The included subjects were all patients with hepatic alveolar echinococcosis diagnosed for the first time, who met the requirements of surgery and did not undergo drug chemotherapy. The tissues were mainly from the surgical specimens of AE patients, and a total of 21 tissue specimens were collected. All 21 (12 females/9 males, mean age was 32 years) AE patients were initially diagnosed by B-ultrasound and were eligible for surgery for the first time. The surgically resected tissues of patients undergoing AE surgery have a complete structure, including lesion tissues, close to lesion tissues (3 cm from the lesion, CLT), and distant from the lesion tissues ($>$3 cm from the lesion, DLT). Tissue samples were analyzed by western blotting (WB), immunohistochemistry, immunofluorescence and Masson. 12 blood specimens were obtained from the patients before the above-mentioned surgeries for ELISA.

50 mL of healthy volunteers’ venous blood was diluted with PBS 1:1, and the diluent was slowly added into the centrifuge tube containing Percoll separation solution (Solarbio, Beijing, China, density 1.130 $\pm$ 0.005 g/mL) at 2:1. After centrifugation, the target cell layer was collected under the Percoll layer. Objective after destroying the red blood cells, the cells were centrifuged at 4 °C, 1500 rpm and 5 min to collect the bottom cells containing eosinophils and neutrophils. PBS was washed and counted. 50 µL anti-CD16 immunomagnetic beads (Cat. #: 130-092-010, Miltenyi biotech, Bergisch Gladbach, Germany) were added to 5 $\times$ 10⁷ cells, mixed well, and incubated in a shaker at 4~8 °C for 30 minutes. After incubation, the cell antibody solution was added to the LS magnetic column (Cat. #: 130-090-544, Miltenyi biotech, Bergisch Gladbach, Germany) and the eluate was collected. At 4 °C, 1500 rpm (400 g), centrifuge for 5 min, discard the supernatant, and resuspend the cells with 1 mL RPMI-1640 (containing 10% FCS), which is the eosinophil (Eos) obtained from separation and purification.

The lesions of Echinococcus multilocularis were isolated from Mongolian gerbils infected with Echinococcus multilocularis preserved in our laboratory. Mongolian gerbils were sacrificed (After anesthesia with isoflurane (concentration: 1.5–2%, Continuous inhalation was maintained for 2–3 minutes to achieve adequate anesthesia prior to the procedure), euthanasia was performed by cervical dislocation (neck fracture) to reduce the suffering of experimental animals), and the metacestode tissues were isolated from the abdominal cavity, and an appropriate amount of sterile normal saline was added to wash the blood water on the cyst surface repeatedly. After being crushed by a tissue homogenizer (OSE-Y50, TIANGEN, Beijing, China), it was centrifuged at 4 °C and 12,000 rpm for 20 min. The supernatant was filtered through a sterile filter (0.22 µm, Cat. #: SLGP033RB, Merck Millipore, Darmstadt, Germany), and the protein was quantified to 20 mg/mL by BCA (Cat. #: A55860, Thermo Fisher, Waltham, MA, USA) and stored at –80 °C. E. multilocularis antigen (E.m-Ag) was prepared for intervention in vitro. All animal experimental procedures were also approved by the Ethics Committee of Qinghai University Affiliated Hospital (Approval No.: P-SL-2019039). All efforts were made to minimize animal suffering and reduce the number of animals used in accordance with the ARRIVE guidelines.

Blood samples were obtained from 12 individuals with hepatic alveolar echinococcosis (HAE) (From patients diagnosed with HAE by B-ultrasound) and 12 healthy control participants (Healthy volunteers from the physical examination center) after obtaining informed consent. Samples were collected using anticoagulated tubes and plasma was separated by centrifugation at 2300 g for 15 minutes. The plasma samples were then stored at –80 °C. ELISA kits (Cat. #: JL19282-48T/JL18366-48T, J&L Biological, Shanghai, China) were used to measure serum levels of secreted ST-2 and IL-33, with a standard curve correlation coefficient of R² = 0.998.

Tissue samples (n = 15) were obtained from individuals with HAE, including close to lesion tissues (3 cm from the lesion, CLT) and distant from the lesion tissues ($>$3 cm from the lesion, DLT). Total protein was extracted and adjusted to a concentration of 3 mg/µL, with 15 µg loaded into each well. After electrophoresis, protein transfer, and blocking with 10% skimmed milk powder at room temperature for 1 hour, membranes were incubated overnight at 4 °C with primary antibodies (antibody concentration: IL-33 (1:1000, ab207737, Abcam, Cambridge, UK), ST-2 (1:2500, ab25877, Abcam, Cambridge, UK)). After equilibration to room temperature, secondary antibody (1:10,000, Cat. #: C31460100, Thermo Fisher, MA, USA) incubation was conducted at 37 °C for 90 minutes. Each step was followed by three washes with PBS containing 0.1% Tween-20. Protein bands were visualized using a Bio-Rad chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA), and band intensity was analyzed using NIH Image software (version 1.53; NIH, Bethesda, MD, USA, https://imagej.nih.gov/ij/).

Surgically excised lesion tissues (n = 15) were fixed in 4% formaldehyde, dehydrated, embedded in paraffin, and sectioned into 3 µm slices. The sections were baked at 60 °C for 30 to 45 minutes, followed by sequential deparaffinization and rehydration using xylene I and II (15 minutes each), absolute ethanol I and II (5 minutes each), 95% ethanol I and II (5 minutes each), 90% ethanol I and II (5 minutes each), and 80% ethanol I and II (5 minutes each). Antigen retrieval was performed using high-pressure or microwave treatment for 10 to 15 minutes in 0.5M EDTA (pH 8.0) retrieval solution. Target areas were circled using a histochemical pen, followed by sequential incubation with an endogenous peroxidase blocker and a non-specific staining blocker (serum or 5% skimmed milk) for 10 minutes at room temperature. Primary antibody (antibody concentration: IL-33 (1:800), ST-2 (1:500)) incubation occurred overnight at 4 °C, followed by secondary antibody incubation at 37 °C for 1 hour after reaching room temperature. Streptavidin-peroxidase was applied for 10 minutes. Each step was followed by three washes with PBS for 3 minutes. DAB (Cat. #: YT075, Biolab, Beijing, China) was used for color development, followed by hematoxylin counterstaining, with staining duration monitored under a microscope (TISSUEFAXS SPECTRA, TissueGnostics, Vienna, Austria). Slides were rinsed with tap water for bluing, sealed, and examined. The following antibodies were utilized: anti-ST-2 antibody (ab25877) (1:500, Abcam, Cambridge, UK) and anti-IL-33 antibody (ab207737) (1:800, Abcam, Cambridge, UK).

Pathological tissue (n = 7) processing, deparaffinization, hydration, and antigen retrieval were conducted as described in the immunohistochemistry protocol. Following antigen retrieval, tissue sections were immersed in a blocking solution for 10 minutes. Primary antibody (antibody concentration: IL-33 (1:2000), ST-2 (1:50)) incubation was carried out for 1 hour at room temperature or overnight at 4 °C, followed by secondary antibody incubation for 10 minutes. Fluorescent dye was applied at room temperature for 10 minutes, followed by antigen retrieval. For multiple fluorescence staining, each staining round was verified using a fluorescence microscope before proceeding to the next step. After all staining procedures were completed, nuclear staining was performed with DAPI working solution at 37 °C for 5 minutes. Slides were mounted with an anti-fading fluorescence quenching mounting medium and examined under a fluorescence microscope (Cytation5, BioTek, Winooski, VT, USA). Eosinophils were defined as CD66⁺CD16⁻ cells. The following antibodies were used: anti-ST-2 antibody (ab25877), anti-IL-33 antibody (ab207737) (Abcam, Cambridge, UK), CD66 (Cat. #: MA5-48180, Thermo Fisher, MA, USA), and CD16 (Cat. #: MA5-36143, Thermo Fisher, MA, USA).

Masson’s trichrome staining was conducted using a staining kit (Cat. #: G1340, Solarbio, Beijing, China). Tissue (n = 15) processing, including hydration, was performed as described for immunohistochemistry. Sections were stained with Weigert’s iron hematoxylin for 5 to 10 minutes and differentiated with acidic ethanol for 1 to 2 seconds. Masson’s blue solution was applied for blue staining, followed by ponceau S staining for 5 to 10 minutes and immersion in phosphomolybdic acid solution for 1 to 2 minutes. After aniline blue staining for 1 to 2 minutes, sections were washed with a weak acid working solution for 1 minute. The sections were then dehydrated, cleared, mounted, and analyzed.

Eosinophils (EOL-1) were cultured in RPMI-1640 complete medium until stabilization. After cell counting, a well-mixed cell suspension was transferred to the upper chamber of Transwell plates (Cat. #: 140620, Thermo Fisher, USA) (450 µL per well, 3 $\times$ 10⁵ cells/mL), while the lower chamber contained complete culture medium with induction factors. The experiment included the following groups: blank control (PBS, 50 µL/mL; n = 5), E.m-Ag (20 mg/mL; n = 5), and IL-33 (3 ng/mL; ab281811, Abcam, Cambridge, UK) treatment groups (n = 5). Cells were incubated for 24-hours in a CO₂ incubator (MCO-20AIC, SANYO, Osaka, Japan). Samples were centrifuged at 110 g for 5 minutes and the supernatant was discarded. An equal volume of PBS was added, followed by DAPI staining. The mixture was thoroughly mixed, and 100 µL was examined. The number of eosinophils in the lower chamber was detected by the multifunctional cell imaging microplate detector (Cytation5, Biotek, Winooski, VT, USA), and the bright field count and DAPI count were carried out at the same time. The average value of the single chamber bright field count and DAPI count was taken as the final value. This experiment was independently repeated three times.

EOL-1 (5 $\times$ 10⁵ cells/mL) were cultured in 10% FBS (Cat. #: A5669701, Thermo Fisher, USA) RPMI-1640 (Cat. #: PM150110A, Procell Life Science & Technology Co., Ltd., Wuhan, Hubei, China) complete medium until stabilization. After counting, the well-mixed cell suspension was evenly divided into three groups (n = 5), each containing five samples. Following a 48-hour stimulation with various factors—recombinant human IL-33 protein (active) (3 ng/mL) (ab281811, Abcam, Cambridge, UK), E.m-Ag, or PBS—cells were incubated with green fluorescent E. coli (1:500) on a shaker for 2 hours. After centrifugation and PBS washing to remove unselected E. coli, the isolated eosinophils were analyzed using flow cytometry (FACSCelesta, Becton, Dickinson & Co., Franklin Lakes, NJ, USA). This experiment was independently repeated three times.

Human hepatic stellate cells (HSCs) LX-2 (Cat. #: CL-0560, Procell Life Science & Technology Co., Ltd., Wuhan, Hubei, China) were cultured in DMEM (Cat. #: PM150210, Procell Life Science & Technology Co., Ltd., Wuhan, Hubei, China) medium until stabilization. After digestion, cells (5 $\times$ 10⁶ cells/mL) were thoroughly mixed and divided into five groups (n = 5), each containing five plates. After 24 hours, the following treatments were applied: blank control (no additions), positive control (E.m-Ag), eosinophil intervention, IL-33 adenovirus intervention (constructed by Cyagen Biosciences Inc., Santa Clara, CA, USA), and eosinophil + IL-33 virus intervention. After 72 hours of culture, the culture medium and suspended eosinophils were removed, and the plates were washed three times with PBS. Adherent HSCs were digested and collected. Total protein was extracted, quantified, and $\alpha$-SMA (1:1500, Cat. #: MA1-06110, Thermo Fisher, MA, USA) expression was analyzed by Western blot. This experiment was independently repeated three times. All cell lines were validated by STR profiling and tested negative for mycoplasma.

Immunohistochemistry images were analyzed using the TISSUE CONSTICS Analysis System (version 7.1; StrataQuest, Austria, https://tissuegnostics.com), following panoramic scanning. Multiple immunofluorescence and Masson’s trichrome staining were analyzed using ImageJ (version 1.53; NIH, Bethesda, MD, USA, https://imagej.nih.gov/ij/). Data are expressed as mean $\pm$ standard deviation (SD). Statistical analysis and graphing were conducted using GraphPad Prism 8.2.1 software (GraphPad, Inc., La Jolla, CA, USA, https://www.graphpad.com). Student’s t-test was used for comparisons between two groups, and one-way analysis of variance (ANOVA) was applied for comparisons among multiple groups. A significance level of $\alpha$ = 0.05 was applied.

Histopathological examination of HAE lesions indicated a distinctive inflammatory fibrous band surrounding the lesion, forming a unique microenvironment with diverse biological functions. To investigate the potential role of IL-33 and ST-2 expression in this region, their expression levels were assessed in tissue sample areas close to the lesion (CLT) (3 cm) and distant from the lesion (DLT) ($>$3 cm). Western blot analysis showed significantly higher expression of ST-2 and IL-33 in CLT compared to DLT (Fig. 1A). Grayscale analysis of protein bands confirmed statistically significant differences (p 0.05), indicating that the IL-33/ST-2 signaling pathway may contribute to local immune regulation in hepatic echinococcosis.

Fig. 1.

Western blot analysis of IL-33 and ST-2 expression in CLT and DLT (n = 15), along with ELISA quantification of serum IL-33 and sST-2 levels in patients with AE and healthy controls (n = 12). (A) Western blot analysis of IL-33 and ST-2 expression in CLT and DLT (n = 15) with grayscale intensity quantified using ImageJ. (B) ELISA-based evaluation of serum IL-33 and ST-2 levels in patients with AE and healthy controls (n = 12). Data are expressed as mean $\pm$ standard deviation. *p 0.05 indicates statistical significance. CLT, close to lesion tissues; DLT, distant from the lesion tissues; ELISA, enzyme-linked immunosorbent assay; IL-33, Interleukin-33; AE, alveolar echinococcosis.

IL-33 is widely recognized as an “alarm” cytokine in inflammatory diseases. To assess its systemic involvement in HAE, plasma levels of IL-33 and sST-2 expression were measured in patients with AE and compared to healthy individuals using ELISA. As shown in Fig. 1B, plasma concentrations of sST-2 and IL-33 were significantly higher in AE patients than in healthy individuals (p 0.05), suggesting that the IL-33/ST-2 signaling pathway is activated in the systemic immune response to hepatic echinococcosis.

Western blot analysis indicated elevated IL-33 and ST-2 expression in CLT. To further investigate the spatial distribution of IL-33 and ST-2, immunohistochemical staining was performed on tissue samples from both CLT and distant from the lesion tissue (DLT) in AE patients. After panoramic scanning, analysis was conducted using the TISSUE CONSTICS Analysis System (Fig. 2). The software classified negative cells (green) and positive cells (red) in DLT and CLT for statistical analysis. Results demonstrated that the proportion of IL-33 (Fig. 2B) and ST-2 (Fig. 2A) positive cells in CLT was significantly higher than in DLT (p 0.05), suggesting that IL-33 and ST-2 expression contributes to immune regulation within the AE lesion microenvironment.

Fig. 2.

Immunohistochemical analysis of IL-33 and ST-2 expression in CLT and DLT (n = 15). (A) Representative immunohistochemical images showing ST-2 expression in DLT and CLT. (B) Immunohistochemical images depicting IL-33 expression in DLT and CLT. Quantification was performed using the TISSUE CONSTICS Analysis System, which identified and measured negative cells (green) and positive cells (red). Scale bar: 100 µm. Data are expressed as mean $\pm$ standard deviation. *p 0.05 indicates statistical significance.

Eosinophils play a crucial role in various parasitic infections, yet their role in AE remains unclear. Since ST-2, the receptor for IL-33, is widely expressed on eosinophils, this study investigated the co-localization of IL-33 and ST-2 with eosinophils within AE lesions. Immunofluorescence staining (Fig. 3) showed higher IL-33 and ST-2 expression (green) in CLT compared to DLT, aligning with immunohistochemistry findings (Fig. 2). Furthermore, analysis of IL-33 expression and eosinophil distribution (Fig. 3A) (yellow) exhibited a strong correlation. The color distribution in the deviation plot generated by ImageJ analysis was relatively uniform (Fig. 3B), indicating a high degree of co-localization between the two groups (R = 0.6835). This co-localized expression in CLT was significantly higher than in DLT (p 0.05). Similarly, ST-2 expression and eosinophil distribution exhibited an even stronger co-localization (R = 0.8127) (Fig. 3B), with significantly greater ST-2 -eosinophil co-localization in CLT compared to DLT (p 0.05). These findings suggest that IL-33 accumulation in the fibrotic infiltration area surrounding AE lesions may enhance eosinophil function through ST-2 binding. However, further in vitro experiments are necessary to confirm its specific effects on eosinophils and their biological functions.

Fig. 3.

Immunofluorescence analysis of ST-2 and IL-33 expression in DLT and CLT and their co-localization with eosinophils (n = 7). (A) Representative immunofluorescence images showing IL-33 and eosinophil co-expression, with yellow indicating regions of overlap. (B) Immunofluorescence images depicting ST-2 and eosinophil co-expression, with yellow indicating co-localization. Quantification of ST-2 and IL-33 expression, along with their co-expression with eosinophils, was performed using Image J. Error analysis plots illustrate the extent of ST-2 and IL-33 co-localization with eosinophils. Scale bar: 100 µm. Data are expressed as mean $\pm$ standard deviation. *p 0.05 indicates statistical significance. The target cells are indicated by the red arrows.

IL-33 plays a key role in fibrosis development and was found to be highly expressed in the tissues surrounding AE lesions (CLT). Given that HAE is characterized by a dense inflammatory fibrous band, fibrosis formation in AE lesions was analyzed. HE staining demonstrated a relatively preserved structure in the DLT compared to CLT (Fig. 4). Masson’s trichrome staining further demonstrated a significantly higher abundance of collagen fibers (blue) in CLT than in DLT. Quantitative analysis using ImageJ software allowed for the identification, isolation, and extraction of collagen fibers (blue) and tissue (red), from which the collagen volume fraction (CVF) was calculated. The results indicated a significantly higher CVF in CLT than in DLT (p 0.05), indicating that IL-33 may contribute to fibrosis formation in AE lesions.

Fig. 4.

Hematoxylin-eosin (HE) and Masson’s trichrome staining analysis of CLT and DLT (n = 15). Representative HE and Masson’s trichrome-stained images of CLT and DLT. Quantitative analysis using Image J software, which identified and extracted collagen fibers (blue) and tissue structures (red). Fibrosis levels in CLT and DLT were quantified by calculating the collagen volume fraction (CVF). Scale bar: 200 µm. Data are expressed as mean $\pm$ standard deviation. *p 0.05 indicates statistical significance.

To assess the effect of IL-33 on eosinophil phagocytic function, eosinophils were divided into three groups: blank control, E.m-Ag, and IL-33 intervention. Flow cytometry results (Fig. 5) indicated that eosinophil phagocytosis of E. coli was significantly enhanced following stimulation with IL-33 and E.m-Ag, with IL-33 inducing a more pronounced increase in eosinophil phagocytic activity compared to the E.m-Ag stimulation (p 0.05). These findings suggest that eosinophil behavioral changes during echinococcosis infection are influenced by the disease state and IL-33 production, with IL-33 playing a dominant regulatory role.

Fig. 5.

Eosinophil phagocytosis of green fluorescent E. coli following different treatments. Phagocytosis assay results for the control group (PBS, 50 µL/mL; n = 5), IL-33-treated group (3 ng/mL; n = 5), and E.m-Ag-treated group (20 mg/mL; n = 5). Flow cytometry to detect GFP signals in eosinophils. Data are expressed as mean $\pm$ standard deviation, *p 0.05.

To evaluate the effect of IL-33 on eosinophil migration, a Transwell assay was performed. The results demonstrated that IL-33 intervention significantly increased eosinophil migration through the Transwell chamber compared to both the blank control and E.m-Ag groups (p 0.05) (Fig. 6A).

Fig. 6.

Eosinophil migration and $\alpha$-SMA expression in HSCs following different treatments. (A) DAPI staining, bright-field imaging, and merged DAPI + bright-field images of eosinophils across three experimental groups (n = 5). Statistical analysis of eosinophil migration through the Transwell membrane under various stimulating conditions. (B) Western blot analysis and quantification of $\alpha$-SMA expression in HSCs following different stimulations (n = 5). Scale bar = 1000 µm. Data are expressed as mean $\pm$ standard deviation. *p 0.05 indicates statistical significance; p $>$ 0.05 denotes no significant difference (ns). The target cells are indicated by the red arrows.

A dense fibrous band is a characteristic feature surrounding AE lesions, and IL-33 has been implicated in fibrosis formation. To further investigate the effect of IL-33 on lesion fibrosis during AE infection, five experimental groups were established: blank control, E.m-Ag, eosinophil intervention, IL-33 inhibition, and eosinophil + IL-33 inhibition combined intervention. Following various interventions on HSCs, Western blot analysis of $\alpha$-SMA expression was performed. Results demonstrated that both eosinophil addition and IL-33 inhibition reduced $\alpha$-SMA expression in HSCs. Notably, the combination of eosinophil addition and IL-33 inhibition led to a more significant decrease in $\alpha$-SMA expression (Fig. 6B), suggesting that IL-33 influences HSC $\alpha$-SMA expression through eosinophil regulation.