Molecular characterization and functional implications on mouse peripheral blood mononuclear cells of annexin proteins from Echinococcus granulosus sensu lato

Background Cystic echinococcosis (CE) is a life-threatening zoonotic disease caused by the larval stage of Echinococcus granulosus sensu lato, which employs various strategies to evade the host immune system for survival. Recent advances have revealed the role of annexins as excretory/secretory products, providing new insights into the immune regulation by these proteins in the pathogenesis of CE. Methods Echinococcus granulosus annexin B proteins EgANXB2, EgANXB18, EgANXB20, and EgANXB23 were cloned, expressed, and analyzed using bioinformatic tools. Membrane binding analysis was used to assess their bioactivity, while their immunoreactivity and tissue distribution characteristics were determined experimentally using western blotting and immunofluorescence staining, respectively. Furthermore, quantitative real-time reverse transcription PCR (qRT-PCR) was used to analyze the mRNA expression profiles of EgANXBs in different developmental stages of E. granulosus. Finally, immunofluorescence staining, cell counting kit 8 assays, flow cytometry, transwell migration assays, and qRT-PCR were used to evaluate the functional effects of rEgANXB18 and rEgANXB20 on mouse peripheral blood mononuclear cells (PBMCs). Results In this study, we identified four EgANXBs with conserved protein structures and calcium-dependent phospholipid binding activities. rEgANXBs were recognized by serum from sheep infected with E. granulosus and distributed in the germinal layer of fertile cysts. Interestingly, transcription levels of the four EgANXBs were significantly higher in protoscoleces than in 28-day strobilated worms. Moreover, we demonstrated that rEgANXB18 and rEgANXB20 were secretory proteins that could bind to PBMCs and regulate their function. Specifically, rEgANXB18 inhibited cell proliferation and migration while promoting cell apoptosis, NO production, and cytokine profile shifting. In contrast, rEgANXB20 showed limited effects on apoptosis but inhibited NO production. Conclusions Our findings suggested that among the four identified EgANXBs, EgANXB2 and EgANXB23 might play a pivotal role for the development of protoscoleces, while EgANXB18 and EgANXB20, as secretory proteins, appeared to participate in the host-parasite interaction by regulating the function of immune cells. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-023-05967-y.


Background
Cystic echinococcosis (CE) is a global zoonosis caused by the larval stage of Echinococcus granulosus sensu lato [1].Currently, CE still poses a significant public health challenge and causes large economic losses.This disease has been officially recognized as a neglected tropical disease by the World Health Organization because of its persistent impact on global health, affecting over 1 million people worldwide [2].Intermediate hosts (mostly herbivores and the human as an accidental intermediate host) and definite hosts (canids) are required in the life cycle of E. granulosus [3].The eggs are released from the definite hosts and subsequently ingested by the intermediate hosts, leading to the development of hydatid cysts within their internal organs, predominantly in the liver and lungs (accounting for > 90% of cases) [4].The protoscoleces (PSCs) within cysts are known to produce a considerable quantity of excretory/secretory products (ESPs), which might play a vital role in immune evasion by the parasites [5].
Some proteins within the ESPs have been identified, and their functions have been further investigated, particularly in relation to their interactions with the host immune cells.For instance, AgB has been found to reduce the H 2 O 2 production of human neutrophils [6] and promote the unconventional maturation of dendritic cells, resulting in reduced secretion of pro-inflammatory cytokines and the polarization of T lymphocytes towards the Th2 lineage [7].Similarly, thioredoxin peroxidase has been found to promote the differentiation of peritoneal macrophages into alternatively activated macrophages in vitro, thereby favoring immune evasion of parasites [8].In summary, certain proteins within the ESPs are of great significance for host-parasite interactions and highlight the potential role of these proteins in facilitating parasite growth within their hosts.
In this study, we cloned and expressed EgANXB2, EgANXB18, EgANXB20, and EgANXB23 of E. granulosus.Then, the calcium-dependent phospholipid binding activities, secretory properties, distribution, and transcription levels of the four EgANXBs were explored.Furthermore, we investigated the regulatory effects of rEgANXB18 and rEgANXB20 on mouse peripheral blood mononuclear cells (PBMCs) to gain a better understanding of the host-parasite interaction.

Parasites and animals
PSCs were collected from hydatid cysts isolated from the liver of sheep infected with E. granulosus s.l.from a slaughterhouse in Sichuan province, China.The PSCs and cyst walls were separated and treated as described in previous reports [22].The viability and molecular genotyping of PSCs were conducted as described in our previously published work [23].PSCs with a viability > 95% were considered for this study.Twenty-eight-day strobilated worms were obtained from the Department of Parasitology of Sichuan Agricultural University.In addition, PSCs, the germinal layer, and the strobilated worms were placed in 4% paraformaldehyde before immunolocalization.Furthermore, specific pathogen-free 6-week-old female SD rats and BALB/c mice were purchased from Chengdu Dashuo Co., Ltd (Chengdu, China).

Cloning, expression, and purification of four EgANXBs
An RNAprep pure Tissue Kit (Tiangen, Beijing, China) was used to extract total RNA from PSCs, and cDNA was subsequently synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA).Specific primers (Additional file 1: Table S1) were designed based on the full-length coding sequences of the four EgANXBs.After PCR, the products were extracted from the gel and cloned into vector pMD19-T (Takara, Dalian, China) for sequencing.Then, the pMD19-T-EgANXBs plasmids were subcloned into vector pET-32a ( +) (Invitrogen, Waltham, MA, USA) and transformed into Escherichia coli BL21 or Rosetta (DE3) cells (Tiangen), followed by induction with 0.24 mg/ml isopropyl β-d-1-thiogalactopyranoside (IPTG) at 16 ℃ for 12 h.The recombinant (r)EgANXBs were purified using a Ni 2+ affinity chromatography column (Bio-Rad, Hercules, CA, USA).The imidazole was removed, and the concentration of each protein was determined using a bicinchoninic acid (BCA) protein quantification kit (Bestbio).The purified proteins (0.5 mg/well) were assessed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gels were stained with coomassie blue.

Ca 2+ -dependent phospholipid binding bioactivity assay
The preparation of liposomes and assessment of Ca 2+ -dependent phospholipid-binding bioactivity of the four rEgANXBs were conducted according to previously published protocols with slight modifications [20].Briefly, three groups (a, b, and c) were included for rEgANXBs, pET32a vector protein (negative control), and bovine serum albumin (BSA) (V) (unrelated control).Both group a and b contained 30 μl of protein (0.5 mg/ml), 20 μl of liposomes (1 mg/ml) (Solarbio, Beijing, China), 30 μl of CaCl 2 (50 mM), and 20 μl of Tris-HCl (50 mM) in a total volume of 100 μl, while group c excluded CaCl 2 .After fully mixing, all groups were incubated at 37 °C for 1 h and then centrifuged at 8000 × g for 10 min to separate the supernatant and precipitate.Then, 30 μl of EDTA (50 mM) and 70 μl of Tris-HCl were added to group b precipitate, incubated, centrifuged, and separated as mentioned above.All the supernatants and precipitates were subjected to 12% SDS-PAGE and Coomassie blue staining for analysis.

Sera and polyclonal antibody preparation
Sheep sera, either positive or negative, were obtained from autopsy-confirmed infected or healthy sheep, respectively.Polyclonal antibodies against the rEgANXBs were produced by immunizing eight rats, with two rats for each protein, as previously described [27].The preimmune rat serum was collected as the negative serum, followed by subcutaneous injection of 0.3 mg protein fully emulsified with an equal volume of complete Freund's Adjuvant (Sigma, St. Louis, MO, USA) for the first immunization.Three booster immunizations with rEgANXBs in incomplete Freund's adjuvant were performed in the following 3 weeks, with 1-week intervals.One week after the last immunization, anti-sera were collected and purified using a Protein G Resin FF Prepacked Column (GenScript, Piscataway, NJ, USA) to obtain anti-rEgANX rat IgGs.

Western blotting
The total proteins of PSCs were extracted using a BBprExtra total protein extraction kit (Bestbio, Shanghai, China).The extracted proteins, along with rEgANXBs and cyst fluid, were separated using 12% SDS-PAGE and transferred to Nitrocellulose Membranes (Biosharp, Hefei, China).Then, the membrane was blocked using 5% skim milk for 2 h.Sheep negative or positive serum diluted 1:100 in phosphate-buffered saline (PBS), rat negative serum, or anti-rEgANXBs rat IgG (dilution 1:400 in PBS) was added and incubated overnight at 4 °C.After washing, horseradish peroxidase (HRP)-conjugated rabbit anti-sheep IgG (dilution 1:1000 in PBS) or HRPconjugated goat anti-rat IgG (H + L) (ABclonal, Wuhan, China) (dilution 1:2000 in PBS) was added for 2 h incubation, and the immunoreactive signals were detected using a Metal Enhanced DAB Substrate Kit (Solarbio, Beijing, China).

Transcription analysis of four EgANXBs
To assess the mRNA expression levels of the four EgANXBs in PSCs and 28-day strobilated worms, quantitative real-time reverse transcription PCR (qRT-PCR) was performed.Total RNA was extracted using a cell total RNA isolation kit (Foregene, Chengdu, China), and cDNA was synthesized.The specific primers are shown in Additional file 1: Table S2, and qPCR was performed using the LightCycler System (Roche, Basel, Switzerland) with TB Green Premix Ex Taq II (Takara, Shiga, Japan).The PCR procedure comprised: 30 s at 95 ℃; 40 cycles of 5 s at 95 ℃ and 30 s at 61 ℃; melting at 95 ℃ for 10 s, 65 ℃ for 5 s, 95 ℃ for 1 s.The GAPDH gene from E. granulosus (encoding glyceraldehyde-3-phosphate dehydrogenase; GenBank: KF894802) was selected as the housekeeping gene.The results were analyzed using the 2 −ΔΔCt method and shown as the relative expression to the gene of strobilated worm [28].

Isolation of mouse PBMCs and endotoxin removal of rEgANXB18 and rEgANXB20 protein preparations
Blood (0.7-1 ml/per mouse) of 60 female BALB/c mice was collected from the tail vein, and the mouse PBMCs were isolated following the manufacturer's instructions of the peripheral blood mononuclear cell separation kit (TBD Science, Inc., Tianjin, China).Cell viability was detected using trypan blue staining.The cells were then resuspended in Roswell Park Memorial Institute (RPMI)1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Newzerum, Christchurch, New Zealand) and 1% penicillin-streptomycin (HyClone, Logan, UT, USA) before being cultured at 37 ℃ in a 5% CO 2 incubator.To avoid the effects of endotoxin on cells, endotoxins in the rEgANXB18 and rEgANXB20 preparations were removed using Endotoxin Removal Beads (Smart-Lifesciences, Changzhou, China) and tested according to the instructions of the ToxinSensor ™ Chromogenic LAL Endotoxin Assay Kit (GenScript).Moreover, rEgANXBs were filtered by 0.45-μm sterile filter membranes and quantified using BCA protein quantification kit (Bestbio).
To assess the effect of rEgANXB18/rEgANXB20 on PBMC migration, a Transwell migration assay was conducted.Specifically, cell chambers with an 8.0-µm pore size (Thermo Fisher) were gently placed in a 24-well plate; RPMI1640 medium (1300 μl) containing 10% inactivated FBS was added to the lower chamber, while 200 μl PBMCs (2 × 10 6 cells) resuspended in RPMI1640 medium without FBS was added to the upper chamber along with rEgANXB18, rEgANXB20 (5, 10, 20, and 40 μg/ml), pET-32a vector protein (10 μg/ml), or 10 μl PBS.PBMCs were incubated for 4 h at 37 °C, and cells that migrated to the lower chamber were collected and counted using a cell counting chamber (Corning Inc., Corning, NY, USA).Cell migration rate (%) was calculated by the formula: migration rate (%) = cells that migrated to the lower chamber/ 2 × 10 6 cells × 100%.The experiments for the migration, proliferation and the apoptosis assays were performed in triplicate.
For cytokine production analysis, PBMCs (1 × 10 6 cells/ ml/well) were seeded into 24-well plates and incubated with rEgANXB18, rEgANXB20 (5, 10, 20, and 40 μg), pET-32a vector protein (10 μg), or 10 μl of PBS at 37 °C for 24 h.The specific primers are shown in Additional file 1: Table S3, and qPCR was performed according to the procedure of qPCR method as above with a different annealing temperature at 61 ℃.The GAPDH (Gen-Bank BC096440.1)gene from mouse was selected as the housekeeping gene, and the relative expression levels of cytokines were analyzed using the 2 −ΔΔCt method.

Statistical analysis
xStatistical analysis was conducted using GraphPad Prism (version 8.0.2;GraphPad Inc., La Jolla, CA, USA), and data are presented as the mean ± standard deviation (SD).An unpaired Student's t-test was used for comparisons between two groups, and one-way analysis of variance (ANOVA) was applied to compare differences among three or more groups.P < 0.05 indicated statistical significance.

Bioinformatic analysis of EgANXBs
The full-length coding sequences of four EgANXBs were successfully cloned and sequenced.EgANXB18, EgANXB20, and EgANXB23 shared the same sequences with their reference sequences, while EgANXB2 (1065 bp) was 21 bp longer than its reference sequence.The molecular weight of four EgANXBs ranged from 35.72 kDa to 39.95 kDa.All four EgANXBs were predicted to be stable, without signal peptides or transmembrane regions (Table 1).
Multiple sequence alignment analysis showed that EgANXB2 had relatively low identity with EgANXB18, EgANXB20, or EgANXB23, and these four EgANXBs exhibited > 97% identity with Echinococcus multilocularis orthologs (Fig. 1).Moreover, all four EgANXBs contained two type II calcium binding sites with the GxGT-{38-40 amino acid residues}-D/E sequence, in accordance with the canonical characteristics of the annexin superfamily [30].The EgANXBs mainly contained α-helices, followed by random coils, β-turns, and β-folds, which was further confirmed by three-dimensional structure prediction (Fig. 2).The four EgANXBs were predicted to form an overall shape of a slightly bent disc, including a convex side and a concave side.The type II calcium binding sites were located on the convex side, except in EgANXB18, which was predicted to lack a calcium binding site.In addition, EgANXB23 was predicted to form dimers.The maximum likelihood phylogenetic tree showed that the four EgANXBs were located in different branches, exhibiting the closest evolutionary relationships with E. multilocularis annexins (Fig. 3).

Expression, purification, and western blotting of rEgANXBs
The rEgANXB2, rEgANXB18, rEgANXB20, and rEgANXB23 were successfully expressed in a soluble form with expected molecular weights of approximately 57 kDa, 56 kDa, 54 kDa, and 53 kDa, respectively (Fig. 4).Western blotting demonstrated that the rEgANXBs could be specifically recognized by their corresponding anti-rEgANXBs rat IgG and positive sera from naturally infected sheep (Fig. 4: Lanes 4, 8), while no signal was detected in the presence of negative sera (Fig. 4: Lanes 5, 9).Moreover, the anti-rEgANXBs rat IgG was able to recognize the four EgANXBs among the extracted total proteins from PSCs (Fig. 4: Lane 6).Furthermore, EgANXB18 and EgANXB20 were detected in cyst fluid (Fig. 5: Lane 2, 4).

Ca 2+ -dependent phospholipid binding bioactivity of rEgANXBs
Our findings showed that all four rEgANXBs were able to bind to liposomes in the presence of Ca 2+ and remained in the precipitate after centrifugation (Fig. 6: a group), while they could not bind to liposomes in the absence of Ca 2+ and thus remained in the supernatant (Fig. 6: c group).Meanwhile, as EDTA was added to competitively bind Ca 2+ , the rEgANXBs were released from liposomes and reappeared in the supernatant (Fig. 6: b group).Notably, rEgANXB2 and rEgANXB23 were not completely released from liposomes after EDTA addition, and some of them remained in the precipitate (Fig. 6A, D).In addition, pET 32a vector expression protein and BSA were analyzed, and no Ca 2+ -dependent phospholipid binding characteristics were observed (Fig. 6E, F), suggesting that the Ca 2+ -dependent phospholipid binding property was solely because of rEgANXBs rather than the pET-32a vector expression protein or other factors.

Localization of EgANXBs by immunofluorescence assays
Immunolocalization analysis showed that both EgANXB2 and EgANXB18 were mainly distributed in the tegument and hooks of PSCs (Fig. 7A, B), while EgANXB20 was predominantly localized in the rostellum of PSCs (Fig. 7C).EgANXB23 was mainly distributed in the tegument and calcareous corpuscles of PSCs (Fig. 7D).In 28-day strobilated worms, EgANXB2 and EgANXB20 were widely distributed, while EgANXB18 was mainly distributed in the tegument and rostellum, and EgANXB23 was only distributed in hooks.Interestingly, all four EgANXBs were found to be similarly distributed in the germinal layer of fertile cysts, with less distribution in the germinal layer of non-fertile cysts.The EgANXB23 showed almost undetectable fluorescence signals.

Transcription levels of EgANXBs in PSCs and 28-day strobilated worms
The qRT-PCR analysis showed that four EgANXBs were transcribed in both the PSCs and 28-day strobilated worms, and the transcription levels of EgANXB2, EgANXB18, EgANXB20, and EgANXB23 in PSCs were significantly higher than those in the 28-day strobilated worms (Fig. 8).

rEgANXBs bind to PBMCs by immunofluorescence assays
When PBMCs were incubated with rEgANXB18 or rEgANXB20, red fluorescence was observed on the cell surface (Fig. 9), while the PBMCs incubated with pET-32a vector expression protein or PBS showed no red fluorescence, suggesting that rEgANXB18 and rEgANXB20 could bind to PBMCs.

Effects of rEgANXBs on PBMC proliferation, apoptosis, and migration
The CCK-8 assay showed that rEgANXB18 inhibited PBMC proliferation in a dose-dependent manner (Fig. 10A).Additionally, we observed that a low concentration (5 μg/ml) of rEgANXB20 had no effect on PBMC proliferation, while high concentrations of rEgANXB20 significantly inhibited it (P < 0.001).As a positive control, ConA could induce the proliferation of PBMCs (P < 0.001).Moreover, we investigated the effect of rEgANXB18 and rEgANXB20 on PBMC apoptosis.Our data demonstrated that rEgANXB18, at high concentrations (20 μg/ml and 40 μg/ml), significantly promoted PBMC apoptosis (P < 0.001).However, rEgANXB20 showed no effects on PBMC apoptosis (Fig. 10B).To assess the influence of rEgANXB18 and rEgANXB20 on PBMC migration, we conducted a Transwell migration assay.The results showed that both rEgANXB18 (P < 0.05) and rEgANXB20 (p < 0.001; except for 5 μg/ml) inhibited PBMC migration (Fig. 10C).

Effects of rEgANXBs on NO production and expression of cytokines in PBMCs
To investigate the effect of rEgANXBs on the production of NO by PBMCs, we measured NO in the cell culture supernatant.The results demonstrated that rEgANXB18 and rEgANXB20 had completely different effects on NO production.Specifically, rEgANXB18 induced NO production in a dose-dependent manner (P < 0.05), while rEgANXB20(10, 20, and 40 μg/ ml) inhibited it (P < 0.05) (Fig. 11A).Meanwhile, qRT-PCR analysis showed that low concentrations of rEgANXB18 (5 μg/ml) could markedly promote the relative mRNA expression levels of cytokines interleukin (IL)-10, IL-17A, transforming growth factor beta 1 (TGF-β1), and interferon gamma (IFN-γ) (P < 0.05).However, with increasing recombinant protein concentration, the expression levels of the cytokines showed no significance compared to those in the PBS group (Fig. 11B).By contrast, rEgANXB20 induced the expression of IL-10, IL-17A, and IFN-γ in a dose-dependent manner.In addition, 5 μg/ ml rEgANXB20 induced the expression of TGF-β1 (P < 0.001) while 20 μg/ml and 40 μg/ml rEgANXB20 inhibited its expression (Fig. 11C).

Discussion
The metacestode of E. granulosus survives within its hosts, producing ESPs that are generally considered to play essential roles in parasite invasion, immune escape, and metabolic adaptation [5].Some annexins from Taenia solium, as secretory proteins discovered in ESPs, were believed to be involved in modulating host immune responses and facilitating parasite immune evasion [31].However, the functional characteristics of E. granulosus annexins have yet to be clearly determined.In this study, we aimed to expand the current understanding of annexins by characterizing four novel EgANXBs and exploring their potential roles in parasite immune evasion.Annexins belong to an evolutionarily conserved protein superfamily characterized by their ability to reversibly interact with membranes in a calcium-dependent manner [30].
Annexins are composed of a C-and an N-terminal domain.The C-terminal domain contains four or eight repeats arranged to form five α helices that finally form the overall shape of a slightly curved disc with a central hydrophilic hole [32,33].Herein, we observed that all four EgANXBs contained the sequence GXGT-{38-40 amino acid residues}-D/E and shared similar conventional three-dimensional structures.Notably, although the GXGT-{38-40 amino acid residues}-D/E sequence was present in EgANXB18, no calcium-binding site was predicted in the protein's three-dimensional structure.
Fig. 4 SDS-PAGE and western blotting analysis of the purified rEgANXB2 (A), rEgANXB18 (B), rEgANXB20 (C), and rEgANXB23 (D).M, protein molecular weight marker; 1, pET32a( +)-vector proteins induced by IPTG; 2, proteins of pET32a( +)-EgANXBs induced by IPTG; 3, purified rEgANXBs; 4, western blotting of purified rEgANXBs probed with anti-rEgANXBs rat IgG; 5, western blotting of purified rEgANXBs probed with negative rat serum; 6, western blotting of extracted total protein of PSCs probed with anti-rEgANXBs rat IgG;7, western blotting of extracted total protein of PSCs probed with negative rat serum; 8, western blotting of purified rEgANXBs probed with serum from E. granulosus infected sheep; 9, western blotting of purified rEgANXBs probed with serum from Echinococcus granulosus non-infected sheep Fig. 5 SDS-PAGE and western blotting analysis of cyst fluid.M, protein molecular weight marker; 1, SDS-PAGE of total proteins within cyst fluid; 2, total proteins within cyst fluid reacted with anti-rEgANXB18 rat IgG; 3 and 5, total proteins within cyst fluid reacted with negative rat serum; 4, total proteins within cyst fluid reacted with anti-rEgANXB20 rat IgG Since EgANXB18 was experimentally shown to bind phospholipids in a Ca 2+ -dependent manner, this unexpected outcome could be attributed to the template selected to construct the model.EgANXB23 was predicted to form a dimer, which is consistent with previous reports indicating that certain annexins, such as ANXA2 and ANXA5, could form dimers and trimers, and even heterotetramers, with other molecules, such as the S100 protein [34][35][36].
The four rEgANXBs were confirmed to bind to phospholipids in a Ca 2+ -dependent manner; intriguingly, although rEgANXB2 and rEgANXB23 could bind to phospholipids, they were not completely released when EDTA was added, likely because of the formation of transmembrane ion channels.Previous studies have shown that several annexins, including ANXA1, ANXA2, ANXA5, ANXA6, and ANXA7 of humans and other species, exhibit transmembrane ion channel activity [37,38].Normally, most of the annexins could detach from the membrane in the absence of Ca 2+ .However, transmembrane ion channels would keep annexins bound to the membrane, and it was observed that ANXA1 and ANXA2 were resistant to EDTA extraction and required detergent treatment for the release from the membrane [39,40].Although more data are needed, we postulated that rEgANXB2 and rEgANXB23 from E. granulosus possibly attach to the membranes and then insert into them to form transmembrane ion channels, such that EDTA failed to extract them from the liposomes.
The distribution of four EgANXBs in different development stages of E. granulosus differed from each other, which was also observed in trematodes [41][42][43][44].Surprisingly, higher transcription levels of the four EgANXBs were detected in PSCs compared with those in 28-day strobilated worms, which contrasted with EgANXB3 and EgANXB38 [20].This suggested a more intimate relationship between EgANXBs in this study and PSCs.Furthermore, EgANXB18 and EgANXB20 were identified as secretory proteins despite lacking signal peptides, suggesting that they were probably secreted through unconventional secretion pathways, such as direct translocation across the plasma membrane or secreted via extracellular vesicles [45].Under the assayed conditions, EgANXB2 and EgANXB23 were not detected in cyst fluid, indicating that they were not secretory proteins, or that their concentrations were too low.Annexins are generally considered to function intracellularly [46].However, several secretory annexins have been identified to be involved in parasite-host interactions including Clonorchis sinensis annexin B30 [41], T. solium annexin B1 [31], and E. granulosus annexins B3, B33, and B38 [20,21].Furthermore, previous evidence has shown that secretory annexins could bind to immune cells via specific receptors, such as formylated peptide receptors (FPR or ALXR) and CD44 of neutrophils for ANXA1 and ANXA2, respectively [47][48][49].Similarly, both rEgANXB18 and rEgANXB20 were observed to bind to PBMCs and inhibit their proliferation, and high concentrations of rEgANXB18 also promoted PBMC apoptosis, while rEgANXB20 showed no effect.This suggested that rEgANXB18 and rEgANXB20 were more likely to suppress the Fig. 9 rEgANXBs bind to mouse PBMCs, as identified by immunofluorescence staining.The rEgANXBs were used as the primary antibody, and DAPI, Cy3 and PBS referred to staining, secondary antibody, and control without primary antibody, respectively immune response by affecting proliferation rather than apoptosis.
Immune cells can be recruited and migrate to specific tissues when an inflammatory response occurs, which is essential for infection control, tissue repair, and immune surveillance [50].Several factors, including annexins, are involved in the modulation of cell migration.For instance, ANXA1 inhibits the activation of integrins induced by CCL5, CCL2, or CXCL1 [51], thereby affecting the migration and aggregation of leukocytes and suppressing the adhesion and migration of neutrophils to vascular endothelial cells [47].In this study, we observed that both rEgANXB18 and rEgANXB20 inhibited the migration of PBMCs, implying that they potentially played a role in the modulation of the immune response in hosts.
NO, a multifunctional bioactive molecule, is generally considered to play an important role in anti-parasite infection by inhibiting cellular energy synthesis or destroying proteins, lipids, and nucleic acids [52].Our study showed that rEgANXB18 and rEgANXB20 exhibited opposite effects on NO production.Specifically, rEgANXB18 promoted NO production, while rEgANXB20 inhibited it, suggesting that both proteins affected NO production, probably by regulating the expression or activity of inducible nitric oxide synthases (iNOS).Previous studies confirmed that iNOS transcription was induced by IFN-γ and lipolysaccharide (LPS) [53].We observed that high concentrations of EgANXB18 do not induce significant IFN-γ production but do induce NO production.In contrast, since rEgANXB20 affected the expression of IFN-γ only at the highest concentration, it is likely to function through a different pathway.
Th1 and Th2 immune responses coexist in patients with CE, with Th1 cytokines playing a role in parasite removal and Th2 cytokines being linked to parasite immune evasion [54].IFN-γ, a Th1 cytokine, has been proven to enhance the activity and antigen-presenting ability of Th1 cells and macrophages while inhibiting the differentiation and function of Tregs and Th2 and Th17 cells [55].Th17/Treg cells are believed to be involved in CE development, with Th17 cells mediating chronic inflammatory responses by secreting IL-17A and Treg cells exerting immunosuppressive effects by producing IL-10 and TGF-β1 [56,57].Our findings demonstrated that low concentrations of rEgANXB18 significantly increased the levels of IL-10, TGF-β1, IL-17A, and IFN-γ mRNA, with the increase of IFN-γ mRNA being markedly higher than those the others.Previous studies have shown that when PSCs were co-cultured PBMCs from CE patients, the addition of IFN-γ promoted the iNOS and NO production, leading to higher PSC mortality [58].Therefore, it is possible that rEgANXB18 participates in anti-parasite infection by inducing IFN-γ expression.In addition, rEgANXB20 promoted the expression of IL-10, IL-17A, and IFN-γ.Moreover, it also significantly increased the expression level of TGF-β1 at low concentrations while showing opposite effects at high concentrations.Taken together, rEgANXB20 induced the expression of both Th1/Th2 and Th17/Treg cytokines, consistent with the cytokine profile observed in patients with CE.

Conclusion
The present study identified four EgANXBs as typical annexins with canonical structures and Ca 2+ -dependent phospholipid binding properties.Notably, EgANXB2 and EgANXB23 were postulated to have ion channel activities when interacting with membranes.Immunolocalization and qRT-PCR analysis revealed that both the distribution and transcription levels of these four EgANXBs in PSCs were higher than in 28-day strobilated worms, indicating their involvement in the growth and development of PSCs.Furthermore, EgANXB18 and EgANXB20 were found to be secretory proteins that could bind to PBMCs to regulate cell proliferation, apoptosis, migration, and NO and cytokine production.These results suggest potential roles of EgANXB18 and EgANXB20 in immune evasion strategies employed by the parasites.

Fig. 10
Fig.10Effects of rEgANX18 and rEgANX20 on mouse PBMCs proliferation (A), apoptosis (B), and migration (C).Statistical analysis between the PBS and other groups was conducted using one-way ANOVA with data displayed as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001; ns: not significant.ANOVA, analysis of variance

Table 1
Bioinformatic analysis of four EgANXBs