Characterisation of extracellular vesicles isolated from hydatid cyst fluid and evaluation of immunomodulatory effects on human monocytes

Abstract Hydatidosis is a disease caused by the larval stage of Echinococcus granulosus, which involves several organs of intermediate hosts. Evidence suggests a communication between hydatid cyst (HC) and hosts via extracellular vesicles. However, a little is known about the communication between EVs derived from HC fluid (HCF) and host cells. In the current study, EVs were isolated using differential centrifugation from sheep HCF and characterized by western blot, electron microscope and size distribution analysis. The uptake of EVs by human monocyte cell line (THP‐1) was evaluated. The effects of EVs on the expression levels of pro‐ and anti‐inflammatory cytokines were investigated using quantitative real‐time PCR (RT‐PCR), 3 and 24 h after incubation. Moreover, the cytokine level of IL‐10 was evaluated in supernatant of THP‐1 cell line at 3 and 24 h. EVs were successfully isolated and showed spherical shape with size distribution at 130.6 nm. After 3 h, the expression levels of pro‐inflammatory cytokine genes (IL1Β, IL15 and IL8) were upregulated, while after 24 h, the expression levels of pro‐inflammatory cytokines were decreased and IL13 gene expression showed upregulation. A statistically significant increase was seen in the levels of IL‐10 after 24 h. The main mechanism of the communication between EVs derived from HCF and their host remains unclear; however, time‐dependent anti‐inflammatory effects in our study suggest that HC may modulate the immune responses via EVs.


| INTRODUC TI ON
Human cystic echinococcosis (Hydatidosis, or hydatid disease) is a zoonotic neglected disease (ZND) caused by the larval stage of Echinococcus granulosus, which is considered as a public health concern, particularly in regions with livestock. 1 Hydatidosis has a worldwide distribution with reports from all continents. The prevalence of this disease can reach 5%-10% in certain endemic regions; however, the surgical incidence rate of human CE in a global scale seems to be less than 10/100000. 3  DALYs per year for CE. 3 Hydatidosis occurs due to the metacestode stage of E. granulosus, which involves almost all organs. 6 The hydatid cyst (HC) structure from the outer to the inner layers consists of a fibrotic layer, which is resulted from host immune responses, laminated layer (LL), germinal layer (GL), which is the origin of brood capsule, and HCs fluid (HCF). 2,6 It was well-documented that HCF is a heterogenic fluid containing several proteins, which are originated from protoscoleces, GL, and even host. 7 Excretory/secretory (E/S) materials, produced by protoscoleces, may permeate across LL and fibrotic layer, and manipulate host immune responses. 8,9 Therefore, it seems that there is a cross-talk between metacestode stage of E. granulosus and its hosts. 9 Extracellular vesicles (EVs) are released from different cell types and microorganisms. EVs play crucial role in cell signalling, modulating of the immune responses, and communication between host cells and infectious agents. [10][11][12][13][14] Based on the biogenesis, EVs have been categorized as exosomes, microvesicles and apoptotic bodies. 15,16 EVs mostly carry a complex of components and show features of their mother cell. EVs contain lipids, proteins, genetic materials (DNA, RNA and micro RNAs) and metabolites. [17][18][19][20] During recent decades and alongside a plenty of studies on characterisation and applications of EVs, many studies have been conducted on the isolation, characterisation and biomedical applications of EVs, which are excreted from parasites. 14

| Parasite preparation
To obtain HCF, a liver full of fertile HCs from a sheep, which was slaughtered in an abattoir located in Guilan province, north of Iran, was aseptically transferred to the Foodborne and Waterborne Diseases

| EVs isolation and characterisation
To isolate EVs from HCF, differential centrifugation was employed on 500 mL of fresh HCF to separate EVs. Isolated EVs were analysed regarding surface markers, shape and size, based on MISEV2018 guideline. 28 Briefly, HCF was centrifuged at 300 × g for 15 min, and supernatant was transferred to sterile 50-mL conical centrifuge tubes for further analyses. Collected supernatant was centrifuged at 3000 × g for 10 min to remove the cell debris. The large vesicles were isolated by high-speed centrifugation at 20,000 × g for 30 min at 4°C. To collect the small EVs, ultracentrifugation at 100,000 × g for 120 min at 4°C in a 45Ti rotor (Beckman Coulter, Inc.) was performed. The resultant pellet was then re-suspended in PBS and spun again at 100,000 × g for 120 min at 4°C. Following the last wash, the EVs were re-suspended in PBS and stored at −80°C. The ultracentrifugation and all characterising processes were carried out in the Department of Stem Cells and Developmental Biology at Royan Institute for Stem Cell Biology and Technology.

| DLS analysis
The suspension of EVs in PBS was loaded into a cuvette. Particle size distribution of the EVs was measured via dynamic light scattering (DLS, Zetasizer nano range). Fluctuations of light scattering intensity were evaluated using a laser to calculate the size of EVs. Data were analysed using Malvern Zetasizer Software (Malvern Instruments, v7.03).

| Western blot
The presence of EV markers (CD63 and CD81) was checked in both parasite lysate and EVs. Protein was extracted from protoscoleces of HC by repeated freeze thawing followed by a sonication in SDS-PAGE sample buffer, as mentioned previously. 29 To extract total protein, EVs were boiled in SDS-PAGE sample buffer followed by sonication for 5 min. BCA assay was employed to estimate the protein content of samples. In addition, the origin of EVs was investigated using analysis of the interaction between HC-positive human serum and EVs via western blot analysis.
To perform western blot, 20 μg (based on BCA assay of protein content) of EVs sample was mixed with SDS-PAGE loading buffer and sonicated for 5 min followed by heating at 95°C for 5 min. The sample was then loaded onto 10% SDS-PAGE gel. Samples were transferred onto polyvinyl difluoride (PVDF) membrane (Millipore), and the membrane was blocked with the solved bovine serum albumin (5%) (BSA) in Tris-buffered saline (TBST) special (Tween 20: 0.1%). The CD63 (1:500, Abcam: Ab8219) and CD81 (1:500, Santa Cruz: SC7637), as positive markers, and Calnexin (1:500, Santacruz: Sc11397), as a negative marker, were incubated overnight with the membrane. Then, the membrane was washed by TBST special (1X) and incubated with a secondary antibody (1:50000, Sigma) for 1 h at room temperature. After washing the membrane with TBST special (1X), the enhanced chemiluminescence (ECL) solution was added.
Then, the protein bands were detected by a chemiluminescence device (Uvitec). All western blot analyses were performed in two repeats and results were same.

| Electron microscopy
The morphology of EVs was evaluated by a scanning electron microscope (SEM). 150 ng suspension of EVs was dried on a glass slide, coated by gold (SCD 005, Bal-Tec), and analysed by the SEM (XL30, Philips).

| EV staining and uptake analysis
In order to reduce the false-positive signal that is common for li-

| RNA isolation, complementary DNA (cDNA) synthesis and quantitative real-time PCR
Total RNA was extracted after 3 and 24 h by total RNA extraction kit (Yekta Tajhiz Azma), according to the manufacturer's protocol.
DNase (Thermo Fisher Scientific™) treatment was performed to remove the probable residual DNA and to improve the quality of the extracted RNA, according to the manufacturer's protocol. The RNA solution was finally collected and stored at −70 ° C until cDNA synthesis. Prior to cDNA synthesis, the concentration of purified RNA samples was determined by NanoDrop (NanoDrop Technologies) apparatus and RNA was adjusted (normalized). cDNA was constructed using cDNA synthesis kit (Yekta Tajhiz Azma), according to the manufacturer's protocol. To study the expression levels of the IL1Β, NLRP3, IL8, IL15, TNFΑ, IFNG, IL4, IL13 and TGFΒ genes, qRT-PCR using specific primers was employed ( Table 1). Real-time PCR was performed using Rotor-Gene Q (Qiagen, Germany) thermocycler in a 20 reaction mixture containing 10 μL SYBR Green qPCR master mix 2X (Ampliqon), 5 μM of primers, and template cDNA, based on MIQE guideline: cycling reactions starting with one cycle of 50°C (2 min) and 95°C (1 min), followed by 40 cycles at 95°C (15 s) and 60°C (1 min). Melting curves were determined with ramping from 75°C to 90°C s −1 , as previously described. 31 The relative expression of targeted genes compared to the housekeeping gene was calculated using 2 (-ΔΔCt) method. All qRT-PCR procedures were performed following the MIQE guideline. Each reaction was performed in duplicate, and the ACTΒ gene was considered as housekeeping gene.

| Cytokine assay by ELISA
Culture supernatants were collected after the treatments and stored at −80°C for cytokine analysis. The secretion level of IL-10, an important anti-inflammatory cytokine, was measured with standard enzyme immunoassays (ELISA) kits, according to the manufacturer's protocols. Raw data were analysed by the web software MyAssays (https://myass ays.com/assay.aspx?id=787), and the concentrations were calculated by a Four Parameter Logistic (4PL) curve.

| Statistical analysis
Student's t-test was applied to analyse relative gene expressions at different time points in comparison to the housekeeping gene. calcification. The HCF contained protoscoleces, daughter cysts and LL ( Figure S1). The genotype of isolated E. granulosus sensu lato was G1. 27

| EVs characterisation and uptake
Western blot analyses confirmed the expression of CD63 marker, while Calnexin and CD81 were absent in EVs samples ( Figure 1A, Figure S2). Our findings confirmed the presence of CD63 and absence of CD81 in protein lysate of protoscoleces, as well ( Figure 1A).
The SEM and morphology revealed round shape vesicles ( Figure 1B).
The DLS analysis showed average size distribution, in which most of EVs (~ 74%) represented a peak at 130.6 nm diameter ( Figure 1C).
HC-positive human serum showed an interaction with protein lysate of protoscoleces and EVs ( Figure 1A, Figures S3 and S4). THP-1 cells were incubated with EVs, which were labelled by Calcein-AM that the results showed an internalization rate over 60% and bright fluorescent signals at 4 h after incubation ( Figure S5).

| Relative gene expression of qReal-time PCR panel
The relative gene expression analysis of inflammatory biomarkers showed statistically significant upregulation of IL1Β, IL15, IL8 and IFNG after 3 h, while TNFΑ and NLRP3 showed statistically significant downregulation (Figure 2A). The anti-inflammatory biomarkers, IL13, TGFΒ and IL4, were significantly downregulated, upregulated and suppressed, respectively ( Figure 2B). Almost all the tested genes showed downregulation after 24 h except NLRP3 and IL15 ( Figure 2C). In addition, the expression changes of anti-inflammatory biomarkers were statistically significant (p = 0.0003), while only IL13 showed upregulation ( Figure 2D).
The expression of IL1Β, compared to the ACTB was increased after 3 h, while it was significantly reduced 24 h after co-incubation with HCF EVs. The comparison between time points showed statistically significant difference (p = 0.0008) ( Figure 3A). Compared to the housekeeping gene, the highest upregulation during 3 h after cocultivation with HCF EVs was seen in the expression of IL15, while it was dramatically decreased after 24 h ( Figure 3B). The expression level of IL8 gene was significantly upregulated and downregulated 3 h and 24 h after the exposure, respectively. ( Figure 3C). The changes of IFNG was not statistically significant at 3 h, but showed downregulation at 24 h. ( Figure 3D). The expression of TNFΑ was significantly decreased at 3 and 24 h after the exposure ( Figure 3E). In addition, the expression of NLRP3, which plays crucial role in innate immunity and the production/ secretion of IL-1β, was significantly decreased at 3 h, while it showed significant overexpression after 24 h ( Figure 3F).

The expression of TGFΒ was significantly increased after 3 h, but
it was significantly downregulated at 24 h ( Figure 3G). Compared to the housekeeping gene, the relative gene expression of IL4 was suppressed in 3 h after the exposure, while it was significantly decreased during 24 h after co-culture ( Figure 3H). Moreover, the IL13 gene was downregulated at 3 h after the exposure, while its expression was increased after 24 h (p = 0.038) ( Figure 3I). All expression changes of studied factors and their significance values are summarized ( Table 2).

| IL-10 detection in the supernatant in THP-1 cell line
The

| DISCUSS ION
EVs contain several molecules that play crucial role in infections caused by parasites. These particles modulate and affect the host immune responses. 22 In the current study, EVs were isolated from HCF using differential centrifugation, which is known as the most common EVs isolation method, 28 The relative expression of investigated factors, compared to the ACTB housekeeping gene, in THP-1 cell line treated by EVs isolated from HC using real-time PCR.

Factors 3 h 24 h Statistical comparisons between time points (p-values)
The mean of relative expression ± SD p-values

| CON CLUS ION
In summary, EVs were successfully isolated from HCF with average

ACK N O WLE D G E M ENTS
The authors thank all members of the Foodborne and Waterborne Diseases Research Center for their supports.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All generated data from the current study are included in the article and its supplementary materials and data.

CO N S E NT FO R PU B LI C ATI O N
All authors declare that they have seen and approved the submitted version of this manuscript.