Studies have shown that both schistosome worms and eggs induce type 2 immunity, which is essential for their maturation, reproduction, and egg excretion (14–17). Deficiency in CD4+ Th2 cells and their effector cytokines IL-4 and IL-13 was shown to substantially decrease or completely abrogate egg excretion as a consequence of impaired worm maturation or failed signaling by type 2 effector cytokines (14, 46–48). Moreover, accumulating evidence suggests that ILC2 and their activating cytokines IL-25, IL-33 and TSLP induce adaptive type 2 immunity independently of IL-4 (22–24). Besides, while there is no report on the induction of IL-25, IL-33 and TSLP release by migrating schistosomula, it has been reported that schistosome eggs induce and/or enhance the production of IL-25 and IL-33 (49, 50), indicating that schistosome eggs may be orchestrating their excretion by inducing the production of alarmin cytokines IL-25, IL-33 and TSLP to trigger type 2 immunity, essential to their excretion. In this study we report that IL-33 deficiency does not affect the maturation of worms as on both early (four weeks after infection) and late (six weeks after infection) worm maturation, the morphology did not differ between worms recovered from IL-33−/− and WT mice, suggesting that IL-33 may not be required for schistosome worm maturation. Consequently, despite a higher number of worm pairs at the ninth week of infection in IL-33−/− mice, the number of eggs per worm pair, as well as the number of tissue eggs did not differ between the mouse genotypes. Although we did not determine the worms’ lengths, the proportion of females in pairs (17, 51), and the number of eggs in feces (14), based on the morphology of worms (34, 52) and the intestinal tissue egg numbers (31, 32) as indications for worm maturation and egg excretion, respectively; to the best of our knowledge, this is the first study that has attempted to look at the role of IL-33, as a potent initiator of type 2 immunity necessary for schistosome worm maturation, in the maturation of S. mansoni worms and the excretion of their eggs.
In a study by Yu et al. (25), it was reported that the injection of S. japonicum-infected mice with exogenous IL-33 increased the number of worms recovered at the sixth week of infection, and exacerbated the liver pathology by increasing the number and size of liver granulomas. This may simply mean that as endogenous IL-33 plays a role in the development of egg-induced liver pathology (25–27), injecting exogenous IL-33 would exacerbate its pathogenic effects. In the present study, we could not find any statistically significant difference in the number of eggs per worm pair between IL-33−/− and WT mice. These results corroborate the ones reported by Yu et al. (25) as they did not find a difference in the number of eggs per female worm. This indicates that IL-33 alone may have a negligible role to play in worm maturation, reproduction, and egg excretion.
While studies related to inflammatory bowel diseases reported controversial roles for IL-33 in the development and/or exacerbation of these diseases, with some reporting a protective role for IL-33 (30, 40), and others incriminating it in the development or exacerbation of these diseases (41, 42), to the best of our knowledge, no report had been made on the role of this cytokine in the intestinal pathology during schistosomiasis. Although IL-33 seemed dispensable for S. mansoni worm maturation and the excretion of their eggs, we sought to know whether it may play a significant role in the development of egg-induced pathology in the intestines of infected mice as it does in the liver (25–27). We found that the absence of IL-33 transiently impaired type 2 immunity in small intestines of IL-33−/− mice, but not in their spleens, characterized by impaired production of IL-5 and IL-13 cytokines in MLNs in response to stimulation with SmSEA and attenuated egg-induced inflammation in IL-33−/− mice ilea at the sixth week of infection. These results are in line with findings by Vannella et al. (45) who, although focused on the role of alarmin cytokines IL-25, IL-33 and TSLP in the development and maintenance of type 2 cytokine-driven inflammation and fibrosis in lungs and liver, found that single ablation of these cytokines had no significant ameliorating effect on the liver pathology. However, when all three cytokines were ablated, a significant improvement of the pathology could be observed in the early phase of the infection, pointing towards the existence of functional redundancy between these cytokines. Findings from the present study tread in the same direction as the absence of IL-33 did not affect the pathology development, nor the number and size of granulomas in the intestines of IL-33−/− mice in time points beyond the sixth week of infection. However, the difference between the study by Vannella et al. (45) and ours is that we started our observation at the sixth week of infection, when the egg-induced type 2 immunity is still at its start, while Vannella et al. (45) started their observation at the ninth week of infection when the egg-induced type 2 immunity has already reached its peak. We think that it could have been possible for them to notice a significant difference between IL-33 deficient mice and WT at an earlier stage of the infection, as observed in our study.
Despite its sharing of functional redundancy with IL-25 and TSLP (45), IL-33 remains the most potent of all three in inducing type 2 immunity (38, 39, 53). In addition to inducing type 2 immunity by itself, IL-33 can also potentiate the type 2 immunity induced by the two other cytokines, IL-25 and TSLP (54). Of all the cells that respond to IL-33, ILC2 and Th2 are the most important as through their production of abundant amounts of type 2 cytokines IL-4, IL-5 and IL-13, they play the most important role in cell-mediated effector type 2 immunity (55, 56), characterized by, among others, accumulation of M2 macrophages and eosinophils in affected tissues. Although dispersed in all tissues, ILC2 are more abundant in lungs and intestinal tissues (57), where they are the first to be activated by IL-33 and migrate to local draining lymph nodes to initiate the adaptive type 2 immunity (24, 58). Thus, it is understandable that the absence of IL-33 in IL-33−/− mice at the early stage of the patent infection might have left them inactivated, leading to impaired type 2 immunity (58) as seen in the present study. In addition to acting through ILC2 and Th2 cells, IL-33 also acts directly on eosinophils, inducing their expansion and activation (59, 60). Therefore, its absence in IL-33−/− mice can explain the small number of eosinophils in the inflammatory infiltrates in the sixth week of infection (61). However, due to the persistence of egg-derived ESP (6–10) as eggs keep accumulating in the tissues, and to the fact that IL-25 and TSLP can induce type 2 immunity independently of IL-33 (50, 62–64), alternative mechanisms leading to activation of both innate and adaptive type 2 immunity, including taking over of ILC2 activation by IL-25 and TSLP and Th2- dependent effector pathways, might have been activated to compensate the absence of IL-33. Together, these alternative mechanisms may have led to improved type 2 immunity in time points beyond the sixth week of infection.
Studies have pointed to the existence of possible interactions between IL-25, IL-33 and TSLP (39, 44). In one study, anti-IL-33 treatment and TSLP receptor deficiency blocked the infection-induced expression of IL-25 in lung epithelial cells, and ex vivo treatment of ILC2 with TSLP increased their expression of IL-25 and IL-33 receptors (44). In another, it was noted that IL-25 shared with IL-33 many activities on macrophages without having additive effects, pointing toward the possible existence of common downstream signaling pathways for their biological activities (39). Therefore, we thought that IL-33 deficiency might be associated with a modified production of IL-25 and TSLP in the intestines of S. mansoni-infected IL-33−/−mice. Our results showed no modification of intestinal production of IL-25 and TSLP as their levels in intestinal tissue homogenates did not differ between mouse genotypes, meaning that although they can, individually or synergistically, induce type 2 immunity, the absence of one may not affect the others in the schistosome infection settings or intestines. The nature and conditions of occurrence of the interactions between IL-25, IL-33 and TSLP pointed out by the above-mentioned studies (39, 44) remain to be clarified.
Studies in humans and mice have reported the increase of IL-33 levels in the sera of individuals and animals infected with S. japonicum infection (26). Also, these increased levels of IL-33 in serum peaked around the eighth week of infection in mice (25), relatively corresponding to the peak of egg-induced immune responses, suggesting that through their ESP, eggs may be the main inducers of IL-33 release in schistosome infections. Indeed, Hams et al. (49, 50) reported that injection of S. mansoni eggs or recombinant form of their derived components, namely ω1, induced the production of IL-25 and IL-33, respectively in the lungs and fat tissue. Whether eggs in intestinal tissues induce the production of IL-33, IL-25 and TSLP is not known. By measuring the levels of these alarmin cytokines in the intestinal tissue homogenates during S. mansoni infection in WT BALB/c mice, we found that IL-33 levels remained constantly higher, even in non-infected mice. In contrast, IL-25 and TSLP levels fluctuated over the infection course, peaking around the tenth week of infection, with TSLP having much lower levels than IL-25. The start of an increase in levels of IL-25 and TSLP tended to correspond to the oviposition, suggesting that this latter might be inducing the release of IL-25 and TSLP, but not of IL-33. In their study, Flamar et al. (58) recently reported that IL-33 expression was high in small intestines of naïve mice, corroborating our findings. This indicates that IL-33 is constantly expressed in high amounts in mouse intestinal tissues.