Atherosclerosis 184 (2006) 330–341

Atheroprotective effect of adjuvants in apolipoproteinE knockout mice

J. Khallou-Lascheta,1, E. Tupina,f,1, G. Caligiurib, B. Poiriera,N. Thieblemontc, A.-T. Gastona, M. Vandaelea, J. Bletone, A. Tchaplae,

S.V. Kaveria, M. Rudlingd, A. Nicolettia,∗a INSERM U681, Institut Biomedical des Cordeliers, 15, rue de l’Ecole de Medecine, Paris, France

b INSERM E00-16, Faculte de Medecine Necker-Enfants Malades, Paris, Francec CNRS FRE2444, Universite Rene Descartes, Paris, France

d Molecular Nutrition Unit, Novum and CME, Department of Medicine, Karolinska University Hospital at Huddinge, Swedene LETIAM, Groupe de Chimie Analytique de Paris Sud, EA 33-43, Orsay, France

f Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden

Received 24 September 2004; received in revised form 6 April 2005; accepted 27 April 2005Available online 26 July 2005

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Strategies aimed at treating atherosclerosis by immunization protocols are emerging. Such protocols commonly use adjuvapecific stimulators of immune responses. However, adjuvants are known to modify various disease processes. The aim of this setermine whether adjuvants alter the development of atherosclerosis.We performed immunization protocols in apolipoprotein E knockout mice (E◦) following chronic administration schedules commo

mployed in experimental atherosclerosis. Our results point out a dramatic effect of several adjuvants on the development of athehree of the four adjuvants tested reduced lesion size. The Alum adjuvant, which is the adjuvant currently used in most vaccination humans, displayed a strong atheroprotective effect. Mechanisms accounting for atheroprotective effect of Freund’s adjuvants inapacity to increase both Th2 responses and anti-MDA-LDL IgM titers, and/or to impose atheroprotective lipoprotein profiles.The present study indicates that adjuvants have potent atheromodulating capabilities, and thus, implies that the choice of adjuva

n long-term immunization protocols in experimental atherosclerosis.2005 Elsevier Ireland Ltd. All rights reserved.

eywords: Immunology; Antibodies; Cytokines; Th balance; Mineral oil

. Introduction

Strategies aimed at evaluating immunization protocols inxperimental atherosclerosis are emerging. Adjuvants aresed as non-specific stimulators to enhance the immuneesponses of immunogens that otherwise would lead to a slownd inefficient process. Adjuvants prevent the catabolism of

he combined antigen and attract appropriate immune cells to

∗ Corresponding author. fax: +33 1 55 42 8262.E-mail address: antonino.nicoletti@upmc.fr (A. Nicoletti).

1 These authors contributed equally to this work.

interact with the immunogen and with each other. Howevis known that adjuvants can per se play a role in various plogical processes[1,2]. The present study aimed to evaluwhether adjuvants modulate atherogenesis.

Freund’s adjuvants[3] are most commonly used becaof their stronger and longer lasting immunogenic respas compared to other adjuvants. They consist of tiny droof water (in which immunostimulants can be incorporastabilized by a surfactant (such as mannide monooleatecontinuous phase of mineral oil or other oils, such as squor squalane. The complete Freund’s adjuvant (CFA)tains killed cells ofMycobacterium tuberculosis while the

021-9150/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.atherosclerosis.2005.04.021

J. Khallou-Laschet et al. / Atherosclerosis 184 (2006) 330–341 331

incomplete Freund’s adjuvant (IFA) does not. CFA promotesboth antibody formation and delayed-type hypersensitivityreactions, while IFA is used to promote only antibody forma-tion.

Alum adjuvants are used as an alternative to Freund’s adju-vants because they have less adverse effects. Alum adjuvantsconsist of aluminum hydroxide or aluminum phosphate-hydrated gels that have the ability to adsorb protein antigensfrom an aqueous solution. AlhydrogelTM (aluminum hydrox-ide gel, referred to as Alum) has been elected as researchstandard in order to minimize inconsistency due to variabil-ity between individual preparations. Alum adjuvants maypresent adsorbed soluble antigens to immunocompetent cellsin a ‘particulate’ form, which would facilitate their uptake.Alum activates complement, attracts eosinophils to the injec-tion site, stimulates the production of IgE but is inefficient ininducing a delayed-type hypersensitivity response[4].

Bacterial DNA, but not vertebrate DNA, has directimmunostimulatory effects on leukocytes in vitro[5,6]. Thisactivation is due to unmethylated CpG dinucleotides[7] thatare present at the expected frequency in bacterial DNA butare under-represented and methylated in vertebrate DNA[8]. CpG DNA induces proliferation of 95% of B cells andtriggers Ig secretion. CpG DNA also directly activates mono-cytes, macrophages, and dendritic cells to secrete a varietyof cytokines.

luma inis-t kout( nd’sa havec ectede colsi vanta roto-c ionp at canb pro-t

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injections of IFA (n = 4) every week. Control mice receivedinjections of PBS (n = 5). Mice were sacrificed at 11 weeksof age.

In the second experiment, mice received i.p. injections ofCFA (n = 3), IFA (n = 3) or PBS (n = 3) once a week.

In the third experiment, mice were injected i.p., everysecond week, with Alum (Superfos, Biosector, Denmark)(30�l/mouse;n = 7), the oligonucleotide CpG 1826 (OperonQuiagen, Alameda, USA; synthesized with a nuclease-resistant phosphorothioate backbone) (500�g/mouse;n = 8)or PBS (n = 9).

2.1.2. Protocol #2Mice were injected i.p. once a week with IFA-OVA (n = 7),

CFA-OVA (n = 7) (100�g OVA/mouse), IFA-PBS (n = 7) orCFA-PBS (n = 7). A group of mice was left untouched (Ctrl)(n = 7).

2.1.3. Protocol #3Mice were injected i.p. once a week with IFA-PBS (n = 7),

or IFA components: mannide monooleate (MMO, ACROS;4.5�l/mouse;n =8) or mineral oil (25.5�l/mouse;n = 7). Themode of preparation of mineral oils makes each lot unique.We arbitrarily chose the mineral oil ref 415080010 fromACROS. The amount of MMO and mineral oil used wasproportional to their respective amount in the IFA prepara-t ;n sc

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Totals g aB einp hro-m an-d uslyd d toc roll

anti-tB oxi-m inedb n/oilr 0f s thep d byl ur-fW rorsc d too ions[

In the present study, we found that Freund’s and Adjuvants exert potent atheroprotective effects when adm

ered to the atherosclerosis-prone apolipoprotein E knocE◦) mice. We have identified the components in Freudjuvant endowed with atheroprotective capacity andharacterized mechanisms that may explain their unexpffect on atherosclerosis. In addition, immunization proto

n experimental atherosclerosis employ a number of adjudministrations for a long period of time. Because such pols differ significantly from the conventional immunizatrotocols, we have determined the immune response the expected from repeated and long-term immunization

ocols.

. Materials and methods

.1. Injection protocols

In all the protocols, 6–7-week-old E◦ male mice from oureeding facility were used. Mice were maintained on alar chow supplemented with 0.15% cholesterol. Adjuvlone or associated with an antigen (50/50, v/v) were inje

n a volume of 100�l per mouse. Mice were kept in standonditions and were sacrificed at 16 weeks of age (exhen indicated). All experiments were approved by our in

utional ethical committee.

.1.1. Protocol #1In the first experiment of this series, mice received a

utaneous injection of CFA followed by intraperitoneal (i

ion. An additional group of mice received LPS (5�l/mouse= 8). A group of untouched animals (n = 6) was used aontrol.

.2. Serum measurements and quantitation oftherosclerotic lesions

Mice were bled and sacrificed under anesthesia.erum cholesterol (TC) level was measured usinoehringer Mannheim kit (CHOD-PAP). The lipoprotrofiles were determined by fast performance liquid catography (FPLC) on the indicated number of romized serum samples from each group as previoescribed[9]. The areas under the curves were useompute the chylomicron/VLDL, LDL and HDL cholesteevels.

The aortic root was dissected and lesions were quated by a blinded observer as previously described[10].riefly, serial cryostat sections were cut from the pral 1 mm of the aortic root. Lesion size was determy computer-assisted morphometry on four hematoxylied-O-stained sections cut at 200, 400, 600, and 80�mrom the cusp origin. Lesion extension is expressed aercentage of the surface area of the aorta occupie

esions and is calculated with the following formula: [(Sace Area of Lesion)/(Surface Area of the Aorta)]× 100.

e have previously shown that this minimizes eraused by oblique sections that could otherwise leaverestimation of the surface area occupied by les10].

332 J. Khallou-Laschet et al. / Atherosclerosis 184 (2006) 330–341

2.3. Cell culture

Spleen cells were prepared as described[10]. Five hun-dred thousand mononuclear cells/well were cultured at37◦C, 5% CO2 into 96-well flat bottom plates (tripli-cates),±concanavalin A (0.5–2.5�g/ml, Sigma),±OVA(50–200�g/ml). After 48 h, 1�Ci of 3H-thymidine wasadded to each well. After another 24 h of incubation, incor-porated3H-thymidine was counted in a Microbeta counter.Results are expressed as cpm.

3. ELISA

3.1. Cytokines

Serum IFN�, IL-12, IL-10, IL-6, IL-4, and IFN� and IL-4from the 48 h supernatants of spleen cell cultures were titratedby ELISA using Pharmingen OptEIA sets. Serum amyloid A(SAA) was tested with a solid-phase sandwich mouse ELISAkit (PhaseTM Range, Tridelta Development Ltd.).

3.2. Antibodies

Total IgG and IgM and specific antibodies to malondi-aldehyde (MDA)-modified LDL and to OVA were quantifiedu ′m IgM( ed asc dies,Owm tor,4 erE cia).M df d thef darya anti-m min-g atica ted

3o

OSr in0 ctedi d ofa d byd rom-e witha ith0 x-a re of

85 kPa. The injector and transfer line temperatures were setto 300 and 250◦C, respectively. Samples (2�l) were injectedin splitless mode (30 s) and the oven programmed as fol-low: 50◦C during 1 min, 50–300◦C at 10◦C/min and 10 minat 300◦C. The operating conditions for EI-MS were sourcetemperature at 150◦C, filament emission current of 750�A,ionising voltage of 70 eV and scan range fromm/z 29 tom/z650 with a periodicity of 1 s.

3.4. Statistical analysis

Results are expressed as means± S.E.M. Simple regres-sion and ANOVA were performed using Statview 5.0 soft-ware (SAS Institute Inc., USA). Differences between groupswere considered significant ifp < 0.05.

4. Results

4.1. Atheroprotective effect of various adjuvants

In order to compare the effect of various adjuvants (IFA,CFA, Alum, and CpG), three experiments were performed(Protocol #1). Animals repeatedly injected with IFA, CFA orAlum adjuvants presented a much less severe disease thant rf te ofa pro-t ivelyi

erol( IFA( pro-t lum

Fc att lesion( on oilr root.B .

sing ELISA. For determination of total Ig, F(ab)2 frag-ents of anti-mouse IgG (1/500; Pierce) or anti-mouse

Pharmingen) were coated on microtiter plates and usapture antibodies. For determination of specific antiboVA (20�g/ml), native (10�g/ml) or MDA-LDL (10 �g/ml)ere coated overnight at 4◦C. LDL were isolated from E◦ouse blood by ultracentrifugation (Beckman 70.1Ti ro5,000 rpm for 18 h at 4◦C). Native LDL was obtained aftDTA removal with a sephadex PD10 column (PharmaDA-LDL was obtained as described[11]. Sera were plate

or 2 h at room temperature, plates were washed, anollowing alkaline phosphatase (AKP)-conjugated seconntibodies were incubated for 1 h at room temperature:ouse IgG1, anti-mouse IgG2a, anti-mouse IgM (Pharen), and anti-mouse IgG (Calbiochem). The AKP enzymctivity was revealed by adding thep-nitrophenylphosphaisodic salt. Plates were read at 405 nm.

.3. Gas chromatography–mass spectrometry analysisf IFA and mineral oil

The two samples, a drop (12.6 mg) of mineral oil (ACRef 41508) and a drop (11.3 mg) of IFA, were diluted.5 ml of hexane (SDS) 99% for trace analysis and inje

n the chromatograph. The GC–MS system consisteHP5890 (Hewlett Packard) chromatograph interface

irect coupling to an INCOS 50 quadrupole mass spectter (Finnigan). The gas chromatograph was equipped30 m× 0.25 mm i.d. fused-silica J&W column coated w.25�m film of DB-5 poly (5% phenyl, 95% methyl silone). The carrier gas was helium with a head pressu

heir respective control littermates (Fig. 1). It became clearom the three experiments of Protocol #1 that the roudministration had no impact with regard to the athero

ection conferred by adjuvants. Thus, they were exclusnjected i.p. in all the subsequent experiments.

A statistically significant decrease in total cholestTC) level was detected in mice injected with CFA andTable 1), pointing to a possible mechanism for the atheroective effect of the Freund’s adjuvants. However, while A

ig. 1. Effect of adjuvants on aortic lesions (Protocol #1). E◦ mice receivedhronic administrations of adjuvants (see Section2) and were sacrificedhe age of 11 weeks (Exp #1) and 16 weeks (Exp #2 and #3). Extent of%) development was analyzed by computer-assisted morphometryed-O-stained and hematoxylin counter-stained sections of the aorticars represent the mean± S.E.M. per group (** p < 0.01;* p < 0.05 vs. Ctrl)

J. Khallou-Laschet et al. / Atherosclerosis 184 (2006) 330–341 333

Table 1Effect of adjuvants on body weight and total cholesterol (TC) levels

TC (mg/dl) r2 (TC vs. SAL) Weight (g) r2 (weight vs. SAL)

Protocol #1Exp #1

Ctrl nd nd}

nd25.7± 1.4 0.4 (p = 0.2574)

}0.5 (p = 0.0462)

IFA/CFA nd nd 32.7± 1.0** 0.6 (p = 0.4132)

Exp #2Ctrl 658.9± 71.5 0.7 (p = 0.1512)

}0.6 (p = 0.0800)

29.8± 1.2 0.9 (p = 0.0520)}

0.4 (p = 0.0535)IFA 532.6± 23.5* 0.2 (p = 0.3182) 28.8± 0.3 0.0 (p = 0.8250)CFA 496.7± 32.1* 0.1 (p = 0.5489) 32.0±0.6* ,†† 0.1 (p = 0.5812)

Exp #3Ctrl 601.2± 51.2 0.3 (p = 0.4150)

}0.1 (p = 0.3097)

29.6± 1.1 0.7 (p = 0.0492)}

0.3 (p = 0.0477)Alum 581.3± 65.9 0.4 (p = 0.2301) 28.5± 1.9 0.2 (p = 0.635l)CpG 623.0± 62.7 0.1 (p = 0.7915) 30.2± 1.6 0.5 (p = 0.3221)

Protocol #2Ctrl 588.9± 43.3 0.3 (p = 0.2313)}

0.4 (p = 0.0003)

29.3± 0.8 0.1 (p = 0.5677)}0.0 (p = 0.3221)

CFA-OVA 451.3± 36.0** 0.2 (p = 0.3545) 28.0± 1.0 0.0 (p = 0.7166)CFA-PBS 378.7± 27.1** 0.4 (p = 0.1364) 29.7± 1.0 0.0 (p = 0.7826)IFA-OVA 482.9± 16.2** 0 3 (p = 0.2487) 30.6± 0.8 0.1 (p = 0.6587)IFA-PBS 487.4± 33.8** 0.0 (p = 0.9931) 29.4± 1.2 0.4 (p = 0.2052)

Protocol #3Ctrl 503.7± 11.6 0.0 (p = 0.6484)}

0.0 (p = 0.2374)

32.7± 0.9 0.0 (p = 0.9903)}0.0 (p = 0.9869)

IFA 357.4± 22.9** 0.7 (p = 0.0358) 30.6± 0.6 0.3 (p = 0.3035)Mineral oil 426.2± 44.3* 0.0 (p = 0.9748) 33.1± 0.7 0.2 (p = 0.3331)MMO 518.0± 34.3†† 0.1 (p = 0.4842) 30.0± 0.9* 0.0 (p = 0.8788)LPS 526.7± 25.9†† 0.6 (p = 0.0165) 31.8± 1.3 0.0 (p = 0.6394)

r2 (TC vs. SAL): coefficient of regression between total cholesterol and the surface area of lesion;r2 (weight vs. SAL): coefficient of regression betweenbody weight and the surface area of lesion. Braces: regression coefficients were also computed on merged groups within each experiment. For the correlationsstatistically significant or close to the statistical significance, numbers in bold indicate a positive correlation, and those in italic, a negative correlation. nd: notdetermined;* p < 0.05,** p < 0.01 vs. Ctrl;†p < 0.05,††p < 0.01 vs. IFA.

was also atheroprotective, it did not reduce TC. Furthermore,a regression analysis indicated that there was no statisticallysignificant correlation between TC level or weight and lesionsize (Table 1), suggesting that the atheroprotection observedfor the Freund’s adjuvants was independent of their effectson TC. Also, in the case of CFA, mice indeed gained weight(Table 1).

The CpG-injected mice displayed lesion surface area inthe same order as the untreated mice (Fig. 1; Exp #3), indi-cating that this adjuvant, unlike all the Freund’s and Alumadjuvants, did not induce any modification in the diseaseprocess. Successive experiments were designed to clarifythe atheroprotection conferred by the widely used Freund’sadjuvants.

4.2. Immune profiles imposed by adjuvants

While it is classically admitted that CFA induces a Th1bias and IFA a Th2 bias[12], the effect of repeated adjuvantinjections on the Th polarization patterns is not known. A dis-tinct Th polarization pattern can be of significant importancefor atherosclerosis[13]. We, thus, assessed whether Freund’sadjuvants could exert protection through modulation of theTh balance.

New groups of mice received chronic administration ofIFA or CFA, with or without ovalbumin as a model antigennot related to atherosclerosis (Protocol #2). As shown by theresults of Protocol #1 (Fig. 1), both IFA and CFA injectionsled to a potent atheroprotective effect (Fig. 2), again associ-ated with a reduction in TC, still not correlated with lesionsize (Table 1). As expected, the presence of OVA did notmodify the Freund’s adjuvant protection (Fig. 2).

4.2.1. Ig profilesMice injected with OVA + Freund’s adjuvant showed

an increase in IgG1, IgG2a, and IgM antibodies directedagainst OVA as compared to animals that were injected withPBS + Freund’s adjuvant or to untouched animals (Fig. 3a).All the groups exhibited similar titers of total IgG and IgM.

Since the humoral immune response directed againstoxidized-LDL might be atheroprotective[14,15], weassessed whether adjuvant injections modify the titer of anti-bodies directed against MDA-LDL. The IgG and IgM reactiv-ities against nLDL were low and similar in all groups (Fig. 3b,insets and data not shown). The titer of anti-MDA LDL IgGwas increased in the CFA-OVA and CFA-PBS-injected mice(Fig. 3b, left). Interestingly, both CFA- and IFA-injected micehad increased anti-MDA-LDL IgM as compared to control

334 J. Khallou-Laschet et al. / Atherosclerosis 184 (2006) 330–341

Fig. 2. Effect of IFA and CFA± OVA on development of lesions (Protocol #2). Mice received an i.p. injection per week of IFA or CFA, with or withoutovalbumin (OVA). Extent of lesion development was analyzed as explained in theFig. 1 legend. The micrographs are representative of the lesions observed ineach group in the aortic root at 400�m from the cusps origin (*** p < 0.001 vs. Ctrl).

Fig. 3. Effect of IFA and CFA± OVA on the Th balance and Ig isotype (Protocol #2). (a) Serum titers of IgG and IgM (at dilutions indicated in parenthesis)were measured by ELISA. (b) Serum titers of IgG and IgM specific for mouse MDA-modified LDL was measured by ELISA. In the insets are shown the IgG(left) and IgM (right) reactivities to MDA-LDL (full line) and to native LDL (broken line) in control mice (*** p < 0.001;** p < 0.01;* p < 0.05 vs. Ctrl; threerandomized samples per group were used for ELISAs in (a) and (b)).

J. Khallou-Laschet et al. / Atherosclerosis 184 (2006) 330–341 335

Fig. 4. In vitro proliferative response and cytokine production (Protocol #2). Spleen cells were cultured either with increasing doses of concanavalin (ConA)or ovalbumin (OVA) for 48 h. Fifty microlitres of supernatant were harvested from each well and3H-thymidine was added. (a) Thymidine incorporation wastested after additional 18 h of culture. (b) IFN� and IL-4 expression levels were evaluated by ELISA on the supernatants harvested at 48h on non-stimulatedcontrol cultures (non-stimulated) or stimulated with the highest dose of OVA (200�g/ml) (** p < 0.01;* p < 0.05 vs. Ctrl;††p < 0.01;†p < 0.05 vs. IFA;n = 7 pergroup).

mice (Fig. 3b, right). This increase was independent of thepresence of OVA.

In mice of the Exp #3 of the Protocol #1, there was no dif-ference for the anti-MDA LDL IgG titers among the threegroups (Ctrl: 0.296± 0.046; Alum: 0.454± 0.066; CpG:0.339± 0.049, 405 nm absorbance± S.E.M., at 1/80 dilu-tion). Anti-MDA-LDL IgM were, however, higher in miceprotected by the Alum injections (Ctrl: 0.233± 0.032; Alum:0.404± 0.048* ; CpG: 0.245± 0.028;* p < 0.05 versus Ctrl, at1/320 dilution).

4.2.2. Cell-mediated responsesProliferative capacities of spleen cells from the different

groups were assessed in response to several stimuli. Unex-pectedly, spleen cells from adjuvant-treated mice displayeda significantly reduced proliferative response to a mitogen,concanavalin A (ConA), as compared to untreated mice(Fig. 4a, left). This reduction was more pronounced in CFA-than in IFA-treated mice. However, despite their hyporespon-siveness to ConA, spleen cells from mice immunized withCFA-OVA proliferated vigorously in response to OVA while

spleen cells from IFA-OVA-injected mice did not (Fig. 4a,right), confirming that the complete but not the incompleteformulation of Freund’s adjuvant can induce delayed-typehypersensitivity reactions.

The OVA immunization protocol was also designed to per-form in vitro detection of T cell-specific cytokines releasedupon antigenic stimulation. Spleen cell culture from CFA-OVA-immunized mice stimulated with OVA produced highamounts of IFN� and of IL-4 (Fig. 4b) indicating againthat chronic CFA administration did not impose a clear Th1polarization. Anti-OVA-specific T cells were actually biasedtowards the Th2 pathway by the IFA since spleen cells derivedfrom IFA-OVA-treated mice produced IL-4 but not IFN� inresponse to OVA (Fig. 4b).

4.3. Identification of the compounds responsible for theFA effects

Experiments described hitherto demonstrated that bothIFA and CFA conferred the same degree of protection indi-cating that theM. tuberculosis cells in CFA do not play a

336 J. Khallou-Laschet et al. / Atherosclerosis 184 (2006) 330–341

Fig. 5. Effect of LPS, MMO, mineral oil, and IFA on lesion development and on serum amyloid A levels (Protocol #3). Mice were injected i.p. once a week withvarious compounds. A group of untouched animals was used as control. (a) Extent of lesion development was analyzed as explained in theFig. 1 legend. Themicrographs are representative of the lesions observed in each group in the aortic root at 400�m from the cusps origin (**** p < 0.0001 vs. Ctrl;††††p < 0.0001;†p < 0.05 vs. IFA). (b) This graph was obtained by plotting the mean (±S.E.M.) of lesions (%) and the mean (±S.E.M.) SAA level for each group of mice.

role and that the components present in IFA are sufficientto confer atheroprotection. IFA is composed of mineral oiland MMO. The disease progression was assessed in MMO,mineral oil or IFA-injected mice. An additional group ofmice was treated with LPS. Analysis of the lesion size inLPS- or MMO-injected mice did not show any differenceas compared to the untouched group of mice (Fig. 5a). Wereconfirmed the atheroprotective effect of IFA that reduced

lesions size by 70% (Fig. 5a). While injection of MMO didnot induce any reduction in lesion size, mineral oil reducedit by 45% compared to control mice. These experimentspoint towards the mineral oil as the candidate componentof IFA that exerts the atheroprotective effect. Both IFAand mineral oil reduced the TC (Table 1) that was againnot correlated to the lesion size. LPS increased the choles-terol level, which was positively correlated to lesion size.

J. Khallou-Laschet et al. / Atherosclerosis 184 (2006) 330–341 337

Table 2Serum cytokine and serum amyloid A (SAA) levels at sacrifice in control mice and mice injected with IFA, mineral oil, MMO or LPS (Protocol #3)

Ctrl (n = 6) IFA (n = 7) Mineral oil (n = 7) MMO (n = 8) LPS (n = 8)

IL-12 (pg/ml) 126± 30 445± 145* 583± 160* 647± 154* 1030± 209*** ,†

IFN� (pg/ml) 823± 42 712± 113 743± 69 677± 34 756± 62IL-4 (pg/ml) 198± 12 156± 6* 178± 11 185± 8 168± 7*

IL-10 (pg/ml) 1316± 69 1272± 150 1205± 78 1211± 84 1125± 48IL-6 (pg/ml) 380± 14 367± 22 346± 8 355± 16 331± 12*

SAA (�g/ml) 36± 16 188± 54** 210± 39** 94± 40 95± 15

SAA: serum amyloid A.* p < 0.05 vs. Ctrl.

** p < 0.01 vs. Ctrl.*** p < 0.001 vs. Ctrl.† p < 0.05 vs. IFA.

Mice that received MMO had a significantly reduced bodyweight.

In order to evaluate the inflammatory status, severalcytokines were assessed in the serum (Table 2). Neither IL-10 nor IFN� were modified by any treatment while IL-6 wasreduced only in LPS-injected mice. As compared to controlmice, serum IL-12 levels were increased in mice that receivedIFA, mineral oil, MMO or LPS. IL-4 serum concentrationswere decreased in IFA- and LPS-treated mice while mineraloil and MMO did not modify it. Interestingly, the only mod-ification that was both common and specific to IFA and tomineral oil groups concerned the SAA levels. Indeed, IFAand mineral oil increased it by 6.3- and 7-fold, respectively.Plotting the extent of lesions against the SAA levels for eachgroup of mice (Fig. 5b) revealed that mice clustered into twogroups: one with extended lesions but low SAA levels (Ctrl,MMO, LPS) and the other having smaller lesions but highSAA levels (IFA, mineral oil).

4.4. Adjuvants and serum lipoprotein patterns

We then analyzed by FPLC whether the serum lipoproteinprofiles were modified by IFA or its compounds. As shownin Fig. 6, MMO did not alter the lipoprotein profile as com-pared to control mice. On the other hand, IFA treatment led toa eo thel les-t erolb

4

IFAa ilityt s. Ag rmedt att orre-s bonsh s (or“ ris-

tane and phytane. Compounds indicated by the symbol *are alkylcyclohexanes but the structure of the alkyl chain isnot exactly determined. Other compounds are mainly methy-lalkanes. At variance with IFA, major compounds of theACROS mineral oil (Fig. 7) are of lower volatility. The mainconstituents are compounds of the sterane series S22–S27(characterized by the ion atm/z 217) and compounds of theterpane series T23–T31 (characterized by the ion atm/z 191).Straight-chain hydrocarbons from C17 to C21, and acyclicisoprenoids pristane and phytane are present in low amounts.Other compounds are chiefly methylalkanes. This analysisclearly showed that the mineral oil and the IFA, while dis-playing some similarities, have clearly distinct and complexcompositions.

5. Discussion

Emerging strategies of immunomodulation based on long-term immunization protocols involving repeated injectionsof adjuvants, which are aimed at modulating atherosclero-sis are currently tested. Given that they can modulate cer-tain immunoinflammatory disorders[1,2], we asked whetheradjuvants could alter the course of atherosclerosis. Our resultsdemonstrate that injection of E◦ mice with CFA, IFA andA Thee s nota SinceC asep vanto ulde leroticp

hatea tion.A d inh pec-u d tot es bye

ex-p tion

substantial decrease (−41%) of the CR/VLDL fraction. Wbserved that mineral oil and IFA had a distinct effect on

ipoprotein pattern. Indeed, mineral oil reduced LDL choerol by 21% and concomitantly increased HDL cholesty 56%.

.5. Mineral oil composition

The fact that for all the parameters tested hitherto,nd mineral oil had distinct effects raised the possib

hat these compounds may indeed have few similaritieas chromatography–mass spectrometry analysis confi

his hypothesis (Fig. 7). MMO is observed in low amounthe end of the chromatogram. The major compounds cpond to the mineral oil. Two series of saturated hydrocarave been clearly identified: straight-chain hydrocarbonn-alkanes”) from C14 to C20, and acyclic isoprenoids p

lum leads to a dramatic decrease in the lesion size.xtent of protection conferred by these adjuvants doeppear to depend upon the disease stage (Procotol #1).pG is the only adjuvant that did not perturb the diserogression, we propose that CpG should be the adjuf choice in long-term immunization protocols. This wonsure that the eventual effect observed on the atheroscrocess is attributable to the immunointervention.

Aluminum adjuvants, together with the calcium phospdjuvant, are the only ones licensed for human applicaluminum-adjuvanted vaccines have been administereuman for more than 50 years. It is fascinating to slate that Alum-based vaccines might have contribute

he reduction of cardiovascular disease these last decadxerting an ‘atheroprotective side-effect’.

The protective effect of CFA in our study was rather unected since other studies with CFA-based immuniza

338 J. Khallou-Laschet et al. / Atherosclerosis 184 (2006) 330–341

Fig. 6. Effect of MMO, mineral oil, and IFA on serum lipoprotein patterns (Protocol #3). FPLC separation was performed on randomized serum samplesfrom untouched control mice (thick full line; Ctrl;n = 6), mice that received chronic administration of MMO (thin full line;n = 6), mineral oil (thin brokenline; n = 4), or IFA (thick broken line;n = 5). Lines represent mean profiles and gray sections S.E.M. A magnification of the HDL profiles is shown in theinset. The chylomicron (CR)/VLDL, LDL, and HDL concentrations were computed from the FPLC profiles (**** p < 0.0001;*** p < 0.001;* p < 0.05 vs. Ctrl;††††p < 0.0001;††p < 0.01 vs. IFA).

protocols involving repeated injections in rabbits[16] andmice[17] have shown that the CFA further fortified by heat-killed preparation ofM. tuberculosis induces a proathero-genic anti-heat shock protein (HSP) immune response. Atpresent, we have no conclusive arguments to explain thisdiscrepancy. Given that IFA had the same atheroprotectiveeffect as CFA, the immune response directed againstM.tuberculosis-derived HSPs is, however, unlikely to be impli-cated in adjuvant-mediated atheroprotection. Furthermore,the fact that atheroprotection was repeatedly found to be con-ferred by Freund’s adjuvants to a similar degree in five inde-pendent experiments in the present work, and that this effectwas incidentally detected in three recent reports[18–20]con-solidates our findings. Importantly, in one of these reports[19], CFA was found to be anti-atherogenic in LDLR◦ miceindicating that the atheroprotective feature of adjuvants is notrestricted to the E◦ mouse model.

While it is admitted that CFA induces a Th1 bias and IFA aTh2 bias[12], the effect of repeated adjuvant injections on theTh polarization patterns was not documented. A distinct Thpolarization pattern can be of significance for atherosclerosis[13]. To evaluate this issue, E◦ mice were immunized with

ovalbumin. Ovalbumin was chosen as a model antigen insteadof oxLDL, HSPs or other antigens related to atherosclero-sis in order not to influence atherogenesis over the effectof adjuvants. First, the atheroprotective effect of Freund’sadjuvants was confirmed. Second, while CFA is described asa Th1-inducing agent in short-term experiments, we foundit to promote Th2 responses as well in chronic immuniza-tion protocols. Indeed, CFA-OVA-treated animals had highlevels of anti-OVA IgG1, attesting that B cells received aTh2 help. This was confirmed in in vitro spleen cell culturesfrom the same animals in which more than 10 pg/ml of IL-4was detected after an OVA challenge. IFA increased the Th2responses as expected. It also increased anti-OVA IgG2a titersindicating that Th1 responses were also stimulated, althoughwe were not able to detect IFN� production in response toOVA in spleen cell cultures from IFA-OVA-treated mice.This indicates that either Th1 cells that helped B cells toproduce IgG2a were eliminated or that the Th1 responsesare rather modest and below our detection limit. Therefore,repeated injections of Freund’s adjuvants do not bias the Thresponse anymore. While adjuvants initially promote the pro-duction of pro-Th1 molecules, a sustained adjuvant-mediated

J. Khallou-Laschet et al. / Atherosclerosis 184 (2006) 330–341 339

Fig. 7. Gas chromatography–mass spectrometry analysis of IFA and mineral oil. (a) Total ion chromatogram obtained from IFA and (b) from mineral oilACROS (ref. 415080010) (Cx: straight-chain saturated hydrocarbons atx carbon atoms; Pr: pristane; Ph: phytane; Sx: steranes atx carbon atoms; Tx: terpanesat x carbon atoms; MMO: mannide monoleate;*: alkylcyclohexanes).

inflammation might rather stimulate a mixed production ofpro-Th1 and pro-Th2 molecules, and therefore, invokes amixed Th1/Th2 response. Our results also raise the possibil-ity that atheroprotective adjuvants are those that can increaseTh2 responses, regardless of their effect on the Th1 responses.Indeed, IFA, CFA, and Alum, all three being atheroprotective,increased the Th2 responses while CpG did not.

While the anti-MDA-LDL IgG titers were increasedonly after CFA administration, both CFA and IFA signifi-cantly increased anti-MDA-LDL IgM. This latter increasecould also contribute to the atheroprotective effect of Fre-und’s adjuvants[14,19,21]. This was corroborated by thefact that the atheroprotective Alum adjuvant also increasedthe anti-MDA-LDL IgM but not IgG. On the other hand,CpG injections had no effect on anti-MDA-LDL antibodytiters. Interestingly, atheroprotection conferred by MDA-LDL-specific Th2 responses relies on the IL-5-dependentenhancement of the innate humoral response to oxLDL,which in aggregate provide protection from atheroscle-rosis [22]. Thus, the increase in Th2 responses and inthe MDA-LDL IgM titers imposed by atheroprotective

adjuvants might be connected through an IL-5-dependentmechanism.

Because they are the most commonly used adjuvantsin experimental studies, we decided to determine whichcompound of the Freund’s adjuvants was endowed withatheroprotective properties. MMO was the most promisingcandidate because of its simple structure and its structuralsimilarities with the lipid A of LPS. Indeed, LPS modulatesexperimental atherogenesis when administered weekly dur-ing 10 weeks at 50�l/mouse/injection[23]. First, we foundthat at 10 times lower dose, though sufficient to dramaticallyincrease serum IL-12 levels, LPS had no effect on lesiondevelopment. The discrepancy between our results and thosereported by Ostos et al.[23] may depend upon different LPSdosage (5�l versus 50�l) and/or diet (0.15% cholesterol ver-sus normal chow diet). Second, we found that MMO hadno atheromodulating effect while mineral oil induced a 45%reduction in lesion size, suggesting that the mineral oil isthe compound responsible for the atheroprotective effect ofFreund’s adjuvants. We were, however, intrigued by severalobservations. We expected the effects of IFA recorded on

340 J. Khallou-Laschet et al. / Atherosclerosis 184 (2006) 330–341

various parameters to be an aggregate of the individual effectsof mineral oil and MMO. This was true for some but not allparameters analyzed. For instance, IFA-reduced serum IL-4levels while neither MMO nor mineral oil did. MMO andmineral oil led to a higher increase of serum IL-12 than IFA.Interestingly, the increase in SAA induced by IFA could bereproduced with mineral oil. While SAA could merely be amarker, these observations raised the possibility that SAAparticipates in the atheroprotection conferred by IFA andmineral oil. Indeed, during the acute-phase reaction, SAAmay transform HDL into an acute-phase (SAA-enriched)proatherogenic HDL[24]. These observations, however, didnot fit with our findings and urged us to further analyze mod-ifications in the lipoprotein profiles eventually induced byadjuvants.

We repeatedly found that IFA and CFA reduced the TClevels; a finding that was counter-balanced by the fact thatTC levels never correlated with lesion size in individualgroups. A significant association between TC and SAL wasfound in certain experiments (Protocol #1, Exp #2 and Pro-tocol #2) when correlations were computed with the datafrom all groups in each experiment but emerged because ofgroup clustering. FPLC analysis performed on serum lipopro-teins from mice included in the Protocol #3 showed thatIFA reduced the CR/VLDL fraction. None of its constituentstested individually could reproduce this effect. MMO had noe theL in-e teinp per-t war-r

A,e factt thata ctrome omA min-e alled“ bil-i nt,t ilara 010)f amed d theh n). Inv thea ts ofm , thef s andt pro-t re ofa ulatei

iousc n in

the atherosclerotic lesion, which, in turn, confers to vascu-lar cells an activated and proinflammatory deleterious phe-notype. However, inflammation is in essence a protectiveprocess. Adjuvants are used for their capacity to stimulatenon-specifically immunoinflammatory responses, an effectthat we have observed in our experiments. However, animalswith higher SAA titers developed smaller lesions. This indi-cates that enhancing the early inflammation can be beneficial.In support of this provocative point of view, a recent reporthas shown that disruption of a key regulator of inflammation,the NF-�B transcription factor, in the macrophages leads toan increase in lesion size in LDLR◦ mice [26]. Therefore,inflammation might not be exclusively proatherogenic. Someinflammatory processes might be atheroprotective.

In conclusion, we have shown that certain adjuvants havepotent atheroprotective properties. The main objective of thisstudy was not to elucidate the mechanism of action of adju-vants since their immunoregulatory effects depend on manyvariables that could not all be tested. However, we provideevidences showing that the atheroprotective effect of adju-vants may rely upon their capacity to increase Th2 responses,to increase anti-MDA-LDL IgM titers, and/or to imposeatheroprotective serum lipoprotein profiles. Adjuvants mayalso instigate an atheroprotective inflammatory profile. Fur-ther studies are required to evaluate whether one of thesemechanisms is predominantly involved in the atheroprotec-t

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ffect on the lipoprotein profile and mineral oil decreasedDL fraction and increased the HDL fraction. IFA and mral oil thus imposed distinct but atheroprotective lipoprorofiles that may contribute to their atheroprotective pro

ies. Complex mechanisms underlying such an effectants further intense investigations.

That mineral oil was not mimicking all effects of IFspecially those on the lipoprotein profile, may rely on the

hat the mode of preparation of mineral oils results in lotsre unique. Indeed, the gas chromatography–mass spetry analysis performed on IFA and on the mineral oil frCROS used in our experiments showed that the tworal oils are complex mixtures and correspond to the so-cmaltene” fraction of petroleum characterized by its soluty in n-pentane[25]. The two fractions are rather differehe second being richer in polycyclic compounds. A simnalysis performed on another mineral oil (ref 124020

rom ACROS related to the one used in this study (sensities, viscosities, and refractive indexes) confirmeigh heterogeneity of these compounds (data not showiew of the complexity in their composition, determiningtheroprotective effect of each of the isolated componenineral oils is beyond the scope of this study. However

act these atheroprotective oils were so heterogeneouhat Alum that does not contain any oil, was also atheroective points to a more general atheromodulating featudjuvants that may be related to their capacity to mod

nflammation.The current dogma proposes that an inflammatory vic

ycle maintains a continuous inflammatory cell infiltratio

-

ive effect of adjuvants.

cknowledgements

This work was supported by the Institut Nationala Sante et de la Recherche Medicale (INSERM) and thwedish Research Council, project no. 71X-15075-01oirier was the recipient of a fellowship from the Groe Reflexion sur la Recherche Cardiovasculaire (GRRCTupin was the recipient of a fellowship from the ARC

oundation and received a research stipend from the Sweart and Lung Foundation.

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