microRNAs: Critical Players during Helminth Infections

microRNAs (miRNAs) are a group of small non-coding RNAs that regulate gene expression post-transcriptionally through their interaction with the 3′ untranslated regions (3′ UTR) of target mRNAs, affecting their stability and/or translation. Therefore, miRNAs regulate biological processes such as signal transduction, cell death, autophagy, metabolism, development, cellular proliferation, and differentiation. Dysregulated expression of microRNAs is associated with infectious diseases, where miRNAs modulate important aspects of the parasite–host interaction. Helminths are parasitic worms that cause various neglected tropical diseases affecting millions worldwide. These parasites have sophisticated mechanisms that give them a surprising immunomodulatory capacity favoring parasite persistence and establishment of infection. In this review, we analyze miRNAs in infections caused by helminths, emphasizing their role in immune regulation and its implication in diagnosis, prognosis, and the development of therapeutic strategies.


Introduction
Helminths are complex organisms that comprise approximately three hundred thousand species that can be either free-living or parasitic [1]. They include some taxonomic groups, such as trematodes (flukes), cestodes (tapeworms), and nematodes (roundworms), associated with infections in animals and humans [2].
Helminths have a complex biological cycle that includes various host organisms in which they experience multiple developmental stages and metabolic adaptations [7,8]. They can be transmitted in a variety of ways; consequently, they have several routes of invasion [9]. However, they all have in common the amazing ability to modulate the host's immune response, suppressing responses that help its elimination and resolution of the infection [10]. This immunomodulatory capacity is related to the release of excretorysecretory (ES) products, such as metabolites, proteins, lipids, and extracellular vesicles (EVs), mediating host-parasite interaction [10][11][12]. EVs loaded with small non-coding RNAs (ncRNAs) are considered an important cross-species communication mechanism and represent a potential therapy for some infectious diseases [13].
MiRNAs are a group of ncRNAs (about 18-25 nucleotides) that regulate gene expression at the post-transcriptional level through their interaction with the 3 untranslated

Modulation of Host Immunity by Helminth Parasites
Although helminths show significant differences in their life cycle and tissue tropism, these parasites have in common that during infection, they carry out different modulatory strategies that affect all phases of the host's immune response, establishing the infection. A wide variety of ES products mediates their ability to manipulate host immunity [61,62] during acute and chronic helminth infection [10]. Although these ES products differ between species, they have a common mechanism of action, the simultaneous promotion of regulatory and proinflammatory Th2 immune responses. This response is also known as a modified Th2 response through pattern recognition receptors (PRRs) such as Toll-like (TLRs) receptors [9].
EVs are particularly important ES since they can transport miRNAs that regulate the host's gene expression after its internalization. Not surprisingly, most of the genes regulated by these EVs are involved in biological processes and pathways associated with pathogenicity and the host immune response [63][64][65].
During infection, dysregulation of miRNAs is related to the pathogenesis of diseases. Thus, host and parasite miRNAs determine the probability, progression, and establishment of the disease; consequently, they are considered master regulators of host-parasite interactions [15].
In the following section, we analyze the possible role of miRNAs in infections caused by helminth parasites, emphasizing their contribution to the immunomodulatory capacity of helminths during pathogen-host interactions.
During infection, dysregulation of miRNAs is related to the pathogenesis of diseases. Thus, host and parasite miRNAs determine the probability, progression, and establishment of the disease; consequently, they are considered master regulators of host-parasite interactions [15].
In the following section, we analyze the possible role of miRNAs in infections caused by helminth parasites, emphasizing their contribution to the immunomodulatory capacity of helminths during pathogen-host interactions.

miRNA in Schistosoma-Host Interaction
Schistosoma's miRNAs play a relevant role during the establishment of infection [72][73][74] (Figure 1). In the parasite, the expression of miRNAs varies at different developmental stages; some are genderbiased. Parasite miRNAs are involved in the regulation of sexual differentiation, maturation, mating, and reproduction; (B) Role of miRNAs in liver injury and hepatic fibrosis. Several miRNAs are dysregulated and documented as pro-fibrogenic (favoring fibrosis; red) or anti-fibrogenic (inhibiting fibrosis; green). Schistosoma eggs produce both responses in liver tissue. Adult Schistosoma parasites accumulate in mesenteric vasculature and release miRNAs-EVs with immunomodulatory effects; (C) Effects of Schistosoma miRNAs released in EVs on target cells. Schistosoma releases miRNAs-EVs to regulate the host's immune response. These miRNAs-EVs are composed of sma-miR-10, sma-miR-125, and sma-bantam in S. mansoni, while miR-125b and bantam are found in S. japonicum EVs. During S. mansoni infection, EVs fuse with T lymphocytes. The release of miR-10 into the T cell cytoplasm modulates the signaling pathway through MAP3K7 and regulates genes negatively depending on NF-kβ activation, diminishing T cell differentiation into Th2 subpopulations. Alternatively, during infection of mice by S. japonicum, EVs fuse with macrophages, and miRNA content inhibits the TLR-mediated inflammation (Sj-miR-125b) or stimulates the production and release of TNF-α to the extracellular environment, a molecule related to parasite development and survival. This process also promotes the increase in the macrophage population and may influence gene expression in Schistosoma. Adobe Illustrator was used to elaborate the figure. In the parasite, the expression of miRNAs varies at different developmental stages; some are genderbiased. Parasite miRNAs are involved in the regulation of sexual differentiation, maturation, mating, and reproduction; (B) Role of miRNAs in liver injury and hepatic fibrosis. Several miRNAs are dysregulated and documented as pro-fibrogenic (favoring fibrosis; red) or anti-fibrogenic (inhibiting fibrosis; green). Schistosoma eggs produce both responses in liver tissue. Adult Schistosoma parasites accumulate in mesenteric vasculature and release miRNAs-EVs with immunomodulatory effects; (C) Effects of Schistosoma miRNAs released in EVs on target cells. Schistosoma releases miRNAs-EVs to regulate the host's immune response. These miRNAs-EVs are composed of sma-miR-10, sma-miR-125, and sma-bantam in S. mansoni, while miR-125b and bantam are found in S. japonicum EVs. During S. mansoni infection, EVs fuse with T lymphocytes. The release of miR-10 into the T cell cytoplasm modulates the signaling pathway through MAP3K7 and regulates genes negatively depending on NF-kβ activation, diminishing T cell differentiation into Th2 subpopulations. Alternatively, during infection of mice by S. japonicum, EVs fuse with macrophages, and miRNA content inhibits the TLR-mediated inflammation (Sj-miR-125b) or stimulates the production and release of TNF-α to the extracellular environment, a molecule related to parasite development and survival. This process also promotes the increase in the macrophage population and may influence gene expression in Schistosoma. Adobe Illustrator was used to elaborate the figure.
Additionally, miR-146 a/b members regulate the differentiation of macrophages into M2 cells [86], which attenuate excessive inflammatory processes. Furthermore, they promote protective responses of the host by secreting cytokines such as IL-10 and TGF-β [87]. IL-10 presents immunosuppressive roles in helminthic infections, and TGF-β promotes tissue fibrosis through the overproduction of type I collagen [88,89].
TGF-β is critical in the development and male-female interactions of Schistosoma as well as during host-parasite interaction [96]. TGF-β signaling pathways are present in S. mansoni, and they are activated by the binding of human ligand TGF-β1 to TGF-β type II receptor (SmTβRII) exposed on the tegument of the parasites. The consequent activation and nuclear translocation of the SMAD multiprotein complex promote the transcription of target genes involved in parasite development and host-parasite interaction [96,97].
In the spleen and lungs, Schistosoma infection also upregulates different miRNAs related mainly to immune response, nutrient metabolism, cell differentiation, apoptosis, and signal pathways [73]. For example, MAPK, insulin, TLRs, and TGF-β signaling pathways are those mainly regulated by differentially expressed miRNAs in response to S. japonicum [98].
In addition to regulating some cellular processes in the parasite, the helminth miRNA-EVs modulate host gene expression and facilitate the dissemination of the pathogen [99,100] ( Figure 1C).
In addition, host TNF-α can induce differential gene expression and protein phosphorylation in schistosomes [105,106] due to TNF-α receptors in S. mansoni [105]. Therefore, the increased proliferation of TNF-producing cells could be a mechanism by which these parasites modulate gene expression in the host [104,105].
In chronic S. mansoni infection, sma-miR-10-5p, sma-miR-125b, and sma-bantam are released in EVs by adult parasites. These miRNAs modulate host T helper cell differentiation. Thus, miRNA-EVs are incorporated by Th cells, where the miRNAs downregulate the Th2-specific transcriptional program [100]. The Th2 pathway is a significant player in response against the helminth parasites and other extracellular parasites [107,108]. Mainly, sma-miR-10-5p is responsible for downregulating the expression of genes under the control of NF-kB, a transcription factor essential for Th2 differentiation, and represses the serine/threonine kinase MAP3K7 expression in the presence of Schistosoma parasites [100] ( Figure 1C).
Therefore, in schistosomiasis, differential expression of miRNAs is observed in the host and in the parasite itself. In parasites, these miRNAs are involved in different aspects of the parasite's biology and are part of a manipulation mechanism of the host's immune system. In the host, in addition to having regulatory roles in infection, it influences the development of the parasite and hepatic disease.

miRNA in Fasciola-Host Interaction
Fasciola parasites are macroscopic organisms living in a hostile environment, such as the mammalian liver and bile ducts, and possess sophisticated mechanisms to evade host immunity. MiRNAs play a crucial role in the development and pathogenesis of this disease [114,115] (Figure 2). 5p and sja-miR-2c-5p can be detected infected individuals with a low parasite load. Others, such as sja-miR-2c-5p, sja-miR-277, and sja-miR-3479, significantly correlate with fecal egg counts and hepatic egg burden [110,112]. Therefore, it has been proposed that these parasite-derived circulating miRNAs could serve as tissue or serum biomarkers for detecting human S. japonicum infection, even in low-intensity infections [109][110][111].
Therefore, in schistosomiasis, differential expression of miRNAs is observed in the host and in the parasite itself. In parasites, these miRNAs are involved in different aspects of the parasite's biology and are part of a manipulation mechanism of the host's immune system. In the host, in addition to having regulatory roles in infection, it influences the development of the parasite and hepatic disease.
Notably, the target genes of Fasciola species miRNAs differ in F. gigantica and F. hepatica (Figure 2A). In the case of F. gigantica, the predicted targets were mostly transcriptional regulators. In contrast, for F. hepatica, the predicted targets are proteins related to reproduction, development processes, response to stimuli, immunomodulation, and locomotion, suggesting different mechanisms of gene regulation between the two parasites [21]. These differences can be attributed to the intermediate hosts, morphological characteristics, geographic distribution, and metabolic adaptations during their life cycle in both species [116,117]. Differences in gene target prediction results could also influence the observed result [118].
In the encysted juvenile stage (NEJs) of F. hepatica, miRNAs play an important role in the invasion process [119]. Moreover, at this stage of development, miRNAs previously reported in the adult stage are also highly expressed (fhe-miR-125b, fhe-miR-bantam, fhe-let-7c, fhe-miR-277, and fhe-miR-71/miR-2 cluster members) [21]. Notably, miR-277 is related to the regulation of enzymes involved in the catabolism of aliphatic amino acids [120], having an essential role in the survival of the parasite under conditions of stress or starvation. These miRNAs are also associated with specific gene regulation expression needs in NEJs [119].
Therefore, during Fasciola infections, changes in miRNA expression occur in the host and parasite. Like Schistosoma, these are key players in the parasite's biology and manipulation of the host's immune responses. In addition, some of these are unique to this parasite, making them targets for possible therapeutic strategies.

miRNA in Brugia malayi-Host Interaction
Filarial parasites employ several strategies to evade the immune response during infection. Most of these strategies are orchestrated by ES, which interferes with the functions of the host's intracellular and extracellular immune machinery [127]. Thus, parasite miRNAs have relevant roles in parasite biology and immune dysfunction in the host [128,129].
In B. malayi, 145 miRNAs have been identified. They are grouped into 99 families, of which 61 are highly conserved with homologs in arthropods, vertebrates, and helminths, and nine appear to be filaria-specific. Several miRNA families differ depending on the development stage and gender [128] (Figure 3). and parasite. Like Schistosoma, these are key players in the parasite's biology and manipulation of the host's immune responses. In addition, some of these are unique to this parasite, making them targets for possible therapeutic strategies.

miRNA in Brugia malayi-Host Interaction
Filarial parasites employ several strategies to evade the immune response during infection. Most of these strategies are orchestrated by ES, which interferes with the functions of the host's intracellular and extracellular immune machinery [127]. Thus, parasite miR-NAs have relevant roles in parasite biology and immune dysfunction in the host [128,129].
Let-7 and miR-2b are key players in expressing some gender-associated genes in S. japonicum [78]. In C. elegans, lin-4, let-7, and miR-84 regulate the temporal events of development and larval-to-adult transition. In this transition, let-7, lin-4, and miR-84 together regulate genes of heterochronous pathways such as the transcription factor HBL-1, the orphan nuclear receptor DAF-12, and the nuclear protein Lin-41 [135,136]. In B. malayi males, these miRNAs also synchronize sex determination pathways, similar to other helminths [137].
For its part, members of the miR-36 family are mainly expressed in female adults and embryogenic stages of B. malayi [143] ( Figure 3A). This family of miRNA appears to be helminth-specific, and its absence in some nematodes is lethal [144,145]. It has been identified both in parasitic worms and free-living ones, and its functions are related to development, sex determination, and tissue regeneration [74,[146][147][148][149].
Notably, miR-71 is one of the most ubiquitous and conserved miRNAs in helminths, including B. malayi, and is associated with longevity, stress resistance, and neuron development [128,152,153] (Figure 3A). Thus, miR-71 modulates the expression of genes involved in the IGF-1/insulin-like pathway (AGE-1, PDK-1, AKT-1) and the genes CHK-1, CDC-25.1, and CDC-25.2, involved in DNA damage checkpoint pathways, which makes it a linker miRNA of both pathways [152]. Additionally, by regulating genes of the IGF-1/insulinlike pathway, miR-71 promotes the expression and activity of the forkhead transcription factor (DAF-16). DAF-16 is a FOXO family transcription factor that modulates antioxidant, antimicrobial, and metabolic enzymes necessary to extend the parasite's lifespan [152,154]. The regulation of longevity by both signaling pathways allows Mf to circulate throughout the host body for a long time until a mosquito ingests it. In addition, the expression of ammonium transport protein and proteins involved in the Lin-12/Notch signaling pathway have also been proposed as targets of bma-miR-71 in B. malayi [155].
MiR-71 also plays a role in host-nematode interactions [156,157]. It is released by EVs and is internalized by immune cells, where it regulates the production of nitric oxide (NO) and the expression of components of RISC and host miRNAs linked to inflammation [157]. Additionally, it has been observed that EVs loaded with miR-71 from Heligmosomoides polygyrus and administered intranasally in BALB/c mice promote the suppression of type 2 cytokines (IL-5 and IL-13) in innate lymphoid cells, as well as the expression of the IL33-receptor and phosphatase DUSP1 in recipients cells [156]. Although DUSP1 regulates the inflammatory response, alterations in its expression dampen the acute inflammatory response by favoring macrophage's arginase expression over nitric oxide synthase [158]. Moreover, reduced expression of DUSP1 results in GATA-2 phosphorylation and inhibition of its ability to promote IL33r transcription [159].
The function of the miR-2 cluster is less known in B. malayi; however, there have been associations with parasite embryogenesis [128]. In other helminths, the miR-2 cluster is involved in apoptosis suppression, development, and stress responses [66,143,145]. In B. pahangi, the miR-2 cluster members pba-miR-2a and pba-miR-2b are temporally expressed and are more abundant in adult worms in a similar way as miR-7 and miR-36 [143]. Interestingly, in Schistosoma, the miR-2 orthologue is clustered with miR-36 and participates in regulating sexual differentiation and maturation [78], evidencing that gene clusters can change the context of their function among helminths.
Infections by adult stages of B. malayi induce the production of IL-4 by the host, promoting the appearance of suppressive cells known as IL-4-dependent F4/80+ macrophages (or nematode elicited macrophages (NeMϕ)), which exert an anti-proliferative effect on lymphocytes, mediated by cell-to-cell contact [164,165], and at the same time act as antigenpresenting cells (APC) to stimulate naïve T cells and induce their differentiation towards Th2 and IL-4-producing cells [166]. This facilitates the activation of suppressive NeMϕ macrophages, originating a positive feedback loop that maintains an environment with abundant unresponsive immune cells that contributes to the establishment of infection ( Figure 2B).
Alternatively, the induction of IL-4 production also would be a mechanism used by this parasite to induce some miRNAs and modulate the immune response. IL-4 induces overexpression of mmu-miR-378-3p in macrophages and suppression of proteins involved in the IL-4-receptor/PI3K/Akt-signaling pathway, exerting a negative regulation on the proliferation of macrophages [160]. It should be noted that the PI3K/Akt-signaling pathway directs various cellular processes in macrophages, so it is likely that other cellular processes are inhibited during the infection process [167] and lead to the immunosuppression seen in lymphatic filariasis [168] (Figure 3B).
For its part, bma-miR-34 could also be vital in immunomodulation during filarial infections [129]. Bma-miR-34 overexpression decreases CXCL10/CXCL11/CXCR3 secretion in immune cells, impairing their migration and activation [176]. In addition, miR-34 overexpression of miR-34a promotes differentiation of a conventional dendritic cell (csDC), producing high amounts of IL-17, which leads to inhibition of CD4+ T cell activation through the repression of transcription factor T cell factor 1 (TCF1) and consequent increases in the orphan nuclear receptor RORγT expression [67]. The latter inhibits cytokine IL-2 secretion in T cells [177,178] (Figure 3B).

Conclusions
Helminth parasites are a globally distributed group of organisms with great clinical relevance, representing a risk for humans and animals. They have an amazing ability to modulate their host's immune response through sophisticated strategies involving miRNAs. In the host, miRNAs modulate signaling pathways, resulting in an altered immune response favoring parasite persistence and tolerance. In the parasite, these molecules regulate their development and virulence. Therefore, studying miRNAs during host-parasite interaction is of great importance to improve prognostic, diagnostic, and therapeutic strategies for those neglected diseases caused by these parasites.

Conflicts of Interest:
The authors declare no conflict of interest.