**2. Parasite identification by the host system**

The first meeting of host and parasite usually breaches the surface (like, epithelium of intestinal or skin) that incites the "alarmin" discharge [10] and is

#### **Figure 1.**

*Recognition of helminth infection by immune system. Innate immune system releases alarmins (IL-33, TSLP) in response to tissue invasion, which might elicit a type 2 immune response; helminth have ability to either inhibit the release of alarmins or block the respective receptors (e.g., IL-33R and ST2). The C-type lectin receptors (CLRs) or Toll-like receptors (TLRs) can recognize pathogen-associated molecular patterns, either presented directly by helminths or by bacteria, moved through damaged epithelium. In the second situation, immune modulators released by helminths suppress the Th1 response, induced by IL-12.*

#### *Helminths Derived Immune-Modulatory Molecules: Implications in Host-Parasite Interaction DOI: http://dx.doi.org/10.5772/intechopen.102927*

recognized through pattern recognition receptors (PRRs), as an example, Toll-like receptors (TLRs) which initiate the production of inflammatory cytokine. Alarmins such as thymic stromal lymphopoietin (TSLP) and interleukin-33 (IL-33) [11, 12], where together stimulate a Type 2 immune response that is anti-helminth and pro-allergic, are strongly related with helminth-mediated tissue damage. Yet, helminths have option to avoid entirely or partially this warning (**Figure 1**); as an example, *Nippostrongylus brasiliensis* compounds effectively prevent dendritic cells (DCs) from responding to TLR ligation and other helminths, with interleukin-12 (IL-12) production being particularly suppressed [13–17]. While the release of IL-33 from the epithelial cell, is directly obstructed through the released products from *Heligmosmoides polygyrus* [18]. Some of the chemical mediators that prevent innate activation are now being identified, as mentioned in the next section.

The prototypical PRRs respond to microbiological substances like lipoteichoic acid and lipopolysaccharide (LPS) by releasing pro-inflammatory cytokines like IL-12, which promote the Th1 response. The consistent ability of various helminth products to inhibit the release of IL-12 in response to TLR stimulation could be a mechanism aimed not so much at blocking anti-parasite immunity as it is at avoiding collateral inflammation at barrier sites where, for example, bacterial translocation may accompany helminth invasion. While the key role of TLRs in pathogen pattern recognition via the host is now well recognized, it is surprising that no analogous recognition mechanism for Th2-inducing species like helminths has yet been defined. However, helminth TLR ligands have been discovered, including the RNA activating TLR3 [19] and the lysophosphatidylserine glycolipid [20] from *Schistosoma mansoni* (*S. mansoni*), and additional receptor like C-type lectin receptors (CLRs) may fulfill the role of innate detection in different situations [21–23].

### **3. Host-parasite molecular interaction**

In the extracellular environment, simple protein-protein interactions, involving with either exposed receptors or fluid-phase host components on the surfaces of host cells can be considered the first stage communication between the host and the parasite. *H. polygyrus*, secretes a functional mimic of the immunomodulatory cytokine TGF-, which binds to mammalian surface receptors and sends an inhibitory signal to the T cells (Johnston et al. submitted to be published). Space blocks further conversation of the numerous singular proteins currently observed to be associated with have helminth cooperation, yet maybe the most fascinating are individuals' superfamily of the cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) (Pfam00188) which are significantly extended across all helminth parasite heredities [24, 25], and profoundly addressed in the emitted protein sections [26, 27]. One member of this family, a hookworm named *Necator americanus*, was one among the first to be identified as a potential partner as NIF, an emitted integrin restriction inhibitor that stops neutrophils [28].

While functional roles for members of the CAP gene family other than NIF are sparse, a homolog attaches to a tomato plant innate defense protein, limiting resistance systems, and triggering infection in a plant-parasitic worm [29]. As a result, helminth-released proteins are not limited to cooperating at the host cell surface, but can also play functions inside cells, raising the question of how they enter the cell.

#### **3.1 Helminth derived proteins and their intracellular functions**

Two well documented helminth glycoproteins infiltrate the host cells and have immense implications. The *S. mansoni* egg-inferred glycoprotein ω1 is a ribonuclease with Lewis X glycan side chains that bind to the surface lectin of dendritic cells, interfering with take-up into the phone and causing the protein moiety to serve as a protein blend obstructer [30, 31]. DCs treated with ω1 activate the type 2 immune pathway, causing immature T cells to mature into Th2 effector cells. The major secreted glycoprotein of the filarial nematode *Acanthocheilionema viteae* (*A. viteae*) is a distinct mediator of ES-62 could be 62-kDa component with N-linked phosphoryl choline (PC) side chains. Through interaction with ES-62 enters the cell via TLR4 on the surface, and the PC moiety disrupts the downstream signaling of both the B cell receptor and TLR4 within the intracellular milieu, effectively blocking cell activation [32]. Although TLR3 is an intracellular pathogen sensor, and the FheCL1 cysteine protease from *Fasciola hepatica* (*F. hepatica*) kills TLR3 in host macrophages, limiting activation; despite the fact that TLR3 is an intracellular pathogen sensor, FheCL1 can reach the endosome and degrade the receptor in situ [17].

The filarial cystatin molecule CPI-2 is used to target a distinct route. This protein contains two blocking sites that are resistant to cysteine proteases and asparagine endo peptidase (AEP) [33]. Human B cells that have been exposed to CPI-2 from *Brugia malayi* (*B. malayi*) (a human filarial parasite) are no longer able to practice presenting protein antigen to T cells, a process that is dependent on AEP activity in the endosome [33]. Advance research on a closely related cystatin from *A. viteae* show that it is taken up by mouse macrophages and activates ERK and p38 kinases, resulting in the production of immune regulation interleukin-10 (IL-10), which is linked to the activation of the CREB and STAT3 signaling pathways [34].

Although the entrance pathway cannot always be determined, other products have been found to modulate intracellular signaling in host cells. The ALT-2 protein, for example, is generated from a large larval transcript of the filarial parasite *B. malayi*, when this protein was given to macrophages or introduced into macrophages via the intracellular protozoan *Leishmania mexicana* (*L. mexicana*), it induced the signaling proteins GATA3 and SOCS1, which are active to generate type 2 responses and inhibit IFN-dependent intracellular inflammatory signaling [35].

#### **3.2 Identification of exosomes and their implications in host-parasite interactions**

Apparently, particularly exosomes and extracellular vesicles appear to play an important role in cellular communication. Exosomes are nano vesicles with a diameter of around 50–100 nm that are secreted by all cells to allow the transfer of specific cargo, primarily lipids, proteins, and RNA species, as well as other phenotypic markers from their cell of origin [36, 37]. Exosomes are formed by the inward budding of multi-vesicular endosomes within a cell, and include components of the original cell, such as RNAs or proteins, that may be trafficked into the same compartment. The extracellular vesicles have been discovered from kinetoplastids growths, and microorganisms' group, the hypothesis that exosome-interceded correspondence could work on a cross-animal categories stage, by which parasite-inferred exosomes could associate with, and conceivably adjust, the host invulnerable framework [38]. Exosomes have just recently been discovered as integral products of extracellular organisms such as helminths [38, 39].

#### *Helminths Derived Immune-Modulatory Molecules: Implications in Host-Parasite Interaction DOI: http://dx.doi.org/10.5772/intechopen.102927*

According to the recent research, exosomes are produced by parasitic helminths. The excretory-secretory portions of the trematodes *F. hepatica*, and *Echinostoma caproni*, which contaminate the liver and gastrointestinal system individually, were the first to disclose this [40], as well as the nematode *H. polygyrus*, which contaminates the small intestinal tract [41]. Information derived from the trematode concentrates additional advises that ES inferred exosomes are fit for arriving at the host climate, as they seem, by all accounts, to be discovered unblemished on the para destinations' covering. The capacity of helminth exosomes to cross-phylum communication between mammals, and helminths is further supported by their uptake by host intestinal epithelial cells.

Exosome formation in free-living nematodes was first demonstrated in helminths, with the demonstration that a novel secretion pathway from the apical membrane of *Caenorhabditis elegans* co-secretes multi vesicular bodies containing exosome-like vesicles with peptides that normally promote cuticle growth [42]. Helminths and protozoa exosomes have similarities in various specific markers. In heat-shock protein 70 (HSP70), endosomal sorting components like surface tetraspanins, and *Alix*, including CD63 and CD9 are all found in mammalian exosomes [37]. As an example, when exosomes secreted by macrophages, which are *Leishmania*-infected experience a series of phenotypic alterations followed by infection, and they hold some exosome markers, with CD63, TsG101, and *Alix* [43]. Furthermore, transcriptome investigation of the cestode, *Echinococcus granulosus*, revealed the existence of other CD63-like tetraspanin family members [44]. Tetraspanins have been chosen as a vaccine against *Echinococcus multilocularis*, a tapeworm that causes alveolar echinococcosis, a highly lethal illness that has spread throughout areas of Central Europe, China, and Siberia [45, 46]. This tetraspanin-targeting vaccination is also being investigated as a potential treatment for the human pathogen *S. mansoni* [47, 48].

Earlier, it was seeming that *H. polygyrus*, a mouse gastrointestinal nematode, has previously been demonstrated to release exosomes containing various miRNA types as well as a significant number of proteins, accounting for around 10% of an adult worm's total protein secretion [41]. The enrichment of a number of important components within the exosomes was also established by a proteomic analysis of the released products represented in the soluble and vesicular fractions using ultracentrifugation separation. Interestingly, some of these proteins have previously been found in the region of *C. elegans'* intestinal epithelial apical membrane cells; electron microscopy revealed multi-vesicular bodies in the intestinal tissues of *H. polygyrus* adults, as well as exosome-like structures freed into the lumen [41], strongly implying that the parasite releases exosomes from its alimentary tract (**Figure 2a**).

Exosomes from external helminths were also found to have immunomodulatory properties. Exosomes from *H. polygyrus* dominate the innate immune response to the fungus *Alternaria alternata*, which is linked to respiratory allergies, mostly through modulating type 2 innate lymphoid cells (ILC2s) [41]. Helminths communicate with host systems by releasing a repertoire of molecules, such as proteins, glycan, and extracellular vesicles/exosomes containing miRNA (**Figure 2b**). Both *in-vitro* and *in-vivo*, *H. polygyrus* exosomes demonstrate a lower expression of IL1RL1/ST2 transcript in mouse cell types. This gene encodes the IL-33 receptor, which is essential for ILC2s to trigger the type 2 immune response, which is compatible with exosomes' in vivo protection from allergic inflammation. The role of the IL-33 ligand-receptor axis in anti-parasite responses is also well established [18, 49]. As a result, our findings support the ability of *H. polygyrus* derived exosomes to evade parasite clearance by altering a critical element of the host immune system.

#### **Figure 2.**

*Host-helminth molecular cross-talk. (a) Helminths communicate with host systems by releasing a repertoire of molecules, such as proteins, glycan, and extracellular vesicles/exosomes containing miRNA. (b) Helminth triggers the Foxp3+ Treg population by producing short-chain fatty acids (SCFAs) and also stimulates the gut-microbes, those secrete SCFAs.*

Exosomes were identified in the culture media of the digenean trematode livestock parasite *Dicrocoelium dendriticum*, which included over 80 protein components and at least 30 miRNA species with similarity or near-identity to known sequences [50]. Despite the lack of functional studies, the scientists noted similarities with the key *Schistosoma* miR-3479, miRNAs Bantam, and miR-10, which are visible indicators in the plasma of infected animals [51].

Moreover, Nowacki et al. identified over 200 miRNAs, 20 tRNA-derived short RNAs, and over 100 proteins in 30–100 nm exosome-like vesicles released by *S. mansoni* schistosomula that are enriched in certain non-coding RNAs and proteins [52]. Furthermore, it was discovered that the *B. malayi* L3 infective stage secretes 50–120 nm vesicles rich in miRNA species, as well as a protein complement that includes not only conventional exosome-associated products, but also those that could interfere with host cell responses, like *Cathepsin L* [53]. Importantly, the adult worm stage was shown to produce less exosomes than the infective stage, which is likely due to the demands of converting from vector to host at this point in the life cycle. Adult *S. mansoni* worms also release 50–130 nm-sized exosome vesicles with approximately 80 identifiable proteins, five of which are tetraspanins, and an abundant saposin-like protein, according to Sotillo et al. [54]. It is also shown that a number of recognized schistosome vaccine candidate antigens, including the tetraspanins, are key components of the exosomes, as previously mentioned. Wang et al. reported that adult worms of the similar parasite *S. japonicum* emit 30–100 nm vesicles after being cultivated *in-vitro* for 5 h, which can be detected using ultracentrifugation of the culture solution [55]. Although this work did not identify the protein cargo of the exosomes, these scientists discovered that *S. japonicum* exosomes boosted the production of nitric oxide in the murine macrophage-like cell line RAW264.7, along with other markers of a Type 1 pathway. The presence of many important proteins and RNA species in secreted vesicles emphasizes both the complexity and diversity of cargo within exosomes, as well as the broad range of potential connections between recipient cells [56].

Investigations of the liver fluke *Opisthorchis viverrini*, a trematode common in regions of Southeast Asia and causally connected to cholangiocarcinoma (bile duct cancer) have revealed a larger chance for helminth exosomes. Exosomes (measured 40–180 nm)

#### *Helminths Derived Immune-Modulatory Molecules: Implications in Host-Parasite Interaction DOI: http://dx.doi.org/10.5772/intechopen.102927*

were found in secretory material from the species mentioned above, along with a similar spectrum of related proteins, including tetraspanins [57]. In the bile fluid of infected hosts, some proteins linked to exosomes were discovered. Anti-tetraspanin antibodies prevented exosomes from entering host cells, implying that this protein is likely to be represented on the vesicular surface in the same way that mammalian exosomes. Suggestively, exosomes from *O. viverrini* were shown to stimulate cell proliferation and induce the generation of the pro-inflammatory cytokine interleukin-6 (IL-6) in a human cholangiocyte cell line in a way that was partially inhibited by an anti-tetraspanin antibody. Taken together, these findings support the theory that *O. viverrini* energies cause tumorigenic alterations in the host bile duct, which could explain the parasite's carcinogenic effects.

#### **3.3 Exosomes contain helminth miRNAs**

It's been a while, all around archived that micro-RNAs and non-coding RNAs specifically, move among cells and life forms through their epitome inside exosomes and other vesicles found outside of the cell [58]. Certainly, this gives a piece of machinery for RNA protection from destruction outside the cell, and appears to provide an absorption pathway to transfer RNA to the recipient's proper cellular compartment. Many of the investigations mentioned above, including those from the nematodes *B. malayi* [53] and *H. polygyrus* [41], as well as the trematodes *D. dendriticum* [50] and *S. mansoni*, identified short RNAs within parasite exosomes [52].

We were able to show a collection of RNA species bundled inside exosomes, including miRNAs such as let-7, miR200, and diminutive [41], which may block the mouse phosphatase Dusp1 using a quantitative measure, thanks to *H. polygyrus*. New information differentiating wide miRNA collections in parasitic helminths is rapidly emerging, albeit the circulation of these released conservative exosomes inside parasitic helminths has yet to be established.

Most importantly, definitive proof for helminth-derived miRNAs acting on host genes has yet to be discovered; however, the circumstantial evidence remains enticing; not only are extensive seed sequences shared between helminth and host miRNAs, but the miRNA-rich exosomes (at least of *H. polygyrus*) also contain worm Argonaut protein [41, 59]. Indicating that a functional gene repression package is being delivered to the targeted cells.
