Calophyllum brasiliense Modulates the Immune Response and Promotes Leishmania amazonensis Intracellular Death

Calophyllum brasiliense is a plant from the Brazilian rain forests and has been used in folk medicine for the treatment of various diseases, including leishmaniasis. This infectious disease depends on the Leishmania sp. and the host immune response. C. brasiliense antileishmanial activity is well known, but the effects on immune response remain to be investigated. This study showed the leishmanicidal and immunomodulatory effects of a 30 μg/mL of hydroalcoholic extract of C. brasiliense in murine macrophages before and after Leishmania (Leishmania) amazonensis infection. The semiquantitative cytokine RNA expression was determined by RT-PCR and the anti-Leishmania activity was measured by infection index (IF). Hydroalcoholic extract of C. brasiliense reduced more than 95% of IF when used before and after Leishmania infection, with 3 and 24 h of treatment (p < 0.05). C. brasiliense inhibited or reduced significantly (p < 0.05) the TNF-α, IL-1β, IL-18, and IL-10 mRNA expression. The antileishmanial and anti-inflammatory effects showed the potential of C. brasiliense as an alternative therapy for leishmaniasis and it must be investigated.


Introduction
Leishmaniases are endemic neglected tropical diseases caused by intracellular parasitic protozoa of the genus Leishmania. The parasite has a complex digenetic life cycle requiring a susceptible vertebrate host and an insect vector, which allow their transmission. Leishmania promastigote forms are inoculated by the phlebotomy vector in the skin of the host. The parasites invade macrophage cells transforming them into amastigote form [1,2]. The initial events involve the invasion of the host cells by Leishmania and their transport to the regional lymph nodes, in which a microenvironment is created from immune response and parasite [1][2][3]. The clinical manifestations depend on complex interactions between the parasite species and the host immune system, which result in different clinical forms of cutaneous, mucocutaneous, diffuse, or visceral diseases [1,2].
The infection caused by Leishmania (Leishmania) amazonensis is characterized by skin and diffuse lesions, and the immune response of the host is essential for the resolution of leishmaniasis. The immune mechanisms involved will define the fate of the infection and its spread or control, as well as the development of the adaptive immune response [3,4]. Leishmania parasites are recognized by immune cells stimulating the T helper 1 lymphocytes (Th1) to produce cytokines that induce the activation of the phagocytic mononuclear system. The macrophage cells produce the microbicidal substances leading to parasite death. The reactive oxygen species (ROS) and oxide nitric are microbicidal agents that promote the death of Leishmania  , previously crushed, underwent cold maceration process in ethanol : water (9 : 1) and successive extractions until all active compounds were retrieved (4 × 61) [29]. Subsequently, the extract was filtered and vacuum evaporated at 35-40°C on a rotating evaporator until total elimination of the organic solvent. The product of this evaporation, a dark green water-insoluble residue, was dissolved in dichloromethane. The organic solvent was then removed by evaporation at room temperature, and the obtained hydroalcoholic extract (≈75.0 g) was stored at −10°C, protected from light until use [10].

Materials and Methods
2.5. Cell Culture and Treatments. Peritoneal macrophages from BALB/c mice were collected with sterile cold RPMI 1640 medium (pH 7.6) and 1.0 × 10 6 macrophages/mL (in RPMI 1640 supplemented with 100 U/ml penicillin, 0.1 mg/mL streptomycin, and 0.05 mg/ml gentamycin) were plated on twelve-well culture plates and incubated at 37°C in an atmosphere of 5% CO 2 for one hour. Subsequently, the nonadherent cells were removed, and the adherent cells were infected with 10 promastigotes of L. (L.) amazonensis by macrophage and incubated for 3 h at 37°C with 5% CO 2 . Hence, cultures were treated with 30 μg/mL of the hydroalcoholic extract of C. brasiliense for 3 h [13]. Alternatively, cultures were pretreated with 30 μg/mL of the hydroalcoholic extract of C. brasiliense for 3 h and then infected with 10 promastigotes per macrophage, for 3 h under the conditions mentioned above. Noninfected macrophages were also treated with 30 μg/mL of hydroalcoholic extract of C. brasiliense for 3 h. Macrophages infected with promastigotes of L. (L.) amazonensis and untreated were used as positive control, and macrophage uninfected and untreated were used as negative control. All procedures were performed in triplicate.
2.6. Antiamastigote Activity (Infection Index). A suspension of 5 × 10 5 macrophages/mL was added on 13 mm-diameter sterile glass coverslips (Glastecnica, São Paulo, SP, Brazil), distributed into 24-well culture plates and then incubated at 37°C with an atmosphere containing 5% CO 2 for 1 h. After that, nonadherent cells were removed by washing with RPMI 1640 medium, while the adherent cells were infected with promastigotes of L. (L.) amazonensis (six parasites per macrophage) [30], and incubated for 3 h. Then, the supernatant was discarded, and the infected cells were treated with 30 μg/mL of hydroalcoholic extract of C. brasiliense for 3 to 24 h. In another condition, macrophages were first treated for 3 h with 30 μg/mL hydroalcoholic extract of C. brasiliense, and after this period, the cells were infected with promastigotes of L. (L.) amazonensis (six parasites per macrophage) incubated for 3 to 24 hours. After these periods of treatment, the supernatant was discarded and the coverslips removed, washed, and stained with Panoptic kit Quick LB® (Laborclin, Curitiba, PR, Brazil), and after, it is fixed on glass slides (24 × 76 mm) with Entellan (Merck, Darmstadt, Germany). The experiments were performed three times in triplicate. The macrophages infected with L. (L.) amazonensis and untreated were used as positive control and macrophages uninfected and untreated as negative control. The antiamastigote activity of C. brasiliense was determined by the infection index (IF) calculated by the percentage of infected macrophages multiplied by the average number of parasites per macrophage.

mRNA Analysis by Semiquantitative Reverse
Transcriptase-Polymerase Chain Reaction (RT-PCR). Total RNA was extracted from macrophages using TRIzol reagent according to the manufacturer's instructions (Invitrogen, Saint Louis, USA). The complementary DNA (cDNA) was synthesized from 1.0 μg of total RNA as a template in a reverse transcription reaction using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). RNA quantification (ng/ml) and the determination of purity were performed using NANODROP 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific Inc., USA) (optimal purity level = 1.8). cDNA samples were amplified by PCR using the specific primer sequences for mRNA: tumor necrosis factor (TNF-α) [31], interleukin (IL) 1β [32], IL-12, IL-18 [31], interferon-γ (IFN-γ) [32], IL-10, enzyme nitric oxide synthase (iNOS) [31], and glyceraldehyde-3 phosphate dehydrogenase (GAPDH and mRNA; Mus musculus) as internal control of the PCR amplification [33] ( Table 1). The primers were chosen according to the BLAST tool that is available in the GenBank database and previous studies. The reaction mixture contained 25 μM of each primer, 0.2 mM dNTP MIX, 1 U Platinum Taq DNA polymerase (Invitrogen), and 1.5 mM MgCl 2 , and DNA (5-10 μL~100 pg/reaction) was added to each test tube in a final volume of 25 μL. The amplified DNA fragments were separated by 1.5% agarose gel electrophoresis and revealed with ethidium bromide in transilluminator. Semiquantitative RT-PCR was performed by quantifying the bands densitometrically using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Each cytokine gene was normalized to GAPDH as a housekeeping gene control (internal control).

Statistical
Analysis. The results were tested for normality of distributions and the data subsequently analyzed by Mann-Whitney U test using STATISTICA 7.0 software and p < 0 05 is considered significant.
Honda et al. [13] observed that pretreatment with dichloromethane extract (10%) or with hexane fraction (5%) of experimentally infected BALB/c mice decreased the volume of the lesions by L. (L.) amazonensis. The animals treated topically during 32 days after the infection revealed healing lesions, and the parasite load in the popliteal lymph nodes was significantly reduced in treated animals. The results showed that crude extract and hexane fraction of C. brasiliense have a significant in vitro and in vivo leishmanicidal activity [13]. These studies indicate the vegetal species' therapeutic potential in obtaining phytotherapeutic drugs [10][11][12][13]. The compounds derivate from C. brasiliense, such as (−) mammea A/BB and 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one, may have antileishmanial activity by acting directly on the depolarization of the Leishmania mitochondria membrane potential, thus leading to parasite death [12]. A topical formulation containing C. brasiliense Camb. extract promoted wound healing in rats with cutaneous leishmaniasis; after 14 days of the treatment start, the animals treated exhibited more than 90% reduction of wound areas. After 21 days of treatment, the treated animals exhibited a significant increase in fibroblasts. Thus, the C. brasiliense emulsion had healing properties in the topical treatment of wounds and accelerated the healing process [13].
C. brasiliense subverted the rise of mRNA expression of these cytokines (TNF-α, IL-1β, IL-18, and IL-10) when used after (Figure 3) or before the Leishmania infection ( Figure 4). Thus, in the present study, C. brasiliense modulated the cytokines mRNA expression by murine macrophages when infected or not with Leishmania, and, when C. brasiliense was tested after and before the infection, it showed the potential of this plant for leishmaniasis treatment. The modulation of cytokines is one of the defense strategies of the host immune system against parasitic infections. In the immune response to Leishmania spp., there is an initial activation of the monocyte-macrophage system through cytokines [2].
Interactions between parasite and immune cells lead to phagocytosis by macrophages that activate trigger mechanisms for their microbicidal response to the production of reactive oxygen metabolites (ROS) and nitric oxide (NO) [2]. As the infection progresses, amastigotes multiply within macrophages, which can present parasite antigens to T and B lymphocytes and these respond by producing specific Th, antibodies, cytokines, and other mediators which, in turn, can activate macrophages to destroy the intracellular Leishmania [35]. The NO is a main leishmanicidal agent produced by the host, and its production depends on iNOS expression [2]. In Leishmania (L.) amazonensis infection, some researchers related that this parasite species can decrease the production of nitric oxide as an escape mechanism of the immune host [2,28,36]. In this study, the iNOS mRNA was not modulated by C. brasiliense during infection, but other studies showed that C. brasiliense can play a role as a potential agent to attenuate the oxidative damage produced by different prooxidants, as ROS and lipid peroxidation [20]. Although the C. brasiliense was not able to modulate iNOS, other immune mechanisms can be investigated, like the cytokines. Cytokines are protein molecules released by various cell types of the immune system and act on the emission of signals between the cells for immune responses. Several studies in murine models have associated a specific cytokine or a combination of cytokines as factors to susceptibility or resistance to Leishmania infection. In this context, a protective immunity for leishmaniasis is related to an efficient Th1 cellular response and the production of cytokines such as IL-12 and IFN-γ [37,38]. In this study, IFN-γ was not modulated by C. brasiliense, and only IL-12 was increased when C. brasiliense was tested before the infection. IL-12 is involved in cell-mediated immunity by stimulating Th1 cells and inflammation control, and it is critical for the dissemination of parasite control [2].
Also, TNF-α expression levels were elevated in macrophages infected by L. (L.) amazonensis, but C. brasiliense was able to subvert it. Another study reported a relationship between increases in TNF-α and the susceptibility to L. (L.) amazonensis infection, inducing the persistent parasitic infection and tissue damage [39]. TNF-α is a proinflammatory cytokine of the Th1 cellular response involved in the occurrence of an intense inflammatory reaction and damage to these tissues and results in the appearance of skin and mucosal ulcers [40]. In the present study, C. brasiliense inhibited the TNF-α mRNA expression completely when used alone or after the Leishmania infection, and it was reduced it when used before Leishmania infection. These results showed a potential of C. brasiliense to reduce the inflammatory cytokines that can lead to the worsening of the leishmaniasis lesions.
C. brasiliense also inhibited IL-18 mRNA expression in infected macrophages. This cytokine is primarily produced by macrophages and dendrites cells and is considered as a cofactor for the IL-12-induced development of the Th1 immune response, which optimizes IFN-γ production by effector Th1 cells [41]. IL-18 inhibition caused by C. brasiliense treatment can explain the IL-12 and IFN-γ inhibition. Thus, hydroalcoholic extract of C. brasiliense has shown potential anti-inflammatory effects.
Beyond Th1 response, Th2 immune response is also crucial to infection evolution, since it inhibits the mechanisms of Leishmania sp. death that are induced by macrophages, leading to disease progression. Th2 cellular response is promoted by IL-10, IL-4, and IL-5 cytokines that are known as downregulators of the immune response [2], and patients infected with L. (L.) amazonensis presented high levels of IL-10, IL-4, and TNF-α [42]. In the present study, Leishmania infection induced TNF-α and IL-10 expression strongly, but C. brasiliense subverted these cytokines, mainly after infection. IL-10 is the main regulator cytokine of Th1 type cellular immune response and acts as a inhibitor of IL-12, IL-2, TNF-α, and IFN-γ synthesis, favoring the parasite persistence [43]. Thus, the results of the present study suggest that the hydroalcoholic extract of the studied plant species, under these conditions, provides an immunomodulatory capacity to inhibit or reduce the mRNA expression of cytokine related to Th2 cell and inflammation, favoring the leishmaniasis resolution.
IL-1β expression was also downregulated by C. brasiliense. First, following inoculation of parasites in the skin and the start of phagocytosis of promastigote forms, it activated mononuclear phagocytes releasing TNF-α and IL-1β cytokines, acting in innate immunity and inflammation, for the purpose of stimulating the recruitment of neutrophils and monocytes to sites of infection. These actions occur through the increased expression of adhesion molecules on the vascular endothelium (ligands for selectins and integrins) and by inducing chemotaxis of leukocytes [2]. Also, the inflammasome-derived IL-1β induces the resistance to Leishmania amazonensis in infected macrophages [44]. Thus, IL-1β can promote the proliferation and activation of Th2 cells and suppress the Th1 response [45]. Therefore, C. brasiliense is a potential IL-1β inhibitor, favoring the leishmaniasis resolution.
In short, our data suggest that the hydroalcoholic extract of C. brasiliense at 30 μg/mL has leishmanicidal activity and immunomodulatory effects. This plant modulated the immune response in murine macrophages infected with L.  (L.) amazonensis or used alone, due to inhibiting completely or reducing significantly mRNA expression of cytokines related to Th1 and Th2 cellular response. Therefore, we suggest that the other inflammatory mediators, such as prostaglandins and leukotrienes, are investigated in animal models submitted to C. brasiliense and Leishmania infection. The cytokine inhibition profile induced by C. brasiliense indicated that this plant has an anti-inflammatory activity. Thus, the antileishmanial and anti-inflammatory immunomodulatory effects showed the potential of this plant as an alternative therapy to cutaneous leishmaniasis caused by L. (L.) amazonensis.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this article.