Adult Hymenolepis nana and its excretory-secretory products elicit mouse immune responses via Tuft/IL-13 signaling pathway

Adult Hymenolepis nana and its excretory-secretory products elicit mouse immune responses via Tuft/IL-13 signaling pathway | 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 Adult Hymenolepis nana and its excretory-secretory products elicit mouse immune responses via Tuft/IL-13 signaling pathway Rong Mou, Xuan-Yin Cui, Yu-Si Luo, Yi Cheng, Qing-Yuan Luo, Zhen-Fen Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5275142/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Mar, 2025 Read the published version in Parasites & Vectors → Version 1 posted 9 You are reading this latest preprint version Abstract Background Hosts typically elicit diverse immune responses to the infection of various parasitic worms, with intestinal tuft cells playing a pivotal role in detecting parasite invasion. Hymenolepis nana ( H. nana ), a zoonotic parasitic worm, resides in the host's intestine. The contribution and underlying mechanisms of tuft cell-mediated immune reactions against H. nana remain unexplored. Methods This study endeavors to examine the immune responses in the mouse intestine elicited by the adult H. nana and its excretory-secretory products (ESP). Detection of various intestinal cell counts and cytokine changes using IHC, IF, RT-qPCR, etc. Results The presence of adult H. nana and its ESP enhances the population of tuft cells and goblet cells while fostering the production of type 2 cytokines, particularly IL-13. Furthermore, the surge in Paneth cells triggered by H. nana aids in maintaining intestinal stem cells homeostasis. Notably, RCM-1, the specific IL-13 inhibitor, dampens intestinal stem cells differentiation and type 2 cytokine secretion, potentially impeding the host's capacity to eliminate H. nana . Conclusions In conclusion, the adult H. nana and its ESP stimulate the immune responses from the mouse intestinal mucosa via the Tuft/IL-13 signaling pathway, facilitating the expulsion of H. nana from the host. Hymenolepis nana tuft cell excretory-secretory products IL-13 RCM-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Highlights 1. Hymenolepis nana ( H. nana ) infection boosts tuft cells in the small intestine, implicating their role in host defense. Meanwhile, H. nana has a dual impact on intestinal stem cells (ISC): Infection reduces ISC, but excretory-secretory products (ESP) promote the proliferation of ISC. ESP's growth stimulus is countered by physical damage, resulting in an overall ISC decrease. 2. After H. nana infection, the host drives increased secretion of mucins, antimicrobial peptides, and cytokines through the Tuft/IL-13 pathway to expel the parasite, whereas the use of RCM-1 (the inhibitor of IL-13), prevented the host from generating effective immune responses to H. nana , thus affecting the killing or excretion of H. nana . 3. H. nana and ESP activate the Tuft/IL-13 pathway, increasing the number of goblet, tuft, and Paneth cells. These cells secrete mucus and antimicrobials, protecting intestinal mucosa, hinting at ‘helminth therapy’ potential for host gut health. 1. Background Parasitic worms are among the most common pathogens in nature. To complete their life cycle, these intestinal worms traverse host tissues, causing severe damage, which imposes a significant burden on global health systems [ 1 ]. The host's resistance to intestinal pathogens relies on the immunoregulatory functions of immune cells. Innate lymphoid cells (ILC) are tissue-resident immune cells that are early responders to infection. The classic ILC subtypes are divided into three groups, group 1 ILC (ILC1) predominantly secretes IFN-γ, group 2 ILC (ILC2) predominantly secretes interleukin (IL)-13, and group 3 ILC (ILC3) predominantly secretes IL-22, and which host ILC predominates depends on the type of intestinal pathogen the host is infected with [ 2 ]. Tissue damage caused by intestinal worm infections triggers epithelial cells to produce alarmin cytokines IL-25 and IL-33, which in turn activate ILC2 [ 3 ]. The cytokines secreted by ILC2 can also directly influence intestinal epithelial cells and various immune cells to drive specific immune responses [ 4 ]. The intestinal epithelium consists of various cell types responsible for nutrient absorption and providing a protective barrier, as well as being able to rapidly change its cellular composition to defend pathogen invasion. Intestinal epithelial cells undergo rapid renewal, with a turnover every 3–5 days [ 5 ]. Intestinal stem cells (ISC) are primarily located in the intestinal crypts, and play a crucial role in the renewal process of intestinal epithelial cells, differentiating into all intestinal epithelial cell types, including enterocytes, enteroendocrine cells, tuft cells, goblet cells, and Paneth cells [ 6 ]. Paneth cells are the only differentiated cells in the crypts and are interspersed with the ISC, and secrete epidermal growth factor (EGF), Wnt3, and the Notch ligand Dll4 to maintain ISC homeostasis [ 7 ]. Intestinal tuft cells are chemosensory epithelial cells that have garnered significant attention in the study of host-parasite interactions. Tuft cells are crucial for defending worms, as their numbers increase sharply during intestinal parasite infections. They are also the primary source of intestinal IL-25, which plays a role in controlling the number of ILC2 during worm infection [ 8 ]. ILC2 can secrete type 2 cytokines, including IL-4, IL-5, IL-9, and IL-13 [ 9 ]. Tuft cells serve as vital sentinels in the gastrointestinal tract, rapidly proliferating after exposure to type 2 cytokines and playing a key role in protecting against worm infections [ 10 ]. This ultimately forms a positive feed-forward loop, where IL-25 produced by tuft cells activates ILC2, and IL-13 produced by ILC2 induces the differentiation of ISC into tuft cells [ 11 ]. IL-13 acts as a crucial player in promoting goblet cell proliferation, mucus secretion, and smooth muscle activity. In response to intestinal parasites residing in the small intestine, IL-13 produced by immune cells increases and induces ISC to differentiate into tuft cells and goblet cells [ 12 ]. Goblet cells are key components of the host's defense against parasites, producing and releasing mucins that form a dense mucus layer on the surface of the intestinal mucosa, working alongside other cells to maintain intestinal homeostasis. After worm infection, the proliferation timelines of tuft cells and goblet cells are synchronized [ 13 ]. The damage caused by worms to the host is primarily divided into mechanical damage induced by the worms and the effects of a series of immunomodulatory molecules they secrete, collectively referred to as excretory-secretory products (ESP) [ 14 ]. Hymenolepis nana ( H. nana ) is a zoonotic parasite that parasitizes the intestines of humans and rodents, causing hymenolepiasis nana. Mild infections of H. nana in humans have no obvious clinical symptoms, while severe infections manifest as abdominal pain, diarrhea, anemia, and fever [ 15 , 16 ]. H. nana infects people of all age groups, with a predominance of infections in children under 10 years old [ 17 ]. It is estimated that the number of infected people worldwide is estimated to be 50 to 75 million and is more pronounced in Asia, Africa, Southern / Eastern Europe, and Central / South America [ 18 ]. After infection with H. nana , the oncosphere in the eggs invades the intestinal villi, develops into cysticercoid on the 4th day of infection, and enters the intestinal lumen, where they mature on about the 12th day [ 19 ]. It is unclear how H. nana relates to the host immune responses and whether tuft cells play a role in defense against H. nana . In the current study, we investigated the effects of H. nana adult worm infection and intraperitoneal injection of adult H. nana -derived ESP on the host. We found that both the adult worms and ESP of H. nana could promote the number of tuft cells, goblet cells, and Paneth cells of the mouse small intestine, while simultaneously activating the Tuft/IL-13 signaling pathway, thereby influencing the immune responses in the mouse intestine. 2. Materials and Methods 2.1 Animal experiments All animal experiments of the current study were approved by the Animal Ethics Committee of Guizhou Medical University (approve Nos. 2100346 and 2100347). 2.1.1 The hamsters and the acquisition of serum from H . nana- infected hamster The 4–6 weeks male hamsters ( n = 30) were purchased from one pet market in Nanming District, Guiyang, China, in March 2024 (Fig. S2A). Hamsters were sacrificed under anesthesia to obtain intestinal parasites. A few parasites were randomly selected and cut with a scissor to obtain eggs. In addition, six parasites were randomly selected from those obtained and stained with carbolic acid red (configured with carmine), eggs and stained adults were observed using the fluorescence microscope (Eclipse 80i, Nikon Ltd., Japan). A few parasites were re-selected and worm DNA was extracted using a MolPure Cell / Tissue DNA Kit (Yesen, Shanghai, China), and PCR amplification for the COX-I gene of H. nana followed by agarose gel electrophoresis. The COX-I primer sequence used in this process was listed in Table S1 . 2.1.2 The C57BL/6J mice experiments The C57BL/6J mice (specific-pathogen-free, female, 6–8 weeks old, weighing approximately 22.0 ± 2.0 g) used in this study were obtained from the Experimental Animal Center of Guizhou Medical University [SCXK (Jing) 2019-0010]. These mice were housed in a standard laboratory environment, devoid of parasitic contamination, with a regulated temperature range of 20–22°C and a controlled 12-h light/dark cycle. After a seven-day period of acclimatization, the experiments commenced. RCM-1 is a known inhibitor of IL-13, and its administration and dosing refer to previous literatures [ 20 , 21 ]. The mice were randomly allocated into five groups: Control (Ctrl, n = 8), ESP ( n = 8), ESP + RCM-1 ( n = 8), H. nana ( n = 8), and H. nana + RCM-1 ( n = 8). All mice received sterilized H 2 O for 14 days. Starting from the first day, the ESP + RCM-1 and H. nana + RCM-1 groups received daily intraperitoneal injections of 1.7 mg/kg RCM-1 (Selleck, Shanghai, China) for seven consecutive days. Our preliminary experiments indicated that 50 µg/day of ESP was more effective than 25 µg/day (Fig. S1 ), hence we selected the dose of 50 µg/day for the ESP and ESP + RCM-1 groups, which were administered with intraperitoneal injections of ESP from the 7th day onwards for seven days. Based on preliminary findings, the H. nana and H. nana + RCM-1 groups were orally inoculated with 2,000 eggs per mouse on the first day, as this dose resulted in the optimal infection rate. All mice were euthanized on the 14th day. 2.2 The extraction and identification of adult H. nana -derived excretory-secretory products (ESP) The H. nana , retrieved from hamsters, underwent rigorous cleaning procedures involving multiple rinses with sterilized PBS. Subsequently, 20–30 adult worms were immersed in RPMI 1640 medium, supplemented with 1% Penicillin/Streptomycin/Amphotericin B. To concentrate the ESP-rich medium, centrifugation at 4,000 x g was executed utilizing a 10 kDa ultrafiltration tube (Millipore, Billerica, MA, USA), with the solvent subsequently exchanged for PBS. Sterility was ensured by passing the ESP solution through a 0.22 µm filter, and the protein content was quantified using a BCA protein assay. The prepared ESP was then stored at -80°C for future applications. In parallel, the protein composition of the ESP was scrutinized through immunoblotting. In brief, equal quantities of ESP samples were loaded into each well, and electrophoresis was performed. The resolved proteins were then transferred onto a PVDF membrane, which was blocked with 5% skim milk to prevent nonspecific antibody interactions. The membrane was probed overnight with a primary antibody (serum from the H. nana -infected hamster, diluted 1:50), followed by the addition of an anti-mouse secondary antibody (diluted 1:10,000) and a 1 h incubation. Finally, the protein bands were visualized using Super ECL Detection Reagent, allowing for the assessment of the number and molecular size of proteins present in the ESP. 2.3 Ileum hematoxylin-eosin (H&E) staining and alien-blue and periodic acid-Schiff (AB-PAS) staining The ileum tissues of the mice were collected and fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 µm thick sections. According to the reagent instructions to stain. 2.4 RT-qPCR Genomic RNA was extracted from mouse ileum tissue using the Trizol protocol. Subsequently, the genomic RNA was reversely transcribed into cDNA, and the amplification was detected using the SYBR RT-qPCR Kit (Yesen, Shanghai, China) in a real-time fluorescence quantitative PCR instrument (CFX96, Bio-Rad, Hercules, CA, USA). The amplification protocol consisted of an initial denaturation step at 95°C for 5 min, followed by 39 cycles of denaturation at 95°C for 10 s, and annealing at 60°C for 30 s. The primer sequences were listed in Table S1 respectively. 2.5 IHC and IF For IHC, the sections were incubated overnight with primary antibodies specific for MUC2 (1:2,000), Dclk1 (1:200), IL-13 (1:200), Olfm4 (1:200), GATA3 (1:200), and lysozyme (lyz) (1:2,000). On the next day, the sections were incubated with the appropriate secondary antibody (Anti-rabbit, 1:500), followed by DAB staining. Finally, the sections were mounted with neutral gum and observed using a slide scanner (Olympus SLIDEVIEW VS200, Japan). For IF, the sections were incubated overnight with primary antibodies with respective specificity for Lgr5 (1:100), Olfm4 (1:200), MUC2 (1:500), Dclk1 (1:200), and lysozyme (lyz) (1:250). The next day, the sections were incubated with fluorescent secondary antibody 488 (1:200) and DAPI solution. Finally, they were mounted with an anti-fade mounting medium and observed using an upright fluorescence microscope (Eclipse 80i, Nikon Ltd, Japan). 2.6 WB The mouse ileum tissues were digested with RIPA lysis buffer. After measuring the concentration, an equal amount of protein samples was loaded and isolated using SDS-PAGE. Next, transfer the proteins onto a PVDF membrane. After blocking with 5% non-fat milk, incubate the membrane with the primary antibody for Lgr5 (1:1,000) at 4°C overnight. Then incubated with the secondary antibody anti-rabbit (1:10,000) for 1 h at room temperature. Finally, Super ECL Detection Reagent was utilized for sensitive detection of the protein and observed using a Chemiluminescence imager (Bio-Rad, Hercules, CA, USA). 2.7 Statistical analysis The statistical analysis and graphical representation were performed using GraphPad Prism (Version 9.5.1). The data were presented as mean + SD. Measurements were first subjected to normality tests, and the Homogeneity of Variance Test was performed between groups. Variance irregularities using the Mann-Whitney U-test. One-way analysis of variance (ANOVA) and t-test were used to compare differences between groups. The p value less than 0.05 means a statistically significant difference. 3. Results 3.1 Experimental design and identification of H. nana Through intraperitoneal injection of ESP in mice, it was found that a dose of 50 µg/day increased the number of goblet cells and tuft cells in the small intestines of mice more than a dose of 25 µg/day (Fig. S1 ). Therefore, this dosage was selected for subsequent experiments to study the interaction between ESP and the host. To investigate the impact of H. nana on the intestinal immune responses in mice, the following experimental approach was adopted (Fig. 1 A). Microscopic examination revealed that the eggs were nearly spherical with a relatively thin eggshell, containing a thicker embryonic membrane within, and inside the membrane, an oncosphere with visible small hooks was observed (Fig. 1 B). After staining the adult worms with carboxyl borate red, the entire worm body appeared red, with the scolex showing a rostellum with distinct hooks, as well as visible circular suckers (Fig. 1 C). The mature proglottid exhibited reproductive systems such as testes and ovaries (Fig. 1 D), and the gravid proglottids were filled with eggs (Fig. 1 E). PCR amplification and agarose gel electrophoresis of the H. nana COX-I gene showed a clear band at 202 bp (Fig. 1 F). In summary, we confirmed that the parasites used in our experiments were H. nana . Successful infection with H. nana was determined by detecting eggs in fecal samples and dissecting adult worms from the intestinal lumen (Fig. S2B & C). We found a 100% infection rate for H. nana , and RCM-1 administration increased the infection load of H. nana (Fig. 1 G). The ESP harvested after culture was analyzed by immunoblotting, revealing a variety of proteins, with the main bands located at 20–25 kDa, 35–45 kDa, 45–60 kDa, and 100–140 kDa, respectively (Fig. S2D). 3.2 RCM-1 exacerbates the pathologic damage in the mouse intestine caused by H. nana To investigate the effects of H. nana infection and ESP on mouse intestinal pathology, we used H&E staining to observe changes in the small intestinal epithelial villi and performed IHC to detect the expression of mucin MUC2. Intestinal parasitic infections can cause atrophy of the small intestinal epithelial villi. We found that H. nana infection resulted in the presence of adult worm segments in the intestinal lumen. Both H. nana infection and intraperitoneal injection of ESP led to the shortening of intestinal epithelial villi. However, following RCM-1 intervention, the shortening of villi induced by ESP showed partial recovery, while the shortening induced by H. nana infection was exacerbated (Fig. 2 A & C). Goblet cell-derived mucin MUC2 plays a crucial role in maintaining intestinal homeostasis. Both H. nana infection and ESP increased the secretion of mucin in the mouse intestine, and this increase could be inhibited by RCM-1. These results indicate that both adult H. nana and its ESP contribute to pathological damage in the mouse intestine, and RCM-1 intervention exacerbates pathological damage caused by H. nana in mice. 3.3 Adult H. nana and its ESP can increase the number of small intestinal goblet cells through IL-13 Goblet cells produce a number of effector molecules including a range of mucins and antimicrobial proteins, which enable these to play a key part in innate defense mechanisms in the gut, against both bacterial and helminth infections [ 22 ]. The effect of H. nana on mouse intestinal goblet cells was investigated using AB-PAS staining of goblet cells, IF, and RT-qPCR methods to detect the goblet cell marker (MUC2). AB-PAS staining revealed goblet cells in the small intestinal epithelial villi stained blue-purple. The results showed that H. nana promoted an increase in the number of goblet cells in the intestine. However, after the RCM-1 intervention, the number of goblet cells decreased. This finding was corroborated by IF staining and RT-qPCR results for MUC2, which also indicated that RCM-1 inhibited the increase in goblet cells induced by both adult H. nana worms and ESP (Fig. 3 ). These results suggest that adult H. nana and its ESP may increase the number of goblet cells through IL-13. 3.4 RCM-1 prevents the involvement of small intestinal tuft cells in defense against H. nana in mice Recent studies have found that tuft cells play a first-line defense role in certain intestinal parasitic infections [ 23 ], but research on tuft cells in H. nana infections is still lacking. IHC and IF were assayed for tuft cell marker double cortin-like kinase-1 (Dclk1), and the results showed that both H. nana adults and ESP significantly increased the number of tuft cells in the intestine, while RCM-1 inhibited this increase. These findings were confirmed by RT-qPCR results (Fig. 4 A-E). The cytokines IL-25 secreted by tuft cells and IL-33 secreted by intestinal epithelial cells act on downstream ILC2 to exert their effects. The results showed that H. nana infection and ESP both promoted an increase in the secretion of IL-25 and IL-33 . These findings indicate that H. nana increases the number of tuft cells in the mouse intestine and suggest that tuft cells are involved in mice's defense against H. nana , and the process can be suppressed by RCM-1. 3.5 H. nana induces ILC2 secretion of type 2 cytokines in mouse small intestine, which is suppressed by RCM-1 Type 2 immune responses are essential for protection against intestinal helminth infections, with inadequate secretion of type 2 cytokines affecting parasite elimination. The transcription factor GATA3 was upregulated in ILC2 [ 24 ], and the number of GATA3 cells was examined by IHC, which revealed that both H. nana and ESP caused an increase in GATA3 cells in the mouse intestine (Fig. 5 A&C). To investigate whether IL-25 and IL-33 promote the secretion of type 2 cytokines by downstream ILC2, we assessed changes in IL-13 expression using IHC and RT-qPCR. The results revealed that both H. nana infection and ESP led to increased secretion of IL-13 (Fig. 5 B&D&E). Decreased expression levels of IL-13 after intervention with the IL-13 inhibitor RCM-1. Additionally, RT-qPCR analysis of other type 2 cytokines, including IL-4 , IL-5 , and IL-9 , the results showed that RCM-1 could inhibit the increase in gene expression induced by H. nana infection or ESP (Fig. 5 F-H). These findings indicate that H. nana infection and ESP may increase ILC2, which stimulates increased secretion of type 2 cytokines, including IL-13. In contrast, RCM-1 causes a decrease in type 2 cytokine secretion, which affects the excretion of H. nana. 3.6 H. nana has a dual effect on the number of ISC: infection reduces and ESP promotes the proliferation, and RCM-1 intervention exacerbates the reduction of ISC The effects of H. nana infection on the number of ISC remain unclear. To assess changes in ISC numbers, we measured the expression of the ISC marker olfactomedin 4 (Olfm4). The results indicated that H. nana infection leads to a reduction in ISC numbers, whereas ESP has the opposite effect, increasing ISC numbers. Additionally, changes in another ISC marker of Lgr5 confirmed these findings (Fig. S3). Following intervention with RCM-1, the changes in ISC numbers induced by both H. nana infection and ESP were consistent, showing a reduction in ISC numbers (Fig. 6 ). This result suggests that H. nana infection may decrease ISC numbers due to mechanical damage caused by the worms, while ESP actually promotes an increase in ISC numbers. After the RCM-1 intervention, the number of ISC was reduced. 3.7 RCM-1 inhibits adult H. nana and its ESP-induced increase in the number of Paneth cells, which disrupts the homeostasis of ISC Paneth cells, located in the intestinal crypts, maintain ISC homeostasis by secreting related growth factors. Lysozyme (Lyz) is the first antimicrobial peptide discovered in Paneth cells and is widely used as a Paneth cell marker in the ileum [ 25 ]. Using IHC, IF, and RT-qPCR to detect Lyz, we found that both adult H. nana worms and ESP increased lysozyme and the number of Paneth cells (Fig. 7 A-E). Further RT-qPCR analysis of Paneth cell-secreted growth factors Wnt3 , EGF , and Dll4 showed increased gene expression (Fig. 7 F-H). IL-13 can promote the secretion of antimicrobial peptides by Paneth cells, and we also observed that inhibition of IL-13 resulted in a reduction in both the number of Paneth cells and the secretion of lysozyme. These results indicate that H. nana promotes an increase in the number of Paneth cells, which in turn secrete growth factors to maintain ISC homeostasis. In contrast, RCM-1 decreases the number of Paneth cells, leading to decreased secretion of antimicrobial peptides and growth factors, thereby disrupting ISC homeostasis. 4. Discussion The intestine is the target organ for most parasitic infections, including those worms and protozoa. These parasites typically activate type 2 immune responses in the host's immune system and have a significant impact on the local tissue microenvironment [ 26 ]. Worms are multicellular parasites that cause a range of adverse effects, such as malnutrition and developmental delays in children, contributing to the global public health burden [ 27 ]. Intestinal worms have evolved mechanisms to evade or suppress host defense responses to ensure their survival and reproduction, laying eggs in the intestinal tissues and being expelled through the host's feces [ 28 ]. H. nana , also known as the dwarf tapeworm, primarily parasitizes the intestines of humans and rodents. H. nana has a global distribution, with an estimated infection rate of 1.2% in humans and up to 13% in rodents [ 17 ]. Conventional diagnostic methods for Hymenolepiasis nana involve fecal examination of eggs or adults. However, PCR-based molecular techniques can improve parasite detection rates and accurately determine species differentiation and genetic characteristics. Mitochondrial cytochrome c oxidase subunit I ( COX-I ) is an important mitochondrial gene with high variability and specificity among different parasites, making it one of the most commonly used molecular genetic markers [ 29 ]. Intestinal worms parasitize the host's intestines, causing a series of damages, including nutrient depletion, mechanical damage from the worm's body, and toxic effects from ESP. In the current study, we identified H. nana through morphological and molecular biology techniques and characterized the main protein components of ESP using immunoblotting. We then explored the immune responses relationship between H. nana and the host using H. nana egg infections and intraperitoneal ESP injections. Our findings showed that adult H. nana worms and ESP could activate the Tuft/IL-13 signaling pathway in the host, leading to an increase in the number of goblet cells, tuft cells, and Paneth cells, as well as an increase in type 2 cytokine secretion, ultimately aimed at clearing H. nana . When RCM-1 was used, the host failed to produce effective immune responses to H. nana , thereby exacerbating H. nana infection in the host. Infection with H. nana in mice can lead to degeneration or destruction of the normal villous structure of the small intestine epithelium, resulting in villous shortening or atrophy [ 30 ]. Infection of rats with H. nana results in enteritis, villous necrosis, and inflammatory infiltration of the mucosa and submucosa [ 31 ]. MUC2 is primarily secreted by goblet cells, forming a mucosal barrier that protects the mucosal surface and plays a crucial role in regulating intestinal immune responses, maintaining mucosal health, and protecting the immune system [ 32 ]. Our study found that both adult H. nana worms and ESP caused the shortening of intestinal epithelial villi and an increase in mucin MUC2 secretion in mice. Intervention with the IL-13 inhibitor RCM-1 could restore villous shortening induced by ESP but exacerbated villous shortening caused by adult H. nana worms. This might be due to RCM-1 increasing the severity of H. nana infection, thereby intensifying damage to the host. The above results indicated that both adult H. nana and ESP caused intestinal pathologic damage, and the greater the worm infection, the more severe the damage. Tuft cells occupy a small proportion of the intestinal epithelium but play a crucial role in recognizing and responding to parasitic infections [ 33 ]. It has been observed that the number of tuft cells in the small intestine increases after intestinal parasite infection. Normally, tuft cells constitute only about 1% of the intestinal epithelial cells in mice, but their numbers can increase several-fold during parasitic infections [ 34 , 35 ]. Tuft cells have become key sentinels in parasitic infections by releasing the alarmin IL-25, which activates ILC2 and enhances the type 2 immune response, signaling the presence of worms [ 11 ]. The type 2 immune responses are protective for the host, not only by directly killing parasites in tissues or reducing their numbers by expulsion from the intestine, but also by protecting the host from tissue damage caused by large extracellular parasites migrating within the body [ 36 ]. The regulation of signaling pathways between tuft cells and ILC2 has garnered significant attention in parasitic intestinal infections. Tuft cells can recognize infections from Nippostrongylus brasiliensis ( N. brasiliensis ), Trichinella spiralis ( T. spiralis ), and Helicotylenchus , releasing IL-25 to increase ILC2 numbers and subsequently IL-13 secretion, which acts on ISC to differentiate into more tuft and goblet cells [ 11 , 34 , 37 ]. IL-33 is a tissue-derived nuclear cytokine that promotes type 2 immune responses during allergy and helminthic infection [ 38 ]. Our study found that both adult H. nana worms and ESP induced increases in the numbers of goblet and tuft cells, along with elevated secretion of cytokines IL-25 and IL-33. Further investigation into the expression changes of type 2 cytokines revealed an increase in type 2 cytokines, including IL-13. The above series of changes can be inhibited by RCM-1, indicating that adult H. nana worms and ESP could activate the signaling pathways between tuft cells and ILC2, and RCM-1-induced reduction in type 2 cytokine secretion affected host excretion of H. nana . ISC are located in the intestinal crypts and possess high proliferative and differentiative capacities. The proliferation and differentiation of ISC ensure the integrity and functional stability of the intestinal epithelium and contribute to the prevention of intestinal diseases [ 39 ]. A recent study has shown that ISC depletion occurs locally in mice infected with Heligmosomoides polygyrus [ 40 ]. ISC can differentiate into mature Paneth cells, unlike other mature intestinal epithelial cells, they migrate downward after differentiation and reside at the base of the crypts, where they are arranged in a cross pattern with ISC [ 41 ]. Following the parasitic invasion of the host intestine, ISC accelerate their proliferation and differentiation, stimulating the proliferation of Paneth cells in response to the infection [ 42 ]. Paneth cells can secrete antimicrobial peptides to maintain intestinal homeostasis [ 43 ] and regulate ISC function through the secretion of growth factors such as Wnt3, EGF, and Dll4 [ 44 ]. Research on the impact of intestinal worm infections on host small intestinal Paneth cells is relatively limited. The study has shown that T. spiralis and N. brasiliensis infections cause a significant increase in Paneth cell numbers [ 45 ]. In addition, IL-13 promotes ISC proliferation and differentiation on the one hand and enhances the secretion of antimicrobial peptides by Paneth cells on the other [ 46 ]. The effects of H. nana infection on ISC and Paneth cells remain unclear. Our research findings indicated that the adults of H. nana reduced the number of ISC, and RCM-1 exacerbated this effect, likely due to mechanical damage caused by H. nana adults in the intestinal lumen. ESP increased the number of ISC, but blocking the IL-13 signaling pathway resulted in a decrease in the number of ISC, suggesting that ESP-induced IL-13 promotes ISC proliferation. Both H. nana adults and ESP increased lysozyme and Paneth cell numbers, consistent with IL-13 changes. This suggested that Paneth cells secrete signaling factors to maintain ISC homeostasis and that IL-13 also promoted Paneth cell proliferation, which increased lysozyme secretion and maintained intestinal homeostasis. Collectively, we found that H. nana adults and its ESP both promoted the increase in goblet cells, tuft cells, Paneth cells, and type 2 cytokines, while IL-13 also facilitated the differentiation of ISC. This finding suggested that the infection with H. nana triggered the activation of the host's Tuft/IL-13 signaling pathway, eliciting the immune responses that facilitated the expulsion of H. nana from the host. 5. Conclusions In this study, we found that H. nana adults and its ESP could activate the Tuft/IL-13 signaling pathway in the mouse intestine, leading to the differentiation of ISC into more goblet and tuft cells, as well as an increase in Paneth cells to maintain the stability of ISC. These changes helped the host to expel the parasites. In contrast, RCM-1 suppressed the host's immune responses to H. nana , resulting in the host's inability to effectively kill or exclude H. nana . (Fig. 8 ). This research preliminarily revealed the role of tuft cells in defending against H. nana and explored the relationship between H. nana and the host's immune responses, providing a foundation for the prevention and treatment of H. nana and its ‘helminth therapy’. Declarations Conflicts of interest The authors have declared that no competing interests. Funding This work was supported by the National Natural Science Foundation of China (No. 82160398). Author Contribution RM, XYC, YSL, and KZ designed the study. RM provided funding and revised the manuscript. XYC drafted the manuscript. XYC, YC, QYL, ZFZ, and WLW performed the experiments. RM, XYC, YSL, and KZ performed data analysis. JFL, YSL, and KZ revised the manuscript. All authors reviewed the manuscript. Data Availability Data is provided within the manuscript or supplementary information files References Kirk MD, Pires SM, Black RE, Caipo M, Crump JA, Devleesschauwer B, et al. World Health Organization Estimates of the Global and Regional Disease Burden of 22 Foodborne Bacterial, Protozoal, and Viral Diseases, 2010: A Data Synthesis. PLoS medicine. 2015;12 12:e1001921; doi: 10.1371/journal.pmed.1001921 . Spits H, Artis D, Colonna M, Diefenbach A, Di Santo JP, Eberl G, et al. 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Small proline-rich protein 2A is a gut bactericidal protein deployed during helminth infection. Science (New York, NY). 2021;374 6568:eabe6723; doi: 10.1126/science.abe6723 . Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx Additional file 1: Figure S1. Effects of different dose of ESP on mouse intestinal goblet cells and tuft cells. ESP1 indicates an intraperitoneal dose of 25 μg/day per mouse and ESP2 indicates a dose of 50 μg/day per mouse. (A) Representative images of AB-PAS-stained with the goblet cells (sharp or deep blue pointed by red arrows, scale bars 100 μm for the upper panel, and 50 μm for the lower panel). (B) Representative images of IF with MUC2 (green) and the nucleus (DAPI, blue) (scale bars 100 μm). (C) Representative images of IHC with Dclk1 (brown pointed by black arrows, scale bars 100 μm for the upper panel, and 50 μm for the lower panel). (D) Representative images of IF with Dclk1 (green) and the nucleus (DAPI, blue) (scale bars 100 μm). Percentages of the statistics of AB-PAS-stained positive area (E), and the number of goblet cells (F), tuft cells (H), and Dclk1-positive area (G) were semi-quantified using Image J software. Data are presented as mean + SD for (E)-(H), n = 6 per group, ** p < 0.01, *** p < 0.001. Additional file 2: Figure S2. H. nana infection model and ESP identification. (A) Representative picture of the hamsters from an urban pet market. (B) The egg of H. nana (detected from the feces of mice) (scale bar 20 μm). (C) Adult worms were dissected from the intestines of H. nana -infected mice. (D) Immunoblotting result of ESP. M: protein marker; Lane 1-4: ESP. Additional file 3: Figure S3. H. nana infection causes a decrease in the number of ISC and ESP causes an increase in ISC. (A) Representative images of IF with Lgr5 (green) and the nucleus (DAPI, blue) (scale bars 100 μm). (B) Representative images of WB presented the expression level of Lgr5. Percentages of the number of ISC (C) and the relative expression of Lgr5 (D) were semi-quantified using Image J software. (E) The transcription levels of Lgr5 and the relative quantification were determined using the 2 -ΔΔCt method normalized to GAPDH . Data are presented as mean + SD for (C)-(E), n =7 per group for (C), n =6 per group for (D)-(E), ** p < 0.01, *** p < 0.001. Additional file 4: Table S1. Primer sequences used for RT-qPCR experiments of current study Cite Share Download PDF Status: Published Journal Publication published 11 Mar, 2025 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 18 Nov, 2024 Reviews received at journal 18 Nov, 2024 Reviews received at journal 05 Nov, 2024 Reviewers agreed at journal 03 Nov, 2024 Reviewers agreed at journal 27 Oct, 2024 Reviewers invited by journal 25 Oct, 2024 Editor assigned by journal 16 Oct, 2024 Submission checks completed at journal 16 Oct, 2024 First submitted to journal 16 Oct, 2024 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. 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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-5275142","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":366824121,"identity":"ec504c61-00a4-4f05-88ac-142a35b2521a","order_by":0,"name":"Rong Mou","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Mou","suffix":""},{"id":366824122,"identity":"addf5f58-f392-4c38-bf34-0b8169e17fb4","order_by":1,"name":"Xuan-Yin Cui","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xuan-Yin","middleName":"","lastName":"Cui","suffix":""},{"id":366824123,"identity":"e9f35cd7-7621-4660-8304-6b82235fabcf","order_by":2,"name":"Yu-Si Luo","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yu-Si","middleName":"","lastName":"Luo","suffix":""},{"id":366824124,"identity":"97812eba-85dd-4499-8257-7e542a6ad07f","order_by":3,"name":"Yi Cheng","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Cheng","suffix":""},{"id":366824125,"identity":"b6f11286-e59c-4b50-84eb-ee84cebc7c22","order_by":4,"name":"Qing-Yuan Luo","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qing-Yuan","middleName":"","lastName":"Luo","suffix":""},{"id":366824126,"identity":"d849f014-7e5c-48ac-9589-d36d74b862c2","order_by":5,"name":"Zhen-Fen Zhang","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhen-Fen","middleName":"","lastName":"Zhang","suffix":""},{"id":366824127,"identity":"0d58d8a2-a882-408b-a7ff-20bc8760d8c2","order_by":6,"name":"Wen-Lan Wu","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wen-Lan","middleName":"","lastName":"Wu","suffix":""},{"id":366824128,"identity":"35dbbe7d-8426-425f-9c2f-a0feece5507f","order_by":7,"name":"Jinfu Li","email":"","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinfu","middleName":"","lastName":"Li","suffix":""},{"id":366824129,"identity":"b72fcecf-8644-4ba3-822b-6978a1eaab12","order_by":8,"name":"Ke Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYBACCQYGNhAtB+GykaDFmHQtiQ1Ea5Fsb3/24OeO2vT5YYcfMHwoO8zAP7sBvxZpngPphr1njuduvJ1mwDjj3GEGiTsH8GuRk0g4JsHbdix34+wcBmbetsMMBhIJBLTIP2yT/Nt2LN0QpOUvMVqkJZjZpHnbahLkpYFaGInRItmTxiYt23bAcIN0msHBnnPpPBI3CGiROH78meTbtjp5+dnJDx/8KLOW459BQAsUAN1zgIEBiBh4iFIPBHUM8g3Eqh0Fo2AUjIIRBwDKhED+ofFXCwAAAABJRU5ErkJggg==","orcid":"","institution":"Guiyang Medical University","correspondingAuthor":true,"prefix":"","firstName":"Ke","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-10-16 10:53:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5275142/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5275142/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-025-06719-w","type":"published","date":"2025-03-11T15:57:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":66960207,"identity":"9de28ff7-0eb2-44b5-b404-697a86aa56c2","added_by":"auto","created_at":"2024-10-18 12:29:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1002508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental flowchart and the identification of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. nana \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eeggs and adults, and RCM-1 increases \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. nana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection load.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) All groups of mice were fed sterilized H\u003csub\u003e2\u003c/sub\u003eO for 14 days, the ESP + RCM-1 group and \u003cem\u003eH.nana\u003c/em\u003e + RCM-1 group were injected intraperitoneally with 1.7 mg/kg/day RCM-1 from the 1\u003csup\u003est \u003c/sup\u003eday for a total of seven injections; The ESP group and ESP + RCM-1 group were injected intraperitoneally with 50 μg/day ESP from the 7\u003csup\u003eth \u003c/sup\u003eday for a total of seven injections; and the \u003cem\u003eH. nana\u003c/em\u003e group and \u003cem\u003eH.nana\u003c/em\u003e + RCM-1 group were gavaged with 2,000 eggs per mouse on the 1\u003csup\u003est\u003c/sup\u003e day. All mice were sacrificed on the 14\u003csup\u003eth\u003c/sup\u003e day. i.p.: intraperitoneal injection; i.g.: intragastric injection. (\u003cstrong\u003eB\u003c/strong\u003e) Representative image of the egg of \u003cem\u003eH.nana\u003c/em\u003e (scale bar 20 μm). (\u003cstrong\u003eC\u003c/strong\u003e) Representative image of the scolex of \u003cem\u003eH. nana\u003c/em\u003e, the sucker and the restellum are indicated by the black and red arrows respectively (scale bar 100 μm). (\u003cstrong\u003eD\u003c/strong\u003e) Representative image of mature proglottids of \u003cem\u003eH.nana\u003c/em\u003e (scale bar 100 μm). (\u003cstrong\u003eE\u003c/strong\u003e) Representative image of gravid proglottids of \u003cem\u003eH.nana \u003c/em\u003e(scale bar 100 μm). (\u003cstrong\u003eF\u003c/strong\u003e) PCR amplification electrophoresis of the \u003cem\u003eCOX-I\u003c/em\u003e of \u003cem\u003eH. nana\u003c/em\u003e, the proposed amplicon was 202 bp. M: DL 2000 marker, Lane 1: The genomic DNA of \u003cem\u003eH. nana\u003c/em\u003e as the PCR template, Lane 2: sterilized H\u003csub\u003e2\u003c/sub\u003eO as the PCR template. (\u003cstrong\u003eG\u003c/strong\u003e) The left panel showed the representative pictures of \u003cem\u003eH. nana\u003c/em\u003e dissected from \u003cem\u003eH. nana\u003c/em\u003e group and \u003cem\u003eH. nana\u003c/em\u003e + RCM-1 group mice. The right panel compared the number of adult worms dissected from the \u003cem\u003eH. nana\u003c/em\u003e group and \u003cem\u003eH. nana\u003c/em\u003e + RCM-1 group mice. For the right panel of (G), data are presented as mean + SD (\u003cem\u003en \u003c/em\u003e= 8 per group), * \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5275142/v1/073e905fe0b73abce8db45b7.png"},{"id":66960210,"identity":"114f3299-a102-41c3-8f6e-6c793dcbb964","added_by":"auto","created_at":"2024-10-18 12:29:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1287650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRCM-1 aggravates pathological damage of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. nana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e to the mouse intestine by inhibiting the secretion of the mucin MUC2\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative images of H\u0026amp;E-stained small intestine (\u003cem\u003eH. nana\u003c/em\u003es pointed by red arrows, scale bars 200 μm for the upper panel and 100 μm for the lower panel). (\u003cstrong\u003eB\u003c/strong\u003e) Representative images of IHC with MUC2 (brown pointed by black arrows, scale bars 100 μm for the upper panel and 50 μm for the lower panel). Mean villus height (μm) (\u003cstrong\u003eC\u003c/strong\u003e) and percentage of MUC2-positive area (\u003cstrong\u003eD\u003c/strong\u003e) were semi-quantified using Image J software. Data are presented as mean + SD for (C) and (D), \u003cem\u003en\u003c/em\u003e = 7 per group, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5275142/v1/7ed5ea3194abe6141a81d7d0.png"},{"id":66960208,"identity":"225c5576-628b-45d7-81a6-22eb4a58319b","added_by":"auto","created_at":"2024-10-18 12:29:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1433085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRCM-1 suppresses the increase in the number of mouse small intestinal goblet cells induced by adult\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e H. nana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and its ESP. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative images of AB-PAS-stained goblet cells (sharp or deep blue pointed by red arrows, scale bars 100 μm for the upper panel and 50 μm for the lower panel). (\u003cstrong\u003eB\u003c/strong\u003e) Representative images of IF with MUC2 (green) and the nucleus (DAPI, blue) (scale bars 100 μm). Percentages of the statistics of AB-PAS-stained positive area (\u003cstrong\u003eC\u003c/strong\u003e) and the number of goblet cells (\u003cstrong\u003eD\u003c/strong\u003e) were semi-quantified using Image J software. (\u003cstrong\u003eE\u003c/strong\u003e) The transcription levels of \u003cem\u003eMUC2\u003c/em\u003e and the relative quantification were determined using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method normalized to \u003cem\u003eGAPDH\u003c/em\u003e. Data are presented as mean + SD for (C)-(E), \u003cem\u003en\u003c/em\u003e = 7 per group, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5275142/v1/3e05938f49810d123b955f67.png"},{"id":66960454,"identity":"8f9002da-f5e0-4bc8-aefe-4b4b09cdbd77","added_by":"auto","created_at":"2024-10-18 12:37:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1334822,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRCM-1 inhibits the increase in the number of small intestinal tuft cells and cytokine secretion induced by adult \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. nana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and its ESP in mice\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative images of IHC with Dclk1 (brown pointed by red arrows, scale bars 100 μm for the upper panel, and 50 μm for the lower panel). (\u003cstrong\u003eB\u003c/strong\u003e) Representative images of IF with Dclk1 (green) and the nucleus (DAPI, blue) (scale bars 100 μm). Percentages of Dclk1-positive area (\u003cstrong\u003eC\u003c/strong\u003e) and the number of tuft cells (\u003cstrong\u003eD\u003c/strong\u003e) were semi-quantified using Image J software. The transcription levels of \u003cem\u003eDclk1\u003c/em\u003e (\u003cstrong\u003eE\u003c/strong\u003e); \u003cem\u003eIL-25\u003c/em\u003e (\u003cstrong\u003eF\u003c/strong\u003e); and \u003cem\u003eIL-33\u003c/em\u003e (\u003cstrong\u003eG\u003c/strong\u003e), the relative quantification was determined using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method normalized to \u003cem\u003eGAPDH\u003c/em\u003e. Data are presented as mean + SD for (C)-(G), \u003cem\u003en\u003c/em\u003e = 7 per group for, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5275142/v1/7b331a982f3fb3e5d914f01e.png"},{"id":66960212,"identity":"7150f98a-2bb9-4595-afbe-1b680c1c3fe5","added_by":"auto","created_at":"2024-10-18 12:29:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1059892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRCM-1 prevents the secretion of type 2 cytokines to expel \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. nana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in mice. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative images of IHC with GATA3 (brown pointed by black arrows, scale bars 50 μm). (\u003cstrong\u003eB\u003c/strong\u003e) Representative images of IHC with IL-13 (brown pointed by red arrows, scale bars 100 μm for the upper panel, and 50 μm for the lower panel). Percentages of positive-area of GATA3 (\u003cstrong\u003eC\u003c/strong\u003e) and IL-13 (\u003cstrong\u003eD\u003c/strong\u003e) were semi-quantified using Image J software. The transcription levels of \u003cem\u003eIL-13\u003c/em\u003e (\u003cstrong\u003eE\u003c/strong\u003e), \u003cem\u003eIL-4\u003c/em\u003e (\u003cstrong\u003eF\u003c/strong\u003e), \u003cem\u003eIL-5\u003c/em\u003e (\u003cstrong\u003eG\u003c/strong\u003e), and \u003cem\u003eIL-9\u003c/em\u003e (\u003cstrong\u003eH\u003c/strong\u003e); The relative quantification was determined using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method normalized to \u003cem\u003eGAPDH\u003c/em\u003e. Data are presented as mean + SD for (C)-(H), \u003cem\u003en \u003c/em\u003e= 7 per group, ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5275142/v1/77df0c792f0c22381192491e.png"},{"id":66960453,"identity":"0aa4503c-8031-4767-b563-b6e584d5ff4e","added_by":"auto","created_at":"2024-10-18 12:37:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":935806,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eH. nana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection causes a decrease in the number of ISC and ESP causes an increase in ISC, injection of RCM-1 decreases the number of ISC in mice.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Representative images of IHC with Olfm4 (brown pointed by red arrows, scale bars 100 μm for the upper panel, and 50 μm for the lower panel). (\u003cstrong\u003eB\u003c/strong\u003e) Representative images of IF with Olfm4 (green) and the nucleus (DAPI, blue) (scale bars 100 μm). Percentages of Olfm4-positive area (\u003cstrong\u003eC\u003c/strong\u003e) and the number of ISC (\u003cstrong\u003eD\u003c/strong\u003e) were semi-quantified using Image J software. (\u003cstrong\u003eE\u003c/strong\u003e) The transcription levels of \u003cem\u003eOlfm4\u003c/em\u003e and the relative quantification were determined using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method normalized to \u003cem\u003eGAPDH\u003c/em\u003e. Data are presented as mean + SD for (C)-(E), \u003cem\u003en\u003c/em\u003e = 7 per group, * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5275142/v1/393b77017f00745a35ae14b1.png"},{"id":66960213,"identity":"470eab6f-260c-4289-a047-f4af8aa8896e","added_by":"auto","created_at":"2024-10-18 12:29:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1723554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRCM-1 suppresses the increase in the number of Paneth cells induced by adult\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e H. nana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and its ESP, leading to a decrease in the secretion of antimicrobial peptides and growth factors.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Representative images of IHC with Lyz (brown by black arrows, scale bars 100 μm for the upper panel, and 50 μm for the lower panel). (\u003cstrong\u003eB\u003c/strong\u003e) Representative images of IF with Lyz (green) and the nucleus (DAPI, blue) (scale bars 100 μm). Percentages of Lyz-positive area (\u003cstrong\u003eC\u003c/strong\u003e) and the number of Paneth cells (\u003cstrong\u003eD\u003c/strong\u003e) were semi-quantified using Image J software. The transcription levels of \u003cem\u003eLyz1\u003c/em\u003e (\u003cstrong\u003eE\u003c/strong\u003e), \u003cem\u003eWnt3\u003c/em\u003e (\u003cstrong\u003eF\u003c/strong\u003e), \u003cem\u003eEGF\u003c/em\u003e (\u003cstrong\u003eG\u003c/strong\u003e), and \u003cem\u003eDll4\u003c/em\u003e (\u003cstrong\u003eH\u003c/strong\u003e), and the relative quantification were determined using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method normalized to \u003cem\u003eGAPDH\u003c/em\u003e. Data are presented as mean + SD for (C)-(H), \u003cem\u003en \u003c/em\u003e= 7 per group, ** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5275142/v1/80c5730746a2a38835de366b.png"},{"id":66960211,"identity":"941a5cc9-1915-4c8b-8722-f85c303d57d6","added_by":"auto","created_at":"2024-10-18 12:29:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":255241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune response pathways of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. nana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e to the mouse intestine. \u003c/strong\u003e\u003cem\u003eH. nana\u003c/em\u003eadults or intraperitoneally injected ESP were recognized by the corresponding receptors on tuft cells, which could secrete IL-25 to act on ILC2, and ILC2 secreted the type 2 cytokine IL-4, 5, 9, 13. IL-13 could act on the differentiation of ISC into more goblet cells and tuft cells, and Paneth cells secreted relevant signaling factors to maintain the homeostasis of ISC to repel parasites. These phenomena were suppressed by injection of RCM-1, an inhibitor of IL-13.\u003c/p\u003e","description":"","filename":"OnlineFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5275142/v1/d5c1471e078eced0f18c9a9b.png"},{"id":78688995,"identity":"60083934-161d-48c9-9ac8-2602808238bb","added_by":"auto","created_at":"2025-03-17 16:09:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13012700,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5275142/v1/6b4d93b4-f6e8-428f-9090-bfb63de01f13.pdf"},{"id":66960214,"identity":"f2b33905-f86e-42e2-ba85-852d82f85c56","added_by":"auto","created_at":"2024-10-18 12:29:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17042859,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: Figure S1.\u003c/strong\u003e \u003cstrong\u003eEffects of different dose of ESP on mouse intestinal goblet cells and tuft cells.\u003c/strong\u003e ESP1 indicates an intraperitoneal dose of 25 μg/day per mouse and ESP2 indicates a dose of 50 μg/day per mouse.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative images of AB-PAS-stained with the goblet cells (sharp or deep blue pointed by red arrows, scale bars 100 μm for the upper panel, and 50 μm for the lower panel).\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Representative images of IF with MUC2 (green) and the nucleus (DAPI, blue) (scale bars 100 μm). (\u003cstrong\u003eC\u003c/strong\u003e) Representative images of IHC with Dclk1 (brown pointed by black arrows, scale bars 100 μm for the upper panel, and 50 μm for the lower panel). (\u003cstrong\u003eD\u003c/strong\u003e) Representative images of IF with Dclk1 (green) and the nucleus (DAPI, blue) (scale bars 100 μm). Percentages of the statistics of AB-PAS-stained positive area (\u003cstrong\u003eE\u003c/strong\u003e), and the number of goblet cells (\u003cstrong\u003eF\u003c/strong\u003e), tuft cells (\u003cstrong\u003eH\u003c/strong\u003e), and Dclk1-positive area (\u003cstrong\u003eG\u003c/strong\u003e) were semi-quantified using Image J software. Data are presented as mean + SD for (E)-(H), \u003cem\u003en\u003c/em\u003e = 6 per group, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2: Figure S2. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. nana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection model and ESP identification.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Representative picture of the hamsters from an urban pet market. (\u003cstrong\u003eB\u003c/strong\u003e) The egg of \u003cem\u003eH. nana\u003c/em\u003e (detected from the feces of mice) (scale bar 20 μm). (\u003cstrong\u003eC\u003c/strong\u003e) Adult worms were dissected from the intestines of \u003cem\u003eH. nana\u003c/em\u003e-infected mice. (\u003cstrong\u003eD\u003c/strong\u003e) Immunoblotting result of ESP. M: protein marker; Lane 1-4: ESP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 3: Figure S3. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. nana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection causes a decrease in the number of ISC and ESP causes an increase in ISC. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative images of IF with Lgr5 (green) and the nucleus (DAPI, blue) (scale bars 100 μm). (\u003cstrong\u003eB\u003c/strong\u003e) Representative images of WB presented the expression level of Lgr5. Percentages of the number of ISC (\u003cstrong\u003eC\u003c/strong\u003e) and the relative expression of Lgr5 (\u003cstrong\u003eD\u003c/strong\u003e) were semi-quantified using Image J software. (\u003cstrong\u003eE\u003c/strong\u003e) The transcription levels of \u003cem\u003eLgr5\u003c/em\u003e and the relative quantification were determined using the 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e method normalized to \u003cem\u003eGAPDH\u003c/em\u003e. Data are presented as mean + SD for (C)-(E), \u003cem\u003en\u003c/em\u003e =7 per group for (C), \u003cem\u003en\u003c/em\u003e =6 per group for (D)-(E), ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 4: Table S1. Primer sequences used for RT-qPCR experiments of current study\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5275142/v1/25acdf311f71edf004f7e52d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Adult Hymenolepis nana and its excretory-secretory products elicit mouse immune responses via Tuft/IL-13 signaling pathway","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. \u003cem\u003eHymenolepis nana\u003c/em\u003e (\u003cem\u003eH. nana\u003c/em\u003e) infection boosts tuft cells in the small intestine, implicating their role in host defense. Meanwhile, \u003cem\u003eH. nana\u003c/em\u003e has a dual impact on intestinal stem cells (ISC): Infection reduces ISC, but excretory-secretory products (ESP) promote the proliferation of ISC. ESP\u0026apos;s growth stimulus is countered by physical damage, resulting in an overall ISC decrease.\u003c/p\u003e\n\u003cp\u003e2. After \u003cem\u003eH. nana\u003c/em\u003e infection, the host drives increased secretion of mucins, antimicrobial peptides, and cytokines through the Tuft/IL-13 pathway to expel the parasite, whereas the use of RCM-1 (the inhibitor of IL-13), prevented the host from generating effective immune responses to \u003cem\u003eH. nana\u003c/em\u003e, thus affecting the killing or excretion of\u003cem\u003e\u0026nbsp;H. nana\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e3. \u003cem\u003eH. nana\u003c/em\u003e and ESP activate the Tuft/IL-13 pathway, increasing the number of goblet, tuft, and Paneth cells. These cells secrete mucus and antimicrobials, protecting intestinal mucosa, hinting at \u0026lsquo;helminth therapy\u0026rsquo; potential for host gut health.\u003c/p\u003e"},{"header":"1. Background","content":"\u003cp\u003eParasitic worms are among the most common pathogens in nature. To complete their life cycle, these intestinal worms traverse host tissues, causing severe damage, which imposes a significant burden on global health systems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The host's resistance to intestinal pathogens relies on the immunoregulatory functions of immune cells. Innate lymphoid cells (ILC) are tissue-resident immune cells that are early responders to infection. The classic ILC subtypes are divided into three groups, group 1 ILC (ILC1) predominantly secretes IFN-γ, group 2 ILC (ILC2) predominantly secretes interleukin (IL)-13, and group 3 ILC (ILC3) predominantly secretes IL-22, and which host ILC predominates depends on the type of intestinal pathogen the host is infected with [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Tissue damage caused by intestinal worm infections triggers epithelial cells to produce alarmin cytokines IL-25 and IL-33, which in turn activate ILC2 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The cytokines secreted by ILC2 can also directly influence intestinal epithelial cells and various immune cells to drive specific immune responses [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The intestinal epithelium consists of various cell types responsible for nutrient absorption and providing a protective barrier, as well as being able to rapidly change its cellular composition to defend pathogen invasion. Intestinal epithelial cells undergo rapid renewal, with a turnover every 3\u0026ndash;5 days [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Intestinal stem cells (ISC) are primarily located in the intestinal crypts, and play a crucial role in the renewal process of intestinal epithelial cells, differentiating into all intestinal epithelial cell types, including enterocytes, enteroendocrine cells, tuft cells, goblet cells, and Paneth cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Paneth cells are the only differentiated cells in the crypts and are interspersed with the ISC, and secrete epidermal growth factor (EGF), Wnt3, and the Notch ligand Dll4 to maintain ISC homeostasis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIntestinal tuft cells are chemosensory epithelial cells that have garnered significant attention in the study of host-parasite interactions. Tuft cells are crucial for defending worms, as their numbers increase sharply during intestinal parasite infections. They are also the primary source of intestinal IL-25, which plays a role in controlling the number of ILC2 during worm infection [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. ILC2 can secrete type 2 cytokines, including IL-4, IL-5, IL-9, and IL-13 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Tuft cells serve as vital sentinels in the gastrointestinal tract, rapidly proliferating after exposure to type 2 cytokines and playing a key role in protecting against worm infections [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This ultimately forms a positive feed-forward loop, where IL-25 produced by tuft cells activates ILC2, and IL-13 produced by ILC2 induces the differentiation of ISC into tuft cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIL-13 acts as a crucial player in promoting goblet cell proliferation, mucus secretion, and smooth muscle activity. In response to intestinal parasites residing in the small intestine, IL-13 produced by immune cells increases and induces ISC to differentiate into tuft cells and goblet cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Goblet cells are key components of the host's defense against parasites, producing and releasing mucins that form a dense mucus layer on the surface of the intestinal mucosa, working alongside other cells to maintain intestinal homeostasis. After worm infection, the proliferation timelines of tuft cells and goblet cells are synchronized [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The damage caused by worms to the host is primarily divided into mechanical damage induced by the worms and the effects of a series of immunomodulatory molecules they secrete, collectively referred to as excretory-secretory products (ESP) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eHymenolepis nana\u003c/em\u003e (\u003cem\u003eH. nana\u003c/em\u003e) is a zoonotic parasite that parasitizes the intestines of humans and rodents, causing hymenolepiasis nana. Mild infections of \u003cem\u003eH. nana\u003c/em\u003e in humans have no obvious clinical symptoms, while severe infections manifest as abdominal pain, diarrhea, anemia, and fever [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. \u003cem\u003eH. nana\u003c/em\u003e infects people of all age groups, with a predominance of infections in children under 10 years old [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It is estimated that the number of infected people worldwide is estimated to be 50 to 75\u0026nbsp;million and is more pronounced in Asia, Africa, Southern / Eastern Europe, and Central / South America [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. After infection with \u003cem\u003eH. nana\u003c/em\u003e, the oncosphere in the eggs invades the intestinal villi, develops into cysticercoid on the 4th day of infection, and enters the intestinal lumen, where they mature on about the 12th day [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It is unclear how \u003cem\u003eH. nana\u003c/em\u003e relates to the host immune responses and whether tuft cells play a role in defense against \u003cem\u003eH. nana\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn the current study, we investigated the effects of \u003cem\u003eH. nana\u003c/em\u003e adult worm infection and intraperitoneal injection of adult \u003cem\u003eH. nana\u003c/em\u003e-derived ESP on the host. We found that both the adult worms and ESP of \u003cem\u003eH. nana\u003c/em\u003e could promote the number of tuft cells, goblet cells, and Paneth cells of the mouse small intestine, while simultaneously activating the Tuft/IL-13 signaling pathway, thereby influencing the immune responses in the mouse intestine.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animal experiments\u003c/h2\u003e \u003cp\u003e All animal experiments of the current study were approved by the Animal Ethics Committee of Guizhou Medical University (approve Nos. 2100346 and 2100347).\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 The hamsters and the acquisition of serum from \u003cem\u003eH\u003c/em\u003e. \u003cem\u003enana-\u003c/em\u003einfected hamster\u003c/h2\u003e \u003cp\u003eThe 4\u0026ndash;6 weeks male hamsters (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;30) were purchased from one pet market in Nanming District, Guiyang, China, in March 2024 (Fig. S2A). Hamsters were sacrificed under anesthesia to obtain intestinal parasites. A few parasites were randomly selected and cut with a scissor to obtain eggs. In addition, six parasites were randomly selected from those obtained and stained with carbolic acid red (configured with carmine), eggs and stained adults were observed using the fluorescence microscope (Eclipse 80i, Nikon Ltd., Japan). A few parasites were re-selected and worm DNA was extracted using a MolPure Cell / Tissue DNA Kit (Yesen, Shanghai, China), and PCR amplification for the \u003cem\u003eCOX-I\u003c/em\u003e gene of \u003cem\u003eH. nana\u003c/em\u003e followed by agarose gel electrophoresis. The \u003cem\u003eCOX-I\u003c/em\u003e primer sequence used in this process was listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 The C57BL/6J mice experiments\u003c/h2\u003e \u003cp\u003eThe C57BL/6J mice (specific-pathogen-free, female, 6\u0026ndash;8 weeks old, weighing approximately 22.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 g) used in this study were obtained from the Experimental Animal Center of Guizhou Medical University [SCXK (Jing) 2019-0010]. These mice were housed in a standard laboratory environment, devoid of parasitic contamination, with a regulated temperature range of 20\u0026ndash;22\u0026deg;C and a controlled 12-h light/dark cycle. After a seven-day period of acclimatization, the experiments commenced.\u003c/p\u003e \u003cp\u003eRCM-1 is a known inhibitor of IL-13, and its administration and dosing refer to previous literatures [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The mice were randomly allocated into five groups: Control (Ctrl, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8), ESP (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8), ESP\u0026thinsp;+\u0026thinsp;RCM-1 (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8), \u003cem\u003eH. nana\u003c/em\u003e (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8), and \u003cem\u003eH. nana\u003c/em\u003e\u0026thinsp;+\u0026thinsp;RCM-1 (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8). All mice received sterilized H\u003csub\u003e2\u003c/sub\u003eO for 14 days. Starting from the first day, the ESP\u0026thinsp;+\u0026thinsp;RCM-1 and \u003cem\u003eH. nana\u003c/em\u003e\u0026thinsp;+\u0026thinsp;RCM-1 groups received daily intraperitoneal injections of 1.7 mg/kg RCM-1 (Selleck, Shanghai, China) for seven consecutive days. Our preliminary experiments indicated that 50 \u0026micro;g/day of ESP was more effective than 25 \u0026micro;g/day (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), hence we selected the dose of 50 \u0026micro;g/day for the ESP and ESP\u0026thinsp;+\u0026thinsp;RCM-1 groups, which were administered with intraperitoneal injections of ESP from the 7th day onwards for seven days. Based on preliminary findings, the \u003cem\u003eH. nana\u003c/em\u003e and \u003cem\u003eH. nana\u003c/em\u003e\u0026thinsp;+\u0026thinsp;RCM-1 groups were orally inoculated with 2,000 eggs per mouse on the first day, as this dose resulted in the optimal infection rate. All mice were euthanized on the 14th day.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2 The extraction and identification of adult \u003cem\u003eH. nana\u003c/em\u003e-derived excretory-secretory products (ESP)\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eH. nana\u003c/em\u003e, retrieved from hamsters, underwent rigorous cleaning procedures involving multiple rinses with sterilized PBS. Subsequently, 20\u0026ndash;30 adult worms were immersed in RPMI 1640 medium, supplemented with 1% Penicillin/Streptomycin/Amphotericin B. To concentrate the ESP-rich medium, centrifugation at 4,000 x g was executed utilizing a 10 kDa ultrafiltration tube (Millipore, Billerica, MA, USA), with the solvent subsequently exchanged for PBS. Sterility was ensured by passing the ESP solution through a 0.22 \u0026micro;m filter, and the protein content was quantified using a BCA protein assay. The prepared ESP was then stored at -80\u0026deg;C for future applications. In parallel, the protein composition of the ESP was scrutinized through immunoblotting. In brief, equal quantities of ESP samples were loaded into each well, and electrophoresis was performed. The resolved proteins were then transferred onto a PVDF membrane, which was blocked with 5% skim milk to prevent nonspecific antibody interactions. The membrane was probed overnight with a primary antibody (serum from the \u003cem\u003eH. nana\u003c/em\u003e-infected hamster, diluted 1:50), followed by the addition of an anti-mouse secondary antibody (diluted 1:10,000) and a 1 h incubation. Finally, the protein bands were visualized using Super ECL Detection Reagent, allowing for the assessment of the number and molecular size of proteins present in the ESP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Ileum hematoxylin-eosin (H\u0026amp;E) staining and alien-blue and periodic acid-Schiff (AB-PAS) staining\u003c/h2\u003e \u003cp\u003eThe ileum tissues of the mice were collected and fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 \u0026micro;m thick sections. According to the reagent instructions to stain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 RT-qPCR\u003c/h2\u003e \u003cp\u003eGenomic RNA was extracted from mouse ileum tissue using the Trizol protocol. Subsequently, the genomic RNA was reversely transcribed into cDNA, and the amplification was detected using the SYBR RT-qPCR Kit (Yesen, Shanghai, China) in a real-time fluorescence quantitative PCR instrument (CFX96, Bio-Rad, Hercules, CA, USA). The amplification protocol consisted of an initial denaturation step at 95\u0026deg;C for 5 min, followed by 39 cycles of denaturation at 95\u0026deg;C for 10 s, and annealing at 60\u0026deg;C for 30 s. The primer sequences were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 IHC and IF\u003c/h2\u003e \u003cp\u003eFor IHC, the sections were incubated overnight with primary antibodies specific for MUC2 (1:2,000), Dclk1 (1:200), IL-13 (1:200), Olfm4 (1:200), GATA3 (1:200), and lysozyme (lyz) (1:2,000). On the next day, the sections were incubated with the appropriate secondary antibody (Anti-rabbit, 1:500), followed by DAB staining. Finally, the sections were mounted with neutral gum and observed using a slide scanner (Olympus SLIDEVIEW VS200, Japan). For IF, the sections were incubated overnight with primary antibodies with respective specificity for Lgr5 (1:100), Olfm4 (1:200), MUC2 (1:500), Dclk1 (1:200), and lysozyme (lyz) (1:250). The next day, the sections were incubated with fluorescent secondary antibody 488 (1:200) and DAPI solution. Finally, they were mounted with an anti-fade mounting medium and observed using an upright fluorescence microscope (Eclipse 80i, Nikon Ltd, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 WB\u003c/h2\u003e \u003cp\u003eThe mouse ileum tissues were digested with RIPA lysis buffer. After measuring the concentration, an equal amount of protein samples was loaded and isolated using SDS-PAGE. Next, transfer the proteins onto a PVDF membrane. After blocking with 5% non-fat milk, incubate the membrane with the primary antibody for Lgr5 (1:1,000) at 4\u0026deg;C overnight. Then incubated with the secondary antibody anti-rabbit (1:10,000) for 1 h at room temperature. Finally, Super ECL Detection Reagent was utilized for sensitive detection of the protein and observed using a Chemiluminescence imager (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis and graphical representation were performed using GraphPad Prism (Version 9.5.1). The data were presented as mean\u0026thinsp;+\u0026thinsp;SD. Measurements were first subjected to normality tests, and the Homogeneity of Variance Test was performed between groups. Variance irregularities using the Mann-Whitney U-test. One-way analysis of variance (ANOVA) and t-test were used to compare differences between groups. The \u003cem\u003ep\u003c/em\u003e value less than 0.05 means a statistically significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Experimental design and identification of \u003cem\u003eH. nana\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThrough intraperitoneal injection of ESP in mice, it was found that a dose of 50 \u0026micro;g/day increased the number of goblet cells and tuft cells in the small intestines of mice more than a dose of 25 \u0026micro;g/day (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Therefore, this dosage was selected for subsequent experiments to study the interaction between ESP and the host. To investigate the impact of \u003cem\u003eH. nana\u003c/em\u003e on the intestinal immune responses in mice, the following experimental approach was adopted (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Microscopic examination revealed that the eggs were nearly spherical with a relatively thin eggshell, containing a thicker embryonic membrane within, and inside the membrane, an oncosphere with visible small hooks was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). After staining the adult worms with carboxyl borate red, the entire worm body appeared red, with the scolex showing a rostellum with distinct hooks, as well as visible circular suckers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The mature proglottid exhibited reproductive systems such as testes and ovaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), and the gravid proglottids were filled with eggs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). PCR amplification and agarose gel electrophoresis of the \u003cem\u003eH. nana COX-I\u003c/em\u003e gene showed a clear band at 202 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In summary, we confirmed that the parasites used in our experiments were \u003cem\u003eH. nana\u003c/em\u003e. Successful infection with \u003cem\u003eH. nana\u003c/em\u003e was determined by detecting eggs in fecal samples and dissecting adult worms from the intestinal lumen (Fig. S2B \u0026amp; C). We found a 100% infection rate for \u003cem\u003eH. nana\u003c/em\u003e, and RCM-1 administration increased the infection load of \u003cem\u003eH. nana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). The ESP harvested after culture was analyzed by immunoblotting, revealing a variety of proteins, with the main bands located at 20\u0026ndash;25 kDa, 35\u0026ndash;45 kDa, 45\u0026ndash;60 kDa, and 100\u0026ndash;140 kDa, respectively (Fig. S2D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 RCM-1 exacerbates the pathologic damage in the mouse intestine caused by \u003cem\u003eH. nana\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo investigate the effects of \u003cem\u003eH. nana\u003c/em\u003e infection and ESP on mouse intestinal pathology, we used H\u0026amp;E staining to observe changes in the small intestinal epithelial villi and performed IHC to detect the expression of mucin MUC2. Intestinal parasitic infections can cause atrophy of the small intestinal epithelial villi. We found that \u003cem\u003eH. nana\u003c/em\u003e infection resulted in the presence of adult worm segments in the intestinal lumen. Both \u003cem\u003eH. nana\u003c/em\u003e infection and intraperitoneal injection of ESP led to the shortening of intestinal epithelial villi. However, following RCM-1 intervention, the shortening of villi induced by ESP showed partial recovery, while the shortening induced by \u003cem\u003eH. nana\u003c/em\u003e infection was exacerbated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA \u0026amp; C). Goblet cell-derived mucin MUC2 plays a crucial role in maintaining intestinal homeostasis. Both \u003cem\u003eH. nana\u003c/em\u003e infection and ESP increased the secretion of mucin in the mouse intestine, and this increase could be inhibited by RCM-1. These results indicate that both adult \u003cem\u003eH. nana\u003c/em\u003e and its ESP contribute to pathological damage in the mouse intestine, and RCM-1 intervention exacerbates pathological damage caused by \u003cem\u003eH. nana\u003c/em\u003e in mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Adult\u003c/b\u003e \u003cb\u003eH. nana\u003c/b\u003e \u003cb\u003eand its ESP can increase the number of small intestinal goblet cells through IL-13\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGoblet cells produce a number of effector molecules including a range of mucins and\u003c/p\u003e \u003cp\u003eantimicrobial proteins, which enable these to play a key part in innate defense mechanisms in the gut, against both bacterial and helminth infections [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The effect of \u003cem\u003eH. nana\u003c/em\u003e on mouse intestinal goblet cells was investigated using AB-PAS staining of goblet cells, IF, and RT-qPCR methods to detect the goblet cell marker (MUC2). AB-PAS staining revealed goblet cells in the small intestinal epithelial villi stained blue-purple. The results showed that \u003cem\u003eH. nana\u003c/em\u003e promoted an increase in the number of goblet cells in the intestine. However, after the RCM-1 intervention, the number of goblet cells decreased. This finding was corroborated by IF staining and RT-qPCR results for MUC2, which also indicated that RCM-1 inhibited the increase in goblet cells induced by both adult \u003cem\u003eH. nana\u003c/em\u003e worms and ESP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results suggest that adult \u003cem\u003eH. nana\u003c/em\u003e and its ESP may increase the number of goblet cells through IL-13.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 RCM-1 prevents the involvement of small intestinal tuft cells in defense against\u003c/b\u003e \u003cb\u003eH. nana\u003c/b\u003e \u003cb\u003ein mice\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRecent studies have found that tuft cells play a first-line defense role in certain intestinal parasitic infections [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], but research on tuft cells in \u003cem\u003eH. nana\u003c/em\u003e infections is still lacking. IHC and IF were assayed for tuft cell marker double cortin-like kinase-1 (Dclk1), and the results showed that both \u003cem\u003eH. nana\u003c/em\u003e adults and ESP significantly increased the number of tuft cells in the intestine, while RCM-1 inhibited this increase. These findings were confirmed by RT-qPCR results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-E). The cytokines \u003cem\u003eIL-25\u003c/em\u003e secreted by tuft cells and \u003cem\u003eIL-33\u003c/em\u003e secreted by intestinal epithelial cells act on downstream ILC2 to exert their effects. The results showed that \u003cem\u003eH. nana\u003c/em\u003e infection and ESP both promoted an increase in the secretion of \u003cem\u003eIL-25\u003c/em\u003e and \u003cem\u003eIL-33\u003c/em\u003e. These findings indicate that \u003cem\u003eH. nana\u003c/em\u003e increases the number of tuft cells in the mouse intestine and suggest that tuft cells are involved in mice's defense against \u003cem\u003eH. nana\u003c/em\u003e, and the process can be suppressed by RCM-1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5\u003c/b\u003e \u003cb\u003eH. nana\u003c/b\u003e \u003cb\u003einduces ILC2 secretion of type 2 cytokines in mouse small intestine, which is suppressed by RCM-1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eType 2 immune responses are essential for protection against intestinal helminth infections, with inadequate secretion of type 2 cytokines affecting parasite elimination. The transcription factor GATA3 was upregulated in ILC2 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and the number of GATA3 cells was examined by IHC, which revealed that both \u003cem\u003eH. nana\u003c/em\u003e and ESP caused an increase in GATA3 cells in the mouse intestine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026amp;C). To investigate whether IL-25 and IL-33 promote the secretion of type 2 cytokines by downstream ILC2, we assessed changes in IL-13 expression using IHC and RT-qPCR. The results revealed that both \u003cem\u003eH. nana\u003c/em\u003e infection and ESP led to increased secretion of IL-13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026amp;D\u0026amp;E). Decreased expression levels of IL-13 after intervention with the IL-13 inhibitor RCM-1. Additionally, RT-qPCR analysis of other type 2 cytokines, including \u003cem\u003eIL-4\u003c/em\u003e, \u003cem\u003eIL-5\u003c/em\u003e, and \u003cem\u003eIL-9\u003c/em\u003e, the results showed that RCM-1 could inhibit the increase in gene expression induced by \u003cem\u003eH. nana\u003c/em\u003e infection or ESP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-H). These findings indicate that \u003cem\u003eH. nana\u003c/em\u003e infection and ESP may increase ILC2, which stimulates increased secretion of type 2 cytokines, including IL-13. In contrast, RCM-1 causes a decrease in type 2 cytokine secretion, which affects the excretion of \u003cem\u003eH. nana.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.6\u003c/b\u003e \u003cb\u003eH. nana\u003c/b\u003e \u003cb\u003ehas a dual effect on the number of ISC: infection reduces and ESP promotes the proliferation, and RCM-1 intervention exacerbates the reduction of ISC\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe effects of \u003cem\u003eH. nana\u003c/em\u003e infection on the number of ISC remain unclear. To assess changes in ISC numbers, we measured the expression of the ISC marker olfactomedin 4 (Olfm4). The results indicated that \u003cem\u003eH. nana\u003c/em\u003e infection leads to a reduction in ISC numbers, whereas ESP has the opposite effect, increasing ISC numbers. Additionally, changes in another ISC marker of Lgr5 confirmed these findings (Fig. S3). Following intervention with RCM-1, the changes in ISC numbers induced by both \u003cem\u003eH. nana\u003c/em\u003e infection and ESP were consistent, showing a reduction in ISC numbers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This result suggests that \u003cem\u003eH. nana\u003c/em\u003e infection may decrease ISC numbers due to mechanical damage caused by the worms, while ESP actually promotes an increase in ISC numbers. After the RCM-1 intervention, the number of ISC was reduced.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.7 RCM-1 inhibits adult\u003c/b\u003e \u003cb\u003eH. nana\u003c/b\u003e \u003cb\u003eand its ESP-induced increase in the number of Paneth cells, which disrupts the homeostasis of ISC\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePaneth cells, located in the intestinal crypts, maintain ISC homeostasis by secreting related growth factors. Lysozyme (Lyz) is the first antimicrobial peptide discovered in Paneth cells and is widely used as a Paneth cell marker in the ileum [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Using IHC, IF, and RT-qPCR to detect Lyz, we found that both adult \u003cem\u003eH. nana\u003c/em\u003e worms and ESP increased lysozyme and the number of Paneth cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-E). Further RT-qPCR analysis of Paneth cell-secreted growth factors \u003cem\u003eWnt3\u003c/em\u003e, \u003cem\u003eEGF\u003c/em\u003e, and \u003cem\u003eDll4\u003c/em\u003e showed increased gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-H). IL-13 can promote the secretion of antimicrobial peptides by Paneth cells, and we also observed that inhibition of IL-13 resulted in a reduction in both the number of Paneth cells and the secretion of lysozyme. These results indicate that \u003cem\u003eH. nana\u003c/em\u003e promotes an increase in the number of Paneth cells, which in turn secrete growth factors to maintain ISC homeostasis. In contrast, RCM-1 decreases the number of Paneth cells, leading to decreased secretion of antimicrobial peptides and growth factors, thereby disrupting ISC homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe intestine is the target organ for most parasitic infections, including those worms and protozoa. These parasites typically activate type 2 immune responses in the host's immune system and have a significant impact on the local tissue microenvironment [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Worms are multicellular parasites that cause a range of adverse effects, such as malnutrition and developmental delays in children, contributing to the global public health burden [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Intestinal worms have evolved mechanisms to evade or suppress host defense responses to ensure their survival and reproduction, laying eggs in the intestinal tissues and being expelled through the host's feces [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. \u003cem\u003eH. nana\u003c/em\u003e, also known as the dwarf tapeworm, primarily parasitizes the intestines of humans and rodents. \u003cem\u003eH. nana\u003c/em\u003e has a global distribution, with an estimated infection rate of 1.2% in humans and up to 13% in rodents [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Conventional diagnostic methods for Hymenolepiasis nana involve fecal examination of eggs or adults. However, PCR-based molecular techniques can improve parasite detection rates and accurately determine species differentiation and genetic characteristics. Mitochondrial cytochrome c oxidase subunit I (\u003cem\u003eCOX-I\u003c/em\u003e) is an important mitochondrial gene with high variability and specificity among different parasites, making it one of the most commonly used molecular genetic markers [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIntestinal worms parasitize the host's intestines, causing a series of damages, including nutrient depletion, mechanical damage from the worm's body, and toxic effects from ESP. In the current study, we identified \u003cem\u003eH. nana\u003c/em\u003e through morphological and molecular biology techniques and characterized the main protein components of ESP using immunoblotting. We then explored the immune responses relationship between \u003cem\u003eH. nana\u003c/em\u003e and the host using \u003cem\u003eH. nana\u003c/em\u003e egg infections and intraperitoneal ESP injections. Our findings showed that adult \u003cem\u003eH. nana\u003c/em\u003e worms and ESP could activate the Tuft/IL-13 signaling pathway in the host, leading to an increase in the number of goblet cells, tuft cells, and Paneth cells, as well as an increase in type 2 cytokine secretion, ultimately aimed at clearing \u003cem\u003eH. nana\u003c/em\u003e. When RCM-1 was used, the host failed to produce effective immune responses to \u003cem\u003eH. nana\u003c/em\u003e, thereby exacerbating \u003cem\u003eH. nana\u003c/em\u003e infection in the host.\u003c/p\u003e \u003cp\u003eInfection with \u003cem\u003eH. nana\u003c/em\u003e in mice can lead to degeneration or destruction of the normal villous structure of the small intestine epithelium, resulting in villous shortening or atrophy [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Infection of rats with \u003cem\u003eH. nana\u003c/em\u003e results in enteritis, villous necrosis, and inflammatory infiltration of the mucosa and submucosa [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. MUC2 is primarily secreted by goblet cells, forming a mucosal barrier that protects the mucosal surface and plays a crucial role in regulating intestinal immune responses, maintaining mucosal health, and protecting the immune system [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our study found that both adult \u003cem\u003eH. nana\u003c/em\u003e worms and ESP caused the shortening of intestinal epithelial villi and an increase in mucin MUC2 secretion in mice. Intervention with the IL-13 inhibitor RCM-1 could restore villous shortening induced by ESP but exacerbated villous shortening caused by adult \u003cem\u003eH. nana\u003c/em\u003e worms. This might be due to RCM-1 increasing the severity of \u003cem\u003eH. nana\u003c/em\u003e infection, thereby intensifying damage to the host. The above results indicated that both adult \u003cem\u003eH. nana\u003c/em\u003e and ESP caused intestinal pathologic damage, and the greater the worm infection, the more severe the damage.\u003c/p\u003e \u003cp\u003eTuft cells occupy a small proportion of the intestinal epithelium but play a crucial role in recognizing and responding to parasitic infections [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. It has been observed that the number of tuft cells in the small intestine increases after intestinal parasite infection. Normally, tuft cells constitute only about 1% of the intestinal epithelial cells in mice, but their numbers can increase several-fold during parasitic infections [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Tuft cells have become key sentinels in parasitic infections by releasing the alarmin IL-25, which activates ILC2 and enhances the type 2 immune response, signaling the presence of worms [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The type 2 immune responses are protective for the host, not only by directly killing parasites in tissues or reducing their numbers by expulsion from the intestine, but also by protecting the host from tissue damage caused by large extracellular parasites migrating within the body [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The regulation of signaling pathways between tuft cells and ILC2 has garnered significant attention in parasitic intestinal infections. Tuft cells can recognize infections from \u003cem\u003eNippostrongylus brasiliensis\u003c/em\u003e (\u003cem\u003eN. brasiliensis\u003c/em\u003e), \u003cem\u003eTrichinella spiralis\u003c/em\u003e (\u003cem\u003eT. spiralis\u003c/em\u003e), and \u003cem\u003eHelicotylenchus\u003c/em\u003e, releasing IL-25 to increase ILC2 numbers and subsequently IL-13 secretion, which acts on ISC to differentiate into more tuft and goblet cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. IL-33 is a tissue-derived nuclear cytokine that promotes type 2 immune responses during allergy and helminthic infection [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our study found that both adult \u003cem\u003eH. nana\u003c/em\u003e worms and ESP induced increases in the numbers of goblet and tuft cells, along with elevated secretion of cytokines IL-25 and IL-33. Further investigation into the expression changes of type 2 cytokines revealed an increase in type 2 cytokines, including IL-13. The above series of changes can be inhibited by RCM-1, indicating that adult \u003cem\u003eH. nana\u003c/em\u003e worms and ESP could activate the signaling pathways between tuft cells and ILC2, and RCM-1-induced reduction in type 2 cytokine secretion affected host excretion of \u003cem\u003eH. nana\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eISC are located in the intestinal crypts and possess high proliferative and differentiative capacities. The proliferation and differentiation of ISC ensure the integrity and functional stability of the intestinal epithelium and contribute to the prevention of intestinal diseases [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. A recent study has shown that ISC depletion occurs locally in mice infected with \u003cem\u003eHeligmosomoides polygyrus\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. ISC can differentiate into mature Paneth cells, unlike other mature intestinal epithelial cells, they migrate downward after differentiation and reside at the base of the crypts, where they are arranged in a cross pattern with ISC [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Following the parasitic invasion of the host intestine, ISC accelerate their proliferation and differentiation, stimulating the proliferation of Paneth cells in response to the infection [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Paneth cells can secrete antimicrobial peptides to maintain intestinal homeostasis [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and regulate ISC function through the secretion of growth factors such as Wnt3, EGF, and Dll4 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Research on the impact of intestinal worm infections on host small intestinal Paneth cells is relatively limited. The study has shown that \u003cem\u003eT. spiralis\u003c/em\u003e and \u003cem\u003eN. brasiliensis\u003c/em\u003e infections cause a significant increase in Paneth cell numbers [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In addition, IL-13 promotes ISC proliferation and differentiation on the one hand and enhances the secretion of antimicrobial peptides by Paneth cells on the other [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The effects of \u003cem\u003eH. nana\u003c/em\u003e infection on ISC and Paneth cells remain unclear. Our research findings indicated that the adults of \u003cem\u003eH. nana\u003c/em\u003e reduced the number of ISC, and RCM-1 exacerbated this effect, likely due to mechanical damage caused by \u003cem\u003eH. nana\u003c/em\u003e adults in the intestinal lumen. ESP increased the number of ISC, but blocking the IL-13 signaling pathway resulted in a decrease in the number of ISC, suggesting that ESP-induced IL-13 promotes ISC proliferation. Both \u003cem\u003eH. nana\u003c/em\u003e adults and ESP increased lysozyme and Paneth cell numbers, consistent with IL-13 changes. This suggested that Paneth cells secrete signaling factors to maintain ISC homeostasis and that IL-13 also promoted Paneth cell proliferation, which increased lysozyme secretion and maintained intestinal homeostasis.\u003c/p\u003e \u003cp\u003eCollectively, we found that \u003cem\u003eH. nana\u003c/em\u003e adults and its ESP both promoted the increase in goblet cells, tuft cells, Paneth cells, and type 2 cytokines, while IL-13 also facilitated the differentiation of ISC. This finding suggested that the infection with \u003cem\u003eH. nana\u003c/em\u003e triggered the activation of the host's Tuft/IL-13 signaling pathway, eliciting the immune responses that facilitated the expulsion of \u003cem\u003eH. nana\u003c/em\u003e from the host.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, we found that \u003cem\u003eH. nana\u003c/em\u003e adults and its ESP could activate the Tuft/IL-13 signaling pathway in the mouse intestine, leading to the differentiation of ISC into more goblet and tuft cells, as well as an increase in Paneth cells to maintain the stability of ISC. These changes helped the host to expel the parasites. In contrast, RCM-1 suppressed the host's immune responses to \u003cem\u003eH. nana\u003c/em\u003e, resulting in the host's inability to effectively kill or exclude \u003cem\u003eH. nana\u003c/em\u003e. (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This research preliminarily revealed the role of tuft cells in defending against \u003cem\u003eH. nana\u003c/em\u003e and explored the relationship between \u003cem\u003eH. nana\u003c/em\u003e and the host's immune responses, providing a foundation for the prevention and treatment of \u003cem\u003eH. nana\u003c/em\u003e and its \u0026lsquo;helminth therapy\u0026rsquo;.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of interest\u003c/h2\u003e \u003cp\u003eThe authors have declared that no competing interests.\u003c/p\u003e \u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 82160398).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRM, XYC, YSL, and KZ designed the study. RM provided funding and revised the manuscript. XYC drafted the manuscript. XYC, YC, QYL, ZFZ, and WLW performed the experiments. RM, XYC, YSL, and KZ performed data analysis. JFL, YSL, and KZ revised the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKirk MD, Pires SM, Black RE, Caipo M, Crump JA, Devleesschauwer B, et al. World Health Organization Estimates of the Global and Regional Disease Burden of 22 Foodborne Bacterial, Protozoal, and Viral Diseases, 2010: A Data Synthesis. 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Science (New York, NY). 2021;374 6568:eabe6723; doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.abe6723\u003c/span\u003e\u003cspan address=\"10.1126/science.abe6723\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hymenolepis nana, tuft cell, excretory-secretory products, IL-13, RCM-1","lastPublishedDoi":"10.21203/rs.3.rs-5275142/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5275142/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHosts typically elicit diverse immune responses to the infection of various parasitic worms, with intestinal tuft cells playing a pivotal role in detecting parasite invasion. \u003cem\u003eHymenolepis nana\u003c/em\u003e (\u003cem\u003eH. nana\u003c/em\u003e), a zoonotic parasitic worm, resides in the host's intestine. The contribution and underlying mechanisms of tuft cell-mediated immune reactions against \u003cem\u003eH. nana\u003c/em\u003e remain unexplored.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study endeavors to examine the immune responses in the mouse intestine elicited by the adult \u003cem\u003eH. nana\u003c/em\u003e and its excretory-secretory products (ESP). Detection of various intestinal cell counts and cytokine changes using IHC, IF, RT-qPCR, etc.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe presence of adult \u003cem\u003eH. nana\u003c/em\u003e and its ESP enhances the population of tuft cells and goblet cells while fostering the production of type 2 cytokines, particularly IL-13. Furthermore, the surge in Paneth cells triggered by \u003cem\u003eH. nana\u003c/em\u003e aids in maintaining intestinal stem cells homeostasis. Notably, RCM-1, the specific IL-13 inhibitor, dampens intestinal stem cells differentiation and type 2 cytokine secretion, potentially impeding the host's capacity to eliminate \u003cem\u003eH. nana\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn conclusion, the adult \u003cem\u003eH. nana\u003c/em\u003e and its ESP stimulate the immune responses from the mouse intestinal mucosa via the Tuft/IL-13 signaling pathway, facilitating the expulsion of \u003cem\u003eH. nana\u003c/em\u003e from the host.\u003c/p\u003e","manuscriptTitle":"Adult Hymenolepis nana and its excretory-secretory products elicit mouse immune responses via Tuft/IL-13 signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-18 12:29:22","doi":"10.21203/rs.3.rs-5275142/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-18T22:45:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-18T16:04:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-05T12:22:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253893792991345410796469838580250778199","date":"2024-11-03T16:41:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"61202574846139470955928556036762650945","date":"2024-10-27T17:33:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-25T22:51:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-16T14:11:14+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-16T14:08:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2024-10-16T10:37:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"665983dd-7c23-47ce-9476-4ecd9fafad03","owner":[],"postedDate":"October 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-03-17T16:02:27+00:00","versionOfRecord":{"articleIdentity":"rs-5275142","link":"https://doi.org/10.1186/s13071-025-06719-w","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2025-03-11 15:57:48","publishedOnDateReadable":"March 11th, 2025"},"versionCreatedAt":"2024-10-18 12:29:22","video":"","vorDoi":"10.1186/s13071-025-06719-w","vorDoiUrl":"https://doi.org/10.1186/s13071-025-06719-w","workflowStages":[]},"version":"v1","identity":"rs-5275142","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5275142","identity":"rs-5275142","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}