Leishmania amazonensis impairs DC function by inhibiting CD40 expression via A2B adenosine receptor activation

Authors

  • Amanda B. Figueiredo,

    1. Laboratório de Imunoparasitologia, Departamento de Ciências Biológicas, ICEB/NUPEB, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil
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  • Tiago D. Serafim,

    1. Laboratório de Imunoparasitologia, Departamento de Ciências Biológicas, ICEB/NUPEB, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil
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  • Eduardo A. Marques-da-Silva,

    Corresponding author
    1. Laboratório de Imunoparasitologia, Departamento de Ciências Biológicas, ICEB/NUPEB, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil
    • Correspondence: Dr. Luís Carlos Crocco Afonso, Laboratório de Imunoparasitologia, Departamento de Ciências Biológicas, ICEB/NUPEB, Universidade Federal de Ouro Preto, Campus do Morro do Cruzeiro, 35400–000 Ouro Preto, Minas Gerais, Brazil Fax: +55-31-3559-1680 e-mail: afonso@nupeb.ufop.br

      Current address: Dr. Eduardo A. Marques-da-Silva, Laboratório de Imunovirologia Molecular, Departamento de Biologia Geral, CCB, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil

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  • José R. Meyer-Fernandes,

    1. Laboratório de Bioquímica Celular, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
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  • and Luís C. C. Afonso

    Corresponding author
    1. Laboratório de Imunoparasitologia, Departamento de Ciências Biológicas, ICEB/NUPEB, Universidade Federal de Ouro Preto, Ouro Preto, Minas Gerais, Brazil
    • Correspondence: Dr. Luís Carlos Crocco Afonso, Laboratório de Imunoparasitologia, Departamento de Ciências Biológicas, ICEB/NUPEB, Universidade Federal de Ouro Preto, Campus do Morro do Cruzeiro, 35400–000 Ouro Preto, Minas Gerais, Brazil Fax: +55-31-3559-1680 e-mail: afonso@nupeb.ufop.br

      Current address: Dr. Eduardo A. Marques-da-Silva, Laboratório de Imunovirologia Molecular, Departamento de Biologia Geral, CCB, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil

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Abstract

Dendritic cells (DCs) play an essential role in the modulation of immune responses and several studies have evaluated the interactions between Leishmania parasites and DCs. While extracellular ATP exhibits proinflammatory properties, adenosine is an important anti-inflammatory mediator. Here we investigated the effects of Leishmania infection on DC responses and the participation of purinergic signalling in this process. Bone marrow-derived dendritic cells (BMDCs) from C57BL/6J mice infected with Leishmania amazonensis, Leishmania braziliensis or Leishmania major metacyclic promastigotes showed decreased major histocompatibility complex (MHC) class II and CD86 expression and increased ectonucleotidase expression as compared with uninfected cells. In addition, L. amazonensis-infected DCs, which had lower CD40 expression, exhibited a decreased ability to induce T-cell proliferation. The presence of MRS1754, a highly selective A2B adenosine receptor antagonist at the time of infection increased MHC class II, CD86 and CD40 expression in L. amazonensis-infected DCs and restored the ability of the infected DCs to induce T-cell proliferation. Similar results were obtained through the inhibition of extracellular ATP hydrolysis using suramin. In conclusion, we propose that A2B receptor activation may be used by L. amazonensis to inhibit DC function and evade the immune response.

Introduction

Leishmaniasis is a group of diseases that are caused by obligate intracellular parasites of the genus Leishmania. Leishmania infections can result in a wide spectrum of clinical manifestations and disease outcome is determined both by the parasite species and by the host immune response. Leishmania amazonensis differs from other species because it is able to cause chronic non-healing lesions in mouse strains genuinely resistant to other Leishmania species, such as Leishmania braziliensis and Leishmania major [[1-3]]. In humans, L. amazonensis causes diffuse cutaneous leishmaniasis, a condition characterised by a lack of antigen-specific T-cell responses against the parasite [[4]].

DCs play a critical role in orchestrating the innate and adaptive components of the immune system [[5, 6]]. Immature DCs capture and process antigens located throughout the body. After contacting microorganisms or other inflammatory substances, these cells initiate their maturation process. DC maturation is characterised by increased expression of major histocompatibility complex (MHC) class II (MHCII) and co-stimulatory molecules, such as CD40, CD80, CD86 and CD54; decreased phagocytic capacity; increased cytokine secretion and expression of different chemokine receptors [[5, 7]].

Extracellular ATP, which is accumulated following cellular injury, has important inflammatory properties characterised by the induction of IL-12 and TNF-α production [[8-11]]. On the other hand, adenosine has been recognised as an immunomodulatory molecule that inhibits the release of proinflammatory cytokines and induces the release of IL-10 [[9, 12, 13]]. The combined action of CD39, which sequentially hydrolyses ATP to ADP and then to AMP, and CD73, which converts AMP to adenosine is essential in controlling extracellular levels of ATP [[9]]. Recently, regulatory T cells have been shown to express CD39 and CD73 on their surface and use adenosine to control effector T-cell activation [[12]].

DCs exhibit surface expression of ectonucleotidases, functional ATP receptors [[14]] and adenosine receptors [[15-17]]; consequently, they are sensitive to nucleotides and nucleosides present in the extracellular environment. Studies have demonstrated that ATP is able to induce increased surface expression of MHCII, CD83, CD86 and CD54 in DCs, thus promoting the establishment of inflammatory responses [[18, 19]]. Adenosine, on the other hand, reduces DC migration and inhibits DC cytokine and chemokine production [[20, 21]].

Several studies have evaluated the interactions between Leishmania parasites and DCs. L. amazonensis promastigotes modulate DC functions by altering MHCII, CD80 and CD86 expression and IL-10 and IL-12 production [[22-25]]. Studies with other Leishmania species have shown that L. braziliensis-infected DCs remained immature and secreted TNF-α [[26]]. L. major was also able to modulate DC function by influencing the balance between Th1 and Th2 responses [[27, 28]]. Although much is known regarding the ability of Leishmania infection to interfere with DC maturation, activation and function, the underlying mechanisms are not completely understood.

Given the known effects of adenosine on DC function, the present study further evaluated the effects of Leishmania infection on the activation and effector functions of DCs by analysing the involvement of purinergic signalling in the process. Our results indicate that the hydrolysis of extracellular ATP and the activation of the A2B adenosine receptor during L. amazonensis infection but not during L. major or L. braziliensis infection, are responsible, at least in part, for the inhibitory effects of the parasite on DC function.

Results

Infection with Leishmania metacyclic promastigotes alters DC surface marker expression

Our initial objective was to assess whether promastigotes of different Leishmania species could infect DCs. Bone marrow-derived dendritic cells (BMDCs) were cultured in the presence of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled L. amazonensis, L. braziliensis or L. major metacyclic promastigotes (1:3 cell to parasite ratio); the infections were then evaluated using flow cytometry. After a 20 h incubation period, approximately 35–45% of DCs were infected; the results were similar among the three parasite species. Importantly, all treatments performed in this work did not interfere with parasite infectivity. Furthermore, DC viability, as assessed using trypan blue exclusion or propidium iodide uptake, was not changed by infection (data not shown).

Different Leishmania species are known to be able to interfere with DC surface marker expression [[22-26]]. However, in most of these studies, bulk effects were analysed and no differentiation was made between cells that became infected and cells that, albeit being in contact with the parasite, remained uninfected was made. To better clarify these findings situation, DCs were incubated with CFSE-labelled parasites for 3 h and then stimulated with LPS. As shown in Fig. 1A, the populations of infected (CFSE+) and uninfected (CFSE) cells were analysed separately. DC surface expression of MHCII and CD86 was significantly decreased in infected cells (Fig. 1B). Reduced surface expression was observed both in terms of the percentage of activated cells (MHCII+CD86+ cells) and in the intensity of expression of these markers in activated infected cells (Fig. 1C and D); these effects were observed regardless of the parasite species examined.

Figure 1.

Leishmania infection impairs DC activation. DCs obtained after 9 days of culture with GM-CSF were infected with CFSE- or PKH26-labelled metacyclic promastigotes (1:3 cell to parasite ratio). After 3 h, the cells were stimulated with 2 μg/mL LPS and then incubated for up to 17 h before flow cytometry analysis. (A) DCs were gated into populations of uninfected (CFSE or PKH26 cells) and infected (CFSE+ or PKH26+ cells) DCs and surface markers MHCII, CD86 and CD40 were analysed in both populations. Dot plots and histograms are representative of L. amazonensis-infected DCs. (B) The percentage of MHCII+CD86+ cells was evaluated, as well as the MFI of (C) MHCII and (D) CD86 in these cells. (E) The percentage of CD40+ cells in populations of uninfected and infected DCs was also examined. (B–E) The results represent the mean + SD from at least three independent experiments performed in triplicate. *p < 0.05 between uninfected and infected DCs, unpaired two-tailed Student's t-test.

We also evaluated CD40 surface expression in infected DCs. Interestingly, our results showed that, despite inhibiting MHCII and CD86 expression, L. braziliensis and L. major infection induced a significant increase in CD40 expression. In contrast, increased CD40 surface expression was not observed in L. amazonensis-infected DCs (Fig. 1E).

These results show that the level of DC functional impairment is dependent on the parasite species and that it is more intense with L. amazonensis compared with L. braziliensis or L. major.

Inhibition of DC activation by L. amazonensis is independent of IL-10

To investigate the mechanisms involved in the inhibition of DC activation by Leishmania parasites, we first examined the influence of autocrine IL-10 production. Several publications have implicated this cytokine as a major factor in DC suppression [[25, 29, 30]]. Therefore, we initially analysed the production of IL-10 in culture supernatants using ELISA and found no differences between control and infected DC cultures (Fig. 2A). However, because changes in DC surface markers were restricted to infected cells, we decided to evaluate the effect of IL-10 on DC activation by blocking the IL-10 receptor using an IL-10R-specific monoclonal antibody prior to DC infection. This treatment was unable to reverse the inhibition of DC activation induced following L. amazonensis infection. However, IL-10 receptor blockade partially restored LPS-induced upregulation of MHCII and CD86 expression in L. braziliensis- and L. major-infected DCs (Fig. 2B). Additionally, IL-10 blockade did not affect CD40 expression in infected DCs regardless of the parasite species examined (data not shown). These results suggest that distinct inhibitory mechanisms are used by different Leishmania species to impair DC activation. While L. braziliensis- and L. major-mediated effects on DC activation were partially dependent on IL-10, this cytokine does not play a role in L. amazonensis-mediated DC inhibition.

Figure 2.

Inhibition of DC activation by L. amazonensis is independent of IL-10. (A) DCs were infected as described in Fig. 1 and IL-10 cytokine levels were measured in the supernatants after 20 h using an ELISA. Control is LPS-stimulated DCs. The results represent the mean + SD from six independent experiments performed in duplicates. (B) DCs were incubated for 30 min with 1B1.3a antibody to block the IL-10 receptor; rat IgG was used as isotype control. DCs were then infected as described in Fig. 1 and analysed using flow cytometry. The percentage of MHCII+CD86+ cells in populations of uninfected and infected DCs in treated groups were expressed in relation to the group of cells incubated with parasites without treatment, set at 100 (dotted line). Data represent the mean + SD from at least three independent experiments performed in duplicates. *p < 0.05 versus untreated group, one-sample t-test with hypothetical value of 100. (C) Representative dot plots from at least three independent experiments of DCs infected with L. amazonensis (top), L. braziliensis (middle) or L. major (bottom) in the absence (left) or presence (right) of 1B1.3a antibody. Numbers in parentheses indicate the percentage of MHCII+CD86+ DCs.

Leishmania infection enhances DC ectonucleotidase expression and enzymatic activity

On the basis of the observations that DCs express ectonucleo-tidases on their surface [[14]] and that these enzymes play important roles in the suppressor functions of regulatory T cells [[8]], we evaluated the expression of these enzymes on the surface of uninfected and infected DCs by examining the surface expression of the ectonucleoside triphosphate diphosphohydrolase (ENTPDase) CD39 and the 5’-nucleoside monophosphate phosphohydrolase (5’-nucleotidase) CD73. Our results show that co-expression of CD39 and CD73 was significantly increased in infected cells, regardless of the parasite species examined (Fig. 3A).

Figure 3.

Leishmania infection increases DC ectonucleotidase expression and activity. (A) DCs were infected as described in Fig. 1 and analysed using flow cytometry. DCs were gated into populations of uninfected (CFSE cells) and infected (CFSE+ cells) DCs and the percentage of CD39+CD73+ cells evaluated in both populations. The results represent the mean + SD from four independent experiments performed in triplicate. *p < 0.05 between uninfected and infected DCs, paired two-tailed Student's t-test. (B–D) DCs were infected as described in Fig. 1, but at a ratio of ten parasites per cell. After 20 h of infection, cells were washed and incubated with (B) ATP, (C) ADP or (D) AMP for 1 h at 30°C, in the absence (untreated) or presence (+ suramin) of suramin. Ectonucleotidase activity was then assessed by measuring Pi release, as described in the Materials and Methods. The results represent the mean + SD from three independent experiments performed in triplicate. #p < 0.05, unpaired two-tailed Student's t-test. *p < 0.05 between (untreated) and (+ suramin) groups, unpaired two-tailed Student's t-test.

To confirm that the increased expression of CD39 and CD73 in infected DCs is accompanied by increased adenosine production, we evaluated the ectonucleotidase activity of these cells after 20 h of incubation in the presence or absence of parasites. Because some of the effects on DC activation were unique to infected cells and less than 50% of cells were infected after 20 h of incubation, we increased the percentage of infected cells to ensure that the effects of the infection process were not diluted by the cells that did not internalise parasites. Therefore, we used a 1:10 cell to parasite ratio that resulted in approximately 80% of cells being infected; this did not alter the parameters of parasite-mediated inhibition of DC activation (data not shown). Hydrolysis of ATP, ADP and AMP was significantly increased in infected DCs, regardless of the parasite species used. Treatment with suramin significantly inhibited the ATP and ADP hydrolysis but had no effect on AMP, which is indicative of its action on CD39 (Fig. 3B to D).

After showing that Leishmania parasites increase ectonucleo-tidase expression in BMDCs, we decided to evaluate the expression of these enzymes in DCs from the draining lymph nodes of infected mice. To achieve this, mice were inoculated on the ears with CFSE-labelled metacyclic promastigotes and 20 h later draining lymph node cells were analysed by flow cytometry. The populations of infected (CFSE+) and uninfected (CFSE) CD11c+ were studied separately, as shown in Fig. 4A. Approximately 12.2 ± 0.9% of lymph node cells were found to be CD11c+ and of these CD11c+ cells 9.4 ± 1.8% were CFSE+; these values were similar regardless of the strain of Leishmania used. Our results showed that CFSE (i.e. uninfected) cells are predominantly CD39lowCD73. On the other hand, CFSE+ (infected) cells are predominantly CD39highCD73low, independent of the parasite species used (Fig. 4B and C). Taken together, our results showed that Leishmania is able to increase the ectonucleotidase expression on DCs during both in vitro and in vivo infections.

Figure 4.

Leishmania infection increases CD39 and CD73 expression on DCs from draining lymph nodes. Draining lymph nodes were isolated from C57BL/6J mice 20 h after inoculation with CFSE-labelled metacyclic promastigotes via the ears and the lymph node cells were analysed using flow cytometry. (A) CD11c+ cells were gated into populations of uninfected (CFSE cells) and infected (CFSE+ cells) cells, and further gated into CD39lowCD73 cells (gate 1) and CD39highCD73low cells (gate 2) in each population. Data are representative of results from six L. amazonensis-infected mice per group. The percentage of cells in (B) gate 1 and (C) gate 2 in mice infected with the indicated Leishmania species was evaluated. Data are the mean + SD of two independent experiments with three animals per group. *p < 0.05 between uninfected and infected DCs, unpaired two-tailed Student's t-test.

These results show a possible correlation between the decreased expression of activation markers on infected DCs and extracellular adenosine production by infected cells; however, they do not explain the difference in CD40 expression between L. amazonensis- and L. braziliensis- or L. major-infected DCs.

L. amazonensis impairs DC functions via A2B receptor activation

The combined action of ENTPDase and 5’-nucleotidase on extracellular ATP is the production of adenosine, which has been shown to decrease DC activation [[15, 17, 31, 32]] by acting through P1 receptors, in particular, A2A and A2B receptors. Thus, our next step was to verify whether the inhibition of these receptors could alter the effects of Leishmania infection on DC activation. First, DCs were infected with L. amazonensis in the presence of ZM241385 or MRS1754, potent A2A and A2B receptor antagonists, respectively. Our data showed that ZM241385 treatment only reversed the parasite-mediated inhibition of DC activation when used in very high concentrations (50 μM) (Fig. 5A). However, treatment with MRS1754 was able to enhance the surface expression of activation markers on infected DCs at all concentrations tested in a dose-dependent manner (Fig. 5B); these results indicate that only stimulation of A2B receptor is important in parasite-mediated DC inhibition.

Figure 5.

L. amazonensis impairs DC activation via A2B receptor activation. DCs were infected as described in Fig. 1 and analysed using flow cytometry. (A, B) DCs and L. amazonensis promastigotes co-cultures were treated with increasing concentrations of adenosine receptor antagonists (A) ZM241385 or (B) MRS1754. The percentage of MHCII+CD86+ cells in populations of uninfected and infected DCs in treated groups were expressed in relation to the group of cells incubated with parasites without treatment, set at 100 (dotted line); results represent the mean + SD from three independent experiments performed in duplicates. (C, D) DCs and parasites co-cultures were treated with 200 μM suramin or 50 μM MRS1754. The percentage of (C) MHCII+CD86+ cells or (D) CD40+ cells in populations of uninfected and infected DCs on treated groups were expressed in relation to the group of cells incubated with parasites without treatment, set at 100 (dotted line); results represent the mean + SD from at least three independent experiments performed in duplicates. *p < 0.05 versus untreated group, one-sample t-test with hypothetical value of 100. (E) Representative dot plots from at least three independent experiments of DCs infected with L. amazonensis, L. braziliensis or L. major in the absence (left) or presence of suramin (middle) or MRS1754 (right). Numbers in parentheses indicate the percentage of MHCII+CD86+ DCs or CD40+ DCs.

To further characterise the participation of purinergic signalling in the impairment of DC activation, we evaluated the roles of ENTPDase, which is responsible for ATP hydrolysis, and A2B receptor in the inhibition of DC function during L. amazonensis, L. braziliensis and L. major infection. For these experiments, DCs were infected with parasites in the presence of 200 μM suramin, an ENTPDase inhibitor, or 50 μM MRS1754, a concentration which resulted in optimal surface activation marker expression in L. amazonensis-infected DCs. Our results show that simultaneous incubation of suramin with the parasite and DCs partially reversed the parasite-induced inhibition of MHCII and CD86 expression on infected cells; these results were not parasite species specific. However, MRS1754 treatment reversed the inhibition of activation markers expression only in DCs infected with L. amazonensis (Fig. 5C). Moreover, both treatments increased CD40 expression in L. amazonensis-infected DCs. However, no effect on CD40 expression was observed in cells infected with L. braziliensis or L. major (Fig. 5D).

Given the effects of Leishmania infection on the expression of activation markers, we next evaluated whether infection could alter the ability of DCs to induce T-cell proliferation. Thus, CD4+ T cells purified from the spleens of L. amazonensis-infected C57BL/6J mice (antigen-specific proliferation) or spleen cells from non-infected BALB/c mice (mixed leukocyte reactions, MLR) were labelled with CFSE and incubated with infected C57BL/6J DCs. Lymphocyte proliferation was evaluated after 4 days using flow cytometry. Our results demonstrated that the decreased activation of DCs induced by L. amazonensis infection resulted in decreased MLR. However, DC infection with L. braziliensis or L. major parasites did not inhibit lymphocyte proliferation (Fig. 6A). Furthermore, Leishmania antigen-specific proliferation was reduced when DCs were infected with L. amazonensis compared with DCs infected with either L. braziliensis or L. major (Fig. 6B). To confirm the role of purinergic signalling on the down-modulation of DC function by L. amazonensis, we treated DCs with suramin or MRS1754 during infection with L. amazonensis. Prior to being incubated with CFSE-labelled lymphocytes, infected DCs were washed to remove the inhibitors. Treatment of DCs with either suramin or MRS1754 resulted in increased MLR and increased Leishmania-specific CD4+ T-cell proliferation when compared with untreated infected DCs (Fig. 6C and D).

Figure 6.

L. amazonensis promastigotes impair DC-stimulated T-cell proliferation by mechanisms dependent on ATP hydrolysis and A2B receptor activation. Spleen cells from non-infected BALB/c mice or CD4+ T-cells from L. amazonensis infected C57BL/6J mice were labelled with CFSE and then co-cultured in the presence of C57BL/6J infected DCs in the presence of 200 μM suramin or 50 μM MRS1754 where indicated. LPS-stimulated DCs served as controls. (A) Gating strategy to determine CD3+ cell proliferation after 4 days. (B–E) Percentage (left axis) and absolute number (right axis) of proliferating cells (mean + SD of three independent experiment performed in quadruplicates) as measured by (B, D) the MLR assay and (C, E) antigen-specific proliferation assay. A total of 100,000 cells for MLR assay or 60,000 cells for antigen-specific proliferation assay were acquired during flow cytometry analysis. *p < 0.05 versus control group (LPS-stimulated DCs), #p < 0.05 versus La group, unpaired two-tailed Student's t-test.

Our results suggest that L. amazonensis, L. braziliensis and L. major increase DC ectonucleotidase expression, which potentially leads to an increase in adenosine production by infected DCs and that these enzymes are important in Leishmania-mediated inhibition of MHCII and CD86 expression following DC activation. However, only L. amazonensis infection of DCs activates the immunosuppressive adenosine receptor A2B that inhibits CD40 expression in addition to MHCII and CD86 expression, consequently inhibiting the ability of DCs to stimulate lymphocyte proliferation.

Discussion

The effect of infection by Leishmania parasites on the activation, maturation and migration of DCs is dependent on several factors, such as parasite species, strain and developmental stage; the subset of DCs analysed and the experimental conditions used in the study (in vivo or in vitro, with or without further stimulation, etc.) (reviewed in [[33]]).

The interaction of Leishmania promastigotes with DCs is generally accepted to cause activation of these cells [[24, 25]], as shown by the enhanced expression of MHCII and co-stimulatory molecules (CD40, CD80, CD83 and CD86) and the ability to stimulate CD4+ T-cell proliferation. However, with the exception of a limited number of studies, evaluation of DC activation was performed using bulk cultures that did not separate infected from uninfected DCs during analysis. When only infected cells were analysed, DC infection was associated with an inhibition of DC activation [[23, 26]]. Our results (Fig. 1A to D) corroborate these findings by showing that Leishmania-infected DCs are refractory to further activation by LPS because MHCII and CD86 expression was decreased compared with uninfected cells. However, while infection with L. braziliensis and L. major stimulated the expression of CD40 in infected DCs, L. amazonensis infection did not alter CD40 expression (Fig. 1E). Discrepancies in CD40 expression in DCs infected with different species of Leishmania have previously been reported. For example, CD40 expression was lower in L. amazonensis-infected DCs compared with L. major-infected DCs [[34]]. Our results confirm these observations and extend these findings to L. braziliensis infection. In addition, decreased CD40 expression has also been observed in Leishmania donovani-infected macrophages [[35]] and DCs infected with Leishmania infantum [[36]].

The mechanisms by which Leishmania infection inhibits DC activation are not completely established. Although increased IL-10 production has been frequently suggested as a possible factor [[25, 30, 37]], neutralisation of IL-10 does not reverse DC down-modulation, at least with regard to IL-12 production [[33]]. In our study, the involvement of IL-10 in suppressing the activation of infected DCs was dependent on the Leishmania species examined. The inhibition of DC activation is partially dependent on IL-10 during L. braziliensis and L. major infection (Fig. 2). However, we did not find any evidence supporting a role for IL-10 in the decreased activation of L. amazonensis-infected DCs (Fig. 2).

Extracellular nucleotides and nucleosides have been shown to affect DC activation and migration. For example, extracellular ATP induces DC maturation and priming of Th1 cells while adenosine has been shown to inhibit DC activation and proinflammatory cytokine production [[18-21]]. Extracellular ATP accumulates during situations of cellular injury or infection and is considered to be a ‘danger signal’ [[10]]. ATP release is thought to be mediated by P2X7 receptors that not only sense extracellular concentrations of ATP but are also involved in ATP secretion [[38]]. Interestingly, L. amazonensis enhances P2X7 receptor expression on murine macrophages [[39]]. The same mechanism may be present in L. amazonensis-infected DCs.

In the mammalian host, the relative concentrations of extracellular ATP and adenosine are controlled by the sequential action of two enzymes: ENTPDase (CD39), which hydrolyses ATP to ADP and then to AMP, and 5’-nucleotidase (CD73), which converts AMP to adenosine [[9, 14]]. Our results showed that CD39 and CD73 expression was significantly increased in infected cells; importantly, enzyme activity was also increased in infected cells (Fig. 3). The expression of ectonucleotidases was also enhanced in infected DCs from draining lymph nodes of infected mice (Fig. 4). The increased expression of ectonucleotidases on the surface of infected cells can be beneficial to the parasite because this mechanism ensures the production of extracellular adenosine and the subsequent inhibition of DC activation.

This hypothesis was initially confirmed for all parasite species used in this work, as the MHCII and CD86 expression was restored in the presence of suramin (Fig. 5C), which prevents ATP hydrolysis by inhibiting ENTPDase (CD39) activity thus decreasing substrate (AMP) availability to 5’-nucleotidase, with a consequent reduction in adenosine production.

Suramin has been shown to decrease DC CD86 expression by acting through ATP receptors [[19]]. In our study, suramin increased rather than decreased MHCII and CD86 expression, suggesting that it predominantly acted on ENTPDase activity. In addition, when we used 30 μM suramin, a concentration sufficient to block the P2 receptor but not inhibit ENTPDases, we found no change in DC activation marker expression (data not shown).

Adenosine exerts its effects by acting on four receptor subtypes, A1, A2A, A2B and A3, that vary in distribution, affinity and function. A2 receptors are responsible for the anti-inflammatory response of adenosine, while A1 and A3 receptors may regulate the action of A2 receptors to prevent the excessive inhibition of immune cells [[40]]. Some studies have demonstrated that the A2B receptor is the main receptor involved in inhibition of DC responses in mice [[16, 17]]. Consistent with the role of adenosine in the inhibition of DC activation in our experiments, A2B receptor blockade was also able to reverse the decreased expression of activation markers on infected DCs similar to suramin (Fig. 5). However, this receptor was involved only in L. amazonensis infection and affected MHCII, CD86 and CD40 expression. These results suggest that the expression of adenosine receptors may differ in DCs infected with L. amazonensis, L. braziliensis and L. major. The role of A2B receptor in the modulation of DC function was confirmed using T-cell proliferation assays (Fig. 6).

As mentioned previously, differential DC CD40 expression induced by distinct Leishmania species has been demonstrated [[34]]. However, the majority of these studies did not evaluate the mechanisms involved in the regulation of CD40 expression. Boggiatto et al. [[34]] demonstrated that inhibition of ERK1/2 phosphorylation restored CD40 expression in L. amazonensis-infected DCs. Interestingly, activation of A2B receptors also induces ERK1/2 phosphorylation [[34, 41]]. Thus, the blockade of A2B receptors in L. amazonensis-infected DCs may have led to inhibition of ERK1/2 phosphorylation with subsequent restoration of CD40 expression.

The presence of adenosine in the extracellular environment may facilitate the persistence of Leishmania in the host due to its immunosuppressive properties [9, 12, 13, 20, 21]. A recent study showed that CD39 expression and adenosine production were increased in a population of tolerogenic DCs, which were characterised by low levels of CD40 expression and IL-12p40 production and a decreased ability to induce T-cell proliferation [[42]]. Other work showed that cAMP, which could result from A2 receptor activation, was the key component in regulatory T-cell-mediated inhibition of DC activation marker expression; further, IL-10 was not involved in the process [[43]]. We observed similar results when DCs were infected with L. amazonensis. Moreover, activation of A2B adenosine receptors has been shown to impair DC maturation and, consequently, reduce their ability to activate CD4+ T cells. A2B receptor activation was also shown to impair the function of other immune cells [[17, 32]]. Our study expands on these findings by demonstrating for the first time that infection of DCs by a parasitic protozoan (L. amazonensis) leads to decreased DC activation by a mechanism dependent on ectonucleotidase expression and A2B receptor activation.

These results contribute to the understanding of the immune evasion mechanisms of L. amazonensis, which is responsible for diffuse cutaneous leishmaniasis, a disease characterised by impaired T-cell activation. On the other hand, infection with L. major and L. braziliensis is usually characterised by a strong cell-mediated immune response that may lead to the control of the parasite. In agreement with these observations, we did not observe the involvement of A2B adenosine receptors in DCs infected by these parasite species. In this case, a less intense down-modulation of DC activation that appeared to be mediated by IL-10 was observed.

In summary, our results demonstrate a new mechanism of L. amazonensis immune evasion mediated through the activation of A2B receptor in infected cells (Fig. 7). Future studies will characterise the expression of adenosine receptors on infected DCs as well as the signalling pathways associated with their possible activation. Further investigation of this pathway may lead to new therapeutic approaches for the control of leishmaniasis.

Figure 7.

Mechanisms used by L. amazonensis, L. braziliensis and L. major to inhibit DC function and evade the immune response. (A) L. amazonensis infection increases CD39 and CD73 expression, ensuring the production of adenosine (ADO). Adenosine activates A2B receptor and impairs the activation of infected DCs, especially by inhibiting CD40 expression. Subsequently, their ability to stimulate lymphocyte proliferation is reduced. (B) Infection with L. braziliensis and L. major, despite increasing the expression of CD39 and CD73, do not activate the A2B receptor. These two species decrease the activation-induced expression of MHCII and CD86, with the participation of IL-10 receptor (IL-10R), but do not affect CD40 expression. Consequently, DC infection with L. braziliensis and L. major does not significantly affect the ability of DC to stimulate T-cell proliferation.

Materials and methods

Animals and parasites

C57BL/6J or BALB/c mice (2–6 months of age) were obtained from the Universidade Federal de Ouro Preto animal facility. Animals were given water and food ad libitum. All animal procedures were approved by the University's Ethical Committee on Animal Experimentation. Leishmania (Leishmania) amazonensis, PH8 strain (IFLA/BR/67/PH8), Leishmania (Viannia) braziliensis, M2903 strain (MHOM/BR/75/M2903) and Leishmania (L.) major, FRIEDLIN strain (MHOM/IL/80/Friedlin) promastigotes were cultured in GRACE's medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated foetal calf serum (FCS) (LGC, Cotia, SP, Brazil), 2 mM L-glutamine (Gibco BRL, Grand Island, NY, USA) and 100 U/mL penicillin G potassium (USB Corporation, Cleveland, OH, USA); the final pH of the media was 6.5. Cultures were incubated at 25°C. Metacyclic promastigotes were purified by gradient centrifugation of parasites at the stationary phase of culture (day 5) over Ficoll 400 (Sigma-Aldrich), as previously described [[3]].

Parasite staining

In in vitro DC infection experiments, metacyclic promastigotes suspended in PBS with 5% FCS were incubated in the presence of 5 μM CFSE (Sigma-Aldrich) at 37°C for 10 min in the dark. The suspensions were centrifuged and the parasites were washed in PBS (pH 7.2) [[44]]. Alternatively, parasites were labelled with PKH26 (Sigma-Aldrich) according to the manufacturer's instructions.

Differentiation of bone marrow-derived dendritic cells

BMDCs were obtained from C57BL/6J bone marrow as previously described [[45]]. Briefly, bone marrow cells were isolated from the femur and tibia of C57BL/6J mice. Bone marrow cell suspensions were centrifuged and cells cultured in RPMI-1640 (Sigma-Aldrich) supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin G potassium and 50 μM β-mercaptoethanol (Pharmacia Biotech AB, Uppsala, Sweden), pH 7.2. Cells were plated in Petri dishes at a concentration of 3 × 105 cells/mL and incubated at 37°C in an atmosphere with 5% CO2. Granulocyte-macrophage colony-stimulating factor (GM-CSF, R&D Systems, Minneapolis, MN, USA) was added to each plate on days 0, 3 and 6 at a concentration of 3 ng/mL (1050 U/mL). Non-adherent DCs were collected on the ninth day of culture. DCs were characterised by CD11b, CD11c, F4/80, Gr-1, MHCII, CD86 and CD40 expression using flow cytometry. Surface marker expression was similar to that reported in the reference protocol.

In vitro DC infection

Metacyclic promastigotes and DCs were co-incubated (1:3 cell to parasite ratio) at 33°C in a 5% CO2 atmosphere for 3 h. To ensure full activation of these cells, LPS (Sigma-Aldrich) at a concentration of 2 μg/mL was added to the cultures; the cells were then incubated at 37°C in a 5% CO2 atmosphere for up to 17 h. Infected DCs were analysed using flow cytometry or used in proliferation assays.

In selected experiments, DCs were incubated for 30 min at 25°C in the presence of 15 μg/mL 1B1.3a antibody (Harlan Bioproducts for Science, Indianapolis, IN, USA) to block the IL-10 receptor prior to being infected; rat IgG (produced in our laboratory) was used as isotype control. In other experiments, suramin (Sigma-Aldrich) or A2A and A2B adenosine receptors antagonists, ZM241385 (4-(2-(7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-α)(1,3,5)triazin-5-ylamino)ethyl)phenol) and MRS1754 (N-(4-cyanophenyl)-2-(4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy)-acetamide), respectively (Tocris Bioscience, Park Ellisville, MO, USA), were added at the moment of DC infection as described in the figure legends. Both ZM241385 and MRS1754 were diluted in dimethylsulfoxide (DMSO), which was added to the control cultures.

Determination of IL-10 production

DC culture supernatants were collected after 20 h and IL-10 cyto-kine levels were measured using an ELISA kit according to the manufacturer's instructions (PeproTech Inc., Rock Hill, NJ, USA).

Determination of ectonucleotidase activity

To analyse ectonucleotidase activity, infections were performed using a 1:10 cell to parasite ratio. The hydrolysis of ATP, ADP and AMP was measured by incubating 5 × 103 (for ATP or ADP) or 5 × 104 (for AMP) intact DCs for 1 h at 30°C in reaction buffer (116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 5 mM MgCl2 and 50 mM HEPES-tris buffer, pH 7.2) in the presence of 5 mM ATP, ADP or AMP (Sigma-Aldrich). The reactions were stopped by the addition of activated charcoal diluted in 0.1 M HCl [[46]]. Non-specific hydrolysis was determined by the addition of cells after the reactions were stopped. The suspensions were pelleted and supernatant aliquots were assessed for released inorganic phosphate (Pi) as previously described [[47]]. Enzymatic activity was expressed as nanomole of Pi released by 106 cells/h.

Isolation of lymph nodes DCs

C57BL/6J mice were inoculated on ears with 1 × 105 CFSE-labelled metacyclic promastigotes. Draining lymph nodes were isolated after 20 h and homogenised in Dulbecco's modified Eagle's medium (DMEM), pH 7.2. Lymph node cells were stained and analysed by flow cytometry as described below.

Proliferation assay

C57BL/6J mice were inoculated on the left hind footpad with 1 × 107 L. amazonensis promastigotes. On the tenth week of infection, mice were euthanised and their spleens were harvested and ground in DMEM (pH 7.2). Red blood cells were lysed with ammonium chloride lysis buffer and the cells were washed with PBS (pH 7.2). Spleen cells were then labelled with biotinylated-GK1.5 antibody (rat anti-mouse CD4, produced in our laboratory) and streptavidin-conjugated microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). CD4-positive selection was performed by passing the cells through a MS column coupled to a MiniMACS separator (Miltenyi Biotec GmbH). The purity of the CD4+ T cells obtained was approximately 90% as assessed using flow cytometry. The lymphocyte concentration was adjusted to 5 × 106 cells/mL in PBS with 5% FCS. This suspension was incubated with 5 μM CFSE at 25°C for 5 min in the dark [[48]]. The cells were washed in PBS (pH 7.2) and resuspended in RPMI supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin G potassium, 25 mM HEPES and 50 μM β-mercaptoethanol, pH 7.2. Lymphocytes (5 × 105 cells) were co-cultured with infected DCs (5 × 104 cells) in 48-well plates in a total volume of 500 μL/well. Co-cultures were incubated at 37°C in a 5% CO2 atmosphere for 4 days; the cells were then collected and analysed using flow cytometry.

Mixed leukocyte reactions

For the MLR, spleen cells from uninfected BALB/c mice were labelled with CFSE as described previously. Leukocytes (5 × 105 cells) were co-cultured with uninfected or infected DCs (5 × 104 cells) in 48-well plates in a total volume of 500 μL/well. Co-cultures were incubated at 37°C in a 5% CO2 atmosphere for 4 days; the cells were then collected and analysed using flow cytometry. The proliferation of CD3+ cells was assessed.

Flow cytometry

Cells at a concentration of 1 × 107 cells/mL in PBS with 1% bovine serum albumin (BSA) were submitted to FcγR blocking in the presence of anti-mouse CD16/CD32 (eBioscience, San Diego, CA, USA). For each stain, 25 μL of the cell suspension was incubated with a combination of desired antibodies at 4°C for 30 min in the dark. The following antibodies were used: anti-mouse CD11c (HL3 clone), anti-mouse CD40 (3/23 clone; BD Pharmingen, San Diego, CA, USA), anti-mouse CD39 (24DMS1 clone), anti-mouse MHCII (M5 114.15.2 clone), anti-mouse CD86 (GL1 clone), anti-mouse CD73 (TY/23 clone; eBioscience), anti-mouse CD3ε (145–2C11 clone; BioLegend, San Diego, CA, USA) and their respective isotype controls. The suspensions were centrifuged and the cells were washed in PBS (pH 7.2) and resuspended in a solution of 1% paraformaldehyde, 47.7 mM sodium cacodylate and 113 mM NaCl, pH 7.2. The samples were analysed using a BD FACSCaliburTM flow cytometer. Cell acquisition was performed using BD CellQuestTM Pro software. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA).

Statistical analysis

Student's t-test and a one-way ANOVA analysis with Tukey's post-test were performed using Prism 5.0 software (GraphPad Software, La Jolla, CA, USA). A p value of <0.05 was considered statistically significant.

Acknowledgements

This work was supported by grants from CAPES, FAPEMIG, CNPq and Rede Mineira de Bioterismo/FAPEMIG. L.C.C.A. is a fellow researcher from CNPq. The authors wish to thank Leandro H. Santos and Marcorelio D. Souza for technical assistance, Dr. Milton A. P. de Oliveira and Dr. Fátima R. Dias for support with DC differentiation, Dr. David Mosser and Dr. Ricardo Gonçalves for helpful discussions, Dr. Leda Q. Vieira for providing the anti-mouse CD40 antibody and PKH26, and Dr. David Sacks for providing the 1B1.3a antibody.

Conflict of interest

The authors declare no financial or commercial conflicts of interest.

Abbreviations
5’-nucleotidase

5’-nucleoside monophosphate phosphohydrolase

BMDC

bone marrow-derived dendritic cell

ENTPDase

ectonucleoside triphosphate diphosphohydrolase

La

Leishmania amazonensis;

Lb

Leishmania braziliensis

Lm

Leishmania major;

MRS1754

N-(4-cyanophenyl)-2-(4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy)-acetamide

Pi

inorganic phosphate

ZM241385

4-(2-(7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-α)(1,3,5)triazin-5-ylamino)ethyl)phenol

Ancillary