Peptide derived from progranulin of the carcinogenic liver fluke, Opisthorchis viverrini stimulates cell hyperproliferation and proinflammatory cytokine production

Purpose Progranulin (PGRN) is a secreted glycoprotein growth factor with roles in wound healing, inflammation, angiogenesis and malignancy. An orthologue of the gene encoding human PGRN was identified in the carcinogenic liver fluke Opisthorchis viverrini. Methods Sequence structure, general characteristics and possible function of O. viverrini PGRN was analyzed using bioinformatics. Expression profiles were investigated with quantitative RT-PCR, western blot and immunolocalization. A specific peptide of Ov-PGRN was used to investigate a role for this molecule in pathogenesis. Results The structure of the gene coding for O. viverrini PGRN was 36,463 bp in length, and comprised of 13 exons, 12 introns, and a promoter sequence. The Ov-pgrn mRNA is 2,768 bp in length and encodes an 846 amino acids with a predicted molecular mass of 91.61 kDa. Ov-PGRN exhibited one half and seven complete granulin domains. Phylogenetic analysis revealed that Ov-PGRN formed its closest relationship with PGRN of liver flukes in the Opisthorchiidae. Transcripts of Ov-pgrn were detected in several developmental stages, with highest expression in the metacercaria, indicating that Ov-PGRN may participate as a growth factor in the early development of O. viverrini. Western blot analysis revealed the presence of detected Ov-PGRN in both soluble somatic or excretory/secretory products, and immunolocalization indicated high levels of expression in the tegument and parenchyma of the adult fluke. Co-culture of a human cholangiocyte cell line and a peptide fragment of Ov-PGRN stimulated proliferation of cholangiocytes and upregulation of expression of the cytokines IL6 and IL8. Conclusion Ov-PGRN is expressed throughout the life cycle of liver fluke, and likely plays a key role in development and growth.


Introduction
Infection with the food-borne liver uke, Opisthorchis viverrini has been classi ed as a group 1 biological carcinogen by the International Agency for Research on Cancer [1,2]. In regions where liver uke infection is endemic, opisthorchiasis is the principal risk factor for cholangiocarcinoma (CCA) [3]. Focusing on the contribution of the excretory and secretory products (ES) of O. viverrini to carcinogenesis, we targeted the O. viverrini granulin-like growth factor, Ov-GRN-1, a prominent component of the ES complement that induces phenotypic hallmarks of cancer. Ov-GRN-1 and other ES components including extracellular vesicles enter cholangiocytes, the epithelial cells that line the biliary tract, and drive cellular signaling that promotes carcinogenesis, including cellular proliferation and migration, angiogenesis and wound healing [4][5][6]. We have con rmed the role of Ov-GRN-1 in driving proliferation of bile duct epithelial cells (cholangiocytes) by genetic manipulation of its expression in the liver uke both by RNA interference and CRISPR gene knockout. Moreover, we have shown that infection of hamsters with the gene-edited, infectious stage of the live uke was feasible and that proliferation of biliary epithelia is markedly suppressed during infection with the ΔOv-grn-1 (Ov-grn-1 knockout) ukes [4,[6][7][8][9].
The genome of O. viverrini encodes three granulin-like genes including progranulin (Ov-PGRN) and two opisthorchiid-speci c granulins (Ov-GRN-1 and Ov-GRN-2) that were identi ed in in silico generated ES products of O. viverrini [10]. Mammalian PGRN has been studied in depth; it is a secreted glycoprotein comprised of multiple granulin domains. Each granulin domain shows 12 conserved cysteine granulin/epithelin modules [11,12]. Cleavage of the signal peptide releases the mature granulin, which can be further cleaved into a slew of active, 6 kDa peptides [13]. The granulin/epithelin module (GEM) itself (contained within each unit) and intact PGRN protein can both regulate cellular proliferation [14,15].
The majority of the granulin family members across the web of life are in a multi-domain form mostly commonly as multiple granulin domains, similar to PGRN.
Opisthorchis and related species are unusual with single domain granulin genes, the most studied form being Ov-GRN-1 which has been shown to promote cell proliferation and wound healing [8, 16-18]. Human PGRN plays a notable role in stimulating cellular proliferation and in ammation, which in turn promotes tumor metastasis [13,19]. Human PGRN is essential for a range of organs, and mutations lead to dementia-like disease in the brain [20].
Notwithstanding that Ov-GRN-1 has been studied extensively, Ov-pgrn from any of the liver ukes has yet to be investigated. Here, we describe the characteristics of the Ov-PGRN protein and investigate the properties of a synthetic peptide of Ov-PGRN-1 that lacks identity to the orthologous human protein and induced proliferation of the H69 cholangiocyte cell line, a model for normal biliary tract epithelium, and induced expression of cholangiocyte proin ammatory cytokines.
O. viverrini metacercariae were isolated from infected cyprinid shes from natural sources by pepsin digestion as described (Pinlaor et al., 2013). Newly excysted-juvenile ukes (NEJ) were prepared from the encysted metacercariae by incubation in 0.25% trypsin in 1´ PBS supplemented with 2´ 200U/ml penicillin, 200 mg/ml streptomycin (Gibco, Thermo Fisher Scienti c, Waltham, MA) for ve min at 37ºC in 5% CO 2 in air, after which NEJs were separated from the discarded cyst walls using mechanical passage through a 24G gauge needle. Flukes were recovered from the biliary tract of Syrian golden hamsters (Mesocricetus auratus) at 2 weeks and 6 weeks after infection with 50-100 metacercariae per hamster by intragastric intubation, to recover juvenile and adult stages of the helminth, respectively [21].
Somatic adult extract (SAE) and excretory-secretory (ES) antigen preparation A soluble lysate of adult worms or somatic adult worm extract (SAE) was prepared from fresh or frozen and homogenized adult worms as described [8]. Brie y, adult worms (~20 worms) recovered from hamsters (above) were washed in cold normal saline solution (NSS) containing 100 µg/ml penicillinstreptomycin. The worms were frozen in liquid nitrogen and homogenized with glasses tissue grinder in PBS containing 1X proteases inhibitor. Lysate was lysed with ultrasonic homogenizer with amplitude 20% at 20 pulses/min for 10 min and centrifuged 10,000×g for 30 min at 4˚C. Supernatant was collected and concentration of protein determined by spectrophotometry at 280 nm. SAE was aliquoted and kept at -80°C until used.
For preparation of the excretory-secretory (ES) products of adult O. viverrini, the live adult worms (~200 worms) collected from experimental hamsters above were washed in sterile NSS containing antibiotics (100 µg/ml penicillin-streptomycin) and transferred into RPMI 1640 culture medium (Gibco, Grand Island, NY) supplemented with 1% glucose, antibiotics and proteinases inhibitors and maintained in culture medium (200 µl/ uke) at 37°C, under 5% CO 2 in air. After incubation for 24 hr, the medium containing ES products was removed, clari ed by centrifugation at 3,000×g at 4˚C for 10 min, the supernatant was concentrated using Amicon 8050 ultra ltration cell (Amicon, Miami, FL) equipped with a YM10 membrane (cutoff, 10,000 daltons), dialyzed against phosphate-buffered saline (PBS) pH 7.4, and sterilized by 0.22 mm ltration. The protein concentration was measured as above after which aliquots were stored at -80°C [22].

Phylogenetic analysis
Progranulin protein sequences were retrieved from GenBank. Sequence homologies were analyzed using the BLAST search program [23]. Signal peptide and cleavage site was predicted with SignalP -5.0 (https://services.healthtech.dtu.dk/service.php?SignalP-5.0). Promoter sequence and transcription site was predicted at https://www.fruit y.org/seq_tools/promoter.html. Asparagines predicted to be Nglycosylation sites were analyzed at https://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0. The protein sequences of PGRN from diverse taxa were retrieved and compared to PGRN of O. viverrini by ClustalW multiple sequences alignment analysis using BioEdit version 7.2.6 [24,25]. The phylogenetic tree of PGRN protein sequences was analyzed and constructed with Maximum likelihood method with Jones-Taylor-Thornton (JTT) model with 1,000 bootstrap replication by using software MEGA version 11.0 [26]. Progranulin protein sequences in phylogenetic tree including THD19177. A liver uke speci c peptide fragment of PGRN The peptide LQSKKDISDAHRMQC (amino acids position 627 -641) designated "Ov-PGRN-L627C641" (Supporting information, Fig. S1) was selected because it appeared to be liver uke PGRN-speci c (does not share identity to human PGRN) and was predicted to be highly immunogenic in rabbits, and therefore suitable for raising a speci c polyclonal antiserum. The peptide was synthesized and was used to generate the polyclonal anti-O. viverrini progranulin antibodies by immunizing New Zealand rabbits (Genscript, Piscataway, NJ).

RNA extraction and quantitative real-time RT-PCR
Total RNA of ukes was extracted with TRIZOL reagents (ThermoFisher Scienti c, Burlington, MA) following the manufacturer's instructions, and contaminating genomic DNA was removed by treatment with DNase I (ThermoFisher Scienti c, Rockfort, IL). The RNA concentration was measured by Nanodrop 2000 (ThermoFisher Scienti c, Wilmington, DE). cDNA was converted from 500 ng of ukes total RNA by using a RevertAid rst strand cDNA synthesis kit (Thermo Fisher Scienti c, Waltham, MA) for qPCR templates. qPCR was performed with biological duplicate samples using a SYBR Green kit (Takara Bio USA, Inc., Mountain View, CA) in a thermal cycler (Light Cycler 480 II, Roche Diagnostics GmbH, Mannheim, Germany). Each qPCR reaction consists of 1 µL of cDNA, 10 µL SYBR Green Master Mix, 10 mM forward and reverse primers for speci c Ov-pgrn gene (forward primer, Ov-PGRN-out-f: 5'-TGTCGGTTCGGGATCCATTG and reverse primer, Ov-PGRN-rev: 5'-ACTTACATTAACGAAAGGACAGC), and distilled water to a nal volume of 20 µL. The thermal cycle was started with a single initiation cycle at 95°C for 10 minutes. The cycle was followed by 40 cycles quanti cation mode of PCR, each PCR cycle consists of denaturation at 95°C for 15 sec, annealing at 53°C, single acquisition mode for 30 sec, and extension at 72°C for 30 sec. After the nal PCR step, the speci city of the real-time PCR reaction was con rmed using melting curve analysis. The evaluation of PGRN expression was performed by relative gene expression analysis using housekeeping genes. The endogenous actin gene (GenBank EL620339.1) was used as a reference control [27]. The control group was prepared the same qPCR reaction as described above except for the difference in a pair of primers for liver uke actin gene instead (forward primer, Ov-actin-F1: 5'-AGCCAACCGAGAGAAGATGA, and reverse primer Ov-actin-R1: 5'-ACCTGACCATCAGGCAGTTC). The relative Ov-pgrn mRNA expression from different developmental stages of O. viverrini, the fold change (R) was calculated by, where ΔCt = Ct value of Ov-pgrn -Ct of value Ov-actin [28].

Western blot analysis
To detect PGRN in somatic adult worm extract and ES product of O. viverrini, 2 mg of SAE and 5 mg ES products were separated in 15 % SDS-PAGE and probed with anti-Ov-PGRN-L627C641 peptide. In brief, electrophoresed proteins were transblotted onto nitrocellulose membrane, after which the membrane was cut into strips and blocked for non-speci c binding with 5 % skim milk in PBST for 1 hr. The membranes were rst incubated overnight with puri ed polyclonal anti-O. viverrini progranulin antibodies generated by immunizing a New Zealand rabbit with the peptide Ov-PGRN-L627C641 (see above) at dilution 1:300 then incubated for 120 min in goat anti-rabbit IgG conjugated with horseradish peroxidase (MerkMillipore, Tumecula, CA) diluted 1:2,000 in PBST. After additional washes with PBST, the membrane was exposed to enhanced chemiluminescence reagent (MerckMillipore, Billerica, MA) and reactive signals visualized using autoradiography using Kodak BioMax lm (Millipore Sigma, Burlington, MA) Immunohistochemistry Para n-embedded sections of adult O. viverrini were de-para nized using xylene. Sections were rehydrated in an ethanol series, serial solution, with two incubations for each stage; 100%, 90%, 80% and 70% ethanol, 5 min each. Sections were immersed in citrate buffer (pH 6) and autoclaved for 10 min for antigen unmasking, followed by blocking with 3% H 2 O 2 in methanol. Thereafter they were incubated overnight at 4°C in rabbit anti-Ov-PGRN-L627C641 peptide sera diluted 1:10, or pre-immunized rabbit IgG in PBS. Sections were probed with anti-rabbit IgG Fc monoclonal secondary antibody (HRP conjugate) (GenScript, Cat. No. A01856, Piscataway, NJ) diluted 1:1,000 in PBS. Peroxidase reaction products were visualized with 3, 3′-diaminobenzidine (DAB) (Sigma-Aldrich, St Louis, MO). Counterstaining was performed with Mayer's hematoxylin for 5 min. A positive signal was indicated by a reddish-brown color under light microscopy.

Cellular proliferation
The human cholangiocyte cell line, H69 was maintained as described [29]. H69 cells were cultured in the presence of the Ov-PGRN-L627C641speci c peptide. Brie y, 1.5x10 3 H69 cells were seeded into wells of a 24 well cell culture plate (SPL Life Sciences, Korea) and cultured with complete medium for 24 hours. Complete medium is de ned as Dulbecco's modi ed Eagle's medium (DMEM)/Ham-F12 supplemented with 100 u/ml penicillin-streptomycin, 25 µg/ml adenine, 5 µg/ml insulin, 1 µg/ml epinephrine, 13.6 ng/ml T3-T, 10 ng/ml epidermal growth factor and 0.62 µg/ml hydrocortisone, sterile-ltered through a 0.22 mm membrane, and then 10% fetal bovine serum was added [30]. Cells were then fasted for 4-6 hours in low growth factor media (DMEM/Ham-F12 supplemented with 100 U/ml penicillin-streptomycin with onetwentieth of the growth factor contents of complete media). The cells were cultured with 0.8 and 1.6 mM PGRN peptide, or 0.2 mM recombinant Ov-GRN-1 [8,9], or vehicle control PBS in low growth factor media for 24 and 48 hours. The viable cell number was determined using an tetrazolium salt (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with absorbance 570 nm as per manufacturer's instructions (Invitrogen, Oregon, USA). The three replicate experiments were assayed for each condition. Cell number was determined at 570 nm using a plate reader (Asys UVM340, Biochrom, Cambridge, UK). The concentrations were established using a standard curve before transforming into relative proliferation compared to control groups. Cell proliferation assays were carried out in triplicate. The data were presented as the mean ± SE of three independent replicates using GraphPad Prism software. One-way ANOVA with a post-hoc tukey test was used for statistical signi cance comparison. p ≤0.05.was considered to be statistically signi cant.
IL-6 and IL-8 IL-6 and IL-8 gene expression levels from the H69 cholangiocyte cell line co-cultured with Ov-PGRN speci c peptide was measured by qRT-PCR. Brie y, H69 cells were treated with Ov-PGRN speci c peptide as described above and subsequently total RNA of harvested cells incubated with 0.8, 1.6 mM of Ov-PGRN speci c peptide was extracted using TriZol®Reagent following company instructions (ThermoFisher Scienti c, Burlington, MA). 500 ng of total RNA was converted to cDNA by using a RevertAid rst strand cDNA synthesis kit (Thermo Fisher Scienti c, Waltham, MA) for qPCR templates. cDNA was ampli ed using PCR with gene-speci c primers designed to amplify a portion of the coding sequences. qPCR was performed with biological duplicate samples using a SYBR Green kit (Maxima SyBr green qPCR master mix, ThermoFisher Scient c, Vilnius, EU) in a thermal cycler (Light Cycler 480 II, Roche Diagnostics GmbH, Mannheim, Germany). Detail of primer sequences for IL-6 (forward; 5'-ACCCCTGACCCAACCACAAAT-3', reverse; 5'-CCTTAAAGCTGCGCAGAATGAGA-3'), IL-8 (forward; 5'-GTGCAGTTTTGCCAAGGAGT-3', reverse; 5'-CTCTGCACCCAGTTTTCCTT-3') [30]. The gene expressions were normalized with internal control using Beta-actin (forward; 5'-TCCCTGGAGAAGAGCTACGA, reverse; 5'AGCACTGTGTTGGCGTACAG) [27].PCR reactions consisted of 12.5 µl of SYBR Green Master Mix (ThermoFisher Scienti c, Vilnius, EU), 0.5 µl (10 mM) each of forward and reverse primers, 1 µl (equivalent to 50 ng of total RNA) of rst-stand cDNA and water to a nal volume of 25 µl. PCR cycling conditions consisted of initiation with pre-heat for one cycle at 95ºC for 10 min followed by 40 cycles of denaturation at 95ºC for30 sec, annealing at 55ºC for 30 sec, extension at 72ºC for 45 sec, and a nal extension at 72ºC for 10 min. Data are presented as the mean ± standard error. Differences between groups were assessed using Student's t-test (GraphPad Prism Software, www.graphpad.com); p ≤0.05.was considered statistically signi cant.
Phylogenetic relationship of Ov-PGRN and other PGRNs from various species indicated that Ov-PGRN formed the closest relationship with PGRN of the closely related carcinogenic ukes Clonorchis sinensis and O. felineus (Fig. 2). PGRN formed major branches in which the Platyhelminthes, Nematoda, and Vertebrata all grouped within their clades. Progranulin is highly expressed in distinct developmental stages of O. viverrini The developmental expression pro le of Ov-pgrn in O. viverrini was evaluated using quantitative RT-PCR, and protein expression pro le was assessed using immunohistochemistry. Ov-pgrn mRNA was expressed in metacercaria, newly excysted juvenile (NEJ), two-week old juvenile (J2W) and adult uke (Fig. 3). The highest Ov-pgrn gene expression level was shown in metacercaria and the lowest levels were in NEJ, J2W and adult, respectively (Fig. 3A). Western blot analysis detected a band of 100-110 kDa, slightly larger than the expected size of Ov-PGRN (92 kDa) in both worm lysate and ES products (Fig. 3B). The greater observed size is likely due to the 10 putative N-linked glycosylation sites identi ed in the predicted protein (Supplemental Fig. 1).
Immunohistochemical localization of the liver of infected hamster probed with rabbit anti-Ov-PGRN-L627C641 peptide revealed expression of Ov-PGRN on the tegument, parenchymal and eggs in the uterus of the adult uke in hamster bile ducts (Fig. 4). The signal also was detected in bile duct cells of the infected hamster (Fig. 4).
Effect of Ov-PGRN peptide on human bile duct cell proliferation H69 cells were incubated with 0.8 and 1.6 µM of Ov-PGRN-L627C641 peptide and cell proliferation was measured with the MTT assay at 24 and 48 h. H69 cells incubated with 0.2 µM of Ov-GRN-1 and culture media were used as a control groups. The results showed that H69 cells incubated with 0.8 and 1.6 µM of Ov-PGRN-L627C641 peptide underwent signi cantly increased cell proliferation only at 48 hrs (160 and 180%, respectively) when compared with culture media alone control (Fig. 5B). H69 cells incubated with 0.2 µM of Ov-GRN-1 recombinant protein also underwent signi cantly increased cell proliferation when compared with culture media alone, as described previously [9].
To evaluate the in ammatory cytokine production that occurs in response to O. viverrini progranulin stimulation. The response of cultured human bile duct cell lines to Ov-PGRN-L627C641 peptide stimulation was investigated. Quantitative RT-PCR was applied to evaluate the mRNA expression level of cytokine from the cells. H69 cells were incubated with 0.8 and 1.6 µM of Ov-PGRN-L627C641 peptide. The relative expression was compared with actin house-keeping gene. The mRNA expression levels of IL-6 and IL-8 were slightly increased in H69 cells incubated with either 0.8 or 1.6 µM of Ov-PGRN -L627C641 peptide at 48 h incubation compared to controls without peptide (Fig. 6).

Discussion
It has been well established that Ov-GRN-1, liver uke granulin, stimulates proliferation of cholangiocytes lining the biliary tract [7,8,31]. The genome of O. viverrini contains two genes encoding single granulin domains termed Ov-grn-1 and Ov-grn-2 and also the multiple granulin domain gene, Ov-pgrn [10]. The Ov-PGRN glycoprotein exhibits homology to human progranulin which has seven and a half conserved granulin domains [11,20]. Here, we described the intact progranulin from the genome of O. viverrini, which we predict consists of one half and seven complete tandem repeats of a 12-cysteine module granulin domain and one incomplete granulin domain. In addition, we describe the predicted structure and likely roles of the liver uke progranulin.
The O. viverrini progranulin showed typical characteristics of the repeat granulin motif. Progranulins from other species stimulate cellular proliferation when intact or when cleaved into single granulin units [20,32]. Detection of progranulin transcripts in larval and adult stages of O. viverrini revealed that intact progranulin is active. Adult liver ukes graze on the biliary epithelium where they secrete ES products and stimulate a biliary mucosal immune response. Cell-mediated immune responses were investigated and showed that the animal infected with O. viverrini has granulomatous in ammation of bile duct by modi ed macrophage [22]. IL-6 and IL-8 were increased in hamster and human with hepatobiliary abnormalities [33][34][35].Treatment with progranulin showed decreasing of liver brosis and in ammation in mice and macrophage [36]. While intact human PGRN showed evidence of suppresses in ammation by blocking TNF-α receptors and signaling [13]. Proteolytic enzymes degrade PGRN to granulin domain peptides which may enhance in ammation by stimulating the secretion of the chemokine interleukin-8 [13]. Our ndings revealed that O. viverrini PGRN peptide increase expression of IL-6 and IL-8 that may involve in ammation of bile duct. We note that access to the genomic structure included the sequences of the introns and exons of O. viverrini progranulin gene will facilitate for programmed gene editing and functional genomics analysis, in like fashion to our studies of Ov-grn-1 [7].
The excretory and secretory molecules of the parasite directly promoting cell proliferation, both innate and adaptive host in ammatory responses in chronic infection contribute to infection-induced malignancy (Sripa et al., 2007). Molecules in ES products stimulate naïve T-cell with Toll-like receptor 4 signaling and express IL6 and IL8 [30]. Upregulation of the proin ammatory transmembrane molecule, TLR4, has been reported in cholangiocytes (H69 cells) cocultured with ES products of O. viverrini [30]. TLR4 overexpression has also been observed in the biliary epithelium of O. viverrini infected humans in situ. The parasite products also induce IkB-a degradation in a MyD88-dependent manner and activate nuclear factor kappa B nuclear translocation, leading to the increased expression and secretion of the strong chemoattractant chemokine IL-8 and proin ammatory cytokine IL-6 [30]. TLR4 is a transmembrane protein, member of the toll-like receptor family, which belongs to the pattern recognition receptor (PRR) family. Its activation leads to an intracellular signaling pathway NF-κB and in ammatory cytokine production which is responsible for activating the innate immune system. These results demonstrated that O. viverrini PGRN stimulated human cholangiocytes and initiate innate mucosal immunity/in ammatory via TLR4 pathway. Given that progranulin of the O. viverrini was identi ed in ES product and seems involved in proliferation and in ammation, future research should explore if Ov-PGRN is involved in carcinogenesis similar to the single granulin protein, Ov-GRN-1 [6-8].

Supplementary Files
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