Table of Contents Author Guidelines Submit a Manuscript
Mediators of Inflammation
Volume 2018, Article ID 6148351, 9 pages
https://doi.org/10.1155/2018/6148351
Research Article

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

1Graduate Program in Bioscience and Physiopathology, Departamento de Análises Clínicas e Biomedicina, The State University of Maringá, Colombo Avenue 5790, 87020-900 Maringá, PR, Brazil
2Departamento de Análises Clínicas e Biomedicina, The State University of Maringá, Maringá, PR, Brazil
3Postgraduate Program in Health Sciences, Centro de Ciências da Saúde, State University of Maringá, Maringá, PR, Brazil

Correspondence should be addressed to I. G. Demarchi; rb.moc.oohay@odrahlag_leb

Received 19 October 2017; Accepted 18 December 2017; Published 13 February 2018

Academic Editor: Elaine Hatanaka

Copyright © 2018 L. Domeneghetti et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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 (). C. brasiliense inhibited or reduced significantly () 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.

1. 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 [13]. 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 sp. [4]. The Th1 immune response is related to clinic cure and therapeutic success [3, 4].

Currently, leishmaniasis has affected 12 million people worldwide, occurring 70,000 deaths every year and there are 350 million people at risk of contracting the disease [5]. Leishmaniasis treatment is based on Glucantime or amphotericin B, but these drugs have shown severe adverse effects, potential toxicity, and therapeutic failure, which lead to the abandonment of the therapy or even its ineffectiveness [5]. Furthermore, antileishmaniasis products that are extracted from nature have increasingly been patented each year, demonstrating the importance of bioprospecting studies to improve the armamentarium of antileishmaniasis drugs [6].

Plants are traditionally used for the treatment of many diseases and are more accessible, as they can be naturally acquired [79], leading them to the position of potential candidates for leishmaniasis treatment [1013]. Calophyllum brasiliense Camb. (Clusiaceae) is a tree popularly known as “guanadi-cedar,” “oil stick,” and “guarandi” [13, 14] and it has extensive natural distribution, on rain forests on wet and marshy soils [15], occurring from Mexico to the south coast of Brazil [16]. The extracts and derivatives from C. brasiliense leaves have been used in folk medicine against various diseases [913, 1727], including leishmaniasis [1013]. Some biological activities of C. brasiliense were reported such as its antibacterial, antifungal, and antiprotozoal activities, its cytotoxicity to tumor cells, and its inhibitory effect on tumor promotion and HIV-1 replication [1013, 1927]. The leishmanicidal activity of C. brasiliense is known [1013], but the other immunomodulatory effects of C. brasiliense remain unclear [20].

Blanco-Ayala and coworkers conducted an investigation suggesting that the xanthones from C. brasiliense played a role as potential agents to attenuate the oxidative damage produced by different prooxidants, such as ROS and lipid peroxidation [20]. ROS and nitric oxide are essential to parasite death [2]. Besides that, L. (L.) amazonensis can escape and survive at the immune environment, requiring the modulation of the components of the immune response as Th1 and Th2 cells and their products [2, 28], but its association with C. brasiliense treatment remains to be investigated. Therefore, this study investigated the potential of hydroalcoholic extract of C. brasiliense in immunomodulating the cytokines’ expression by murine macrophages infected with L. (L.) amazonensis.

2. Materials and Methods

2.1. Culture and Maintenance of Parasite

Promastigotes forms of L. (L.) amazonensis (MHOM/BR/1977/LTB0016), held in cryopreservation, were thawed and maintained at 25°C in 199 culture medium supplemented with fetal bovine serum 10% (v/v), human urine (1%), L-glutamine (20 mM), and antibiotics (penicillin 100 IU/ml and streptomycin 0.1 mg/ml). Weekly, subcultures were made of 199 medium cultures and parasite inoculum was adjusted according to each experiment.

2.2. Animals

BALB/c isogenic mice, female, 8–10 weeks, were obtained from the Central Animal Laboratory at the State University of Maringá, after approval by the Ethics Committee on the Use of Experimental Animals of the State University of Maringá (Paraná State, Brazil) (warrant number 133/2012). The animals were kept in collective cages with water and food ad libitum with 12 h light-dark cycles.

2.3. Plant Material

The leaves of C. brasiliense were collected between January 2009 to December 2010, in Parque Estadual da Ilha do Cardoso in Cananéia, located on the southern coast of São Paulo state (Brazil). This tree is found in all coastal Atlantic forest vegetation (https://nossosparques.org.br/). The plant was identified by Dr. Maria CM Young, and a voucher specimen was deposited (SP 363818) in the herbarium of the Botanical Institute of São Paulo, SP, Brazil. The plant sample was weighed, dried in a circulating air oven (Quimiss Q-31, Diadema, SP, Brazil) at 35°C, crushed in a Wiley mill (Tecnal Marconis, TE048), and subsequently stored in a dry place, away from light.

2.4. Hydroalcoholic Extract of C. brasiliense

The sample (1.3 kg), 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].

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 × 106 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% CO2 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% CO2. 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 × 105 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% CO2 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.

2.7. 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 MgCl2, 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).

Table 1: Gene and PCR conditions.
2.8. 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 is considered significant.

3. Results and Discussion

C. brasiliense plant is a folk medicine used in many infections, such as leishmaniasis [1013], in Brazil and other countries [913, 1727]. In the present study, we showed anti-Leishmania activity and immunomodulatory effects of C. brasiliense during infection. Murine macrophages infected with L. (L.) amazonensis and treated with 30 μg/mL hydroalcoholic extract of C. brasiliense showed a significant reduction in the infection index (IF) () after 3 h (IF: 16.5, 95.3% of reduction) and after 24 h (IF: 33.0, 95.9% of reduction), when compared to untreated cells (IF: 351/3 h and 799/24 h) (Figure 1(a)). The leishmanicidal results are similar to those of Brenzan et al. [11], who related the leishmanicidal activity of C. brasiliense for promastigote and axenic amastigote L. (L.) amazonensis in a concentration between 20 and 40 μg/mL and did not observe cytotoxic activity.

Figure 1: Leishmanicidal activity of Calophyllum brasiliense: 5 × 105 macrophages/mL on glass coverslips were (a) infected with L. (L.) amazonensis promastigotes (6 parasites per macrophage) during 3 h and treated with hydroalcoholic extract of C. brasiliense (30 μg/mL) and infection index was obtained after 3 and 24 h of treatment. (b) Cultures were treated with C. brasiliense alcoholic extract (30 μg/mL) for 3 h then they were infected with L. (L.) amazonensis, and the infection index was obtained after 3 and 24 hours. Macrophages infected with L. (L.) amazonensis and without treatment were controls. compared to controls.

In subsequent experiments, macrophages were pretreated with C. brasiliense extract and infected with L. (L.) amazonensis for 3 and 24 h (Figure 1(b)). The IF was also significantly reduced () compared to controls, from 374.5 to 15 (96%) after 3 h and from 822.3 to 11.6 (98.6%) after 24 h.

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 [1013].

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].

Besides antileishmanial activity, researches have been conducted to discover new drugs with lower toxicity and immunomodulatory effects for leishmaniasis therapy [28, 34]. C. brasiliense treatment modulated the mRNA cytokine expression produced by murine macrophages infected with Leishmania and did not modify iNOS mRNA expression. From all cytokines investigated, C. brasiliense inhibited TNF-α mRNA expression (100%) and reduced in 18% IL-1β and 23% IL-10 by uninfected macrophages, compared to untreated and uninfected macrophages (negative control) () (Figure 2). The infection increased 60% of TNF-α mRNA expression, IL-1β (48%), and IL-10 by macrophages, compared to the negative control () (Figure 2). TNF-α, IL-1β, and IL-10 expression by infected and treated macrophages was significantly lower than infected macrophages (, Figure 2). Macrophages (negative control) did not express iNOS and IFN-γ mRNA in all period (Figures 2-4) and IL-12 and IL-18 in 3 h (Figure 2).

Figure 2: Expression levels of cytokines and iNOS mRNA by macrophages infected with L. (L.) amazonensis or treated with hydroalcoholic extract of C. brasiliense. (a) L.L.a: L. (L.) amazonensis; C.b.: C. brasiliense. L.L.a + C.b.: peritoneal macrophages from BALB/c mice were infected with promastigotes of L. (L.) amazonensis (10 parasites : 1 macrophage) for 3 h and then treated with hydroalcoholic extract of C. brasiliense (30 μg/ml) for 3 h; L.L.a.: macrophages infected and untreated; control: macrophages uninfected and untreated were incubated only with RPMI during 6 h (negative control) significantly compared with negative control; compared to infected macrophages. (b) In 1.5% agarose gel electrophoresis, the products of reverse transcriptase-polymerase chain reaction for conditions above were revealed with ethidium bromide in a transilluminator. M: molecular marker; C−: amplification negative control (H2O); C+: DNA obtained from macrophages stimulated with 5 μg/mL of lipopolysaccharide (LPS); MO: macrophages (negative control, uninfected and untreated); Cb: macrophages treated with C. brasiliense; LLa: macrophages infected with L. (L.) amazonensis.

In infected and after treated with C. brasiliense macrophages, the hydroalcoholic extract inhibited 100% mRNA expression of TNF-α, IL-10, IL-12, and IL-18 and reduced 51% IL-1β, compared to the positive control (Figure 3). L. (L.) amazonensis infection induced an increase of TNF-α (10%), IL-1β (30%), and IL-18 (6%) expression, in comparison to the negative control (Figure 3). The C. brasiliense treatment subverted the cytokine expression induced by infection since it inhibited TNF-α, IL-12, IL-18, and IL-10 expression and reduced the IL-1β expression after infection and treatment (Figure 3).

Figure 3: Expression levels of cytokine and iNOS mRNA by macrophages infected with L. (L.) amazonensis and treated with hydroalcoholic extract of C. brasiliense. (a) L.L.a: L. (L.) amazonensis; C.b.: C. brasiliense. L.L.a + C.b.: peritoneal macrophages from BALB/c mice were infected with promastigotes of L. (L.) amazonensis (10 parasites: 1 macrophage) for 3 h and then treated with hydroalcoholic extract of C. brasiliense (30 μg/ml) for 3 h; L.L.a.: macrophages infected and untreated; control: macrophages uninfected and untreated were incubated only with RPMI during 6 h (negative control). significantly compared to negative control; compared to infected and untreated macrophages. (b) Cytokine mRNA expression by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) DNA fragments was separated in 1.5% agarose gel electrophoresis and revealed with ethidium bromide in a transilluminator. M: molecular marker; C−: amplification negative control (H2O); C+: DNA obtained from macrophages stimulated with 5 μg/mL of LPS (positive control); 1 and MO: macrophages (negative control, uninfected and untreated); 2 and L.L.a. + C.b.: macrophages infected with L. (L.) amazonensis and treated with C. brasiliense; 3 and L.L.a.: macrophages infected with L. (L.) amazonensis.

In macrophages pretreated with C. brasiliense and after infected with Leishmania, the hydroalcoholic extract reduced TNF-α expression (9%), IL-12 (6%), IL-18 (15%), and IL-10 (7%) and increased IL-1β, compared to the negative control (Figure 4) (). The parasite infection promoted the increase of IL-1β and reduced TNF-α, IL-12, IL-18, and IL-10, compared to the negative control (Figure 4). The previous C. brasiliense treatment reduced significantly these same cytokines () even after the infection and increased IL-12 (Figure 4), but it did not influence IL-18 expression.

Figure 4: Expression levels of cytokine and iNOS mRNA by macrophages treated with hydroalcoholic extract of C. brasiliense and infected with Leishmania (L.) amazonensis. (a) L.L.a.: L. (L.) amazonensis; C.b.: C. brasiliense. Peritoneal macrophages from BALB/c mice were treated with hydroalcoholic extract of C. brasiliense (30 μg/mL) for 3 h and infected with promastigotes of L. (L.) amazonensis (10 parasites : 1 macrophage) by more than 3 h. Macrophages were only infected with promastigotes of L. (L.) amazonensis (10 : 1) and untreated. As a negative control, macrophages were incubated only with RPMI for 6 h. significantly compared to negative control; compared to infected and untreated macrophages. (b) Cytokine mRNA expression by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) DNA fragments was separated in 1.5% agarose gel electrophoresis and revealed with ethidium bromide in a transilluminator. M: molecular marker; C−: amplification negative control (H2O); C+: DNA obtained from macrophages stimulated with 5 μg/mL of LPS; MO: macrophages (negative control, uninfected and untreated); 4 and C.b. + L.L.a.: macrophages treated with C. brasiliense for 3 h and infected with L. (L.) amazonenis; 5 and L.L.a.: macrophages infected with L. (L.) amazonensis; 6: macrophages uninfected and untreated.

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.

Acknowledgments

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil.

References

  1. Brasil, Ministério da Saúde, Manual de Vigilância da Leishmaniose Tegumentar Americana, Editora do Ministério da Saúde, Brasília, Brazil, 2 edition, 2007.
  2. W. N. Oliveira, L. E. Ribeiro, A. Schrieffer, P. Machado, E. M. Carvalho, and O. Bacellar, “The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of human tegumentary leishmaniasis,” Cytokine, vol. 66, no. 2, pp. 127–132, 2014. View at Publisher · View at Google Scholar · View at Scopus
  3. WHO, World Health Organization. Control of the Leishmaniasis: Report of a Meeting of the WHO. Expert Committee on the Control of Leihmaniases. “WHO Technical Report Series”, World Health Organization, Geneva, Switzerland, 2010, 949.
  4. K. J. Gollob, A. G. Viana, and W. O. Dutra, “Immunoregulation in human American leishmaniasis: balancing pathology and protection,” Parasite Immunology, vol. 36, no. 8, pp. 367–376, 2014. View at Publisher · View at Google Scholar · View at Scopus
  5. WHO, World Health Organization, “Control of the Leishmaniasis: Report of a Meeting of the WHO Expert Committee on the Control of Leishmaniases,” WHO Technical Report Series, World Health Organization, Geneva, Switzerland, 2010.
  6. A. Machado-Silva, P. P. Guimarães, C. A. Tavares, and R. D. Sinisterra, “New perspectives for leishmaniasis chemotherapy over current anti-leishmanial drugs: a patent landscape,” Expert Opinion on Therapeutic Patents, vol. 25, no. 3, pp. 247–260, 2014. View at Publisher · View at Google Scholar · View at Scopus
  7. M. Iriti, S. Vitalini, G. Fico, and F. Faoro, “Neuroprotective herbs and foods from different traditional medicines and diets,” Molecules, vol. 15, no. 5, pp. 3517–3555, 2010. View at Publisher · View at Google Scholar · View at Scopus
  8. P. M. Cheuka, G. Mayoka, P. Mutai, and K. Chibale, “The role of natural products in drug discovery and development against neglected tropical diseases,” Molecules, vol. 22, no. 1, article E58, 2017. View at Publisher · View at Google Scholar · View at Scopus
  9. V. C. Filho, C. Meyre-Silva, and R. Niero, “Chemical and pharmacological aspects of the genus Calophyllum,” Chemistry & Biodiversity, vol. 6, no. 3, pp. 313–327, 2009. View at Publisher · View at Google Scholar · View at Scopus
  10. B. M. Cardoso, T. F. De Mello, D. S. Lera et al., “Antileishmanial activity of Calophyllum brasiliense leaf extract,” Planta Medica, vol. 83, no. 01/02, pp. 57–62, 2017. View at Publisher · View at Google Scholar · View at Scopus
  11. M. A. Brenzan, C. V. Nakamura, B. P. D. Filho, T. Ueda-Nakamura, M. C. Young, and D. A. G. Cortez, “Antileishmanial activity of crude extract and coumarin from Calophyllum brasiliense leaves against Leishmania amazonensis,” Parasitology Research, vol. 101, no. 3, pp. 715–722, 2007. View at Publisher · View at Google Scholar · View at Scopus
  12. M. A. Brenzan, A. O. Santos, C. V. Nakamura et al., “Effects of (−) mammea a/BB isolated from Calophyllum brasiliense leaves and derivatives on mitochondrial membrane of Leishmania amazonensis,” Phytomedicine, vol. 19, no. 3-4, pp. 223–230, 2012. View at Publisher · View at Google Scholar · View at Scopus
  13. P. A. Honda, I. C. P. Ferreira, D. A. G. Cortez et al., “Efficacy of components from leaves of Calophyllum brasiliense against Leishmania (Leishmania) amazonensis,” Phytomedicine, vol. 17, no. 5, pp. 333–338, 2010. View at Publisher · View at Google Scholar · View at Scopus
  14. C. M. O. Simões, E. P. Schenkel, G. Gosmann, J. C. P. Mello, L. A. Mentz, and P. R. Petrovick, Farmacognosia: da Planta ao Medicamento, Editora da Universidade UFRGS, Porto Alegre/Florianópolis, 2002.
  15. H. Lorenzi, Árvores Brasileiras: Manual de Identificação e Cultivo de Plantas Arbóreas Nativas do Brasil, vol. 352, Instituto Plantarum, Nova Odessa: Plantarum, 1992.
  16. M. C. M. Marques and C. A. Joly, “Germinação e crescimento de Calophyllum brasiliense (Clusiaceae), uma espécie típica de florestas inundadas,” Acta Botanica Brasilica, vol. 14, no. 1, pp. 113–120, 2000. View at Publisher · View at Google Scholar · View at Scopus
  17. L. C. Klein-Júnior, D. Zambiasi, G. R. Salgado, F. Delle Monache, V. C. Filho, and F. de Campos Buzzi, “The validation of Calophyllum brasiliense (“guanandi”) uses in Brazilian traditional medicine as analgesic by in vivo antinociceptive evaluation and its chemical analysis,” Naunyn-Schmiedeberg’s Archives of Pharmacology, vol. 390, no. 7, pp. 733–739, 2017. View at Publisher · View at Google Scholar · View at Scopus
  18. T. V. Lordani, M. A. Brenzan, L. E. Cortez et al., “Effect of a topical formulation containing Calophyllum brasiliense Camb. extract on cutaneous wound healing in rats,” Natural Product Research, vol. 29, no. 10, pp. 953–957, 2015. View at Publisher · View at Google Scholar · View at Scopus
  19. C. Ruiz-Marcial, R. Reyes Chilpa, E. Estrada, J. Reyes-Esparza, G. G. Fariña, and L. Rodríguez-Fragoso, “Antiproliferative, cytotoxic and antitumour activity of coumarins isolated from Calophyllum brasiliense,” The Journal of Pharmacy and Pharmacology, vol. 59, no. 5, pp. 719–725, 2007. View at Publisher · View at Google Scholar · View at Scopus
  20. T. Blanco-Ayala, R. Lugo-Huitrón, E. M. Serrano-López et al., “Antioxidant properties of xanthones from Calophyllum brasiliense: prevention of oxidative damage induced by FeSO4,” BMC Complementary and Alternative Medicine, vol. 13, no. 1, p. 262, 2013. View at Publisher · View at Google Scholar · View at Scopus
  21. F. Abe, S. Nagafuji, and H. Okabe, “Trypanocidal constituents in plants 3. Leaves of Garcinia intermedia and heartwood of Calophyllum brasiliense,” Biological & Pharmaceutical Bulletin, vol. 27, no. 1, pp. 141–143, 2004. View at Publisher · View at Google Scholar · View at Scopus
  22. V. F. Noldin, D. B. Isaias, and V. C. Filho, “Gênero Calophyllum: importância química e farmacológica,” Química Nova, vol. 29, no. 3, pp. 549–554, 2006. View at Publisher · View at Google Scholar · View at Scopus
  23. C. T. Pires, R. B. d. L. Scodro, M. A. Brenzan et al., “Anti-Mycobacterium tuberculosis activity of Calophyllum brasiliense extracts obtained by supercritical fluid extraction and conventional techniques,” Current Pharmaceutical Biotechnology, vol. 17, no. 6, pp. 532–539, 2016. View at Publisher · View at Google Scholar
  24. C. T. Pires, M. A. Brenzan, R. B. Scodro et al., “Anti-Mycobacterium tuberculosis activity and cytotoxicity of Calophyllum brasiliense Cambess (Clusiaceae),” Memórias do Instituto Oswaldo Cruz, vol. 109, no. 3, pp. 324–329, 2014. View at Publisher · View at Google Scholar · View at Scopus
  25. J. B. Pretto, V. Cechinel-Filho, V. F. Noldin, M. R. Sartori, D. E. Isaias, and A. B. Cruz, “Antimicrobial activity of fractions and compounds from Calophyllum brasiliense (Clusiaceae/Guttiferae),” Zeitschrift fur Naturforschung C, vol. 59, no. 9-10, pp. 657–662, 2004. View at Google Scholar
  26. S. Kimura, C. Ito, N. Jyoko et al., “Inhibition of leukemic cell growth by a novel anti-cancer drug (GUT-70) from Calophyllum brasiliense that acts by induction of apoptosis,” International Journal of Cancer, vol. 113, no. 1, pp. 158–165, 2005. View at Publisher · View at Google Scholar · View at Scopus
  27. M. Huerta-Reyes, M. C. Basualdo, F. Abe, M. Jimenez-Estrada, C. Soler, and R. Reyes-Chilpa, “HIV-1 inhibitory compounds from Calophyllum brasiliense leaves,” Biological and Pharmaceutical Bulletin, vol. 27, no. 9, pp. 1471–1475, 2004. View at Publisher · View at Google Scholar · View at Scopus
  28. I. G. Demarchi, M. V. Thomazella, M. de Souza Terron et al., “Antileishmanial activity of essential oil and 6,7-dehydroroyleanone isolated from Tetradenia riparia,” Experimental Parasitology, vol. 157, pp. 128–137, 2015. View at Publisher · View at Google Scholar · View at Scopus
  29. L. N. Prista, A. A. Correia, and R. Morgado, Técnica Farmacêutica e Farmácia Galênica, Fundação Calouste Gulbenkian, Lisboa, 4 edition, 1996.
  30. I. G. Demarchi, M. De Souza Terron, M. V. Thomazella et al., “Antileishmanial and immunomodulatory effects of the essential oil from Tetradenia riparia (Hochstetter) Codd,” Parasite Immunology, vol. 38, no. 1, pp. 64–77, 2016. View at Publisher · View at Google Scholar · View at Scopus
  31. H. Kolodziej, A. Burmeister, W. Trun et al., “Tannins and related compounds induce nitric oxide synthase and cytokines gene expressions in Leishmania major-infected macrophage-like RAW 264.7 cells,” Bioorganic & Medicinal Chemistry, vol. 13, no. 23, pp. 6470–6476, 2005. View at Publisher · View at Google Scholar · View at Scopus
  32. T. Chen, X. Zhao, Y. Liu, Q. Shi, Z. Hua, and P. Shen, “Analysis of immunomodulating nitric oxide, iNOS and cytokines mRNA in mouse macrophages induced by microcystin-LR,” Toxicology, vol. 197, no. 1, pp. 67–77, 2004. View at Publisher · View at Google Scholar · View at Scopus
  33. S. N. Byrne, C. Beaugie, C. O’Sullivan, S. Leighton, and G. M. Halliday, “The immune-modulating cytokine and endogenous alarmin Interleukin-33 is upregulated in skin exposed to inflammatory UVB radiation,” The American Journal of Pathology, vol. 179, no. 1, pp. 211–222, 2011. View at Publisher · View at Google Scholar · View at Scopus
  34. B. Monge-Maillo and R. Lopez-Velez, “Therapeutic options for Old World cutaneous leishmaniasis and New World cutaneous and mucocutaneous leishmaniasis,” Drugs, vol. 73, no. 17, pp. 1889–1920, 2013. View at Publisher · View at Google Scholar · View at Scopus
  35. K. R. Gantt, T. L. Goldman, M. L. McCormick et al., “Oxidative responses of human and murine macrophages during phagocytosis of Leishmania chagasi,” The Journal of Immunology, vol. 167, no. 2, pp. 893–901, 2001. View at Publisher · View at Google Scholar
  36. S. O. Amoo, A. O. Aremu, M. Moyo, and J. Van Staden, “Antioxidant and acetylcholinesterase-inhibitory properties of long-term stored medicinal plants,” BMC Complementary and Alternative Medicine, vol. 12, no. 1, p. 87, 2012. View at Publisher · View at Google Scholar · View at Scopus
  37. C. Dye, “The logic of visceral leishmaniasis control,” The American Journal of Tropical Medicine and Hygiene, vol. 55, no. 2, pp. 125–130, 1996. View at Publisher · View at Google Scholar
  38. E. Pinelli, R. M. Gonzalo, C. J. Boog et al., “Leishmania infantum-specific T cell lines derived from asymptomatic dogs that lyse infected macrophages in a major histocompatibility complex-restricted manner,” European Journal of Immunology, vol. 25, no. 6, pp. 1594–1600, 1995. View at Publisher · View at Google Scholar · View at Scopus
  39. G. A. Duque, M. Fukuda, S. J. Turco, S. Stager, and A. Descoteaux, “Leishmania promastigotes induce cytokine secretion in macrophages through the degradation of synaptotagmin XI,” Journal of Immunology, vol. 193, no. 5, pp. 2363–2372, 2014. View at Publisher · View at Google Scholar · View at Scopus
  40. P. R. L. Machado, L. Carvalho, M. I. A. S. Araújo, and E. M. Carvalho, “Mecanismos de resposta imune às infecções,” Anais Brasileiros de Dermatologia, vol. 79, no. 6, pp. 647–662, 2004. View at Publisher · View at Google Scholar
  41. Y. Li, K. Ishii, H. Hisaeda et al., “IL-18 gene therapy develops Th1-type immune responses in Leishmania major-infected BALB/c mice: is the effect mediated by the CpG signaling TLR9?” Gene Therapy, vol. 11, no. 11, pp. 941–948, 2004. View at Publisher · View at Google Scholar · View at Scopus
  42. T. T. Espir, L. de Paula Figueira, M. de Farias Naiff et al., “The role of inflammatory, anti-inflammatory, and regulatory cytokines in patients infected with cutaneous leishmaniasis in Amazonas state, Brazil,” Journal of Immunology Research, vol. 2014, Article ID 481750, 10 pages, 2014. View at Publisher · View at Google Scholar · View at Scopus
  43. L. P. Carvalho, M. Soto, and S. Jerônimo, “Characterization of the immune response to Leishmania infantum recombinant antigens,” Microbes and Infection, vol. 5, no. 1, pp. 7–12, 2003. View at Publisher · View at Google Scholar · View at Scopus
  44. D. S. Lima-Junior, D. L. Costa, V. Carregaro et al., “Inflammasome-derived IL-1beta production induces nitric oxide-mediated resistance to Leishmania,” Nature Medicine, vol. 19, no. 7, pp. 909–915, 2013. View at Publisher · View at Google Scholar · View at Scopus
  45. D. R. Neill, S. H. Wong, A. Bellosi et al., “Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity,” Nature, vol. 464, no. 7293, pp. 1367–1370, 2010. View at Publisher · View at Google Scholar · View at Scopus