Natural Products: Insights into Leishmaniasis Inflammatory Response

Leishmaniasis is a vector-borne disease that affects several populations worldwide, against which there are no vaccines available and the chemotherapy is highly toxic. Depending on the species causing the infection, the disease is characterized by commitment of tissues, including the skin, mucous membranes, and internal organs. Despite the relevance of host inflammatory mediators on parasite burden control, Leishmania and host immune cells interaction may generate an exacerbated proinflammatory response that plays an important role in the development of leishmaniasis clinical manifestations. Plant-derived natural products have been recognized as bioactive agents with several properties, including anti-protozoal and anti-inflammatory activities. The present review focuses on the antileishmanial activity of plant-derived natural products that are able to modulate the inflammatory response in vitro and in vivo. The capability of crude extracts and some isolated substances in promoting an anti-inflammatory response during Leishmania infection may be used as part of an effective strategy to fight the disease.


Introduction
Human leishmaniasis is an infectious disease caused by 20 different Leishmania species reported in 98 countries and territories spread across four continents (Africa, Americas, Asia, and Europe). Leishmaniasis is considered a major public health issue as it currently affects 12 million people [1]. The anthroponotic and zoonotic forms of transmission may occur. In the last case, the primary reservoirs of Leishmania are sylvatic mammals such as forest rodents, hyraxes, and wild canids. However, urban or domestic dogs are the most relevant species in the epidemiology of this disease [2].
Leishmania infection occurs during the hematophagy of female sand flies belonging to Phlebotomus (Old World) and Lutzomyia (New World) genus. The metacyclic promastigote forms present in the foregut of the sand flies are inoculated in the dermis-epidermis junction of the vertebrate host, infecting cells of the mononuclear phagocyte system [3]. The interaction between parasites and host immune cells leads to an inflammatory response essential for parasite control. However, an exacerbated proinflammatory response may cause tissue damage, such as those easily observed in cutaneous leishmaniasis cases [4,5]. On the other hand, the lack of an effective inflammatory response may promote increased 2 Mediators of Inflammation parasite burden. In this scenario, a moderate inflammatory response would be ideal for an effective control of the disease.
Plants have been long recognized as a rich source of biologically active extracts, essential oils, and isolated substances. In fact, research laboratories around the world search in plants for active substances against diverse illnesses such as microbial and protozoal infections, cancer, diabetes, and inflammatory processes [6]. Indeed, plant-derived natural products such as phenolic compounds, steroids, quinones, coumarins, terpenoids, and alkaloids have been widely investigated for their antileishmanial potential [7,8].
In the present review, we start with an introduction about the current scenario of leishmaniasis epidemiology and treatment, followed by some highlights on the inflammatory response generated by Leishmania infection. The last part of this work focuses on the modulatory effects of plantderived natural products over inflammatory mediators and their impact on parasite burden in vivo and in vitro.

Available Chemotherapy for Leishmaniasis
Chemotherapy is the current method for human leishmaniasis treatment since there are no vaccines available. Usually, the therapeutic approach starts with the use of pentavalent antimonials such as sodium stibogluconate and meglumine antimoniate. However, when these drugs exhibit low efficacy or simply cannot be prescribed for leishmaniasis treatment, second-line drugs are indicated [12,15,16].
Several Leishmania-killing mechanisms have been attributed to pentavalent antimonials including apoptosis, disturbance of fatty acids -oxidation, adenosine diphosphate phosphorylation, and redox balance. In addition, antimonials inhibit the glycolysis pathway and are able to directly act on infected macrophages eliciting an oxidative/nitrosative stress against internalized parasites [17,18]. Despite the variety of antileishmanial targets, the use of pentavalent antimonials has been extensively discussed due to their toxic effects to liver and heart tissues. Regarding the use of amphotericin B, this drug targets ergosterol, an essential plasma membrane sterol found in Leishmania spp. Also, amphotericin B recognizes cholesterol in mammalian cells, which leads to high toxicity and severe side effects, including kidney failure, anemia, fever, and hypokalemia [19].
Miltefosine and paromomycin are two other drugs that have been introduced for the treatment of leishmaniasis. Miltefosine was the first orally administered drug effective against VL. The mechanism of antileishmanial action of miltefosine remains unclear but apoptosis preceded by drug intracellular accumulation has been described. Other possible mechanisms include cytochrome c oxidase inhibition, which leads to mitochondrial dysfunction and immunomodulation [20]. The recommended dose of miltefosine for VL treatment is approximately 2.5 mg/kg/day for 4 weeks. The long term therapy in conjunction with miltefosine long halflife (about 150 h) can accelerate the onset of drug resistance. Moreover, recent studies have pointed out that miltefosine has a potential teratogenic and abortifacient effect, preventing its prescription during pregnancy [18,21]. Paromomycin is an aminoglycoside antibiotic that has shown important results in leishmaniasis treatment, mainly for the cutaneous form of the disease [22]. However, in vitro studies have already reported the emergence of paromomycin-resistant parasites, compromising its use as a wide antileishmanial agent in the future [23]. In addition, the toxicity of miltefosine and paromomycin has also been described [12].
In summary, the current chemotherapy scenario urges for more efficient and secure antileishmanial treatments, encouraging the search for new bioactive compounds such as those from natural origin. In fact, plant-derived natural products represent a promising class of drug candidates against leishmaniasis.

Inflammatory Response to
Leishmania Infection Parasite-host interaction is a complex process that modulates Leishmania infection and the immunological response to it, including inflammation. Several molecules are involved in inflammation during leishmaniasis, such as cytokines and the lipid mediator leukotriene B4 (LTB4). Many of the molecules that promote inflammation also activate phagocytes leading to the production of nitric oxide (NO), the main effector molecule in parasite killing. However, an exacerbated production of these molecules may also lead to tissue damage. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) are cytokines produced by macrophages after the recognition of pathogens, including Leishmania. They promote inflammation by inducing the expression of adhesion molecules (selectin and integrin ligands) on the endothelial surface. TNF-or TNF-receptor 1-(TNFR1-) deficient mice are able to control L. major replication but develop larger lesions [24,25]. The role of IL-1 in leishmaniasis is controversial, as IL-1 contributes to Th1 priming at early infection but worsens the disease outcome in established infection [26].
IL-10 is an important anti-inflammatory cytokine responsible for peripheral tolerance to self-antigens and preventing exacerbated immune responses to foreign antigens. However, when expressed in large quantities, IL-10 may have deleterious effects during leishmaniasis, leading to an early suppression of innate and acquired immune responses, pathogen proliferation, and aggravation of the disease [27]. In leishmaniasis, phagocytes are stimulated to produce IL-10, which leads to a reduced production of cytokines related to the Th1 profile, such as IL-12 and interferon gamma (IFN-) [28]. This causes a reduction in NO production that consequently reduces the microbicidal capacity of macrophages. IL-10 may be secreted by numerous cells, including macrophages, T cells, and B cells.
The cytokines IL-12 and IL-4 also play an important role during Leishmania infection. They define the cell profile through the polarization of CD4+ T cells and modulate the response from other cells [29,30]. IL-12 activates NK cells and CD8+ T cells, leading to IFN-production [31]. In addition, IL-12 induces the differentiation of CD4+ T cells to the Th1 profile, which also produces IFN-, a potent inducer of NO production in macrophages. Thus, IL-12 possesses an indirect microbicidal action. In contrast, IL-4 induces the differentiation of CD4+ T cells to a Th2 profile, which produces IL-4, IL-5, and IL-13. This profile suppresses NO production and leads to an increase in eosinophils [32].
LTB4 is an eicosanoid with chemotactic function synthesized from leukotriene A4 by leukotriene-A4 hydrolase. In vitro, LTB4 contributes to the microbicidal action of macrophages through the production of NO and reactive oxygen species while, in vivo, LTB4 reduces the parasite load and the footpad swelling [33,34]. The importance of the type of immune response, if Th1 or Th2, lies in the fact that Th1 immune response characterizes the resistance mechanism to Leishmania infection, while Th2 response has been associated with susceptibility to parasite infection. The Th1 immune response is associated with production of proinflammatory cytokines such as IFN-, TNF-, and IL-12, while the susceptibility profile of Th2 response is characterized by anti-inflammatory cytokines expression such as IL-10 and IL-4 ( Figure 1) [35].
In humans, protection against VL is mediated by Th1 immune response whereas pathogenesis is associated with Th2 response. Most studies suggest that poor Th1-type responses are associated with severe clinical forms of leishmaniasis [36]. Some studies have demonstrated the importance of proinflammatory cytokines IFN-, TNF-, and IL-12 in L. donovani infection. Depletion of these cytokines aggravated the disease progression or made hosts susceptible to infection by L. donovani [37].
However, studies about CL showed that higher frequency of proinflammatory cytokine production leads to larger lesions. Some studies pointed that high production of IFN-, TNF, and NO is not always beneficial [38]. Thus, inadequately controlled immune responses could potentially lead to pathological manifestations and tissue damage. This is contradictory since many studies pointed out that the Th1-mediated response is important for disease control. The activation of type effector cells that produce the macrophageactivating cytokines (i.e., IFN-) is necessary for host control over parasite replication [39]. Increasing evidence suggests that the paradigm established about the necessity of a Th1 response for a better prognosis of leishmaniasis is not a rigid concept and the balance between proinflammatory and anti-inflammatory cytokines determines the outcome of the infection [40][41][42].

Natural Products Effects on Host Immunological Response
As mentioned earlier, leishmaniasis treatment is primarily based on antimonial compounds followed by amphotericin B as a second choice drug. However, high toxicity, severe side effects, and elevated costs hinder the use of these drugs in countries where leishmaniasis is endemic. In many instances, traditional medicines are the alternative for accessible treatments against parasitic diseases [41]. Unfortunately, most of them are hardly explored and their mechanisms of action are mainly unknown. Plants possess a large repertoire of secondary metabolites that display a wide variety of pharmacological activities. Indeed, numerous plantderived bioactive compounds have been described, such as terpenoids, flavonoids, alkynes, alkaloids, saponins, sterols, phenylpropanoyl esters, lactones, tannins, and coumarins [43][44][45].
Traditional herbal medicines are gaining increased attention as they can reduce the risk of chronic diseases and act as antibiotics, antioxidants, and/or immunomodulators. Several studies have described the effects of plant extracts or isolated compounds in immune cells and cytokine production [43]. Thus, the study of active compounds obtained from plants used in traditional medicine plays a pivotal role in the search for new antileishmanial molecules [39,41].
Several raw extracts from different plants have been shown to exhibit antileishmanial activity, which may not only be due to their direct action on the parasite, but also due to a concomitant effect on the host immune response [41]. Therefore, the search for plant extracts with a wide spectrum of antileishmanial and immunomodulatory activities may enable the discovery of substances suitable for the disease control. Some studies have focused on the effects of leishmanicidal essential oils and plant extracts in the production of pro-and anti-inflammatory soluble mediators. Altogether, these studies suggest that the induction or inhibition of cytokine production is a critical factor for effective parasite destruction without producing excessive tissue damage. Table 1 summarizes the currently known plant extracts and their effects on inflammatory mediators.
The plant popularly known as Evanta (Angostura longiflora (Krause) Kallunki) is used for the treatment of leishmaniasis and other parasitic diseases in Bolivia [41]. In addition to having direct activity against L. braziliensis, Evanta extracts also interfere with the activation of both mouse and human T cells. Calla-Magarinos et al. (2009) [41] showed that the alkaloid-rich extract from Evanta barks (AEE) reduced INFexpression in J774 and spleen cells, despite its lack of effect on TNF-and NO production. Similar effects were observed in human peripheral blood mononuclear cells (PBMCs). The major compound in the alkaloid-rich extract from Evanta barks is 2-phenylquinoline. Interestingly, the isolated substance ( Figure 2) showed a similar effect to that observed for AEE. Moreover, 2-phenylquinoline reduced INF-production and cell proliferation in vitro, suggesting that it may contribute to the control of the chronic inflammatory reaction that characterizes Leishmania infection.

Recently, Calla-Magariños et al. (2013) demonstrated that the alkaloid-rich Evanta extract interferes with in vitro
antigen-specific lymphocyte activation [40]. When spleen cells from L. braziliensis-immunized mice were pretreated with AEE and stimulated with Leishmania lysate or Leishmania-infected bone marrow macrophages (L-BMM), the levels of IFN-decreased. In addition, in vivo treatment with the Evanta extract affected reactivation of primed lymphocytes, reducing the production of IFN-, IL-12, and TNF-by spleen cells induced with L-BMM. AEE treatment also affected the kinetics of infection. Mice infected with L. braziliensis promastigotes in the left hind footpad showed a more effective decrease in the footpad thickness when treated with AEE than those treated with meglumine antimoniate. These results suggest that AEE can control both Leishmania infection and the inflammatory reaction against it.
The leaf methanol extract and the essential oil from Xylopia discreta display antileishmanial activity and immune stimulatory effects over infected murine macrophages [42]. To evaluate the effects of the methanol extract and the essential oil from X. discreta, López et al. (2009) infected J774 cells with L. panamensis and measured the levels of proinflammatory mediators. IL-12, IL-10, IL-6, MCP-1, and TNF-were quantified after treatment with different concentrations of X. discreta extract or essential oil. No statistical differences in the production of interleukins and TNFwere observed between treated and untreated cells. However, a significant increase in MCP-1 production was observed after cell treatment. Surprisingly, no differences in cytokine production were detected when pentamidine was used as antileishmanial drug [42].
The extract produced from the leaf of Neem (Azadirachta indica) presents antileishmanial and immunomodulatory activities [46]. The leaf and seed extracts of A. indica were shown to possess immunomodulatory, insecticidal, antiseptic, anticancer, antiviral, antifungal, and antiprotozoal properties. Its oil, bark, and leaf extracts have therapeutic efficacy against leprosy, intestinal helminthiasis, and respiratory disorders in children [47]. Similar to the X. discreta extract [42], the ethyl acetate extract fraction of Neem also induces a Th1 response. Cytokine production was evaluated by real time quantitative PCR (RT-qPCR) on THP-1 and PBMCs infected with L. donovani strain Dd8. Cells treated with Neem extract showed a significant increase in TNF-, IL-8, and IL-1 production, while IL-10 expression was unaltered, indicating a strong Th1 response. However, the expression of TNFand IFN-was unaltered in spleen tissue (in vivo analysis), whereas the expression of Th2 cytokines (IL-10, IL-4, and TGF-) was significantly reduced [46]. These results suggest that the leaf extract of Neem induces a protective immune polarization during leishmaniasis.  Glycyrrhiza glabra   Increased the IL-12 and IFNproduction in mice [51] Sambucus nigra Commercial preparation (Sambucol) -L. major 25 L twice a day Increased the production of IL-1 , IL-6, IL-8, and TNF-by human monocytes [65]  Artemisia annua  donovani were analyzed for IgG2a (induced by INF-) and IgG1 (induced by IL-4) levels. Highest levels of IgG2a are indicative of Th1 response, while IgG1 indicate Th2 activation. ALE and ASE stimulated the production of high levels of IgG2a and low levels of IgG1. As expected, ALE and ASE treatment induced NO generation by macrophages primed with SLA. Confirming these results, Th1/Th2 cytokine levels were quantified in culture supernatants of spleen cells from animals treated with ALE and ASE. The extracts significantly increased the levels of Th1 cytokines, such as INF-, TNF-, and IL-2, and decreased the IL-10 and IL-4 levels [47]. Although  and Dayakar et al. (2015) have used different parts of A. indica, both of them showed the proinflammatory effects of bioactive molecules derived from this plant.
The genus Laennecia and the correlated genus Conyza are known to produce bioactive substances displaying antimicrobial, antiparasitic, antidiarrhoeal, antinociceptive, antioxidant, and anti-inflammatory activities. Aiming to evaluate the potential of L. confusa, Ruiz et al. (2012) investigated the inhibitory effect of different extracts from its stems against several pathogenic microorganisms. In addition, the antiinflammatory activity of these extracts was evaluated. The aqueous and chloroform extracts, as well as a chloroform fraction, named, CE2, presented antiparasitic activity against L. donovani. However, these extracts and fractions did not affect the production of proinflammatory cytokines (IL-6) in THP-1 cells [48].
A similar approach was conducted by Bolivar et al. (2011) with Galium mexicanum and by Paredes et al. (2013) with Lopezia racemosa, with both of them being traditional medicinal plants used in Mexico [49,50]. Flavonoids, iridoid glycosides, iridoid acids, triterpene saponins, and anthraquinones have been isolated from the Galium genus. Among the G. mexicanum extracts and fractions analyzed, the hexane fractions HE 5 and HE 14b presented anti-L. donovani promastigotes activity, while the hexane fraction HE 5 and methanol fractions ME 13-15 reduced the LPSinduced macrophage production of IL-6, suggesting an antiinflammatory character of these samples [49].
The aerial parts of L. racemosa were submitted to extraction with various solvents and the extracts were fractionated. The hexane fractions HF 11-14b, methanol fractions MF 28-36, and the chloroform extract were able to inhibit L. donovani growth. In relation to the reduction of IL-6 production by macrophages exposed to LPS, the fractions HF 11-14b showed significant anti-inflammatory activity by reducing the secretion of the aforementioned cytokine [50].
Croton caudatus leaves extract is a promising extract against visceral leishmaniasis. Stems and leaves of C. caudatus have been used for the treatment of rheumatic arthritis, malaria, convulsions, ardent fever, numbness, worm-infested animals, vomiting, and dysentery in India [39]. Terpenes as crotocaudin, isocrotocaudin, crotoncaudatin, and crocaudatol have been isolated from this extract. Dey et al. (2015) demonstrated that the semipurified hexane extract of C. caudatus leaves (JDHex) inhibited the proliferation of L. donovani promastigotes (IC 50 = 10 g/mL) and intracellular amastigotes (IC 50 = 2.5 g/mL). To evaluate the immunomodulatory activity of JDHex, the production of proinflammatory cytokines, such as IL-12 and TNF-, as well as anti-inflammatory cytokines, IL-10 and TGF-, was investigated in vitro and in vivo. L. donovani-infected murine peritoneal macrophages treated with JDHex showed an increase in intracellular IL-12 (p70 fraction) and a reduction in TGF-and IL-10 production. In addition, JDHex induced an increase in NO that could be directly correlated with the induction of TNF-expression in infected macrophages. These results suggest that the immunomodulatory activity of JDHex occurs via a Th1 response. In vivo experiments performed with mice infected with L. donovani and treated orally with different concentrations of JDHex for 5 days after 1 month of infection showed that treated mice had an induction in IFN-production. In addition, the parasite load in spleen was reduced dose-dependently. As JDHex was efficient against L. donovani intracellular amastigotes, the authors suggested that the proinflammatory activity of JDHex may be useful for antileishmanial therapy [39]. L. amazonensis-infected mice were treated with a triterpene-rich fraction of Bacchari suncinella during five days. The analysis of immune response revealed that treated mice presented higher levels of IL-12 and IFNthan the control group. Treatment with the triterpenic fraction reduced the size of lesions, as well as the parasitism and the parasite load [51]. It is worth noting that the triterpenic fraction of B. suncinella stimulated the inflammatory process while reducing the size of mice lesions.
The flavonoid-rich Artemisia annua L. extract has been shown to possess antioxidant, antimicrobial, and antiinflammatory activities [52]. Studies carried out with the leaves and seeds of A. annua against L. donovani-infected mice caused increased production of Th1 cytokines (IFN-) and a simultaneous decrease in Th2 cytokines (IL-4 and IL-10). Moreover, A. annua extracts resulted in higher CD4+ and CD8+ T cell numbers, lymphoproliferation, upregulation of costimulatory molecules (CD80 and CD86) on APCs, and generation of NO [53].

Conclusion
Promising drug candidates for leishmaniasis treatment should be able to eliminate the parasite but also elicit an appropriate immune response. Plant-derived natural products such as crude extracts, purified fractions, or isolated substances have demonstrated their effectiveness as immunomodulatory agents. The anti-inflammatory activity of the natural products pointed here could be useful for the control of an exacerbated proinflammatory response, ameliorating leishmaniasis clinical symptoms, such as tissue damage.

Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.