Microbes and Infection 8 (2006) 2745e2755www.elsevier.com/locate/micinf

Original article

A DNA vaccine encoding CCL4/MIP-1b enhances myocarditisin experimental Trypanosoma cruzi infection in rats

Ester Roffe a, Adriano L.S. Souza a, Braulia C. Caetano a, Patrıcia P. Machado a,Lucıola S. Barcelos a, Remo C. Russo a, Helton C. Santiago a, Danielle G. Souza a,

Vanessa Pinho a, Herbert B. Tanowitz b, Elisabeth R.S. Camargos c,Oscar Bruna-Romero d, Mauro M. Teixeira a,*

a Departamento de Bioquımica e Imunologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais,

Avenida Antonio Carlos 6627, Pampulha, Belo Horizonte, Minas Gerais 31270-901, Brazilb Department of Pathology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA

c Departamento de Morfologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Avenida Antonio Carlos 6627,

Pampulha, Belo Horizonte, Minas Gerais 31270-901, Brazild Departamento de Microbiologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Avenida Antonio Carlos 6627,

Pampulha, Belo Horizonte, Minas Gerais 31270-901, Brazil

Received 26 June 2006; accepted 7 August 2006

Available online 30 August 2006

Abstract

Chagas’ disease, caused by Trypanosoma cruzi, is a major cause of cardiovascular disease in Latin America. Exacerbated inflammation dis-proportional to parasite load characterizes chronic myocardial lesions in chagasic patients. Chemokines and their receptors are expected to ac-count for the renewed inflammatory processes after the inoculation of the parasite, but their potential unique functions are far from being clear.Herein, we evaluated the effect of a DNA vaccine encoding CCL4/MIP-1b, a CC-chemokine, in T. cruzi-elicited myocarditis in rats. Holtzmanrats were given intramuscularly cardiotoxin and the CCL4/MIP-1b DNA-containing plasmid (100 mg) was delivered in this muscular site fourtimes. Fourteen days after last immunization, animals were inoculated with a myotropical CL-Brener T. cruzi clone. Peak of parasitism wasobserved at day 15 after infection, preceding the peak of myocardial inflammation at day 20. Myocarditis was still intense at day 30, but theinflammatory infiltrates showed a more focal distribution. The expression of CCL2/MCP-1 and CCL4/MIP-1b correlated closely with the ki-netics of myocardial inflammation. The CCL4/MIP-1b DNA vaccine induced an increase of the levels of the anti-CCL4/MIP-1b observed inT. cruzi-infected animals. This was associated with an exacerbation of myocardial inflammation and fibrosis, although alterations in parasitemiaand myocardial parasitism were not observed. Our data suggest that CCL4/MIP-1b plays a role in preventing excessive inflammation and pa-thology rather than in controlling parasite replication.� 2006 Elsevier Masson SAS. All rights reserved.

Keywords: Trypanosoma cruzi; Myocarditis; Chemokine; DNA vaccine; CCL4/MIP-1b

1. Introduction

Chagas’ disease is caused by the protozoan Trypano-soma cruzi and affects around 15 million people in Latin

* Corresponding author. Tel./fax: þ55 31 3499 2651.

E-mail address: mmtex@icb.ufmg.br (M.M. Teixeira).

1286-4579/$ - see front matter � 2006 Elsevier Masson SAS. All rights reserv

doi:10.1016/j.micinf.2006.08.004

America [1]. Chronic inflammation and fibrosis characterizethe chronic cardiac form of the disease [2]. The mainpathological manifestation of chronic cardiomyopathy isa fibrosing and progressive disease, with structural disar-rangement that leads to the loss of function of the heartand results in cardiac failure, ventricular arrhythmias andhypertrophy [3].

ed.

2746 E. Roffe et al. / Microbes and Infection 8 (2006) 2745e2755

Chemokines are mediators of inflammation thought to playa role in the pathogenesis of several inflammatory and infec-tious diseases, including Chagas disease [4]. T. cruzi-infectedcardiomyocytes produce CC and CXC-chemokines in vitroand treatment with CXCL1-2/MIP-2, CCL2/MCP-1 andCXCL10/IP-10 alone or in combination induces synthesis ofnitric oxide and inhibit parasite multiplication [5]. However,at the chronic phase of infection, when parasite load is scarce,T. cruzi-infected mice are still expressing considerableamounts of CCL5/RANTES, CCL3/MIP-1a and IFNg-in-duced CXC-chemokines [6,7]. More recently, treatment withMet-RANTES, a selective CCR1 and CCR5 antagonist wasdescribed to improve survival, decrease heart inflammationand reduce cardiac deposition of an extracellular matrix com-ponent involved in fibrosis, without enhancement of parasite-mia and myocardial parasitism in infected mice [8]. This isin contrast to in vitro data showing the importance of CC-che-mokines to control T. cruzi multiplication [5,9]. Also, T. cruziinfection of CCR5 knock-out mice was strongly deleterious.Indeed, myocardial inflammation was dramatically inhibitedwhereas parasitism was exacerbated and mortality of CCR5deficient infected mice was strongly increased, suggestingthat CCR5 receptor could play an important protective rolein control of T. cruzi infection [10]. Thus, whereas late phar-macological blockade of CCR1 and CCR5 prevented pathol-ogy and lethality, absence of CCR5 was associated with lossof the ability to mount an inflammatory response and loss ofcontrol of infection. The role of individual chemokines hasnot been evaluated in detail in experimental T. cruzi infection.Moreover, all studies to date evaluating the expression and roleof chemokines have been carried out in mice. Infection of ratswith T. cruzi represents an alternative model to study diseasepathogenesis as this animal appears to clear the infectionmore effectively and develop a more silent disease, resemblingthe chronic indeterminate form observed in humans [11,12].

Chemokine-based DNA vaccines have been evaluated inexperimental models of tissue-destructive autoimmune pro-cesses. The rationale underlying such studies is that immuni-zation with a chemokine gene in an immunogenic plasmidfacilitates the induction of anti-chemokine antibodies whichhave the ability to block the action of the chemokine of inter-est. For example, DNA vaccines encoding chemokine geneswere shown to partially or totally inhibit pathology, or reverseestablished disease in models of experimental allergic enceph-alomyelitis and chronic poly-adjuvant-induced arthritis[13,14].

In the present study, we have evaluated the expression ofcytokines, chemokines, tissue pathology and parasitism inrats infected with the CL-Brener clone of T. cruzi. Our studiesshowed a marked expression of several chemokines, includingCCL4/MIP-1b. CCL4/MIP-1b is a CCR5-acting chemokinewhich exerts chemotactic and migratory effect in monocytes,T lymphocytes, dendritic cells and NK cells [15]. There areno reported studies evaluating the role of CCL4/MIP-1b in ex-perimental T. cruzi infection. Here, we investigated whetheradministration of a DNA vaccine encoding CCL4/MIP-1bhad any effect in T. cruzi-elicited myocarditis in rats.

2. Materials and methods

2.1. Animals and infection

Holtzman rats, 90 days old and 300e400 g of body weight,were obtained from CEBIO/UFMG (Minas Gerais, Brazil) andmaintained in the animal facilities of the Laboratorio de Imu-nofarmacologia, with filtered water and food ad libitum. Ani-mals were inoculated intraperitoneally with 104 blood forms ofthe CL-Brener clone of T. cruzi/50 g body weight. All animalprocedures had prior approval from the local animal ethicscommittee (CETEA/UFMG). The myocardium was obtainedin different days corresponding to the acute (15, 20 and 30)and chronic (65 and 130) phases of infection.

Parasitemia was estimated according to Brener’s methodevery other day in 5 mL of peripheral blood sampled fromthe tail vein of T. cruzi-infected mice [16].

2.2. Cytokines and chemokine detection by ELISA

The upper part of the heart was processed in a solutioncontaining protease inhibitors and centrifuged at 10,000 rpmfor 10 min in 4 �C, as previously described [17]. The superna-tant was stocked at �20 �C until used to detect IFN-g, IL-4(Duoset, R&D Systems), TNF-a, IL-10 (gently donated byDr. Stephen Poole, England) and the chemokines CCL2/MCP-1 and CCL5/RANTES (Peprotech, USA) by ELISA.

2.3. Histopathological analysis

The middle part of the heart was washed in sterile PBS,smoothly dried and immediately fixed in 4% buffered parafor-maldehyde. The tissue was embedded in paraffin, cut in 7 mmsections and stained with hematoxylin and eosin, to quantifyinflammation and infection, and with Picro-syrius, to evaluatefibrosis. Cardiac parasitism and inflammation of hearts from5e7 animals per group were analyzed with a Zeiss integratingeyepiece with 100 hits (Oberkohen, Germany) at a final mag-nification of 320�. A total of 4000 hits were evaluated in eachsection of cardiac tissue. The infection and inflammation indi-ces represent the number of hits covered by amastigote nestsand inflammatory cells, respectively.

2.4. RT-PCR

The RNA was extracted by using RNAzol (Sigma, USA).The RNA obtained was resuspended in diethylpyrocarbonate-treated water and stocked at �70 �C until use. The RNAwas quantified and 1 mg was utilized in the reverse transcrip-tion reaction. The resulting cDNA was used for amplificationof the desired gene with the following primer sequences andspecific conditions (annealing temperature and cycles):CCL2/MCP-1 sense 50-ATG-CAG-GTC-TCT-GTC-ACG-CTT-CTG-GGC-30, anti-sense 50-CTA-GTT-CTC-TGT-CAT-ACT-GGT-CAC-30 (55 �C, 35 cycles); CCL4/MIP-1b sense50-ATG-AAG-CTC-TGC-GTG-TCT-GCC-TTC-30, anti-sense50-TCA-GTT-CAA-CTC-CAA-GTC-ATT-CAC-30 (50 �C, 33

2747E. Roffe et al. / Microbes and Infection 8 (2006) 2745e2755

cycles); CCL5/RANTES sense 50-ATG-AAG-ATC-TCT-GCA-GCT-GCA-TCC-30, anti-sense 50-CTA-GCT-CAT-CTC-CAA-ATA-GTT-G-30, (50 �C, 26 cycles); GAPDH sense50-CTC-AAG-ATT-GTC-AGC-AAT-GC-30; anti-sense 50-CAG-GAT-GCC-CTT-TAG-TGG-GC-30 (60 �C, 26 cycles).

2.5. Quantification of parasite tissue loads byreal-time PCR

Real-time PCR for parasite quantification was performed asdescribed previously [18], with minor modifications. Briefly,on different days after infection, the hearts were digestedwith proteinase K, followed by a phenol-chloroform-isoamylalcohol affinity extraction. Real-time PCR using 50 ng of totalDNA was performed on an ABI PRISM 7900 sequence detec-tion system (Applied Biosystems) using SYBR Green PCRMaster Mix according to the manufacturer’s recommenda-tions. Purified T. cruzi DNA (American Type Culture Collec-tion) was sequentially diluted for curve generation in aqueoussolution containing equivalent amounts of DNA from unin-fected mouse tissues. The following primers were used:T. cruzi-specific-primers (TCZ gene e a tandemly repeatedgenomic sequence, known as ‘‘satellite’’ DNA) GCTCTTGCCCACAMGGGTGC, where M ¼ A or C (S35-forward), andCCAAGCAGCGGATAGTTCAGG (S36-reverse).

2.6. Hydroxyproline quantification

Fragments of 100 mg of myocardium were removed forhydroxyproline determination as an indirect measure of colla-gen content and as previously described [19]. This methoduses Chloramine T as an oxidizing agent and compares valuesof hydroxyproline with a standard curve.

2.7. Rat CCL4/MIP-1b cloning and construction of thevaccination plasmid

The cloning of the CCL4/MIP-1b gene (279 bp) was ac-complished from the product resulting from the CCL4/MIP-1b specific amplification by RT-PCR reaction in myocardialsamples from T. cruzi-infected rats 20 days after infection.The DNA was extracted from agarosis gel by a DNA purifica-tion kit from agarosis (Promega, USA). The cloning was per-formed with p-TopoTA cloning kit (Invitrogen, San Diego,CA), as recommended by the supplier. The p-Topo recombi-nant plasmid was used to transform chemically competent E.coli XL1-blue bacteria. To confirm the CCL4/MIP-1b cloningthe plasmid was purified using a miniprep kit (Qiagen, USA),digested with EcoR1 (Promega, USA) and sequenced (MEG-ABACE�). The confirmed CCL4/MIP-1b gene was insertedinto the vaccination pcDNA 3.1 plasmid (Invitrogen, USA)with T4 DNA ligase enzyme and transfected into E. coliXL1-blue bacteria. The vaccination construct was in purified(midiprep kit) and sequenced. The correct recombinant colo-nies were grown in 2,5 L of LB-ampicilin medium and plas-mid purification was performed by Endofree gigaprep kit.

The DNA resulting was measured at 260 and 280 nm in spec-trophotometer and stocked at �70 until use.

2.8. Immunization schedule

Cardiotoxin (10 mM, Sigma) was injected intramuscularlyand 7 days later animals received an intramuscular injectionof 100 mg of DNA in 300 ml of apyrogenic ultra-pure water,divided in two injections of 150 ml of DNA into each tibialmuscle. Animals received 4 boosters every 7 days. To investi-gate whether DNA immunization causes any effect per se,uninfected animals were immunized with CCL4/MIP-1b. Asthere were no differences in all myocardial parameters studiedin comparison to non-vaccinated uninfected rats (data notshown), data in uninfected rats were pooled for facilitationof presentation.

2.9. Detection of serum anti-chemokine antibodies

Commercial recombinant rat CCL4/MIP-1b (Peprotech)was coated onto ELISA plates at 10 ng/well. Serial dilutionsof rat sera in PBS-BSA 1% were added. Anti-rat IgG streptoa-vidin-conjugated antibody (Sigma) was used at 1:1000. Absor-bance was measured at 492 nm in spectrophotometer.

2.10. In vivo readout assay for screening the neutralizingactivity of the rat anti-CCL4/MIP-1b antibodies

To address the in vivo neutralizing action of antibodieselicited by CCL4/MIP-1b-encoding DNA vaccination, serumwas obtained at 20 days after infection of CCL4/MIP-1b- orcontrol plasmid-vaccinated animals (n ¼ 6). The sera werepooled, diluted at 1:20 in PBS and given subcutaneously torats. After 1 h, an optimal dose of CCL4/MIP-1b (300 ng/cav-ity) or sterile PBS was injected intraperitoneally and animalskilled after 18 h. Cells were harvested with 15 ml PBS, totalcell counts performed in a modified Neubauer chamber usingTurk’s stain and differential cell counts on cytospin prepara-tions (Shandon III) stained with May-Grumwald-Giemsa usingstandard morphologic criteria to identify cell types. There wasno effect of the sera on the baseline recruitment of PBS (datanot shown).

2.11. Statistical analysis

Results are shown as means � S.E.M. Differences betweengroups were compared using Student’s t test (two sets of data)or one-way ANOVA (three or more sets of data), followed bythe Student-NewmaneKeuls post hoc test. Differences wereconsidered significant at p < 0.05.

3. Results

3.1. Parasitemia and myocardium inflammation in rats

Infected rats had low parasitemia. Parasites were firstdetected in blood at day 6 and levels peaked at day 12 after

2748 E. Roffe et al. / Microbes and Infection 8 (2006) 2745e2755

infection (around 2 � 104 parasites/mL of blood). The parasi-temia subsided rapidly and parasites could not be detected inblood at day 19 after infection or later (data not shown). Therewas not lethality in the present group of experiments.

Myocardial alterations after T. cruzi infection are shownin Fig. 1. Intense myocarditis was already observed at day15 and peaked at day 20 after infection (Fig. 1A). At thisstage, inflammatory infiltrates were composed predominantlyby mononuclear cells with a disperse distribution in the

tissue. Amastigote pseudocysts, frequently accompanied byinflammatory infiltrates, were also observed in the myocar-dium at days 15 and 20 after infection (Fig. 1C). The peakof parasitism was observed at day 15, thus preceding thepeak of myocardial inflammation (Figs. 1A,B). At 20 daysafter infection, there were increased numbers of infiltratesthat were more intense and showed a more disperse distribu-tion in the cardiac tissue (Fig. 1C). The myocarditis was stillintense at day 30 after infection, but the inflammatory

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Fig. 1. Histopathological alterations in myocardium of T. cruzi-infected animals. Quantification of (A) inflammation; and (B) infection correspond to the quan-

tification of points containing inflammatory cell nuclei or amastigote pseudocysts, respectively. Quantification was performed by using an ocular containing 100

points/microscopic field in a final magnification of 200�. A total number of 40 microscopic fields were analyzed by section, in a total of 4000 points. The insert

shows the quantification of T. cruzi DNA by Real time-PCR. Results are the means � S.E.M. of 5e7 animals in each group. The myocardium from infected an-

imals was excised at days 20 (C); 30 (D); and 65 (E) after infection. The heart of a non-infected animal is shown in (F). ND, not determined. *, ** and *** for

P < 0.05, 0.01 and 0.001, respectively, when comparing infected versus non-infected animals.

2749E. Roffe et al. / Microbes and Infection 8 (2006) 2745e2755

infiltrates showed a more focal distribution (Fig. 1D). Para-site nests were uncommon at day 30, but few amastigotescould be observed inside the infiltrates. By day 65, therewas a significant reduction in the myocarditis and parasiteswere not found (Fig. 1E).

3.2. Expression of chemokines and cytokines in themyocardium of T. cruzi-infected rats

The RT-PCR results are shown in Fig. 2A and the mean ofthe densitometric analysis on Figs. 2BeD. CCL2/MCP-1mRNA was already expressed at day 12 after infection andpeaked at day 20 (Fig. 2). Thereafter, expression returned tobackground levels. The expression of CCL4/MIP-1b mRNAfollowed a similar pattern but the chemokine was onlydetected at days 15 and 20 after infection (Fig. 2C). Myocardial

expression of CCL5/RANTES mRNA was slightly increasedthroughout the observation period after infection (Figs. 2A,C).

The expression of CCL2/MCP-1 and CCL5/RANTESprotein in the myocardium of T. cruzi-infected animals isshown in Fig. 3. Overall, there was a good correlation betweenthe expression of mRNA, as detected by RT-PCR, and protein,as detected by ELISA, for the chemokine CCL2/MCP-1. Thetrend was also similar for CCL5/RANTES in both assays.Thus, CCL2/MCP-1 expression was noticed from days 12 to30 whereas CCL5/RANTES was not detected above levelsfound in non-infected rats (Figs. 3A,B). A commercial kitfor CCL4/MIP-1b was not available at the time these experi-ments were conducted.

The expression of several cytokines thought to play a rolein the pathophysiology of T. cruzi infection was also evalu-ated. IFN-g, TNF-a, IL-4 and IL-10 are increased during the

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Fig. 2. Kinetics of mRNA expression of CCL4/MIP-b in cardiac tissue of T. cruzi-infected animals. A) RT-PCR bands; BeD) densitometry of RT-PCR bands. The

organs were excised and submitted to RNA extraction by Phenol-chloroformium technique. The resulting RNA was quantified by spectrophotometry and used to

reverse transcription. The cDNA was amplified by PCR reaction using specific primer to the chemokine CCL4/MIP-b. The dotted lines across the bars represent

background values in non-infected animals. The result was expressed in arbitrary units according to the constitutively expressed GAPDH gene. Results are the

means of 3 animals in each group. ND; Not done.

2750 E. Roffe et al. / Microbes and Infection 8 (2006) 2745e2755

CCL2/MCP-1

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Fig. 3. Kinetics of chemokine protein production in cardiac tissue of T. cruzi-infected animals. The organs were excised and processed for the measurement of (A)

CCL2/MCP-1; (B) CCL5/RANTES; (C) IFN-g; (D) TNF-a; (E) IL-4; and (F) IL-10 by ELISA. The dotted lines across the bars represent background values in

non-infected animals. Results are the means � S.E.M. of 5e7 animals in each group. *, ** and *** for P < 0.05, 0.01 and 0.001, respectively, when comparing

infected versus non-infected animals.

acute phase (Figs. 3CeF). The peak of IFN-g production wasearlier than that of IL-4 (15 vs 20 days after infection) and wasnot detected 30 days after infection (Figs. 3C,E). Moreover,during the chronic phase, levels of these cytokines were nearthose found in non-infected rats (Fig. 3).

3.3. Effects of the vaccination with a DNA vaccineencoding for CCL4/MIP-1b in the course ofT. cruzi infection in rats

The functional role of CCL4/MIP-1b was investigated byusing a CCL4/MIP-1b-encoding DNA vaccine. Anti-chemo-kine antibodies are commonly elevated in autoimmune dis-eases, such as experimental autoimmune encephalomyelitis andarthritis, and vaccination with DNA coding chemokines bythe schedule used here has been known to induce an increasein levels of anti-chemokine antibodies [13,14]. In comparisonto non-infected rats, higher levels of anti-CCL4/MIP-1b

antibodies were detected in infected animals at 30 days butnot at 20 days after infection. Moreover, levels of antibodieswere enhanced in T. cruzi-infected animals that were vacci-nated (Fig. 4A). We also addressed whether the antibodies eli-cited by the CCL4/MIP-1b vaccination could indeed preventthe function of CCL4/MIP-1b. Pre-treatment with sera fromCCL4/MIP-1b-immunized animals (diluted to 1:20) waseffective at blocking the migration of leukocytes to the perito-neal cavity after injection of CCL4/MIP-1b. In contrast, serafrom animals immunized with control plasmid failed to affectthe recruitment induced by CCL4/MIP-1b (Fig. 4B).

The immunization with empty or CCL4/MIP-1b plasmidsdid not cause any significant effect on parasitemia neither ininfection-associated lethality (data not shown). There was anexacerbation of the inflammatory response in the heart ofCCL4/MIP-1b immunized rats at 20 and 30 days after infec-tion, as compared to non-immunized and plasmid control im-munized animals (Figs. 5A,C vs B,D and E). There did not

2751E. Roffe et al. / Microbes and Infection 8 (2006) 2745e2755

appear to be any major change in the composition ofinflammatory lesions in CCL4/MIP-1b- and vector control-immunized animals (data not shown). However, theinflammatory infiltrates in the heart of CCL4/MIP-1b-immu-nized animals were multi-focal (Figs. 5A,C) in comparisonwith a more focal response that was observed in plasmidcontrol-immunized animals (Figs. 5B,D). Myocardial parasit-ism was scanty and not different in CCL4/MIP-1b and plasmidcontrol-immunized rats (Fig. 5F).

An increase in collagen content, an index of tissue fibrosis,was evidenced in heart sections of CCL4/MIP-1b-immunizedanimals at days 20 (data not shown) and 30 after infection(Fig. 6A), as shown by picro-syrius staining. In non-immu-nized (data not shown) and in plasmid control-immunized

Anti-CCL4/MIP-1 antibodies in serum

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Fig. 4. Quantification and in vivo effects of anti-CCL4/MIP-b antibodies in se-

rum of T. cruzi-infected animals previously immunized with CCL4/MIP-

b (black bar) or control plasmid (grey bar), and non-infected (open bar) ani-

mals. In (A), the serum was diluted to 1:20 in PBS-BSA 1% and used in direct

ELISA for determination of levels. Results are the means � S.E.M. of 5e7 an-

imals in each group. ** and ## for P < 0.01 when comparing infected versus

non-infected animals, and CCL4/MIP-b- versus control plasmid-immunized

animals, respectively. (B), Blocking activity of antibodies elicited by CCL4/

MIP-1b-encoding DNA vaccination. Animals were pre-treated with sera

from T. cruzi-infected animals that were previously immunized with CCL4/

MIP-1b- or control plasmid. The sera was pooled from 6 animals and diluted

to 1:20 in PBS and given subcutaneously 1 h before the injection of CCL4/

MIP-1b (300 ng/cavity). Results are shown as number of cells � 104 per cav-

ity. Mononuclear cells (black bar) and neutrophils (open bar) are shown. The

dotted lines across the bars represent background values in animals given PBS.

Results are the means � S.E.M. of 5 animals in each group. * and ** for

P < 0.5 and 0.01 when comparing CCL4/MIP-1b- versus control plasmid-im-

munized animals, respectively.

and infected animals (Fig. 6B), there was staining of collagenfibers dispersed in the cardiac tissue, especially around inflam-matory infiltrates (Figs. 6A,B) and blood vessels (data notshown). Blood vessels were stained in hearts of non-infectedanimals also, but no staining of fibers in this tissue wasobserved (data not shown). In CCL4/MIP-1b-vaccinatedanimals, the same profile of staining was observed, but theintensity and area of the staining was greater (Fig. 6A) andthere was an increased number of inflammatory infiltrateswhich were stained (data not shown). Quantification of colla-gen content by hydroxyproline measurement concurred withthe staining analysis (Figs. 6C,D). Immunization with CCL4/MIP-1b had no significant effect on the infection-inducedincrease in chemokine and cytokine levels in myocardium atday 20 and day 30 after infection (data not shown).

4. Discussion

Expression of inducible chemokines is thought to be a ver-tebrate cellular ‘‘SOS response’’ to recruit leukocytes to anarea of injury [20]. Although certain chemokines expressedin T. cruzi-infected hearts may be important for parasitic con-tainment, their expression may extend injury through severalmechanisms, including the maintenance of chronic inflamma-tion [21]. So, chemokines are suggested to play an ambiguousrole in T. cruzi infection: in the acute phase, these mediatorsmay be important to facilitate movement of cells and in theacquisition of a parasite-specific immune response that willcontrol parasite replication; in contrast, in the chronic phase,parasite-induced IFN-g and chemokines may play a role inthe maintenance of inflammation. In such way, it is crucialto identify which chemokines are essential for parasite controlin the acute phase of infection and which chemokines may bedeleteriously produced during the chronic phase. Such knowl-edge may aid in the development of novel pharmacologicalinterventions to control unwanted chronic inflammation.

Inoculation of rats with T. cruzi represents an experimentalmodel of resistance to infection [22]. Indeed, we found verylow parasitemia, and other studies suggest that parasitemiaintensity correlates directly to resistance in different inbredstrains of rats [23]. Despite the low levels of myocardial par-asitism, there is significant acute myocardial pathology. Thecardiac disease does not progress to heart dilatation and fail-ure, but infected rats manifest electrocardiographic alterationsin the chronic phase, probably associated with the focal myo-carditis and myocardial fibrosis [24]. Thus, despite continuedinfection and a degree of heart damage, infected rats manageto regulate the immune and inflammatory response and pre-vent severe disease after infection with certain strains of T.cruzi. On the other hand, T. cruzi-elicited myocarditis in ratsshare important features with the more commonly studied mu-rine model, including the predominance of mononuclear cellsin inflammatory lesions and the predominance of TNF-a andtype 1 cytokines in the heart (IFN-g, CCL5/RANTES).Thus, it appears that T. cruzi infection in rats is a valid modelto study the immune response involved in myocarditis causedby this protozoan parasite.

2752 E. Roffe et al. / Microbes and Infection 8 (2006) 2745e2755

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A B

DC

Fig. 5. Histopathological alterations in myocardium of T. cruzi-infected animals previously immunized with CCL4/MIP-b (solid bar and symbol) or control plas-

mid (open bar and symbol). The dotted lines across the bars represent background values in non-infected animals. The myocardium was excised and evaluated 20

(A, B, E, F) or 30 days (C, D, E, F) after infection. Animals were immunized previously with CCL4/MIP-1b-encoding DNA vaccine (A, C) or control plasmid

pcDNA3 (B, D) prior to infection. The organs were included in paraffin and sectioned in microtome to posterior hematoxylin and eosin staining. The inflammation

(E) and infection (F) indices were quantified using an ocular containing 100 points/microscopic field in a final magnification of 400�. A total number of 40 mi-

croscopic fields were analyzed by section, in a total of 4000 points. ND, non-determined. Results are the means � S.E.M. of 5e7 animals in each group. * and # for

P < 0.05 when comparing infected versus non-infected animals, and CCL4/MIP-1b- versus control plasmid-immunized animals, respectively.

Holtzman rats infected with the CL-Brener clone of T. cruzihad low parasitemia. The peak of myocardial parasitism pre-ceded the peak of myocardial inflammation (day 15 vs 20,respectively). Myocarditis was still intense at day 30 afterinfection, but the inflammatory infiltrates showed a more focaldistribution. Parasite nests were uncommon at day 30, but fewamastigotes could be observed inside the infiltrates. By day 65,there was a significant reduction in the myocarditis and para-sites were not found. The expression of several chemokinesand cytokines correlated closely with the kinetics of

myocardial inflammation and both inflammation and cyto-kine/chemokine levels subsided during the chronic phase ofinfection.

In T. cruzi-infected C57BL/6 mice, it was observed that cy-tokine expression was dominated by the presence of IFN-gmRNA in the acute phase of infection, whereas the balanceof type 1 and type 2 cytokines was switched in favor of IL-4 and IL-10 mRNAs in chronic phase [6]. In C3H/He mice,a susceptible mouse strain that present chronic myocarditis, in-creased expression of TNF-a and IFN-g mRNA was observed

2753E. Roffe et al. / Microbes and Infection 8 (2006) 2745e2755

1.0

1.1

1.2

1.3

#

OH

-p

ro

lin

e

(fo

ld

in

crease o

ver

T. cru

zi-in

fected

an

im

als)

C

1.0

1.1

1.2

1.3

1.4

1.5 #

OH

-p

ro

lin

e

(fo

ld

in

crease o

ver

T. cru

zi-in

fected

a

nim

als)

D

A B

Fig. 6. Quantification of collagen content in myocardium of T. cruzi-infected animals previously immunized with CCL4/MIP-b (solid bar) or control plasmid (open

bar), and non-infected animals. The myocardium was excised and evaluated 20 after infection. Animals were previously immunized with CCL4/MIP-b-encoding

DNA vaccine (A); or control plasmid pcDNA3 (B) prior to infection. The myocardium was included in paraffin, sectioned and stained with Picrus-syrius. Final

magnification of 400�. Quantification of hydroxyproline was carried out 20 days (C); and 30 days (D) after infection. Results are the means � S.E.M. of 5e7

animals in each group. # for P < 0.05 when CCL4/MIP-b- versus control plasmid-immunized animals.

in the cardiac tissue in acute and chronic infection. Interest-ingly, Talvani et al. noticed an increased expression of IL-4mRNA only during the chronic infection, when focal inflam-matory infiltrates persisted [7]. In the present work we foundan increased production of TNF-a, IFN-g and IL-4 only inthe acute phase of infection. The latter findings are consistentwith the ability of rats to resolve the chronic infection andinfection-associated inflammation.

CCL4/MIP-1b is a CCR5-acting chemokine which exertschemotactic and migratory effect in monocytes, T lympho-cytes, dendritic cells and NK cells [15]. There are no reportedstudies evaluating the role of CCL4/MIP-1b in experimentalT. cruzi infection. The presented results show that administra-tion of a CCL4/MIP-1b encoding DNA vaccine to T. cruzi-infected animals did not affect the parasitism, but resulted inexacerbation of the inflammatory process and collagen deposi-tion in myocardium, alterations more evident 30 days post-infection, at the end of acute phase. Consistent with ourstudies, in an EAE model in which a CCL4/MIP-1b encodingDNA vaccine was used, there was increased mononuclear cellinfiltrates in the central nervous system and significant aggra-vation of clinical disease; as opposed to CCL2/MCP-1 andCCL3/MIP-1a DNA vaccines that induced protection [13].Evidences show that CCL4/MIP-1b can recruit regulatoryCD4þ CD25þ regulatory T cells (Tregs) [25]. This cell pop-ulation is characterized by production of high levels of IL-10

and may be involved in the regulation of immune responses, asdemonstrated in diverse inflammatory models, as autoimmune[26], transplants [27] and allergies [28]. One study has sug-gested that Tregs are expressed preferentially in patientswho do not develop severe Chagas disease [29], but no studieshave formally demonstrated a role for these cells in experi-mental models of T. cruzi infection. Thus, an interesting pos-sibility raised from our studies is that CCL4/MIP-1b may berelevant for the recruitment of Tregs to the cardiac tissue ofinfected rats. Blockade of CCL4/MIP-1b with vaccine mayprevent migration of these cells and, hence, worsen infection.Further studies evaluating the role of Tregs and the role ofCCL4/MIP-1b for Tregs recruitment are necessary to resolvethese issues.

CCL4/MIP-1b binds only to CCR5. However, CCR5 is alsoa receptor for the chemokines CCL3/MIP-1a and CCL5/RANTES [30]. Moreover, CCL3/MIP-1a and CCL5/RANTESbind to additional receptors, including CCR1 [31]. In contrastto our findings, T. cruzi infection was much more severe inCCR5-deficient animals when compared to their wild-typecontrols [10]. It is unclear whether the apparent discrepantrole of CCL4/MIP-1b (in rats) versus CCR5 (in mice) block-ade represents a differential usage of chemokines and theirreceptors in different species. Alternatively, in the absenceof CCL4/MIP-1b, other chemokines, such as CCL3/MIP-1aand CCL5/RANTES, would still be available for activating

2754 E. Roffe et al. / Microbes and Infection 8 (2006) 2745e2755

the CCR5 receptor, which appears to be essential for leukocyteinflux during T. cruzi infection [10]. Preliminary data from ourlaboratory using DNA vaccination with plasmids containingCCL5/RANTES and CCL3/MIP-1a demonstrate that ratsindeed have greater heart parasitism after infection (our un-published observations). It is of note that the latter chemokinescan also activate other receptors, including CCR1. So, absenceof CCR5 and unopposed activation of CCR1 could be relevantto infection outcome. Indeed, treatment with Met-RANTES,a CCR1 and CCR5 blocker, resulted in reduced myocarditiswithout an increase in parasitism and parasitemia [8]. InCCR5�/� mice, T cell-mediated hepatitis was exacerbatedand accompanied by an increase in infiltration of CCR1þ cellsin the liver [32]. Thus, CCR5 activation appears essential forT. cruzi-associated inflammatory and regulatory cell influx.Whereas the influx of regulatory cells appears to be mediatedmainly by CCL4/MIP-1b, co-activation of CCR1 and CCR5by CCL3/MIP-1a and CCL5/RANTES are relevant for inflam-matory cell influx, control of parasite replication, and associ-ated with chronic fibrosis. Altogether, these results suggestthat an imbalance between the chemokines and chemokinereceptors expressed in the cardiac tissue may be a preponderantfactor in the perpetuation of the inflammatory process.

In conclusion, we have shown here that CCL4/MIP-1b isexpressed during the acute phase of T. cruzi infection ofrats. The neutralization of CCL4/MIP-1b using a DNA vaccineencoding for CCL4/MIP-1b was deleterious to the host, evi-denced by the increase in myocardial inflammation and highercollagen deposition. In contrast, CCL4/MIP-1b appeared notto be relevant for the ability of the host to deal with the acuteinfection. Our results suggest that CCL4/MIP-1b plays a rolein preventing excessive inflammation and pathology ratherthan in controlling parasite load.

Acknowledgements

We thank Mariana Sim~oes and Dr. Guilherme Correa Oli-veira (CPqRR/Fiocruz, Belo Horizonte, Brazil) for sequencingthe constructs, and Valdineria Borges, Luıza Silva and CarlosHenrique Silva, for technical support. This work receivedfinantial support from Fundac~ao de Amparo a Pesquisa doEstado de Minas Gerais (FAPEMIG, Brazil), Conselho Nacio-nal de Desenvolvimento Cientıfico e Tecnologico (CNPq,Brazil), Coordenac~ao de Aperfeicoamento de Pessoal de NıvelSuperior (CAPES, Brazil) and Fogarty International ResearchCollaboration Award (NIH/FIRCA, 1 R03-TW006857-01A1,USA).

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