**2. Host cytokines' effects on platyhelminths**

Cytokines are small proteins (5–25 kDa) produced by many cells (especially from the immune system), which exert a signaling effect (at an autocrine, paracrine, or

endocrine level) in a broad range of tissues [19]. Generally, cytokine studies focus on the immune system's regulation when faced with an infection; however, we will describe how the host cytokines can modulate platyhelminths' biological/physiological processes and their possible signaling pathways.

### **2.1 Interleukins (12, 2, 7, 4, and 1)**

Interleukin-7 (IL-7) is a cytokine secreted by bone-marrow, endothelial, and epithelial-stromal cells essential in the hematopoietic system for the proliferation, differentiation, and development of B cells [20]. It is also involved in the thymic development of mature T lymphocytes, natural killer (NK), and lymphokine-activated killer (LAK) cells [21].

IL-7 interferes in the development of *S. mansoni* (in murine infection) [22]. Female knockout mice for the IL-7 gene (IL-7−/−) infected with *S. mansoni* showed significant differences in parasite development, egg-induced pathology, and worm recovery rate. It was also observed that fewer eggs were laid *in vivo,* and more dead eggs were detected without IL-7. The decreasing egg burden ameliorated the liver pathology, and morphological differences in the length of male and female worms in the IL-7−/− mice were observed [22].

Studies with radiolabeled IL-7 suggested that this cytokine did not bind directly on the parasite surface; hence, the observed effects of IL-7 deficient mice could be attributed to the cytokine's interactions with the host's immune and or endocrine responses [23, 24].

Interleukin-2 (IL-2) is a cytokine with autocrine and paracrine effects secreted by activated T-CD4 + cells [25]. Further investigations interrogated the modulation of *S. mansoni* development by IL-7 and IL-2 through the influence of these cytokines on CD4+ T (T helper) cells [26]. With the use of knockout mice for IL-7 receptor (IL-7Rα−/−) and IL-2 (IL-2−/−) for *S. mansoni* infection, the morphology of adult worms is affected. In both knockout mice, the infected mice produced smaller male worms than the control group. In the IL-7 receptor knockout mice, the parasite's egg production was drastically reduced [26]. Studies with IL-2 receptor knockout mice (IL-2Rα−/−) infected with *S. mansoni* revealed a similar impact on the parasite's development, using the knockout mice for the cytokine. Thus, it could be concluded that the modulation of IL-7 and IL-2 in *S. mansoni* development (adult growth and egg-laying and granuloma formation) is indirect; the cytokines act on the host's CD4+ T cells [26].

Interleukin-12 (IL-12) and interleukin-4 (IL-4) reciprocally regulate differentiation of naïve CD4+ T lymphocytes and directly promote the development of CD4+ Th1 cells and the CD4+ T-cell differentiation in the Th2 phenotype (which also produces IL-4). In 2012, Cheng et al. [27] used an approach with hybridoma cells injected into different mice groups to evaluate the effect of monoclonal antibodies against IL-12 and IL-4 on parasite infection. The effect of IL-12 and IL-4 on worm development and granuloma formation in a murine infection by *Schistosoma japonicum* (*S. japonicum*) was evaluated. It was observed that, 24 days post-infection, the group of anti-IL-12 had a significant increase in the number of eggs per couple and eggs in the liver. The granuloma size and fibrosis in the liver in the anti-IL-12 mice were significantly more prominent on day 42. The decreasing of T helper 1 (Th1) cytokine expression through the blocking of IL-12 promotes the T helper 2 (Th2) cytokine expression and reduces Interferon-γ (IFN-γ) and Interferon-α (IFN-α) cytokine levels.

The length of worms in the anti-IL-12 group was increased; however, the degree of increase was different in males and females. The female size was higher in anti-IL-12 than in anti-IL-4 and control groups at 28 and 42 days post-infection, while the male size was higher just at 28 days in the anti-IL-12 [27]. The data in this study suggest that IL-12 deficiency benefits *S. japonicum* worm development in the early days of infection, indicating the action of cytokine against the schistosome. At the same time, its effect was reduced at 42 days post-infection, revealing a transitory effect. It is important to note that IL-12 promotes a Th1 response.

Finally, interleukin-1 (IL-1) from *Biomphalaria glabrata* (*B. glabrata)*, the intermediate host of *S. mansoni,* also affects the parasite. In the vertebrate's immune system, the cytokine IL-1 mediates cytotoxic, humoral, and inflammatory responses, induces leukocyte recruitment to the inflammation site, and is involved in the cytotoxic reactive oxygen intermediate (ROI) production mechanism in the effector cells [28]. In the immune defense system of the invertebrate *B. glabrata*, molecules with functional homology to the vertebrate IL-1 (SnaIL-1) have been detected and isolated. It was observed that, in response to schistosome infection's primary sporocysts, susceptible snail strains exhibit a decrease in plasmatic SnaIL-1 levels, while the SnaIL-1 cytokine levels rise in resistant strains. Also, when the susceptible snails are treated with a recombinant human Interleukin-1β (rhIL-1β), there is a rapid phagocytosis activation and ROI production at the same levels found in the resistant snail strains [29].

In 1998, Connors et al. [30] investigated if the treatment of two strands of susceptible *B. glabrata* with rhIL-1β had effect on the infection of the invertebrate host (directly or mediating by hemocyte's activity). After 15 days of exposure, the authors observed a significant decrease of 50% in the number of counted miracidia on snails from both treated groups. Histological analysis of snail tentacles performed 3 days after exposure revealed a significantly higher percentage of dead or disrupted parasites compared to the control. *In-vitro* assays using hemocytes-free cultured parasites in contact with the plasma from rhIL-1β -injected snails showed an immediate killing effect on the parasites.

These results imply that the cytokine may stimulate *in-vivo* induction of a cell-free killing mechanism in the *B. glabrata*. Parasites' killing seems to have no connection with the hemocyte's encapsulation of the parasites, suggesting the presence of both a humoral cytotoxic molecule and a cellular signaling mechanism. Injection of the recombinant human IL-1β on the susceptible snails has activated the otherwise reduced cytotoxic capabilities of the snail's immune system; however, other factors such as reduced number of hemocytes compared to resistant strains may limit the killing of the parasites. Further, invertebrates' cytokine-like activity may occur in molecules with substantially different structures from their mammalian counterparts, obviating the need for more analysis of the elements involved in the host-parasite signaling mechanisms.
