Echinococcus multilocularis Calreticulin Interferes with C1q-Mediated Complement Activation

As a zoonotic disease caused by Echinococcus multilocularis larvae, alveolar echinococcosis (AE) is one of the most severe forms of parasitic infection. Over a long evolutional process E. multilocularis has developed complex strategies to escape host immune attack and survive within a host. However, the mechanisms underlying immune evasion remain unclear. Here we investigated the binding activity of E. multilocularis calreticulin (EmCRT), a highly conserved Ca2+-binding protein, to human complement C1q and its ability to inhibit classical complement activation. ELISA, Far Western blotting and immunoprecipitation results demonstrated that both recombinant and natural EmCRTs bound to human C1q, and the interaction of recombinant EmCRT (rEmCRT) inhibited C1q binding to IgM. Consequently, rEmCRT inhibited classical complement activation manifested as decreasing C4/C3 depositions and antibody-sensitized cell lysis. Moreover, rEmCRT binding to C1q suppressed C1q binding to human mast cell, HMC-1, resulting in reduced C1q-induced mast cell chemotaxis. According to these results, E. multilocularis expresses EmCRT to interfere with C1q-mediated complement activation and C1q-dependent non-complement activation of immune cells, possibly as an immune evasion strategy of the parasite in the host.


Introduction
Echinococcosis is a serious zoonotic disease caused by the infections of Echinococcus multilocularis (Em) and Echinococcus granulosus (Eg) larvae, causing alveolar echinococcosis (AE) and cystic echinococcosis (CE), respectively. Of these, AE is more serious and a lifethreatening disease with high mortality and poor prognosis if not well treated [1]. The main prevalence of AE is in regions of the Northern Hemisphere, such as Asia, Europe and North America. There are an estimated 18,235 new AE infections or cases each year worldwide [2]. It is considered to be one of the most deadly helminthic diseases in humans [3]. Human AE infection is caused by the accidental ingestion of food contaminated with eggs, which form microcystic metacestode vesicles in the liver [4]. The vesicles remain indefinitely proliferative in infected livers, like a tumor, invading surrounding tissues or even metastasizing to distant organs, such as the spleen and brain. E. multilocularis metacestode vesicles that are exposed to host immune system [5] have developed sophisticated strategies to evade host immune responses, including innate and adaptive immunity. Understanding of the parasite-developed immune evasion mechanisms would facilitate the identification of vaccine and drug targets to control E. multilocularis and other helminth infections.
The complement system acts as the primary line of defense against invading pathogens and plays a crucial role in the innate and acquired immune responses [6,7]. Many pathogens Trop. Med. Infect. Dis. 2023, 8,47 3 of 12 as metacestode vesicles as described [31]. The crude somatic extracts of protoscoleces (PSCs) and metacestode vesicles, and vesicle fluid protein were obtained according to our previously established protocol [29]. His-tagged recombinant EmCRT (rEmCRT) was expressed in E. coli BL21 under induction of 0.4 mM IPTG and purified by Ni-affinity chromatography (Beyotime Biotechnology, Shanghai, China) as described [29].

Cell Culture
HMC-1, the human mast cell line obtained from Qingqi (Shanghai Biotechnology Development Co., Ltd., Shanghai, China), was grown in DMEM medium containing 1 × streptomycin, penicillin and amphotericin B and 10% FBS in 5% CO 2 at 37 • C.
Far Western blotting: The same amount of C1q (5 µg) and BSA (5 µg) were separated in 15% polyacrylamide gel and transferred onto PVDF membrane (Merck, Darmstadt, Germany). After being blocked with 3% BSA, the membrane was incubated with rEmCRT at 5 µg/mL in binding buffer at 37 • C for two hours. The binding of rEmCRT to C1q was detected with the same antibodies as mentioned above.
Immunoprecipitation: To further determine whether C1q was able to bind to nondenatured rEmCRT or native EmCRT derived from different stages of worm extracts, Protein A + G Agarose (Thermo, Waltham, MA, USA) were incubated with 5 µg of anti-His antibody + 5 µg rEmCRT, or 5 µL anti-EmCRT antisera + crude extracts of metacestode vesicles, PSCs or metacestode vesicle fluid (each 20 µg) overnight at 4 • C. Total 10 µg of human C1q was added to incubate overnight at 4 • C. After being centrifuged at 1000× g at 4 • C for 3 min, the supernatant was discarded and the agarose bead-protein complex was washed three times with radioimmunoprecipitation assay (RIPA) lysis buffer (BOSTER Biological Technology Co., Ltd., Chengmai, China). Finally, 30 µL RIPA buffer was added and the protein complex in the supernatant was separated by SDS-PAGE and then transferred onto PVDF membrane. The C1q pulled down by the non-denatured recombinant and native EmCRT protein was determined using rabbit anti-human C1qA antibody (Abcam, Cambridge, UK) at 1:4000 dilutions.

C3 and C4 Deposition Assay
As a C1q activator, human IgM (Sigma, St. Louis, MO, USA) was coated on 96-well plates at 2 µg/mL overnight at 4 • C, then blocked with 5% BSA in PBS at 37 • C for 2 h. On each well of the plates, 2 µg of C1q was added that had been pre-incubated with different amounts of rEmCRT (0, 2, 4 µg) or BSA (4 µg, as a comparison) in total volume of 100 µL for 2 h at 37 • C and incubated for 1 h at 37 • C. After washing with PBST, C1q-D diluted at 1:100 in 1 × Veronal buffer (VB, Lonza, Basel, Switzerland) containing 0.05% Tween-20 and 0.1% gelatin was added into each well for 1 h at 37 • C to finish the classical complement activation. NHS at dilution of 1:50 was used as a positive control. After being washed for three times with PBST, each well was added with 100 µL goat anti-human C4 mAb (1:1000 Abcam, Cambridge, UK) or rabbit anti-human C3 polyclonal antibodies (1:100, BOSTER Biological Technology Co., Ltd., Chengmai, China) to determine C4 and C3 intermediate product of classical complement activation. HRP-conjugated rabbit anti-goat or goat antirabbit IgG (1:5000 or 1:1000, Affinity Biosciences, Liyang, China) was used as the secondary antibody and OPD (Beyotime Biotechnology, Shanghai, China) was used as the substrate.

Hemolytic Assays
To determine whether rEmCRT inhibited classical complement-activation-mediated hemolysis of sheep red blood cells (SRBC), fresh SRBC at 5×10 8 cells/mL in 1×HBSS ++ (Hank's balanced salt solution supplemented with 0.15 mM CaCl 2 and 1 mM MgCl 2 , Solarbio, Beijing, China) was sensitized with rabbit anti-erythrocyte antibody (Zhengzhou Baiji Biotechnology Co., Ltd., Zhengzhou, China) at 37 • C for 30 min. After being washed with 1×HBSS ++ , the antibody-sensitized SRBC were incubated with 1 µg of C1q preincubated with 0, 1, 2 or 4 µg of rEmCRT or 4 ug or BSA in total volume of 100 µL followed by adding C1q-D (8% in HBSS ++ ) for 1 h at 37 • C to complete the complement classical activation. The reaction was stopped by adding cold HBSS ++ containing 10 mM EDTA. The supernatants were collected by centrifuging at 1200× g for 10 min, and OD 412 was measured. The erythrocyte lysis rate was calculated with total hemolysis in water as control.

Inhibition of rEmCRT on the Binding of C1q to IgM
To determine whether rEmCRT inhibited C1q binding to IgM, the plates were coated with human IgM (2 µg/mL) and then blocked with 2% BSA in PBS for 2 h at 37 • C. C1q (1 µg) was pre-incubated with rEmCRT or BSA (0, 0.5, 1, 1.5, 2, 2.5 and 3 µg in 100 µL binding buffer) at 37 • C for two hours. The reaction complex was added to the IgM-coated plates overnight at 4 • C. Anti-C1q polyclonal antibody (Abcam, Cambridge, UK) at 1:1000 dilution was used to detect remaining C1q on IgM-coated plates.

Cell Immunofluorescence Labeling
To evaluate whether rEmCRT inhibited C1q binding to the C1q receptor on mast cells, HMC-1 cells were adhered on glass slides, then fixed for 20 min at room temperature with paraformaldehyde 4%. The fixed HMC-1 cells were blocked with normal goat serum (BOSTER Biological Technology Co., Ltd., Chengmai, China) for 30 min at room temperature. Human C1q at concentration of 80 µg/mL was pre-incubated with different amounts of rEmCRT (0, 30, 60 and 80 µg/mL) for one hour at 37 • C, then transferred to the HMC-1 cells on the slides. Rabbit anti-C1q mAb (Abcam, Cambridge, UK) diluted at 1:100 in PBS was used to measure the binding of C1q to HMC-1 cells. DAPI staining was performed to show nuclei of the cells (Solarbio, Beijing, China). Confocal laser scanning microscope (Nikon, Tokyo, Japan) was used to acquire the images.

Transwell Chemotaxis Assay
The inhibitory effect of rEmCRT on the C1q-induced chemotactic migration of HMC-1 cells was identified using an 8-µm-pore transwell chamber (Corning, New York, NY, USA). Total 200 µL of DMEM medium containing 2% FBS was added to the upper chamber with 3 × 10 5 HMC-1 cells. The 10 nM human C1q was pre-incubated with various concentrations of rEmCRT (0, 3, 6 µg) in 500 µL DMEM medium with 5% FBS, then transferred to the lower chamber to initiate chemotactic migration of HMC-1 cells at 5% CO 2 , 37 • C for 24 h. The cells that migrated through the membrane to the lower chamber were counted using a flow cytometer (Beckman Coulter, Brea, CA, USA) [21]. LPS (100 ng/mL) was used as a positive control and BSA (6 µg/0.5 mL) as a negative control for the chemotaxis assay.

Statistical Analysis
All data were shown as mean ± standard deviation and one-way ANOVA was performed. All statistical analysis was performed by GraphPad Prism 7 (San Diego, CA, USA). p < 0.05 was considered as statistically significance.

Recombinant EmCRT Binds to Human C1q
The interaction between rEmCRT and human complement C1q was determined by different immunological assays. ELISA results demonstrated that rEmCRT was capable of binding to C1q in a dose-dependent way ( Figure 1A(a)). Under the same conditions, there was no apparent binding of rEmCRT to the BSA-coated plate ( Figure 1A(b)). SDS-PAGE separation results showed that C1q contained mainly A, and weak B and C chains under reduced condition ( Figure 1B(a)). The Far Western blotting demonstrated that rEmCRT mainly bound to A chain, weakly bound to C chain of complement C1q, but did not bind to BSA as the non-relative control ( Figure 1B(b)). Further, to confirm whether recombinant EmCRT bound to human C1q under non-denatured condition, the natural form of C1q was pulled down by rEmCRT bound to anti-His antibody ( Figure 1C). Anti-His antibody alone without rEmCRT could not pull down C1q. These findings demonstrated that rEmCRT was able to bind to the A and C chains of complement C1q.

Native EmCRT from Worm Extracts Binds to Human C1q
Immunoprecipitation and Western blotting were performed to investigate the binding of native EmCRT derived from E. multilocularis larval stages to human C1q (Figure 2). The results distinctly showed that C1q could bind to native EmCRT derived from worm extracts or vesicle fluid, pulled down by the anti-EmCRT polyclonal antibody and detected by anti-C1q antibody. No C1q was seen in the anti-EmCRT alone control, suggest-

Native EmCRT from Worm Extracts Binds to Human C1q
Immunoprecipitation and Western blotting were performed to investigate the binding of native EmCRT derived from E. multilocularis larval stages to human C1q ( Figure 2). The results distinctly showed that C1q could bind to native EmCRT derived from worm extracts or vesicle fluid, pulled down by the anti-EmCRT polyclonal antibody and detected by anti-C1q antibody. No C1q was seen in the anti-EmCRT alone control, suggesting that C1q specifically binds to native EmCRT derived from worm extracts or fluid.

rEmCRT Inhibits the Classical Complement Activation Pathway and Hemolysis
To evaluate whether the binding of rEmCRT to C1q interferes with C1q-initiated classical complement activation, the activation intermediate products C4 and C3 were measured. The results displayed that classical complement activation can be completed in C1q-D serum by supplementing with C1q at a similar level to NHS based on the levels of C4 or C3 deposited in the plates. However, the addition of rEmCRT to C1q significantly decreased C4 and C3 deposition in a dose-dependent way (Figure 3), indicating that the binding of rEmCRT to C1q interfered with C1q/IgM-initiated classical complement activation. There was no inhibitory effect in the presence of BSA as a control.
To further determine whether rEmCRT inhibits C1q-dependent classical pathway, antibody-sensitized SRBC were incubated with C1q pre-incubated with different amounts of rEmCRT. As shown in Figure 4, rEmCRT inhibited C1q-initiated classical complementactivation-mediated hemolysis in a dose-dependent manner. There was no obvious hemolysis observed in the presence of C1q-D serum alone, since the classical pathway could not be activated in the absence of C1q. BSA as a control protein did not show an inhibitory effect.

rEmCRT Inhibits the Classical Complement Activation Pathway and Hemolysis
To evaluate whether the binding of rEmCRT to C1q interferes with C1q-initiated classical complement activation, the activation intermediate products C4 and C3 were measured. The results displayed that classical complement activation can be completed in C1q-D serum by supplementing with C1q at a similar level to NHS based on the levels of C4 or C3 deposited in the plates. However, the addition of rEmCRT to C1q significantly decreased C4 and C3 deposition in a dose-dependent way (Figure 3), indicating that the binding of rEmCRT to C1q interfered with C1q/IgM-initiated classical complement activation. There was no inhibitory effect in the presence of BSA as a control.
To further determine whether rEmCRT inhibits C1q-dependent classical pathway, antibody-sensitized SRBC were incubated with C1q pre-incubated with different amounts of rEmCRT. As shown in Figure 4, rEmCRT inhibited C1q-initiated classical complementactivation-mediated hemolysis in a dose-dependent manner. There was no obvious hemolysis observed in the presence of C1q-D serum alone, since the classical pathway could not be activated in the absence of C1q. BSA as a control protein did not show an inhibitory effect.
antibody-sensitized SRBC were incubated with C1q pre-incubated with different amounts of rEmCRT. As shown in Figure 4, rEmCRT inhibited C1q-initiated classical complementactivation-mediated hemolysis in a dose-dependent manner. There was no obvious hemolysis observed in the presence of C1q-D serum alone, since the classical pathway could not be activated in the absence of C1q. BSA as a control protein did not show an inhibitory effect.  plates (2 µ g/mL). After being washed, C1q-D serum was added as a supplement of other complement components to trigger classical pathway activation. Anti-C4 or -C3 antibodies were used to detect the deposits of C4 and C3 on the plates. The results are shown as the means ± SDs for three independent experiments. * p < 0.05, **** p < 0.0001. ns, no significant difference. . Complement-mediated hemolysis was inhibited by rEmCRT. C1q-mediated antibody-sensitized sheep blood cells hemolysis was inhibited by rEmCRT in a dose-dependent manner. Three independent experiments were conducted and the results were presented as means ± SDs. * p < 0.05 and **** p < 0.0001. ns, no significant difference.

rEmCRT Competitively Inhibits the Binding of Human C1q to IgM
To understand how rEmCRT inhibits C1q-mediated classical complement activation, the competitive inhibition assay was carried out in the presence of IgM. C1q was preincubated with various amounts of rEmCRT before being transferred to IgM-coated plates. The results showed that pre-treatment with rEmCRT significantly inhibited C1q's binding ability to IgM, and the inhibition was dose dependent. No inhibitory effect was observed in BSA control group ( Figure 5). The results suggest that rEmCRT competes with IgM to bind to C1q. . Complement-mediated hemolysis was inhibited by rEmCRT. C1q-mediated antibodysensitized sheep blood cells hemolysis was inhibited by rEmCRT in a dose-dependent manner. Three independent experiments were conducted and the results were presented as means ± SDs. * p < 0.05 and **** p < 0.0001. ns, no significant difference.

rEmCRT Competitively Inhibits the Binding of Human C1q to IgM
To understand how rEmCRT inhibits C1q-mediated classical complement activation, the competitive inhibition assay was carried out in the presence of IgM. C1q was preincubated with various amounts of rEmCRT before being transferred to IgM-coated plates. The results showed that pre-treatment with rEmCRT significantly inhibited C1q's binding ability to IgM, and the inhibition was dose dependent. No inhibitory effect was observed in BSA control group ( Figure 5). The results suggest that rEmCRT competes with IgM to bind to C1q.
To understand how rEmCRT inhibits C1q-mediated classical complement activation, the competitive inhibition assay was carried out in the presence of IgM. C1q was preincubated with various amounts of rEmCRT before being transferred to IgM-coated plates. The results showed that pre-treatment with rEmCRT significantly inhibited C1q's binding ability to IgM, and the inhibition was dose dependent. No inhibitory effect was observed in BSA control group ( Figure 5). The results suggest that rEmCRT competes with IgM to bind to C1q. Figure 5. rEmCRT inhibited human C1q to bind to IgM. Total 10 µg/mL of C1q was pre-incubated with 0.5 to 3 times excess (w:w) of rEmCRT or BSA and then transferred to the plates coated with IgM. After being washed, anti-C1q polyclonal antibody was used to detect C1q binding to IgM in presence of rEmCRT. Data are expressed as mean ± SDs from three independent experiments and statistical analysis was performed using one-way ANOVA. **** p < 0.0001 compared to BSA control.

rEmCRT Inhibits C1q Binding to Mast Cells
To assess whether rEmCRT could inhibit the binding of C1q to the C1q receptor on mast cells, C1q (80 µg/mL) was mixed with rEmCRT at concentration of 0, 30, 60 and 80 µg/mL. Then, it was transferred into HMC-1 cells. The C1q binding on the mast cells was detected by immunofluorescence staining with anti-C1q antibody. rEmCRT inhibited C1q binding to mast cells and the inhibition was dose dependent. No obvious fluorescence was observed in the rEmCRT or PBS alone control group ( Figure 6). Thus, the results indicate that rEmCRT binds to C1q, which interferes with C1q's binding to mast cells. . rEmCRT inhibited human C1q to bind to IgM. Total 10 μg/mL of C1q was pre-incubated with 0.5 to 3 times excess (w:w) of rEmCRT or BSA and then transferred to the plates coated with IgM. After being washed, anti-C1q polyclonal antibody was used to detect C1q binding to IgM in presence of rEmCRT. Data are expressed as mean ± SDs from three independent experiments and statistical analysis was performed using one-way ANOVA. **** p < 0.0001 compared to BSA control.

rEmCRT Inhibits C1q Binding to Mast Cells
To assess whether rEmCRT could inhibit the binding of C1q to the C1q receptor on mast cells, C1q (80 μg/mL) was mixed with rEmCRT at concentration of 0, 30, 60 and 80 μg/mL. Then, it was transferred into HMC-1 cells. The C1q binding on the mast cells was detected by immunofluorescence staining with anti-C1q antibody. rEmCRT inhibited C1q binding to mast cells and the inhibition was dose dependent. No obvious fluorescence was observed in the rEmCRT or PBS alone control group ( Figure 6). Thus, the results indicate that rEmCRT binds to C1q, which interferes with C1q's binding to mast cells. Figure 6. Recombinant EmCRT inhibited the binding of C1q to HMC-1 cells. HMC-1 cells were adhered on glass slides and fixed with 4% paraformaldehyde, and incubated with C1q (80 µ g/mL) that was pre-incubated with different amounts of rEmCRT (0, 30, 60 or 80 µ g/mL). The binding of C1q on HMC-1 cells was detected with anti-C1q antibody and FITC-conjugated goat anti-rabbit IgG (green). Nuclei were dyed with DAPI (blue). The magnitude is 400× and one amplified cell located at the lower right corner is 1000×.

rEmCRT Inhibits C1q-Induced Mast Cells Chemotaxis
To determine the effect of rEmCRT on C1q-induced chemotaxis of mast cells, a migration assay using a transwell chamber was conducted. The results revealed that both LPS and C1q significantly attracted HMC-1 cells migration through the membrane ( Figure   Figure 6. Recombinant EmCRT inhibited the binding of C1q to HMC-1 cells. HMC-1 cells were adhered on glass slides and fixed with 4% paraformaldehyde, and incubated with C1q (80 µg/mL) that was pre-incubated with different amounts of rEmCRT (0, 30, 60 or 80 µg/mL). The binding of C1q on HMC-1 cells was detected with anti-C1q antibody and FITC-conjugated goat anti-rabbit IgG (green). Nuclei were dyed with DAPI (blue). The magnitude is 400× and one amplified cell located at the lower right corner is 1000×.

rEmCRT Inhibits C1q-Induced Mast Cells Chemotaxis
To determine the effect of rEmCRT on C1q-induced chemotaxis of mast cells, a migration assay using a transwell chamber was conducted. The results revealed that both LPS and C1q significantly attracted HMC-1 cells migration through the membrane (Figure 7). The C1q-induced mast cells migration through the membrane was inhibited by the preincubation with rEmCRT in a dose-dependent manner (**** p < 0.0001). BSA protein had no effect on HMC-1 cells attraction by C1q.
Trop. Med. Infect. Dis. 2023, 8, 47 10 of 13 Figure 7. The inhibition of rEmCRT on C1q-induced migration of mast cells was conducted in a transwell 24-well plate. In the upper chamber, 3 × 10 5 HMC-1 cells were seeded per well. An amount of 10 nM of C1q was pre-incubated with rEmCRT (0, 6 or 12 μg/mL) and then transferred into the lower chamber. LPS (100 ng/mL) was added as a positive control. The number of cells was counted by a flow cytometer. Data are shown as the mean ± SDs from three independent tests, each test was carried out in triplicate. **** p < 0.0001. ns, no significant difference.

Discussion
Helminths are multicellular pathogens that generate many macromolecules to regulate host immune responses as an evasion strategy. The complement system is the primary line of immune defense against pathogen invasion, and works by attacking pathogens directly and enhancing and opsonizing the functions of antibodies and immune effectors (neutrophils, eosinophils, mast cells, macrophages, etc.) to eliminate the invaded pathogens [32]. Therefore, blocking complement attack is crucial to pathogens' survival in the invaded host [6,7,13]. Many pathogens, such as viruses, bacteria and parasites, apparently share comparable strategies or mechanisms to avoid complement attack [9]. However, there is a lack of knowledge about the complement escape mechanism in the parasitism of cestodes.
Calreticulin (CRT) is a well conserved Ca 2+ binding protein and molecular chaperone. CRT is involved in a spectrum of processes, such as Ca 2+ homeostasis, antigen presentation and process, cellular adhesion and motility of organisms [33,34]. Available studies demonstrated that CRTs from some parasites, such as protozoa, helminths, and arthropods, have the ability to bind to complement component C1q to interfere with the host complement activation [25]. Furthermore, the binding domain of C1q was located in the S-domain of the protein [35]. In our previous study, sequence alignment reveals that Em-CRT shares 49-56% sequence identity with other helminth CRTs, including N. americanus [28], H. contortus [27] and B. malayi [26], indicating that it is genetically conserved among helminths [36].
Our previous studies have identified that EmCRT was identified on the surface of the In the upper chamber, 3 × 10 5 HMC-1 cells were seeded per well. An amount of 10 nM of C1q was pre-incubated with rEmCRT (0, 6 or 12 µg/mL) and then transferred into the lower chamber. LPS (100 ng/mL) was added as a positive control. The number of cells was counted by a flow cytometer. Data are shown as the mean ± SDs from three independent tests, each test was carried out in triplicate. **** p < 0.0001. ns, no significant difference.

Discussion
Helminths are multicellular pathogens that generate many macromolecules to regulate host immune responses as an evasion strategy. The complement system is the primary line of immune defense against pathogen invasion, and works by attacking pathogens directly and enhancing and opsonizing the functions of antibodies and immune effectors (neutrophils, eosinophils, mast cells, macrophages, etc.) to eliminate the invaded pathogens [32]. Therefore, blocking complement attack is crucial to pathogens' survival in the invaded host [6,7,13]. Many pathogens, such as viruses, bacteria and parasites, apparently share comparable strategies or mechanisms to avoid complement attack [9]. However, there is a lack of knowledge about the complement escape mechanism in the parasitism of cestodes.
Calreticulin (CRT) is a well conserved Ca 2+ binding protein and molecular chaperone. CRT is involved in a spectrum of processes, such as Ca 2+ homeostasis, antigen presentation and process, cellular adhesion and motility of organisms [33,34]. Available studies demonstrated that CRTs from some parasites, such as protozoa, helminths, and arthropods, have the ability to bind to complement component C1q to interfere with the host complement activation [25]. Furthermore, the binding domain of C1q was located in the S-domain of the protein [35]. In our previous study, sequence alignment reveals that EmCRT shares 49-56% sequence identity with other helminth CRTs, including N. americanus [28], H. contortus [27] and B. malayi [26], indicating that it is genetically conserved among helminths [36].
Our previous studies have identified that EmCRT was identified on the surface of the E. multilocularis and in the excretory-secretory (ES) products of larval stages as well, suggesting its accessibility to host immune systems including complement components, which provides a biological basis to study its interaction with host immune and complement systems. In this study, we demonstrated that both the recombinant EmCRT and the natural protein from the parasite were able to bind to C1q, mainly to A chain, indicating the possibility of its function as an inhibitor of classical pathway of complement activation. In fact, we subsequently observed a significant reduction in C4b and C3b intermediate products after C1q was pre-incubated with rEmCRT. The antibody-sensitized sheep red blood cells hemolysis was also inhibited as a result of the unsuccessful formation of the membrane attack complex (MAC) due to the binding of EmCRT to C1q. Further investigation in this study identified that EmCRT competed with IgM to bind to C1q [37]. It explains how EmCRT inhibits the IgM-initiated classical complement activation. Except for being involved in the classical complement pathway, C1q also possesses multiple biological functions as a versatile pattern recognition molecule [17]. In response to foreign pathogen invasion, C1q acts as an initiator for classical complement activation and a non-complement immune activator as well. In this study, we investigated the effect of EmCRT on the C1qmediated mast cell activation except for its inhibitory effect on the complement classical activation pathway. Mast cells as innate immune cells take an active part in the innate immune responses to a number of pathogens and enhance the earliest processes in the development of acquired immune responses [1,2,21,38]. During chronic atopic disease or helminthiasis, mature mast cells are infiltrated at the sites of inflammation. Two specific C1q receptors, cC1q-R (binding to the collagen-like stalk of C1q) and gC1q-R (binding to the globular heads of C1q), were found on mast cells. C1q binds to the C1q receptors on the mast cells to act as an attractant to induce mast cells migration to infectious or inflammatory sites [21]. In the present study, we demonstrated that C1q could bind to the surface of HMC-1 mast cells, the addition of rEmCRT decreased the C1q binding to mast cells in a dose-depend manner, possibly through blocking the binding ability of C1q to the C1q receptor on mast cells. The addition of rEmCRT also inhibited C1q-induced HMC-1 cells chemotaxis detected by transwell chamber assays. All in vitro experimental results in this study suggest that E. multilocularis produces calreticulin to interfere with C1q-initiated classical complement activation and C1q-induced chemotaxis of mast cells, possibly as a survival strategy of the parasite in the host. However, the actual effect of calreticulin on the complement activation on E. multilocularis metacestodes in vivo, or on the protoscoleces in vitro, needs to be further investigated.
Combined with our previous study, our results suggest that E. multilocularis produces calreticulin during infection to inhibit C1q-mediated activation of the classical complement pathway and C1q-dependent immune cell activation, possibly as a survival strategy in the hostile immune environment of the host. Therefore, EmCRT could be a good vaccine candidate and drug target against E. multilocularis infection. The actual impact of EmCRT on the complement activation on E. multilocularis metacestodes in vivo is under investigation.
Author Contributions: L.Z. and S.X. designed and conceived the study. S.X. and L.C. conducted the experiments. S.X., B.Z., L.C., Y.Y., J.C., G.Y. and Y.S. analyzed the data. S.X. wrote the paper. L.Z., B.Z., S.X. and Y.W. revised the manuscript. All authors have read and agreed to the published version of the manuscript.