DNA Immunization Perturbs Lipid Metabolites and Increases Risk of Atherogenesis Fu Yang,†,4 Shikai Yan,§,4 Fang Wang,† Ying He,† Yingjun Guo,† Qi Zhou,† Yue Wang,† Xiaoying Zhang,† Weidong Zhang,*,‡,§ and Shuhan Sun*,† Department of Medical Genetics, Second Military Medical University, Shanghai, China, School of Pharmacy, Second Military Medical University, Shanghai, China, and School of Pharmacy, Shanghai Jiaotong University, Shanghai, China Received October 14, 2007
In addition to conventional vaccination, DNA-mediated immunization has been developed as an alternative approach in the prevention and treatment of different infectious diseases, including hepatitis B. To define sets of serum protein and metabolite biomarkers that could be employed to determine the efficacy and safety of DNA vaccines, an integrated multiple systems biology approach was undertaken on mice immunized with DNA vaccine, recombinant protein, plasmid vector, and phosphatebuffered solution. Their sera were analyzed by two-dimensional electrophoresis and HPLC coupled with time-of-flight mass spectrometry. We detected an increase in phytosphingosine, dihydrosphingosine, palmitoylcarnitine, and ceramide in the sera of DNA-vaccinated mice. Several protein molecules were found to be altered in DNA-vaccinated mice, including apolipoprotein A-I precursor. Taken together, these results indicated that DNA vaccine stimulated hepatic sphingolipid synthesis, which may have altered the structure of circulating lipoproteins and promoted atherogenesis. This study also underscores the power of metabolomics and proteomics in the definition of DNA-vaccine-mediated metabolic phenotypes. Keywords: DNA immunization • metabolomics • proteomics • apolipoprotein • sphingolipid
Introduction DNA-mediated immunization has been recognized as a new approach for prevention and treatment of hepatitis B virus (HBV). Previous reports have indicated that DNA-mediated immunization in mice by a single intramuscular injection of plasmid DNA expressing HBV surface antigen (HBsAg) can induce a strong and sustained humoral response for at least 74 weeks.1,2 Many previous clinical trials have indicated that DNA vaccination is safe, and there is no evidence of systemic toxicity.3,4 However, a recent study has shown that long-term persistence of HBsAg and anti-HBs antibody in the serum of DNA-immunized mice leads to liver and kidney lesions due to the formation and deposition of circulating immune complexes.5 Additionally, some studies have demonstrated that the ease with which immune responses are raised in mice does not necessarily translate into those in some larger animals, particularly for antibody responses. The mechanism and safety * To whom correspondence should be addressed. Prof. Shuhan Sun, Department of Medical Genetics, Second Military Medical University, 800 Xiang-Yin Road, Shanghai 200433, P. R. China. Tel.: +86-21-25070331. Fax: +86-21-25070331. Email:
[email protected]. Or Prof. Weidong Zhang, School of Pharmacy, Second Military Medical University, No. 325, Guo He Road, Shanghai, 200433, P. R. China. Tel.: + 86-21-25070386. Fax: + 86-2125070386. E-mail:
[email protected]. † Department of Medical Genetics, Second Military Medical University. 4 Both authors contributed equally to the work. § School of Pharmacy, Shanghai Jiaotong University. ‡ School of Pharmacy, Second Military Medical University. 10.1021/pr700663q CCC: $40.75
2008 American Chemical Society
of DNA vaccines are insufficiently understood. Therefore, an overall appraisal of DNA vaccines is still needed. Traditionally, studies on DNA vaccines have been performed by measuring and comparing the concentrations of one or a few immunologic parameters in blood plasma/serum or tissues pre- and postvaccination.5–7 These parameters are usually preselected based on some expected hypothesis, and the aim is to either verify or disprove this hypothesis. However, as a means of unbiased global screening of physiological perturbations, this approach is limited. This means that detection of unexpected or novel mechanistic phenomena or markers is almost impossible to obtain. For this purpose, a robust method that can simultaneously quantify and identify a large number (hundreds to thousands) of molecules is needed. The integrated multiple systems biology approach has been employed in recent years and has turned out to be an efficient approach for improving our understanding of systems as a whole.8,9,13 In this paper, we present a strategy for a hypothesis-free global metabolite and protein screening in mouse serum in relation to DNA vaccination. The goals were as follows: (i) to establish and validate HPLC/MS-based metabolic differences among mice vaccinated with DNA vaccine (pVAX-s), recombinant protein (rHBsAg), plasmid vector (pVAX1), and PBS; (ii) to establish concentration ranges for different metabolite markers that were highly specific for DNA vaccine; and most importantly (iii) to identify metabolic pathways that might be disturbed by DNA vaccine. Journal of Proteome Research 2008, 7, 741–748 741 Published on Web 01/01/2008
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Figure 1. (a) Anti-HBsAg antibody induced by immunization of pVAX-s, pVAX1, rHBsAg, and PBS. Mouse sera were collected at baseline and at 3, 6, 9, and 12 weeks after initial immunization, and anti-HBsAg antibody levels were detected by ELISA. Each point represents the mean titer value. Error bars represent SEM (b) INF-γ ELISPOT response in groups of C57BL/6 mice immunized with pVAX-s, pVAX1, rHBsAg, and PBS. For all animals, splenocytes were separately restimulated in vitro with rHBsAg. The results are expressed as the mean number of INF-γ-secreting cells (spots) per 106 splenocytes. *P < 0.05.
Materials and Methods DNA Vaccine and Recombinant Protein Preparation. The plasmid pVAX-s encoding HBV small envelope protein and rHBsAg were constructed as previously described.10 Annexin B1, designated as cC1 antigen when it was first identified from Taenia solium cysticerci,11 is a 37.8 kDa cytosolic protein that consists of 346 amino acids. pcDNA-B1 and pVAX-B1 were constructed by cloning cDNA of annexin B1 protein into the EcoRI restriction sites of pcDNA3 and pVAX1 downstream of the cytomegalovirus early promoter. Plasmids used in this study were prepared using the alkaline lysis method, followed by Triton X-114 treatment to remove endotoxin. Mouse Handling and Sample Collection. All the C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China). They were bred and humanely cared for under SPF conditions in the Laboratory Animal Center of the Second Military Medical University (Shanghai China). Female mice (13 per group, 8 weeks old) were chosen at the start of three rounds of vaccination. They were fed with a certified standard diet and tap water ad libitum. Temperature and humidity were regulated at 21–22 °C and 35–15%, respectively. A light cycle of 12 h on/12 h off was established. PBS, pVAX-s, pVAX1 vector, pcDNA-B1, pVAX-B1, and rHBsAg were injected three times at 4-week intervals. Intramuscular injections of 100 µg of pVAX-s, pVAX1, pcDNA-B1, or pVAX-B1 diluted in 100 µL of PBS were administered to the in quadriceps muscle. For protein vaccination, mice were midline subcutaneously immunized with 10 µg of purified rHBsAg. No adjuvant was used because we did not want to introduce any interference for the vaccination, so that the analysis would reflect the differences only caused by the vaccines. Intramuscular injection of 100 µL of PBS was performed in the quadriceps as a negative control. At the indicated time (0, 3, 6, 9, and 12 weeks after the first injection), 100 µL of blood was obtained from the retro-orbital venous plexus under light diethyl ether anesthesia. The blood of pcDNA-B1 and pVAX-B1 vaccinated mice was only collected at 12 weeks after the first injection. These blood samples were collected into standard vials and allowed to clot at room 742
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Yang et al. temperature for 30 min, and then the serum was separated by centrifugation and stored at -80 °C until analysis. Specific Antibody Analysis and ELISPOT Assay. Total HBsAg-specific antibody was detected by ELISA (Fosun LongMarch; Shanghai, China) following its introduction. The number of antigen-specific interferon (IFN)-γ-producing cells was measured as follows. Spleens from vaccinated mice were harvested and made into single cell suspensions by filtering through a 70-µm mesh filter (Becton Dickinson, San Jose, CA, USA). Splenocytes were cultured at 5 × 106 cells per well in 24-well tissue culture dishes in RPMI 1640 medium (Biowhittaker) which contained 2.0 µg/mL of rHBsAg for 18 h. ELISPOT assays were performed using a commercially available kit (R&D Systems, Minneapolis, MN, USA). Splenocytes were plated on precoated ELISPOT plates at 1.25 × 104 to 1 × 105 CD8+ cells per well in triplicate. After incubation at 37 °C for 16 h, cells were removed and plates processed according to the manufacturer’s instructions. Resulting spots were counted with a stereomicroscope (Carl Zeiss, Thornwood, NY, USA) at × 20 to × 40 magnification. Only brown-colored spots with fuzzy borders were scored as spot-forming cells. Metabolomic Analysis. Sample Preparation. Sera were thawed before analysis. Triplicate volumes of acetonitrile were added to 80 µL of serum and shaken vigorously (30 s), and the mixture was allowed to stand for 10 min. Then they were centrifuged at 12 000 rpm for 10 min. The supernatant was filtered through a syringe filter (0.2 µm) before HPLC/MS analysis. HPLC/MS Analysis. Analysis was performed on an Agilent1200 liquid chromatography series (Agilent, MA, USA), coupled to a 6510 Q-TOF mass spectrometer (Agilent) equipped with an electrospray source. A C18 RP-ODS column (4.6 mm × 50 mm, 1.8 µm; Agilen) and a C18 guard column (4.6 mm × 7.5 mm, 3.5 µm; Agilent) were used. The mobile phases were composed of water (A) and acetonitrile (B). The gradient was as follows: 0 min, 85% A, 15% B; 6–10 min, 35% A, 65% B; 12 min, 12% A, 88% B; 18 min, 10% A, 90% B; 23–30 min, 5% A, 95% B. Elution was performed at a solvent flow rate of 0.8 mL/ min. The column compartment was kept at a temperature of 25 °C, and the sample injection volume was 2 µL. One quarter of the column effluent (0.2 mL/min) was delivered into the MS ion source. The conditions of the electrospray ionization source were as follows: positive ion mode, drying gas N2 8 L/min, temperature 320 °C, pressure of nebulizer 35 psi, capillary voltage 4000 V, fragment voltage 160 V, and scan range 50–1000 u. Multivariate Statistical Analysis. Metabolite profiling detected by liquid chromatography coupled with Q-TOF spectrometry produces complex data sets that require significant preprocessing before multiple samples can be analyzed statistically. The data were preprocessed as follows: first, an MHD format file for each sample, which included information on mass value, retention time, and peak area, was generated by a molecular feature extraction algorithm; then, all the MHD files were imported to GeneSpring software (Agilent). Retention time alignment and peak matching algorithm were applied for all sample sets. Finally, the results were exported as a CSV file, which included information on mass value, retention time, and peak area across all samples. The preprocessing results were read into the MATLAB platform for further analysis. One-way analysis of variance was applied to pick out significant variables among different groups. Then, principle component analysis (PCA), which is widely
DNA Immunization Effects on Lipid Metabolites and Atherogenesis
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Figure 2. Scores (a) and their corresponding loadings (b) plots of PCA performed on the HPLC/QTOF/MS profile of sera of mice from immune groups of PBS (up triangles filled with magenta), pVAX1 (down triangles filled with blue), protein (diamonds filled with red), and pVAX-s (squares filled with green). Scores (c) and their corresponding loadings (d) plots of PCA performed on the HPLC/QTOF/MS profile of sera data of mice from various sampling times of 0 week (up triangles filled with magenta), 3 weeks (squares filled with green), 6 weeks (down triangles filled with blue), and 9 weeks (diamonds filled with red) after the first injection. Scores (e) and loadings (f) plots of PCA performed on groups of pcDNA-B1 (magenta up triangles), PBS (green squares), and pVAX-B1 (blue down triangles) immunized mice. Significant variables responsible for this separation were found with the aid of Hotelling’s T2 test, and these variables were labeled with retention time (min) and mass value in the loadings plot.
applied in metabolomic studies, was utilized to extract and display the systematic variation in the data set. By means of PCA, an overview of all samples can be visualized in the scores plot, and some significant metabolites can be statistically identified in the loadings plot by Hotelling’s T2 test. Prior to performing PCA, CS (normalization to a constant sum) and pareto scaling were used. To validate the biomarkers in which we were interested, standards of ceramide, phytosphingosine, dihydrosphingosine, and palmitoycarnitine were subjected to LC/MS/MS under the same conditions. The result was eventually confirmed by comparing the retention times and spectra between samples and standards. Standards were purchased from Sigma-Aldrich (USA). Proteomic Analysis. Two-dimensional electrophoresis (2DE), image analysis, and protein identification were performed as described previously.12
Results Specific Antibody Analysis and ELISPOT Assay. To assess the efficacy of injected pVAX-s and rHBsAg in inducing a humoral immune response, anti-HBsAg antibody in the sera was detected by ELISA at baseline and at 3, 6, 9, and 12 weeks after the first injection (Figure 1a). Titers of the antibody to HBsAg in the pVAX-s- and rHBsAg-vaccinated mouse sera
began to increase on day 21 postimmunization and reached a maximum at the end of the observation period. To explore whether immunization with pVAX-s plasmid DNA and rHBsAg induced a CTL response, we detected by ELISPOT assay the presence of IFN-γ-producing lymphocytes in the spleen. The rHBsAg was used as a specific antigen (Figure 1b). Based on data comparison for all groups, it was ascertained that pVAX-s was capable of inducing a specific CTL response in immunized animals. These results demonstrated that pVAX-s induced both HBVspecific antibody and T-cell responses to HBsAg in mice. Specific antibody and T-cell responses to annexin B1 were also induced in our mice (data not shown). Metabolomic Analysis. In this study, HPLC/MS detected numerous metabolites in the serum extracts. Approximately 2000 peaks (defined by a pair of m/z value and retention time) were resolved for each serum sample using the above method. The PCA scores plot of the pVAX-s-, pVAX1-, PBS-, and rHBsAgvaccinated groups could be readily divided into four clusters (Figure 2a), which suggests that different immunogens cause different metabolic phenotypes in serum. To investigate the progression of some of these metabolic features with immunization, a time-course phenotype study was performed in pVAX-s-vaccinated mice. The PCA scores of pVAX-s-vaccinated mice demonstrated a time dependency of the serum metabolic Journal of Proteome Research • Vol. 7, No. 2, 2008 743
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Table 1. Postulated Metabolites Associated with Different Vaccinate Approaches MS fragments* chemical no.
retention time (min)
MS1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
9.353 3.2910 8.6140 9.5830 14.0580 14.2460 10.4670 13.677 13.325 0.892 13.9810 6.6650 7.4570 7.076 9.353
524.3648 316.3139 318.3932 288.2829 330.3297 302.2985 149.0158 400.3405 443.3274 188.0628 122.0899 415.2060 118.0795 531.2702 467.0937
a
MS2
184.0729, 86.0967 298.3079, 106.0901 300.2866, 257.2759 169.2143 312.3256, 268,2463 284.2897, 95.0967 122.0368 382.3850, 313.3059 403.34070, 261.1243 143.1167, 84.0818 105.0654 135.0788, 198.0892 74.0902 385.1394, 146.9810 441.1366, 311.0862
Confirmed by standard retention time and spectra.
b
identity
formula
stearoylglycerophosphocholine decanoylcarnitine phytosphingosine prosyclidine Val Val Leu dihydrosphingosinea citramalic palmitoylcarnitinea testosterone decanoate indoleacrylic acid phenylethylamine diltiazem valine TrpTyrTyr (p-azidophenacyl)glutathione
C26H54NO7P C17H33NO4 C18H39NO3 C19H29NO C16H31N3O4 C18H39NO2 C5H8O5 C23H45NO4 C29H46O3 C11H9NO2 C8H11N C22H26N2O4S C5H11NO2 C20H20N2O6 C18H22N6O7S
mass
ratiob
523.3648 0.0125 315.2409 0.0519 317.2939 3.3367 287.2249 133.8859 329.2315 0.1872 301.2981 3.388 148.0372 0.2764 399.3349 2.369 442.3447 1.0456 187.633 1.2925 121.0891 1.0425 414.1613 1.0250 117.0790 3.203 530.2166 3.1975 466.1271 0.879
The ratio of relative amounts of group pVAX-s to group PBS.
Figure 3. (a) Tandem MS of the quasimolecular ion: 302.2985. (b) Possible fragmentation mechanism of dihydrosphingosine and MS of the commercial standard dihydrosphingosine.
phenotype (Figure 2c). Corresponding loadings plots (Figure 2b and Figure 2d) revealed potential biomarkers that contributed most to the separation of groups. Twenty-six potential biomarkers were selected out by Hotelling’s T2 test. The identification of biomarkers has been fully described in our previous study.13 In the present study, 15 of these biomarkers were structurally proposed (shown in Table 1). Dihydrosphingosine and palmitoycarnitine were confirmed by standards (Figure 3). Among these markers, phytosphingosine, dihydrosphingosine, and palmitoycarnitine were mapped into the pathway 744
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of sphingolipid metabolism (Figure 4). Moreover, as shown in Figure 5 the concentration of dihydrosphingosine and palmitoycarnitine increased along with the process of DNA vaccine inoculation and reached a peak at the 12th week. All these results indicated that pVAX-s inoculation promoted sphingolipid metabolism. To identify whether the perturbation of sphingolipid metabolism was universal to DNA vaccination, we performed metabolomic analysis on pcDNA-B1-, pVAX-B1-, and PBSvaccinated mouse serum. The scores and loadings plots of PCA are given in Figure 2e and f. From the scores plots, it was found
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spots that were identified successfully). Numbered spots were excised and subject to in-gel tryptic digestion. Proteins identified by MALDI-TOF MS are listed in Table 3. According to previous studies, DNA vaccines may induce myocyte injury in inoculated quadriceps. Histological analysis of muscle 12 weeks after DNA immunization demonstrated significant inflammatory infiltration in inoculated quadriceps (data not shown).
Discussion
Figure 4. Scheme illustrating sphingolipid biosynthesis and metabolism. For details, please refer to the text. Levels of phytosphingosine, dihydrosphingosine, palmitoylcarnitine, and ceramide increased visibly in our metabolomic data.
that the three groups were clearly separated from each other. Those potential biomarkers that contributed mostly to the separation of groups were also identified (Table 2). It was interesting that palmitoylcarnitine and ceramide were among those biomarkers. Proteomic Analysis. Average 2-DE gels for pVAX-s- and PBSimmunized mouse sera were obtained from three different animals per group. Approximately 800 spots were detected in the silver-stained gels using ImageMaster (Amersham USA), most of which were reproducible. The overall spot patterns were largely similar between the two groups. The proteins in the high-molecular-weight region of the 2-DE gels could not be separated clearly. In a selected medium and low-molecularweight region, a mean of 500 spots were matched. Compared with the control group, 13 spots in the pVAX-s mice were demonstrated with a relative concentration changed of more than 3-fold, including eight up-regulated and five downregulated proteins. Enlarged silvers-stained gels highlight quantitative differences in Figure 6(here we only showed the
DNA vaccine pVAX-s induced both specific antibody and T-cell responses to HBsAg in mice. Specific antibody and T-cell responses to annexin B1 were also induced in our experiment mice (data not show). We found that phytosphingosine, dihydrosphingosine, decanoylcarnitine, palmitoylcarnitine, and ceramide increased obviously in sera of DNA-vaccinated mice. Additionally, we also found that apolipoprotein A-I precursor increased markedly in the sera of pVAX-s- compared with PBSimmunized mice. All of these are involved in the pathways of lipid metabolism. Apolipoprotein A-I contains several 22-residue repeats, which form a pair of R helices. This family includes: apolipoproteins A-I, A-IV, and E. In addition to their role in lipid transport, they have in vitro immunomodulatory properties.14,15 Palmitoycarnitine, a zwitterionic lipid derivative, is synthesized in cell from carnitine and palmitoyl-CoA due to the activity of palmitoycarnitine transferase I. Also palmitoycarnitine exits in serum with the style of binding with apolipoprotein. Therefore, it was not unexpected that we discovered fatty acyl carnitine and apolipoprotein increased in the sera of pVAX-s-immunized mice at the same time (we also found that apolipoprotein M increased in the serum of pcDNA-B1-immunized mice12). This means that the lipid metabolism was disturbed by DNA vaccine. Other studies have confirmed that altering the availability of exogenous and/or endogenous lipids affects antigen processing and/or presentation.16 The increase of apolipopro-
Figure 5. Panel b shows that the relative concentration of palmitoylcarnitine increased in the pVAX-s compared with other immunization groups, and its concentration increased gradually with the time course after the first injection of pVAX-s (a). The variation in relative concentration of dihydrosphingosine is shown in (c) and (d), which is very similar to that of palmitoylcarnitine. *P < 0.05. Journal of Proteome Research • Vol. 7, No. 2, 2008 745
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Table 2. Postulated Metabolites Associated with Different Vectors of DNA Vaccine MS fragments chemical no.
retention time (min)
MS1
1 2 3 4 5 6
7.86 7.41 7.88 9.84 7.95 29.26
496.3320 520.3320 518.3130 400.3332 522.3475 429.3651
478.3280, 419.2800 502.3258, 443.2546 459.2473, 313.2726 340.8989, 385.3885 504.3467 401.3435, 347,6232
7 8 9 10
0.90 1.43 10.08 9.70
160.0682 175.1114 426.3502 580.2822
101.0589, 83.0495 157.0682, 130.0978 338.1516 515.7773
11
5.92
356.2715
249.7341
identity
formula
mass
ratiob
ratioc
palmitoyllysophosphatidylcholine linoleoylphosphatidylcholine linolenoyllysolecithin palmitoylcarnitinea oleoylglycerophosphocholine 4-hydroxymethyl-4-methyl-5cholesta-8,24-dienol indolleacetaldehyde arginnine ceramidea 3-hydroxy-24-oxo-7-sulfooxycholanylamino ethanesulfonic acid tetracosahexaenoic acid
C24H50NO7P C26H50NO7P C26H48NO7P C23H45NO4 C26H52NO7P C29H48O2
495.3325 519.3325 517.3168 399.3330 521.3471 428.3654
4.365 3.687 1.075 2.569 0.276 1.375
2.357 4.763 2.78 3.679 0.145 1.346
C10H9NO C6H14N4O2 C26H51NO3 C26H45NO9S2
159.0684 174.1114 425.3869 579.2535
1.459 3.759 2.379 0.450
2.874 1.763 7.359 0.213
C24H36O2
355.2715
0.136
0.782
MS2
a Confirmed by standard retention time and spectra. group pcDNA-B1 to group PBS.
b
The ratio of relative amounts of group pVAX-B1 to group PBS. c The ratio of relative amounts of
Figure 6. Representative silver-stained 2-DE gels of serum proteins of pVAX-s- (left) and PBS- (right) immunized mice. Sera were collected at 12 weeks after the first immunization. Proteins (200 µg) were loaded and separated first on IPG strips with a pH range of 4–7 and then on 16 × 18-cm glass plates. The positions of identified proteins are marked by arrows. Table 3. Identification of the Differentially Expressed Proteins between the Sera of p-VAX-s and PBS Immunized Mice no.
protein
Mr
PI
1 2 3 4 5
Fetuin-A Chain-L ApoA-I precursor albumin growth factor receptor bound protein 2
37302 23882 30358 23609 23687
6.04 4.98 5.52 5.48 5.68
tein and fatty acyl carnitine may affect antigen processing and presentation, T-lymphocyte activation, inflammation, and potentially the nature and production of cytokines by Tlymphocytes in DNA-vaccinated mice. They may serve as potential surrogate markers of successful vaccination and provide targets for research on the molecular mechanisms of DNA vaccines. The biosynthesis of sphingolipid (Figure 4) is initiated by the condensation of serine and palmitoyl-CoA, which results in the formation of 3-ketosphinganine (3-ketodihydrosphingosine), which is subsequently reduced to dihydrosphingosine. The first and rate-limiting enzyme in sphingolipid synthesis is serine palmitoyltransferase (SPT), which catalyzes the condensation of serine with palmitoyl-CoA.17 Dihydroceramide is formed by the amide linkage of fatty acyl groups to dihydrosphingosine. 746
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accession no of NCBI
gi gi gi gi gi
231466 18655521 109571 26986064 123228035
fold change
sequence soverage/score
3.76 4.32 4.37 3.65 0.18
30%/68 69%95 45%/92 63%/119 36%/151
Radiolabeling and pulse-chase studies have indicated that ceramide is formed from dihydroceramide by the introduction of a trans-4,5-double bond.18 Once formed, ceramide serves as a precursor for all other complex sphingolipids, such as galactosylceramide, glucosylceramide, and sphingomyelin. In this metabolic network, we demonstrated an increase in phytosphingosine, dihydrosphingosine, palmitoylcarnitine, and ceramide after vaccination with pVAX-s, pcDNA-B1, and pVAXB1. More importantly, the concentration of dihydrosphingosine and palmitoylcarnitine was increased by DNA vaccination. This result illustrated that DNA vaccine facilitated the process of sphingolipid biosynthesis, no matter what the antigens and plasmid vectors were. According to the previous studies, DNA vaccines may induce myocytes injury in inoculated quadriceps or long-term lesions
DNA Immunization Effects on Lipid Metabolites and Atherogenesis 19,20
of the liver and kidney in immunized mice. In our histological analysis of DNA-vaccinated mice, hematoxylin and eotin-stained muscle sections demonstrated significant inflammatory infiltration in inoculated quadriceps (data not shown). Metabolism of sphingolipids can be altered during inflammation. LPS stimulates hepatic ceramide and sphingomyelin synthesis by increasing mRNA expression and activity of SPT.21 It is reasonable to suppose that inflammatory infiltration in inoculated quadriceps increases mRNA expression and activity of SPT, which up-regulates hepatic sphingolipid synthesis. An increase in these sphingolipid levels in lipoproteins can have a number of consequences that may increase the atherogenicity of lipoprotein particles.22–24 For example, it has been shown that increased ceramide levels in low-density lipoprotein facilitate its aggregation, which in turn enhances its uptake by macrophages and leads to foam-cell formation.25 Moreover, sphingomyelin inhibits the activity of Lecithin-cholesterol acyltransferase, which may decrease the reverse cholesterol transport pathway, thereby increasing the risk of atherogenesis.26,27 Taken together, these findings support the concept that DNA-vaccine-induced inflammatory infiltrations in quadriceps muscle stimulate hepatic sphingolipid synthesis, which results in an altered structure of circulating lipoproteins and may increase the risk of atherogenesis. It may be that the increase in sphingolipid synthesis in the liver is a safety concern of DNA vaccines. Moreover, metabolites of sphingolipids, particularly those of sphingomyelin catabolism (ceramide, sphingosine, and sphingosine-1-phosphate), are also bioactive lipids that mediate essential biological functions such as chemotactic motility, calcium homeostasis, cell growth, cell death, and differentiation.28,29 It is therefore possible that the DNA-vaccine-induced increase in hepatic sphingolipid synthesis provides additional substrates (i.e., sphingomyelin and ceramide) for these cytokine signaling pathways. Other studies have emphasized the importance of sphingolipids in additional immune cell processes such as differentiation of monocytes into macrophage or granulocyte lineages, regulation of apoptotic cell death, and survival of lymphocytes and macrophages.30,31 Generally, it can be concluded that sphingolipids and metabolites can intervene in the regulation of multiple aspects of immune cell function.32,33 We believe that the changes of phytosphingosine, dihydrosphingosinea, palmitoylcarnitine, and ceramide in our study provide an opportunity to elucidate the obscure role of DNA vaccines. In conclusion, we identified tens of metabolites and several proteins in mice inoculated with DNA vaccines by using a strategy of integrating HPLC/MS-based metabolomics and 2DE-based proteomics. The results gave a better understanding of the physiological changes in mice inoculated with DNA vaccines, which may be important for understanding the mechanism and safety of DNA vaccines. With this method, we are able to obtain more information from the samples and to identify potential biomarkers. We believe that metabolomics and proteomics are powerful tools in the area of DNA vaccine research.
Acknowledgment. This work was funded by the Key Program (no. 30530660) from the National Natural Science Foundation of China, the Basic Research Program (no. 04JC14004, 06QA14064) from the Science and Technology Commission of Shanghai, the Program for Changjiang Scholars and Innovative Research Team in University
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(PCSIRT), and the National High Technology Research and Development Program (863 Program, no. 2006AA02Z338).
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