**2.2 Transforming growth factor-β (TGF-β)**

TGF-β plays an essential role in wound healing, angiogenesis, immunoregulation, and cancer development. These cytokine's effects are dual-sided, contributing to the differentiation of regulatory (suppressive) T cells (Tr cells) and inflammatory Th17 cells. In mammals, all leukocytes produce at least one isoform of TGF-β [31]. TGF-β is locally produced by the host's immune system cells in response to the presence of helminth parasites.

*Perspective Chapter: Molecular Crosstalk and Signal Transduction between Platyhelminths… DOI: http://dx.doi.org/10.5772/intechopen.103776*

In the genome of *Taenia solium* (*T. solium*) and *Taenia crassiceps* (*T. crassiceps*, a canine tapeworm), protein-coding genes for the pivotal signaling elements were identified [32]. TsTGFβR1, TsTGFβR2, TGF-β Type I, Bone Morphogenetic Protein (BMP) Type-I receptor Tr-3, and activin (TGF-ligand) were identified and had a high identity with *Echinococcus* sp. The expression of TsTGFβR1, TsTGFβR2 at mRNA, and protein level was detected in *T. solium* and *T. crassiceps* cysticerci. It showed that both TGF receptors are expressed in the parasite's teguments more prominently in the tegument of *T. crassiceps* and the periphery of *T. solium* cysticerci from the brain than in the cysticerci from skeletal muscle of infected pigs. It is interesting because the TGF-β levels in the cerebrospinal fluid are higher than in serum, suggesting that the exposure to the host's molecule could be involved in cysticerci growth and differentiation [32].

It is interesting to note that *T. solium* and *T. crassiceps* cysticerci were *in vitro* exposed to three concentrations of recombinant human TGFβ-1 (0.001, 0.01, and 0.1 ng/mL). The human cytokine caused a significant increment in the size of cysticerci in *T. crassiceps*. In the *T. solium* cysticerci, a considerable improvement in the survival rate was observed with no effect on its size. The parasite could internalize the host's TGF-β via endocytosis as a regulatory event. However, these effects may be mediated by the direct interaction of the host's cytokine with the parasite receptors. The results observed on the parasites in an *in vitro* treatment and the antibody recognition of receptors are lower when TGF- β incubation occurs. The lower antibody recognition of both Type-I and Type-II parasite receptors when cysticerci were cultured with increasing levels of TGF-β suggests that TGF-βcould bind the Type-II receptor (avoiding the recognition of antibody), then the complex TGF-β-TsTGFβR2 receptor would recruit the Type-I receptor, forming a complex which would prevent the bounding of Type-I receptor antibody. These results point to hTGF-β as a factor in cysticerci growth and survival, which could also play a role in the lack of effectiveness of cysticidal treatment of patients.

In *S. mansoni,* several TGF-β signaling pathway elements have been identified and described, including two TGF-β receptors (SmTβR1 and SmTβR2), one homolog gene to Inhinbin/Activin (SmInAct), a homolog to the BMP (SmBMP), Smp300/CBP, Smad2, and Smad4. In female worms, these elements could play a role in vitelline cell development and egg embryogenesis, as these molecules' expression is detected in these organs (reviewed in [33].

Oliveira et al. [34] studied the effects of the human TGF-β (hTGF-β) on the gene expression profile of *S. mansoni* adult worms. Microarray experiments were performed with RNA extracted from adult worms that were *in vitro* treated with the human cytokine. This experiment revealed that changes in the expression influence the pattern of treated worms. With this approach, 381 genes were detected as differentially expressed, with 316 down-regulated and 65 up-regulated. These genes are related to biological functions such as muscular system development and function, tissue morphology, cellular assembly and organization, organ development, tissue development and cellular growth, and proliferation. Some functions, such as contractile fiber and myosin complex, hydrolase activity, and adenyl ribonucleotide binding, are related to the down-regulated genes. It correlates with the already described TGF-β induction of cytoskeleton remodeling through the myosin chain and Rho GTPase [35, 36].

**Figure 2** summarizes host cytokines' direct or indirect effect on the parasites.

#### **Figure 2.**

*Influences of mammals and invertebrate host's cytokines on platyhelminths. (A) Cytokines. (B) Vertebrate immune system cells. (C) Platyhelminthes. (D) Cytokine. (E) Invertebrate effector cells. (F) S. mansoni primary sporocysts. Some cytokines (TNF-α and TGF-β) exert direct effects on the parasite; on the other hand, other cytokines (such as interleukins) exert effects on immune system cells and these cells regulate parasites´ biological processes.*

#### **2.3 The example of human Tumor Necrosis Factor-α (TNF- α) on** *S. mansoni*

Human TNF-α and its effect on *S. mansoni* are excellent examples of how the comprehension of the molecular crosstalk between host and pathogen has increased in the last decades. Some studies have described the influence of the pro-inflammatory cytokine TNF-α on the fecundity and metabolism of the parasite *S. mansoni*. Amiri et al. [37] described that human TNF-α induces the formation of granulomas and causes a positive effect on the parasite's egg-laying. Controversially, it was shown that egglaying decreases and induces changes in the uptake of tyrosine [38] and methionine [39] on *S. mansoni* in the presence of the human cytokine. It was also documented that parasites' egg-laying and fecundity occurred later when immunodeficient mice (SCID) were used for infection with the parasite [40]. Finally, Davies et al. [41] reported that host TNF-α promotes the parasite survival and the development of adult worms.

In this context, the molecular mechanism started to be elucidated by searching for *S. mansoni* homologous gene to the human TNF-α receptor. A homolog gene was identified and characterized (SmTNFR) [42] and generated a transcript of 1967 nucleotides that encodes a receptor composed of 599 amino acids. The predicted protein has an extracellular portion that contains four TNF-α conserved domains (cysteine-rich domains), the main characteristic of the TNF receptor family. Extracellular domains' modular architecture is similar to the neural growth factor receptor (NGFR). The first analysis of the intracellular portion revealed no conserved domains, which is not expected in a homologous gene to NGFR characterized by Death Domain (DD), which makes it similar to TNF-R2, a non-death domain. The transcript expression level (mRNA) is detected in all developmental stages, but the highest expression level is detected in cercaria [42].

Parallel to the description of SmTNFR, other homolog genes for a possible signaling pathway were also identified. It is interesting to highlight that all elements required and activated by the human TNF-R2 signaling pathway (which does not have DD and, therefore, is not related to the activation of apoptosis) were found [42].

Recently, through *in-silico* analysis, 29 genes of homologous receptors to SmTNFR in other species of parasitic flatworms were identified. The homologs may evidence

#### *Perspective Chapter: Molecular Crosstalk and Signal Transduction between Platyhelminths… DOI: http://dx.doi.org/10.5772/intechopen.103776*

conservation of the TNF-α signaling pathway in a part of helminths. Additionally, highly conserved homologs of endogenous TNF-α ligands only in free-living flatworms were also identified. This suggests that the loss of the endogenous ligand (observed in parasitic flatworms) and the consequent use of the host's ligand was an event that occurred throughout the evolutionary process, as a cause or consequence of the parasitism [43].

Further, TNFR homologs identified in platyhelminths had conserved DD, which was concluded after the analysis of the secondary structure of intracellular regions. The intracellular portion of all receptors was reanalyzed, and evidence of the presence of DD was found in most SmTNFR homologs in platyhelminths but with different levels of conservation. Generally, cestodes have a more conserved DD than trematodes. This urges us to rethink the possible signaling pathway triggered by SmTNFR, since this receiver was initially classified as without DD [42].

Parallelly, Oliveira et al. [42] investigated the effect of human TNF-α on the gene expression profile in newly transformed schistosomula (NTS) and adult worms. NTSs (3 h after transformation) were treated with human TNF-α for 1 h (at the concentration of 20 ng/mL), and adult worms treated with human TNF-α for 1 h and 24 h. Microarray experiments revealed 548 genes with altered expression in NTSs after treatment with the human cytokine (309 up-regulated and 239 down-regulated). These genes are involved in biological functions related to the regulation of gene expression, cell proliferation and growth, and cell development. Two groups of differentially expressed genes were identified in adult worms treated for 1 h and 24 h. One group had transient changes in expression, that is, an inverse change pattern within 24 h compared to the pattern obtained within 1h. This group comprises 1365 genes, 821 of which have their expression level increased in 1 h of treatment and decreased in 24 h, and 544 have the opposite expression pattern. The second group has sustained changes in its expression level in 1 h and 24 h; this group comprises 492 genes, 337 being with the expression level increased by treatment with human TNF-α and 155 with the expression level decreased. These differentially expressed genes were organized in gene expression networks, and the most significantly enriched network interacts with TNF-α in other organisms. The network suggests that the parasite response to the human cytokine is conserved and similar to the reaction in humans [42]. Interestingly, the enzyme lactate dehydrogenase (responsible for producing lactate) was differentially expressed in schistosomula and adult worms treated with human TNF-α.

Thus, it was described that human TNF-α induces the phosphorylation of different proteins in adult male worms after *in vitro* treatment for 15 min. Differentially phosphorylated proteins were related to muscle contraction, cytoskeletal remodeling, cell signaling, and metabolism. Lactate dehydrogenase and a subunit of ATP synthase are differentially phosphorylated proteins. These results indicate that this enzyme, in the glycolytic pathway of the parasite, is being potentially regulated by the host's TNF-α in its expression level (mRNA) and activity [44].

Since the literature description of egg-laying is contradictory, and lactate dehydrogenase is differentially expressed and phosphorylated, the effect of TNF-α on egglaying and metabolism was investigated in the adult parasites, in an *in vitro* treatment with doses of the human cytokine (5, 20, and 40 ng/mL) during 5 days [45].

The average number of eggs/couple increased on the second day in the treatment with 40 ng/mL. On the third day, there was a significant decrease in the average of eggs/couple for the treatments with 20 and 40 ng/mL; besides, there was a decrease for the doses of 5 and 40 ng/mL on the fourth day of incubation with the cytokine. The most important observation is that the total number of eggs was not different between treatments and control over the 5 days of treatment. The conclusion is that although egg-laying dynamics were affected, the fecundity was not. The host's TNF-α causes a decrease in the half-life of the egg-laying; therefore, when faced with the stimulus, couples lay eggs more quickly, but not in greater or lesser amounts than the respective negative control [45].

The TNF-α treatment induced significant changes in lactate concentration or possibly the glucose uptake when there was also a change in egg-laying. On the third day of treatment, for example, when lactate production decreased, the number of eggs laid was also reduced, indicating that energy metabolism is a relevant actor regulated by human TNF-α and interferes in the production dynamics and egg-laying [45].

**Figure 3.** *Timeline of main discoveries described in the literature about host TNF-α and its effects on S. mansoni.*

*Perspective Chapter: Molecular Crosstalk and Signal Transduction between Platyhelminths… DOI: http://dx.doi.org/10.5772/intechopen.103776*

In addition, the increase in the accumulation of adenosine triphosphate (ATP) in adult worms on the fifth day was observed. The compromised egg-laying can explain it at this time: the high demand for ATP is destined for oogenesis and, when not necessary, this molecule can accumulate, especially against the modulation induced by the human cytokine [45]. It is also interesting to note that one subunit of ATP synthase is regulated by human TNF-α [44].

**Figure 3** summarizes the history of the characterization of the effects of TNF-α on Schistosoma mansoni. It is an exciting example of how a cytokine effect can be elucidated like a puzzle, piece by piece.
