MicroRNAs: Biological Regulators in Pathogen–Host Interactions

An inflammatory response is essential for combating invading pathogens. Several effector components, as well as immune cell populations, are involved in mounting an immune response, thereby destroying pathogenic organisms such as bacteria, fungi, viruses, and parasites. In the past decade, microRNAs (miRNAs), a group of noncoding small RNAs, have emerged as functionally significant regulatory molecules with the significant capability of fine-tuning biological processes. The important role of miRNAs in inflammation and immune responses is highlighted by studies in which the regulation of miRNAs in the host was shown to be related to infectious diseases and associated with the eradication or susceptibility of the infection. Here, we review the biological aspects of microRNAs, focusing on their roles as regulators of gene expression during pathogen–host interactions and their implications in the immune response against Leishmania, Trypanosoma, Toxoplasma, and Plasmodium infectious diseases.


Introduction
The discovery of noncoding RNAs (ncRNAs) has revolutionized the field of molecular biology. These ncRNAs do not code for proteins but globally impact genome maintenance and gene expression [1][2][3][4]. These RNAs can be categorized according to their length, localization, and function, such as ncRNAs that regulate gene expression, as long noncoding RNAs (lncRNAs), microRNAs (miRNAs), small interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs); RNA maturation, as small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs); and protein synthesis, as ribosomal RNA (rRNAs) and transfer RNAs (tRNAs) [5,6]. miRNAs (typically 21 nucleotides) are a class of ncRNAs which interact with the 3 untranslated region (3 UTR) of messenger RNAs (mRNAs), leading to mRNA degradation or translational repression [7][8][9][10]. The involvement of miRNAs in a variety of biological processes, such as chronic pathologies and infectious diseases, illustrates their complexity as they have been described as being able to simultaneously interact with diverse molecules, such as RNA binding proteins (RBPs), making the miRNA recognition site more accessible to the RNA-induced silencing complex (RISC), which also enables the processing of pri-miRNAs [11,12] or lncRNAs [1,13], controlling different points of the gene expression flux.
Host-pathogen interactions result in signaling and physiological modifications in host cells that induce the miRNA-mediated post-transcriptional regulation of genes involved in the inflammatory response during the induction of the immune response [14,15]. These miRNAs are involved in the modulation of both innate and adaptive immune responses [16]. In recent years, the alteration of marrow [48,49]. This disease is considered a neglected tropical disease, endemic in 98 countries around the world, with the annual incidence for cutaneous leishmaniasis estimated to be 0.7-1.2 million and 0.2-0.4 million for visceral leishmaniasis [47,50]. Leishmania has a dimorphic life cycle, alternating between extracellular replicative promastigote forms that live in the digestive tract of the sand fly (Phlebotomus and Lutzomyia) and amastigote forms that live in the interior of the phagolysosomal compartment in mammalian host phagocytic cells, such as macrophages, neutrophils, and dendritic cells (DCs) [50,51]. The initial steps of the inflammatory response occur during the phagocytosis of Leishmania and its establishment inside the phagolysosome and it is able to modulate the immune response by reducing the efficiency of inflammation and the development of an adaptive immune response [52][53][54][55]. Recent studies demonstrated that infections caused by L. amazonensis, L. infantum, L. major, and L. donovani can induce alteration in the miRNA profile from macrophages and dendritic cells of humans, murines, and dogs, thereby implicating the recognition and activation mechanisms of the immune response against parasites [56][57][58][59][60][61][62][63].
Interestingly, some virulence factors of parasites interfere with the miRNA machinery, regulating host mRNA expression. As observed for L. donovani, the parasite glycoprotein gp63 targets the host Dicer1, cleaving Dicer to downregulate pre-miR-122 and its processing to miR-122, resulting in post-transcriptional regulation of host mRNA/miRNA interactions, leading to increased parasite burden in mouse liver [59], as shown in Figure 2. The activity of L. amazonensis arginase, an enzyme that uses l-arginine to produce ornithine and urea, can impact the regulation of macrophage miRNA expression during infection [61]. The use of l-arginine by both host and parasite arginases could alter the amino acid availability for host activation of the microbicidal response once l-arginine is used by NOS2 to produce NO [61]. Indeed, l-arginine deprivation leads to the downregulation of the CD3ζ chain in T cells, thereby suppressing the T cell response [76,77]. Indeed, miR-122 repression of the cationic amino acid transporter (CAT1) can regulate intracellular l-arginine availability [78]. Besides, miR-155 represses the expression of arginase 2 in DC, preventing l-arginine depletion and allowing the activation of T cells [79]. In addition, parasites can affect the activation of microbicidal mechanisms, such as NO production, and also cell recruitment to the location of the lesion. Our group showed that L. amazonensis induces upregulation of miR-294-3p and miR-721 in BALB/c-BMDM, which binds to Nos2 3′UTR, reducing the levels of NOS2 and NO production and increasing infectivity [61]. Also, miRNAs that are deregulated during infection, such as miR-30e and miR-302d, interfere with Nos2 mRNA expression and NO production; miR-294 and miR-302d regulate Tnf mRNA levels and miR-294 alters Ccl2/Mcp-1 mRNA, implicating the expression of these miRNAs in controlling infectivity [80]. Indeed, for a set of miRNAs, let-7a, miR-25, miR-26a, miR-132, miR-140, miR-146a, and miR-155, their upregulation in L. major-infected human macrophages was negatively correlated with the Once the parasites are phagocytized by these immune cells, they are differentiated into amastigote forms and begin to multiply within those cells. Amastigotes have a reduced flagellum and ovaleted morphology and survive inside the phagolysosomal compartment. Parasites in promastigote form express noncoding RNAs (ncRNAs) that can regulate the invertebrate host-sandfly-metabolic pathways. However, sandflies also express miRNAs, which can affect the parasite biology, influencing its success in the colonization of sandflies' digestive tract and differentiation to the infective forms and the vertebrate host. Not only do the invertebrate hosts express miRNAs, but the vertebrate one also does. The experimental models using human Monocytes Derived-Macrophages (MDM) or murine Bone Marrow-Derived Macrophages (BMDM), and also in the Peripheral Blood Cells (PBMCs) from human and dogs show that infection of mammalian hosts can interfere with the profile of host miRNAs. Also, the plasma samples or the cutaneous lesion site from leishmaniasis patients show modifications in the miRNA profile.
In addition, parasites can affect the activation of microbicidal mechanisms, such as NO production, and also cell recruitment to the location of the lesion. Our group showed that L. amazonensis induces upregulation of miR-294-3p and miR-721 in BALB/c-BMDM, which binds to Nos2 3 UTR, reducing the levels of NOS2 and NO production and increasing infectivity [61]. Also, miRNAs that are deregulated during infection, such as miR-30e and miR-302d, interfere with Nos2 mRNA expression and NO production; miR-294 and miR-302d regulate Tnf mRNA levels and miR-294 alters Ccl2/Mcp-1 mRNA, implicating the expression of these miRNAs in controlling infectivity [80].
Indeed, for a set of miRNAs, let-7a, miR-25, miR-26a, miR-132, miR-140, miR-146a, and miR-155, their upregulation in L. major-infected human macrophages was negatively correlated with the expression of their corresponding chemokine targets, CCL2, CCL5, CXCL10, CXCL11, and CXCL12, thereby corroborating the data that indicate the downregulation of chemokines CCR2, CCL5, and CXCL10 during infection [81]. Likewise, the activation of hypoxia inducible factor-1 (HIF-1α) partially controls miR-210 during the L. major infection of human macrophages, linking HIF-1α overexpression to susceptibility to both L. donovani and L. amazonensis [82]. This mechanism occurs via the downregulation of the NF-κB mediating transcription of pro-inflammatory cytokine genes, such as TNF-α and IL-12, thereby altering the immune response to parasite survival inside the macrophages [83]. Interestingly, miR-21 is upregulated in peripheral blood mononuclear cells (PBMCs) infected with L. donovani, and miR-blocking increases the expression of IL-12 mRNA in murine-DCs infected with L. donovani, leading to a proliferation of CD4 + T cells [84]. Further, miRNA-361-3p, a regulator of TNF, was observed to be negatively correlated with localized cutaneous leishmaniasis (LCL) skin lesions caused by L. braziliensis, which is resistant to pentavalent antimonial and healing of parasites [85], while the expression of miR-193b and miR-671 was observed to be correlated with that of their respective target genes, CD40 and TNFR, in lesions of L. braziliensis-infected patients.
Furthermore, canine visceral leishmaniasis in symptomatic dogs who were naturally infected with L. infantum showed a differential modulation in the expression of miR-150, miR-451, miR-192, miR-194, and miR-371 in PBMCs, which can target genes to regulate the immune response and pathogenesis, such as NF-κB, TNF-α, CD80, and IFN-γ, which are important molecules related to resistance against the disease [62]. The L. infantum infection of human U937 and THP-1-derived macrophages upregulates miR-346, which decreases the mRNA level of genes initially associated with decreased MHC class I and II antigen presentation, TAP1, RFX1, and BCAP31.
Parasites can incite anti-inflammatory responses and modify antigen presentation by deregulating the host's autophagy machinery via mimicking apoptosis signals, such as those known as apoptotic-like Leishmania, and reducing the proliferation of CD4 T cells [86,87]. The miRNAs miR-101c, miR-129-5p, miR-155, and miR-210-5p, which are differentially expressed in L. major-infected BMDM, were related to the activation of the autophagic machinery and interfere with parasite clearance [56]. The L. donovani infection of THP-1 and human-MDM upregulates miR-30a levels and its inhibition increases the autophagic mechanism via negative regulation of Beclin 1 (BECN1), thereby decreasing infectivity [88]. Likewise, the L. donovani-infected murine macrophage expression of miRNA-3473f inhibits autophagy [63].
Moreover, miR-511 is upregulated in DCs infected with L. donovani and can interfere in Toll-like receptor 4 (TLR4) signaling, which is an important molecule in the recognition and activation of the immune response to Leishmania infections [89]. Also, L. donovani strain, which is resistant to sodium stibogluconate, a pentavalent antimonial that is used for visceral leishmaniasis treatment in the Indian subcontinent, upregulates the expression of miR-466i in murine macrophages that target MyD88, increasing the levels of IL-10 production and the severity of disease [57]. Our group showed that the upregulation of let-7e, let-7f, and let-7g occurs in a MyD88, TLR2, or TLR4 dependent way during L. amazonensis infection and let-7e inhibition increases the expression of NOS2 mRNA, the NOS2 protein amount, and NO production, thus impacting infectivity [90]. Also, let-7e inhibition during L. amazonensis infection upregulates the levels of the validated targets, Tnfpaip3, Map2k4, Tbk1, and Tnf, as well as in the predicted targets, Traf 6, Ppara, Mapk8ip3/Jip3, Map3k1, and Ube2n, globally impacting TLR signaling gene expression [90]. It is interesting that the infection of macrophages with Mycobacterium and Neisseria upregulate the levels of let-7e [91,92]. Indeed, let-7e targets p65 NF-κB activation and phosphoinositide-3 kinase/serine-threonine protein kinase (Pi3k/Akt) and TLR4, reducing the expression of pro-inflammatory cytokines, such as TNF, IFN-α, IL-6, and the chemokines MCP-1, MIP-1, and IP-10.
Some studies corroborate the integration of the transcription factors and miRNA regulation of host immune responses. L. donovani increases the levels of the c-Myc transcription factor in human MDMs, leading to an upregulation of DROSHA and a downregulation of miRNAs let-7a, miR-151, miR-34a, miR-98, miR-148b, and miR-378a, while c-Myc knockdown reverts the expression regulation pattern of these molecules and attenuates parasite survival [93]. c-Myc overexpression sustains M2 polarization, a characteristic of human tumor-associated macrophages (TAMs), and regulates the expression of vascular endothelial growth factor (VEGF), matrix metallopeptidase 9 (MMP9), HIF-1α, and transforming growth factor beta (TGF-β).
Geraci and coauthors elegantly showed that let-7a, let-7b, and miR-103 are upregulated in DCs and macrophages that are infected with L. donovani, but downregulated in L. major infections [60]; miRNAs that target genes involved in inflammation and their expression can also modulate interferon regulation, demonstrating the importance of Leishmania species in determining the outcome of an infection and the miRNA profile. Overall, Leishmania species can induce a wide-ranging variance in the miRNA profile, which is correlated with the target transcript and host cell types, supporting species-specificity in miRNA regulation and the host recognition of pathogen guides miRNA-mRNA interactions during Leishmania infection.
Regarding the invertebrate host, phlebotomine sandflies are well recognized as vectors for species of Leishmania and a wide range of pathogens, such as bacteria and viruses, with the regulation of miRNAs favoring pathogen survival and transmission [94][95][96]. Yang and Wu discriminated miRNAs from other noncoding RNAs in Lutzomya longipalpis, a vector of leishmaniasis in the Americas and also suggest the role of miRNA targeting genes involved in Leishmania infection, such as llo-miR-9388-5p and llo-miR-3871-5p [97] in Phlebotomus perniciosus, a major vector of Leishmania infantum in Europe [98].
Computational algorithm prediction and RNA-seq analysis support the evidence of ncRNAs in Leishmania, as shown for L. amazonensis, L. braziliensis, L. donovani, L. infantum, and L. major, indicating that ncRNA may have a role in gene expression modulation and could help us understand genomic organization, as well as transcriptional expression and regulation in promastigote and amastigote forms. However, only L. braziliensis possesses a functional RNAi pathway and AGO1 [99][100][101][102].

Trypanosoma-Host Interactions
Chagas disease is another neglected tropical disease caused by the parasite Trypanosoma cruzi. It is a significant public health problem, affecting 6 to 7 million people worldwide (WHO) [103]. Although the impact of Chagas disease has decreased over the years, it is still a public health problem in Latin America. Additionally, the disease has been described in non-endemic areas, such as Canada, the United States, Europe, Australia, and Japan, mainly due to its transmission by blood transfusion, organ transplantation, vertical transmission, or accidental ingestion [103][104][105][106]. In South America, T. cruzi is mainly transmitted by the feces of infected Triatominae bugs during their blood-meal [103-106]. The disease is characterized by parasite invasion in the bloodstream, infecting many cell types in different tissues, including muscle cardiac cells. The disease presents two clinical phases, acute and chronic, varying from asymptomatic with non-specific symptoms to cardiomyopathy, dysfunction of the digestive tract, or alterations in the nervous system [107,108]. In both phases, there is an activation of the immune system. In the acute phase, the immune system acts to control the infection and parasite replication. By contrast, during the chronic phase, several cascades are regulated and related to the autoimmune and/or to the inflammatory response sustained by parasite persistence [109]. The progression and severity of the disease can vary according to the individual, geographic region, and/or parasite tissue tropism, presenting specific genotype and phenotype characteristics that can affect host-pathogen interactions [110]. Benznidazole and nifurtimox, the treatments that are currently available, began to be used over 40 years ago. Moreover, both drugs have several limitations, such as long treatment times and being primarily effective for acute forms of disease [111]. Thus, new effective therapies and prognosis markers are required.
Gene expression profiling of myocardial tissue and the heart have shown differential gene expression to be modulated among chronic Chagas patients, as well as acute and chronic T. cruzi-infected mice. The differentially expressed genes are related to immune response, energy and metabolism, and cell stress response [112]. The inflammatory response comprises the activation of the TLRs cascade signaling, the induction of IFN-γ production in T cells [112,113], and antibody production by B cells to reduce parasitaemia, which is counterbalanced by IL-10. Despite an innate and adaptative response, low-grade chronic infection was established to infiltrate Th1 cells, thereby producing IFN-γ in myocardial tissue and leading to heart damage [112,114,115].
The involvement of miRNAs in Chagas disease has also been described to act in the gene expression modulation of physiological and pathophysiological factors. The interplay between heart tissue cells, including cardiomyocytes, fibroblasts, endothelial, and infiltrating inflammatory cells, and the possibility of miRNA transfer across these cells represents a complex connection between miRNAs and mRNAs, impacting the resistance to infection and pathogenesis.
The modulation of miRNAs from the hearts of T. cruzi-infected mice is positively correlated with a parasitemia peak at 30 days post-infection (dpi), as observed for miR-146b, miR-21, miR-142-3p, and miR-142-5p, while a negative correlation is observed for miR-145-5p and miR-149-5p and also suggests the regulation of genes involved in the pathophysiological conditions of experimental infection, such as calcium and potassium channels and electrocardiography (ECG) parameters [116], as shown in Figure 3. The dysregulation of miR-133 and miR-208 have been correlated with heart genes that are related to cardiovascular disease in chronic Chagas patients and acute T. cruzi-infected mice [112,114,115]. Ferreira et al. showed the upregulation of miRNAs in whole heart tissue, such as miR-155-5p in the acute (15 dpi) and chronic phase (30 and 45 dpi) and let-7a-5p in the chronic phase of infection with T. cruzi in the murine experimental model. This pattern of miRNA modulation was correlated with Nrf2 transcriptional response and oxidative stress [117]. In addition, miR-149-5p, miR-138-5p, and miR-16-5p appeared to be differentially expressed during T. cruzi infection through modulation of IFN-γ and NFR2-modulated genes in infiltrating cells and, consequently, the control of parasitism and tissue damage [117].
The infection of B6 mice by T. cruzi leads to increased levels of miR-10a in thymic epithelial cells (TEC) involved in intrathymic T cell differentiation, which can be caused by TGF-β signaling and impact thymus atrophy during infection [124,125].
T. cruzi genome and transcriptome analysis showed the presence of ncRNA, such as small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and transfer RNA (tRNA) [126][127][128] (Figure 3). Indeed, sncRNAs can be delivered into HeLa cells by extracellular vesicles from parasites, increasing the susceptibility to infection [129]. miR-155-5p in the acute (15 dpi) and chronic phase (30 and 45 dpi) and let-7a-5p in the chronic phase of infection with T. cruzi in the murine experimental model. This pattern of miRNA modulation was correlated with Nrf2 transcriptional response and oxidative stress [117]. In addition, miR-149-5p, miR-138-5p, and miR-16-5p appeared to be differentially expressed during T. cruzi infection through modulation of IFN-γ and NFR2-modulated genes in infiltrating cells and, consequently, the control of parasitism and tissue damage [117].  Regarding T. brucei gambiense and T. b. rhodesiense, these cause a fatal human disease known as African sleeping sickness chronic illness, which is prevalent in West Africa and East Africa, respectively [130]. T. brucei is transmitted by the bite of infected tsetse flies (genus Glossina), alternating the bloodstream-form in the mammalian host (man, domestic animals, and ruminants) and insect-form of life [131]. Parasites injected in hosts cause a local inflammatory reaction, a tender reddish swelling, and the multiplication of parasites in the plasma and interstitial fluid begin an acute febrile illness. T. b. gambiense classically causes an enlargement of cervical lymph nodes and can progress to neurological diseases, such as meningoencephalitis, lethargy, and coma. Besides, T. b. rhodesiense mostly causes acute systemic diseases, such as haemolymphatic alteration, swollen lymph nodes, fever, and rapid weight loss. T. brucei resides in the bloodstream and evades the host immune response by antigenic variation [131][132][133]. Interestingly, the miR-193b and miR-338 were upregulated in blood samples from patients and miR-199a-3p and miR-27b were seropositive for T. b. gambiense [134]. The T. brucei transcriptome profile identified ncRNAs, and a description of these molecules could improve our knowledge of parasite biology [135,136]. Interestingly, T. brucei present a total of 881 predicted miRNAs, including the genomic cluster of miRNAs that codify identical precursors, such as miR-1-2 and miR-4 to miR-12, as well as miR-84 to miR-106, which can impact virulence factors, such as variant surface glycoprotein (VSG), in different stages of the transformation and proliferation of parasites [137]. However, T. brucei possesses a functional RNAi as well as AGO1 machinery, but the functional RNAi machinery was not found in T. cruzi, L. major, and L. donovani [99].

Toxoplasma-Host Interaction
Toxoplasma gondii is a ubiquitous parasite that can infect a large range of hosts, including mice, humans, pigs, birds, sheep, and cats, the last of which is the definitive host where sexual replication takes place. Its life cycle is complex depending on the host. When it replicates asexually, the parasite presents two forms: tachyzoites and bradyzoites-a fast replicant and a slow replicant, respectively. In addition, bradyzoites can encyst and remain dormant for a long period of time until the host becomes immunocompromised [138]. Infection in the main range of hosts starts with the ingestion of raw infected meat or the ingestion of oocysts that are shed in cat feces. The parasite then starts its odyssey of conquering as many cells as possible and replicating. T. gondii is a highly successful parasite due, in part, to its silent colonization of its host [139,140]. Almost all types of immune cells are recruited to control infection with T. gondii: macrophages, neutrophils, dendritic cells, lymphocytes, and NK cells [139,[141][142][143][144]. Although, almost all infections are silent and this parasite is able to change the host's miRNA profile, modifying the way that it responds to the infection. Moreover, the parasite also presents its own miRNA processing machinery and miRNAs.
The first published study on this subject, released in 2010, correlated the miRNA profile of primary HFFs cells (human foreskin fibroblasts) with T. gondii infection. This study demonstrated the upregulation of two clusters: miR-17-92 (miR-17, miR-18, miR-19a, miR-20a, miR-19-b1, and miR-92-1) and miR-106b-25 (miR-106b, miR-93, and miR-25) in infected cells [145], as shown in Figure 4. Interestingly, part of the miRNAs in both clusters share the same seed sequence, suggesting that these miRNAs are related with the regulation of the same kinds of processes in HFF cells. [145]. Later, in 2014, another group showed that the expression of the miR-17~92 cluster in human macrophages is dependent on STAT3 (signal transducer and activator of transcription) and these miRNAs can regulate apoptosis due to the inhibition of BIM, which is a pro-apoptotic molecule [146]. This mechanism for inhibiting apoptosis is a known pathway that enables the T. gondii to evade an immune response [147]. There are many studies on the role of the miR-17-92 cluster in relation to several cellular processes, including cancer, the immunopathogenesis of autoimmunity, inflammation, or even chronic diseases. This cluster is encoded in humans via MIR17HG (miR-17 host gene) [148,149]. This open reading frame has its transcription regulated by c-Myc [150], HIF1 [149], and many other transcription factors related to inflammation. In addition, the functions of this miRNA cluster are related with proliferation as well as B and T cell differentiation [149,151]. miR-17~92 are found to regulate immune cell proliferation. B and T cells are examples of this phenomenon [149,151]. miR-17~92 helps T cells to differentiate, proliferate, and polarize into T helper cells [151], however this cluster is also related to the differentiation of regulatory T cells, as described by Xiao et al. in 2008, who demonstrated that a mutation in the mice miR17hg host gene leads to lymphoproliferative disease and autoimmunity because of a decrease in the differentiation of this kind of cell [152]. For B cell biology, the conditional deletion of the Dicer in early B cell progenitors blocked the differentiation of pro-B cells to pre-B cells and upregulated miR-17~92 targets, especially the pro-apoptotic Bim molecule [153], suggesting, again, that this cluster is related to apoptosis regulation. Oocysts are released from infected cats' feces, sporulate in the environment, and become infective. Samples of kittens' liver present modifications in miRNA expression. Humans can also become infected by eating undercooked meat of infected animals or while nursing infected cats. In the human host, this infection can lead to toxoplasmosis, a disease that affects various organs' tissues such as skeletal muscle, myocardium, and the brain. As a result, neuronal cells, monocyte/macrophage, and fibroblasts from infected patients present modifications in miRNA expression. Also, porcine alveolar macrophages and splenocytes samples have altered miRNA profiles in infected pigs. To study toxoplasmosis in vivo, mice models are commonly used because mice can naturally be infected by the parasites and affect multiple organs while changing their miRNA profile. Mice are largely used to study the disease outcomes and treatments, they can also be naturally infected and have multiple organs affected with the infection, and it is already known that the spleen, plasma, and brain have their miRNA profile changed during the infection. The box color represents the increase (red) or decrease (green) of miRNAs expression. Oocysts are released from infected cats' feces, sporulate in the environment, and become infective. Samples of kittens' liver present modifications in miRNA expression. Humans can also become infected by eating undercooked meat of infected animals or while nursing infected cats. In the human host, this infection can lead to toxoplasmosis, a disease that affects various organs' tissues such as skeletal muscle, myocardium, and the brain. As a result, neuronal cells, monocyte/macrophage, and fibroblasts from infected patients present modifications in miRNA expression. Also, porcine alveolar macrophages and splenocytes samples have altered miRNA profiles in infected pigs. To study toxoplasmosis in vivo, mice models are commonly used because mice can naturally be infected by the parasites and affect multiple organs while changing their miRNA profile. Mice are largely used to study the disease outcomes and treatments, they can also be naturally infected and have multiple organs affected with the infection, and it is already known that the spleen, plasma, and brain have their miRNA profile changed during the infection. The box color represents the increase (red) or decrease (green) of miRNAs expression.
Although miR-17-92 and miR-106-25 are not the only miRNAs that are modified when infection with a parasite is established, many other studies have demonstrated that hosts can have their miRNA expression patterns modified. Toxoplasma infection in pigs has been described with global miRNA changes in cells infected with the parasite-one showing the changes in splenocytes infected with the YZ-1 Chinese strain (type IX) and the other showing changes in alveolar macrophages. Comparing the type I and type II strains, both assessed the miRNA profile by sequencing small RNAs. Between these three Toxoplasma strains, miR-17 presented different patterns of regulation, for example, when porcine alveolar macrophages (PAMs) were infected with the RH strain, their expression was downregulated. On the other hand, infection with the Me49 strain upregulated the expression of this miRNA. In addition, when porcine splenocytes were infected with YZ-1, miR-17 was upregulated after 25 dpi [154]. However, PAMs also presented changes in the miRNAs that regulate TNFα, indicating a predictive link between some novel miRNAs. Thus, it is practical to imagine that this inflammatory pathway might be modified due to infection, since the more silent the infection, the more successful it is, and TNFα signaling leads to an inflammatory response that can destroy all parasites. It is also interesting to observe that in chronic infection of pig spleen (50 dpi), there is a massive downregulation of miRNAs.
Mice are the main study models for many infectious diseases such as toxoplasmosis. Thus, understanding how these animals respond to infection remains of interest. For example, Canella et al. used paired tests between infected HFF and mice (in vivo) to compare different Toxoplasma strains, with different virulence results [155]. They observed that miR-146a was upregulated in HFF infection with Me49 and downregulated in infection with the RH strain. They also demonstrated that the expression of ROP16 was closely related to the upregulation of that miRNA. They also demonstrated that the knockout of miR-146a made the animal resistant to a virulent Toxoplasma strain and the deletion of ROP16 increased the expression of miR-146a in a strain-specific fashion, thereby demonstrating that there is a balance between parasite protein expression and the host miRNA profile. T. gondii RH and Me49 strain infections of BALB/c mice upregulated miRNAs miR-712-3p, miR-511-5p, and miR-217-5p in the host plasma in a specific way as these miRNAs are not found in samples from mice infected with Plasmodium berghei, P. yoelii, P. chabaudi, Cryptosporidium parvum, mouse hepatitis virus (MHV), or Staphylococcus aureus [156]. Also, Jia and coworkers proposed using circulating miRNA as biomarkers for the detection of T. gondii infection, even in the early phase of infection [156].
Infected mice brains were also studied to compare the global miRNA changes during infection with T. gondii PRU strain oocysts during acute and chronic infection [157]. This kind of comparison was also made using the spleen of mice infected with the T. gondii RH strain, comparing the acute and chronic phases of infection [158]. In addition, the human neuroepithelial cell line was infected with RH-2F, PRU, or CTG to compare how those strains could modify the miRNA profile of neuronal cells, as well as the normal functions of neurons. It was observed that the upregulation of miR-132 during infection with all studied strains was responsible for regulating the expression of genes involved in dopamine metabolism [159]. In 2017, a North America-based cohort study revealed that infection with T. gondii affects the human brain in many pathways related to epilepsy, neurodegeneration, and cancer. This study presented the complex interaction between the proteomic and miRNomic profiles in the brain of congenitally infected patients and the following strains: type I-RH and GT1; type II-Me49 and PRU; and type III-VEG. The authors also evaluated the polymorphisms involved in the susceptibility/resistance to infection in the families of these patients. They observed that NF-κB and TGFβ were the central nodes of the complex net of interactions among all those molecules; the authors also observed allelic variants in NFκB and TGFβ among the studied families [160].
Changes in the miRNA profiles of cats were also an object of study since cats are the definitive host of Toxoplasma. The global miRNA profile of cat liver infected with the PRU Toxoplasma strain (type II) showed 82 modulated miRNAs, with 48 downregulated. Moreover, miR-17 appeared to be upregulated, demonstrating that this miRNA/cluster expression is closely related to infection with type II strains of T. gondii [161].

Plasmodium-Host Interactions
Malaria is an infectious disease that is caused by Plasmodium spp. parasites and remains the most devastating illness in tropical countries, which is responsible for the death of over a million people every year, mainly in sub-Saharan Africa, Asia, and Latin America [162]. Children under 5 years of age are the most affected [162]. The parasite life cycle involves the infection of humans through the bites of infected female Anopheles mosquitoes who inject sporozoite forms that migrate to the liver, thereby infecting hepatocytes. Rupture of the infected hepatocytes initiates the asexual intraerythrocytic cycle [162]. Hepatosplenomegaly is a hallmark of malaria and the Plasmodium asexual stage causes this pathology and subverts protective immunity, whereas splenocytes and liver Kupffer cells are able to remove/eliminate senescent and infected red blood cells (iRBCs) [163,164]. Splenic macrophages are the principal elements in the clearance of malaria-causing parasites from the blood-stage and have been the focus of many studies concerning protective immunity; nevertheless, miRNA profiles in these populations of cells have not been studied. However, the miRNA expression in plasma, liver, and cerebral samples are well studied [165][166][167].
Several studies have shown changes in the miRNA profile in infection by P. falciparum and P. vivax, which cause malaria in humans, as well as in experimental malaria models of self-healing P. chabaudi infections of mice and P. berghei ANKA, which causes cerebral malaria in mice and alters the development of acquired protective immunity against Plasmodium [165,168,169]. Chloroquine therapy, a drug which is recommended for malaria treatment, alters the miRNA profile and reduces the levels of NOD-like receptor (NLR) family pyrin domain-containing 1 and 3 (NLRP1 and NLRP3) genes related to inflammasome signaling [170].
The levels of miR-451 and miR-16 are downregulated in plasma samples from patients with P. vivax infection [169]. However, the miR-146a rs2910164 polymorphism, which could affect the expression level of mature miR-146a, is associated with increased susceptibility to P. falciparum infection in first-time pregnant women [171]. miR-146a is involved in the regulation of molecules of TLR signaling, such as TNF receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 genes (IRAK1) [172] (Figure 5).
Cerebral malaria in P. berghei ANKA-infected mice increases the levels of miR-27a, miR-142, and miR-223 when compared to P. yoelii-infected mice; these miRNAs are implicated in TNF signaling and monocyte sequestration in cerebral microvessels in cerebral malaria [167]. The upregulation of let-7i, miR-27a, miR-150, miR-126, miR-210, and miR-155 are correlated with immunomodulator signals, apoptosis, and leukocyte adhesion, and are characteristic of the pathogenesis of experimental cerebral malaria.
aga-miR-1174 [182,183]. Also, the Drosha, Dicer I, and Argo1 knockdown strategy favored P. berghei survival [183]. Indeed, sugar-feeding of A. stephensi upregulated miRNAs, such as ast-miR-263a, ast-miR-283, and ast-miR-210 [184]. Moreoever, A. stephensi feeding of parasite-infected mammals altered the expression of the following miRNAs: ast-miR-2944a-5p, ast-miR-92b, ast-miR-989, ast-miR-275, ast-miR-281-3p, ast-miR-281-5p, ast-miR-306, ast-miR-263a-5p, ast-miR-7, ast-miR-309, and ast-miR-305-3p [184]. Human malaria infection begins with the bite of infected female mosquito Anopheles (invertebrate host), delivering sporozoite forms that migrate to the liver and differentiate into merozoites. These forms leave the liver and start the blood-stage asexual replication. In this stage, Plasmodium can differentiate into gametocytes, which can be ingested by the mosquito and develop the sexual phase of the life cycle. Afterward, the mosquito can infect another individual. The species P. falciparum and P. vivax are anthropophilic, infecting only humans; both of them can induce modifications in the miRNA profile in human plasma samples. Besides, mice infections with P. chaubadi and P. beghei have been used as malaria experimental models. In the murine infection, the parasite changes the circulating miRNA profile and it is seen in the same way in the liver of infected mice with P. chaubadi Figure 5. Plasmodium induces miRNA profile modifications during pathogen-host interaction. Human malaria infection begins with the bite of infected female mosquito Anopheles (invertebrate host), delivering sporozoite forms that migrate to the liver and differentiate into merozoites. These forms leave the liver and start the blood-stage asexual replication. In this stage, Plasmodium can differentiate into gametocytes, which can be ingested by the mosquito and develop the sexual phase of the life cycle. Afterward, the mosquito can infect another individual. The species P. falciparum and P. vivax are anthropophilic, infecting only humans; both of them can induce modifications in the miRNA profile in human plasma samples. Besides, mice infections with P. chaubadi and P. beghei have been used as malaria experimental models. In the murine infection, the parasite changes the circulating miRNA profile and it is seen in the same way in the liver of infected mice with P. chaubadi as it is seen in the brain of mice infected with P. berghei ANKA. Although, the invertebrate host presents modifications in the miRNA profile when infected with Plasmodium. The red boxes represent the upregulation of miRNAs related to these models. The arrows represent the upregulation (↑) or downregulation (↓) of miRNA.
Some ncRNAs are described in the malaria parasite P. falciparum, which differs in expression during different stages of the parasite life cycle, which is associated with regulation of the expression of virulence-related genes, such as the var gene family [180,181].

Novel Perspectives: MicroRNAs as Diagnostic Markers and Therapeutic Targets
miRNAs have been emerging as biomarkers and treatment assets of many diseases [185,186].
Why not think about them as a profitable approach to better diagnose and properly treat parasitic diseases? Here, we revised how miRNAs are related to parasitic diseases and included not only the vertebrate hosts, but also the invertebrate ones. Moreover, parasitic diseases affect animals that provide economical and emotional benefits to people. It is inarguable that the loss of support pets or livestock animals causes socio-economic and psychological consequences for people. Nevertheless, a deep gap persists in the development of therapeutic strategies for neglected diseases.
Although, there is an increasing number of studies that are being done in miRNA, however translational research of miRNA still remains a challenge. Earlier this year, Hanna et al. reviewed the role of miRNAs in clinical studies. They observed that findings in miRNA research are limited to academia, often do not make it through phase 3 or 4 of clinical trials, and there is a gap between basic science and clinical application [185,186].
Regarding the neglected diseases, researchers have dedicated decades to the development of new drugs and identifying new biomarkers of disease progression. Despite this, the use of miRNAs as a biomarker and its potential for treatment are poorly explored when compared with hotspot diseases like cancer, cardiac and circulatory diseases, and neurological disorders. Currently, there are only two clinical trials regarding parasitosis and both are cohort studies. The first one was developed in Chicago city, USA, correlating the miRNA profile with the protein expression in toxoplasmosis patients [160]. The other study is focused on figuring out what miRNAs are modified in plasma samples from patients who suffer from acute myocarditis caused by the Chagas disease from Salvador city in Brazil. This study is available in the clinical trials database to consult (<https://clinicaltrials.gov/ ct2/show/NCT01842880?term=miRNA&cond=Trypanosomiasis&draw=2&rank=1>).
Thus, the myriad of processes regulated by miRNAs makes them a very profitable approach to be studied, not only as biomarkers, but also as drugs in the treatment of pathologies [186]. Diversity of cell culture platforms, animal models, and the evaluation of circulating fluids (saliva, plasma, and serum) are now available to assess the treatment mechanisms, toxicity, and potential therapeutic efficacy of miRNA candidates in vitro [185,186]. Also, miRNAs can be used in the association of conventional treatments to target a large number of genes and pathways that deliver the host susceptibility to disease and ameliorate the prognosis. Further, the impact of specific miRNAs or a combination of some miRNAs on the regulation of immune responses in the hosts remains a significant challenge.

Conclusions
The distinct regulation of miRNA-mRNA interaction during host-pathogen interactions highlights the importance of species-specificity, cell type, and a host's genetic background in determining the details of post-transcriptional regulation of gene expression mediated by miRNAs. Nevertheless, the regulation of the same miRNAs, such as miR-146a and/or miR-155, modulated in host cells that are infected with T. gondii infection, cerebral malaria, and L. major infection [56,81,165] leads to the identification of the signature of the host cell response to parasites and can help in the identification of common characteristics that are implicated in the subversion of the immune response.