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

doi:10.21203/rs.3.rs-5275142/v1

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

https://doi.org/10.21203/rs.3.rs-5275142/v1

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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

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.

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.

2.1 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 H₂O 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.

3.1 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. 1A). 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. 1B). 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. 1C). The mature proglottid exhibited reproductive systems such as testes and ovaries (Fig. 1D), and the gravid proglottids were filled with eggs (Fig. 1E). PCR amplification and agarose gel electrophoresis of the H. nana COX-I gene showed a clear band at 202 bp (Fig. 1F). 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. 1G). 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. 2A & 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. 4A-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. 5A&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. 5B&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. 5F-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. 7A-E). Further RT-qPCR analysis of Paneth cell-secreted growth factors Wnt3, EGF, and Dll4 showed increased gene expression (Fig. 7F-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.

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.

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’.

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

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No competing interests reported.

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

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

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Abstract

Background

Methods

Results

Conclusions

Figures

Highlights

1. Background

2. Materials and Methods

2.1 Animal experiments

2.1.1 The hamsters and the acquisition of serum from H. nana-infected hamster

2.1.2 The C57BL/6J mice experiments

2.2 The extraction and identification of adult H. nana-derived excretory-secretory products (ESP)

2.3 Ileum hematoxylin-eosin (H&E) staining and alien-blue and periodic acid-Schiff (AB-PAS) staining

2.4 RT-qPCR

2.5 IHC and IF

2.6 WB

2.7 Statistical analysis

3. Results

3.1 Experimental design and identification of H. nana

3.2 RCM-1 exacerbates the pathologic damage in the mouse intestine caused by H. nana

4. Discussion

5. Conclusions

Declarations

Conflicts of interest

Funding

Author Contribution

Data Availability

References

Additional Declarations

Supplementary Files

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Version 1