NLRP3 plays a key role in antihelminth immunity in the enteral and parenteral stages of Trichinella spiralis-infected mice

NLRP3 plays a key role in antihelminth immunity in the enteral and parenteral stages of Trichinella spiralis-infected mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article NLRP3 plays a key role in antihelminth immunity in the enteral and parenteral stages of Trichinella spiralis-infected mice Tian-Xu Pan, Hai-Bin Huang, Guang-Xun Zhao, Yu Quan, Jun-Yi Li, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1951416/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Trichinellosis is an important food-borne zoonosis, and no effective treatments are yet available. Nod-like receptor plays a critical role in the host response against nematodes. Therefore, we aimed to explore the role of the NLRP3 inflammasome (NLRP3) during the adult, migrating, and encysted stages of Trichinella spiralis (T. spirali s) infection. Methods: The mice were treated with the specific NLRP3 inhibitor MCC950 after inoculation with T. spiralis. Then, NLRP3 plays the role in T. spirali s-infected mice were evaluated using ELISA, Western blotting, Flow cytometry, Histopathological evaluation, Bone marrow-derived macrophage (BMDM) stimulation and immunofluorescence Results: The in vivo results showed that NLRP3 enhanced the Th1 immune response in the adult stage and the migrating stage and weakened the Th2 immune response in the encysted stage. NLRP3 promoted the release of proinflammatory factors (INF-γ) and suppressed the release of anti-inflammatory factors (IL-4). Pathological changes were also improved in the absence of NLRP3 in mice during T. spiralis infection. Importantly, a significant reduction in adult worm burden and muscle larvae burden at 7 and 35 days post infection was observed in mice treated with the specific NLRP3 inhibitor MCC950. In vitro, we first demonstrated that NLRP3 in macrophages can be activated by T. spiralis proteins and promotes IL-1β and IL-18 release. Conclusions: This study revealed that the NLRP3 is involved in the host response to T. spiralis infection and that targeted inhibition of NLRP3 enhanced the Th2 response and accelerated T. spiralis expulsion. These findings may help in the development of protocols for controlling trichinellosis. T. spiralis NLRP3 Immune response Th1/Th2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Trichinosis is an important food-borne parasitic zoonosis mainly caused by ingesting raw or undercooked meat infected with the encapsulated muscle larvae of Trichinella spiralis ( T. spiralis ) [1, 2] . T. spiralis can infect a wide range of mammalian hosts, including humans and more than 150 mammalian species, which not only leads to great threat to human health worldwide but also represents an economic problem in the livestock industry [3] . Although various countries around the world have taken measures to control and suppress the occurrence of the disease, it has not been effectively controlled because its immune response process is complex [4]. Therefore, a good understanding of the immune mechanisms may facilitate the discovery of approaches to control trichinosis. Increasing attention has been given to defining the role of the host immune response in T. spiralis infection. The innate immune system plays an important role in recognizing pathogens and triggering biological mechanisms to control infection and eliminate pathogens [5] . It is activated when pattern-recognition sensor proteins, such as Toll-like receptors (TLRs) or nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), detect the presence of pathogens, their products, or danger signals [6] . NLR inflammasomes play a central role in innate immunity [7] . The NOD-like receptor protein 3 inflammasome (NLRP3) is a protein complex consisting of the NLRP3 scaffold, ASC adaptor, and procaspase-1. NLRP3, which includes mature caspase-1, can promote the activation of cytokines such as IL-1β and IL-18 and promote the occurrence of an inflammatory response [8] . NLRP3 inflammasome complexes are the most intensively studied and are activated by a broad range of stimuli. Recently, considerable evidence has suggested that NLRP3 activation is essential for the control of different parasitic infections, as it plays a protective role in reducing Toxoplasma gondii and Neospora caninum infection loads [9, 10] . In addition, NLRP3 has been involved in the inhibition of eosinophil influx that results from the type 2 response to the parasite in mice infected with Nippostrongylus brasiliensis [11] . Despite the critical role of inflammasomes in the immune response to parasites, their functions in T. spiralis chronic infection immunity appear less clear. The parasitic process can be divided into adult, migrating, and encysted stages [12] . With the transition of larvae from the intestinal phase to the encysted stage in the host, the host’s immune type gradually changes from Th1 to Th2 and evokes concurrent inflammation-related factor changes [13, 14] . CD4 + T cells play a key role in parasite immunity and can differentiate into different subpopulations, including Th1, Th2, Th17 and Treg cells. T. spiralis and its secretory products can induce Th2 immune responses to suppress inflammatory responses [15] . Recent studies have shown that Th2 immune responses may help reduce tissue damage and strengthen tissue repair [16] . The changes in Th1 and Th2 immune types mainly rely on the secretion of cytokines [17] . In this study, to explore whether NLRP3 participates and plays a role during T. spiralis infection, we established a mouse model of T. spiralis infection and then intraperitoneally injected an NLRP3 inhibitor (MCC950) at different time points. The aim of the study was to detect the effects of NLRP3 on parasitism and immunity of T. spiralis in adult, migrating, and encysted stages. 2. Materials and methods 2.1 Parasites and animals The T. spiralis isolate ISS534 used in this study was maintained by serial infection of SD rats from the Animal Parasite Laboratory of Jilin Agricultural University. BALB/c mice (female, 4–6 weeks old) and SD rats were purchased from Beijing Fukang Biotechnology company. All animal husbandry was maintained in Jilin Agricultural University Animal Experiment Center under the care of a professional breeder with a light/dark cycle of 12 hours (h) and with sterile food and water. Furthermore, all experiments in this study were performed in accordance with the Chinese Animal Management Ordinance. The animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Jilin Agricultural University. 2.2 Isolation of T. spiralis adults, new-born larvae and muscle larvae Muscle larvae, adults and new-born larvae were obtained from rats as previously described [18] . In brief, BALB/c mice were orally inoculated with 300 muscle larvae of T. spiralis and sacrificed 7 days post-infection (dpi). The intestine was exposed, and the entire small intestine was harvested and washed by flushing in sterile saline to remove any intestinal contents before the tissue was cut into 3-cm pieces. The small intestine sections were transferred to a separation screening cloth of a nematode larva separator (200 mesh), which was put in a small beaker containing sterile saline (preheated at 37 °C) and incubated at 37 °C for 4 h. T. spiralis adults were collected from the bottom of the beaker and counted. The adults were incubated and washed in Roswell Park Memorial Institute-1640 medium (RPMI-1640, Gibco) with 20% foetal bovine serum (FBS, Gibco), 100 U/mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco) at 37 °C in a humidified incubator with 5% CO 2 . The culture solution was filtered with a 280-mesh sterile mesh so that the new-born larvae were collected. Muscle larvae of T. spiralis were obtained by standard pepsin digestion (1% pepsin and 1% HCl at 37 °C for 2 h) to release larvae from the muscles of the rats infected at 35 days. 2.3 Animal experimental design Forty-five healthy female BALB/c mice were randomly divided into three groups: TS+MCC950, TS and control. TS+MCC950 mice were intraperitoneally (IP) injected daily with the specific NLRP3 inhibitor MCC950 (10 mg/kg) after inoculation with T. spiralis (350 larvae per mouse), whereas the TS group mice were injected with an equal volume of PBS (i.p.). The mice in the control group received PBS solution. Five mice in each group were killed on the 7th, 14th and 35th days after infection with T. spiralis by cervical dislocation. The blood samples were placed in a sterile EP tube for 1 h at 37 ℃ and stored overnight in a 4 ℃ refrigerator. Serum was obtained by centrifuging the EP tube at 3000 rpm for 10 min at 4 ℃. Single-cell suspensions of the spleen (SPL) or mesenteric lymph nodes (MLN) were analysed by flow cytometry as previously described [19] . The small intestinal and masseter muscles were extracted as pathological sections to detect inflammation. Western blotting was utilized to detect changes in NLRP3 protein levels. 2.4 Enzyme-linked immunosorbent assay The production of IgG1, IgG2, IL-1β and IL-18 in the serum of infected mice was determined in the control group, TS group, and TS+MCC950 group at 7, 14, and 35 dpi. Enzyme-linked immunosorbent assay (ELISA) kits (Abcam) were used. 2.5 Western blot Small intestinal was pulverized using a mortar and pestle in liquid nitrogen and homogenized in ice-cold RIPA buffer (Thermo Scientific™). The protein concentration of the extracts was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific™). Total protein was resolved on 12% SDS–PAGE gels and then transferred to polyvinylidene difluoride membranes (PVDF). The membrane was blocked in TBST (10 mM Tris HCl, 0.15 M NaCl containing 0.05% Tween 20) with 5% nonfat skim milk for 3 h at RT and incubated with the corresponding primary antibody at 4 °C overnight. After washing for 10 min with TBST three times, the membranes were incubated with secondary antibodies at room temperature for 1 hour and washed again. Bands were visualized with ECL chromogenic reagent. The primary antibodies used were anti-NLRP3 (Abcam, 1:2000 dilution) and anti-mouse β-actin ( Proteintech, China,1:2000 dilution). 2.6 Flow cytometry The relative ratios of IL-4 and INF-γ on CD3 + CD4 + T cells in the spleen (SPL) and mesenteric lymph nodes (MLN) were analysed by flow cytometry. Single-cell suspensions of the SPL and mesenteric lymph nodes (MLN) of all groups of mice were prepared as previously described (Shi et al., 2016) at Days 7, 14, and 35 after infection. Briefly, single cells were counted and seeded in 48-well plates at 2.0 × 10 6 cells/well in 500 µl RPMI-1640 medium (100 U/ml penicillin, 100 μg/ml streptomycin, 10% heat-inactivated FBS) containing ionomycin (1 µg/mL), Golgi plug (10 µg/mL) and PMA (20 ng/mL) and were then incubated at 37 °C in a 5% CO 2 incubator for 6 hours. After treatment, the cells were collected into EP tubes by centrifugation (2000 rcf for 5 min at 4 °C) and discarded from the supernatant. Harvested cells were resuspended in 100 μL of cold PBS and incubated with various combinations of fluorochrome-labelled antibodies (CD3, CD4 along with isotype controls) according to the manufacturer's instructions (BD Stemflow). Anti-mouse CD3 (PerCP-CY5.5, BD Biosciences) and anti-mouse CD4 (FITC, BD Biosciences) were used to label CD3 + T cells and CD4 + T cells at 4 °C for 1 h in the dark. After staining for cell surface markers, the cells were fixed and permeabilized with a Cytofix/Cytoperm kit (BD Biosciences) following the manufacturer’s instructions and washed two times with 500 μl of cold BD Perm/Wash TM buffer (BD Biosciences). Intracellular staining was required to determine the percentage of IL-4 + and INF-γ + cells. Next, treatment with anti-mouse IL-4 (APC, BD Biosciences) and anti-mouse INF-γ (PE, BD Biosciences) was performed at 4 °C for 1 h in the dark. Following this, all stained cells were washed three times with cold PBS to remove unbound antibodies, suspended in 300 µl of PBS, and then examined using a LSR Fortessa TM (BD Biosciences). Data were analysed with Flow Jo software (ver 7.6.1, Tree Star Inc., USA). 2.7 Histopathological evaluation and immunofluorescence At 7, 14, and 35 dpi, lingual muscle and small intestine samples were obtained from each group and fixed in 4% formalin for 48 h. The tissue was subjected to washing, dehydrated in gradual ethanol (70-100%), made transparent with xylene, processed, embedded with paraffin wax, sliced (3 μm), placed in warm water (42 ℃) for spreading, collected using slides, baked, and prepared into paraffin tissue sections. The tissue slices were baked at 80 ℃ for 1 h and placed in xylene for 8 min twice. For histopathological evaluation, the sections were stained with haematoxylin and eosin and visualized with a microscope (Leica). The number of goblet cells per ten randomly selected villus–crypt units (VCU) was determined by microscopy from at least two sections per animal as previously described [20] . Twenty nonoverlapping representative fields of the tissue were examined microscopically using a 400X objective, and the number of inflammatory cells infiltrating the masseter muscle was observed [21] . 2.8 Bone marrow-derived macrophage (BMDM) isolation, culture and stimulation Mouse bone marrow-derived macrophages (BMDMs) were generated from five-week-old BALB/c mice as previously described [22] . In the in vitro experiments, the BMDMs were randomly divided into the PBS negative control, ATP positive control, adult protein group, neonatal larvae protein group, muscle larvae protein group, and MCC950 group. ELISA was used to detect changes in IL-18 and IL-1β levels, and indirect immunofluorescence was used to detect the expression of NLRP3. BMDMs were primed with LPS for 3 h and then treated with DMSO (Beyotime) or DMSO+MCC950 (7.5 nm/mL) for 30 min, followed by stimulation with T. spiralis protein (50 mg/mL) of new-born larvae, muscle larvae and adult stages for 6 hours. Protein extraction was performed as previously described (Gómez-Morales, et al., 2018). After treatment, the BMDMs were washed with PBS, fixed with 80% cold acetone for 30 min at RT and then washed three times with PBS. After blocking with 3% BSA in PBS for 30 min at RT, the BMDMs were incubated with an antibody against NLRP3 (Sigma) for 1 h at 4 °C. After washing with PBST, the BMDMs were incubated with secondary antibody (Alexa Fluor® 488 (green). Proteintech) for 40 min at RT. After washing, nuclei were stained with DAPI (blue. Sigma) for 10 min in the dark. Imaging analysis was performed using a fluorescence microscope. 2.9 Statistical analysis Data were statistically analysed by using GraphPad Prism 9.0 software. The Shapiro–Wilk test was used to analyse normality. For comparisons of only two groups, data were analysed using Student’s t test, while for comparisons of three or more groups, we performed one-way ANOVA with Bonferroni’s multiple comparison test as indicated. The Mann–Whitney U test was used for nonnormally distributed data. The statistically significant differences between the means are indicated by asterisks (* P< 0.05, ** P< 0.01, *** P< 0.001). Statistical data are expressed as the mean value ± SD. 3. Results 3.1 NLRP3 increases the Th1 response during T. spiralis infection To determine whether the NLRP3 inflammatory body is involved in T. spiralis infection. The contents of the cytokines IL-1β and IL-18 and host immune types IgG1 and IgG2 were detected by ELISA kits in the adult stage (7 D), migrating stage (14 D), and encysted stage (35 D), as shown in Fig. 1. With respect to IL-1β, the TS+MCC950 group showed a significant decrease compared with the TS group in the adult, migrating and encysted stages [7D: ANOVA, F (2.12) = 70.650, P < 0.001; 14D: ANOVA, F (2.12) = 23.840, P < 0.001; 35D : ANOVA, F (2.12) = 10.220, P = 0.0025] (Fig. 1A). IL-18 excretion was also significantly decreased in the adult stage and the migrating stage [7D : ANOVA, F (2.12) = 15.520, P < 0.001; 14D: ANOVA, F (2.12) = 88.160, P < 0.001], but was not significantly different in encysted stages[35D: ANOVA, F (2.12) = 4.733, P =0.229] (Fig. 1, B). For IgG1, the excretion was not significantly different in the adult stage[7D: ANOVA, F (2.12) = 7.717, P > 0.05], and the TS+MCC950 group showed a significant decrease in the migrating stage compared with the TS group[14D: ANOVA, F (2.12) = 8.462, p = 0.0076] but was increased significantly in the encysted stage[35D: ANOVA, F(2.12)=163.2, P < 0.001](Fig. 1C). IgG2 was significantly decreased in the migrating and adult stages [7D: ANOVA, F (2.12) = 147.9, p < 0.001; 14D: ANOVA, F (2.12) = 64.69, p 0.05] (Fig. 1D). These results suggest that NLRP3 participates in T. spiralis infection and that inhibiting NLRP3 promotes the host Th1-type immune response in the adult stage and the migrating stage and inhibits the Th2-type immune response in the encysted stage. 3.2 T. spiralis infection promotes the expression of NLRP3 in the small intestine The protein expression of NLRP3 in the intestine was detected by western blotting (Fig. 2). The results showed that the expression of NLRP3 was significantly elevated in the TS group compared with the TS+MCC950 group or the control group in the adult stage (7D), and the expression levels of NLRP3 had no evident change in the different groups in the migrating stage (14D). In the encysted stage, the expression of NLRP3 in the small intestine in the TS group was significantly higher than that in the control group and the TS+MCC950 group. This illustrated that T. spiralis could promote the expression of NLRP3 in the adult and encysted stages. 3.3 Effects of NLRP3 on IFN-γ and IL-4 during T. spiralis infection We detected the impact of NLRP3 expression on changes in the expression of proinflammatory factors (INF-γ) and anti-inflammatory factors (IL-4). In adults, flow cytometry assays showed that the INF-γ expression levels in the MLN and SPL in the TS+MCC950 group were significantly decreased compared with those in the TS group[MLN: ANOVA, F (2.12) = 96.69, P < 0.001]; SPL: ANOVA, F (2.12) = 372.8, P < 0.001] , however, the IL-4 expression levels in the MLN were increased significantly [MLN: ANOVA, F (2.12) = 138.4, P < 0.001] (Fig. 3B). In the migrating stage, there was no difference in INF-γ expression among the three groups, while the levels of IL-4 increased significantly in the SPL and MLN of the TS+MCC950 group compared with the TS group [MLN: ANOVA, F (2.12 ) = 40.57, P < 0.001]; SPL: ANOVA, F (2.12) = 123.6, P < 0.001] (Fig. 3C). In the encysted stage, compared with the TS group, there was a significant decrease in the expression levels of INF-γ in the MLN of the TS+MCC950 group [MLN: ANOVA, F (2.12) = 19.87, P = 0.006], and the expression of INF-γ levels tended to gradually decrease in the SPL, with no statistical significance (Fig. 3D). There was no significant difference in the expression of IL-4 between the two infection groups. 3.4 NLRP3 enhances the burden of intestinal worms and muscle larvae during T. spiralis infection Five mice were randomly selected from the TS group and the TS+MCC950 group in the adult stage (7 D) and encysted stage (35 D), and the number of worms was obtained by separating muscle larvae and adults of T. spiralis . The results showed that the number of worms in the TS+MCC950 group was significantly reduced compared with that in the TS group [t (8) = 13.47, P < 0.001](Fig. 4A), and the number of muscle larvae was significantly reduced [t ( 8) = 8.166, P < 0.001] (Fig. 4B). It was concluded that NLRP3 could enhance the survival of T. spiralis in the host. 3.5 Effects of NLRP3 on the pathological damage to intestinal and masseter muscle in mice during T. spiralis infection To investigate how NLRP3 enhances the burden of intestinal worms and muscle larvae during T. spiralis infection, we observed goblet cell hyperplasia of the intestinal epithelium and inflammatory cell infiltration of masseter muscle, which are characteristic of intestinal nematode infection. The number of intestinal goblet cells increased on Days 7 and 14 in T. spiralis- infected mice compared with noninfected mice. Compared with mice in the TS group, mice in the TS+MCC950 group showed goblet cell hyperplasia in the intestinal mucosa in the adult and migrating stages [7D: ANOVA, F (2.12) = 120, P < 0.001;14D: ANOVA, F (2.12) = 112.6, P < 0.001] (Fig. 5B).The mucosal epithelium was integral, and the morphology of the villi was normal. There was no significant difference in the encysted stage (Fig. 5A). Histological observation of H&E-stained T. spiralis- infected masseter muscle clearly showed inflammatory cell infiltration around the encystation (Fig. 5C). The level of inflammatory cells was significantly increased sharply in the migrating and encysted stages. In addition, the TS+MCC950 group displayed a significant reduction in inflammatory cellular infiltration and parasite cysts in the muscle when compared with the TS group [14D: ANOVA, F (2.12) = 25.96, P = 0.0025; 35D: ANOVA, F (2.12) = 107.7, P < 0.0012] (Fig. 5D). Suppressing NLRP3 could increase goblet cells in the intestine and ameliorate inflammatory cell infiltration in the masseter muscle during T. spiralis infection. 3.6 T. spiralis protein activates NLRP3 in BMDMs To determine the role of proteins of three different stages of T. spiralis associated with the activation of the NLRP3 inflammasome, in vitro culture of mouse BMDMs was performed, and all groups of BMDMs were pretreated with LPS. The results showed that the activation of NLRP3 in the stimulation group was higher than that in the PBS group. Moreover, compared with the T. spiralis protein stimulation group (adult protein, neonatal larvae protein, and muscle larvae protein), there was a significant decrease in the activation of NLRP3 in the MCC950 group (Fig. 5A). The secretion of IL-18 and IL-1β in the T. spiralis protein stimulation group was lower than that in the positive control group but significantly higher than that in the MCC950 group [IL-18:( adult protein: ANOVA, F (3.8) = 295.4, P < 0.001; neonatal larvae protein: ANOVA, F (3.8) = 357.1, P < 0.001; muscle larvae protein: ANOVA, F (3.8) = 252, P< 0.001); IL-1β:(adult protein: ANOVA, F (3.8) = 219.4, P < 0.001; neonatal larvae protein: ANOVA, F (3.8) =248.0, P < 0.001; muscle larvae protein: ANOVA, F (3.8) =316.1, P < 0.001)] (Fig. 5D, E). these results suggest that different life cycle stages of T. spiralis proteins promote the activation of NLRP3 and that NLRP3 promotes the secretion of IL-18 and IL-1β in BMDMs. 4. Discussion Parasitic helminth infestation is the most common chronic disease among humans and animals. At present, due to the complexity of the life cycle of worms, the mechanisms by which antigens induce Th2-type immune responses and initiate inflammation are not entirely clear, and no effective immunological approach is used to control worms [23, 24] . Although there is evidence that inflammasome signalling and inflammasome-dependent cytokines are important for host defence against T. spiralis, further studies are needed to establish how the inflammasome affects adaptive immune responses in the context of T. spiralis infections [25] . In the present study, we identified that NLRP3 is involved in both innate and adaptive immune responses and the conversion of immune response patterns by regulating the secretion of IL-1β and IL-18 during T. spiralis infection. Additionally, we first demonstrated that NLRP3 in macrophages can be activated by T. spiralis proteins and promotes IL-1β and IL-18 release. MCC950 inhibits the activation of NLRP3 to speed up Th2-type immune type-mediated T. spiralis clearance. NLRP3 is associated with the Th1 immune response and mediates host protection against pathogen invasions [26] . Recent studies have also demonstrated that NLRP3 plays an important role in the promotion of Th2 immune responses [27] . We confirmed that NLRP3 promotes a Th1 immune response during T. spiralis infections, and this effect may be caused by cytokine secretion of IL-1β and IL-18. IL-18 is one of the best-characterized inflammasome-dependent cytokines whose maturation requires cleavage by caspase-1 from its inactive intracellular precursor pro-IL-18, which can induce IFN-γ production in Th1-type cells and can promote T-cell proliferation [28, 29] . Our data support an important role for NLRP3-dependent IL-18 in regulating immunity and inflammation following T. spiralis clearance. Moreover, we demonstrated that IL-1β was similarly regulated by NLRP3 during T. spiralis infection. NLRP3 activation leading to IL-1β production is critical for the induction of a Th2 response [30] . IL-1β-deficient mice are susceptible to chronic Trichuris muris infection, and the inability to expel the worms is associated with a defect in the development of a Th2 response, suggesting the critical role of IL-1β in regulating the Th2 response during gastrointestinal nematode infection [25, 31] . We demonstrated that the decrease in the expression of IL-18 and IL-1β may lead to an increased Th2-type response and weakening of the Th1-type response when NLRP3 is inhibited after T. spiralis infection. Similar results were obtained in a study using dendritic cells of NLRP3 −/− mice [25] . Inflammasomes have a regulatory role in the infection of early innate responses and can promote the maturation and production of inflammatory cytokines. There is a large body of evidence related to the involvement of inflammasomes in the innate immune response [32] . We demonstrated that NLRP3 induced an inflammatory response and mediates protective immunity to T. spiralis infection, possibly via adaptive immune responses. Th1 subset cells mediate cellular immunity by secreting IFN-γ, and Th2 subset cells mediate cellular immunity by secreting IL-4 [33, 34] . In the present study, NLRP3-inhibited mice displayed decreased IFN-γ levels, which may promote a shift in the Th1/Th2 balance towards Th2-dominant immunity during the early stage of infection (adult stage, migrating stage). The IFN-γ levels changed with time since infection, leading to low levels of IL-4. In contrast, T. spiralis infection shifted the Th1/Th2 balance towards a dominant Th1 immune response during the encysted stage. IFN-γ can mediate and regulate immunity and invasiveness to resist T. spiralis infection, and parasite expulsion is IL-4 dependent [35, 36]. Based on the above, inhibition or deletion of NLRP3 expression could provide increased immunity to T. spiralis infection. Previous studies have shown that NLRP3 plays a key role in pathogenic bacteria-induced progression of acute inflammation [37] . Reports have demonstrated that NLRP3 is involved in the inhibition of eosinophil influx to the parasite in mice infected with Nippostrongylus brasiliensis , and IL-1β secretion is mediated by P2X7R in small intestinal epithelial cells in response to Toxoplasma gondii infection [6, 11] . T. spiralis infections in host intestinal acute mucosal inflammation, after larvae enter the skeletal muscle tissue, induce a relevant inflammatory reaction that is responsible for myositis and can cause systemic inflammatory manifestations all over the body before entering the striated muscles [38] . In this study, we identified that T. spiralis enhances NLRP3-dependent IL-18 and IL-1β secretion, which may contribute to the inflammatory cell infiltration and histological changes observed in the muscle and intestinal tissue. In the acute phase of the infection, the parasite load dictates the magnitude of the inflammatory response and tissue damage [39] . In this study, a significant reduction in adult worm burden and muscle larvae burden at 7 and 35 days postinfection was observed in mice treated with the specific NLRP3 inhibitor MCC950. It has also been found to be effective in the treatment of Trichuris muris [40] . NLRP3 inhibition may influence the functions of nonhaematopoietic cells in the intestine that promote worm expulsion, including epithelial cell turnover, goblet cell expansion, mucus production and smooth muscle hypercontractility [40] . This is consistent with the increase in goblet cells observed in the absence of NLRP3 in mice during T. spiralis infection in this study. It is conceivable that targeting the NLRP3 pathway in T. spiralis -infected hosts using MCC950 may be a rational approach for lowering worm burdens. However, people living in helminth endemic regions are often exposed to coinfections with other parasites, and previous studies have demonstrated a protective role for NLRP3 in these infection models. Some studies have shown that the absence of NLRP3 results in increased parasite burden, such as with Toxoplasma gondii and Neospora caninum [10, 41] . Further studies are necessary to assess the impact of such therapeutic strategies on other preventive and curative interventions. 5. Conclusion Taken together, these findings demonstrate that NLRP3 regulates immunity and inflammation in T. spiralis infections and that targeted inhibition of NLRP3 enhanced the Th2 response and accelerated T. spiralis expulsion. Therefore, NLRP3 is an important target for controlling T. spiralis infection. Abbreviations NLRP3: NOD-like receptor protein 3 inflammasome; Th: CD4+ T helper cells; ELISA: Enzyme-linked immunosorbent assay; FBS: Fetal bovine serum; IL: Interleukin; IFN-γ: Interferon gamma; HRP: Horseradish peroxidase; PBS: Phosphate-buffered saline; PVDF: Polyvinyl difluoride; SDS-PAGE: Sodium dodecyl sulphate–polyacrylamide gel electrophoresis; BMDMs: Mouse bone marrow-derived macrophages. Declarations Acknowledgements The authors gratefully acknowledge the teachers of Jilin Agricultural University. A special thanks to Dan-Wang for the precious contribution with Enzyme-linked immunosorbent assay. Funding This work was supported by the National Natural Science Foundation of China (32072888, 31941018, and U21A20261) and the Science and Technology Development Program of Jilin Province (20180201040NY, 20190301042NY, and YDZJ202102CXJD029). Availability of data and materials The data supporting the conclusions of this article are included within the article and the additional files. Authors’ contributions Gui-Lian Yang and Chun-Feng Wang conceived and designed the experiments. Tian-Xu Pan, Hai-Bin Huang, Yu Quan, Jun-Yi Li, Ying Xue, Hui-Nan Lu, Zhi-Yu Zhu, Yue Wang, Chun-Wei Shi, and Nan Wang carried out the research and performed data processing and analyses. Tian-Xu Pan, Guang-Xun Zhao, and Hai-Bin Huang performed the statistical analysis. Tian-Xu Pan drafted the manuscript with input from all other authors. All authors have read and approved the final manuscript. Ethics approval and consent to participate The animal experimental procedures were performed based on the regulations of the Administration of Affairs Concerning Experimental Animals in China. All animal experiments in this study were approved by the constitution of the Experimental Animal Welfare and Ethics Committee of Jilin Agricultural University. Consent for publication All the authors agreed to publish the manuscript Competing interests The authors declare that they have no financial or other competing interests. References Gu Y, Sun X, Huang J, Zhan B, Zhu X. Trichinella spiralisA Multiple Antigen Peptide Vaccine Containing CD4 T Cell Epitopes Enhances Humoral Immunity against Infection in Mice. Journal of immunology research. 2020;2020:2074803; doi: 10.1155/2020/2074803. Long S, Wang Z, Liu R, Liu L, Li L, Jiang P, et al. Molecular identification of Trichinella spiralis nudix hydrolase and its induced protective immunity against trichinellosis in BALB/c mice. Parasites & vectors. 2014;7:600; doi: 10.1186/s13071-014-0600-9. Wang Z, Hao C, Huang J, Zhuang Q, Zhan B, Zhu X. Mapping of the complement C1q binding site on Trichinella spiralis paramyosin. Parasites & vectors. 2018;11 1:666; doi: 10.1186/s13071-018-3258-x. Pratesi F, Bongiorni F, Kociecka W, Migliorini P, Bruschi F. Heart- and skeletal muscle-specific antigens recognized by trichinellosis patient sera. Parasite immunology. 2006;28 9:447-51; doi: 10.1111/j.1365-3024.2006.00889.x. Chu J, Shi G, Fan Y, Choi I, Cha G, Zhou Y, et al. Toxoplasma gondiiProduction of IL-1β and Inflammasome with Up-Regulated Expressions of NOD-Like Receptor Related Genes in -Infected THP-1 Macrophages. The Korean journal of parasitology. 2016;54 6:711-7; doi: 10.3347/kjp.2016.54.6.711. Quan J, Huang R, Wang Z, Huang S, Choi I, Zhou Y, et al. P2X7 receptor mediates NLRP3-dependent IL-1β secretion and parasite proliferation in Toxoplasma gondii-infected human small intestinal epithelial cells. Parasites & vectors. 2018;11 1:1; doi: 10.1186/s13071-017-2573-y. de Almeida R, Clendenon S, Richards W, Boedigheimer M, Damore M, Rossetti S, et al. Transcriptome analysis reveals manifold mechanisms of cyst development in ADPKD. Human genomics. 2016;10 1:37; doi: 10.1186/s40246-016-0095-x. Du RH, Tan J, Sun XY, Lu M, Ding JH, Hu G. Fluoxetine Inhibits NLRP3 Inflammasome Activation: Implication in Depression. International Journal of Neuropsychopharmacology. 2016:pyw037. Wang X, Gong P, Zhang X, Wang J, Tai… L. NLRP3 inflammasome activation in murine macrophages caused by Neospora caninum infection. Parasites & Vectors. 2017;10 1. Abreu MSAC, Coutinho A-d-SCL, Prado RT, Costa RGD, Maria B, Sim?Es ZD, et al. The P2X7 Receptor Mediates Toxoplasma gondii Control in Macrophages through Canonical NLRP3 Inflammasome Activation and Reactive Oxygen Species Production. Frontiers in Immunology. 2017;8. Chenery A, Alhallaf R, Agha Z, Ajendra J, Parkinson J, Cooper M, et al. Inflammasome-Independent Role for NLRP3 in Controlling Innate Antihelminth Immunity and Tissue Repair in the Lung. Journal of immunology (Baltimore, Md : 1950). 2019;203 10:2724-34; doi: 10.4049/jimmunol.1900640. Aranzamendi C, Sofronic-Milosavljevic L, Pinelli E. Helminths: Immunoregulation and Inflammatory Diseases-Which Side Are Trichinella spp. and Toxocara spp. on? Journal of parasitology research. 2013;2013:329438; doi: 10.1155/2013/329438. Sofronic-Milosavljevic L, Ilic N, Pinelli E, Gruden-Movsesijan A. Secretory Products of Trichinella spiralis Muscle Larvae and Immunomodulation: Implication for Autoimmune Diseases, Allergies, and Malignancies. Journal of immunology research. 2015;2015:523875; doi: 10.1155/2015/523875. Mosmann TR. Cytokine secretion patterns and cross-regulation of T cell subsets. Immunologic Research. 1991;10 3-4:183-8. Alisa, Gruden-Movsesijan, Ljiljana, Sofronic-Milosavljevic, Natasa, Ilic, et al. Secretory Products of Trichinella spiralis Muscle Larvae and Immunomodulation: Implication for Autoimmune Diseases, Allergies, and Malignancies. Clinical & Developmental Immunology. 2015. Maizels RM, Gause WC. How helminths go viral. Science. 2014;345 6196:517-8. Tymoshok NO, Lazarenko LM, Bubnov RV, Shynkarenko LN, Babenko LP, Mokrozub VV, et al. New aspects the regulation of immune response through balance Th1/Th2 cytokines. EPMA Journal, The. 2014;5 Suppl 1:A134. Despommier D, Campbell W, Blair L. The in vivo and in vitro analysis of immunity to Trichinella spiralis in mice and rats. Parasitology. 1977;74 1:109-19; doi: 10.1017/s0031182000047570. Huang H, Yang W, Hu J, Jiang Y, Wang J, Shi C, et al. Antitumour metastasis and the antiangiogenic and antitumour effects of a Eimeria stiedae soluble protein. Parasite immunology. 2021:e12825; doi: 10.1111/pim.12825. Yu YR, Liu XC, Zhang JS, Ji… C. Taurine drinking attenuates the burden of intestinal adult worms and muscle larvae in mice with Trichinella spiralis infection. Parasitology Research. 2013;112 10:3457-63. Mi-Kyung P, Yun-Jeong K, Jin-Ok J, Kyung-Wan B, Hak-Sun Y, Hyun CY, et al. Effect of Muscle Strength by Trichinella spiralis Infection during Chronic Phase. International journal of medical sciences. 2018;15 8:802-7. Sugata R, Sebastian S, Erik A, Tanvir A, Parihar SP, Mumin O, et al. Redefining the transcriptional regulatory dynamics of classically and alternatively activated macrophages by deepCAGE transcriptomics. Nucleic Acids Research. 2015;43 14:6969-82. Gu Y, Wei J, Yang J, Huang J, Yang X, Zhu X. Protective immunity against Trichinella spiralis infection induced by a multi-epitope vaccine in a murine model. PloS one. 2013;8 10:e77238; doi: 10.1371/journal.pone.0077238. van der Werf N, Redpath S, Azuma M, Yagita H, Taylor M. Th2 cell-intrinsic hypo-responsiveness determines susceptibility to helminth infection. PLoS pathogens. 2013;9 3:e1003215; doi: 10.1371/journal.ppat.1003215. Jin X, Bai X, Yang Y, Ding J, Shi H, Fu B, et al. NLRP3 played a role in Trichinella spiralis-triggered Th2 and regulatory T cells response. Veterinary research. 2020;51 1:107; doi: 10.1186/s13567-020-00829-2. Silva G, Costa R, Silveira T, Caetano B, Horta C, Gutierrez F, et al. Apoptosis-associated speck-like protein containing a caspase recruitment domain inflammasomes mediate IL-1β response and host resistance to Trypanosoma cruzi infection. Journal of immunology (Baltimore, Md : 1950). 2013;191 6:3373-83; doi: 10.4049/jimmunol.1203293. Bruchard M, Rebé C, Derangère V, Togbé D, Ryffel B, Boidot R, et al. The receptor NLRP3 is a transcriptional regulator of TH2 differentiation. Nature Immunology. 2015;16 8:859-70. Schlaeger J, Cai H, Steffen A, Angulo V, Shroff A, Briller J, et al. Acupuncture to Improve Symptoms for Stable Angina: Protocol for a Randomized Controlled Trial. JMIR research protocols. 2019;8 7:e14705; doi: 10.2196/14705. Zhang T. Interleukin-12 (IL-12) and IL-18 synergistically induce the fungicidal activity of murine peritoneal exudate cells against Cryptococcus neoformans through production of gamma interferon by natural killer cells. Infection & Immunity. 1997;65. Besnard A, Guillou N, Tschopp J, Erard F, Couillin I, Iwakura Y, et al. NLRP3 inflammasome is required in murine asthma in the absence of aluminum adjuvant. Allergy. 2011;66 8:1047-57; doi: 10.1111/j.1398-9995.2011.02586.x. Helmby H, Grencis RK. Interleukin 1 plays a major role in the development of Th2-mediated immunity. Eur J Immunol. 2004;34 12:3674-81; doi: 10.1002/eji.200425452. Ciraci C, Janczy JR, Sutterwala FS, Cassel SL. Control of innate and adaptive immunity by the inflammasome. Microbes & Infection. 2012;14 14. Jia X, Li X, Shen Y, Miao J, Liu H, Li G, et al. MiR-16 regulates mouse peritoneal macrophage polarization and affects T-cell activation. Journal of cellular and molecular medicine. 2016;20 10:1898-907; doi: 10.1111/jcmm.12882. Zhu F, Liu X, Sun Z, Yu C, Liu L, Yang S, et al. Immune-Enhancing Effects of Taishan Pinus massoniana Pollen Polysaccharides on DNA Vaccine Expressing Bordetella avium ompA. Frontiers in microbiology. 2016;7:66; doi: 10.3389/fmicb.2016.00066. Song Y, Xu J, Wang X, Yang Y, Sun S. Regulation of host immune cells and cytokine production induced by Trichinella spiralis infection. Parasite. 2019;26:74. Lawrence CE, Paterson JCM, Higgins LM, Macdonald TT, Kennedy MW, Garside P. IL-4-regulated enteropathy in an intestinal nematode infection. European Journal of Immunology. 2010;28 9:2672-84. Chen C, Yang C, Tsai Y, Liaw C, Chang W, Hwang T. Ugonin U stimulates NLRP3 inflammasome activation and enhances inflammasome-mediated pathogen clearance. Redox biology. 2017;11:263-74; doi: 10.1016/j.redox.2016.12.018. Bruschi F, Chiumiento L. Trichinella inflammatory myopathy: host or parasite strategy? Parasites & vectors. 2011;4:42; doi: 10.1186/1756-3305-4-42. Lafuse W, Wozniak D, Rajaram M. Role of Cardiac Macrophages on Cardiac Inflammation, Fibrosis and Tissue Repair. Cells. 2020;10 1; doi: 10.3390/cells10010051. Alhallaf R, Agha Z, Miller CM, Robertson AAB, Sotillo J, Croese J, et al. The NLRP3 Inflammasome Suppresses Protective Immunity to Gastrointestinal Helminth Infection. Cell Reports. 2018;23 4:1085. Wang X, Gong P, Zhang X, Li S, Lu X, Zhao C, et al. Neospora caninumNLRP3 Inflammasome Participates in Host Response to Infection. Frontiers in immunology. 2018;9:1791; doi: 10.3389/fimmu.2018.01791. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1951416","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":128151190,"identity":"d847191d-3945-4165-9164-65a9cf4ea05a","order_by":0,"name":"Tian-Xu Pan","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tian-Xu","middleName":"","lastName":"Pan","suffix":""},{"id":128151191,"identity":"d44e8378-a00e-4332-8f3c-da361f971245","order_by":1,"name":"Hai-Bin Huang","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hai-Bin","middleName":"","lastName":"Huang","suffix":""},{"id":128151192,"identity":"695c489c-919e-4ccc-a1df-0a2aa1997d12","order_by":2,"name":"Guang-Xun Zhao","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guang-Xun","middleName":"","lastName":"Zhao","suffix":""},{"id":128151194,"identity":"5db52ab3-66f5-41e7-af75-0c459a3da009","order_by":3,"name":"Yu Quan","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Quan","suffix":""},{"id":128151196,"identity":"b328e76a-3c8a-43f9-9dbb-505e03aafb5b","order_by":4,"name":"Jun-Yi Li","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jun-Yi","middleName":"","lastName":"Li","suffix":""},{"id":128151197,"identity":"e6e646a3-4a38-4e48-99c2-507c8516b998","order_by":5,"name":"Ying Xue","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Xue","suffix":""},{"id":128151200,"identity":"6ba8d4dc-6add-41bb-be64-73f011624106","order_by":6,"name":"Hui-Nan Lu","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hui-Nan","middleName":"","lastName":"Lu","suffix":""},{"id":128151202,"identity":"25f84988-3054-4f30-989b-0a2b35e19044","order_by":7,"name":"Zhi-Yu Zhu","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhi-Yu","middleName":"","lastName":"Zhu","suffix":""},{"id":128151204,"identity":"d293ef26-df77-435f-96a5-8e7520c57d4b","order_by":8,"name":"Yue Wang","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Wang","suffix":""},{"id":128151205,"identity":"768e7e9c-4a51-4bf9-9d36-05bd3f45f27d","order_by":9,"name":"Chun-Wei Shi","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chun-Wei","middleName":"","lastName":"Shi","suffix":""},{"id":128151206,"identity":"91330123-8f01-4ac8-a957-66746584c732","order_by":10,"name":"Nan Wang","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Wang","suffix":""},{"id":128151207,"identity":"bf9a9745-5df2-4264-b765-399d81cfc014","order_by":11,"name":"Gui-Lian Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIie3OMUvDQBTA8RcOkuWo6yta40d4cnAECn6WBiHTUQpCKVj0oJC1n6VL50IgXaxdMzjoIgg3NIjQ0YtkvZpR8P5wRx7c7y4APt/fDS9CgOAVJz8TdSLcEkZI3Qlwu0KELoS2u+LjK094L9qVs4RuUh0t1gjzFzd5GmfDQW5/jI+zCuk21bycIpTvbrJRUvQbAkpawlKNSmKgCzfZm5acGTlBekx1bH4hlRJvdUPs5YBU2Ff4adKvjGTw3BAjEGkrcp7dJaPSTXp7Jerj9OEyXqrrT5zdD5ZRsaoOcze52kB4ztuBod3C5mvkBACxBlYf2yE4nDjp8/l8/7dv33tL1lJuQvsAAAAASUVORK5CYII=","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Gui-Lian","middleName":"","lastName":"Yang","suffix":""},{"id":128151208,"identity":"adc02ccb-b957-4574-805b-4c0c6f592663","order_by":12,"name":"Chun-Feng Wang","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chun-Feng","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2022-08-11 06:15:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1951416/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1951416/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":25269801,"identity":"069f395a-99c7-47a9-9209-3e694b5fcea1","added_by":"auto","created_at":"2022-08-16 15:38:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":171476,"visible":true,"origin":"","legend":"\u003cp\u003eThe content of the cytokines IL-1β, IL-18, IgG1 and IgG2 in the serum of various groups of mice at 7, 14, and 35 dpi were detected by ELISA. (A) The levels of IL-1β. (B) The levels of IL-18. (C) The levels of IgG1. (D) The levels of IgG2. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001. Statistical data are expressed as the mean value ± SD for 5 mice per group.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1951416/v1/1e51c8dfae6194b8699a1bf8.png"},{"id":25269803,"identity":"0785e011-7776-45d9-9040-5f621bc0216f","added_by":"auto","created_at":"2022-08-16 15:38:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":258275,"visible":true,"origin":"","legend":"\u003cp\u003eDetection of NLRP3 protein by western blot analysis for NLRP3 protein levels in the adult, migrating and cycle stages. Statistical data are expressed as the mean value ± SD for 3 mice per group.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1951416/v1/ac6636e3fc13e0cbc92b9b26.png"},{"id":25269802,"identity":"16d577e6-b8b7-4655-a24a-58e5723820e4","added_by":"auto","created_at":"2022-08-16 15:38:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1670505,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of IFN-γ and IL-4 in the SPL and MLN of mice of various groups was analysed by flow cytometry on Days 7, 14, and 35. (A) Gating strategy for the T-cell subset. (B) In the adult stage, (C) in the migrating stage, and (D) in the cycle stage. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001. Statistical data are expressed as the mean value ± SD for 5 mice per group.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-1951416/v1/0e2c910ef911d7bc8b7c0764.png"},{"id":25269804,"identity":"9e02e37e-5a91-46c6-a80a-a1c1334f8da0","added_by":"auto","created_at":"2022-08-16 15:38:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":201666,"visible":true,"origin":"","legend":"\u003cp\u003eAdult and muscle larvae burden\u003c/p\u003e\u003cp\u003e(A) Intestinal adult burden at Day 7 after infection with \u003cem\u003eT. spiralis. \u003c/em\u003e(B) Muscle larval burden in muscle at Day 35 after infection with \u003cem\u003eT. spiralis\u003c/em\u003e. Mice were infected with 400 muscle larvae. Statistical data are expressed as the mean value ± SD for 5 mice per group.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1951416/v1/6f5b50355077ce6ca84c9b58.png"},{"id":25270523,"identity":"862b6918-14f2-48a4-ae55-33f9a893fb8a","added_by":"auto","created_at":"2022-08-16 15:43:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3576061,"visible":true,"origin":"","legend":"\u003cp\u003eHistological analysis of \u003cem\u003eT. spiralis\u003c/em\u003e-infected small intestine and masseter muscle\u003c/p\u003e\u003cp\u003e(A) Representative images of H\u0026amp;E-stained small intestine. (B) Effect of NLRP3 on goblet cell hyperplasia of intestinal epithelium after infection with \u003cem\u003eT. spiralis\u003c/em\u003e. The number of goblet cells in ten VCU was counted. (C) Representative images of H\u0026amp;E-stained masseter muscle tissue. (D) Inflammatory cell infiltration of masseter muscle after infection with \u003cem\u003eT. spiralis\u003c/em\u003e. Twenty nonoverlapping representative fields of tissue were imaged under a light microscope using a 200X objective, and the amount of inflammation in a field was counted. The histological test revealed 5-10 sections per muscle tissue. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001. Statistical data are expressed as the mean value ± SD for 5 mice per group.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-1951416/v1/7d69c5273cdf027c00b7490a.png"},{"id":25269806,"identity":"99d53fd3-7754-4a33-b04d-3687c3932431","added_by":"auto","created_at":"2022-08-16 15:38:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3096033,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eT. spiralis\u003c/em\u003e proteins activate NLRP3 in BMDMs. \u003c/p\u003e\u003cp\u003eAll group of BMDMs was pretreated with LPS. (A) BMDMs were stimulated with adult \u003cem\u003eT. spiralis \u003c/em\u003eprotein. (B) BMDMs were stimulated with neonatal larval proteins of\u003cem\u003e T. spiralis\u003c/em\u003e. (C) BMDMs were stimulated with muscle larvae protein of \u003cem\u003eT. spiralis\u003c/em\u003e. Scale bars: 20 μm. (D, E) The contents of the cytokines IL-1β and IL-18 in BMDMs were measured by ELISA. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.001. All data shown are representative of three independent experiments.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-1951416/v1/4eb87349d3945647c6e4cb1b.png"},{"id":25270525,"identity":"b46394fe-c774-4ac0-8a18-787a82281004","added_by":"auto","created_at":"2022-08-16 15:43:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3181604,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1951416/v1/84e47625-a2f5-455e-a458-a67c3b5d97ba.pdf"},{"id":25270524,"identity":"c9687b3e-aa2d-49f8-907f-0d5682119815","added_by":"auto","created_at":"2022-08-16 15:43:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":581566,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1951416/v1/19e7714f-7603-494e-85c0-6b5ffe8909b2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"NLRP3 plays a key role in antihelminth immunity in the enteral and parenteral stages of Trichinella spiralis-infected mice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTrichinosis is an important food-borne parasitic zoonosis mainly caused by ingesting raw or undercooked meat infected with the encapsulated muscle larvae of \u003cem\u003eTrichinella spiralis\u0026nbsp;\u003c/em\u003e(\u003cem\u003eT. spiralis\u003c/em\u003e)\u003csub\u003e\u0026nbsp;\u003c/sub\u003e\u003csup\u003e[1, 2]\u003c/sup\u003e. \u003cem\u003eT. spiralis\u003c/em\u003e can infect a wide range of mammalian hosts, including humans and more than 150 mammalian species, which not only leads to great threat to human health worldwide but also represents an economic problem in the livestock industry\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[3]\u003c/sup\u003e. Although various countries around the world have taken measures to control and suppress the occurrence of the disease, it has not been effectively controlled because its immune response process is complex [4]. Therefore, a good understanding of the immune mechanisms may facilitate the discovery of approaches to control trichinosis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIncreasing attention has been given to defining the role of the host immune response in \u003cem\u003eT. spiralis\u003c/em\u003e infection. The innate immune system plays an important role in recognizing pathogens and triggering biological mechanisms to control infection and eliminate pathogens \u003csup\u003e[5]\u003c/sup\u003e. It is activated when pattern-recognition sensor proteins, such as Toll-like receptors (TLRs) or nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), detect the presence of pathogens, their products, or danger signals \u003csup\u003e[6]\u003c/sup\u003e. NLR inflammasomes play a central role in innate immunity \u003csup\u003e[7]\u003c/sup\u003e. The NOD-like receptor protein 3 inflammasome (NLRP3) is a protein complex consisting of the NLRP3 scaffold, ASC adaptor, and procaspase-1. NLRP3, which includes mature caspase-1, can promote the activation of cytokines such as IL-1\u0026beta; and IL-18 and promote the occurrence of an inflammatory response\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[8]\u003c/sup\u003e. NLRP3 inflammasome complexes are the most intensively studied and are activated by a broad range of stimuli. Recently, considerable evidence has suggested that NLRP3 activation is essential for the control of different parasitic infections, as it plays a protective role in reducing \u003cem\u003eToxoplasma gondii\u0026nbsp;\u003c/em\u003eand \u003cem\u003eNeospora caninum\u0026nbsp;\u003c/em\u003einfection loads \u003csup\u003e[9, 10]\u003c/sup\u003e. In addition, NLRP3 has been involved in the inhibition of eosinophil influx that results from the type 2 response to the parasite in mice infected with \u003cem\u003eNippostrongylus brasiliensis\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e[11]\u003c/sup\u003e. Despite the critical role of inflammasomes in the immune response to parasites, their functions in \u003cem\u003eT. spiralis\u0026nbsp;\u003c/em\u003echronic infection immunity appear less clear.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe parasitic process can be divided into adult, migrating, and encysted stages \u003csup\u003e[12]\u003c/sup\u003e. With the transition of larvae from the intestinal phase to the encysted stage in the host, the host\u0026rsquo;s immune type gradually changes from Th1 to Th2 and evokes concurrent inflammation-related factor changes \u003csup\u003e[13, 14]\u003c/sup\u003e. CD4\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells play a key role in parasite immunity and can differentiate into different subpopulations, including Th1, Th2, Th17 and Treg cells. \u003cem\u003eT. spiralis\u0026nbsp;\u003c/em\u003eand its secretory products can induce Th2 immune responses to suppress inflammatory responses \u003csup\u003e[15]\u003c/sup\u003e. Recent studies have shown that Th2 immune responses may help reduce tissue damage and strengthen tissue repair \u003csup\u003e[16]\u003c/sup\u003e. The changes in Th1 and Th2 immune types mainly rely on the secretion of cytokines\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[17]\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, to explore whether NLRP3 participates and plays a role during \u003cem\u003eT. spiralis\u0026nbsp;\u003c/em\u003einfection, we established a mouse model of \u003cem\u003eT. spiralis\u0026nbsp;\u003c/em\u003einfection and then intraperitoneally injected an NLRP3 inhibitor (MCC950) at different time points. The aim of the study was to detect the effects of NLRP3 on parasitism and immunity of \u003cem\u003eT. spiralis\u003c/em\u003e in adult, migrating, and encysted stages.\u003c/p\u003e"},{"header":"2.\tMaterials and methods","content":"\u003ch2\u003e2.1 Parasites and animals\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe \u003cem\u003eT. spiralis\u003c/em\u003e isolate ISS534 used in this study was maintained by serial infection of SD rats from the Animal Parasite Laboratory of Jilin Agricultural University. BALB/c mice (female, 4\u0026ndash;6 weeks old) and SD rats were purchased from Beijing Fukang Biotechnology company. All animal husbandry was maintained in Jilin Agricultural University Animal Experiment Center under the care of a professional breeder with a light/dark cycle of 12 hours (h) and with sterile food and water. Furthermore, all experiments in this study were performed in accordance with the Chinese Animal Management Ordinance. The animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Jilin Agricultural University.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.2 Isolation of \u003cem\u003eT. spiralis\u003c/em\u003e adults, new-born larvae and muscle larvae\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eMuscle larvae, adults and new-born larvae were obtained from rats as previously described \u003csup\u003e[18]\u003c/sup\u003e. In brief, BALB/c mice were orally inoculated with 300 muscle larvae of \u003cem\u003eT. spiralis\u003c/em\u003e and sacrificed 7 days post-infection (dpi). The intestine was exposed, and the entire small intestine was harvested and washed by flushing in sterile saline to remove any intestinal contents before the tissue was cut into 3-cm pieces. The small intestine sections were transferred to a separation screening cloth of a nematode larva separator (200 mesh), which was put in a small beaker containing sterile saline (preheated at 37 \u0026deg;C) and incubated at 37 \u0026deg;C for 4 h. \u003cem\u003eT. spiralis\u003c/em\u003e adults were collected from the bottom of the beaker and counted. The adults were incubated and washed in Roswell Park Memorial Institute-1640 medium (RPMI-1640, Gibco) with 20% foetal bovine serum (FBS, Gibco), 100 U/mL penicillin (Gibco), and 100 \u0026mu;g/mL streptomycin (Gibco) at 37 \u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. The culture solution was filtered with a 280-mesh sterile mesh so that the new-born larvae were collected. Muscle larvae of \u003cem\u003eT. spiralis\u003c/em\u003e were obtained by standard pepsin digestion (1% pepsin and 1% HCl at 37 \u0026deg;C for 2 h) to release larvae from the muscles of the rats infected at 35 days.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.3 Animal experimental design\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eForty-five healthy female BALB/c mice were randomly divided into three groups: TS+MCC950, TS and control. TS+MCC950 mice were intraperitoneally (IP) injected daily with the specific NLRP3 inhibitor MCC950 (10 mg/kg) after inoculation with \u003cem\u003eT. spiralis\u0026nbsp;\u003c/em\u003e(350 larvae per mouse), whereas the TS group mice were injected with an equal volume of PBS (i.p.). The mice in the control group received PBS solution. Five mice in each group were killed on the 7th, 14th and 35th days after infection with \u003cem\u003eT. spiralis\u003c/em\u003e by cervical dislocation. The blood samples were placed in a sterile EP tube for 1 h at 37 ℃ and stored overnight in a 4 ℃ refrigerator. Serum was obtained by centrifuging the EP tube at 3000 rpm for 10 min at 4 ℃. Single-cell suspensions of the spleen (SPL) or mesenteric lymph nodes (MLN) were analysed by flow cytometry as previously described\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[19]\u003c/sup\u003e. The small intestinal and masseter muscles were extracted as pathological sections to detect inflammation. Western blotting was utilized to detect changes in NLRP3 protein levels.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.4 Enzyme-linked immunosorbent assay\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe production of IgG1, IgG2, IL-1\u0026beta; and IL-18 in the serum of infected mice was determined in the control group, TS group, and TS+MCC950 group at 7, 14, and 35 dpi. Enzyme-linked immunosorbent assay (ELISA) kits (Abcam) were used.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.5 Western blot\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eSmall intestinal was pulverized using a mortar and pestle in liquid nitrogen and homogenized in ice-cold RIPA buffer (Thermo Scientific\u0026trade;). The protein concentration of the extracts was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific\u0026trade;). Total protein was resolved on 12% SDS\u0026ndash;PAGE gels and then transferred to polyvinylidene difluoride membranes (PVDF). The membrane was blocked in TBST (10 mM Tris HCl, 0.15 M NaCl containing 0.05% Tween 20) with 5% nonfat skim milk for 3 h at RT and incubated with the corresponding primary antibody at 4 \u0026deg;C overnight. After washing for 10 min with TBST three times, the membranes were incubated with secondary antibodies at room temperature for 1 hour and washed again. Bands were visualized with ECL chromogenic reagent. The primary antibodies used were anti-NLRP3 (Abcam, 1:2000 dilution) and anti-mouse \u0026beta;-actin ( Proteintech, China,1:2000 dilution). \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.6 Flow cytometry\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe relative ratios of IL-4 and INF-\u0026gamma;\u0026nbsp;on CD3\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e T cells in the spleen (SPL) and mesenteric lymph nodes (MLN) were analysed by flow cytometry. Single-cell suspensions of the SPL and mesenteric lymph nodes (MLN) of all groups of mice were prepared as previously described (Shi et al., 2016) at Days 7, 14, and 35 after infection. Briefly, single cells were counted and seeded in 48-well plates at 2.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well in 500 \u0026micro;l RPMI-1640 medium (100 U/ml penicillin, 100 \u0026mu;g/ml streptomycin, 10% heat-inactivated FBS) containing ionomycin (1 \u0026micro;g/mL), Golgi plug (10 \u0026micro;g/mL) and PMA (20 ng/mL) and were then incubated at 37 \u0026deg;C in a 5% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eincubator for 6 hours. After treatment, the cells were collected into EP tubes by centrifugation (2000 rcf for 5 min at 4 \u0026deg;C) and discarded from the supernatant.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHarvested cells were resuspended in 100 \u0026mu;L of cold PBS and incubated with various combinations of fluorochrome-labelled antibodies (CD3, CD4 along with isotype controls) according to the manufacturer\u0026apos;s instructions (BD Stemflow). Anti-mouse CD3 (PerCP-CY5.5, BD Biosciences) and anti-mouse CD4 (FITC, BD Biosciences) were used to label CD3\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells and CD4\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cells at 4 \u0026deg;C for 1 h in the dark. After staining for cell surface markers, the cells were fixed and permeabilized with a Cytofix/Cytoperm kit (BD Biosciences) following the manufacturer\u0026rsquo;s instructions and washed two times with 500 \u0026mu;l of cold BD Perm/Wash\u003csup\u003eTM\u0026nbsp;\u003c/sup\u003ebuffer (BD Biosciences). Intracellular staining was required to determine the percentage of IL-4\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eand INF-\u0026gamma;\u003csup\u003e+\u0026nbsp;\u003c/sup\u003ecells. Next, treatment with anti-mouse IL-4 (APC, BD Biosciences) and anti-mouse INF-\u0026gamma; (PE, BD Biosciences) was performed at 4 \u0026deg;C for 1 h in the dark. Following this, all stained cells were washed three times with cold PBS to remove unbound antibodies, suspended in 300 \u0026micro;l of PBS, and then examined using a LSR Fortessa\u003csup\u003eTM\u003c/sup\u003e (BD Biosciences). Data were analysed with Flow Jo software (ver 7.6.1, Tree Star Inc., USA).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.7 Histopathological evaluation and immunofluorescence\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eAt 7, 14, and 35 dpi, lingual muscle and small intestine samples were obtained from each group and fixed in 4% formalin for 48 h. The tissue was subjected to washing, dehydrated in gradual ethanol (70-100%), made transparent with xylene, processed, embedded with paraffin wax, sliced (3 \u0026mu;m), placed in warm water (42 ℃) for spreading, collected using slides, baked, and prepared into paraffin tissue sections. The tissue slices were baked at 80 ℃ for 1 h and placed in xylene for 8 min twice. For histopathological evaluation, the sections were stained with haematoxylin and eosin and visualized with a microscope (Leica). The number of goblet cells per ten randomly selected villus\u0026ndash;crypt units (VCU) was determined by microscopy from at least two sections per animal as previously described\u003csup\u003e[20]\u003c/sup\u003e. Twenty nonoverlapping representative fields of the tissue were examined microscopically using a 400X objective, and the number of inflammatory cells infiltrating the masseter muscle was observed\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[21]\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003e2.8 Bone marrow-derived macrophage (BMDM) isolation, culture and stimulation\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eMouse bone marrow-derived macrophages (BMDMs) were generated from five-week-old BALB/c mice as previously described \u003csup\u003e[22]\u003c/sup\u003e. In the in vitro experiments, the BMDMs were randomly divided into the PBS negative control, ATP positive control, adult protein group, neonatal larvae protein group, muscle larvae protein group, and MCC950 group. ELISA was used to detect changes in IL-18 and IL-1\u0026beta; levels, and indirect immunofluorescence was used to detect the expression of NLRP3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBMDMs were primed with LPS for 3 h and then treated with DMSO (Beyotime) or DMSO+MCC950 (7.5 nm/mL) for 30 min, followed by stimulation with \u003cem\u003eT. spiralis\u003c/em\u003e protein (50 mg/mL) of new-born larvae, muscle larvae and adult stages for 6 hours. Protein extraction was performed as previously described (G\u0026oacute;mez-Morales, et al., 2018). After treatment, the BMDMs were washed with PBS, fixed with 80% cold acetone for 30 min at RT and then washed three times with PBS. After blocking with 3% BSA in PBS for 30 min at RT, the BMDMs were incubated with an antibody against NLRP3 (Sigma) for 1 h at 4 \u0026deg;C. After washing with PBST, the BMDMs were incubated with secondary antibody (Alexa Fluor\u0026reg; 488 (green). Proteintech) for 40 min at RT. After washing, nuclei were stained with DAPI (blue. Sigma) for 10 min in the dark. Imaging analysis was performed using a fluorescence microscope.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e2.9 Statistical analysis\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eData were statistically analysed by using GraphPad Prism 9.0 software. The Shapiro\u0026ndash;Wilk test was used to analyse normality. For comparisons of only two groups, data were analysed using Student\u0026rsquo;s t test, while for comparisons of three or more groups, we performed one-way ANOVA with Bonferroni\u0026rsquo;s multiple comparison test as indicated. The Mann\u0026ndash;Whitney U test was used for nonnormally distributed data. The statistically significant differences between the means are indicated by asterisks (*\u003cem\u003eP\u0026lt;\u003c/em\u003e 0.05, **\u003cem\u003eP\u0026lt;\u003c/em\u003e 0.01, ***\u003cem\u003eP\u0026lt;\u003c/em\u003e 0.001). Statistical data are expressed as the mean value \u0026plusmn; SD.\u003c/p\u003e"},{"header":"3. Results","content":"\u003ch2\u003e3.1 NLRP3 increases the Th1 response during \u003cem\u003eT. spiralis\u003c/em\u003e infection\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eTo determine whether the NLRP3 inflammatory body is involved in \u003cem\u003eT. spiralis\u003c/em\u003e infection. The contents of the cytokines IL-1\u0026beta; and IL-18 and host immune types IgG1 and IgG2 were detected by ELISA kits in the adult stage (7 D), migrating stage (14 D), and encysted stage (35 D), as shown in Fig. 1. With respect to IL-1\u0026beta;, the TS+MCC950 group showed a significant decrease compared with the TS group in the adult, migrating and encysted stages [7D: ANOVA, F \u003csub\u003e(2.12)\u0026nbsp;\u003c/sub\u003e= 70.650, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; 14D: ANOVA, F \u003csub\u003e(2.12)\u003c/sub\u003e = 23.840, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; 35D : ANOVA, F\u003csub\u003e(2.12)\u0026nbsp;\u003c/sub\u003e= 10.220, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e= 0.0025] (Fig. 1A). IL-18 excretion was also significantly decreased in the adult stage and the migrating stage [7D : ANOVA, F \u003csub\u003e(2.12)\u003c/sub\u003e = 15.520, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; 14D: ANOVA, F\u003csub\u003e\u0026nbsp;(2.12)\u003c/sub\u003e = 88.160, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001], but was not significantly different in encysted stages[35D: ANOVA, F\u003cem\u003e(2.12)\u0026nbsp;\u003c/em\u003e= 4.733, \u003cem\u003eP\u003c/em\u003e =0.229] (Fig. 1, B). For IgG1, the excretion was not significantly different in the adult stage[7D: ANOVA, F \u003csub\u003e(2.12) \u0026nbsp;\u003c/sub\u003e= 7.717, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05], and the TS+MCC950 group showed a significant decrease in the migrating stage compared with the TS group[14D: ANOVA, F \u003csub\u003e(2.12)\u0026nbsp;\u003c/sub\u003e= 8.462, \u003cem\u003ep =\u0026nbsp;\u003c/em\u003e0.0076] but was increased significantly in the encysted stage[35D: ANOVA, F(2.12)=163.2, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001](Fig. 1C). IgG2 was significantly decreased in the migrating and adult stages [7D: ANOVA, F \u003csub\u003e(2.12)\u0026nbsp;\u003c/sub\u003e= 147.9, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; 14D: ANOVA, F \u003csub\u003e(2.12)\u0026nbsp;\u003c/sub\u003e= 64.69, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001], with no significant differences in the encysted stages [35D: ANOVA, F \u003csub\u003e(2.12)\u003c/sub\u003e = 2.878, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026gt; 0.05] (Fig. 1D). These results suggest that NLRP3 participates in \u003cem\u003eT. spiralis\u003c/em\u003e infection and that inhibiting NLRP3 promotes the host Th1-type immune response in the adult stage and the migrating stage and inhibits the Th2-type immune response in the encysted stage.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.2 \u003cem\u003eT. spiralis\u003c/em\u003e infection promotes the expression of NLRP3 in the small intestine\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe protein expression of NLRP3 in the intestine was detected by western blotting (Fig. 2). The results showed that the expression of NLRP3 was significantly elevated in the TS group compared with the TS+MCC950 group or the control group in the adult stage (7D), and the expression levels of NLRP3 had no evident change in the different groups in the migrating stage (14D). In the encysted stage, the expression of NLRP3 in the small intestine in the TS group was significantly higher than that in the control group and the TS+MCC950 group. This illustrated that T. spiralis could promote the expression of NLRP3 in the adult and encysted stages.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.3 Effects of NLRP3 on IFN-\u0026gamma; and IL-4 during\u003cem\u003e\u0026nbsp;T. spiralis\u003c/em\u003e infection\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eWe detected the impact of NLRP3 expression on changes in the expression of proinflammatory factors (INF-\u0026gamma;) and anti-inflammatory factors (IL-4). In adults, flow cytometry assays showed that the INF-\u0026gamma; expression levels in the MLN and SPL in the TS+MCC950 group were significantly decreased compared with those in the TS group[MLN:\u0026nbsp;ANOVA, F\u003csub\u003e\u0026nbsp;(2.12)\u003c/sub\u003e = 96.69, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001]; SPL: ANOVA, F \u003csub\u003e(2.12)\u003c/sub\u003e = 372.8, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001] , however, the IL-4 expression levels in the MLN were increased significantly [MLN: ANOVA, F\u003csub\u003e\u0026nbsp;(2.12)\u003c/sub\u003e = 138.4, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001] (Fig. 3B). In the migrating stage, there was no difference in INF-\u0026gamma; expression among the three groups, while the levels of IL-4 increased significantly in the SPL and MLN of the TS+MCC950 group compared with the TS group [MLN: ANOVA, F \u003csub\u003e(2.12\u003c/sub\u003e) = 40.57, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001]; SPL: ANOVA, F (2.12) = 123.6, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001] (Fig. 3C). In the encysted stage, compared with the TS group, there was a significant decrease in the expression levels of INF-\u0026gamma; in the MLN of the TS+MCC950 group [MLN: ANOVA, F \u003csub\u003e(2.12)\u003c/sub\u003e = 19.87, \u003cem\u003eP\u003c/em\u003e = 0.006], and the expression of INF-\u0026gamma; levels tended to gradually decrease in the SPL, with no statistical significance (Fig. 3D). There was no significant difference in the expression of IL-4 between the two infection groups.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.4 NLRP3 enhances the burden of intestinal worms and muscle larvae during \u003cem\u003eT. spiralis\u003c/em\u003e infection\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eFive mice were randomly selected from the TS group and the TS+MCC950 group in the adult stage (7 D) and encysted stage (35 D), and the number of worms was obtained by separating muscle larvae and adults of \u003cem\u003eT. spiralis\u003c/em\u003e. The results showed that the number of worms in the TS+MCC950 group was significantly reduced compared with that in the TS group [t \u003csub\u003e(8)\u003c/sub\u003e = 13.47, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001](Fig. 4A), and the number of muscle larvae was significantly reduced [t\u003csub\u003e( 8)\u003c/sub\u003e = 8.166, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001] (Fig. 4B). It was concluded that NLRP3 could enhance the survival of \u003cem\u003eT. spiralis\u003c/em\u003e in the host.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.5 Effects of NLRP3 on the pathological damage to intestinal and masseter muscle in mice during \u003cem\u003eT. spiralis\u003c/em\u003e infection\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eTo investigate how NLRP3 enhances the burden of intestinal worms and muscle larvae during \u003cem\u003eT. spiralis\u0026nbsp;\u003c/em\u003einfection, we observed goblet cell hyperplasia of the intestinal epithelium and inflammatory cell infiltration of masseter muscle, which are characteristic of intestinal nematode infection. The number of intestinal goblet cells increased on Days 7 and 14 in \u003cem\u003eT. spiralis-\u003c/em\u003einfected mice compared with noninfected mice.\u0026nbsp;Compared with mice in the\u0026nbsp;TS group,\u0026nbsp;mice\u0026nbsp;in\u0026nbsp;the TS+MCC950 group showed goblet cell hyperplasia in\u0026nbsp;the\u0026nbsp;intestinal mucosa in\u0026nbsp;the\u0026nbsp;adult and migrating\u0026nbsp;stages\u0026nbsp;[7D:\u0026nbsp;ANOVA, F\u003csub\u003e\u0026nbsp;(2.12)\u003c/sub\u003e = 120, \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001;14D: ANOVA, F\u003csub\u003e(2.12)\u0026nbsp;\u003c/sub\u003e= 112.6, \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001] (Fig. 5B).The mucosal epithelium was integral, and the morphology of the villi was normal. There was no significant difference in the encysted stage (Fig. 5A). Histological observation of H\u0026amp;E-stained \u003cem\u003eT. spiralis-\u003c/em\u003einfected masseter muscle clearly showed inflammatory cell infiltration around the encystation (Fig. 5C). The level of inflammatory cells was significantly increased sharply in the migrating and encysted stages. In addition, the TS+MCC950 group displayed a significant reduction in inflammatory cellular infiltration and parasite cysts in the muscle when compared with the TS group [14D: ANOVA, F \u003csub\u003e(2.12)\u003c/sub\u003e = 25.96, \u003cem\u003eP =\u0026nbsp;\u003c/em\u003e0.0025; 35D: ANOVA, F\u003csub\u003e(2.12)\u003c/sub\u003e = 107.7, \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.0012] (Fig. 5D). Suppressing NLRP3 could increase goblet cells in the intestine and ameliorate inflammatory cell infiltration in the masseter muscle during \u003cem\u003eT. spiralis\u003c/em\u003e infection.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.6 \u003cem\u003eT. spiralis\u003c/em\u003e protein activates NLRP3 in BMDMs\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eTo determine the role of\u0026nbsp;proteins\u0026nbsp;of three different stages of\u003cem\u003e\u0026nbsp;T. spiralis\u003c/em\u003e associated with the activation of the NLRP3 inflammasome, in vitro culture of mouse BMDMs was performed, and all groups of BMDMs were pretreated with LPS. The results showed that the activation of NLRP3 in the stimulation group was higher than that in the PBS group. Moreover, compared with the \u003cem\u003eT. spiralis\u003c/em\u003e protein stimulation group (adult protein, neonatal larvae protein, and muscle larvae protein), there was a significant decrease in the activation of NLRP3 in the MCC950 group (Fig. 5A). The secretion of IL-18 and IL-1\u0026beta; in the \u003cem\u003eT. spiralis\u003c/em\u003e protein stimulation group was lower than that in the positive control group but significantly higher than that in the MCC950 group [IL-18:( adult protein: ANOVA, F \u003csub\u003e(3.8)\u003c/sub\u003e = 295.4,\u003cem\u003e\u0026nbsp;P \u0026lt;\u003c/em\u003e 0.001; neonatal larvae protein: ANOVA, F\u003csub\u003e\u0026nbsp;(3.8)\u003c/sub\u003e = 357.1, \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001; muscle larvae protein: ANOVA, F \u003csub\u003e(3.8)\u0026nbsp;\u003c/sub\u003e= 252, \u003cem\u003eP\u0026lt;\u003c/em\u003e 0.001); IL-1\u0026beta;:(adult protein: ANOVA, F\u003csub\u003e(3.8)\u003c/sub\u003e = 219.4,\u003cem\u003e\u0026nbsp;P \u0026lt;\u003c/em\u003e 0.001; neonatal larvae protein: ANOVA, F\u003csub\u003e\u0026nbsp;(3.8)\u003c/sub\u003e =248.0, \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001; muscle larvae protein: ANOVA, F \u003csub\u003e(3.8)\u003c/sub\u003e=316.1, \u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001)] (Fig. 5D, E). these results suggest that different life cycle stages of \u003cem\u003eT. spiralis\u0026nbsp;\u003c/em\u003eproteins promote the activation of NLRP3 and that NLRP3 promotes the secretion of IL-18 and IL-1\u0026beta; in BMDMs.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eParasitic helminth infestation is the most common chronic disease among humans and animals. At present, due to \u003cem\u003ethe complexity\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eof the life cycle of worms, the mechanisms by which antigens induce Th2-type immune responses and initiate inflammation are not entirely clear, and no effective immunological approach is used to control worms \u003csup\u003e[23, 24]\u003c/sup\u003e. Although there is evidence that inflammasome signalling and inflammasome-dependent cytokines are important for host defence against \u003cem\u003eT. spiralis,\u003c/em\u003e further studies are needed to establish how the inflammasome affects adaptive immune responses in the context of \u003cem\u003eT. spiralis\u0026nbsp;\u003c/em\u003einfections \u003csup\u003e[25]\u003c/sup\u003e. In the present study, we identified that NLRP3 is involved in both innate and adaptive immune responses and the conversion of immune response patterns by regulating the secretion of IL-1\u0026beta; and IL-18 during \u003cem\u003eT. spiralis\u0026nbsp;\u003c/em\u003einfection. Additionally, we first demonstrated that NLRP3 in macrophages can be activated by \u003cem\u003eT. spiralis\u0026nbsp;\u003c/em\u003eproteins and promotes IL-1\u0026beta; and IL-18 release. MCC950 inhibits the activation of NLRP3 to speed up Th2-type immune type-mediated \u003cem\u003eT. spiralis\u003c/em\u003e clearance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNLRP3 is associated with the Th1 immune response and mediates host protection against pathogen invasions \u003csup\u003e[26]\u003c/sup\u003e. Recent studies have also demonstrated that NLRP3 plays an important role in the promotion of Th2 immune responses \u003csup\u003e[27]\u003c/sup\u003e. We confirmed that NLRP3 promotes a Th1 immune response during \u003cem\u003eT. spiralis\u003c/em\u003e infections, and this effect may be caused by cytokine secretion of\u003cem\u003e\u0026nbsp;\u003c/em\u003eIL-1\u0026beta; and IL-18. IL-18 is one of the best-characterized inflammasome-dependent cytokines whose maturation requires cleavage by caspase-1 from its inactive intracellular precursor pro-IL-18, which can induce IFN-\u0026gamma; production in Th1-type cells and can promote T-cell proliferation \u003csup\u003e[28, 29]\u003c/sup\u003e. Our data support an important role for NLRP3-dependent IL-18 in regulating immunity and inflammation following \u003cem\u003eT. spiralis\u0026nbsp;\u003c/em\u003eclearance. Moreover, we demonstrated that IL-1\u0026beta; was similarly regulated by NLRP3 during \u003cem\u003eT. spiralis\u003c/em\u003e infection. NLRP3 activation leading to IL-1\u0026beta; production is critical for the induction of a Th2 response\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[30]\u003c/sup\u003e. IL-1\u0026beta;-deficient mice are susceptible to chronic\u003cem\u003e\u0026nbsp;Trichuris muris\u0026nbsp;\u003c/em\u003einfection, and the inability to expel the worms is associated with a defect in the development of a Th2 response, suggesting the critical role of IL-1\u0026beta; in regulating the Th2 response during gastrointestinal nematode infection\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[25, 31]\u003c/sup\u003e. We demonstrated that the decrease in the expression of IL-18 and IL-1\u0026beta; may lead to an increased Th2-type response and weakening of the Th1-type response when NLRP3 is inhibited after \u003cem\u003eT. spiralis\u003c/em\u003e infection. Similar results were obtained in a study using dendritic cells of NLRP3\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice \u003csup\u003e[25]\u003c/sup\u003e. Inflammasomes have a regulatory role in the infection of early innate responses and can promote the maturation and production of inflammatory cytokines. There is a large body of evidence related to the involvement of inflammasomes in the innate immune response \u003csup\u003e[32]\u003c/sup\u003e. We demonstrated that NLRP3 induced an inflammatory response and mediates protective immunity to \u003cem\u003eT. spiralis\u003c/em\u003e infection, possibly via adaptive immune responses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTh1 subset cells mediate cellular immunity by secreting IFN-\u0026gamma;, and Th2 subset cells mediate cellular immunity by secreting IL-4 \u003csup\u003e[33, 34]\u003c/sup\u003e. In the present study, NLRP3-inhibited mice displayed decreased IFN-\u0026gamma; levels, which may promote a shift in the Th1/Th2 balance towards Th2-dominant immunity during the early stage of infection (adult stage, migrating stage). The IFN-\u0026gamma; levels changed with time since infection, leading to low levels of IL-4. In contrast, \u003cem\u003eT. spiralis\u003c/em\u003e infection shifted the Th1/Th2 balance towards a dominant Th1 immune response during the encysted stage. IFN-\u0026gamma; can mediate and regulate immunity and invasiveness to resist \u003cem\u003eT. spiralis\u003c/em\u003e infection, and parasite expulsion is IL-4 dependent [35, 36]. Based on the above, inhibition or deletion of NLRP3 expression could provide increased immunity to \u003cem\u003eT. spiralis\u003c/em\u003e infection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that NLRP3 plays a key role in pathogenic bacteria-induced progression of acute inflammation \u003csup\u003e[37]\u003c/sup\u003e. Reports have demonstrated that NLRP3 is involved in the inhibition of eosinophil influx to the parasite in mice infected with \u003cem\u003eNippostrongylus brasiliensis\u003c/em\u003e, and IL-1\u0026beta; secretion is mediated by P2X7R in small intestinal epithelial cells in response to \u003cem\u003eToxoplasma gondii\u0026nbsp;\u003c/em\u003einfection\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[6, 11]\u003c/sup\u003e. \u003cem\u003eT. spiralis\u003c/em\u003e infections in host intestinal acute mucosal inflammation, after larvae enter the skeletal muscle tissue, induce a relevant inflammatory reaction that is responsible for myositis and can cause systemic inflammatory manifestations all over the body before entering the striated muscles \u003csup\u003e[38]\u003c/sup\u003e. In this study, we identified that \u003cem\u003eT. spiralis\u003c/em\u003e enhances NLRP3-dependent IL-18 and IL-1\u0026beta; secretion, which may contribute to the inflammatory cell infiltration and histological changes observed in the muscle and intestinal tissue.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the acute phase of the infection, the parasite load dictates the magnitude of the inflammatory response and tissue damage \u003csup\u003e[39]\u003c/sup\u003e. In this study, a significant reduction in adult worm burden and muscle larvae burden at 7 and 35 days postinfection was observed in mice treated with the specific NLRP3 inhibitor MCC950. It has also been found to be effective in the treatment of \u003cem\u003eTrichuris muris\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e[40]\u003c/sup\u003e. NLRP3 inhibition may influence the functions of nonhaematopoietic cells in the intestine that promote worm expulsion, including epithelial cell turnover, goblet cell expansion, mucus production and smooth muscle hypercontractility \u003csup\u003e[40]\u003c/sup\u003e. This is consistent with the increase in goblet cells observed in the absence of NLRP3 in mice during \u003cem\u003eT. spiralis\u003c/em\u003e infection in this study. It is conceivable that targeting the NLRP3 pathway in \u003cem\u003eT. spiralis\u003c/em\u003e-infected hosts using MCC950 may be a rational approach for lowering worm burdens. However, people living in helminth endemic regions are often exposed to coinfections with other parasites, and previous studies have demonstrated a protective role for NLRP3 in these infection models. Some studies have shown that the absence of NLRP3 results in increased parasite burden, such as with \u003cem\u003eToxoplasma gondii\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Neospora caninum\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e[10, 41]\u003c/sup\u003e. Further studies are necessary to assess the impact of such therapeutic strategies on other preventive and curative interventions.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eTaken together, these findings demonstrate that NLRP3 regulates immunity and inflammation in \u003cem\u003eT. spiralis\u003c/em\u003e infections and that targeted inhibition of NLRP3 enhanced the Th2 response and accelerated \u003cem\u003eT. spiralis\u003c/em\u003e expulsion.\u0026nbsp;Therefore, NLRP3 is an important target for controlling\u003cem\u003e\u0026nbsp;T. spiralis\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eNLRP3: NOD-like receptor protein 3 inflammasome; Th: CD4+\u0026thinsp;T helper cells; ELISA: Enzyme-linked immunosorbent assay; FBS: Fetal bovine serum; IL: Interleukin; IFN-\u0026gamma;: Interferon gamma; HRP: Horseradish peroxidase; PBS: Phosphate-buffered saline; PVDF: Polyvinyl difluoride; SDS-PAGE: Sodium dodecyl sulphate\u0026ndash;polyacrylamide gel electrophoresis; BMDMs: Mouse bone marrow-derived macrophages.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the teachers of Jilin Agricultural University. A special thanks to Dan-Wang for the precious contribution with Enzyme-linked immunosorbent assay.\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32072888, 31941018, and U21A20261) and the Science and Technology Development Program of Jilin Province (20180201040NY, 20190301042NY, and YDZJ202102CXJD029). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the conclusions of this article are included within the article and the additional files. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGui-Lian Yang and Chun-Feng Wang conceived and designed the experiments. Tian-Xu Pan, Hai-Bin Huang, Yu Quan, Jun-Yi Li, Ying Xue, Hui-Nan Lu, Zhi-Yu Zhu, Yue Wang, Chun-Wei Shi, and Nan Wang carried out the research and performed data processing and analyses. Tian-Xu Pan, Guang-Xun Zhao, and Hai-Bin Huang performed the statistical analysis. Tian-Xu Pan drafted the manuscript with input from all other authors. All authors have read and approved the final manuscript. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experimental procedures were performed based on the regulations of the Administration of Affairs Concerning Experimental Animals in China. All animal experiments in this study were approved by the constitution of the Experimental Animal Welfare and Ethics Committee of Jilin Agricultural University. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors agreed to publish the manuscript \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no financial or other competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGu Y, Sun X, Huang J, Zhan B, Zhu X. Trichinella spiralisA Multiple Antigen Peptide Vaccine Containing CD4 T Cell Epitopes Enhances Humoral Immunity against Infection in Mice. Journal of immunology research.\u003cem\u003e \u003c/em\u003e2020;2020:2074803; doi: 10.1155/2020/2074803.\u003c/li\u003e\n\u003cli\u003eLong S, Wang Z, Liu R, Liu L, Li L, Jiang P, et al. Molecular identification of Trichinella spiralis nudix hydrolase and its induced protective immunity against trichinellosis in BALB/c mice. Parasites \u0026amp; vectors.\u003cem\u003e \u003c/em\u003e2014;7:600; doi: 10.1186/s13071-014-0600-9.\u003c/li\u003e\n\u003cli\u003eWang Z, Hao C, Huang J, Zhuang Q, Zhan B, Zhu X. Mapping of the complement C1q binding site on Trichinella spiralis paramyosin. Parasites \u0026amp; vectors.\u003cem\u003e \u003c/em\u003e2018;11 1:666; doi: 10.1186/s13071-018-3258-x.\u003c/li\u003e\n\u003cli\u003ePratesi F, Bongiorni F, Kociecka W, Migliorini P, Bruschi F. Heart- and skeletal muscle-specific antigens recognized by trichinellosis patient sera. Parasite immunology.\u003cem\u003e \u003c/em\u003e2006;28 9:447-51; doi: 10.1111/j.1365-3024.2006.00889.x.\u003c/li\u003e\n\u003cli\u003eChu J, Shi G, Fan Y, Choi I, Cha G, Zhou Y, et al. Toxoplasma gondiiProduction of IL-1\u0026beta; and Inflammasome with Up-Regulated Expressions of NOD-Like Receptor Related Genes in -Infected THP-1 Macrophages. The Korean journal of parasitology.\u003cem\u003e \u003c/em\u003e2016;54 6:711-7; doi: 10.3347/kjp.2016.54.6.711.\u003c/li\u003e\n\u003cli\u003eQuan J, Huang R, Wang Z, Huang S, Choi I, Zhou Y, et al. P2X7 receptor mediates NLRP3-dependent IL-1\u0026beta; secretion and parasite proliferation in Toxoplasma gondii-infected human small intestinal epithelial cells. Parasites \u0026amp; vectors.\u003cem\u003e \u003c/em\u003e2018;11 1:1; doi: 10.1186/s13071-017-2573-y.\u003c/li\u003e\n\u003cli\u003ede Almeida R, Clendenon S, Richards W, Boedigheimer M, Damore M, Rossetti S, et al. Transcriptome analysis reveals manifold mechanisms of cyst development in ADPKD. Human genomics.\u003cem\u003e \u003c/em\u003e2016;10 1:37; doi: 10.1186/s40246-016-0095-x.\u003c/li\u003e\n\u003cli\u003eDu RH, Tan J, Sun XY, Lu M, Ding JH, Hu G. Fluoxetine Inhibits NLRP3 Inflammasome Activation: Implication in Depression. International Journal of Neuropsychopharmacology.\u003cem\u003e \u003c/em\u003e2016:pyw037.\u003c/li\u003e\n\u003cli\u003eWang X, Gong P, Zhang X, Wang J, Tai\u0026hellip; L. NLRP3 inflammasome activation in murine macrophages caused by Neospora caninum infection. Parasites \u0026amp; Vectors.\u003cem\u003e \u003c/em\u003e2017;10 1.\u003c/li\u003e\n\u003cli\u003eAbreu MSAC, Coutinho A-d-SCL, Prado RT, Costa RGD, Maria B, Sim?Es ZD, et al. The P2X7 Receptor Mediates Toxoplasma gondii Control in Macrophages through Canonical NLRP3 Inflammasome Activation and Reactive Oxygen Species Production. Frontiers in Immunology.\u003cem\u003e \u003c/em\u003e2017;8.\u003c/li\u003e\n\u003cli\u003eChenery A, Alhallaf R, Agha Z, Ajendra J, Parkinson J, Cooper M, et al. Inflammasome-Independent Role for NLRP3 in Controlling Innate Antihelminth Immunity and Tissue Repair in the Lung. Journal of immunology (Baltimore, Md : 1950).\u003cem\u003e \u003c/em\u003e2019;203 10:2724-34; doi: 10.4049/jimmunol.1900640.\u003c/li\u003e\n\u003cli\u003eAranzamendi C, Sofronic-Milosavljevic L, Pinelli E. Helminths: Immunoregulation and Inflammatory Diseases-Which Side Are Trichinella spp. and Toxocara spp. on? Journal of parasitology research.\u003cem\u003e \u003c/em\u003e2013;2013:329438; doi: 10.1155/2013/329438.\u003c/li\u003e\n\u003cli\u003eSofronic-Milosavljevic L, Ilic N, Pinelli E, Gruden-Movsesijan A. Secretory Products of Trichinella spiralis Muscle Larvae and Immunomodulation: Implication for Autoimmune Diseases, Allergies, and Malignancies. Journal of immunology research.\u003cem\u003e \u003c/em\u003e2015;2015:523875; doi: 10.1155/2015/523875.\u003c/li\u003e\n\u003cli\u003eMosmann TR. Cytokine secretion patterns and cross-regulation of T cell subsets. Immunologic Research.\u003cem\u003e \u003c/em\u003e1991;10 3-4:183-8.\u003c/li\u003e\n\u003cli\u003eAlisa, Gruden-Movsesijan, Ljiljana, Sofronic-Milosavljevic, Natasa, Ilic, et al. Secretory Products of Trichinella spiralis Muscle Larvae and Immunomodulation: Implication for Autoimmune Diseases, Allergies, and Malignancies. Clinical \u0026amp; Developmental Immunology.\u003cem\u003e \u003c/em\u003e2015.\u003c/li\u003e\n\u003cli\u003eMaizels RM, Gause WC. How helminths go viral. Science.\u003cem\u003e \u003c/em\u003e2014;345 6196:517-8.\u003c/li\u003e\n\u003cli\u003eTymoshok NO, Lazarenko LM, Bubnov RV, Shynkarenko LN, Babenko LP, Mokrozub VV, et al. New aspects the regulation of immune response through balance Th1/Th2 cytokines. EPMA Journal, The.\u003cem\u003e \u003c/em\u003e2014;5 Suppl 1:A134.\u003c/li\u003e\n\u003cli\u003eDespommier D, Campbell W, Blair L. The in vivo and in vitro analysis of immunity to Trichinella spiralis in mice and rats. Parasitology.\u003cem\u003e \u003c/em\u003e1977;74 1:109-19; doi: 10.1017/s0031182000047570.\u003c/li\u003e\n\u003cli\u003eHuang H, Yang W, Hu J, Jiang Y, Wang J, Shi C, et al. Antitumour metastasis and the antiangiogenic and antitumour effects of a Eimeria stiedae soluble protein. Parasite immunology.\u003cem\u003e \u003c/em\u003e2021:e12825; doi: 10.1111/pim.12825.\u003c/li\u003e\n\u003cli\u003eYu YR, Liu XC, Zhang JS, Ji\u0026hellip; C. Taurine drinking attenuates the burden of intestinal adult worms and muscle larvae in mice with Trichinella spiralis infection. Parasitology Research.\u003cem\u003e \u003c/em\u003e2013;112 10:3457-63.\u003c/li\u003e\n\u003cli\u003eMi-Kyung P, Yun-Jeong K, Jin-Ok J, Kyung-Wan B, Hak-Sun Y, Hyun CY, et al. Effect of Muscle Strength by Trichinella spiralis Infection during Chronic Phase. International journal of medical sciences.\u003cem\u003e \u003c/em\u003e2018;15 8:802-7.\u003c/li\u003e\n\u003cli\u003eSugata R, Sebastian S, Erik A, Tanvir A, Parihar SP, Mumin O, et al. Redefining the transcriptional regulatory dynamics of classically and alternatively activated macrophages by deepCAGE transcriptomics. Nucleic Acids Research.\u003cem\u003e \u003c/em\u003e2015;43 14:6969-82.\u003c/li\u003e\n\u003cli\u003eGu Y, Wei J, Yang J, Huang J, Yang X, Zhu X. Protective immunity against Trichinella spiralis infection induced by a multi-epitope vaccine in a murine model. PloS one.\u003cem\u003e \u003c/em\u003e2013;8 10:e77238; doi: 10.1371/journal.pone.0077238.\u003c/li\u003e\n\u003cli\u003evan der Werf N, Redpath S, Azuma M, Yagita H, Taylor M. Th2 cell-intrinsic hypo-responsiveness determines susceptibility to helminth infection. PLoS pathogens.\u003cem\u003e \u003c/em\u003e2013;9 3:e1003215; doi: 10.1371/journal.ppat.1003215.\u003c/li\u003e\n\u003cli\u003eJin X, Bai X, Yang Y, Ding J, Shi H, Fu B, et al. NLRP3 played a role in Trichinella spiralis-triggered Th2 and regulatory T cells response. Veterinary research.\u003cem\u003e \u003c/em\u003e2020;51 1:107; doi: 10.1186/s13567-020-00829-2.\u003c/li\u003e\n\u003cli\u003eSilva G, Costa R, Silveira T, Caetano B, Horta C, Gutierrez F, et al. Apoptosis-associated speck-like protein containing a caspase recruitment domain inflammasomes mediate IL-1\u0026beta; response and host resistance to Trypanosoma cruzi infection. Journal of immunology (Baltimore, Md : 1950).\u003cem\u003e \u003c/em\u003e2013;191 6:3373-83; doi: 10.4049/jimmunol.1203293.\u003c/li\u003e\n\u003cli\u003eBruchard M, Reb\u0026eacute; C, Derang\u0026egrave;re V, Togb\u0026eacute; D, Ryffel B, Boidot R, et al. The receptor NLRP3 is a transcriptional regulator of TH2 differentiation. Nature Immunology.\u003cem\u003e \u003c/em\u003e2015;16 8:859-70.\u003c/li\u003e\n\u003cli\u003eSchlaeger J, Cai H, Steffen A, Angulo V, Shroff A, Briller J, et al. Acupuncture to Improve Symptoms for Stable Angina: Protocol for a Randomized Controlled Trial. JMIR research protocols.\u003cem\u003e \u003c/em\u003e2019;8 7:e14705; doi: 10.2196/14705.\u003c/li\u003e\n\u003cli\u003eZhang T. Interleukin-12 (IL-12) and IL-18 synergistically induce the fungicidal activity of murine peritoneal exudate cells against Cryptococcus neoformans through production of gamma interferon by natural killer cells. Infection \u0026amp; Immunity.\u003cem\u003e \u003c/em\u003e1997;65.\u003c/li\u003e\n\u003cli\u003eBesnard A, Guillou N, Tschopp J, Erard F, Couillin I, Iwakura Y, et al. NLRP3 inflammasome is required in murine asthma in the absence of aluminum adjuvant. Allergy.\u003cem\u003e \u003c/em\u003e2011;66 8:1047-57; doi: 10.1111/j.1398-9995.2011.02586.x.\u003c/li\u003e\n\u003cli\u003eHelmby H, Grencis RK. Interleukin 1 plays a major role in the development of Th2-mediated immunity. Eur J Immunol.\u003cem\u003e \u003c/em\u003e2004;34 12:3674-81; doi: 10.1002/eji.200425452.\u003c/li\u003e\n\u003cli\u003eCiraci C, Janczy JR, Sutterwala FS, Cassel SL. Control of innate and adaptive immunity by the inflammasome. Microbes \u0026amp; Infection.\u003cem\u003e \u003c/em\u003e2012;14 14.\u003c/li\u003e\n\u003cli\u003eJia X, Li X, Shen Y, Miao J, Liu H, Li G, et al. MiR-16 regulates mouse peritoneal macrophage polarization and affects T-cell activation. Journal of cellular and molecular medicine.\u003cem\u003e \u003c/em\u003e2016;20 10:1898-907; doi: 10.1111/jcmm.12882.\u003c/li\u003e\n\u003cli\u003eZhu F, Liu X, Sun Z, Yu C, Liu L, Yang S, et al. Immune-Enhancing Effects of Taishan Pinus massoniana Pollen Polysaccharides on DNA Vaccine Expressing Bordetella avium ompA. Frontiers in microbiology.\u003cem\u003e \u003c/em\u003e2016;7:66; doi: 10.3389/fmicb.2016.00066.\u003c/li\u003e\n\u003cli\u003eSong Y, Xu J, Wang X, Yang Y, Sun S. Regulation of host immune cells and cytokine production induced by Trichinella spiralis infection. Parasite.\u003cem\u003e \u003c/em\u003e2019;26:74.\u003c/li\u003e\n\u003cli\u003eLawrence CE, Paterson JCM, Higgins LM, Macdonald TT, Kennedy MW, Garside P. IL-4-regulated enteropathy in an intestinal nematode infection. European Journal of Immunology.\u003cem\u003e \u003c/em\u003e2010;28 9:2672-84.\u003c/li\u003e\n\u003cli\u003eChen C, Yang C, Tsai Y, Liaw C, Chang W, Hwang T. Ugonin U stimulates NLRP3 inflammasome activation and enhances inflammasome-mediated pathogen clearance. Redox biology.\u003cem\u003e \u003c/em\u003e2017;11:263-74; doi: 10.1016/j.redox.2016.12.018.\u003c/li\u003e\n\u003cli\u003eBruschi F, Chiumiento L. Trichinella inflammatory myopathy: host or parasite strategy? Parasites \u0026amp; vectors.\u003cem\u003e \u003c/em\u003e2011;4:42; doi: 10.1186/1756-3305-4-42.\u003c/li\u003e\n\u003cli\u003eLafuse W, Wozniak D, Rajaram M. Role of Cardiac Macrophages on Cardiac Inflammation, Fibrosis and Tissue Repair. Cells.\u003cem\u003e \u003c/em\u003e2020;10 1; doi: 10.3390/cells10010051.\u003c/li\u003e\n\u003cli\u003eAlhallaf R, Agha Z, Miller CM, Robertson AAB, Sotillo J, Croese J, et al. The NLRP3 Inflammasome Suppresses Protective Immunity to Gastrointestinal Helminth Infection. Cell Reports.\u003cem\u003e \u003c/em\u003e2018;23 4:1085.\u003c/li\u003e\n\u003cli\u003eWang X, Gong P, Zhang X, Li S, Lu X, Zhao C, et al. Neospora caninumNLRP3 Inflammasome Participates in Host Response to Infection. Frontiers in immunology.\u003cem\u003e \u003c/em\u003e2018;9:1791; doi: 10.3389/fimmu.2018.01791.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"T. spiralis, NLRP3, Immune response, Th1/Th2","lastPublishedDoi":"10.21203/rs.3.rs-1951416/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1951416/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Trichinellosis is an important food-borne zoonosis, and no effective treatments are yet available. Nod-like receptor plays a critical role in the host response against nematodes. Therefore, we aimed to explore the role of the NLRP3 inflammasome (NLRP3) during the adult, migrating, and encysted stages of \u003cem\u003eTrichinella spiralis (T. spirali\u003c/em\u003es) infection. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThe mice were treated with the specific NLRP3 inhibitor MCC950 after inoculation with \u003cem\u003eT. spiralis.\u003c/em\u003e Then, NLRP3 plays the role in \u003cem\u003eT. spirali\u003c/em\u003es-infected mice were evaluated using ELISA, Western blotting, Flow cytometry, Histopathological evaluation, Bone marrow-derived macrophage (BMDM) stimulation and immunofluorescence\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe in vivo results showed that NLRP3 enhanced the Th1 immune response in the adult stage and the migrating stage and weakened the Th2 immune response in the encysted stage. NLRP3 promoted the release of proinflammatory factors (INF-γ) and suppressed the release of anti-inflammatory factors (IL-4). Pathological changes were also improved in the absence of NLRP3 in mice during \u003cem\u003eT. spiralis\u003c/em\u003e infection. Importantly, a significant reduction in adult worm burden and muscle larvae burden at 7 and 35 days post infection was observed in mice treated with the specific NLRP3 inhibitor MCC950. In vitro, we first demonstrated that NLRP3 in macrophages can be activated by \u003cem\u003eT. spiralis \u003c/em\u003eproteins and promotes IL-1β and IL-18 release. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThis study revealed that the NLRP3 is involved in the host response to \u003cem\u003eT. spiralis\u003c/em\u003e infection and that targeted inhibition of NLRP3 enhanced the Th2 response and accelerated \u003cem\u003eT. spiralis\u003c/em\u003e expulsion. These findings may help in the development of protocols for controlling trichinellosis.\u003c/p\u003e","manuscriptTitle":"NLRP3 plays a key role in antihelminth immunity in the enteral and parenteral stages of Trichinella spiralis-infected mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-08-16 15:38:31","doi":"10.21203/rs.3.rs-1951416/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f072ff7d-189c-43dd-af10-a7740839a50e","owner":[],"postedDate":"August 16th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-08-16T15:43:33+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-16 15:38:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1951416","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1951416","identity":"rs-1951416","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}