Immunostimulatory DNA from Paracoccidioides brasiliensis

Acts as T-Helper 1 Promoter in Susceptible Mice

M. C. SOUZA, M. CORREÃ A, S. R. ALMEIDA, J. D. LOPES & Z. P. CAMARGO

Department of Microbiology, Immunology and Parasitology ± Federal University of SaÄo Paulo (UNIFESP), SaÄo Paulo, SP, Brazil

(Received 5 December 2000; Accepted in revised form 4 April 2001)

Souza MC, CorreÃa M, Almeida SR, Lopes JD, Camargo ZP. Immunostimulatory DNA from Paracocci-

dioides brasiliensis Acts as T-Helper 1 Promoter in Susceptible Mice. Scand J Immunol 2001;54:348±356

Th1 immune responses afford protection against some pathogens like the fungus P. brasiliensis (P.b.),

ethiological agent of Paracoccidioidomycosis (PCM). It is well known that nonmethylated CpG sequences

from bacterial DNA have immunomodulatory properties and can be used as a Th1-promoting adjuvant. By

analyzing the available gene sequences of P.b. we observed a high number of unmethylated CpG

dinucleotides. In a murine model of the PCM infection, the isogenic mouse strain known to be susceptible

presents a predominant Th2 pattern. In order to access the possibility of the genomic DNA to act as a Th1-

promoting adjuvant, in vitro assays were made and indicated a significant increase in phagocytosis when the

macrophages were stimulated with DNA from P.b. and in vivo assays of a decreased production of antibodies

antigp43, the main antigen of the PCM system. The analysis of the antibody isotypes and the cytokine

production suggested a Th1 modulation in the susceptible animals. Thus, when mice were infected with

fungus plus synthetic oligodeoxynucleotide (ODN), made from the available sequence of gp43, a decrease in

the fungus dissemination was observed. Results herein described suggest that genomic DNA from P.b. could

have a immunostimulatory function as a Th-1-promoting adjuvant in susceptible mice.

Dr Z. P. Camargo, Universidade Federal de SaÄo Paulo, Disciplina de Biologia Celular, 04023±062, Rua

Botucatu, 862, 88 andar, SaÄo Paulo, S.P., Brazil. E-mail: zoilo@ecb.epm.br

INTRODUCTION

The PCM is a deep mycosis characterized by a suppurative

granulomatous inflammation, suppression of cellular immunity

and high antibody titters [1, 2]. The disease may develop in a

multiple of forms, ranging from benign and localized to severe and

disseminated forms, depending on the extension of depression of

cellular immunity [2±4]. The etiological agent of this mycosis is

P.b., a thermally dimorphic fungus. Airborne fungal propaguli start

the infection with micelial forms that undergo conversion to the

yeast phase, the infective stage of P.b, in the lungs [4, 5].

Most exposed individuals develop an asymptomatic infection

(PCM infection) with well-organized granulomatous lesions in

the lungs and lymphonodes called primary complexes. In some

individuals these lesions contain many viable fungi that can

remain in a latent stage and, after a few years, can disseminate to

other organs and tissues. The acute form occurs when

dissemination happens quickly, producing high levels of humoral

immune response and depression of cell-mediated immunity

[4, 6]. The chronic forms present preserved cellular immunity and

moderate specific antibody response. Correlation between the

clinical forms and the pattern of immune response has been

investigated in both the human and the mouse model [2, 7].

Calich et al. (1985) [8] have established a genetically

controlled murine model of the PCM infection. The protective

immune responses have been shown to vary in the different

isogenic mouse strains after infection with P.b. Amongst these

strains A/Sn and B10A are known models of resistance and

susceptibility, respectively [4, 8, 9], and show diverging

immune responses, where the resistance mechanism was

associated with a cellular immune response and the activation

of phagocytes, whereas susceptibility was associated with the

preferential activation of B cells and an inefficient macrophage

activation [2, 10]. These B10.A susceptible mice present low

levels of interferon (IFN)-g concomitant with the production of

Th2-inhibitory cytokines that lead to a progressive disease with

many disorganized granulomatous lesions [2, 4, 11].

The cells that stimulate the functions of the Th1 and Th2 type

Scand. J. Immunol. 54, 348±356, 2001

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of the responses can be regulated by many factors such as dose

of antigen, antigen-presenting cells (APCs) and type of

adjuvant. It is known that Th1 response confers protection

against some pathogens and also against P.b., and that

macrophages have a fundamental role acting as the first line

of defence. This function depends on their activation state,

which will lead them to perform microbicidal activities [12, 13].

On the other hand, recent assays have shown stimulatory

properties of bacterial DNA through nonmethylated CG

sequences. These sequences stimulate mouse B cells and APCs,

enhance intradermal gene immunization and can be used as

Th1-promoting adjuvants [14, 15]. These properties of bacterial

DNA are not unique to bacteria and apply to a wide spectrum of

nonvertebrates including insects, nematodes, mollusks and

yeast, in contrast with DNA from various vertebrates such as

frogs and fishes, that are nonstimulatory [16, 17]. Methylation of

the cytosine from CG dinucleotides is critical for the immune

stimulation because treatment of the bacterial DNA or synthetic

oligonucleotides with DNA methylase abrogates in vitro

mitogenic activity and its stimulatory characteristics. The motifs

responsible for this phenomenon have the basic structure 5 0-Pu-

Pu-CpG-Pyr-Pyr-3 0 and are called CpG motifs or immune

stimulatory sequences (ISS) [15, 18, 19]. These ISS present in

microbial DNA enhance the immunogenicity of APCs [14, 20]

and lead to the induction of interleukin (IL)-1 and nitric oxide

(NOi) in IFN-g-treated macrophages [20].

Evidences concerning the immunostimulatory properties of

bacterial DNA are well known, but there are scarce information

about pathogenic fungi DNA such as P.b. In the present work,

we analyzed the available gene sequences from P.b. and

observed that CpG motifs were present in large numbers.

Results herein presented show that these sequences induced a

Th1 response with the increase of IFN-g and IgG2a, and with a

decrease of specific humoral response during the infection of

susceptible mice. In addition, susceptible mice infected with P.b.

and stimulated by synthetic oligodeoxynucleotides (ODN),

containing unmethylated CpG motifs, resulted in the control

of the infection, and fungus was found only in the lungs. In

contrast, the group infected with the fungus and unstimulated

with ODN evolved with dissemination to the spleen and liver.

MATERIALS AND METHODS

Animals and infection. B10.A male mice, 8±12-weeks-old, were

obtained from the SaÄo Paulo University animal facilities. Groups of

five B10.A mice were used, infected intraperitoneally with 5 � 105

yeast forms of P.b. (isolate 1914) suspended in phosphate-buffered

saline (PBS), stimulated or unstimulated with genomic nondenatured

DNA from P.b (100 mg/animal) or synthetic oligonucleotides (ODN

2960) at a concentration of 20 mg/animal. Blood was collected weekly

and stored at 2 70 8C until use.

Preparation of DNA. E. coli DNA was purchased from sigma (St.

Louis, MO, USA) and herring sperm DNA were purchased from Life

Technologies (Bethesda, MD, USA). DNA from P.b. was obtained as

described elsewhere [21]. Briefly, frozen cell mass (equivalent to 10 g

wet weight) was obtained from 7 days of filtrated culture of yeast forms

was transferred to a mortar containing liquid N2. The frozen cell mass was

ground to a fine powder. The contents were subsequently mixed with a lysis

buffer (50 mm Tris-Cl, 100 mm EDTA, 5% sarcosyl and 0.5 mg/ml of

proteinase K), the cell lysate was transferred to a Potter-Elvehjem (Tomas,

Philadelphia, USA) and homogenized for 15±30 min in an ice bath. The tube

with lysate was then placed in a water bath (56 8C) for 2 h and the cell debris

were precipitated by centrifugation at 3000 � g before collecting the

supernatant. The supernatant was extracted with an equal volume of

phenol : choroform: isoamyl alcohol (50 : 49 : 1) for 30 min and

centrifuged for 30 min at 12 000 � g at 4 8C to remove the cell wall

insoluble components. 0.1 volume of 3 m sodium acetate, pH 5.2, and 2

volumes of ice cold ethanol supernatant was then added and it was kept

overnight at a temperature of 2 20 8C. The large DNA molecules

precipitated were centrifuged for 30 min at 12 000 � g and the pellet was

washed with 70% ethanol, air dried, resuspended with 10 mm Tris-Cl, 1 mm

EDTA and digested with Rnase (Sigma) (100 mg/ml). The last procedure

was to extract once with phenol : choroform (1 : 1, v/v), twice with

choroform : isoamyl alcohol (49 : 1) and finally precipitating the DNA with

ethanol as described above. The DNA concentrations were determined

spectrophotometrically by optical density (OD) analysis. Purified DNA

(100 mg) was treated with Dnase for 2 h at 37 8C following the

manufacturer's instructions and stored at 2 20 8C until use.

Oligodeoxynucleotides. Oligodeoxynucleotide (ODN 2960: CATA

5 0GTATGAGAGCGTTAGGCA-3 0) was made of available sequences

of the gp43 gene containing unmethylated CpG motifs and was

purchased from the Biophysics Department of SaÄo Paulo Federal

University. The oligodeoxynucleotide was purified by precipitation

with 3 m sodium acetate and absolute ethanol, air-dried and

resuspended in sterilized PBS until use.

Phagocytic assays. Peritoneal macrophages from naive mice were

obtained as described elsewhere [22]. Briefly, the macrophages were

isolated by selective adherence to glass slides (5 � 105 cells/well) for

30 min at 37 8C with RPMI 1640 (Sigma Chemical) containing 10%

heat-inactivate fetal bovine serum (FBS; Gibco, Paisley, UK) and 1024M

2-mercaptoethanol (Sigma). The glass coverslips were rinsed with PBS

for removal of nonadherent cells and cultured with fresh medium for 8 h

with 2 � 106 P.b. cells/well stimulated or unstimulate with P.b. DNA (50

and 100 mg/well). Subsequently, non internalized P.b. were removed, and

the glass coverslips were fixed in 1.5% glutaraldehyde and stained with

Giemsa. An average of at least 100 macrophages were counted to

determine the phagocytic index (PI), calculated as the number of

phagocytic cells times mean number of internalized particles.

Measurement of nitrite concentration. The concentration of nitrite

(NO22) in peritoneal macrophages (3 � 106/ml) supernatants was

measured by a microplate Griess assay as described previously [23].

Briefly, 100 ml of the supernatants cultured at 37 8C in an atmosphere of

5% CO2 with RPMI plus FBS for 24, 48 and 72 h and P.b. cells (1.5 � 106/

ml) in the presence or absence of 100 mg of P.b. DNA, 50 mg of E. coli

DNA or 500 U/ml of IFN-g were incubated with an equal volume of the

Griess reagent at room temperature. The absorbance was determinated with

a Multiskan MCC/340 II EIA reader (Flow Laboratories Inc., McLean,

Virginia, USA). The NO22 concentration was determined using a standard

curve of 1±200 mm NaNO2. For in vivo assays, mice were immunized

intraperitoneally with 100 mg of P.b. DNA, macrophages were harvested

and NO measured as described above.

Fungicidal activity of macrophages. Peritoneal macrophages were

obtained and cultured with P.b. in plate dishes for 24, 48 and 72 h as

described above. Non-phagocytosed fungi were removed and fungicidal

activity was measured by the disruption of these cells with 200 ml of

distilled and sterilized water for 2 min with the volume, then completed

q 2001 Blackwell Science Ltd, Scandinavian Journal of Immunology, 54, 348±356

Immunostimulatory DNA from P. brasiliensis 349

with 800 ml of PBS. The cellular suspensions were plated on BHI agar

plus 1% of streptomicyn and penicilyn antibiotics supplemented with 5%

FBS and 5% P.b. culture filtrate (growth-factor), as described elsewhere

[24]. The plates were incubated at 37 8C and the colonies were counted

from the day 5 until no increase in the counts was observed (day 30).

ELISA assays for specific humoral response and isotyping of antibody

production in infected animals. During week 8 of the mice infection the

antibody response (IgG antigp43) was measured by an ELISA assay as

described previously [25]. Briefly, polystyrene plates (Nunc, Roskilde,

Denmark; MaxiSorpe surface) were coated with 250 ng/well of purified

gp43 as antigen and 5% fat-free dehydrated skim milk in PBS was used

for blocking. Serum (1 : 50 dilution) from mice infected with P.b. yeast

stimulated or unstimulated with P.b. DNA and P.b. DNA alone (control)

were quantified with an antimouse Ig-biotin-streptavidin-peroxidase

system (Sigma Chemical). For isotyping of these antibodies an ELISA

isotyping panel was used according to the manufacturer's instructions

(Bio-Rad Laboratories, Richmond, CA, USA).

Cytokine determination. IFN-g and IL-4 were determined in those

sera after the week 8 of the infection of mice by ELISA according to the

manufacturer's instructions (Pharmingen, San Diego, CA, USA).

Cytokine activities were determined employing standard curves with

serial dilutions of mouse recombinant IFN-g and IL-4.

Fungal loads in organs of infected animals. At the week 12 the groups of

infected animals were killed and the fungal loads in the lungs, liver and

spleen were determinate by colony-forming units (CFU). Briefly, organs

were collected, weighed and disrupted using a tissue grinder (Coler-Parmer

International, Vernon Hills, Illinois, USA). The cellular suspensions were

plated on BHI agar plus antibiotics and 5% FBS and growth factors

according to Singer-Vermes et al. (1992) [24]. Colonies were counted from

day 7 until no increase in the counts was observed. The results were

expressed in logarithmic values of the CFU counts/g of tissue.

Statistical analysis.. Statistical data were obtained by analysis of

variance (anova) and by the Turkey±Kramer test. All values were

reported as the mean ^ standard deviation error of the mean with

significance in the range of P , 0.05.

RESULTS

In vitro assays

In order to verify the CpG methylation of the P.b. DNA, we

examined the sensitivity of DNA to digestion with the restriction

enzyme, HpaII, as previously described [16]. Results showed

that P.b. CpG DNA was sensitive to this restriction enzyme,

confirming that cytosine in P.b. DNA is unmethylated and could

be immunestimulatory in the PCM infection (data not shown).

Phagocytic assays

Effects of P.b. DNA in the phagocytic activity was evaluated

and the phagocytosis index (PI) observed was significantly

higher (P , 0.001) for macrophages stimulated with P.b. DNA

in both concentrations assayed (50 and 100 mg). Figure 1 shows

that the macrophages stimulated with P.b. DNA had higher

phagocytic indexes than unstimulated cultures (culture medium

only). When macrophages were incubated with P.b. DNA

treated with Dnase, no stimulation was observed.

Inhibition of macrophages NO production by P.b. DNA

In these experiments, we initially stimulated murine peritoneal

macrophages by P.b. DNA, bacterial DNA [(E. coli (EC DNA)]

and herring sperm DNA (Her DNA) for 24, 48 and 72 h and

then measured the NO22 contents in the culture supernatants.

The stimulation of macrophages with P.b. DNA resulted in a

decreased NO production, especially at a concentration of

50 mg, while, as shown in Fig. 2 macrophages stimulated by E.

coli DNA, Her DNA or unstimulated had very similar results.

These data suggest that the increase of PI did not correlate with

an increase in NO production by P.b. DNA.

Fungicidal activity of macrophages

To investigate whether the NO inhibition correlated with the

fungicidal activity of macrophages, we studied its fungicidal

capacity by viable yeast recaptured from disrupted cells.

Figure 3 shows that the decreased levels of NO in supernatants

were correlated with an increase of yeast colonies. These assays

showed increased numbers of colonies at 48 and 72 h of

Fig. 1. Phagocytosis of P. brasiliensis by

macrophages stimulated with 50 mg e

100 mg P.b. DNA and Dnase treated P.b.

DNA after 8 h. The results are representative

of three independent experiments and

expressed as arithmetic mean. *P , 0.001

when compared with the unstimulated group.

350 M. C. Souza et al.

q 2001 Blackwell Science Ltd, Scandinavian Journal of Immunology, 54, 348±356

phagocytosis in macrophage cultures stimulated with P.b. DNA,

especially at 50 mg of concentration, when compared with those

that were unstimulated. These data correlate with data of NO

production by P.b. DNA, because a higher decrease was

observed at 50 mg.

In vivo assays

Humoral specific response in stimulated and unstimulated

mice. Considering that the cellular immune response is

correlated with the resistance of infection by P.b. and

susceptible animals present high antibody levels in the PCM

infection, Fig. 4 shows decreased antibody levels in animals

stimulated by P.b. DNA and infected intraperitoneally with P.b.

cells. Sera from these animals were evaluated weekly by ELISA

and at week 8 a decrease in antibody production by the

stimulated group was observed, in contrast to the high antibody

level observed in the unstimulated group. Levels of specific

isotyping antibody production were assessed in stimulated and

unstimulated animals during the course of disease. Figure 5(A)

shows that in mice stimulated with P.b. DNA., the levels of

IgG2a, a Th1 marker, increased significantly. On the other hand,

the unstimulated group presented high levels of IgG2b

(Fig. 5B).

Cytokines production by susceptible animals stimulated or

unstimulated with P.b. DNA and infected with P. brasiliensis

yeast cells. Figure 6(A) shows a significant increase of IFN-g

production in mice stimulated with P.b. DNA when compared to

the unstimulated group, suggesting a switch to Th1. On the other

hand, no significant difference in the Th2 cytokine (IL-4) was

observed (Fig. 6B). Taken together, these results, and others on

the decreased antibody production as well as presence of

specific Th1 antibody described above, suggest that the immune

response in susceptible mice has a strong tendency to Th1 type

of immune response, instead of the previous Th2 type.

Fungal loads in organs of the infected animals. To assess in

vivo effects of mice stimulated by P.b. DNA in PCM infection,

assays to determine fungal loads in organs were made by CFU

counting. As shown in Fig. 7, mice stimulated by synthetic

oligodeoxynucleotides (ODN 2960) and infected with P.b.

presented fungal CFUs only in the lungs. In contrast,

unstimulated animals showed high amounts of fungal CFUs in

the lungs, and also fungal CFUs in the liver and spleen. These data

show a protective role of CpG motifs in P.b. DNA, because ODN

2960 represents the gp43 gene sequences, a main antigen of P.b.

NO production by macrophages stimulated with IFN-g and

P.b. DNA. This experiment was carried out in order to

investigate whether macrophages previously stimulated with

Fig. 2. Production of the nitric oxide (NO) at

supernatants macrophages culture stimulated

with 30, 50 and 100 mg of the P.b. DNA,

50 mg E. coli DNA (EcDNA) and 50 mg of

the Herring sperm DNA (HerrDNA) at

different times. The results are representative

of three independent experiments and

expressed as arithmetic mean.

Fig. 3. Fungicidal activity of macrophages

stimulated with P.b. DNA (50 and 100 mg)

50 mg E. coli DNA and 500 U of IFN-g. The

results are representative of three

independent experiments and expressed as

arithmetic mean.

q 2001 Blackwell Science Ltd, Scandinavian Journal of Immunology, 54, 348±356

Immunostimulatory DNA from P. brasiliensis 351

IFN-g could increase NO production when incubated with P.b.

DNA. Figure 8(A) shows an increase of NO secretion when

macrophages were stimulated in vitro with 500 U of IFN-g. In

order to investigate the effect of P.b. DNA in the NO production

in vivo, mice were stimulated with P.b. DNA and their

macrophages were then harvested and the NO measured.

Figure 8(B) shows an increased NO production in mice

immunized with P.b. DNA when compared to naive

macrophages.

DISCUSSION

The resistance against certain diseases including PCM, is

correlated with the lymphokines produced by Th1 lymphocytes

[26, 27]. The murine model of resistance showed a sustained

secretion of IL-2 and IFN-g as well as a preferential IgG2a

production. In contrast, susceptible B10.A mice presented a

negligible IFN-g secretion and preferentially produced IgG2b

[9]. On the other hand, CpG DNA (ISS motifs) from bacteria

and other invertebrates have shown the ability to modulate the

differentiation of Th responses. In pathogenic fungi, the

presence and stimulatory properties of CpG DNA have not yet

been reported. In the present work, we show that ISS motifs are

present in P.b. and can have stimulatory effects during PCM

infection in B10.A susceptible mice.

The ability of P.b. ISS motifs to induce in vitro stimulation of

macrophages was studied, and the results showed that these ISS

were able to increase the phagocytic capacity of macrophages

from B10.A mice, reaching the best stimulation at 50 and

100 mg of P.b. DNA. In order to verify whether this stimulation

was really owing to the ISS present in P.b. DNA, purified DNA

was treated with Dnase and the macrophage stimulation was the

same as that observed in the unstimulated cells. These results

provided the first direct evidence of the immunomodulation of

cells from susceptible animals by P.b. CpG DNA, because these

animals normally present an impaired macrophage activation

[28].

Previous investigations have already shown that protection

against bacterial [29, 30], protozoan [31] and fungal [32±34]

infections correlate directly with the expression of cell-mediated

immune response and parasite killing by activated macrophages

[28]. So, in our study, the next step was to verify the fungicidal

activity (dependent of the NO production) of macrophages

stimulated by P.b. DNA, EC DNA or IFN-g in macrophage

culture supernatants after P.b. yeast phagocytosis. We observed

a significant decrease in NO concentration at 24, 48 and 72 h in

the group stimulated with P.b. DNA, mainly at a 50 mg

concentration. On the other hand, macrophages stimulated with

E. coli DNA or IFN-g increased NO production. These results

showed different ways of action in P.b. DNA and bacterial DNA.

Different mechanism of bacterial DNA and other DNAs in

mitogenicity to B cells have been studied and these studies

showed that DNA from various organisms varied considerably,

i.e. E. coli DNA was 100-fold more potent than yeast DNA (S.

pombe) [16]. In such assays made by us P.b. DNA was unable of

induce B-cell stimulation (data not shown). They showed that

the actions of bacterial DNA and P.b. DNA were different, at

least in what concerns NO production as well as B-cell

stimulation. Therefore, the fungicidal activity of stimulated

macrophages decreased and resulted in higher amounts of CFUs

(at 50 mg and 100 mg of P.b. DNA) than in unstimulated

macrophages. These data indicated that the decreased NO was

dose-dependent, because only at 50 mg or higher concentrations

correlated with decreased NO production and increased amounts

of CFUs.

It has been accepted that NO or related Nos produced by

activated macrophages have an important role in the killing of

different pathogens [35]. A high production of NO has been

associated with the suppression of the proliferative response to

ConA in cultures of splenocytes from P.b.-infected mice [40].

These data suggest that NO may modulate the immune response

to the P.b. infection in the murine model [13] and our findings

suggest that these ISS motifs present in P.b. DNA could decrease

the macrophage NO production in the first step of the infection,

increasing the Th proliferative cellular immune response.

Previous studies demonstrated that monocyte-derived cells

were directly activated by CpG motifs to secrete the cytokine

IL-12 and IFN type I, and natural killer (NK) cells respond with

increased lytic activity and IFN-g secretion, enhancing the

protective immune response [46, 47]. Thus, CpG motifs can

activate other cells such as NK cells and these cells could

secrete IFN-g, an important cytokine to promote the Th1 shift.

In our model it is tempting to speculate that other cells, such

as NK cells, could activate macrophages and stimulate the NO

secretion, increasing their fungicidal activities as well as

promoting protective immune response. Our results confirmed

this hypothesis, because in macrophages from mice that were

stimulated with P.b. DNA a significant increase of NO

production was observed after 48 h, and these cells activated

in vitro with IFN-g and stimulated with P.b. DNA, increased the

NO production.

On the other hand, in our study susceptible mice stimulated

Fig. 4. Inhibition of antigp43 titres in mice stimulated with P.b. DNA

and infected with yeast form of P. brasiliensis at week 8 of the infection.

The results are representative of three independent experiments and

expressed as arithmetic mean. *P , 0.001 when compared with

unstimulated mice.

352 M. C. Souza et al.

q 2001 Blackwell Science Ltd, Scandinavian Journal of Immunology, 54, 348±356

with P.b. DNA showed a decreased antibody production when

compared to the group of unstimulated mice, showing an in vivo

immunomodulation capacity, because these animals presented a

higher level of antibody production in the course of the PCM

infection.

Results presented here suggest a shift of immune response to

Th1, which could be confirmed by verifying gp43-antigen-

specific antibody isotypes and cytokines production. Mice

infected with fungus and stimulated with P.b. DNA presented

a significant increase of IgG2a levels, which is an Th1-

associated isotype, while unstimulated animals showed in-

creased IgG2b levels.

Analyses of cytokine production profiles and immunopatho-

logical features displayed by susceptible mice indicate that the

susceptibility is linked to an early production of inhibitory

mediators that downregulate the IFN-g production and suppress

the development of protective Th1 type of response [41].

When the levels of cytokines (IFN-g and IL-4) in the sera of

these animals were analyzed, a significant increase of IFN-g

was observed in those animals stimulated with P.b. DNA, but no

significant differences were observed in the IL-4 production.

These results showed that in susceptible mice, which presented a

predominant Th2 response, it was possible to switch the immune

response pattern by using P.b. DNA in the beginning of the

infection, where a Th1 response was predominant with higher

production of IFN-g and IgG2a isotype.

In vitro assays have showed that IFN-g plays a pivotal role

against several pathogens [42, 43] including P.b. [44] through

macrophage activation. Treatment of infected mice with high

amounts of anti-IFN-g resulted in an increased fungal burden [28].

In a well-studied murine leishmaniasis model, the predomi-

nant Th1 response in C57BL/6 mice confer resistance to

infection with Leishmania major, while in BALB/c mice, Th2

responses lead to susceptibility. Recently, Zimmerman et al.

(1998) used the CpG ODN from an antigenic component of

L. major for the induction of protection in susceptible mice, and

they showed a switch to a Th1 response with a high production

of IFN-g.

In order to verify the ability of synthetic CpG ODN, from

available sequences of gp43, in conferring resistance in vivo,

mice were infected with fungus and stimulated with ODN and

fungal load in different organs was determined by CFU method.

In the group of mice stimulated with P.b. DNA, the infection

was limited to the lungs. In contrast, when unstimulated mice

were infected with P.b., a disseminated infection with high

amounts of CFUs in the lungs and spleen and lower fungal

CFUs in the liver was observed.

In several infectious diseases, the ability to direct the Th1 or

Th2 differentiation of the antigen-specific immune response has

significant implications for the therapy. In murine leishmaniasis,

susceptible mice can be made resistant by the administration of

IL-12 [48] or ODN [49]. Thus, CpG ODN may be useful as an

adjuvant in order to induce the protective Th1 immunity in

several disease models.

Fig. 5. Anti-gp43 IgG2a (A) and IgG2b (B)

isotype production in mice stimulated and

infected with P. brasiliensis at week 8 of the

infection. The results are representative of

three independent experiments and expressed

as arithmetic mean. *P , 0.01 when

compared with the unstimulated mice.

q 2001 Blackwell Science Ltd, Scandinavian Journal of Immunology, 54, 348±356

Immunostimulatory DNA from P. brasiliensis 353

It is clear from our findings that P.b. DNA or CpG ODN has a

highly efficient role in inducing the protection in the experi-

mental model of the susceptible mice, and that this protection

was mediated by the production of IFN-g. Although it is too

early to propose a definitive role for the P.b. DNA in promoting

a more effective immune response against PCM infections,

results shown here suggest that such a response may be induced

by using this novel kind of adjuvant.

ACKNOWLEDGMENTS

We are indebt to Creuza Rosa de Oliveira and Maria de Jesus

Silva Barbosa for the technical assistance.

This work was supported by Conselho Nacional para o

Desenvolvimento CientõÂfico e TecnoloÂgico (CNPq), and Funda-

cËaÁs de Amparo aÁ Pesquisa de Estade de SaÄo Paulo (FAPESP).

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Fig. 6. (A) Increase of IFN-g concentration in sera of mice stimulated

with DNA and infected with P. brasiliensis. (B) Concentration of IL-4

cytokine in sera of stimulated/infected mice. The results are

representative of three independent experiments and expressed as

arithmetic mean. *P , 0.01 when compared with the unstimulated

mice group.

Fig. 7. Protective effect of immunization with synthetic

oligodeoxynucleotides against intraperitoneal infection by

P. brasiliensis. The individual CFUs are shown with averages as

horizontal bars.

Fig. 8. (A) Production of NO at supernatants macrophages culture

stimulated with P.b. DNA (100 mg), IFN-g (500 U) or P.b. DNA plus

IFN-g in different times. (B) Production of NO at supernatants

macrophages culture from naive and immunized mice (100 mg DNA).

The results are representative of three independent experiments and

expressed as arithmetic mean.

354 M. C. Souza et al.

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