of cationic liposome was further analyzed using flow cytometry as mean fluorescence intensities of the cells.The values are expressed as the mean ± SEM of duplicate cultures from three independent experiments.Significance was evaluated by an unpaired t-test with Welch's correction: *p < 0.05.

Figure 8 .
Figure 8. Coexistence of FITC-OVA and the cationic liposomes is crucial for enhanced uptake of FITC-OVA.(a) For co-culture experiment, DC2.4 cells were treated with vehicle (black), FITC-OVA (50 μg/mL) (orange), and FITC-OVA (50 μg/mL) in combination with cationic liposomes (20 nmol/mL) (blue) for 1 h at 37˚C.For wash-out experiment, DC2.4 cells were treated with cationic liposomes (20 nmol/mL) for 4 h at 37˚C.After washing the liposomes out, DC2.4 cells were incubated with FITC-OVA (50 μg/mL) for 1 h at 37˚C (green).FITC-OVA uptake and the cationic liposome uptake was then analyzed using flow cytometry as mean fluorescence intensities of the cells.The values are expressed as the mean ± SEM of duplicate cultures from three independent experiments.Significance was evaluated by an unpaired t-test with Welch's correction: *p < 0.05, (b) confocal microscopy images for wash-out and co-culture experiments.FITC-OVA = green; DiI-liposome = red; DAPI = blue; co-localization of FITC-OVA and DiI-liposome = yellow.

Table 1 .
Chemical inhibitors for endocytic pathways used in this study.