Parasite Immunology, 2015, 37, 340–348

DOI: 10.1111/pim.12188

Plasma IgG autoantibody against actin-related protein 3 in liver fluke Opisthorchis viverrini infection R. RUCKSAKEN,1,2 O. HAONON,1,2 P. PINLAOR,2,3 C. PAIROJKUL,2,4 S. ROYTRAKUL,5 P. YONGVANIT,2,6 C. SELMI7,8 & S. PINLAOR1,2 1 Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 2Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 3Centre for Research and Development in Medical Diagnostic Laboratory, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand, 4Department of Pathology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 5Proteomics Research Laboratory, Genome Institute, National Center for Genetic Engineering and Biotechnology, Pathumthani, Thailand, 6Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, 7Division of Rheumatology and Clinical Immunology, Humanitas Clinical and Research Center, Rozzano, Milan, Italy, 8BIOMETRA Department, University of Milan, Milan, Italy

SUMMARY Opisthorchiasis secondary to Opisthorchis viverrini infection leads to cholangiocellular carcinoma through chronic inflammation of the bile ducts and possibly inducing autoimmunity. It was hypothesized that plasma autoantibodies directed against self-proteins are biomarkers for opisthorchiasis. Plasma from patients with opisthorchiasis was tested using proteins derived from immortalized cholangiocyte cell lines, and spots reacting with plasma were excised and subjected to LC-MS/MS. Seven protein spots were recognized by IgG autoantibodies, and the highest matching scored protein was actin-related protein 3 (ARP3). The antibody against ARP3 was tested in plasma from 55 O. viverrini-infected patients, 24 patients with others endemic parasitic infections and 17 healthy controls using Western blot and ELISA. Immunoreactivity against recombinant ARP3 was significantly more prevalent in opisthorchiasis compared to healthy controls at Western blotting and ELISA (P < 005). Plasma ARP3 autoantibody titres were also higher in opisthorchiasis compared to healthy individuals (P < 001) and other parasitic infections including Strongyloides stercoralis (P < 0001), echinostome (P < 005), hookworms (P < 0001) and Taenia spp. (P < 005). It was further characterized in that the ARP3 autoantibody titre had a sensitivity of 7818% and specificity of 100% for opisthorchiasis. In conclusion, it may be suggested that plasma anti-ARP3

Correspondence: Somchai Pinlaor, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand (e-mail: [email protected]). Received: 5 November 2014 Accepted for publication: 16 March 2015

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might represent a new diagnostic antibody for opisthorchiasis. Keywords actin-related protein 3, autoimmunity, diagnostic antibody, opisthorchiasis

INTRODUCTION Human liver fluke Opisthorchis viverrini infection represents a major public health issue in South-East Asia, especially in the north-eastern regions of Thailand where about 6 million people are currently infected (1, 2). In endemic communities, although the prevalence appears to be declining, the rate may reach 60% in some regions (2). O. viverrini infection is associated with a number of hepatobiliary benign diseases, including cholangitis, obstructive jaundice, hepatomegaly, cholecystitis and cholelithiasis as well as the aggressive bile duct cancer, cholangiocellular carcinoma (CCA) (1) through chronic inflammation and fibrosis (3) inducing oxidative/nitrative stresses (4). Such stress is associated with the appearance of plasma autoantibodies in several cancers and other chronic diseases (5), and this may also apply to chronic O. viverrini infection. If this was the case, plasma autoantibodies would represent ideal biomarkers to discriminate the progression (6), severity (7, 8) and perpetuation (9) of disease. Furthermore, plasma autoantibodies are apparent in several parasitic diseases including protozoan and helminth infections, as in Schistosoma mansoni (10), Schistosoma haematobium (11), Schistosoma japonicum (12), Toxocara canis (13), Onchocerca volvulus (7), Toxoplasma gondii (14), Trypanosoma cruzi (15) and malaria (16). In some cases, © 2015 John Wiley & Sons Ltd

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autoantibody titres were associated with the severity of infections (7, 10, 17) and high titres may protect against severe Plasmodium falciparum infection (18). Therefore, it was hypothesized that an autoantibody against O. viverrini infection could be used as a new disease-specific marker for opisthorchiasis and possibly represents a future therapeutic target (19). This report herein proposes that plasma from patients with O. viverrini is positive for different autoantibodies directed at self-proteins and that actin-related protein 3 (ARP3) has the highest specificity and sensitivity.

MATERIALS AND METHODS Subjects Plasma samples were utilized from 55 patients with O. viverrini infection, 27 with and 28 without periductal fibrosis at abdominal ultrasonography, and none of these patients had developed cholangiocarcinoma at the time of blood collection. Their mean age was 587  12. Selection was based on the presence of parasite eggs in the stool samples as examined by the formalin ethyl acetate concentration technique and O. viverrini antibody detection. Seventeen healthy individuals (mean age 521  9) with negative faecal samples for O. viverrini eggs, normal urinalysis and normal hepatobiliary tract assessed by ultrasonography were selected as controls. Further, 24 patients with different parasitic infections were included as controls and included 10 with Strongyloides stercoralis (mean age 585  18), five with echinostome (mean age 588  9), five with hookworms (mean age 583  25) and four with Taenia spp. (mean age 573  4). Ten millilitre peripheral blood samples was obtained by sterile venipuncture collected in tubes containing EDTA. Blood was centrifuged at 3000 g for 15 min at 4°C. Plasma samples were stored at 80°C until analysis. Written informed consent was obtained from all participants, and the study protocols (HE551303 and HE531320) were approved by the Human Research Ethics Committee, Khon Kaen University, Thailand (HE561290).

Autoantibody in O. viverrini infection

medium, FBS and antibiotics were purchased from Gibco (Invitrogen, Auckland, New Zealand).

Two-dimensional (2D) gel electrophoresis Two-dimensional gel electrophoresis was carried out as described previously (21). Briefly, pellets from MMNK1 cell lines were lysed and added to 150 lL of sample preparation solution in a cocktail of 8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2% (v/v) immobilized pH gradient (IPG) buffer pH 3–10, 40 mM dithiothreitol (DTT) and protease inhibitors (GE Healthcare, Piscataway, NJ, USA). Protein concentrations were measured using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA), and 150 lg was loaded onto 7-cm Immobiline DryStrips (pH 3-10; GE Healthcare). Isoelectric focusing was carried out using Ettan IPGphor II (GE Healthcare) at 20°C at the following steps 30 min, 300 V; 30 min, 1000 V gradient; 2 h, 5000 V gradient; 30 min, 5000 V. The 2D run was performed using 12% SDS-PAGE, and gels were stained with Coomassie brilliant blue (CBB) or transferred to polyvinylidene difluoride membranes (PVDF; Amersham Bioscience, Piscataway, NJ, USA) for Western blot analyses.

Immunoblotting PVDF membranes were blocked with 5% nonfat skim milk/01% Tween 20 in phosphate buffer saline (PBST) for 1 h at room temperature and incubated with the 1 : 2000 diluted pooled plasma samples from healthy individuals (n = 10) as controls or pooled plasma samples from patients with opisthorchiasis (n = 10) in 2% nonfat skim milk/PBST at room temperature for 1 h. After the washing step, the PVDF membranes were incubated with antihuman IgG HRP conjugate dilution 1 : 15000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h. Finally, signals were visualized with enhanced chemiluminescence (ECL) solution (GE Healthcare) and PVDF membranes were stained in parallel with CBB to match spots.

Proteins identification by mass spectrometry Cell lines and cultures The immortalized cholangiocyte (MMNK1) cell line was established as previously described (20), and cells were cultured in HAM’s F-12 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS), 2 mmol/L glutamine, 15 mmol/L HEPES and 14 mmol/L sodium bicarbonate, 100 U/mL penicillin G and 100 U/mL streptomycin. Cells were then maintained at 37°C in a 5% humidified CO2 incubator. Consumables including

Protein spots on gels stained with CBB which corresponded to positive spots on Western blot membranes were cut and digested with trypsin for liquid chromatography–tandem mass spectrometry (LC-MS/MS) as previously described (21). In brief, the gel spots were destained, reduced, alkylated, and then subjected to in-gel tryptic digestion (10 ng/ lL trypsin in 50% acetonitrile/10 mM ammonium bicarbonate; Promega, Madison, WI, USA). For LC-MS/MS analyses, nanoscale LC separation of tryptic peptides was

© 2015 John Wiley & Sons Ltd, Parasite Immunology, 37, 340–348

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performed using a NanoAcquity system (Waters Corp., Milford, MA, USA) and samples were analysed together using a SYNAPT-HDMS mass spectrometer (Waters Corp.). Tandem mass spectrometry (MS/MS) spectra were generated in a micromass file (PKL) supported for MS/MS ion search using the MASCOT search engine (Matrix Science, London, UK). Mass spectrometry data were searched against the NCBI database for human proteins and proteins exhibiting a Mascot score exceeding 66 that were considered as statistically significant (P < 005).

ARP3 autoantibody testing by Western blotting ARP3 autoantibody was searched in individual plasma from five representative healthy controls and seven representative opisthorchiasis patients using Western blot. Briefly, 01 lg of recombinant human full-length ARP3 produced by an in vitro wheat germ protein expression system (Abnova, Walnut, CA, USA) was mixed with sample buffer and directly loaded for electrophoresis without heating. Samples were run on 15-well, 10% SDS-PAGE gels. After being transferred to PVDF membranes for 1 h at 035A, membranes were cut into single strips to incubate with individual plasma samples of healthy controls or plasma samples from patients with opisthorchiasis dilutions 1 : 250 (lanes 1–12, Fig. 2). Membrane strips in lanes 13 and 14 were incubated with pooled healthy controls and pooled opisthorchiasis samples, while one lane was cut and stained with CBB. All membranes were then incubated with anti-human IgG HRP conjugate dilution 1 : 2500 (Santa Cruz Biotechnology) diluted in 2% nonfat skim milk/PBST at room temperature for 1 h. The membranes were developed using ECL solution (GE Healthcare). Relative band intensities between healthy controls and opisthorchiasis patients were analysed using ImageQuant TL software v7.0 (GE Healthcare).

ARP3 autoantibody testing by ELISA ARP3 IgG autoantibody titres were tested using indirect ELISA in 17 plasma samples from healthy controls, 55 plasma samples from O. viverrini-infected patients (O. viverrini infected with or without fibrosis) and 24 plasma samples from patients with other parasitic infections. Briefly, the 96-well MaxiSorp immunoplates (Nunc, Roskilde, Denmark) were coated with 100 lL of recombinant human full-length ARP3 (1 lg/mL in 19 phosphate buffer saline, PBS) at 4°C overnight and washed once with 19 PBST. The plates were blocked with 100 lL of 5% nonfat skim milk/PBST at room temperature for 2 h and washed twice with 19 PBST. Then, 100 lL of plasma samples diluted 1 : 1000 in dilution buffer (5% nonfat skim milk/

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PBST) was added and incubated at 37°C for 1 h. After washing three times with 19 PBST, 100 lL of goat antihuman IgG-HRP conjugated antibody dilution 1 : 4000 was added and incubated at room temperature for 1 h. Plates were washed five times with 19 PBST. Finally, 100 lL of 3,30 ,5,50 -tetramethylbenzidine (TMB) substrate (Thermo Scientific, West Palm Beach, FL, USA) was added and stopped the reactions with 3MH2SO4.

Statistical analysis Statistical analyses were performed using SPSS version 17.0 (IBM, Armonk, NY, USA). Continuous variables were expressed as the mean  SD. To compare the expression levels of ARP3 autoantibody between opisthorchiasis patients and healthy controls, the statistical significance band intensities of the immunoblots were measured and compared using Student’s t-test and autoantibodies levels were measured by ELISA for each patient group and compared using Student’s t-test or the nonparametric Mann– Whitney test. The diagnostic accuracy of ARP3 antibodies was assessed using the receiver operating characteristic (ROC) curve which was constructed by plotting the sensitivity vs. (100%)-specificity, and area under curves (AUCs) with 95% confidence intervals were calculated for the marker.

RESULTS Immunoreactive protein identification In healthy controls, faint reactive spots were seen in control membranes incubated with pooled plasma samples from healthy individuals (Fig. 1a). In O. viverrini-infected patients, although several reactive spots were found in 2D Western blot membranes after incubation with pooled plasma of opisthorchiasis patients; only seven immunoreactive spots were successfully matched with the CBB gels, and these immunoreactive spots did not match with the membranes incubated with pooled plasma samples of healthy individuals (Fig. 1b,c). Spot numbers 1–7 were identified, and protein data including their GI numbers, biological processes and protein scores are summarized in Table 1. Among all statistically significant matched proteins, ARP3 showed the highest protein score and was selected for further validation.

Validation of anti-ARP3 in opisthorchiasis using Western blot Recombinant ARP3 proteins were detected as 70 kDa bands in all plasma samples from patients with opisthorchiasis, © 2015 John Wiley & Sons Ltd, Parasite Immunology, 37, 340–348

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(a)

(b)

Figure 1 Representative 2-dimensional gel electrophoresis and Western blotting of immortalized cholangiocyte cell line lysates separated and transferred to PVDF membranes. Membranes were incubated with (a) pooled plasma samples from healthy individuals as controls or (b) pooled plasma samples from patients with opisthorchiasis and incubated with secondary anti-human IgG, (c) parallel gels were stained with Coomassie brilliant blue (CBB). Matching between membranes incubated with pooled plasma from patients with opisthorchiasis and CBB-stained gel was performed. IgG autoantibody reactive spots are indicated by numbers 1–7 in both Western blot membranes and CBB gels, proteins names and other details for spot numbers 1–7 are shown in Table 1.

(c)

Table 1 Proteins identified by autoantibodies from O. viverrini-infected patients on MMNK1 cells

Spot no.

Protein name

Biological process

GI number

Protein score

Coverage (%)a

Peptide match

P-value

Species

1 2 3

Triadin Abnormal spindle protein Actin-related protein 3 isoform 1 F-actin-capping protein subunit alpha-1 PRP38 pre-mRNA processing factor 38 SRp46 splicing factor Glyceraldehyde-3-phosphate dehydrogenase

Ion transport Cell cycle Cytoskeleton

gi|28950585 gi|24061713 gi|5031573

92 105 287

27 19 18

27 38 10

Plasma IgG autoantibody against actin-related protein 3 in liver fluke Opisthorchis viverrini infection.

Opisthorchiasis secondary to Opisthorchis viverrini infection leads to cholangiocellular carcinoma through chronic inflammation of the bile ducts and ...
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