microRNAs: Are they Important in the Development of Resistance in Leishmaniasis?

Leishmaniasis is an infectious and parasitic disease of great importance in public health. Numerous studies indicate that biochemical and molecular mechanisms are factors that contribute to the emergence of antileishmanial drug resistance. Currently, miRNAs have been identified as targets for the invasion of pathogens to control the immune response and imply resistance to treatments. Considering the alarming growth in drug resistance, new possibilities for controlling leishmaniasis have been emerging. Natural compounds originating from medicinal plants are being increasingly explored as promising antileishmanial alternatives. The chapter aims to provide a brief review on mechanisms of action associated with traditional agents used to treat leishmaniasis, focusing mainly on molecular bases associated with the resistance of Leishmania spp. to current drugs and identifying the possible miRNAs involved in this process. In addition, we seek to describe some of the promising plant molecules that can be used as potential antileishmanial agents and their possible mechanisms of action.


Introduction
Leishmaniasis is an infectious and parasitic disease of major importance to public health worldwide due to its difficult control, mainly because the commonly used antileishmanial drugs have contributed to the increasing increase in parasite resistance [1,2].
Biochemical, molecular and/or genetic mechanisms are factors that contribute to the emergence of resistance [3][4][5]. Among these mechanisms, we highlight microRNAs (miRNAs), small non-coding RNAs functionally involved in various biological processes of an organism [6][7][8]. Currently, these structures have been identified as the main targets for invasion by pathogens, including Leishmania spp., aiming to control the immune response and possibly implying resistance to treatments [9,10].

Traditional therapeutic agents and mechanisms of action
Leishmaniasis presents different clinical manifestations, ranging from skin lesions to severe visceral forms, depending on the species of the parasite of the genus Leishmania [4].
Chemotherapy is the only effective alternative to treat all forms of the disease. Therapeutic approaches to control leishmaniasis comprise five main drugs: pentavalent antimony, amphotericin B, miltefosine, paromomycin and pentamidine, however, these drugs are associated with serious problems, such as toxicity and especially with the emergence of resistant strains, making treatment difficult [18].
Although, current antileishmanial drugs have been used for decades, their mechanisms of action remain obscure, however, some assumptions are accepted. Pentavalent antimonials, for example, eliminate parasites through the process of apoptosis and also inhibit trypanothione reductase, an important enzyme for the survival of the parasite in the host [19]. Miltefosine, in turn, also induces cell death by apoptosis, in addition to inducing various immunological and inflammatory effects on macrophages [20]. The mechanism of action of amphotericin B may involve interaction with membrane sterols, resulting in membrane disorganization, increased permeability and cell damage in the parasite [21,22]. Paromomycin inhibits protein synthesis and modifies the fluidity and permeability of the membrane and also decreases mitochondrial potential [3,23]. While pentamidine acts as a DNA-binding drug, causing the parasite mitochondrial membrane potential collapse and induction of kinetoplast DNA destruction [24,25].
The prolonged use of these drugs has contributed to the acceleration of leishmaniasis resistance; however, little is known about the cellular and molecular mechanisms, especially about the importance of miRNAs involved in the emergence of this resistance (Figure 1).

miRNAs and drug resistance
MicroRNAs (miRNAs) are a small class of non-coding RNAs composed of 18-24 nucleotides, present in a variety of organisms, including parasites, plants, animals and humans [7,8,26,27]. The first description of these structures occurred after studies with the nematode parasite Caenorhabditis elegans [28].
The canonical biosynthesis of a miRNA starts with a primary miRNA (pri-miRNA) that is transcribed and recognized by a microprocessor complex. This process includes the double-stranded RNA-specific endoribonuclease enzymes (DROSHA) and the DiGeorge syndrome critical region gene 8 (DGCR8), which are cleaved to form a miRNA precursor (pre-miRNA). The pre-miRNAs are then exported to the cytoplasm for further processing by the DICER enzyme and cofactors such as protein kinase R activator (PACT) or transactivation response RNA binding protein (TRBP) [29].
The mature duplex miRNA is finally loaded into an RNA-induced silencing complex (RISC) that is multiprotein and a corrected miRNA strand (−5p or -3p) that binds to the Argonaute protein (AGO) guiding the complex to its mRNA target [30]. Both biogenesis and maturation of miRNAs are tightly regulated processes. The first level of adjustment is represented by single nucleotide polymorphisms (SNPs) and epigenetic control of transcription through mechanisms of acetylation, DNA methylation and histones. The biosynthesis and maturation of miRNAs can also be influenced by RNA binding proteins (RBPs), which can interact with key enzyme processes such as DROSHA, DGCR8, DICER and the complex RISC [31].
In addition to impacting the actions of miRNAs on their targets, editing pri and pre-miRNAs is also an important mechanism that modulates the biosynthesis and maturation of specific miRNAs. Degradation of the miRNA strand (−5p or -3p strand) or incorporation into the RISC complex (lead strand) determines the pool of target mRNAs. The complementarity between the miRNA response elements (MRE) in the mRNA and the seed sequence in the miRNA strand determines the specificity of the action of the RISC complex for mRNAs. Furthermore, the degree of complementarity between the MRE and the seed sequences generally dictates whether the mRNA is degraded or its translation is blocked [32]. The mechanism of biogenesis and maturation of miRNA is described in Figure 2.
miRNAs are involved in important biological processes in organisms, including the control of gene and pathological expression, cell and organ development, differentiation and homeostasis, tumor suppression and stem cell regulation [26]. miRNAs are also known to be involved in the development of human parasitic diseases. For example, in Schistosoma mansoni, the causative agent of schistosomiasis, it was shown that miRNAs can control the development of the parasite and also of liver pathology [33]. While in Chagas disease, caused by Trypanosoma cruzi, there are reports of the involvement of miRNAs in cardiovascular disorders caused by illness [34][35][36].
In toxoplasmosis, a disease caused by the agent Toxoplasma gondii, some miR-NAs may be related to the degree of virulence, inhibition of apoptosis and immune response [37,38]. While in malaria (Plasmodium spp.), evidence has shown that the agent manipulates the host miRNA expression, supporting its growth and survival, and also regulates important genes for the host immune response [39][40][41].
In leishmaniasis, miRNAs play an important role in the biology of infection, pathogenicity, recognition and activation of the immune response by macrophages and dendritic cells, host-parasite interaction and drugs resistance [42][43][44]. Although, research emphasizes the importance of studying miRNAs during infection or pathogenesis by different parasites, the role of these structures in the development of antiparasitic resistance remains unclear. Drug resistance is a growing problem worldwide. Resisting a drug means saying that there was a reduction in the drug effectiveness in the total elimination of the pathogen responsible for the disease [45]. miRNAs have been identified as targets for pathogen invasion and immune response control, in addition to resistance to chemotherapy in a variety of organisms and diseases (Figure 3) [6,10].
In recent years, studies have shown that miRNAs are involved in tumor cell resistance to chemotherapy [46][47][48]. According to the studies, miRNAs regulate different target genes, especially genes that affect the cell response to chemotherapy drugs. In insects, some miRNAs are involved in resistance to the insecticide diamide in the control of Plutella xylostella, mainly miR-7a and miR-8519 [49]. Other studies have reported the importance of miR-2, miR-13, miR-7, miR-92a and miR-13,664 expression in the Culex pipiens insect resistance to the drug deltamethrin [50][51][52].
The resistance of some parasites is also related to miRNAs. In the nematode C. elegans, miR-1 down-regulates the expression of two nicotinic acetylcholine receptor subunits (nAChR), unc-29 and unc-63 [53]. According to the authors, when the expression of these subunits is increased, it corresponds to a decrease in muscle sensitivity to acetylcholine by levamisole, that is, the drug action is decreased. In Toxocara spp., the causative agent of toxocariasis in humans and other animals, computational studies predict that some miRNAs function as targets associated with drug resistance, such as Tc-miR-2861, Tc-miR-2881 and Tc-miR -5126 [54]. Additionally, miR-9551 is regulated in Teladorsagia circumcincta, the most prevalent parasite in United Kingdom animals with proven multiple drug resistance [55]. Recently, miR-9551 expression was also correlated with an anthelmintic resistance phenotype of Haemonchus contortus to the drug ivermectin [9].
The role of miRNAs in drug resistance is clear for a wide variety of organisms, for protozoan parasites, mainly Leishmania spp. it's still not well elucidated. However, the expression of miRNAs in leishmaniasis can be used as a strategy to escape the host immune system [13,14]. For example, the pentavalent antimony-resistant Leishmania donovani species exploits miR-466i to regulate levels of inflammatory cytokines produced by macrophages during infection, and in other cases, it decreases miR-122 expression by hepatocytes to facilitate infection, ensuring its differentiation and intracellular multiplication [56,57].
For years, pentavalent antimony was the main treatment against leishmaniasis and with this, the parasites acquired resistance and the drug lost its effectiveness [58]. The resistance mechanism of Leishmania donovani to pentavalent antimonals can be explained by the increased production of IL-10 and TGF-β inducing the upregulation of the multiresistant protein-1 (MDR1) and causing the expulsion of the drug by macrophages [58]. Also, according to the authors, during infection by the resistant parasite, miRNAs control the expression of pro and anti-inflammatory cytokines in macrophages internally regulated by the proteins Ago2, PP2A and HuR. HuR positively inhibits PP2A expression by activating the modulatory function of HuR and PP2A antagonist miRNAs and acting as a balancing factor for immune responses in macrophages to interrupt or prevent infection by pentavalent antimonal resistant L. donovani [59].
Tiwari et al. [10] pointed out that Leishmania parasites possibly induce macrophages to express miRNA regulatory proteins that help expel drugs from cells. This drug resistance has been associated with the downregulation of miRNA-763, −1264, and -3473f by parasitized macrophages that induced upregulation of ABC transporters [10]. Recent studies have revealed that miRNA deficiency can also influence the emergence of host resistance to leishmaniasis. According to Varikuti et al. [60], miR-21 is positively expressed in dendritic cells and macrophages and in organs such as the spleen and liver infected by L. donovani. During infection, the lack of miR-21 increases IL-12 production and the Th1 immune response and consequently increases host resistance. miR-21 plays an essential role in the pathogenesis of the disease and is considered a potential target in the treatment of leishmaniasis. miRNAs alter transporters, receptors and ion channels drug, reducing sensitivity and consequently developing resistance [61,62]. The change in the miR profile may be a strategy of infection or drug resistance of parasites.
Thus, these studies highlight the importance of these structures in the development of leishmaniasis resistance to currently available drugs and indicate that the identification of miRNAs can provide strategies for controlling leishmaniasis.
To contain the acceleration of resistance by parasites, the search for new treatments has emerged. The discovery of new drugs with fewer side effects and more advantageous cost-benefits than current treatments has gained strength, in this sense, the use of natural products in the treatment of leishmaniasis has become an excellent alternative.

Natural products as an anti-Leishmania source
Natural products originating from plants are being increasingly explored as promising therapeutic alternatives, and the action of these products is mainly attributed to existing bioactive phytochemicals [63,64].
The concentration of these compounds depends on the nature of the chemical used as a solvent during the extraction and handling process, as well as the storage conditions [65]. Terpenoids, phenolic compounds, alkaloids and flavonoids are the most important phytochemical groups. Terpenoids from essential oils are extracted by hydrodistillation and mechanical pressing techniques, while phenolic compounds, alkaloids and flavonoids are extracted using organic solvents, mainly dichloromethane, methanol, ethanol, ethyl acetate, n-butanol, chloroform, ether and benzene [65][66][67]. After extraction, the phytochemical analysis to identify the main classes of compounds is performed using chromatographic and colorimetric techniques [68]. Approximately 10,000 phytochemicals have been identified so far and their numerous medicinal properties are being investigated. Leaves, seeds, fruits and flowers are the plant organs richest in phytochemicals [69,70].
Currently, these groups of phytochemicals have anti-Leishmania properties and mechanisms of action already described. Terpenoids, aromatic and lipophilic substances, possibly can cross the plasma membrane, interfering with cell composition, inducing mitochondrial rupture and chromatin condensation and causing parasite death by apoptosis [71,72]. The glycyrrhizic acid, a triterpene extracted from the root of Glycyrrhiza glabra, was found to be responsible for the change in the host immune system, inducing an increase in the release of inflammatory cytokines in macrophages infected by Leishmania, facilitating a more efficient response against the parasite [73].
Phenolic compounds primarily function as antioxidants and free radical scavengers [74]. Its action on Leishmania is attributed to its ability to induce cell death through apoptosis and inhibition of cell replication through iron chelation [75][76][77]. Recently, Antwi et al. [78] also suggested that rosmarinic acid exerts an antileishmanial effect through iron chelation, however, its mechanism of action results in morphological alterations and interruption of the parasite cell cycle.
Alkaloids are alkaline compounds and can be classified based on the presence and activity of specific amino acids [79]. Many alkaloids have been described as having biological activities against Leishmania spp., however, the mechanism of action of these compounds is not fully understood. According to Fournet et al. [80] alkaloids can inhibit an essential antioxidant enzyme in the parasite, trypanothione reductase. Additionally, a study showed that different synthetic quinoline alkaloids caused ultrastructural changes in Leishmania, such as loss of cell membrane integrity in the parasites, in addition to inducing apoptosis [81].
Flavonoids, in turn, are a class of natural compounds with several known biological activities, including anti-Leishmania. Flavonoids subdivide into classes such as chalcones, flavones, isoflavones, flavonols and anthocyanidins [82]. Quercetin, a flavonol extracted from Kalanchoe pinnata, showed activity against Leishmania amazonensis promastigotes [83]. According to the authors, the compound increased the levels of reactive oxygen species, causing mitochondrial damage and leading to parasite death. Recently, Araújo et al. [84] demonstrated that flavonoids isolated from Solanum paludosum had an antiproliferative effect on Leishmania parasites inducing cell cycle blockage and autophagy.
In general, plants contain biomolecules with high active potential against leishmaniasis. However, the process of discovering these molecules is considered complex and time-consuming, since it involves phases such as isolation, identification, optimization of their properties and selection of safe and effective compounds for drug development [64,70].
Studies have identified that the expression of miRNAs in different types of cancer is specifically regulated by phytochemicals [15]. In the treatment of breast cancer, several phytochemicals have been identified as tumor suppressors, directly controlling the expression of miRNAs [15][16][17]. According to Baselga-Escudero et al. [85] polyphenols can bind directly to miRNAs and that the chemical structure of these compounds influences the expression of miRNAs. Recently, through in vitro and in vivo studies, quercetin was able to modulate miRNAs and, consequently, suppress oncogenes or stimulate tumor suppressor genes by altering the expression of miRNAs [86].
These studies show the importance of natural compounds in the regulation of miRNAs and the possible influence of these in their action on diseases. However,

Author details
Sandra Alves de Araújo 1,2,3 , Tatiane Aranha da Penha-Silva 4 *, Jaqueline Diniz Pinho 2 , Marcelo de Souza Andrade 1,3 * and Ana Lucia Abreu-Silva 1,2 it remains unclear whether miRNAs can interact with phytochemicals so that they are not absorbed and/or metabolized by the parasites and thus contribute to the resistance mechanism of leishmaniasis (Figure 3).

Conclusions
In conclusion, miRNAs have a wide range of important biological functions for parasites, including leishmaniasis. Although, the role of these structures in the development of antiparasitic resistance remains unclear, this work provides suggestive information about the strong contribution of miRNAs in this process. Furthermore, natural compounds with anti-Leishmania activity may play an important role in the regulation of miRNAs, influencing their action on the parasite. Therefore, the identification and characterization of specific miRNAs, as well as the clarification of their interaction with natural compounds are crucial for understanding the resistance mechanism of leishmaniasis.