Role of Raf Kinase Inhibitor Protein in Modulating Echinococcal Cyst Fluid-Induced Degranulation in Bone Marrow-Derived Mast Cells

Echinococcus granulosus invades and parasitizes human tissues and organs, most commonly affecting the liver and lungs, where it forms cysts of varying sizes and causes cystic echinococcosis (CE) [1]. The rupture of these cysts releases echinococcal cyst fluid (EgCF), severe allergic reactions, including potentially fatal anaphylactic shock [2]. Traditional anti-shock treatments are often ineffective due to the long-term state of specific immune tolerance observed in patients with CE [3]. As this allergic reaction is immunoglobulin E (IgE)-dependent, bone marrow-derived mast cells (BMMCs) play a crucial role in initiating and regulating allergic responses by releasing stored bioactive substances through degranulation in IgE-mediated allergic reactions [4].

Raf kinase inhibitor protein (RKIP) is a multifunctional protein that regulates multiple signalling pathways, including MAPK and NF-κB [5, 6]. Previous studies have demonstrated that RKIP negatively regulates IgE- Fc-epsilon Receptor Ⅰ (FcεRI)-mediated responses in BMMCs, particularly in the context of asthma pathogenesis [7]. Therefore, this study investigated the role of RKIP in EgCF-induced BMMC degranulation, aiming to provide a theoretical basis and experimental evidence for understanding CE-induced allergic reactions. Additionally, it explored the potential of RKIP as a therapeutic target, addressing existing research gaps and advancing the development of targeted treatments for CE-associated allergies.

RKIP gene knockout (KO) mice were obtained from the Shanghai Model Organisms Center, Inc., while wild-type (WT) C57BL/6 mice were sourced from the Animal Research Laboratory at Xinjiang Medical University.

The study utilized RPMI-1640 (Gibco), foetal bovine serum (FBS) (Wuhan Pricella Biotechnology Co., Ltd.), penicillin-streptomycin double antibiotics (Cytiva), IL-3, SCF (PeproTech), FITC-CD117, APC-Fc-epsilon Receptor I α (FcεRIα) (BioLegend, USA), and ELISA kits for β-hexosaminidase, IL-4, IL-6, and TNF-α (Shanghai JianglaiBio Co., Ltd.). Fresh sheep livers naturally infected with Echinococcus granulosus were obtained from the Urumqi Hualing Slaughterhouse in Xinjiang. The liver surface was disinfected with 75% ethanol, and cyst fluid was extracted with sterile syringes and transferred into 50 mL centrifuge tubes. After natural precipitation, the supernatant was centrifuged at 12,000 g for 10 min at 4°C. The supernatant was filtered through 0.22 μm filters and stored at −80°C. For the preparation of serum from E. granulosus-infected mice, WT mice were intraperitoneally injected with 3,000 E. granulosus. Blood samples were collected via orbital sampling after 3 months, allowed to clot at room temperature, centrifuged at 4°C to separate the serum, and stored at −80°C. A CO₂ cell culture incubator (Thermo Fisher Scientific, USA), a vertical electrophoresis apparatus, an electrophoresis tank and related equipment, a wet transfer apparatus (Bio-Rad, USA), and a flow cytometer (BD) were utilized.

Eight specific-pathogen-free KO and WT mice, aged 4–6 weeks and weighing 12–15 g, were euthanized through cervical dislocation. The bodies were disinfected in 75% ethanol for several seconds before femurs, and tibias were harvested in a laminar flow hood. Bones were placed in phosphate-buffered saline (PBS) containing 10% penicillin-streptomycin. Bone marrow was extracted into 10 cm culture dishes using a 1 mL syringe filled with RPMI-1640 medium supplemented with 10% FBS. The tissue was filtered through a 100 μm strainer to eliminate bone debris, centrifuged at 1,000 rpm for 5 min at room temperature, and the supernatant was discarded. The red blood cell lysis buffer was applied and allowed to stand for 2 min, followed by centrifugation and removal of the supernatant. Cells were washed twice with PBS, centrifuged, and resuspended in RPMI-1640 medium containing 10% FBS. Cells were then stimulated with SCF (final concentration 10 ng/mL) and IL-3 (final concentration 10 ng/mL), seeded in six-well plates at a density of 1 × 10⁶ cells/mL, and cultured in a 37°C incubator with 5% CO₂ for 4 weeks. The medium was refreshed by replacing half the volume every 3 days and fully replaced every 7 days.

The morphological characteristics of BMMCs were observed and imaged using an inverted light microscope at weeks 1 and 4 of the culture period.

BMMCs cultured for 4 weeks were harvested by centrifugation at room temperature, and the supernatant was discarded. The cells were washed 2–3 times with PBS and resuspended in staining buffer at a cell concentration of 1 × 10⁷ cells/mL. The cell suspension was mixed and equally distributed into EP tubes at 100 μL per tube. FITC-CD117 and APC-FcεRIα flow cytometry antibodies were added in appropriate concentrations, and the tubes were incubated on ice in the dark for 20 min. Subsequently, 1 mL of PBS was added to each tube, mixed by vortexing, and centrifuged. The supernatant was discarded and the cells were resuspended in 300 μL of staining buffer, filtered through a 70 μm strainer, transferred to flow cytometry tubes, and analysed using a flow cytometer.

Suspended cells from RKIP KO and WT groups were collected by centrifugation and washed 2–3 times with pre-cooled PBS. The cell pellets were resuspended in RIPA lysis buffer containing a protease inhibitor at a 100:1 ratio. Following complete lysis on ice, the cells were centrifuged, and the supernatant containing extracted proteins was collected. Western blot analysis was performed to confirm the KO of the RKIP gene.

BMMCs from KO and WT groups were harvested by centrifugation at room temperature, resuspended in RPMI-1640 medium at a concentration of 1 × 10⁶ cells/mL, and seeded into 6-well plates. The non-sensitized and sensitized groups were cultured in RPMI-1640 medium containing 10% serum of granuloma echinococcus-infected mice to simulate the in vivo environment of infected tissues, and the control group was only added with the same volume of RPMI-1640 medium and cultured for 24 h at 37°C in a 5% CO₂ incubator. The sensitized group was treated with a crude concentrated vesicle solution at a final concentration of 40 µg/mL, whereas the non-sensitized and control groups were treated with an equal volume of PBS (online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000545176). Centrifugation collected the supernatants at different times after stimulation to detect cytokine secretion. Some cells were lysed with 0.1% Triton X-100, and the supernatant was centrifuged to detect β-aminohexosidase release, while some cells were treated with 1 mL of TRIzol, mixed thoroughly, and stored at −80°C for RNA extraction, in which WT cells were added with RIPA lysate for protein extraction.

Proteins were extracted from WT BMMCs, and Western blot analysis was performed to detect changes in RKIP Expression during the BMMC allergic reaction.

Samples and reagents were prepared according to the ELISA kit instructions, and optical density was measured at 450 nm using a microplate reader. The β-hexosaminidase release rate was calculated using the following formula: β-hexosaminidase release rate = (extracellular β-hexosaminidase content)/(intracellular β-hexosaminidase content + extracellular β-hexosaminidase content) × 100%.

The collected cell supernatants were analysed following the instructions provided with the respective mouse IL-4, IL-6, and TNF-α ELISA kits.

Cellular RNA was extracted using TRIzol reagent, followed by reverse transcription to cDNA. PCR amplification was conducted for IL-4, IL-6, and TNF-α genes, with β-actin serving as the internal reference for evaluating mRNA expression levels.

Statistical analyses were conducted using GraphPad Prism 8.0.2 software. The experimental data are expressed as mean ± standard deviation (x ± s). Initially, normality and homogeneity of variance tests were performed. For data that met the assumptions of normal distribution and homogeneous variance, a two-way ANOVA was applied, followed by the Bonferroni multiple comparison test for pairwise comparisons when significant differences were observed. A p value of less than 0.05 (p < 0.05) was considered statistically significant.

After 1 week of induction culture, a large number of adherent cells were observed, while suspension cells initially exhibited round shapes but displayed variations in size and morphology. After 4 weeks of culture, with regular medium changes, adherent cells were almost completely absent, and the suspension cells appeared consistently round.

Flow cytometry analysis demonstrated that, after 4 weeks of culture, both KO and WT BMMCs displayed double-positive rates for CD117 and FcεRIα exceeding 98% (Fig. 1a, b).

Western blot analysis validated the successful KO of RKIP in cultured KO BMMCs (Fig. 1c).

The β-hexosaminidase release rate was evaluated in KO and WT BMMCs following EgCF sensitization. As shown in Figure 1d, at 3 h post-sensitization, the KO group exhibited a significantly higher BMMC degranulation rate compared to the WT group (p < 0.05).

RKIP protein expression levels on WT BMMC surfaces were monitored at 1, 2, and 3 h post-sensitization. As shown in Figure 1e, f, RKIP expression progressively decreased during the degranulation reaction, with statistically significant changes (p < 0.05).

The release of cytokines from KO and WT BMMCs was evaluated. The absence of RKIP resulted in a significant increase in the release of IL-4, IL-6, and TNF-α from BMMCs, with statistical significance (Fig. 2a).

The qRT-PCR results showed that the absence of RKIP led to a significant increase in the mRNA expression levels of IL-4, IL-6, and TNF-α in BMMCs, with statistical significance (Fig. 2b).

Mast cells, originating from the bone marrow, are found in the submucosa of various human tissues and organs, including the skin, lungs, intestines, and blood vessels [8‒11]. They express surface molecules such as FcεRI, which is occupied by IgE molecules [12, 13], G protein-coupled receptors, complement receptors, and cytokine receptors like IL-4 [14]. Additionally, mast cells store intracellular granular substances, including leukotrienes, histamine, prostaglandins, IL-4, IL-6, and TNF-α [13, 14]. Upon cross-linking of FcεRI-IgE complexes occupying the surface of basophils or mast cells, these mediators are released, influencing not only the circulatory, respiratory, and digestive systems but also recruiting other inflammatory cells, thereby promoting inflammation [15‒19]. Therefore, mast cells were selected as the primary focus of the investigation into the inflammatory mechanisms underlying CE-induced allergic reactions.

In humans, FcεRI is usually a tetramer (αβγ₂) containing one α-chain (IgE binding), one β-chain (signalling amplification), and two γ-chains (signalling). The presence of the β-chain enhances receptor stability and signalling efficiency, and the tetramer can be expressed at high levels in mast cells and basophils and at low levels in dendritic cells. In contrast, in mice, FcεRI is predominantly trimeric (αγ₂) and lacks the β-chain, an absence that may affect the level of receptor expression and signalling intensity at the cell surface, and trimeric (αγ₂) is only present in mast cells and basophils [20‒22]. A previous study has shown that RKIP negatively regulates FceRI-mediated mast cell function in asthma and exerts a protective effect [7]. In the present study, we treated BMMCs with serum from mice infected with granuloma echinococcus to simulate an in vivo environment for mast cells during infection. For the first time, we explored the role of RKIP in EGCF-induced mast cell activation through in vitro experiments. However, there is a lack of in-depth studies on the mechanism of action of RKIP in EgCF-induced mast cell activation, but based on the known function of RKIP in other inflammatory models, we can speculate on the potential mechanism of RKIP in EgCF-induced mast cell activation. RKIP may inhibit Raf-1 kinase activity by binding to Raf-1 kinase, and inhibiting ERK1/2 RKIP may inhibit the activity of Raf-1 kinase by binding to Raf-1 kinase, inhibit the downstream phosphorylation of ERK1/2, and reduce the secretion of inflammatory factors, such as TNF-α and IL-6 [23]. RKIP may also inhibit the phosphorylation and degradation of IκB by interacting with the IKKβ subunit of the IKK complex, inhibiting the nuclear translocation of the p65 subunit of NF-κB and reducing the production of inflammatory factors [24, 25]. In addition, previous studies have shown that RKIP binds to the p85 subunit of PI3K, regulates its activity, affects the downstream Akt signalling pathway, and negatively regulates IgE-FcεRI-mediated activation of BMMCs in an asthma model [7]. The results of the present experiment are also consistent with previous studies of RKIP as a negative regulator; thus, this mechanism may also be present in EgCF-induced mast cell activation, and RKIP may inhibit mast cell activation by regulating the PI3K/Akt signalling pathway.

In conclusion, the findings suggest that RKIP plays a critical inhibitory role in EgCF-induced BMMC degranulation (online suppl. Fig. 2). Targeting RKIP through agonists or mimetic compounds could represent a promising therapeutic strategy for managing E. granulosus-induced allergic reactions.

However, since the present study only performed in vitro cellular experiments, it could not fully simulate the complex physiological environment and multiple cellular interactions in vivo. Therefore, next, the results are to be further validated in in vivo experiments to fully understand the mechanism of action and physiological functions of RKIP in living organisms. In addition, we used murine cells, which may not fully reflect the response of human cells. Future studies should explore more advanced experimental techniques and consider using human cells to improve the applicability of our findings.

We would like to acknowledge the hard and dedicated work of all the staff that implemented the intervention and evaluation components of the study. Yu-Qian Li: formal analysis and writing – review and editing. Li-Wei Cao: conceptualization, data curation, and formal analysis. Bayina Batesurong: acquisition of data and statistical analysis.

All experiments were evaluated and approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (No. IACUC-20200318-04) and complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

The authors declare that they have no competing interests.

This study was funded by National Natural Science Foundation of China (No. 82060581) and Xinjiang Perioperative Organ Protection Laboratory (XJDX1411).

Shan-Shan Li: acquisition of data and critical revision of the manuscript for intellectual content. Xue-Li Pu: conception and design of the research and writing of the manuscript. Jing-Ru Zhou: data curation, formal analysis, and writing – original draft. Chun-Sheng Wang: analysis and interpretation of the data, resources, software, and formal analysis. Jia-Ling Wang: statistical analysis, conceptualization, and data curation. Xilizati Kulaixi: data curation, formal analysis, and writing – original draft. Jian-Rong Ye: conception and design of the research, obtaining financing, and critical revision of the manuscript for intellectual content. All authors read and approved the final draft.

Edited by: H.-U. Simon, Bern.Shan-Shan Li and Xue-Li Pu contributed equally to this study.

All data generated or analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.