Ulcerative colitis and Crohn’s disease are two major clinical entities included in IBD, which may affect 0.5% of Western world population (1 million people in the United States, and 2.5–3 million people in Europe), with 37 new cases per 10⁵ inhabitants in Europe (Molodecky et al., 2012; Kaplan and Ng, 2017). IBD is also spreading in developing countries, where the prevalence is lower than in developed ones (Molodecky et al., 2012; Ng et al., 2013). Most of IBD patients are affected in their productive age, originating not only important social problems, but also economic losses (Kaplan, 2015).

The etiology of the disease is not well understood and because of the complexity of this pathology, the development of effective treatments will require studies using multidisciplinary approaches, where animal models have proved to be highly informative (Lin and Hackam, 2011).

Due to the absence of a curative treatment for IBD, there have been numerous attempts to incorporate new therapies. Some of these include new drugs or nutritional supplements to correct or enhance the effectiveness of the currently authorized treatments. In fact, the complexity of the pathogenesis of these diseases has led to empirically evolved current drug therapies, which primarily treat inflammation. The latest therapies attempt to restore the intestinal immune balance by efficiently combining immunosuppressant agents, biological therapy and modulators of the microbiota (Vanhove et al., 2016).

Interestingly, previous epidemiologic studies and clinic trials suggested that helminths, either by natural or artificial infections, might protect people from IBD (Smallwood et al., 2017). This has been confirmed by Ramanan et al. (2016), which have demonstrated that alteration of commensal and pathogenic bacteria produced by gastrointestinal helminths infection in turn protects the host against IBD (Ramanan et al., 2016). Still, major concerns of the helminthic therapy are related to ethical issues and the ability of controlling the course of the infection. In this sense, the identification of helminth-derived molecules that ultimately mediate host immune modulation is attracting much attention.

Extracellular vesicles, including exosomes and microvesicles, have been described as participating in intercellular communications with important roles in physiological and pathological processes, where they can be used as diagnostic and therapeutic weapons (Yáñez-Mó et al., 2015; Barile and Vassalli, 2017). EVs have been described to participate in inflammation, like EVs from granulocytes, which have been used for IBD treatment in a mice model (Wang et al., 2016; Song et al., 2017).

Fasciola hepatica is a flatworm that excretes/secretes a large number of molecules to the host-parasite interplay, including immune modulators (Dalton et al., 2013). Our group has identified some of these molecules in EVs released by parasite adults in culture (Marcilla et al., 2012; Cwiklinski et al., 2015; Fromm et al., 2015). To assess the functional role of these EVs in the host and exploring their usefulness as therapeutic agents against IBD, we have studied their potential in preventing ulcerative colitis in a DSS induced colitis model in C57BL/6 mice. Our data support that F. hepatica EVs (FhEVs) can protect from IBD in this model, since their inoculation prevents intestinal damage in acute colitis by altering the local immune response. Thus, FhEVs might be employed for preventing relapses in IBD patients and could be explored as a potential new therapy for treating IBD.

Intestinal bowel disease is one of the most important diseases affecting developing countries, and it is currently spreading in undeveloped ones (Molodecky et al., 2012; Ng et al., 2013). The etiology of the disease is not well understood, with a complex pathology which has no effective treatment available. In fact, the development of effective treatments require studies using multidisciplinary approaches, where animal models have proven highly useful (Lin and Hackam, 2011). DSS-induced colitis model has been widely used to explore new therapeutic options (Wirtz and Neurath, 2007; Kiesler et al., 2015).

Among the new treatments, the use of biologicals is gaining attention (Rogler, 2015; Maizels, 2016). In this context, and following the Hygiene hypothesis (Strachan, 1989), clinical trials with parasitic helminths have been initiated, and some are underway (Smallwood et al., 2017). Some concerns about using helminths to treat IBD have arisen, with the possibility of producing malignancy (Bonovas et al., 2016). In fact, a recent report has shown that the treatment with the parasitic nematode Heligmosomoides polygyrus can induce tumor progression in a DSS-induced colitis in Balb/c mice (Pastille et al., 2017).

Along with those potential clinical problems, the use of parasites to treat IBD also face ethical problems (McSorley and Maizels, 2012), so the alternative of using parasite defined products, like excretory/secretory products (ESP), or isolated molecules seem to be a good option. In this context, studies with a crude extract from the laminated layer of Echinococcus granulosus (a tapeworm parasite) showed not only its preventive effect but also played a beneficial role in maintaining the integrity of the intestinal mucosal barrier against DSS-induced injury (Soufli et al., 2015). Those results supported the role of EVs as promoters of the epithelial barrier function (Xu et al., 2016).

Previous studies had shown that EVs administered either orally or injected could reach the colon of mice and exert their therapeutic effect on induced colitis (Ju et al., 2013; Yang et al., 2015). Very recently, Wang et al. (2017) have reported a protective effect in a similar mice model when using EVs produced by dendritic cells previously exposed to eggs from the parasitic trematode Schistosoma japonicum (Wang et al., 2017). In this regard, our data highlight that the parasite F. hepatica EVs have a potent modulatory effect on the immune response in DSS-induced colitis in mice. We provide evidence that F. hepatica EVs can prevent DSS-induced colitis by down regulating pro-inflammatory cytokines like TNFα, IL-6, and IL17A, as well as suppressing MAPK/NF-κB signaling pathways. Accordingly, FhEVs treatment decreased MPO activity, indicating a reduction in neutrophil infiltration in damaged colon and which has also been corroborated in the histological sections. In accordance, reduce levels of cytokines TNF-α and IL-17A have been detected in the colon of FhEVs-treated mice, which are inducers of neutrophil transmigration (Kolls and Lindén, 2004; Griffin et al., 2012). DSS colitis can be exacerbated by granulocyte recruitment (Natsui et al., 1997; Williams and Parkos, 2007; Sander et al., 2008; Chami et al., 2014; Kishida et al., 2015). The activity of many enzymes and chemicals produced by neutrophils is not specific to pathogens, so they can damage host tissues when released extracellularly, contributing to the aggravation of mucosal inflammation. Likewise, in UC, unrestricted neutrophil activation may cause significant tissue damage that further leads to chronic pathology and the extent of neutrophil infiltration correlates with the severity of the disease (Bressenot et al., 2015). Therefore, the reduced neutrophil infiltration in FhEVs-treated mice, would provide for the protection effect detected in this model of acute colitis induced by DSS, and may be crucial for the treatment of UC patients. In Rag1^-/- mice, a decreased neutrophil infiltration is also observed in the histology sections after FhEVs treatment, in accordance with the results obtained in C57B/L6 mice. It is important to remark that though Rag1^-/- mice have no mature T and B lymphocytes (Mombaerts et al., 1992), develop totally normal granulocytes, like WT mice, as has been demonstrated in reports which use these mice in infection models (Smith et al., 2009; Seymour et al., 2015; Carey et al., 2016; Papp et al., 2016).

Furthermore, our results suggest that neither lymphocytes nor IL-10 are involved in the protective effect by FhEVs in DSS-induced colitis mice model. This seems to be at odds with previous reports showing that either splenic B cells or EVs from granulocytic myeloid-derived suppressor cells, attenuate DSS-induced colitis by promoting Tregs expansion, and inhibit Th1 cells proliferation (Reyes et al., 2015; Wang et al., 2016).

The accurate identification of the molecules responsible for the anti-inflammatory effect is underway, but suitable candidates are molecules previously identified in FhEVs, like FABPs (Cwiklinski et al., 2015), which have been shown to induce anti-inflammatory response in animal models by diminishing the levels of TNFα (Ramos-Benitez et al., 2017). Other exosome-contained molecules like miRNAs represent potential candidates as they may act as immunomodulators of the intestinal innate immune response (Buck et al., 2014). Interestingly, a repertoire of miRNAs with immune-regulatory function have been found in F. hepatica EVs previously by our group (Fromm et al., 2015).

Resident intestinal macrophages and colonic dendritic cells have been reported to have a high anti-inflammatory phenotype and are hypo-responsive to microbial stimuli in rodents and humans (Bilsborough and Viney, 2004; Kelsall and Leon, 2005; Smith et al., 2005). In line with this, it has been described that depletion of both dendritic cells and macrophages from the intestinal lamina propria in C57BL/6, BALB/c, and SCID mice (without T and B cells) increased DSS colitis severity. These mice had increased neutrophilic inflammation, epithelial injury, and enhanced mucin depletion from globet cells (Qualls et al., 2006). On the other hand, helminth infections are associated with the induction of immunosuppressive M2/Alternatively Activated Macrophages (AAMs) (Loke et al., 2000; Herbert et al., 2004). It is therefore conceivable that FhEVs are enhancing the anti-inflammatory functions of these innate cell populations as they are protective in Rag1^-/- mice that lack T and B cells populations. Other helminth infections have been shown to prevent colitis development independently of Tregs stimulation, through the inhibition of prostaglandins by AAMs (Ledesma-Soto et al., 2015) or recruitment of a novel macrophage population distinct to AAMs or Gr1⁺ macrophages (Smith et al., 2007). Moreover, in addition to their immunosuppressive role, lamina propria macrophages play an important role in the regeneration of damaged epithelium by controlling the epithelial progenitor niche after DSS-induced colitis (Pull et al., 2005). On the other hand, the reduced neutrophil infiltration detected in FhEVs-treated mice might be a consequence of the decreased epithelial barrier damage and diminished production of pro-inflammatory cytokines from the resident myeloid cell populations in the gut as observed in the foregoing reports, because of the preventive role of these exosomes. Moreover, some reports described that subcutaneously injected exosomes can be detected in several organs including the gastrointestinal tract even after 24 h of administration, and cleared from the bloodstream in a few minutes (Wiklander et al., 2015). However, in our experimental settings the exosomes are administered 12 days before DSS administration, which makes us to think that they are exerting their role in the gut resident populations. A plausible mechanism may be epigenetic reprogramming of these innate cell populations, a process that has been recently termed as trained immunity (Netea et al., 2016), or even of the intestinal epithelial cells. Whether, in addition, FhEVs could be exerting their function by altering granulopoiesis at the bone marrow level, or influencing in endothelial cells the level of expression of adhesion molecules, requires further experimentation and biodistribution analyses.

Apart from the induction of strong Th2 responses and IgE production, which can be discarded in our experimental setting due to the maintenance of the protective effect in Rag1^-/- mice that lack mature T and B lymphocytes, helminths also promote the expansion of eosinophils, mast cells and basophils which could not be discarded. Levels of these cell populations can be measured by flow cytometry to evaluate if there were differences among FhEVs-treated versus non-treated mice. Interestingly, among these cell populations, the only one which have been described to promote a protective role in acute DSS colitis are the eosinophils, due to the production of anti-inflammatory lipid mediators (Masterson et al., 2015). To further elucidate which innate cell population is mediating the protection, adoptive transfer experiments could be performed by injecting specific sorted cell populations derived from FhEVs-immunized Rag1^-/- mice into non-immunize ones. Our most likely candidates are macrophages, as stated in the foregoing works. Besides, macrophages are highly phagocytic cells that could be acquiring a highest number of exosomes. Moreover, the role of dendritic cells is typically focused on T cell polarization, which are not involved. Specific deletion of macrophages can be performed by the use of liposomal clodronate agent. If they turn to be involved in the conferred protection to DSS-colitis, it would be very informative to evaluate the role of FhEVs in M1 and M2 polarization, which could be performed by flow cytometry or RNA-seq.

Our findings suggest that other immune cells aside lymphocytes are involved in the protective response (Masterson et al., 2015; Coakley et al., 2017; Wang et al., 2017; Campbell et al., 2018). Future studies will address the identification of the immune cells involved in the FhEVs protective effect and the mechanisms behind it. The fact that the conferred protection against intestinal inflammation is mediated by EVs is of great importance to move this research forward into translational applications.

AM, MT, DB, MR, RG, and FS-M: participated in the study concept and design. JR, AG, MS, FC, CM, DB, and MT: acquisition of data. JR, AG, MS, EG, CM, MR, RG, DB, FS-M, and AM analyzed and interpreted data. MR, RG, DB, FS-M, and AM drafted and critically revised the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer PX and handling Editor declared their shared affiliation.