Functional Characterization of an Interferon Gamma Receptor-Like Protein on Entamoeba histolytica

E. histolytica culture.E. histolytica HM-1:IMSS trophozoites were grown axenically in TYI-S-33 medium containing 10% bovine serum and supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% heat-inactivated adult bovine serum, as previously described (43). To maintain virulence, trophozoites were regularly passed through golden hamster (Mesocricetus auratus) livers as described previously (44). Trophozoites were harvested in the logarithmic phase of growth (48 to 72 h) by cooling the culture tubes on ice and centrifuging the cell suspensions at 300 × g for 15 min. Recovered E. histolytica trophozoites were suspended in culture medium without serum, pH 7.2, for the assays.

Immunofluorescence detection of IFN-γ fixed on E. histolytica trophozoites.For double immunofluorescence assays, E. histolytica trophozoites (2 × 10⁵) were cultured in coverslips in a petri dish with TYI-S-33 medium (Trypticase, yeast extract, iron serum) for 15 min at 37°C. Coverslips were washed three times with phosphate-buffered saline (PBS), medium supplemented with IFN-γ (100 ng/ml; Peprotech, Rocky Hill, NJ, USA) was added, and the trophozoites were incubated for 20, 60, or 180 min at 37°C. Coverslips were washed three times with PBS, and the trophozoites were fixed with 4% paraformaldehyde for 20 min and blocked with 10% fetal bovine serum in PBS for 1 h at 37°C. The preparations were incubated overnight at 4°C with primary antibodies anti-IFN-γ antibody (1:100) (catalog number 500-P32; Peprotech) and anti-IFN-γR1 antibody (1:100) (catalog number MA5-16583; Thermo Fisher Scientific). After three washes with PBS, secondary antibodies (1:1,000) Alexa Fluor 594-conjugated goat anti-mouse IgG(H+L) (catalog number A11005; Thermo Fisher Scientific) and Alexa Fluor 488-conjugated goat anti-rabbit IgG(H+L) (catalog number A11008; Thermo Fisher Scientific) were added and the preparations were incubated overnight at 4°C. Nuclei were stained with Hoechst stain (1 μg/ml; Thermo Fisher Scientific) in PBS for 10 min at room temperature. Coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) and analyzed in an LMS700 microscope (Zeiss). Images were processed with ZEN 2009 Light Edition software (Zeiss). To quantify colocalization, 1-μm z-stacks of entire cells or an area around the plasma membrane were analyzed according to the Costes methodology, which includes the elimination of autofluorescence with the Coloc2 plugin of Fiji software (ImageJ). Graphing was done with GraphPad Prism 7 software (45).

Western blot analysis for detecting IFN-γR1 on E. histolytica trophozoite membranes.E. histolytica trophozoites were cultured in the presence or absence of 100 ng/ml of recombinant IFN-γ (Peprotech, Rocky Hill, NJ, USA) for 20 min at 37°C. After incubation, trophozoites were washed with PBS. For protein extraction, approximately 1 × 10⁶ cells were resuspended in 1 ml of lysis buffer with protease inhibitors (50 mM Tris-HCl, pH 6.8, 5 mM N-ethylmaleimide, 3 mM iodoacetamide, 1 mM phenylmethanesulfonyl fluoride, and 3 mM tosyl-l-lysine chloromethyl ketone). All inhibitors were purchased from Sigma-Aldrich. The cells were homogenized with −70°C/37°C incubation cycles. The lysates were centrifuged at 40,000 × g for 1 h at 4°C. The supernatant and pellet correspond to the cytosolic and membrane fraction, respectively. Positive-control human leukocyte total lysates were collected as well. Then, the pellets were suspended in 200 μl lysis buffer and 2.5% Triton X-100. Protein quantification was performed with the Bradford method (46). For Western blotting, 50 μg of each protein extract was separated in a 10% SDS-PAGE gel, and proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked with Tris-buffered saline (TBS) and 5% skimmed milk for 1 h at room temperature. For immunodetection, the membranes were incubated for 24 h at 4°C with the primary antibody, a mouse anti-IFN-γR1 monoclonal antibody (1:1000; Thermo Fisher Scientific, Waltham, MA, USA). Blots were incubated with goat anti-mouse IgG conjugated with Alexa Fluor 594 (1:5,000; Millipore, Burlington, MA, USA). After the incubation, the membranes were washed with TBST (Tris-buffered saline–0.05% Tween 20) and blots were developed with Clarity Western ECL substrate (Bio-Rad, Hercules CA, USA) for chemiluminescence imaging.

Isolation of total RNA and qPCR.Total RNA was isolated from 1 × 10⁶ E. histolytica trophozoites incubated or not with IFN-γ for 0, 10, 20, 30, and 60 min, using the SV total RNA isolation system (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Reverse transcription was performed with 50 ng of total RNA from E. histolytica trophozoites using the GoScript reverse transcription system (catalog number a5001; Promega), followed by real-time quantitative PCR (qPCR) analysis using qPCR GreenMaster with UNG, clear (Jena Bioscience, Jena. Germany) in a StepOne machine (Applied Biosystems) with the following program: 50°C for 2 min, 95°C for 3 min, and 40 cycles of 95°C for 30 s and 60°C for 30 s. Oligonucleotides were designed to target genes encoding E. histolytica cysteine proteases 1, 2, 4, and 5, amebapore, Cox-1, Gal/GalNAc lectin, and peroxiredoxin (Table S1 in the supplemental material). Relative expression levels were normalized against the expression of the respective E. histolytica genes from cells cultured without stimuli as an internal control, and differences were determined by employing the ΔΔC_T relative method. The rRNA gene was used as the housekeeping gene, using the StepOne machine (Applied biosystem).

Erythrophagocytosis.E. histolytica trophozoites were cultured in glass tubes under axenic conditions. Trophozoites (2 × 10⁶) were harvested by chilling the culture tubes at 4°C in a water-ice bath for 10 min and then centrifuging them at 300 × g for 5 min and washing with sterile Dulbecco’s PBS (DPBS) (catalog number D8537; Sigma-Aldrich). To abrogate signaling via IFN-γR1, trophozoites were pretreated with the STAT1 inhibitor fludarabine at 50 μM (catalog number Sc-204755; Santa Cruz) for 30 min before exposure to IFN-γ (100 ng/ml) for 20 min. Following incubation, trophozoites were washed twice with DPBS before the assay. Fresh human erythrocytes in DPBS solution were stained with PKH26 (catalog number MINI26; Sigma-Aldrich), counted, and used at a 1:100 (trophozoites/erythrocytes) ratio. To establish the interaction, erythrocytes were added, and the interaction was carried out for 20 min at 37°C in DPBS, after which they were washed twice with DPBS. Lysis buffer (red blood cell [RBC] lysing buffer) (catalog number R7757; Sigma-Aldrich) was added for 1 min at room temperature. Fetal bovine serum (FBS; 0.5 ml) (catalog number 10437-028; Gibco) was added for 1 min at room temperature, and the cells were washed once with DPBS. The cells were fixed with 4% paraformaldehyde for 20 min at room temperature and washed with DPBS, removing as much liquid as possible from the sample. To each tube was added a drop of FluorSave reagent (catalog number 345789; Calbiochem), and the contents were mixed carefully, placed on a clean slide, and stored in the dark at 4°C for at least 24 h before being observed under a microscope.

Immunoprecipitation.E. histolytica trophozoites were cultured in glass tubes under axenic conditions. Trophozoites were harvested by chilling the culture tubes at 4°C in a water-ice bath for 10 min, and then they were centrifuged at 300 × g for 5 min and washed with sterile DPBS (Sigma-Aldrich). Trophozoites were treated with fludarabine (50 μM) at 37°C for 30 min and then stimulated with IFN-γ (100 ng/ml) at 37°C for 20 min. Lysates were prepared from trophozoites by using a modified protocol (47). E. histolytica trophozoites were washed twice in cold PBS and suspended in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (Sigma), phenylmethylsulfonyl fluoride (PMSF), sodium fluoride, and sodium orthovanadate. Lysates were centrifuged at 10,000 × g for 10 min. Immunoprecipitation was carried out using 200 μg of protein, and the primary antibody (anti-phospho-Tyr antibody, 1.5 μg; BD transduction laboratories) was incubated with the supernatant for 8 h at 4°C, followed by 12 h at 4°C with protein A/G plus-agarose (Santa Cruz Biotechnology). Samples were run on 7.5% SDS-PAGE gels and probed with antibodies as follows: anti-STAT1 antibody (Santa Cruz) and anti-phospho-STAT1 antibody (Abcam).

Chemotaxis.Chemotaxis of E. histolytica trophozoites toward IFN-γ was monitored in Transwell migration chambers (catalog number 3464; Corning) as previously reported (48). The chemoattractant gradients were generated by placing 600 μl of culture medium without serum and containing 100 ng/ml of IFN-γ in the lower chamber of the migration units and 30,000 E. histolytica trophozoites on the filter of the upper Transwell chamber. Cells were maintained under 0.05% CO₂ conditions at 37°C for 30 min. Cells that migrated toward IFN-γ and reached the lower chamber were recovered, stained with crystal violet, and quantified in an inverted microscope. To inhibit chemotaxis toward IFN-γ, trophozoites were treated with cytochalasin D, which depolymerizes actin/myosin filaments, as previously described (21). A coverslip chemotaxis gradient assay was used to visualize individual migrating E. histolytica trophozoites; for this, 180 μl of serum-depleted medium containing 100 ng/ml of IFN-γ was injected into a 0.75% agarose strip placed along one edge of each coverslip. On the opposite edge, trophozoites were placed along a narrow band and allowed to attach to the glass for 20 min. Chemotaxis of trophozoites toward IFN-γ (100 ng) was visualized using phase-contrast video microscopy. Video registers were made with a Carl Zeiss Axiovert 40CFL inverted microscope and the digital camera used in the immunofluorescence assays. Time-lapse videos were generated from 114 spaced frames, with 2 s between each frame acquired for 4-min real-time registers. Each video was processed with AxioVision 40V 4.6.3.0 software. To register random motility of trophozoites in the absence of IFN-γ, agarose strips not containing IFN-γ and only injected with serum-free culture medium were placed on one edge of coverslips, and migration was recorded as described above. The data analysis was performed using ImageJ 1.51n (http://imageJ.nih.gov/ij).

Cytotoxicity assay.Human adenocarcinoma Caco-2 cells and human liver cancer HepG2 cells from ATCC were grown to confluent monolayers in Dulbecco’s modified Eagle’s medium (DMEM) (catalog number 11995-040; Thermo Fisher) with 10% serum, penicillin, and streptomycin for 3 days (modified from the procedures in references 49 and 50). Cells grown in 24-well plates to 80 to 90% confluence were washed twice with DPBS, and 500 μl of DMEM medium was added per well. E. histolytica trophozoites (1 × 10⁵ per well) were treated or not with the STAT1 inhibitor fludarabine (50 μM) (catalog number Sc-204755; Santa Cruz) for 30 min prior to IFN-γ (100 ng/ml) stimulation for 20 min. For the interaction, trophozoites were added to each well for 1 h. After incubation, the cells were placed on ice, washed with DPBS, and fixed with 2.5% paraformaldehyde for 10 min. The monolayer was stained with 0.1% methylene blue in 0.1 M borate buffer (pH 8) for 10 min at room temperature. The plates were washed with 0.1 M borate buffer twice to remove excess stain. Finally, 1 ml of 1 N HCl was added to each well for 30 min at 37°C to extract the stain, and the absorbance read on a spectrometer at 655 nm (optical density at 655 nm [OD₆₅₅]). The percentage of monolayer destruction was calculated as follows: [OD₆₅₅ (control wells) − OD₆₅₅ (experimental wells)/OD₆₅₅ (control wells)] × 100.

Animals.Male golden hamsters (Mesocricetus auratus) weighing 140 to 160 g were used in this study. The animals were maintained on standard diet with free access to drinking water. All animals received humane care according to the guidelines of the Committee on Bioethics in the animal facilities of the Autonomous University of Aguascalientes, Aguascalientes, Mexico, which are based on the guidelines for animal research published by the National Institutes of Health (51).

Experimental hepatic amebiasis.E. histolytica trophozoites (5 × 10⁵) were incubated in the presence or absence of 100 ng/ml of recombinant IFN-γ (Peprotech, Rocky Hill, NJ, USA) for 20 min. After incubation, trophozoites were washed with PBS and inoculated into the left liver lobe of hamsters in 100 μl of culture medium as previously described (44, 52). After 4 days, the animals were anaesthetized with sodium pentobarbital (50 mg/kg of body weight intraperitoneally), the liver was excised for macroscopic evaluation, and several specimens that included ALA were taken, fixed in 4% paraformaldehyde, and processed for histological analysis.

H&E staining.To visualize ALA development, we performed hematoxylin and eosin (H&E) staining as described in the Manual of Histologic Staining Methods of the Armed Forces (53). The sample tissues were analyzed to determine the percentages of necrotic and inflammatory areas using ImageJ software.

Immunohistochemistry.To visualize the presence of E. histolytica trophozoites in liver tissue, sections were subjected to immunohistochemistry as described previously (53). For E. histolytica immunodetection, the samples were incubated with the primary antibody rabbit anti-E. histolytica antibody, raised in our laboratory, diluted 1:400 at 4°C. The secondary antibody, goat anti-rabbit IgG, was diluted 1:500 (Sigma-Aldrich) and incubated for 2 h at room temperature. Slides were washed with PBS-Tween 20 for 10 min, and peroxidase activity was developed with diaminobenzidine (DAB) (Pierce Biotechnology, Inc., Rockford, IL, USA) for 5 min.

Statistical analysis.Statistical analyses were performed using GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA, USA). Student’s t test and one-way analysis of variance (ANOVA) with Tukey’s post hoc test were used. Differences between groups were assessed as significant at a P value of <0.05. Experimental results are represented in the figures as the mean values from three independent experiments ± standard deviations (SD).