Unsaturated Squalene Content in Emulsion Vaccine Adjuvants Plays

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Unsaturated Squalene Content in Emulsion Vaccine Adjuvants Plays a Crucial Role in ROS-Mediated Antigen Uptake and Cellular Immunity Chung-Hsiung Huang, Chiung-Yi Huang, and Ming-Hsi Huang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00800 • Publication Date (Web): 13 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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Molecular Pharmaceutics

Unsaturated Squalene Content in Emulsion Vaccine Adjuvants Plays a Crucial Role in ROS-Mediated Antigen Uptake and Cellular Immunity

Chung-Hsiung Huang,*,† Chiung-Yi Huang,† and Ming-Hsi Huang*,†,‡

†

National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes,

35053 Miaoli, Taiwan ‡

Graduate Institute of Biomedical Sciences, China Medical University, 40402 Taichung, Taiwan

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Content Graphic

ABSTRACT: Emulsion-based adjuvants have been demonstrated to be an effective tool in increasing vaccine efficacy. Here, we aimed to launch a mechanistic study on how emulsion adjuvants interacting with immune cells and to elucidate the roles of the core oil in vaccine immunogenicity. Our results showed that treatment of dendritic cells (DCs) and splenocytes with a squalene-based emulsion (referred as SqE) induced reactive oxidative species (ROS) production and resulted in an increase in apoptotic and necrotic cells in a concentration- and time-dependent manner. Furthermore, DCs co-cultured with cellular debris of SqE-pretreated splenocytes resulted in a higher level of ovalbumin (OVA) antigen uptake by DCs than those cocultured with untreated splenocytes. Interestingly, the potency was rather attenuated when splenocytes pretreated with N-acetyl-cysteine, an antioxidant. Notably, SqE possesses a high impact on eliciting ROS-mediated antigen uptake compared with a squalane-based emulsion (SqA). Concordantly, immunogenicity studies have shown that SqE is better able than SqA to activate antigen-presenting cells, and to enhance antigen-specific T-cell immunity. Taken together, our results show that unsaturated squalene oil cored within emulsions plays a crucial role in ROS-mediated antigen uptake and cellular immunity, providing a basis for the design and development of vaccine adjuvant. KEYWORDS: emulsion adjuvant, reactive oxidative species (ROS), squalene


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INTRODUCTION Squalene, a branched-chain isoprenoid hydrocarbon with six unconjugated double bonds, is currently used as a skin moisturizer in cosmetics, a component of over-the-counter medicines and health supplements, and an excipient (adjuvant) in vaccine preparation.1,2 It is important to note that squalene alone is not an adjuvant, but emulsions of squalene are formulated into vaccines to increase their immunogenicity and efficacy.2 Recently, squalene-based oil-in-water emulsions are used to adjuvant both seasonal and pandemic influenza vaccines and have already been approved in humans for the prevention of influenza illness.2 In the context of a vaccine, it is important to evaluate whether co-culturing candidate adjuvants can promote antigen uptake by antigen-presenting cells (APCs) and lead to enhance vaccination feasibility. Unlike water-in-oil (W/O) emulsions, there is no oily barrier for oil-inwater (O/W) emulsions that isolate the encapsulated materials from external ones; therefore, several studies have emerged that the potential mechanisms of action of O/W adjuvants include enhancement of antigen presentation at the injection site, recruitment and activation of APCs, and direct stimulation of cytokine and chemokine production by macrophages and granulocytes.3-5 However, the enhancement of vaccine immunogenicity by stepwise release of antigens within O/W is ambiguous. Recently, it has been proposed that the cell death induced by vaccine adjuvants may play an important role in triggering immune responses.6,7 To test this hypothesis, some studies have essentially investigated the possible apoptotic and necrotic effects of the surface-active agents on immune cells;7,8 however, the core oil is indeed a key constituent of the emulsions, and the relationship of the selected oil with cell viability and adjuvanticity has not been extensively studied.



Reactive oxygen species (ROS) are oxygen-containing reactive chemical species and are formed as a natural byproduct of the normal metabolism of oxygen.9,10 ROS have been known to be an essential component in activating signaling pathways to initiate biological processes and immune responses.11,12 However, excessive levels of ROS can activate the signaling pathways of death receptors and even promote the peroxidation of lipids, proteins and DNA, which consequently results in cell apoptosis and necrosis.13,14 Currently, the relationship between ROS production and antigen uptake is still unclear. Some studies have been demonstrated that treating J774S.1 macrophages with emulsion adjuvants resulted in a substantial amount of ROS production, may leading to cell apoptosis and necrosis.14,15 Therefore, we hypothesized that augment of antigen uptake into APCs requires co-incubation emulsion adjuvants with ROSmediated cellular debris. In the present study, we explored mechanistic reasoning toward embracing the interface between the physicochemical and biological signatures for core oil selection in vaccine immunogenicity. We used ovalbumin (OVA) as a model antigen to elucidate the roles of unsaturated squalene oil within emulsion adjuvants in the induction of cell death and generation of ROS by primary immune cells, which facilitate antigen uptake into APCs. Bone marrowderived dendritic cells (DCs, the major class of professional APCs) and cells harvested from the spleen (the major lymphocytes rich organ) were directly exposed to these emulsions, and the analyses of concentration- and time-course of cell death and ROS production were performed. The level of Alexa 647-conjugated ovalbumin (OVA*) uptake by DCs co-cultured with squalenebased emulsion (SqE)-treated splenocytes was examined to investigate whether ROS-mediated cell death facilitates antigen uptake. Next, splenocytes were pretreated with a typical ROS inhibitor, N-acetyl-cysteine (NAC),16 to confirm that ROS production is a necessary process in


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emulsion-induced cell death and antigen uptake. Finally, the immunological evaluation of OVA after formulated with SqE was determined in mice for the activation of lymphoid APCs and splenic T cells by assessing the expression of activation markers, cytokines and transcription factors. The results were compared with those obtained with emulsions consisting of squalane (SqA), the saturated form of squalene.

Materials and Methods Materials. All chemicals and reagents were purchased from Sigma Chemical (St. Louis, MO, USA) unless otherwise stated. Antibodies and an Annexin V/PI detection kit were purchased from BioLegend Inc. (San Diego, CA, USA). 10% neutral buffered formalin and a decalcifying solution were purchased from Leica Biosystems Richmond Inc. (Richmond, IL, USA). The cellpermeant 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) cellular ROS detection assay kit was purchased from Abcam, Inc. (Cambridge, MA, USA). Alexa 647-conjugated ovalbumin (OVA*), Alexa 568-conjugated anti-mouse IgG (IgG*), Hoechst 33342 nucleic acid stain, and DiD' lipophilic carbocyanine dye were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). LysoTrackerTM Green DND-26 was purchased from Life technologies Co. (Eugene, OR, USA). An In-Situ Cell Death Detection Kit was purchased from Roche Life Science (Indianapolis, IN, USA). Squalene, squalane and Span®85 (sorbitantrioleate) were purchased from Sigma-Aldrich (Steinheim, Germany). PyroGene™ Recombinant Factor C Endotoxin Detection Assay was purchased from LONZA Walkersville Inc. (MD, USA). The bioresorbable emulsifier PEG-b-PLACL was synthesized by the ring-opening polymerization of lactide (LA) and ε-caprolactone (CL) in the presence of polyethylene glycol 5,000 monomethyl ether and Tin(II) 2-ethylhexanoate.17



Preparation of SqE and SqA. SqE was emulsified with PEG-b-PLACL and Span®85, as previous described.17 Briefly, 120 mg of PEG-b-PLACL, 0.8 mL of phosphate-buffered saline (PBS), and 1.1 mL of oily solution consisting of squalene and Span®85 (85/15 v/v) were emulsified using a homogenizer at 6,000 rpm for 5 min. The emulsion stocks were stored at 4°C and diluted with sterile PBS before further use. SqE-formulated OVA was investigated by redispersing 200 µL of SqE stock into 1,800 µL of OVA-containing solution and was mixed with a test-tube rotator (Labinco LD-79, Netherlands) at 5 rpm for at least 1 h. Squalene was replaced with the same volume of squalane to yield SqA. Endotoxin was measured prior to in vitro and in vivo experiments by Endotoxin Detection Assay, and the amount of exdotoxin in emulsion samples was lower than 0.01 EU/mL. Physicochemical characterization of SqE and SqA. Particle size and ζ-potential were measured by dynamic light scattering (DLS) technique (Zetasizer Nano ZS, Malvern Instruments Ltd., Worcestershire, UK). The samples were reproduced following the above procedures using purified water instead of PBS. The measurements were performed in triplicate and the data are expressed as the mean ± standard error of the mean (SEM). Mice and ethics statement. Five-week-old female C57BL/6 mice were obtained from the National Laboratory Animal Center. All mice were housed for at least one week at the Laboratory Animal Facility of the NHRI in Miaoli County, Taiwan. All experiments were conducted in accordance with the guidelines of Laboratory Animal Center of NHRI. All animal studies were approved by the NHRI Institutional Animal Care and Use Committee (NHRIIACUC-105139-AC). Isolation and culture of primary cells. The spleen and the bone marrow were isolated from C57BL/6 mice. The DC suspensions were prepared by flushing out the bone marrow and


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incubating the cells (2×106 cells/dish) in cultured medium consisting of RPMI 1640 (HyClone, Logan, Utah, USA) containing 2 mM L-glutamine and supplemented with 25 mM HEPES (Gibco Invitrogen, Grand island, NY, USA), 0.05 mM 2-mercaptoethanol (Sigma, St. Louis, MO, USA), 10% heat-inactivated fetal bovine serum (HyClone, Logan, Utah, USA) and 1% antibiotics and in the presence of 20 ng/mL GM-CSF for use in further experiments. After 72 h, 20 µL of fresh GM-CSF (10 µg/mL) was added to each culture dish. On day 6, culture dishes were washed, and DCs were collected by centrifugation for further experiments. Splenocyte suspensions were prepared by mashing the spleen through a cell strainer using a syringe plunger. To remove the red blood cells, the harvested suspensions were suspended in ACK buffer (155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) and then washed with cultured medium for use in further experiments. Analysis of cell apoptosis and necrosis. Splenocytes (5×106 cells/mL) and DCs (1×106 cells/mL) were pretreated with 1 mM NAC for 30 min followed by treatment with either SqE or SqA for 16 h. After washed with PBS, the cells were stained with Annexin V and propidium iodide (PI) following the supplier’s instruction and analyzed by flow cytometry. Apoptotic and necrotic cells were denoted as the percentage of Annexin V+ and Annexin V-/PI+ cells, respectively. Measurement of intracellular ROS production. Splenocytes (5×106 cells/mL) and DCs (1×106 cells/mL) were pretreated with 1 mM NAC for 30 min followed by treatment with either SqE or SqA for 16 h. After washed with PBS, the cells were incubated in the presence of 20 µM DCFDA for 30 min. The generation of ROS (fluorescent green) was quantified by flow cytometry.



Detection of emulsion uptake by DCs. SqE and SqA were fluorescent-labeled with DiD' dye (1 mM) and IgG* (10 µg/mL) prior to incubation. DCs (1×106 cells/mL; 1 mL) were cultured with the fluorescent-labeled emulsions (1,000 nL/mL) in 35-mm dish for 4 h. The free fluorescent dye and labeled emulsions were then washed away with PBS, and the cells were stained with Hoechst 33342 (5 µg/mL) and LysoTrackerTM Green DND-26 (50 nM) for 10 min to label cell nuclei and lysosomal vehicles, respectively. The fluorescence images were acquired from live cells utilizing a Leica TCS SP5 confocal microscope. Detection of OVA* uptake by DCs. Splenocytes (5×106 cells/mL) were pretreated either with or without NAC (1 mM) for 30 min. The pretreated splenocytes were then treated without or with either SqE or SqA (1,000 nL/mL) for 16 h. Splenocytes treated with 50 µM H2O2 were used as a positive control for ROS-induced cell death. Subsequently, DCs (5×105 cells/mL) were co-cultured with the treated splenocytes (5×105 cells/mL) in the presence of OVA* (20 µg/mL) for 1 h. After washed with PBS, the cells were stained with FITC-conjugated anti-CD11c antibody, and the uptake of OVA* by CD11c+ cells was analyzed by flow cytometry. To detect the internalization of OVA*, DCs were stained with Hoechst 33342 (5 µg/mL) and LysoTrackerTM Green DND-26 (50 nM) for 10 min, and the fluorescence images were acquired as described above. Vaccination and in situ cell death detection. C57BL/6 mice (three mice per group) were injected s.c. in both hindlimbs with 10 µg of OVA alone or adjuvanted with either SqE or SqA (10 % v/v in PBS, total volume of 100 µL). Three days after injection, the tissues at the injection site were excised and sectioned by the Pathology Core Laboratory of NHRI. The slides were subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays by using the In-Situ Cell Death Detection Kit following the manufacturer's instructions. The number


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of TUNEL-positive signals was quantified using the ImageJ image processing and analysis program (Bethesda, MD, USA). Three measurements per tissue section and 6 sections per group were analyzed at 400-fold magnification. Analysis of lymphoid APCs activation. Three days after vaccination, the lymphoid cells from the inguinal and popliteal lymph nodes (LNs) were collected from the vaccinated mice. The cell suspensions were harvested and re-suspended in ACK buffer for 1 min. The cells were collected and stained with FITC-conjugated CD11c, PE-conjugated CD40, PeCy7-conjugated CD86 and Alexa 647-conjugated MHC II antibodies and then subjected to flow-cytometry analysis (LSRII; BD Immunocytometry Systems, CA, USA). Analysis of T-cell immunity. Seven days after vaccination, the spleens were collected from the vaccinated mice, and 5 × 106 splenocytes/mL were cultured in the absence or presence of OVA (10 µg/mL) for 72 h. The supernatants were collected for IFN-γ and IL-17 measurement by ELISA (DueSet® ELISA Development kit, R&D Systems, Inc., MN USA) following the supplier’s instruction. After incubation for 24 h, total RNA was extracted from the splenocytes in each group using the TRI Reagent (Sigma) according to the manufacturer’s instructions. The steady-state mRNA expression levels of T-bet, RORγt and β-actin were measured by reverse transcription-polymerase chain reaction (RT-PCR). All isolated RNA samples were confirmed to be free of DNA contamination by the absence of product after PCR amplification in the absence of reverse transcription. For reverse transcription, 10 µg of total RNA was reverse-transcribed into cDNA using the Maxime RT PreMix Kit (iNtRON Biotechnology, Inc.) and random primers. The reverse transcription proceeded at 45 °C for 60 min and then at 95 °C for 5 min. Next, 2x PCR Master Mix Solution (iNtRON Biotechnology, Inc.) and 10 pmol of each forward and reverse primer specific for the gene of interest were added to each cDNA sample for PCR. The



primers used are summarized in Table S1. The samples were heated to 94 °C for 2 min and cycled 30-37 times at 94 °C for 30 seconds, 55 °C for 45 seconds, and 72 °C for 60 seconds, followed by an additional step at 72 °C for 5 min. The PCR products were electrophoresed in 2% agarose gels and stained with 0.1 µg/mL SYBR® Green (Thermo Fisher Scientific, Inc., CA, USA) for visualization. Quantification was performed by assessing the optical density of the DNA bands using the ImageJ image processing and analysis program (MD, USA). The results are expressed as the density ratio between the gene of interest and the reference standard (βactin). Statistical analysis. The data are expressed as the mean ± standard error of mean (SEM) for each treatment group. The treated groups and the control group were compared by one-way analysis of variance (ANOVA). Dunnett’s two-tailed t-test was used to assess the statistical difference between the treatment groups and the control groups. The level of significance was set at p < 0.05.

RESULTS Physicochemical characterization of SqE and SqA emulsions. SqE emulsion contains squalene oil and two surfactants, PEG-b-PLACL and Span®85, mixing with the aqueous. The emulsion was denominated to be SqA when squalene was replaced with squalane. Before administration, the stock emulsions were re-dispersing into OVA antigenic media, resulting homogeneous suspensions entrapping OVA at the surface. Table I shows the physicochemical characteristics of the emulsions considered in this study. Both SqE and SqA stocks were white and isotropic, and the densities were measured 0.90 g/mL and 0.85 g/mL, respectively. Of note, the squalene oil has a density of 0.85 g/mL, while the squalane oil has that of 0.81 g/mL.18 SqE


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had a mean diameter of 365 nm and a ζ-potential of -34.9 mV; while the mean diameter of SqA slightly increased to 450 nm, and the ζ-potential to -32.2 mV. The presence of OVA is not a key point to the particle size and ζ-potential, probably due to small amount of OVA content in the vaccine dosage. It is noteworthy that O/W emulsions with submicron size facilitate the induction of appropriate immunity.19 The two emulsions were then investigated for their ability to interact with the immune cells and to enhance the vaccine immunogenicity. Emulsion treatment induced apoptotic and necrotic effects in DCs and splenocytes. First, we investigated whether treatment with SqE induced apoptosis and necrosis in DCs. After a 16-h incubation, the percentage of Annexin V+ and/or PI+ DCs was increased in the SqE group in a dose-dependent manner in the range of 10-1,000 nL/mL (Figure 1A, upper panel), indicating that SqE can induce apoptosis and necrosis in DCs. As previous studies have demonstrated different efficacies of saturated and unsaturated fatty acids on inducing cell apoptosis,20,21 we further investigated the effect of different core oils (SqE and SqA) on cell death. Interestingly, the potency was rather reduced when the core oil within the emulsion was replaced as squalane. To confirm this, cell viability was also detected in DCs incubated with the same amount (1,000 nL/mL) of either SqE or SqA for 0-16 h. The data showed that the SqE treatment induced a higher percentage of apoptotic and necrotic DCs compared to SqA treatment at each time point (Figure 1A, lower panel). We also attempted to study whether treatment with SqE and SqA induce apoptosis and necrosis in splenocytes. The data showed that SqE is more potent than SqA in inducing cell death (Figure 1B). Emulsion treatment induced ROS production in DCs and splenocytes. As excessive ROS would result in cell apoptosis and even necrosis,14,15 we further investigated whether emulsion treatment induces ROS production in DCs. Consistent with the findings observed in the



induction of cell death, the effect of emulsions on ROS production is a function of the strength (concentration-dependent manner) and duration of exposure (time-dependent manner) (Figure 2A). As SqE and SqA showed distinct efficacies in inducing cell death, we further compared the efficacy of SqE and SqA in inducing ROS production. Concordantly, SqE induced a high level of ROS production in DCs; however, the potency was reduced when the squalene core oil was replaced as squalane (Figure 2A). Similar results were also observed in splenocytes (shown in Figure 2B), using unsaturated squalene as the core oil of the emulsion resulted in an increased ability to induce ROS production from splenocytes compared to that of emulsions with saturated squalane as the core oil. Furthermore, the higher concentrations of the emulsions, the more pronounced levels of ROS production were induced. The effect of cell death induced by the emulsion adjuvants was highly correlated with ROS production in DCs and in splenocytes as well. Collectively, we identified the apoptotic and necrotic effects of emulsion adjuvants on primary immune cells and suggested that ROS production may play a crucial role in emulsioninduced cell death. Emulsion-induced cell death and ROS production were impaired by pretreatment with NAC. The apoptotic and necrotic activities of emulsion adjuvants in DCs and splenocytes have been identified; however, the effect of cell death induced by emulsion-induced ROS production was not ascertained. Therefore, we further investigated whether pretreatment of DCs and splenocytes with an antioxidant could prevent emulsion-induced cell death. NAC was selected as the antioxidant because it is the precursor of the endogenous antioxidant glutathione, and administration of NAC replenishes glutathione stores.16 In addition, NAC also acts as a scavenger of oxygen free radicals and interacts with ROS and nitrogen species.16 The results show that pretreatment with NAC led to a reduction of emulsion-induced cell death in DCs in a


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concentration-dependent manner (Figure 3A). Furthermore, pretreatment with NAC significantly attenuated emulsion-induced ROS production (Figure 3A). Emulsion-induced cell death and ROS production were also impaired by pretreatment the splenocytes with NAC (Figure 3B). Interestingly, it appears that replacing the squalene core oil with squalane did not significantly alter the ability of the emulsion to induce apoptosis and necrosis in NAC-pretreated splenocytes (Figure 3B). These results clearly substantiated that the ROS production is a necessary process to promote emulsion-induced cell death. Antigen uptake by DCs requires cellular debris issued from emulsion-treated splenocytes. In a preliminary study, we attempted to address whether the treatment of the emulsions can directly promote antigen uptake by DCs. After incubation with DCs, neither SqE nor SqA could increase the intensity of fluorescence signal corresponding to OVA* (Figure 4A). We also investigated whether emulsions could be taken by DCs. To this, the oily and the aqueous phases of the emulsions were labeled with fluorescent dye DiD' and IgG*, respectively. Confocal microscopic images showed that co-culturing DCs with labeled emulsions exhibited fluorescent signals (purple: DiD'; red: IgG*) within discreet lysosomal vehicles of DCs (green) (Figure 4B). The mean fluorescence intensity (MFI) of DiD’ in emulsion-treated DCs was detected 952 ± 23 for SqE, and 920 ± 40 for SqA, indicating that SqE and SqA internalized by DCs were quantitatively similar. These results show that while emulsions per se could be taken into lysosome, they were not capable of accelerating antigen uptake. We hypothesized that a potential competition happens between emulsions and OVA* for the uptake by DCs. We then investigated whether the presence of apoptotic cellular debris can facilitate antigen uptake by DCs. Compared to those co-cultured with untreated splenocytes, DCs co-cultured with emulsion-treated splenocytes resulted in the enhancement in OVA* uptake (Figure 5A). Notably, DCs co-cultured



with SqE-treated splenocytes resulted in higher level of OVA* uptake than that with SqA-treated splenocytes. Similar results were found that a great deal of OVA* was contained in the lysosomal vehicles of DCs (Figure 5B). However, the potency was rather attenuated when splenocytes pretreated with NAC (Figure 5A), indicating that ROS-mediated cell death is an essential action mechanism of emulsion adjuvants to facilitate antigen uptake. Cellular apoptosis and necrosis on activation of APCs. TUNEL staining is a common method for detecting DNA fragmentation by labeling the terminal end of nucleic acids those results from apoptotic signaling cascades.22 We next conducted a histological TUNEL staining study of the tissues at the injection site to investigate the apoptotic effect of emulsion adjuvants in vivo. As shown in Figure 6A, subcutaneous vaccination of mice with SqE-adjuvanted OVA recruited cell infiltration around the emulsion depot, and a numerous percentage of those cells were TUNEL-positive apoptotic cells. Although vaccination with SqA-adjuvanted OVA also recruited cell infiltration around the emulsion depot, the number of TUNEL-positive cells was lower than that around the SqE depot (Figure 6A). Note that the use of squalene as the core oil showed more potency to induce cell apoptosis at the injection site than the use of squalane, which agrees with the cell culture study. Because antigen-loaded DCs in peripheral tissues then migrate to local draining lymph nodes (LNs) where they present antigen to lymphocytes, we further investigated the expression of MHC class II and co-stimulatory molecules on APCs harvested from the LNs of vaccinated mice. Consistently, high levels of CD40, CD86 and MHC II expression on CD11c+ LN cells were observed for mice vaccinated with SqE-adjuvanted OVA compared with OVA alone (Figure 6B). However, the potency was rather reduced when the adjuvant replaced as SqA.


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SqE is more effective than SqA to enhance T-cell immune responses. For antigen-specific T-cell activation, this is through the processing of peptide epitopes of protein antigens into major histocompatibility complex (MHC) molecules.23 We further investigated whether ROS-mediated antigen uptake and activation of APCs leads to the promotion of antigen-specific T-cell responses. As shown in Figure 6C, the elevated level of IFN-γ and IL-17 secretion by splenocytes re-stimulated with OVA was observed in mice vaccinated with OVA adjuvanted with emulsions compared with OVA alone. Notably, higher level of cytokines secretion was observed in mice vaccinated with OVA adjuvanted with SqE than that with SqA, indicating SqE was a more potent emulsion adjuvant than SqA to induce antigen-specific T-cell immune responses. Subsequently, the expression of T-cell transcription factors in splenocytes was measured to confirm the activation of T helper (TH) cell subsets induced by emulsion adjuvants. As shown in Figure 6C, the elevated mRNA expression of T-bet (TH1-type) and RORγt (TH17-type) was observed in mice vaccinated with OVA adjuvanted with emulsions compared to that with OVA alone, in agreement with our previous study which showed that SqE induced TH1- and TH17dominant immune responses.17 Collectively, both emulsions were able to induce high levels of cytokine secretion and transcription factor expression, while SqE was more potent than SqA to induce antigen-specific cellular immunity.

DISCUSSION Necrotic cells have been demonstrated to be endogenous activators of DCs and function as natural adjuvants to stimulate a primary immune response.24 On the other hand, the exposure of phosphatidyl serine (PS) on the outer leaflet of the plasma membrane of apoptotic cells results in PS-mediated phagocytosis, which enhances antigen uptake.25 Several studies have elucidated the



role of cell death on activating macrophages;26,27 however, little is known about the effects of cell death on facilitating antigen uptake by DCs, which are the most potent APCs that perform the cross-presentation of exogenous antigens.28 Emulsion adjuvant-induced cell death has been suggested as a potential mechanism to enhance antigen delivery, and the role of emulsifiers in emulsion-induced cell death and antigen uptake has also been investigated.7,8 For example, Pluronic L121-stabilized emulsions were demonstrated to be a potent adjuvant to induce significant cell death and to elicit strong antigen-specific immune responses compared with Tween®- and Span®-stabilized emulsions.7 In our previous study, we demonstrated that bioresorbable polymeric emulsifiers started to lose the lipophilic units in the main-chain polymer from several weeks to months and thereby affected the stability of the emulsions.17 We propose that the major adjuvant effect of the emulsified particles attributes to the feasibility of the antigen presenting to APCs, and also the minor effect to degradation mechanisms and emulsion breaks. However, little information is available as to the role of core oil in the action mechanism of emulsion adjuvants. In this study, we provided the first evidence that the core oil selection plays a critical role in emulsion-induced ROS-mediated cell death and antigen uptake. This notion is substantiated by several lines of evidence. Firstly, direct exposure to SqE induced higher levels of ROS-mediated necrotic and apoptotic effects in DCs and splenocytes than that to SqA. Secondly, emulsions alone were incapable facilitating antigen uptake, whereas emulsion-treated splenocytes, especially SqE-treated splenocytes, facilitated antigen uptake into DCs. Finally, pretreatment with antioxidant not only prevented emulsion-induced ROS-mediated cell death but also attenuated antigen uptake. In line with the in vitro studies, emulsions comprised unsaturated core oil was a more potent adjuvant to elicit the apoptosis of recruited cells at the injection site, LN


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APC activation and antigen-specific T-cell immune responses. Our data clearly show that the unsaturated squalene plays an important role in the activity of emulsion adjuvants, and these observations provide critical insights to the underlying mechanisms of action of emulsion adjuvants at the cellular and molecular level. Some studies have compared the effects of saturated and unsaturated fatty acids on the induction of ROS production and cell death.20,21,29,30 Nevertheless, accumulated evidence shows that saturated fatty acids, but not unsaturated ones, can induce apoptotic cell death.20,21 Antagonism between saturated and unsaturated fatty acids in ROS production has been demonstrated as one of the mechanisms to induce or prevent cytotoxicity.29 It has also been reported that fatty acids induce the apoptosis and necrosis of macrophages, but this cytotoxicity was not strictly related to the degree of saturation of the fatty acids.30 Regarding the core oil selection, squalane is highly stable and less susceptible to oxidation.31 To date, there is no available information pertaining to the effects of squalene and/or squalane on inducing ROS production and cell death. It has been reported that squalene initially shows antioxidant properties after isolation; nevertheless, squalene also easily become oxidized after exposure to air and subsequently act as an oxidizing agent.32 As summarized in Figure 7, we suggest that SqE is more easily oxidized than SqA and become more potent pro-oxidant to induce intracellular ROS production. Accordingly, the distinct efficacy of SqE and SqA as inducers of ROS-mediated antigen uptake and antigen-specific T-cell immune responses suggested a role of the degree of saturation of the core oil in the activities of emulsion adjuvants. Regarding the mechanism of emulsion-mediated adjuvanticity, some hypotheses regarding the mechanism of emulsion-mediated adjuvanticity have been proposed, including particulate depot effect and immunomodulatory activity.2-7 In contrast to the former which are thought to



prolong the presence of antigen in tissues, the latter mainly mimic specific signals to activate the immune cells. However, these parameters are strongly influenced by the excipients of oil components and the surfactant system. Our previous study demonstrated that vaccine antigen could be absorbed onto emulsified particles and released from the matrix in a sustained manner; subsequently driving the production of antigen-specific antibodies.17 Here we demonstrated that unsaturated squalene core oil plays a critical role in the promotion of ROS-mediated antigen uptake, providing another potential mechanism for the adjuvant activity of emulsions.

CONCLUSIONS In this study, we reported the mechanisms underlying emulsion adjuvants interact with immune cells in vitro and elucidated their roles in vaccine immunogenicity in vivo. We demonstrated that emulsion comprised of unsaturated squalene oil as the core is better able than saturated squalane to elicit ROS-mediated antigen uptake and APC activation, leading to the promotion of antigenspecific T-cell immunity. Accordingly, our results highlight the importance of unsaturated squalene core oil in the adjuvant activity of emulsions and offer insight into the design and development of vaccine adjuvants. ASSOCIATED CONTENT Supporting Information. Table S1 Primers used for RT-PCR analysis. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors


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* Correspondence should be addressed to Chung-Hsiung Huang ([email protected]) or Ming-Hsi Huang ([email protected]) ORCID Chung-Hsiung Huang: 0000-0002-2295-6412 Ming-Hsi Huang: 0000-0002-9670-3821

Notes The authors declare no conflict of interest. ACKNOWLEDGMENT This work was supported by grant 106A1-IVPP23-014 from the National Health Research Institutes of Taiwan and grant MOST 106-2314-B-400-016-MY3 from the Ministry of Science and Technology of Taiwan. The authors are grateful to Optical Biology Core Laboratory of NHRI for the help in confocal microscope analysis and Miss Tzu-Ying Yeh for her help in preparing the materials.

REFERENCES (1) Kelly, G. S. Squalene and its potential clinical uses. Altern. Med. Rev. 1999, 4, 29–36. (2) O'Hagan, D. T.; Tsai, T. F.; Brito, L. A. Emulsion based vaccine adjuvants. Hum. Vaccin. Immunother. 2013, 9, 1698–1700.



(3) Seubert, A.; Monaci, E.; Pizza, M.; O'Hagan, D. T.; Wack, A. The adjuvants aluminum hydroxide and MF59 induce monocyte and granulocyte chemoattractants and enhance monocyte differentiation toward dendritic cells. J. Immunol. 2008, 180, 5402–5412. (4) Tritto, E.;Mosca, F.; De Gregorio, E. Mechanism of action of licensed vaccine adjuvants. Vaccine 2009, 27, 3331–3334. (5) O'Hagan, D. T.; Ott, G. S.; De Gregorio, E.; Seubert, A. The mechanism of action of MF59 - an innately attractive adjuvant formulation. Vaccine 2012, 30, 4341–4348. (6) Wu, C. A.; Yang, Y. W. Induction of cell death by saponin and antigen delivery. Pharm. Res. 2004, 21, 271–277. (7) Yang, Y. W.; Wei, A. C.; Shen, S. S. The immunogenicity-enhancing effect of emulsion vaccine adjuvants is independent of the dispersion type and antigen release rate-a revisit of the role of the hydrophile-lipophile balance (HLB) value. Vaccine 2005, 23, 2665–2675. (8) Yang, Y. W.; Shen, S. S. Enhanced antigen delivery via cell death induced by the vaccine adjuvants. Vaccine 2007, 25, 7763–7772. (9) Hayyan, M.; Hashim, M. A.; AlNashef, I. M. Superoxide ion: generation and chemical implications. Chem. Rev. 2016, 116, 3029–3085. (10) Devasagayam, T. P.; Tilak, J. C.; Boloor, K. K.; Sane, K. S.; Ghaskadbi, S. S.; Lele, R. D. Free radicals and antioxidants in human health: current status and future prospects. J. Assoc. Physicians India. 2004, 52, 794–804. (11) Schieber, M.; Chandel, N. S. ROS function in redox signaling and oxidative stress. Curr. Biol. 2014, 24, 453–462.


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(12) Schmielau, J.; Finn, O. J. Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of t-cell function in advanced cancer patients. Cancer Res. 2001, 61, 4756–4760. (13) Fortes, G. B.; Alves, L. S.; de Oliveira, R.; Dutra, F. F.; Rodrigues, D.; Fernandez, P. L.; Souto-Padron, T.; De Rosa, M. J.; Kelliher, M.; Golenbock, D.; Chan, F. K.; Bozza, M. T. Heme induces programmed necrosis on macrophages through autocrine TNF and ROS production. Blood 2012, 119, 2368–2375. (14) Hampton, M. B.; Orrenius, S. Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis. FEBS Lett. 1997, 414, 552–556. (15) Ozben, T. Oxidative stress and apoptosis: impact on cancer therapy. J. Pharm. Sci. 2007, 96, 2181–2196. (16) Zafarullah, M.; Li, W. Q.; Sylvester, J.; Ahmad, M. Molecular mechanisms of Nacetylcysteine actions. Cell Mol. Life Sci. 2003, 60, 6-20. (17) Huang, C. H.; Huang, C. Y.; Cheng, C. P.; Dai, S. H.; Chen, H. W.; Leng, C. H.; Chong, P.; Liu, S. J.; Huang, M. H. Degradable emulsion as vaccine adjuvant reshapes antigen-specific immunity and thereby ameliorates vaccine efficacy. Sci. Rep. 2016, 6, 36732. (18) Vogel, F. R.; Powell, M. F. In Vaccine Design: The subunit and Adjuvant Approach; Powell, M. F.; Newman, M. J., Eds.; Plenum, New York, 1995;Chapter 7, pp 141-228. (19) Reddy, S. T.; Swartz, M. A.; Hubbell, J. A. Targeting dendritic cells with biomaterials: developing the next generation of vaccines. Trends Immunol. 2006, 27, 573–579.



(20) Mei, S.; Ni, H. M.; Manley, S.; Bockus, A.; Kassel, K. M.; Luyendyk, J. P.; Copple B. L.; Ding, W. X. Differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes. J. Pharmacol. Exp. Ther. 2011, 339, 487-498. (21) Fürstova, V.; Kopska, T.; James, R.F.; Kovar, J. Comparison of the effect of individual saturated and unsaturated fatty acids on cell growth and death induction in the human pancreatic beta-cell line NES2Y. Life Sci. 2007, 82, 684-691. (22) Gavrieli, Y.; Sherman, Y.; Ben-Sasson, S. A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 1992, 119, 493-501. (23) Song, Y. C.; Chou, A. H.; Homhuan, A.; Huang, M. H.; Chiang, S. K.; Shen, K. Y.; Chuang, P. W.; Leng, C. H.; Tao, M. H.; Chong, P.; Liu, S. J. Presentation of lipopeptide by dendritic cells induces anti-tumor responses via an endocytosis-independent pathway in vivo. J. Leukoc. Biol. 2011, 90, 323-332. (24) Gallucci, S.; Lolkema, M.; Matzinger, P. Natural adjuvants: endogenous activators of dendritic cells. Nat. Med. 1999, 5, 1249-1255. (25) Hoffmann, P. R.;de Cathelineau, A. M.; Ogden, C. A.; Leverrier, Y.; Bratton, D. L.; Daleke, D. L.; Ridley; A. J.; Fadok, V. A.; Henson, P. M. Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells. J. Cell Biol. 2001, 155, 649-659. (26) Mosser, D. M.; Edwards, J. P. Exploring the full spectrum of macrophage activation. Nat. Rev. Immunol. 2009, 8, 958-969.


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(27) Konttinen, Y. T.; Pajarinen, J.; Takakubo, Y.; Gallo, J.; Nich, C.; Takagi, M.; Goodman, S. B. Macrophage polarization and activation in responses to implant debris: influence by “particle disease” and ion disease”. J. Long Term Eff. Med. Implants 2014, 24, 267-281. (28) den Haan, J. M.;Arens, R.; van Zelm, M. C. The activation of the adaptive immune system: cross-talk between antigen-presenting cells, T cells and B cells. Immunol. Lett. 2014, 162, 103-112. (29) Gehrmann, W.; Würdemann, W.;Plötz, T.; Jörns, A.; Lenzen, S.; Elsner, M. Antagonism between saturated and unsaturated fatty acids in ROS mediated lipotoxicity in rat insulinproducing cells. Cell Physiol. Biochem. 2015, 36, 852-865. (30) Martins de Lima, T.; Cury-Boaventura, M. F.; Giannocco, G.; Nunes, M. T.; Curi, R. Comparative toxicity of fatty acids on a macrophage cell line (J774). Clin. Sci. 2006, 111, 307317. (31) Jenkins, J. W. Cosmetics: Science and technology. J Am. Oil Chem. Soc. 1973, 50, A54. (32) Sobel, H.;Marmorston, J. The possible role of squalene as a protective agent in sebum. Cancer Res. 1956, 16, 500-503.



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Table I. Physicochemical characteristics of SqE and SqAa Acronym

a

Core oil

Density (g/mL)

Particle size (nm)

ζ-potential (mV)

blank

with OVA

blank

with OVA

SqE

Squalene

0.90±0.03

365±36

344±11

-34.9±1.2

-39.6±0.2

SqA

Squalane

0.85±0.04

450±19

457±8

-32.2±0.7

-39.8±0.1

The aqueous being purified water.


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Figure 1. Apoptosis and necrosis of DCs and splenocytes induced by emulsion-based adjuvants. (A) DCs (1×106 cells/mL) and (B) splenocytes (5×106 cells/mL) were either treated with different concentrations of emulsions (-●- SqE, -○- SqA; 0-1,000 nL/mL) for 16 h or incubated with 1,000 nL/mL of emulsions for the indicated time courses (0-16 h). After incubation, the cells were harvested, stained with Annexin V/PI and analyzed by flow cytometry. Apoptosis and necrosis were denoted as the percentage of Annexin V+ and Annexin V-/PI+ cells, respectively. The data are expressed as the mean ± SEM from triplicate culture. The results are representative of three independent experiments.



Figure 2. The production of ROS from emulsion-stimulated DCs and splenocytes. (A) DCs (1×106 cells/mL) and (B) splenocytes (5×106 cells/mL) were either treated with different concentrations of emulsions (-●- SqE, -○- SqA; 0-1,000 nL/mL) for 16 h or incubated with 1,000 nL/mL of emulsions for the indicated time courses (0-16 h). After incubation, the cells were harvested, stained with DCFDA and analyzed by flow cytometry to detect ROS generation. The flow cytometric histograms represent SqE-induced ROS production. The data are expressed as the mean ± SEM from triplicate culture. The results are representative of three independent experiments.


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Figure 3. Emulsion-induced ROS production and cell death were impaired by pretreatment with NAC. (A) DCs (1×106 cells/mL) and (B) splenocytes (5×106 cells/mL) were pretreated with 0-1 mM of NAC for 30 min, and then cultured in the absence or presence of emulsions (-●- SqE, -○SqA; 1,000 nL/mL) for 16 h. After incubation, the cells were harvested and stained with DCFDA or Annexin V/PI, and subjected to flow cytometry analysis. Apoptosis and necrosis were denoted as the percentage of Annexin V+ and Annexin V-/PI+ cells, respectively. The data are expressed as the mean ± SEM from triplicate culture. The results are representative of three independent experiments.



Figure 4. Cellular uptake of OVA* and emulsions by DCs. (A) Representative flow cytometric histograms of OVA* uptake in DCs. DCs (1×106 cells/mL) were incubated with a concentration (0, 10 or 1,000 nL/mL) of SqE or SqA. After incubating for 16 h, the DCs were washed with PBS and cultured in the presence of OVA* (20 µg/mL). After culturing for 1 h, the cells were washed with PBS and stained with CD11c-FITC, and the uptake of OVA* by CD11c+ DCs was analyzed by flow cytometry analysis. (B) Representative confocal microscopic images of emulsion uptake in DCs. DCs (1×106 cells/mL) were treated with DiD'- and IgG*-labeled emulsions for 4 h, followed by stained with Hoechst 33342 (5 µg/mL; blue) and LysoTrackerTM Green DND-26 (50 nM; green) for 10 min. After washed with PBS, the cells were subjected to fluorescence confocal microscope.


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Figure 5. Emulsion-treated splenocytes were required for the uptake of OVA* into DCs. Naïve splenocytes were pretreated without or with NAC (1 mM) for 30 min followed by treatment with either SqE or SqA (1,000 nL/mL). Splenocytes treated with H2O2 (50 µM) were used as a positive control. After incubation for 16 h, the splenocytes (5×105 cells/mL) were washed with PBS and co-cultured with DCs (5×105 cells/mL) in the presence of Alexa 647-conjugated ovalbumin (OVA*; 20 µg/mL) for 1 h. After free OVA* was washed away with PBS, DCs were stained with FITC-conjugated anti-CD11c antibody and subjected to flow cytometry analysis. All data shown were gated on CD11c+ cells. The change in the mean fluorescence intensity (MFI) from the group of untreated splenocytes to each treatment group is indicated. The results are representative of three independent experiments. The data are expressed as the mean ± SEM from triplicate culture. (B) Representative confocal microscopic images of emulsion uptake in DCs. Naïve splenocytes were treated with either SqE or SqA (1,000 nL/mL). After incubation for 16 h, the splenocytes (5×105 cells/mL) were washed with PBS and co-cultured with DCs (5×105 cells/mL) in the presence of OVA* (20 µg/mL) for 1 h. The cultured DCs were then stained with



Hoechst (5 µg/mL; blue) and LysoTrackerTM Green DND-26 (50 nM; green) for 10 min. After washed with PBS, the cells were subjected to fluorescence confocal microscope. i, ii, iii, and iv represent DCs cultured with OVA* alone (red), OVA* plus untreated splenocytes, OVA* plus SqE-treated splenocytes and, OVA* plus SqA-treated splenocytes, respectively.


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Figure 6. Impact of emulsion-mediated apoptotic cells on the activation of lymphoid APCs and antigen-specific splenic T-cell immunity. C57BL/6 mice (n=3 per group) were vaccinated s.c. in both hindlimbs with i.) OVA alone, or ii.) SqE-adjuvanted OVA, or iii.) SqA-adjuvanted OVA. (A) TUNEL staining images of the injection site at day 3 post-vaccination (original magnification, ×400). Brown signals indicate dead cells (black arrows); #indicates the emulsion. The positive cells were quantified and expressed as the mean ± SEM. (B) Draining LN cells were harvested on day 3 post-vaccination and stained with FITC-conjugated CD11c, PEconjugated CD40, PeCy7-conjugated CD86 and Alexa 647-conjugated MHC II antibodies. The mean fluorescence intensity (MFI) of CD40, CD86 and MHC class II on the gated CD11c+ cells were determined by flow cytometry. (C) Analysis of T-cell immunity. Seven days after the vaccination, splenocyte suspensions (5×106 cells/mL) were incubated in the presence or absence of OVA protein (10 µg/mL) for 72 h. Supernatants from the cultures were collected to measure the concentrations of cytokines IFN-γ and IL-17 by ELISA via paired antibodies. The data are presented as cytokine release in the presence of OVA minus release in the presence of medium



only. The mRNA expression levels of T-bet and RORγt were measured by RT-PCR. The data are normalized to the β-actin mRNA and graphed as the fold change over the non-adjuvanted OVA control. The results are representative of two independent experiments, with each experiment performed in triplicate. The data are expressed as the mean ± SEM from triplicate samples. * indicates a significant difference between the comparative groups (p < 0.05, one-way ANOVA). N.D.: not detected.


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Figure 7. Schematic presentation of the respective mechanism by which the unsaturated squalene oil plays a critical role in ROS-mediated antigen uptake and cellular immunity of the emulsion-based adjuvants. Following vaccination, squalene was more forceful than squalane to induce ROS production in DCs and splenocytes, leading to cellular apoptosis and necrosis. In the presence of cellular debris, antigen uptake by DCs was then promoted. Subsequently, the APCs were activated and migrated to the lymph nodes, where the antigen-loaded APCs processed the antigen into peptide epitopes for presentation to T cells through the MHC pathways.



Table of Content 88x35mm (300 x 300 DPI)


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Figure 1. Apoptosis and necrosis of DCs and splenocytes induced by emulsion-based adjuvants. (A) DCs (1×106 cells/mL) and (B) splenocytes (5×106 cells/mL) were either treated with different concentrations of emulsions (-●- SqE, -○- SqA; 0-1,000 nL/mL) for 16 h or incubated with 1,000 nL/mL of emulsions for the indicated time courses (0-16 h). After incubation, the cells were harvested, stained with Annexin V/PI and analyzed by flow cytometry. Apoptosis and necrosis were denoted as the percentage of Annexin V+ and Annexin V-/PI+ cells, respectively. The data are expressed as the mean ± SEM from triplicate culture. The re-sults are representative of three independent experiments. 203x90mm (300 x 300 DPI)



Figure 2. The production of ROS from emulsion-stimulated DCs and splenocytes. (A) DCs (1×106 cells/mL) and (B) splenocytes (5×106 cells/mL) were either treated with different concentrations of emulsions (-●SqE, -○- SqA; 0-1,000 nL/mL) for 16 h or incubated with 1,000 nL/mL of emulsions for the indicated time courses (0-16 h). After incubation, the cells were harvested, stained with DCFDA and analyzed by flow cytometry to detect ROS generation. The flow cytometric histograms represent SqE-induced ROS production. The data are expressed as the mean ± SEM from triplicate culture. The results are representative of three independent experiments. 203x110mm (300 x 300 DPI)


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Figure 3. Emulsion-induced ROS production and cell death were impaired by pretreatment with NAC. (A) DCs (1×106 cells/mL) and (B) splenocytes (5×106 cells/mL) were pretreated with 0-1 mM of NAC for 30 min, and then cultured in the absence or presence of emulsions (-●- SqE, -○- SqA; 1,000 nL/mL) for 16 h. After incubation, the cells were harvested and stained with DCFDA or Annexin V/PI, and subjected to flow cytometry analysis. Apoptosis and necrosis were denoted as the percentage of Annexin V+ and Annexin V/PI+ cells, respectively. The data are expressed as the mean ± SEM from triplicate culture. The results are representative of three independent experiments. 203x135mm (300 x 300 DPI)



Figure 4. Cellular uptake of OVA* and emulsions by DCs. (A) Representative flow cytometric histograms of OVA* uptake in DCs. DCs (1×106 cells/mL) were incubated with a concentration (0, 10 or 1,000 nL/mL) of SqE or SqA. After incubating for 16 h, the DCs were washed with PBS and cultured in the presence of OVA* (20 µg/mL). After culturing for 1 h, the cells were washed with PBS and stained with CD11c-FITC, and the uptake of OVA* by CD11c+ DCs was analyzed by flow cytometry analysis. (B) Repre-sentative confocal microscopic images of emulsion uptake in DCs. DCs (1×106 cells/mL) were treated with DiD'- and IgG*labeled emulsions for 4 h, followed by stained with Hoechst 33342 (5 µg/mL; blue) and LysoTrackerTM Green DND-26 (50 nM; green) for 10 min. After washed with PBS, the cells were subjected to fluorescence confocal microscope. 169x113mm (300 x 300 DPI)


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Figure 5. Emulsion-treated splenocytes were required for the uptake of OVA* into DCs. Naïve splenocytes were pretreated with-out or with NAC (1 mM) for 30 min followed by treatment with either SqE or SqA (1,000 nL/mL). Splenocytes treated with H2O2 (50 µM) were used as a positive control. After incubation for 16 h, the splenocytes (5×105 cells/mL) were washed with PBS and co-cultured with DCs (5×105 cells/mL) in the presence of Alexa 647-conjugated ovalbumin (OVA*; 20 µg/mL) for 1 h. After free OVA* was washed away with PBS, DCs were stained with FITC-conjugated anti-CD11c antibody and subjected to flow cytometry analysis. All data shown were gated on CD11c+ cells. The change in the mean fluorescence intensity (MFI) from the group of un-treated splenocytes to each treatment group is indicated. The results are representative of three independent experiments. The data are expressed as the mean ± SEM from triplicate culture. (B) Representative confocal microscopic images of emulsion up-take in DCs. Naïve splenocytes were treated with either SqE or SqA (1,000 nL/mL). After incubation for 16 h, the splenocytes (5×105 cells/mL) were washed with PBS and co-cultured with DCs (5×105 cells/mL) in the presence of OVA* (20 µg/mL) for 1 h. The cultured DCs were then stained with Hoechst (5 µg/mL; blue) and LysoTrackerTM Green DND-26 (50 nM; green) for 10 min. After washed with PBS, the cells were subjected to fluorescence confocal microscope. i, ii, iii, and iv represent DCs cultured with OVA* alone (red), OVA* plus untreated splenocytes, OVA* plus SqE-treated splenocytes and, OVA* plus SqA-treated splenocytes, respectively. 203x93mm (300 x 300 DPI)



Figure 6. Impact of emulsion-mediated apoptotic cells on the activation of lymphoid APCs and antigenspecific splenic T-cell im-munity. C57BL/6 mice (n=3 per group) were vaccinated s.c. in both hindlimbs with i.) OVA alone, or ii.) SqE-adjuvanted OVA, or iii.) SqA-adjuvanted OVA. (A) TUNEL staining images of the injection site at day 3 post-vaccination (original magnification, ×400). Brown signals indicate dead cells (black arrows); #indicates the emulsion. The positive cells were quantified and expressed as the mean ± SEM. (B) Draining LN cells were harvested on day 3 post-vaccination and stained with FITC-conjugated CD11c, PE-conjugated CD40, PeCy7-conjugated CD86 and Alexa 647-conjugated MHC II antibodies. The mean fluorescence intensity (MFI) of CD40, CD86 and MHC class II on the gated CD11c+ cells were determined by flow cytometry. (C) Analysis of T-cell immunity. Seven days after the vaccination, splenocyte suspensions (5×106 cells/mL) were incubated in the presence or absence of OVA protein (10 µg/mL) for 72 h. Supernatants from the cultures were collected to measure the concentrations of cytokines IFN-γ and IL17 by ELISA via paired antibodies. The data are presented as cytokine release in the presence of OVA minus release in the presence of medium only. The mRNA expression levels of T-bet and RORγt were measured by RT-PCR. The data are normalized to the β-actin mRNA and graphed as the fold change over the nonadjuvanted OVA control. The results are representative of two independent experiments, with each experiment performed in triplicate. The data are expressed as the mean ± SEM from triplicate samples. * indicates a significant difference between the comparative groups (p < 0.05, one-way ANOVA). N.D.: not detected. 203x101mm (300 x 300 DPI)


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Figure 7. Schematic presentation of the respective mecha-nism by which the unsaturated squalene oil plays a critical role in ROS-mediated antigen uptake and cellular immunity of the emulsion-based adjuvants. Following vaccination, squalene was more forceful than squalane to induce ROS production in DCs and splenocytes, leading to cellular apop-tosis and necrosis. In the presence of cellular debris, antigen uptake by DCs was then promoted. Subsequently, the APCs were activated and migrated to the lymph nodes, where the antigen-loaded APCs processed the antigen into peptide epitopes for presentation to T cells through the MHC path-ways. 101x95mm (300 x 300 DPI)


Unsaturated Squalene Content in Emulsion Vaccine Adjuvants Plays

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