Female BALB/c (wild type; WT) and BALB/c-A_2AR^−/− (A_2AR^−/−) mice that weighed between 18 and 22 g were housed in the animal facility of the Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo (Brazil) in temperature-controlled rooms (22–25°C) and received water and food ad libitum. All experiments were conducted in accordance with the National Institutes of Health (NIH) guidelines on the welfare of experimental animals and with the approval of the Ethics Committee of the School of Medicine of Ribeirão Preto (No 196/2011).

Isolate HU-UFS14 of L. infantum was cultured in Schneider medium supplemented with 20% heat-inactivated fetal bovine serum (Gibco^®, Life Technologies, Carlsbad, CA, USA), 5% penicillin and streptomycin (all from Sigma-Aldrich, St. Louis, MO, USA), and 2% human male urine. The mice were intravenously infected with 10⁷ of the promastigote form of L. infantum parasites in the stationary growth phase. The hepatic and splenic parasite burdens were determined using a quantitative limiting dilution assay (51, 52).

The mice were euthanized 0, 4, and 6 weeks after infection, and their livers were removed. The tissues were fixed in formalin, dehydrated in graded ethanol, and embedded in paraffin. Serial sections (5 µm) were cut and mounted on glass slides that had been precoated with 0.1% poly-l-lysine (Sigma-Aldrich). Histological assessment was performed after routine hematoxylin-eosin staining. The extent of granuloma formation was analyzed in 50 fields per animal, being classified as: none granuloma, which is characterized by some parasitized cells but in the absence of inflammatory cells surrounding them; developing granuloma, which is characterized as parasitized cells surrounded by some inflammatory leukocytes; mature granuloma, in which the fused parasitized cells are surrounded by a mantle of mononuclear and polymorphonuclear cells; and empty granuloma, when no parasites could be seen inside the areas of the granulomatous reaction (53). For immunohistochemical reactions, the paraffin was removed from the tissues, and antigenic recovery was performed by heating in citrate buffer (pH 6.0), for 30 min at 37°C. Endogenous peroxidase was blocked using 3% H₂O₂, the cells were permeabilized with 0.5% Triton, and non-specific reactions were blocked with 1% bovine serum albumin. The sections were incubated overnight with rat anti-mouse Ly6G (clone 1A8) (Biolegend, San Diego, CA, USA), or isotype control antibodies (Abcam, Cambridge, MA, USA), followed by incubation with a biotinylated secondary antibody and avidin-biotin complex (Vector Laboratories, ON, Canada). The reaction was detected with diaminobenzidine, and the sections were counterstained with Mayer’s hematoxylin. For the intracellular staining of iNOS, the liver sections were permeabilized with 0.01% saponin and incubated with rabbit anti-mouse iNOS (clone sc-649) (Santa Cruz Biotechnology, Dallas, TX, USA). Afterward, the sections were incubated with an avidin-biotin-peroxidase complex (Vector Laboratories, ON, Canada), and the color was developed using 3,3′-diaminobenzidine (Vector Laboratories). The slides were counterstained with Mayer’s hematoxylin. The areas positive for iNOS staining in the hepatic tissue were quantified using IHC Toolbox Software ImageJ (NIH, MacBiophotonics, Boston, MA, USA). The isotype control from iNOS and LY6G staining by immunohistochemistry is showed as Figure S1 in Supplementary Material.

The liver leukocytes were recovered using Ficoll-Paque PLUS gradient centrifugation. After processing, the viability was assessed using Trypan blue exclusion and the cell concentration was determined. For cytokine staining, the cells were preincubated with 20 ng/ml of PMA, 500 ng/ml of ionomycin, and Golgi Plug for 6 h; permeabilized using a Cytofix/Cytoperm kit according to the manufacturer’s instructions; and stained with α-IL-17A conjugated with Alexa700 and α-IFN-γ conjugated with APC-CY7. For FoxP3 labeling, the Foxp3 Staining Kit was used according to the manufacturer’s recommendations. For each sample, data from a minimum of 200,000 cells were acquired using a FACSCanto II flow cytometer and analyzed using FlowJo software (Tree Star, OR, USA).

Single-cell suspensions from the spleens of the A_2AR^−/− or WT mice at various time points of infection were prepared aseptically, diluted to a concentration of 2 × 10⁶ cells/ml, and dispensed into 48-well plates in a total volume of 500 µl of complete RPMI-1640 medium (1 × 10⁶ cells/well; Gibco) with or without soluble Leishmania antigen (SLA) (5 µg/ml). The cell culture supernatants were harvested after 72 h of culture at 37°C in 5% CO₂, and levels of IFN-γ and IL-10 were determined using ELISA with commercial kits (BD Biosciences and R&D Systems, Minneapolis, MN, USA).

For leukocyte identification, the inflammatory cells were gated based on their characteristic size (FSC) and granularity (SSC), and the T lymphocytes (CD4⁺ CD3⁺) and neutrophils (Ly6G^highMHCII⁻) were individually identified. For intracellular staining, the cells were previously cultured with PMA (50 ng/ml) and ionomycin for 4 h, permeabilized with a Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s guide and stained with APC-Cy7-labeled α-IFN-γ or Alexa700-labeled α-IL-17 and surface-stained with FITC-labeled α-CD3 and PerCP-labeled α-CD4. For the neutrophil activation analysis, the cells were stained with antibodies to α-Ly6G, α-CD11b, α-CXCR2, and α-CD69 with APC, FITC, PERCP, and PEcy7, respectively. Foxp3 labeling were carried out using the Mouse Foxp3 Buffer Set (BD Pharmigen™) and Foxp3 (Alexa 647) antibodies. The isotype controls used were rat IgG2b and rat IgG2a. All antibodies were from BD Biosciences or eBiosciences (San Diego, CA, USA). The cell acquisition was performed using a FACSort flow cytometer. The data were plotted and analyzed using the FlowJo software (Tree Star, Ashland, OR, USA). Gating strategies were determined represented the leukocyte counts were determined by measuring the relative proportions of the leukocyte subpopulations that stained with the specific antibodies in a total of 200,000 acquired events relative to the total leukocyte numbers obtained in a Neubauer chamber. The Strategy gate for identification of inflammatory leukocytes during L. infantum infection was showed as Figure S2 in Supplementary Material.

WT and A_2AR^−/− mice were treated (i.p.) with the monoclonal antibody anti-mouse IFN-γ clone XMG1.2 (BioXCell, West Lebanon, New Hampshire, EUA). Briefly, the mice were given 20 µg of antibody 1 day before and a second dose 24 h after the infection. From the fourth week post infection, the animals were treated with 10 µg every 3 days for 2 weeks thereafter. The control group was treated with an irrelevant mab IgG following the same schedule.

Spleens from naïve WT or A_2AR^−/− mice and the cells were filtered through a cell strainer, centrifuged at 500 × g at 4°C for 10 min, and resuspended in RPMI-1640 medium at 2.5 × 10⁶ cells/ml. The cells were stained with CSFE and stimulated with plate-bound α-CD3 mAb (2 µg/ml) and α-CD28 (1 µg/ml), or medium for 4 days in a total volume of 500 µl per condition. The lymph proliferation was determined by CFSE staining by flow cytometry assay. For Th1 differentiation, CD4⁺ T cells were isolated from spleen cell suspensions of naïve WT or A_2AR^−/− and stimulated with plate-bound α-CD3 (2 µg/ml), α-CD28 (1 µg/ml) (both from BioXCell) for 4 days in RPMI-1640 medium supplemented with 5% FBS (Gibco), 100 U/ml penicillin/100 µg/ml streptomycin, 1 mM sodium pyruvate, non-essential amino acids, l-glutamine and 50 µM 2-mercaptoethanol in the presence of recombinant cytokines. For Th1, differentiation was included IL-12 (5 ng/ml) plus anti-IL-4 (10 µg/ml) in addition to IL-2 (25 U/ml). All recombinant cytokines were obtained from RD and neutralizing antibodies were obtained from BioXCell. After 4 days of culture, differentiated cells were reestimulated with PMA (50 ng/ml), ionomycin (500 ng/ml) (Sigma-Aldrich), and brefeldin A for 4 h.

The total RNA was extracted from the tissues using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and the SV Total RNA Isolation System Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Complementary DNA was synthesized using Transcriptase Reverse SuperScript III (Invitrogen). SYBR Green Mix-based quantitative PCR assays were performed using the StepOnePlus Real-time PCR System (Applied Biosystems, Singapore, Malaysia). The mean threshold cycle (C_t) values of duplicate measurements were used to calculate the expression of the target gene, which was normalized to the housekeeping genes Actb, B2m and Gapdh using the 2−ΔΔCt formula. The standard PCR conditions were as follows: 50°C for 2 min, 95°C for 2 min and 40 cycles of 15 s at 95°C, 30 s at 58°C and 30 s at 72°C, followed by a standard denaturation curve.

The data are expressed as the means ± SD and are representative of two to four independent experiments. The results from individual experiments were not combined because they were analyzed individually. The means of the different groups were compared by ANOVA followed by Tukey’s honest significant difference test. Comparisons between two groups were performed using Student’s t-test. The analyses were performed using Prism 5.0 software (GraphPad). Statistical significance was set at P < 0.05.

To investigate the relevance of A_2AR in the course of L. infantum infection, A_2AR^−/− mice and control littermates were intravenously infected with 10⁷ promastigote forms of L. infantum, and the parasite loads in the spleens and livers were quantified at various times after the infection. Strikingly, and in contrast to the WT counterparts, the spleens of the mice lacking A_2AR harbored reduced parasite loads in both periods (Figure 1A); whereas, in the livers, fewer parasites were observed only at the sixth wpi (Figure 1B). This result suggested that A_2AR participates in the establishment of visceral Leishmania infection. Surprisingly, the A_2AR^−/− mice exhibited increased weights of both spleen and liver compared to the WT mice (Figures 1C,D), which may be consequence of an inflammatory reaction.

One feature of VL is the formation of granulomas in an attempt to control the spreading of parasite (2). The granulomas can be pathologically classified as: no granuloma reaction, developing granulomas, mature granulomas, and empty granulomas (53). Histopathological analysis demonstrated that the A_2AR^−/− mice exhibited larger areas with well-formed granulomas at sixth wpi than those observed in the WT mice. These areas included mature granulomas (Figure 2C) and empty granulomatous reactions (Figure 2D). These results were consistent with the lower parasite numbers detected in the livers (Figure 1B) and dearth areas with none granuloma reaction (Figure 2A). Moreover, we could not detect any difference with respect to the developing granuloma reactions during the fourth and sixth wpi between the WT and A_2AR^−/− mice (Figure 2B). These data demonstrated that activation of A_2AR results in the failure of BALB/c mice to control the L. infantum infection possibly due to the generation of a weaker cellular immune response.

Because the development of IFN-γ-producing CD4⁺ T helper cells is crucial for the control of parasite replication in the target organs of VL, we investigated whether this response could be affected in A_2AR-dependent manner. The Ifng gene expression was upregulated in the livers (Figure 3B), but it not altered into spleen (Figure 3A), of the A_2AR^−/− mice at the sixth wpi compared to the control littermates. In terms of protein, IFN-γ production was measured in response to restimulation of the spleen cells from A_2AR^−/− or WT mice with the SLA. Stimulation of the cells from the WT mice with SLA induced the release of significant amounts of IFN-γ into culture supernatant compared with WT control (medium-without SLA) Figure 3C. However, the supernatants of the spleen cells from the A_2AR^−/− mice contained higher levels of this cytokine by in comparison with supernatants stimulated WT cells. This profile was weakly observed at the fourth wpi, but it was pronounced at the sixth wpi. Moreover, there is a significant difference in the amounts of IFN-γ into culture supernatants from antigen-stimulated A_2AR^−/− compared with respective A_2AR^−/− control (medium- without SLA). The Leishmania antigen had minimal effects on the basal IFN-γ release in either strain of non-infected mice.

By flow cytometry, we observed that infection with L. infantum promoted a significant induction of IFN-γ-producing CD4⁺ T cells in both BALB/c and A_2AR^−/− mice compared to their counterparts in terms of both percentages (Figures 3D,E) and total number of cells (Figure 3F). However, it must be noted that the previously mentioned Th1 profile enhancement was heightened in A_2AR^−/− mice. Moreover, the infection promoted the expansion of the CD4⁺ IL-17⁺ gated on CD3⁺ cells, but such population was not affected in the absence of the receptor. There were no differences between the WT and A_2AR^−/− mice in the frequencies and absolute numbers of the CD4⁺ IL-17⁺ gated on CD3⁺ (Figures S3A,B in Supplementary Material). We also could not see any difference in the frequency and number of IL-17 production by other CD3⁺ population, herein characterized as CD4⁻ IL-17⁺ gated on CD3⁺, that could be CD8⁺ T, NK⁺ T, or γδ⁺ T cells between WT and A_2AR^−/− (Figures S3A,C in Supplementary Material).

A considerable body of evidence has shown that the Th1 subset produces IFN-γ, which in turn induces expression of iNOS (NOS2) in infected phagocytes, which generates NO (54). The resulting production of NO by iNOS represents an important tool to kill Leishmania sp parasites (5, 6). By qPCR, we detected greater Nos2 mRNA expression in the spleens of the A_2AR^−/− mice than in the WT group at both the fourth and sixth weeks post infection (Figure 3H). Likewise, greater areas positive for iNOS staining were observed in the livers of the knockout mice at sixth week post infection than in the WT group (Figure 3G). These results suggested that A_2AR activation downregulated the Th1 responses which could favor the parasite spreading.

Neutrophils are recruited to Leishmania inoculation foci (55) and participate in the restriction of the parasites during VL (56, 57). Because neutrophils both produce and respond to adenosine (58), we addressed whether A_2AR signaling affected the neutrophilic inflammation in target organs of the disease.

On the basis of the size (FSC) and granularity (SSC) characteristics, we found a higher frequency of leukocytes with a high side-scatter height, a classical gate for granulocytes, in the infected A_2AR^−/− mice compared to the infected WT mice (Figure 4A). Using specific antibodies to identify the neutrophils, we observed differences in absolute numbers of Ly6G⁺ CD11b⁺ cells (neutrophils markers) at fourth wpi and the most notable difference was that the neutrophilic influx observed at sixth wpi that was greater both in percentage (Figures 4B,C) and total number (Figure 4D) of cells in the infected A_2AR^−/− mice than in the littermate controls. Interestingly, the strong neutrophilic influx in the absence of A_2AR was associated with the increased expression of cxcl1, which codes for an important neutrophil chemotactic mediator, by the spleen cells (Figure 4E). Subsequently, we used flow cytometry to examine the surface molecules expressed on surface of CD11b⁺ Ly6G⁺ cells to determine whether differences in expression of CXCR2 or CD69, relative to neutrophil migration (59–61) and activation (62, 63), respectively, could account for the strong inflammatory process encountered in the tissues of the infected A_2AR^−/− mice. Consistent with these observations, the neutrophils from the A_2AR^−/− mice exhibited an enhanced expression of CXCR2, the CXCL1 receptor (Figure 4F; Figure S4 in Supplementary Material). Among the neutrophil activation marker, we observed that the integrated median fluorescence intensity (iMFI) for CD69 from the A_2AR^−/− neutrophils was significantly higher than the iMFI for these markers on the WT neutrophils (Figure 4G; Figure S4 in Supplementary Material). Likewise, consistent with the spleen results, the liver sections from the A_2AR^−/− mice showed a marked increase in the stained neutrophils that infiltrated into the hepatic granulomas (Figure 4J). By flow cytometry, higher neutrophils were detected into liver of A_2AR^−/− mice (Figures 4H,I), which suggests that both neutrophil migration and activation may be affected by the adenosine receptor during VL.

It has been determined that IFN-γ directly modulates neutrophil behavior to favor the expression of molecules involved with cell adhesion, migration, activation, and killing activity (64). To assess whether IFN-γ could be responsible for the neutrophilic influx into the target organs of the A_2AR^−/− mice, we blocked IFN-γ using a specific antibody in the infected mice. The treatment with the α-IFN-γ antibody abrogated the neutrophilic inflammation, as observed by the reduction of percentage (Figures 5A,B) and total number (Figure 5C) of CD11b⁺ LY6G⁺ cells in the spleens of the A_2AR^−/− mice. In addition, a smaller surface expression of CXCR2 (Figure 5D) and CD69 (Figure 5E) was observed in the A_2AR^−/− mice treated with α-IFN-γ than with respective group treated with α-IgG. Interestingly, none of these parameters differed between the WT mice that were treated with α-IFN-γ or anti-IgG control. Together, these data suggested that A_2AR may modulate the Th1 subset that is capable of inducing neutrophilic inflammation and controlling the parasite spreading into tissue.

To determine the mechanism by which A_2AR modulates Th1 responses during L. infantum infection, we first examined whether T cell-intrinsic A_2AR is involved in either T cells proliferation or Th1 generation. CFSE-labeled CD4⁺ T cells isolated from the spleens of naïve WT or A_2AR^−/− mice cultured under Th0 and Th1 condition were stimulated with α-CD3 + α-CD28 for 4 days. The proliferation assay was analyzed by CFSE-positivity and Th1 differentiation by either intracellular stained-IFN-γ production or T-bet mRNA expression. The frequency of CFSE-labeled CD4⁺ T cells between A_2AR^−/− in the presence of polyclonal stimuli was similar to that in WT cells (Figures S5A,B in Supplementary Material). Moreover, the IFN-γ-production by A_2AR^−/− CD4⁺ T cells maintained under Th1 condition were induced in similar levels that those in WT cells (Figure S5C in Supplementary Material). There was no significant difference in T-bet mRNA expression by WT and A_2AR^−/− CD4⁺ T cells under Th1 polarizing condition (Figure S5D in Supplementary Material), indicating that T cell-intrinsic A_2AR does not affect the either proliferation or differentiation of Th1 cells.

Taking into account that A_2AR signaling can restore homeostasis by promoting Tregs generation and immunosupression (27) and inflammatory mediators such as IFN-γ can limit the Treg function and differentiation (65) and that IFN-γ was upregulated in the infectious foci of L. infantum (Figures 3A–F), we next evaluated whether the Th1-induced inflammation observed in the A_2AR^−/− mice could be related to compromised Treg functions during infection. The results showed that following the infection, the frequency of CD4⁺ T Foxp3⁺ cells were reduced in both infected groups at fourth wpi compared with respective naïve littermate control group. However, the reduction of CD4⁺ T Foxp3⁺ cells was more pronounced on A_2AR^−/− mice when compared to infected WT mice (Figures 6A,B). In terms of total cells, despite the infection promoting CD4⁺ T Foxp3⁺ cells expansion on both infected groups at fourth wpi, it was reduced on infected A_2AR^−/− mice when compared to infected WT compared with respective non-infected littermate controls (Figures 6A,B). Moreover, foxp3 mRNA expression was reduced in the livers of A_2AR^−/− mice (Figure 6C). We previously demonstrated that adenosine provided an anti-inflammatory activity through a mechanism that was dependent on PGE₂-induced IL-10 release (29). Consistent with the observed Treg reduction, the transcripts of IL-10, an important anti-inflammatory cytokine that is released through A_2AR signaling, was reduced in the spleens at fourth wpi (Figure 6E) and livers at sixth wpi (Figure 6F) of the knockout mice. We observed a similar inhibition in the IL-10 release into supernatants of the L. infantum antigen-stimulated spleen leukocytes from the A_2AR^−/− mice compared with the stimulated cells from the infected WT mice (Figure 6D).