Study design and patients
This cross-sectional study was performed at a tertiary public hospital in Rio Grande do Sul, Brazil (Hospital de Clínicas de Porto Alegre, HCPA). Patients diagnosed with RA according to the American College of Rheumatology/American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria 45, with active disease and with indication of knee arthrocentesis by the attending rheumatologist were included in study. Patients with positive infection serology tests (HIV, HBV, HCV, syphilis, tuberculosis) and indication of arthrocentesis on other joints were excluded. The disease activity score (DAS) was calculated with DAS28-erythrocyte sedimentation rate. The study was approved by the HCPA Hospital Research Ethics Committee (CEP) and written informed consent was obtained from each patient, complying with all methods in accordance with CEP guidelines and regulations.
Collection procedure of synovial liquid
The arthrocentesis procedure was performed by a rheumatologist. Briefly, local anesthetic was used in the area of the joint and a needle with a syringe attached is inserted within the joint (joint injection) and joint fluid is drawn back under suction (aspirated) into the syringe. The synovial liquid was placed in a 15 ml falcon tube and stored in ice until processing.
Isolation and Culture of FLS cells
The synovial fluid of RA patients was processed for the isolation of FLS. The liquid was centrifuged at 2000 x g for 5 min, resuspended in Dulbecco’s Modified Eagle’s Medium - High glucose (DMEM-HG, Gibco by Life Technologies, USA) supplemented with 15% fetal bovine serum (FBS, Gibco), 200 mM L-glutamine (Gibco), 100 units/mL penicillin (Gibco), 100 mg/mL streptomycin (P/S) (Gibco), 50 mg/mL of gentamicin and transferred to a 6-well plate for culture (Thermo Fisher Scientific, USA). Cells were allowed to attach during three days before replacing the growth medium. The cultures were kept at 37°C in a 5% CO2 atmosphere, and the medium was replaced every 3 days. The initial culture was monitored until 70–80% of confluence and then cells were detached with trypsin-EDTA (Gibco) and transferred to a culture flask for the tests. FLS between 5 and 11 passages were used for experiments.
Preparation of F. hepatica extract
Mature F. hepatica was extracted from the bile ducts of bovine livers obtained at local abattoirs and extensively washed at 37ºC in 10 mM phosphate-buffered saline (PBS) pH 7.3. Only clean and viable parasites were used for further downstream procedure. Adult liver flukes F. hepatica extract was obtained by homogenization (1g of adult parasite/10 mL PBS) using a glass tissue grinder and then sonicated in ice bath five times with 60 s bursts at 20% power followed by 30 s pauses. After centrifugation at 20 000 g for 30 min at 4 °C, supernatant was stored at −80 °C until use 46. Protein concentration was determined by using Qubit fluorimetric assay (Invitrogen).
Morphology and Characterization of FLS
Morphological analysis of FLS was performed by phalloidin staining of actin filaments (Sigma-Aldrich by Merck, St. Louis, EUA). FLS (0.7×104 cells/ml) were cultured over microscopy coverslips (18 x 18 mm) and placed into 12-well culture plates. After 48 h, adhered cells were fixed for 15 min with 4% paraformaldehyde at RT in dark. Then, the cells were washed three times with PBS and blocked with 3% bovine serum albumin (BSA) (Sigma-Aldrich) for 1 h at RT in dark. Staining was performed with 50 nM Alexa 488 conjugated phalloidin (Sigma-Aldrich) for 1 h at RT in dark. Nuclei were stained with (300 nM) of 4′,6-diamidino-2-phenylindole solution (DAPI; Sigma-Aldrich, Missouri, USA). Finally, cells were photographed in a fluorescence microscope with appropriate filters (Olympus IX-71, Olympus Corporation, Tokyo, Japan). For phenotyping characterization, FLS were used in passage 5. Cells were centrifuged (2000 x g for 5 min), the supernatant was aspirated, and the antibodies were applied directly onto the pellet. After, the FLS were incubated with the following monoclonal antibodies: anti-Thy-1 (CD90), anti-CD55 (both FLS positive) and anti-CD68 (macrophage positive) (BD Biosciences, New Jersey, EUA). After incubation for 15 min at dark and room temperature (RT), cells were washed with PBS, centrifuged, resuspended and analyzed by flow cytometry (Attune, Applied Biosystems, Life Technologies). Unstained cells were used as controls.
Viability assay (MTT)
FLS viability was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The FLS were seeded in 96-well plates at a density of 5×103 cells/ml and exposed to different doses of F. hepatica extract (60, 80, and 100 µg/ml) for 24, 48, and 72 h at 37 °C in 5% CO2. Parallel controls were carried out with PBS (vehicle control) or untreated group (control). The extract were replaced every day. Then, the culture medium was replaced with MTT (0.5 mg/ml) and incubated for 4 h. The supernatants were removed and dimethyl sulphoxide (DMSO, Sigma Aldrich) was added to dissolve the MTT formazan crystals. The absorbance of each well was measured at 570 nm and 690 nm (SpectraMax M3) spectrophotometer and background was discounted. Cell viability was expressed as percent of average of control (or vehicle control). Eight experiments were performed independently (n=8).
Nuclear Morphometric Analysis (NMA)
A nuclear morphometric analysis (NMA) was performed as described by Filippi-Chiela et al. (2012). FLS were seeded in 24-well plates at a density of 1×104 cells/ml. The following day, FLS were treated with 100µg/mL F. hepatica extract for 48 h. After 24 h, cells were fixed with 4% paraformaldehyde and stained with 100 µL (300 nM) of 4′,6-diamidino-2-phenylindole solution (DAPI; Sigma-Aldrich, Missouri, USA). Then, cells were photographed in a fluorescence microscope with appropriate filters (Olympus IX-71, Olympus Corporation, Tokyo, Japan) and a total of 50 nuclei was obtained from random fields. The images were analyzed using Image Pro-Plus 6.0 software (Media Cybernetics, Maryland, USA), for the acquisition of the nuclear area and the following parameters of nuclear shape: roundness, aspect, radius ratio and area/box. These four variables of shape were grouped in an index, named the Nuclear Irregularity Index (NII). The Area versus NII plot classified each nuclei according to its morphometric. Three experiments were performed independently (n=3).
Apoptosis and necrosis assay (Annexin V/Propidium Iodide [PI])
FLS were plated in a 24-well plate at a density of 5x104 cells/ ml for apoptosis and cell death detection. FLS were treated with 100 µg/mL of F. hepatica extract or PBS for 48 h. The test was performed by the Annexin V-fluorescein isothiocyanate (FITC)/Propidium Iodide (PI) kit (Gibco| Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, FLS were washed and resuspended in Annexin V binding buffer, centrifuged at 1600 x g for 5 min, and labeled with Annexin V-FITC and PI. After 10 min at RT in dark, Annexin V binding buffer was added and centrifuged one more time. The resulting fluorescence was detected by flow cytometry (Attune, Applied Biosystems, Life Technologies). Two experiments were performed independently (n=4).
To evaluate the adherence potential of FLS, cells were seeded in 96-well plate at a density of 1×102 cells/ml and exposed to 100 µg/ml F. hepatica extract or PBS for 6 h. After 6 h, cells were counted immediately in an optical microscopy for adherence to polystyrene plate evaluation. Adherence was expressed by the number of adhered cells divided by the number of seeded cells. Four experiments were performed independently (n=4).
Wound healing assay
The wound closure motility assay was performed by seeding 3.5x104 cells/ml in 24-well plates overnight and by treating them with 100 µg/mL F. hepatica extract or PBS for 48 h. A linear wound was created using a 200 µL micropipette tip and then washed with medium to remove unattached cells. Light microscopy images were taken immediately 0 and 24 h after wounding and, at the end of the experiment, cells were fixed with 4% paraformaldehyde and stained with crystal violet. The distance of the wound was measured in “inch” by ImageJ software and was expressed as percent of the average of control. Four experiments were performed independently (n=4).
To perform the invasion capacity analysis, FLS were treated with 100 µg/mL of F. hepatica or PBS for 48 h in T75 culture flasks. After, the FLS were seeded in matrigel inserts (collagen matrix) Corning (24 well plates, 8-mm pore diameter; Bedford, MA, USA) at a density of 1×105 cells/well. The test was performed according to the manufacturer's specifications. To measure cell invasion, FLS were seeded in medium free of FBS in the upper chamber. Medium supplemented with 15% FBS was used as an attractant in the lower chamber. After 24 h, cells that invaded through the matrix were stained with Crystal Violet, photographed by microscope, and analyzed in the ImageJ software. Two experiments were performed independently (n=3).
Enzyme-linked immunosorbent assay (ELISA)
To evaluate the TNF-α expression of FLS, a density of 1x104 cells/ml were seeded in a 24-well plate for ELISA assay. When cells reached the confluence of 80%, the culture medium was suplemmented with 2 µL/mL of interferon-gamma (INF-γ - Thermo Fisher) for 3 days to induce an inflammatory condition in vitro. After that, FLS were treated with 100µg/mL F.hepatica extract or PBS for 48 h. Then, the medium was replaced and the supernatant was collected after 24 h and stored at -80°C until use. The concentration of TNF-α in supernatants was measured by human TNF-α Elisa MaxTM Standard Set (BioLegend, San Diego, CA, USA). The absorbance of each well was measured at 450 nm and 570 nm in SpectraMax M3 spectrophotometer. Data analyses were performed in My Assays software (“Four Parameter Logistic Curve” online data analysis tool, MyAssays Ltd, Sussex, UK) and they are representative at pg/mL. Three experiments were performed independently (n=3).
Balb/C mice (male, 8–12 weeks, 20–25 g) were used for AIA model, while DBA/1 J mice (male, 8–12 weeks, 18–22 g) were used for CIA model. All animals were kept under a 12 h light-dark cycles, 40–60% relative humidity, and temperature of 22 ± 2 °C with free access to water and food. The study complies with the principles of the 3Rs: Replacement of animals by alternatives wherever possible, Reduction in number of animals used, and Refinement of experimental conditions and procedures to minimize the harm to animals. Moreover, all experimental procedures were performed following recommendations of the National Institute of Health Guide for Care and Use of Animals and with the approval by the Ethics Committee for the Use of Animals (CEUA) of the Hospital de Clínicas de Porto Alegre (project identification code: 17-0310, approval date: 26/07/2017), complying with all methods in accordance with CEUA guidelines and regulations. All animals were anesthetized with isoflurane (Abbott, Abbott Park, IL, USA) for arthritis induction and euthanasia and were monitored twice a week to assess any behavioral change and loss of quality of life, observing signals of inactivity, suffering, stress, unbearable pain and no food or water intake.
Induction of AIA
Fourteen Balb/c mice were sensitized on day 0 with a subcutaneous injection of 200 µl of a solution containing an equal ratio of 500 µg methylated bovine serum albumin (mBSA) (Sigma-Aldrich, Saint Louis, MO, USA) diluted in saline and complete Freund’s adjuvant (CFA) (Sigma-Aldrich). On day 7 and day 14 mice were subjected to booster subcutaneous injections of mBSA with incomplete Freund’s adjuvant (IFA) (Sigma-Aldrich). Arthritis was induced on day 21, with an intra-articular (ia) injection of 30 µg of mBSA into the left knee joint. The right knee joint was injected with saline 0.9% as a negative control of arthritis (sham group) 47. Animals were randomly divided into two groups and then were treated with F. hepatica extract in the dosage of 200 µg (extract group) or PBS (control group). Treatment was performed intraperitoneally in 100 µl 24 h and 30 min prior to mBSA joint injection.
Evaluation of joint nociception and leukocyte migration in AIA
Nociception was measured at 0 h, 3 h, 6 h, and 24 h after mBSA injection using a digital analgesiometer (Insight Instruments, Ribeirão Preto, SP, Brazil). After environmental adaptation, a force was applied to the hind paws of mice and the equipment measured the intensity of the force when the paw was withdrawn, with results expressed as the flexion-elicited withdrawal threshold in grams. Leukocyte migration was evaluated 24 h after mBSA injection. After euthanasia, the knee cavities were washed three times with 5 µl of PBS-EDTA and then diluted in a final volume of 90 µl. The recovered liquid was mixed in Turk’s solution (1:1) and the total number of leukocytes was counted using a Neubauer Chamber (HBG, Giessen-Lützellinden, Germany) under an optical microscope (Olympus, Tokyo, Japan).
Induction of CIA
Sixteen DBA/1 J mice were immunized with 70 μl of an emulsion containing an equal volume of 2 mg/ml bovine collagen type II (CII) (Chondrex, Redmond, WA, USA) and CFA with 2 mg/ml of heat-inactivated Mycobacterium tuberculosis (Strain H37 RA) (Difco Laboratories, Lawrence, KS, USA) by intradermal injection in the base of the tail on day 0. After eighteen days, a booster injection was performed in another site of the tail with 70 μl of an emulsion with CII and IFA 38. Animals were randomized into two groups: arthritis-induced animals treated with PBS (control group) and arthritis-induced animals treated with F. hepatica extract at the dosage of 200 µg (extract group). Animals were treated once a day by intraperitoneal injection and treatment started after booster lasting for 28 days (46 days of experimentation).
Clinical arthritis scoring of CIA
After booster injection, animals were monitored every other day for clinical signs of arthritis using the following score: 0–normal, 1–mild swelling and erythema, 2–moderate swelling and erythema, 3–severe swelling and erythema extending from the ankle to metatarsal joints and 4–severe erythema and swelling with loss of function. The total score of the animal is presented as the sum of the score in each paw (range: 0–16) and the score of hind paws of the animals is presented as the mean of both hind paws (range: 0–4) 38.
Data are presented as mean ± standard deviation (SD). Groups were compared by one-way or two-way analysis of variance (ANOVA) and by Student's t-test using GraphPad Prism 6.0. Statistical differences were considered significant with a p value<0.05.