Parasitology Research https://doi.org/10.1007/s00436-020-06884-0 IMMUNOLOGY AND HOST-PARASITE INTERACTIONS - ORIGINAL PAPER Immunomodulatory effect of different extracts from Angiostrongylus cantonensis on airway inflammation in an allergic asthma model Vanessa Fey Pascoal 1 & Aline Andrea da Cunha 2 & Alessandra Loureiro Morassutti 1 & Géssica Luana Antunes 2 & Keila Abreu da Silveira 2 & Josiane Silva Silveira 2 & Nailê Karine Nuñez 2 & Rodrigo Godinho de Souza 2 & Carlos Graeff-Teixeira 1 & Paulo Márcio Pitrez 2 Received: 20 August 2019 / Accepted: 13 September 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020 Abstract This study aimed to evaluate the effects of early-life exposure to different extracts of Angiostrongylus cantonensis (A. cantonensis) on airway inflammation in an allergic asthma model. The total soluble extract (TE) and the soluble extracts of the digestive (AcD), reproductive (AcR), and cuticle (AcC) systems of A. cantonensis were used for immunisation before ovalbumin (OVA)-sensitisation/challenge in an OVA-induced allergic asthma model. The initial hypothesis of the study was that some soluble extract of the systems (AcD, AcR, or AcC) could be more potent to the modulation of inflammation than the TE. Our data, however, shows that immunisation with the TE is more promising because it decreased the high influx of inflammatory cells on airways and promoted an increase of interferon-γ (IFN-ɣ) and interleukin-10 (IL-10) levels. Besides this, the immunisation with the TE also led to a reduction of goblet cells and mucus overproduction in the lung tissue of asthmatic mice. We believe that the extracts have a distinct capacity to modulate the immune system, due to the TE possessing a greater variability of molecules, which together leads to control of airway inflammation. In conclusion, this is the first study to reveal that the TE of A. cantonensis adult worms has a greater potential for developing a novel therapeutic for allergic asthma. Keywords Angiostrongylus cantonensis . Asthma . Immunomodulation . Parasites . Cytokines . Hygiene hypothesis Introduction Epidemiological studies have shown an increase in allergic diseases in parallel to a decrease in the incidence of infectious diseases (Cooper et al. 2003; Pereira et al. 2007; van der Werff et al. 2012). This increase has arisen along with improved living conditions, represented by improved basic sanitation and economic conditions occurring in countries such as Section Editor: Sabine Specht * Alessandra Loureiro Morassutti almorassutti@gmail.com 1 Laboratory of Molecular Parasitology, School of Sciences, Pontifical Catholic University of Rio Grande do Sul, 6690 Ipiranga Ave., Porto Alegre, RS 90610-000, Brazil 2 Laboratory of Pediatric Respirology, Infant Center, School of Medicine, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Brazil Sweden, Finland, and Germany where there is a high incidence of allergic diseases (Seiskari et al. 2007). According to the hygiene hypothesis, exposure to pathogens, such as helminths, may be beneficial for the development of the immune system (Vatanen et al. 2016). The helminths are a diverse group of organisms that can infect every organ and organ system (Daniłowicz-Luebert et al. 2011). Many studies have provided support for the hygiene hypothesis. In animal models of arthritis and allergic asthma, immunomodulatory effects were shown (Song et al. 2011; Pitrez et al. 2015). Furthermore, the therapeutic efficacy has been observed in clinical trials of inflammatory diseases including rhinitis and multiple sclerosis (Croft et al. 2012; Correale and Farez 2007). Helminths have molecules that have potential therapeutic action against inflammatory diseases. Allergic asthma is an inflammatory airway disease manifested clinically by recurrent episodes of wheezing, dyspnea, chest tightness, and cough (de Sousa et al. 2011). The inflammatory response that occurs in airways is generally mediated by CD4+ T helper (Th) 2 cells that Parasitol Res are programmed to produce specific cytokines such as interleukin (IL) IL-4, IL-5, and IL-13 (Moser et al. 1992). IL-4 regulates allergen-specific immunoglobulin E (IgE) synthesis, IL-5 recruits and activates eosinophils, and IL-13 induces mucus overproduction (Lambrecht and Hammad 2015). A study by Araujo et al. showed that patients with asthma living in an area of poly-helminthic endemicity have a decreased Th2 inflammatory response and suggest that this modulation occurs through the increase of anti-inflammatory IL-10 levels (Araujo et al. 2004). It has been found that besides IL-10, other cytokines, such as IFN-γ, can directly inhibit the Th2 response and, thus, can mediate a reduction in inflammation that leads to allergic diseases (Araujo et al. 2004). In a recent study, we have demonstrated that early-life administration of Angiostrongylus cantonensis (A. cantonensis) extract decreases allergic lung inflammation in a murine model (Pitrez et al. 2015). A. cantonensis is a parasitic nematode of rodents (mainly Rattus norvegicus) and is the main etiologic agent of human eosinophilic meningoencephalitis. Adult worms live inside the pulmonary arteries of rats and produce the infective stage larvae (L1) which are released from faeces. Intermediate hosts, such as molluscs, become infected both by the ingestion of L1 or by tissue penetration through the mollusc tegument. The larvae undergo two molts and produce the third stage (L3) (Morassutti et al. 2014). Humans acquire A. cantonensis infection by eating raw or undercooked foods and the infection may also occur by accidental ingestion during hand manipulation of molluscs in fisheries and/or during garden upkeep. After ingestion, L3 larvae penetrate the intestinal walls, gain access to the bloodstream, migrate to the central nervous system where they molt twice (L4 and L5), and eventually die in the meninges (Morassutti et al. 2014). Helminth infections can also cause pathologies and diseases in humans; the use of specific extracts for the therapy of inflammatory diseases has generated substantial interest. Assays using crude extracts from A. cantonensis have shown that female reproductive tract extracts are more immunogenic than male reproductive tract extracts (Bender et al. 2003). One possible explanation is because the cuticle and the digestive products from the worm feeding are in constant contact with the immune system (Morassutti et al. 2013). In this way, we hypothesise that the soluble extract of digestive (AcD), reproductive (AcR), and cuticle (AcC) systems could represent a more responsive strategy than the total soluble extract (TE) of A. cantonensis. Thus, considering that the identification of an extract with a greater immunomodulatory capacity is a crucial approach for developing a novel therapeutic for allergic asthma, this study aims to evaluate the effects of early-life exposure to different extracts of A. cantonensis on airway inflammation in an allergic asthma model. Materials and methods Animal use This study was performed with female 21-day-old BALB/c mice acquired from the Centre for Experimental Biological Models (CeMBE), Pontifical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, Brazil. Mice received a balanced chow diet and water ad libitum, were housed in ventilated cages with the temperature maintained at 21 ± 1 °C, and an illumination schedule 12 h/12 h light-dark. The animals were divided into six groups: (1) negative control (PBS), (2) animals submitted to allergic asthma model with ovalbumin (OVA), (3) animals immunised with the TE of A. cantonensis and submitted to allergic asthma model with OVA (TE/OVA), (4) animals immunised with AcD extract and submitted to allergic asthma model with OVA (AcD/ OVA), (5) animals immunised with AcR extract and submitted to allergic asthma model with OVA (AcR/OVA), and (6) animals immunised with AcC extract and submitted to allergic asthma model with OVA (AcC/OVA). In the last three groups, the animals were immunised with the proteins present in the TE of the digestive, reproductive, and cuticle systems of A. cantonensis worms. Ten to fourteen animals were used per group. Preparation of the TE and extract of the systems (AcD, AcR, and AcC) from A. cantonensis worms Adult female A. cantonensis worms were isolated from infected rats and obtained from the cycle maintained at the Laboratory of Molecular Parasitology, School of Sciences, PUCRS. In order to obtain the TE, forty female A. cantonensis worms were homogenised in liquid nitrogen in 1 mL of phosphate-buffered saline (PBS), sonicated by three pulses of 15 min each with 30 min intervals, on ice, and at 40 A (Ultrasonic Processor 75021, Bioblock Scientific, Strasbourg, France). After sonication, the extract was centrifuged (Harrier 18/80R, Sanyo Gallenkamp PLC, East Sussex, UK) at 403×g and 4 °C for 20 min. Protease inhibitors were added to the soluble fraction, as instructed by the manufacturer (Protease Inhibitor Mix, GE Healthcare, Chicago, IL, USA), and the precipitate was discarded. The total protein concentration of the TE was determined using a Qubit® apparatus (Invitrogen, Carlsbad, CA, USA). For the AcD, AcR, and AcC production, the separation of the digestive, reproductive, and cuticle systems of female A. cantonensis worms was done manually using a stereomeasuring microscope (Stemi DV4, Zeiss, Oberkochen, Germany) with a refrigerated base and two 1.20 × 40 mm gauge needles. The worm parts were separately macerated in liquid nitrogen and homogenised in 1 mL of PBS with subsequent processing similar to that used for the TE preparation. Parasitol Res Immunisation with the TE and system (AcD, AcR, and AcC) proteins from A. cantonensis Twenty-one days before the start of the asthma protocol, three immunisations were performed with TE, AcD, AcR, and AcC in their respective groups, at a concentration of 200 μg/animal, intraperitoneally making a total of 600 μg of protein per animal with a 7-day interval between the immunisations. Analysis of cytokines in BAL IL-4, IL-5, IL-10, IFN-ɣ, and eotaxin levels were measured by the multiplex technique (Milliplex Map, MAGPIX® Technology, Austin, TX, USA) according to the manufacturer’s instructions, and the analyses were performed using the Milliplex® Analyst software 5.1. OVA-specific IgE in serum Immunisation and induction of allergic asthma model Seven days after the third immunisation with TE, AcD, AcR, or AcC, the allergic asthma protocol was initiated. The animals were sensitised subcutaneously with 20 μg OVA (OVAGrade V, Sigma, St Louis, Missouri, USA) in the absence of adjuvant/alum-free and diluted in PBS in two sensitisations (days 0 and 7 of the protocol). Intranasal challenges were performed with a solution of OVA (50 μg) on three consecutive days (days 14, 15, and 16 of the protocol). In the negative control, only PBS was administered in the sensitisation and intranasal challenges. Twenty-four hours after the third intranasal challenge, the mice were anesthetised with ketamine (0.4 mg/g) and xylazine (0.2 mg/g) and euthanised by cardiac puncture exsanguination (day 17 of the protocol). Serum was obtained by centrifugation of the blood sample obtained during the cardiac puncture exsanguination at 360×g for 5 min (Fanem, São Paulo, Brazil) and stored in a freezer at − 80 °C for the subsequent measurement of OVA-specific IgE levels. A bronchoalveolar lavage (BAL) was also performed through a tracheostomy with the injection and aspiration of 1 mL of PBS. The sample was centrifuged at 717×g for 4 min, and the supernatant was frozen at − 80 °C for further analysis of IL-4, IL-5, IL-10, IFN-ɣ, and eotaxin. The precipitate from the cells was resuspended and used for total and differential cell counts. Finally, the lung tissue was removed and kept in formaldehyde for 24 h for preparation of the paraffin block for histopathological analysis. A summary of the protocol is presented in Fig. 1. Total and differential cell counts in BAL The precipitate was resuspended with 350 μL of PBS for total cell counts (TCC), and trypan blue exclusion test with a Neubauer chamber (BOECO, Hamburg, Germany) was used for analysis. Differential cytology slides were prepared with 80 μL of the suspension in a cytocentrifuge (Fanem) at 360×g for 5 min. The slides were dried in ambient air and stained with Panótico rápido (Laborclin, Sao Paulo, Brazil). The cells were analysed according to their morphology under optical microscopy and the data expressed in absolute counts (counting a total of 400 cells). The sensitisation to OVA was analysed by measuring OVAspecific IgE in serum through enzyme-linked immunosorbent assay (ELISA) (MD Bioproducts®, MD Biosciences, Zurich, Switzerland) according to the manufacturer’s instructions. Histopathological analysis Histological sections of 4 μm were prepared from paraffin blocks of lung tissue and stained with hematoxylin and eosin (H&E) (Pró-Cito Soldan Cytological Products, Brazil) for the analysis of peribronchial inflammatory infiltrates and with alcian blue (InLab, Sao Luis, Brazil) for the analysis of goblet cells. Statistical analysis The data are presented as the mean ± standard deviation (SD). The data were analysed by one-way analysis of variance (ANOVA) followed by a Tukey post hoc test. Statistical differences were considered to be significant at p = < 0.05. The statistical analyses and graphs were performed using Graphpad Prism software (version 8.0, San Diego, CA, USA). Results Immunisations with the TE and with soluble extract of systems (AcD, AcR, and AcC) was capable of decreasing the influx of inflammatory cells on BAL in an allergic asthma model To verify if the model of acute asthma used in the study promoted an increase in the cellular count in BAL, we initially evaluated the TCC. We observed that OVA promoted an increase in cellularity compared with the PBS group (p = < 0.001). On the other hand, when the animals received helminth extracts, we observed a significant reduction in the TCC, in relation to the OVA group (p = < 0.001) (Fig. 2a). According to the inflammatory profile, especially the eosinophil count (Fig. 2b), the OVA group presented a significant increase when compared with the PBS group (p = < 0.001). The increase in the count was also observed in macrophages (p = < 0.001), neutrophils (p = < 0.01), and lymphocytes (p = Parasitol Res Fig. 1 Scheme of the protocol used in the study. Three immunisations with TE and soluble extracts of the systems (AcD, AcR, and AcC) were performed with a 7-day interval between each immunisation. Seven days after the third immunisation (day 21 of the protocol), the acute asthma protocol was initiated (day 0). Abbreviations: TE, total soluble extract of A. cantonensis worms; OVA, ovalbumin; I.P., intraperitoneal; S.C., subcutaneous; I.N., intranasal; TCC, total cell counts < 0.001). However, we observed a reduction of eosinophils (p = < 0.001; Fig. 2b), macrophages (p = < 0.01 and p = < 0.05; Fig. 2c), and lymphocytes (p = < 0.01 and p = < 0.05; Fig. 2e) in the groups immunised with TE, AcD, AcR, and AcC, respectively, when compared with the OVA group. Except for neutrophils, there was no statistically significant difference (Fig. 2d). TE immunisation decreased inflammatory infiltrates and goblet cell hyperplasia whilst soluble extracts of systems (AcD, AcR, and AcC) have no effects on these parameters in an allergic asthma model Immunisation with the TE increased IL-10 and IFN-ɣ levels whilst immunisation with soluble extracts of systems (AcD, AcR, and AcC) did not alter these cytokine levels on BAL in an allergic asthma model In addition to the profile of inflammatory cells evaluated in BAL, the levels of IL-4, IL-5, eotaxin, IL-10, and IFNɣ were also evaluated (Fig. 3). The OVA group presented an increase in the levels of IL-4 (p = < 0.01; Fig. 3a), IL-5 (p = < 0.05; Fig. 3b), and eotaxin (p = < 0.001; Fig. 3e) in BAL when compared with the PBS group. However, early immunisation with the TE, AcD, AcR, and AcC of A. cantonensis did not show changes in the cytokine levels evaluated. IFN-ɣ and IL-10 levels were not significantly different between the OVA and PBS groups (Fig. 3c, d). However, in the group pre-immunised intraperitoneally with the TE of A. cantonensis, there was an increase in the levels of IFN-ɣ and IL-10 (p = < 0.05; Fig. 3c, d), whereas there were no significant changes in the groups that received the immunisation with the system extracts (AcD, AcR, and AcC) of A. cantonensis (p = > 0.05). We also investigated the IgE levels in mice serum. We observed significantly elevated levels of IgE in the OVA group compared with the control group (p = < 0.001). Nevertheless, immunisation with the TE and extract of systems (AcD, AcR, and AcC) showed no significant changes when compared with the OVA group (Fig. 3f). In the histopathological examination, the lung sections stained with H&E showed an evident increase in inflammatory cells located in the peribronchial and perivascular areas in the OVA group. Nevertheless, both peribronchial and perivascular inflammatory infiltrates were reduced in mice that were immunised with the TE. The immunisation with soluble extracts of systems (AcD, AcR, and AcC) did not show changes in peribronchial and perivascular inflammatory infiltrates (Fig. 4a). In the histological sections of lung tissue stained with alcian blue (Fig. 4b), we observed an increase in goblet cells and mucus overproduction in the OVA group. On the other hand, the early immunisation with the TE decreased the number of goblet cells and mucus overproduction compared with the OVA group. The early immunisation with AcD, AcR, and AcC also did not alter this parameter (Fig. 4b). Discussion Parasitic infections appear to inhibit allergic and inflammatory immune responses (Maizels et al. 2004; Fallon and Mangan 2007). Helminths produce immunomodulatory molecules to suppress immune responses at various levels, from innate immunity to end-effector mechanisms in adaptive responses, offering potential opportunities to treat a range of human diseases (Daniłowicz-Luebert et al. 2011; Song et al. 2011; Pitrez et al. 2015; Croft et al. 2012; Correale and Farez 2007). Our study revealed that early immunisation with the TE and specific systems (AcD, AcR, and AcC) of A. cantonensis adult worms protects against an inflammatory response in an allergic asthma model. These extracts from A. cantonensis have Parasitol Res Fig. 2 Total and differential cell count in BAL between the groups studied (n = 10–14 animals per group). TCC (a), absolute eosinophil count (b), absolute macrophage count (c), absolute neutrophil count (d), and absolute lymphocyte count (e). #p = < 0.05, significant difference between the negative control (PBS) and positive control (OVA); *p = < 0.05, significant difference between the positive control (OVA) and animals with asthma immunised with the TE and soluble extract of the systems (AcD, AcR, and AcC) of A. cantonensis; +p = < 0.05, significant difference between the negative control (PBS) and animals with asthma immunised with TE and soluble extract of the systems (AcD, AcR, and AcC) of A. cantonensis. Data from two independent experiments (oneway ANOVA followed by Tukey test) shown a different capacity to modulate the immune system. We demonstrated for the first time, that the TE showed more potential for developing a novel therapeutic. First, we observed a high influx of inflammatory cells in airways in the allergic asthma group. However, the early immunisation with different extracts of A. cantonensis has shown a reduction in the influx of inflammatory cells. One hallmark feature of allergic asthma is the eosinophilia and in our differential count, the decrease in these cells was evident in mice that received immunisation with the different extracts. Furthermore, the immunisation with the extracts also reduced macrophage infiltration where these cells would release proteolytic enzymes and generate oxidants, which cause tissue damage and can potentiate inflammation (Tetley 2005). One possible explanation for these results is that the immunisation with different extracts could increase the influx of anti-inflammatory cytokines, such as IFN-ɣ and IL-10. The immunisation with the soluble extract of the AcD, AcR, and AcC systems did not alter the levels of these cytokines. The importance of the anti-inflammatory role of IL-10 obtained by the administration of the TE from A. cantonensis in our model of allergic asthma is in agreement with other studies that evaluated mice infected with Heligmosomoides polygyrus (H. polygyrus) and Schistosoma mansoni (S. mansoni) (Kitagaki et al. 2006; Mangan et al. 2004). We have previously shown that mice exposed to Ascaris lumbricoides (A. lumbricoides) extract also show an increased IL-10 production in the lungs in an OVA-induced allergic response and Parasitol Res Fig. 3 IL-4 (a), IL-5 (b), IFN-ɣ (c), IL-10 (d), eotaxin (e) levels in BAL, and OVA-specific IgE levels (f) in serum between the groups studied (n = 10–14 animals per group). #p = < 0.05, significant difference between the negative control (PBS) and positive control (OVA); *p = < 0.05, significant difference between the positive control (OVA) and animals with asthma immunised with the TE and soluble extract of the systems (AcD, AcR, and AcC) of A. cantonensis; +p = < 0.05, significant difference between the negative control (PBS) and animals with asthma immunised with the TE and soluble extract of the systems (AcD, AcR, and AcC) of A. cantonensis. Samples were collected from two independent experiments (oneway ANOVA followed by Tukey test) the same did not occur with the A. cantonensis extract (Pitrez et al. 2015). Cytokine measurements seem to show a great deal of variability in BAL, which may explain the differences between the results from the different studies. However, other studies have demonstrated that helminth-induced protection in allergic diseases may have multiple immunological mechanisms, such as immunological hyporesponsiveness and immune system shift to modified Th2 responses, among others (Fallon and Mangan 2007). Moreover, different helminths seem to show very specific and complex immune system interactions with the hosts. S. mansoni and Nippostrongylus braziliensis (N. brasiliensis) antigens seem to inhibit an allergic lung response by mechanisms other than the IL-10 response (Trujillo-Vargas et al. 2007; Cardoso et al. 2010). Interestingly, in infections such as toxoplasmosis and schistosomiasis, Th1 cells or B cells are important sources of IL-10 (Cardoso et al. 2010; Jankovic et al. 2007). Our findings show elevated levels of IFN-ɣ only in the TE group. It has been shown that the lack of suppression of the Th2 response in atopic children is associated with the inability of neonates to produce sufficient amounts of IFN-ɣ (Bradding et al. 2006). However, it has been suggested that the increased incidence of atopic diseases may be associated with a decrease in the prevalence of infections that induce Th1 responses in early life. It is believed that these early infections modify the Th2 response to Th1 profile responses by inducing the production of IL-12, IL-18, and IFN-ɣ, thus, offering protection from allergic diseases, corroborating with the hygiene Parasitol Res Fig. 4 Histopathological analysis in lung tissue between the groups studied (n = 10–14 animals per group). Representative photomicrographs of stained sections with H&E (× 200 magnification, arrow indicates inflammatory infiltrate cells) (a) and alcian blue (× 200 magnification, arrow indicates globet cells) (b). Negative control (PBS), positive control (OVA), animals with asthma immunised with total soluble extract of A. cantonensis (TE) and SE soluble extract (digestive, reproductive, and cuticle systems). Scale bars, 100 μm hypothesis originally proposed by Strachan in the late 1980s (Bradding et al. 2006; Prescott et al. 1999; Shirakawa et al. 1997; Shaheen et al. 1996; Matricardi et al. 2000). In allergic diseases, Th2 lymphocytes differentiate through contact with allergens or parasites. These cells are programmed to produce specific cytokines such as IL-4, IL-5, and IL-13 and eotaxins, thus, playing a central role in airway remodelling in patients with asthma (Moser et al. 1992; Lambrecht and Hammad 2015). IL-4 regulates allergenspecific IgE synthesis, the IL-5 recruits and activates eosinophils, producing chemokines, such as eotaxins. The Th1 cells coordinate the immune response to intracellular pathogens and stimulate the inhibition of the Th2 response (Rankin et al. 2000). In our analyses, the levels of IL-4, IL-5, and Parasitol Res eotaxin did not show a significant reduction when exposed to helminth extracts. One possible explanation for this is that the analysis of cytokines and mediators in BAL of murine models may not present an adequate sensitivity for mechanistic studies. The measurement of biological markers in BAL, particularly in murine animal models, has limitations, often due to dilutive factors of the method or an insufficient number of animals studied, which are usually methodologically difficult to control. The results of previous studies on Th2 cytokine responses, such as IL-5, are discrepant in murine models of asthma with infection or exposure to parasitic antigens. Some studies have shown that IL-5 is unaltered or may be elevated following exposure to helminths in asthma models (Trujillo-Vargas et al. 2007; Wang et al. 2001). One of our previous studies showed that exposure to the crude extract of A. cantonensis did not reduce IL-5 in the BAL of mice with asthma, unlike exposure to extracts of A. costaricensis and A. lumbricoides (Pitrez et al. 2015). Although the crude extract of A. cantonensis did not reduce IL-5, it was evident that there was a decrease in eosinophil infiltration. The TE can reduce the inflammatory response without a change in the levels of pro-inflammatory cytokines, but with a direct effect on antiinflammatory cytokines levels. Another factor related to allergic lung responses is the role played by IgE. The levels of OVA-specific IgE found in the sera from animals immunised with the TE and specific systems (AcD, AcR, and AcC) from A. cantonensis were not inhibited by the exposure to helminth extracts. However, Trujillo-Vargas and colleagues showed that mice immunised with excretory and secretory products of N. brasiliensis reduced the levels of IgE specific for OVA (Trujillo-Vargas et al. 2007). On the other hand, H. polygyrus infection inhibited asthma in the murine model showing no reduction of the allergen-specific IgE response (Illi et al. 2001). As with the cytokine response, the discrepant immune response results among different helminths in previous studies shows that helminths appear to interact in a complex and distinct way with the host, probably triggering different immune responses. Airway remodelling, referring to structural changes in the airway wall, in asthma is another important feature of the disease with prominent thickening of a layer underlying the fibrillar epithelium, associated with types III and V collagen deposition (Pelly et al. 2016), as well as airway remodelling with smooth muscle layer thickening (Jeffery 2001; James and Carroll 2000). Another characteristic feature of airway remodelling is the increase in the number of goblet cells secreting mucin in the superficial epithelium due to hyperplasia and metaplasia (Jones et al. 2016). The present experimental model is an acute asthma protocol where airway remodelling has not developed so far. Our histological sections of lung tissue stained with alcian blue illustrate that asthmatic mice have shown an increase in the number of goblet cells and mucus overproduction. However, only the TE was capable of decreasing this mucus overproduction. Our study has one limitation that warrants discussion. The natural presence of bacterial products in the helminth extract (lipopolysaccharides (LPS)) may influence the inhibitory effect of the allergic lung response. However, previous studies have demonstrated that helminth-specific proteins and LPS also inhibit the allergic lung response in mice (TrujilloVargas et al. 2007; Ordoñez et al. 2001). Since these products are difficult to remove from a parasite extract without removing helminth proteins in the process, we believe that this factor does not invalidate our results since the inhibitory effect of a crude helminth extract on asthma can be associated with several components that are contained therein, potentially elevating its ultimate modulating effect. From a translational point of view, in the search for novel preventive therapies in asthma, this factor may not be an important issue once a crude helminth extract has been the aim of the investigation. Although helminths are prototypical inducers of type 2 immunity, they have been correlated with a reduced reactivity to an allergen skin prick test and to some degree with asthma protection (Cruz Filho et al. 2017). A general explanation for this non-intuitive association is that helminths induce a ‘modified Th2’ response with immunoregulatory cells, such as regulatory T cells (Tregs), complementing the Th2-arm of immunity and regulating the response to bystander antigens such as aeroallergens. Therefore, several groups have tried to find helminth-derived products with immunomodulatory properties that could be used to suppress Th2 immunity (Navarro et al. 2016). Our findings show that early immunisation with the TE and specific systems (AcD, AcR, and AcC) of A. cantonensis adult worms decreased the high influx of inflammatory cells on airways in allergic asthmatic mice. The initial hypothesis of the study was that the extract of specific systems (AcD, AcR, and AcC) could be more potent to the modulation of inflammation than the TE. Our results, however, show that immunisation with the TE are more promising as there was a decreased influx of inflammatory cells in airways and it promoted an increase in IFN-ɣ and IL-10 production, where these cytokines are involved in anti-inflammatory responses and immunomodulation. Furthermore, only the immunisation with the TE leads to reduced goblet cells and mucus overproduction in the lung tissue of asthmatic mice. This distinct capacity to modulate the immune system can be explained due to the TE having a greater variability of molecules, which together promote a better effect on the airway inflammatory response. In summary, this is the first study to reveal that the TE of A. cantonensis adult worms has a greater potential for developing a novel therapeutic for allergic asthma. Funding This study was funded by the National Council for Scientific and Technological Development (CNPq, 307005/2014-3). Parasitol Res Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the Animal Ethics Committee (11/00238) from PUCRS. References Araujo MIA, Hoppe B, Medeiros M Jr, Alcântara L, Almeida MC, Schriefer A et al (2004) Impaired T helper 2 response to aeroallergen in helminth-infected patients with asthma. 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