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Fasciola hepatica extract suppresses fibroblast-like synoviocytes in vitro and alleviates experimental arthritis

Advances in Rheumatology volume 62, Article number: 43 (2022) Cite this article

Abstract

Background

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial inflammation, fibroblast-like synoviocytes (FLS) activation and joint destruction. Fasciola hepatica is a platyhelminth that releases excretory-secretory immunomodulatory products capable of suppressing the Th1 immune response. Despite the effectiveness of available treatments for inducing disease remission, current options are not successful in all patients and may cause side effects. Thus, we evaluated the therapeutic potential of F. hepatica extract on FLS from RA patients and arthritis models.

Methods

FLS were isolated from synovial fluid of RA patients, cultured, and exposed to F. hepatica extract (60, 80, and 100 µg/ml) for different time points to assess cell viability, adherence, migration and invasion. For in vivo experiments, mice with antigen (AIA) and collagen (CIA) induced arthritis received a 200 µg/dose of F. hepatica extract daily. Statistical analysis was performed by ANOVA and Student's t-test using GraphPad Prism 6.0.

Results

In vitro assays showed that extract decreased FLS cell viability at concentration of 100 µg/ml (83.8% ± 5.0 extract vs. 100.0% ± 0.0 control; p < 0.05), adherence in 20% (92.0 cells ± 5.8 extract vs. 116.3 cells ± 7.9 control; p < 0.05), migratory potential (69.5% ± 17.6 extract vs. 100.0% control; p < 0.05), and cell invasiveness potential through the matrigel (76.0% ± 8.4 extract vs. 100.0% control; p < 0.01). The extract reduced leukocyte migration by 56% (40 × 104 leukocytes/knee ± 19.00) compared to control (90.90 × 104 leukocytes/knee ± 12.90) (p < 0.01) and nociception (6.37 g ± 0.99 extract vs. 3.81 g ± 1.44 control; p < 0.001) in AIA and delayed clinical onset of CIA (11.75 ± 2.96 extract vs. 14.00 ± 2.56 control; p = 0.126).

Conclusion

Our results point out a potential immunomodulatory effect of F. hepatica extract in RA models. Therefore, the characterization of promising new immunomodulatory molecules should be pursued, as they can promote the development of new therapies.

Trial registration Collection of synovial liquid and in vitro procedures were approved by the Ethics Committee with Certificate of Presentation of Ethical Appreciation in Plataforma Brasil (CAAE: 89044918.8.0000.5327; date of registration: 26/07/2018).

Background

Rheumatoid arthritis (RA) is a chronic inflammatory disease that occurs in 0.5–1.0% of the adult population worldwide [1] and is characterized by synovial hyperplasia with invasion and destruction of cartilage and bone [2]. Synovial joints cover the articular space and produces the synovial fluid for the joints [2]. The synovial membrane mainly consists of fibroblast-like synoviocytes (FLS) and during the progression of RA, a subpopulation of these cells acquire a modified phenotype that is aggressive and locally invasive [3]. In addition to contributing to the inflammatory environment, the ability to invade and promote cartilage destruction stands out as a main characteristic of FLS in RA. Thus, these cells are considered one of the main tissue components in the initiation and perpetuation of RA, as well as in the process of joint destruction [2, 4].

Although the available treatments are relatively effective for inducing and maintaining disease remission, there is no known cure for RA [5, 6]. In addition to the steroid and non-steroid anti-inflammatory drug options, disease-modifying antirheumatic drugs (DMARDs) can alter the course of RA [7]. The DMARDs can be conventional and targeted synthetic DMARDs, as well as biologic DMARDs. However, these options have not been successful in all patients and could cause a number of side effects, some of which involve significant health care costs [8]. Thus, new therapeutic alternatives are needed for RA.

In this context, parasite-secreted proteins with the potential to modulate the immune system could be explored as a therapeutic approach for RA and other autoinflammatory diseases [9]. In fact, parasite-mediated regulatory effects on the immune system have been reported in immune mediated diseases, such as RA [10, 11], multiple sclerosis [12], inflammatory bowel diseases, such as Crohn's disease [13, 14] and ulcerative colitis [13], as well as in celiac disease [11], type 1 diabetes [15], and allergies [16]. Among the helminths with immunomodulatory characteristics, Fasciola hepatica has been studied for this purpose. The tegument covering this parasite has a layer rich in secretory inclusions that is externally limited by a dense glycocalyx plasma membrane [17, 18]. F. hepatica tegument is highly glycosylated, with the presence of oligosaccharide motifs and glycoproteins, which have immunomodulatory properties [19]. The interaction with the host immune cells occurs through constant tegument shedding [20, 21].

To the best of our knowledge, no studies have assessed the effect of F. hepatica extract on FLS derived from RA patient or animal models of arthritis. In this study, we evaluated the therapeutic potential of F. hepatica extract in FLS from patients with RA [9] and in acute and chronic mice models of arthritis.

Materials and methods

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.

Collection procedure of synovial liquid

All procedures of arthrocentesis were performed by the same 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 × 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 37 °C 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 (1 g 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 × 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 × 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. [47]. FLS were seeded in 24-well plates at a density of 1 × 104 cells/ml. The following day, FLS were treated with 100 µg/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 5 × 104 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 × 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).

Adherence assay

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 3.5 × 104 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).

Invasion assay

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 with 100 µg/mL of F. hepatica or PBS 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 1 × 104 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 100 µg/mL F.hepatica extract or PBS for 48 h. Concentrations of TNF and IL-6 in supernatants were measured by human TNF-α Elisa Max™ Standard Set and by human IL-6 Elisa Max™ Deluxe 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).

Animals

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. 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) [48]. 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].

Statistical analyses

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.

Results

Clinical and serological characteristics of RA patients

Synovial fluid was collected from a total of thirty-two patients with established RA. Of the 32 FLS isolates, 12 cellular cultures grew appropriately in vitro and were used in this study. These were derived from 7 females and 5 males, mean age 65 years, mean duration of disease 15 years. Four of the patients were positive for anti-cyclic citrullinated peptides (anti-CCP) antibodies and presented a disease activity score (DAS-28) of 3,61 (IQR: 3.3–4.7) (2 high, 9 moderate, and 1 low disease activity). Most of the patients (33.3%) received treatment with DMARD and concurrent glucocorticoids. The baseline demographic, clinical, and laboratory parameters can be found in Additional file 1: Table S1 online.

Morphology and Characterization of FLS

To evaluate the morphology of FLS, cells collected from RA patients were cultured and stained with Alexa 488 conjugated phalloidin and analyzed by fluorescence microscopy. The FLS cells showed the classic FLS elongated phenotype (Fig. 1A). To determine the purity of FLS cultures, the cells were tested with flow-cytometric analysis using FITC- Mouse Anti-Human CD90, PE- Mouse Anti-Human CD55 (both FLS positive) and FITC- Mouse Anti-Human CD68 (macrophage positive) monoclonal antibodies (Fig. 1B). Most cells expressed the surface markers for FLS, such as CD55 (87 ± 4.5%) and CD90 (92 ± 6.0%) and they were negative for CD68 (1% ± 1.1) (Fig. 1C).

Fig. 1
figure 1

Characterization of FLS cultures. A Representative image of FLS morphology labeled with Phalloidin for actin filamentous, F-actin (green), and DAPI for nuclei (blue). B Percentage of positive cells for CD55, CD90 (both FLS markers), and CD68 (macrophage marker). (C) Representative of flow cytometry analysis for CD55, CD90, and CD68. Data are expressed as mean ± SD

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F. hepatica extract decreases the cellular viability of FLS

To analyze FLS viability after treatment with F. hepatica extract, the FLS were exposed to different doses (0, 60, 80, and 100 µg/mL) and time points (24, 48, and 72 h). The F. hepatica extract decreased the cell viability of FLS at concentration of 100 µg/ml after 48 h (83.8% ± 5.0 extract vs. 100.0% ± 0.0 control; p < 0.05) and at concentrations of 80 µg/ml (88.4% ± 3.0 extract vs. 100.0% ± 0.0 control; p < 0.05), and 100 µg/ml (89.8% extract ± 3.8 extract vs. 100.0% ± 0.0 control; p < 0.05) after 72 h, when compared with control group without treatment (n = 8). There was no significant difference in cell viability at 60 µg/ml and between the time of 48 h and 72 h at 100 µg/ml. Based on these results, the 100 µg/ml dose and 48 h of exposure were chosen as the treatment protocol for the subsequent in vitro analyses (Fig. 2).

Fig. 2
figure 2

F. hepatica extract decreased the FLS viability. F. hepatica extract was tested at different concentration (0, 60, 80, 100 µg/mL) and timepoints (24, 48 and 72 h) in FLS cultures. Cell viability was evaluated by MTT test. A decrease in cell viability was observed at concentration of 100 µg/mL after 48 h and at concentrations of 80 µg/mL and 100 µg/mL after 72 h, when compared with the control group without treatment. *p < 0.05; ANOVA two-way. (n = 8). Data were normalized as percent of control at each time point and is expressed as mean ± SD

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F. hepatica extract did not alter nuclear morphometrics and did not cause cell death or senescence of FLS

Nuclear size and shape can indicate several cell fates, including mitosis, apoptosis, senescence, or mitotic catastrophe. To evaluate the nuclear morphometrics, the cells were treated with F. hepatica extract for 48 h, followed by DAPI staining and analysis in Image Pro Plus 6.0. Treatment did not alter the nuclear morphometrics of FLS in area, roundness, aspect, radius ratio, or area box (Fig. 3A/ Fig. 3B) (n = 3). To assess apoptosis and necrosis, FLS were labeled with annexin and propidium iodide and were analyzed by flow cytometry (Fig. 3C). The F. hepatica extract did not induce apoptosis or cell death (Fig. 3D) (n = 4).

Fig. 3
figure 3

F. hepatica extract did not alter parameters of nuclear morphometry, apoptosis, cell death of FLS. A Nuclear morphometric analysis was performed in FLS cell after 48 h culture with F. hepatica extract (100 mg/mL). Representative analysis in parameters of area, roundness, aspect, radius ratio, area box, and the nuclear irregularity index NII in parameters of nuclear irregularity when compared with the untreated group. Red dots: control; blue dots: F. hepatica extract; (n = 3) B Percentage of positive cells for viability parameters (n = 3). C Representative flow cytometry analysis of apoptosis (Annexin V-FITC, BL1 axis) and necrosis (propidium iodide, BL3 axis); Control (left) and treated (right). Nonstained control is also showed. Q1 (Annexin-/PI-): viable cells; Q2 (Annexin + /PI-): early apoptotic cells; Q3 (Annexin-/PI +): necrotic cells; Q4 (Annexin + /PI +): late apoptotic cells; D Percentage of viable, apoptotic, and dead cells (n = 4). Data are expressed as mean ± SD

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F. hepatica extract decreased FLS adherence efficiency, invasion, and migratory potential

To analyze FLS adherence efficiency, the cells were seeded in a culture plate, immediately treated with F. hepatica extract and analyzed after 6 h by optical microscopy. F. hepatica extract decreased the FLS capacity to adhere in 20% compared with the control group (92.0 cells ± 5.8 extract vs. 116.3 cells ± 7.9 control; p < 0.05); (n = 4) (Fig. 4A). To evaluate the invasion potential of FLS, the cells were treated with F. hepatica extract for 48 h and then cultured in matrigel inserts for 24 h. Treatment with extract reduced FLS invasion potential through the matrigel compared with the control group (76.0% ± 8.4 extract vs. 100.0% control; p < 0.01) (n = 3) (Fig. 4B). To determine the FLS migratory potential, FLS were treated with the F. hepatica extract for 48 h and then a linear wound was created in the well. Light microscopy images were taken immediately 0 and 24 h after wounding and images were analyzed by ImageJ. The distance of the wound was measured in “inch” and expressed as percent of the average of control. F.hepatica extract reduced migratory potential of FLS compared with the control group (69.5% ± 17.6 extract vs. 100.0% control; p < 0.05) (n = 4) (Fig. 4C). TNF and IL-6, two important cytokines involved in the RA, were analyzed on FLS supernatants after stimulation with INF-© for 72 h to induce an inflammatory condition. After treatment, the cell supernatant was placed in an ELISA plate for TNF and IL-6 quantification. Although F. hepatica treatment tended to decrease TNF and IL-6 release compared to the control group, this difference was not statistically significant (TNF: 8.55 ± 0.51 pg/mL control vs. 7.01 ± 0.52 pg/mL extract; IL-6: 747.8 ± 126.2 pg/mL control vs. 615.0 ± 131.6 pg/mL extract).

Fig. 4
figure 4

F. hepatica extract reduced the adherence efficiency, the migratory, and invasion potentials of FLS. A Adherence capacity of FLS was analyzed after 6 h of F. hepatica extract incubation (100 µg/mL) (n = 4). B Migratory potential of FLS after wound healing (n = 3). C Representative microscopy images of FLS migration assay. D Invasive capacity of FLS was evaluated by matrigel assay (n = 3). E Representative images of stained cells that invaded through matrigel invasion chambers. F TNF-α secretion by FLS after INF-© stimulation (n = 3) G IL-6 secretion by FLS after INF-γ stimulation (n = 3). Original magnification: 20×. T Test; Data are expressed as mean ± SD; p < 0.05

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F. hepatica extract reduces nociception and leukocyte chemotaxis in acute antigen-induced arthritis

To analyze the in vivo effects of F. hepatica extract in mouse models of RA, we first evaluated the analgesic potential of F. hepatica extract in acute methylated bovine serum albumin (mBSA) antigen-induced arthritis (AIA) (Fig. 5A), considering increased pain in response to the mBSA injection and inflammatory process. Nociception in control group mice increased 3 h after the mBSA injection, peaking at 24 h. F. hepatica extract treatment reduced nociception, with treated mice enduring 1.7 times more nociceptive stimulus than control (6.37 g ± 0.99 extract vs. 3.81 g ± 1.44 control; p < 0.001) 24 h after the mBSA injection (Fig. 5B).

Fig. 5
figure 5

F. hepatica extract decreased nociception and leukocytes chemotaxis in AIA. A Illustration of experimental design B Nociception was determined at 0, 3, 6 and 24 h after mBSA injection C Counting of leukocytes in the knee of mice 24 h after injection with mBSA. ANOVA followed by Bonferroni post hoc test; Data are expressed as mean ± SEM (n = 7) *p < 0.05 **p < 0.01 ***p < 0.001 (control vs. extract) ### p < 0.001 (control vs. sham)

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Leukocyte migration to the knee joint is a consequence of the inflammatory response to mBSA antigen, being an important marker of local inflammatory activity 22. Therefore, this parameter is a useful predictor of anti-inflammatory effect. As expected, mBSA injection provoked massive recruitment of leukocytes to the knee joint cavity of the control group, compared to saline injection in the contralateral joint of the same mice (sham group). Treatment with F. hepatica extract inhibited leukocyte chemotaxis by 56% (40 × 104 leukocytes/knee ± 19.00) compared to control (90.90 × 104 leukocytes/knee ± 12.90) (p < 0.01) (Fig. 5C).

Treatment with F. hepatica extract delayed the beginning of clinical signs and prevented body weight loss in collagen-induced arthritis

After confirming the efficacy of F. hepatica extract treatment in the initial process of arthritis development, we analyzed its effects in the chronic phase of the disease by performing collagen-induced arthritis (CIA) model (Fig. 6A). Clinical signs of arthritis in the CIA mice treated with PBS started to be observable on day 25. The incidence of CIA was more than 50% on day 31 and all animals were affected 40 days after disease induction. F. hepatica did not reduce the clinical arthritis score (11.75 ± 2.96 extract vs. 14.00 ± 2.56 control; p = 0.126) (Fig. 6B). However, it changed the time-course development of the disease since mice treated with F. hepatica extract presented a later beginning of clinical signs of arthritis that started to be observable only at day 31, with more than 50% of mice affected only by day 36. Only on day 43 all mice presented clinical signs of arthritis (Fig. 6C). On the final day of the experimental period (day 46 after CIA induction), mice treated with F. hepatica extract had a lower mean clinical score in the hind limbs (2.94 ± 0.82 extract vs. 3.62 ± 0.52 control; p = 0.065) (Fig. 6D). Additionally, the treatment group weighed more at the end of the experimental period (1.68% ± 2.32 extract vs. − 1.76% ± 3.83 control; p < 0.05) (Fig. 6E, F).

Fig. 6
figure 6

F. hepatica delayed the beginning of clinical signs of arthritis and prevented body weight loss in CIA. A Illustration of experimental design B Clinical score of arthritis during CIA development C Incidence of arthritis D Clinical score of arthritis in the hind limbs at the last day of the CIA experiment E Body weight change F Body weight change at the last day of experimentation. Data are expressed as mean ± SEM (n = 8). (B, C, E) Two-way ANOVA followed by Bonferroni post hoc test; (D, F) Student’s T-test. **p < 0.01

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Discussion

Despite the treatments available to induce and maintain RA remission, there is no cure. In addition to causing side effects and significant health care costs, conventional treatments are not effective for all patients. In vitro culture and animal models are very useful for assessing new RA therapies, allowing control of genetic and environmental factors and testing of several doses at different time points. In this study, we evaluated F. Hepatica extract as a new strategy for RA treatment and found that it decreased FLS cell viability, adherence, migratory potential, and cell invasiveness in vitro. It reduced leukocyte migration and nociception in AIA and delayed the clinical onset of CIA, while preventing weight loss.

FLS cells are involved in the initiation and perpetuation of RA, as well as in the joint degradation process [4, 23]. Despite the wealth of knowledge gained in recent years, there are still many open questions that must be answered to fully understand the role of FLS in the development of destructive RA. It is not clear how FLS become phenotypically altered and activated in RA or at which stage of the disease this occurs [4]. Therefore, strategies targeting these cells could be pursued to further improve outcomes in RA [6].

We found decreased in the viability of FLS treated with F. hepatica extract, which indicates that the extract could affect the synovial cell layer. As a consequence of persistent local inflammation, the thickness of the synovial membrane increases to 10–15 cell layers, which mainly consist of FLS [22]. Sharaf et al. [19] demonstrated that products excretory-secretory (ESPs) from F. hepatica had significant suppressive effects on lymphoproliferation, up to 74%. Reducing FLS viability could decrease the progression of RA.

Besides increased viability, RA FLS have increased resistance to apoptosis, which contributes to an accumulation of cells in the rheumatoid lesion. Despite the observed effect of F. hepatica extract on cell viability, there was no notable effect on apoptosis and cell death according to morphometric parameters and cell surface markers. One mechanism for this resistance could be the antiapoptotic factors in synovial fluid [24]. Impaired apoptosis contributes to the development of the microenvironment and maintaining an inflammatory response that characterizes established RA [22, 25]. Moreover, altering the profile of the cells involved in RA is a delicate process since it can modify/eliminate other cells that are also sensitive to the drug. Hence, the selective elimination of a subpopulation of cells such as FLS may affect other synovial cells or lead to other diseases [26].

FLS has a key role in pannus formation and joint destruction [27] due to the ability to adhere, invade and degrade cartilage and bone. Accordingly, our findings demonstrated that F. hepatica extract modifies parameters associated with the aggressive profile of FLS such as adherence, migration, and invasion potential. Adherence and mechanical forces can affect FLS via the extracellular matrix, modulating the migration and adhesion of FLS [28]. RA FLS adhere significantly more to collagen type IV, fibronectin, and laminin than normal FLS. Another important characteristic is the invasive profile of FLS. The in vitro FLS invasion phenotype is correlated with histological joint damage in rats [29] and radiological joint destruction in RA patients [30]. The suppression or modification of these phenotypes may indicate a potential inhibition of the processes involved in cartilage and bone damage.

TNF and IL-6 are cytokines with both pro-inflammatory and immune-regulatory properties. In our study, we observed decreased release of TNF and IL-6 by FLS treated with F hepatica extract under inflammatory conditions, although the difference was not statistically significant, which could have been related to insufficient sample size. Clinical trials and animal studies have indicated the direct involvement of both TNF and IL-6 in the pathogenesis of RA [31]. Previous study showed that F. hepatica can suppress pro-inflammatory cytokines [32]. Another study demonstrated this same effect in a septic shock model by administering a preparation of antigens present in the F. hepatica tegument [18].

To corroborate our in vitro findings, we analyzed the therapeutic effect of F. hepatica extract in AIA and CIA, the most frequently tested animal models of arthritis in drugs that have been approved for RA treatment [33]. We demonstrated that F. hepatica extract was able to reduce leukocyte chemotaxis and acute nociception in the AIA model. Acute AIA is characterized by joint inflammation 24 h after induction, including swelling, leukocyte infiltration, and pronounced hyperalgesia. In RA patients, leukocyte migration is an important pathological feature of the disease, being a marker of local inflammatory activity and pain induction [34]. Different mechanisms are highlighted as fundamental in this process, such as autoantigen presentation, cytokine signaling, and leukocyte activation [23]. F. hepatica produces molecules that can modulate these mechanisms, such as the antigens present in its tegument, which reduced IFN-γ and TNF levels in mice stimulated with lipopolysaccharide [18, 35] as well as the cathelicidin-like helminth defense molecule 1 (FhHDM-1), which impairs macrophage antigen processing and presentation by inhibiting lysosomal acidification [36, 37].

In addition to pain, the initial process of leukocyte chemotaxis leads to hypercellularity, edema, and, with time, the establishment of an inflammatory environment in the joint that contributes to subsequent synovial fibroblast activation, pannus formation, and joint destruction [34]. All these human features of the disease are also present in the CIA model [38] which can be divided into three developmental stages: the induction phase, the pre-arthritis phase with the presence of autoimmunity in the absence of clinical symptoms, and the established arthritis phase [39]. We found that treatment with F. hepatica extract delayed disease onset by delaying the beginning of clinical signs for 6 days thus prolonging the pre-arthritis phase of the disease. Moreover, although it did not significantly diminish the global clinical score of arthritis, it reduced the clinical score in the hind paws and reduced body weight loss. These data indicate that F. hepatica extract as an adjuvant treatment with a DMARD can lead to an additive effect in improving clinical symptoms in immune-mediated arthritis. Interestingly, Carranza and collaborators demonstrated that F. hepatica extract induces a tolerogenic phenotype in dendritic cells, and when these cells were injected in mice with CIA they were able to reduce the symptom incidence and severity [40].

As previously mentioned, since F. hepatica extract has many different molecules with potential pharmacological effects, such as cystatins, glycoproteins, and antioxidant proteins [19, 41,42,43], detailed data on the concentrations of these components is unknown and it is not possible to determine which one exerts a specific effect on the studied parameters. Hence, further studies are necessary to clarify the exact concentration of each factor. Based on our results, F. hepatica extract acts on targets that affects cell chemotaxis, actin cytoskeletal reorganization, focal adhesion and activity of enzymes linked to extracellular matrix degradation, such as cysteine proteases and metalloproteinases. Therefore, we believe that additional efforts should be directed not only to clarify the exact concentration of each component, but to assess the molecular mechanisms of action and the absorption, distribution, metabolism, and excretion (ADME) properties of the F. hepatica extract. Moreover, the extract consists mainly of proteins, and it may induce the production of neutralizing antibodies that can interfere in its efficacy over time [44]. Therefore, the characterization of promising new immunomodulatory molecules found in helminth-released products should be pursued, as they can promote the development of new therapies.

Conclusions

To our knowledge, this is the first study to evaluate the therapeutic effect of F. hepatica extract on the FLS of RA patients and in animal models of arthritis. Treatment with F. hepatica extract affected the viability of FLS, but did not induce apoptosis or necrosis. However, the extract reduced FLS adherence, migration potential, and invasion capacity. In addition, it showed a tendency to decrease TNF release by FLS, which indicates an immunomodulatory effect in FLS. In vivo, treatment with F. hepatica extract reduced the initial events of arthritis, such as leukocyte migration and acute nociception. Moreover, it showed immunomodulatory ability by delaying the appearance of arthritis while preventing body weight loss. Therefore, the characterization of promising new immunomodulatory molecules found in helminth-released products should be pursued, as they can promote the development of new therapies. Our results indicate that F. hepatica extract is a potential therapy for RA due to its ability to reduce the aggressive and invasive profile of FLS, reduce leukocyte chemotaxis, and extend the time for the clinical onset of arthritis in mice models.

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ACR:

American College of Rheumatology

AIA:

Antigen-induced arthritis

Anti-CCP:

Anti-cyclic citrullinated peptides

BSA:

Bovine serum albumin

CII:

Bovine collagen type II

CIA:

Collagen-induced arthritis

CFA:

Complete Freund’s adjuvant

DMARDs:

Disease-modifying antirheumatic drugs

ELISA:

Enzyme-linked immunosorbent assay

ESPs:

Products excretory-secretory

EULAR:

European league Against Rheumatic

FBS:

Fetal Bovine serum

IFA:

Incomplete Freund’s adjuvant

INF-γ:

Interferon-gamma

PBS:

Phospate-buffered saline

mBSA:

Methylated bovine serum albumin

NMA:

Nuclear Morphometric Analysis

NII:

Nuclear Irregularity Index

TNF:

Tumor necrosis fator-alpha

V-FITC:

V-fluorescein isothiocyanate

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Acknowledgements

The authors acknowledge the staff from Serviço de Reumatologia, Unidade de Experimentação Animal, Unidade de Pesquisa Laboratorial and Unidade de Patologia Experimental from Hospital de Clínicas de Porto Alegre.

Funding

The authors acknowledge the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação de Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE-HCPA) for providing funding.

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Authors and Affiliations

  1. Laboratório de Doenças Autoimunes, Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil

    Suelen Pizzolatto Dalmolin, Renata Ternus Pedó, Thales Hein da Rosa, Jordana Miranda de Souza Silva, Mirian Farinon, Maria Luísa Gasparini, Rafaela Cavalheiro do Espírito Santo & Ricardo Machado Xavier

  2. Programa de Pós-Graduação em Medicina: Ciências Médicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

    Renata Ternus Pedó, Thales Hein da Rosa, Jordana Miranda de Souza Silva, Mirian Farinon, Maria Luísa Gasparini, Rafaela Cavalheiro do Espírito Santo & Ricardo Machado Xavier

  3. Programa de Pós-Graduação Ciências em Gastroenterologia e Hepatologia, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

    Eduardo Cremonese Filippi Chiela & Ana Helena Paz

  4. Departamento de Ciências Morfológicas, ICBS, Serviço de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil

    Eduardo Cremonese Filippi Chiela & Ana Helena Paz

  5. Centro de Pesquisas Experimental, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil

    Eduardo Cremonese Filippi Chiela & Ana Helena Paz

  6. Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

    Martín Pablo Cancela Sehabiague & Henrique Bunselmeyer Ferreira

  7. Laboratório de Genômica Estrutural e Funcional, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

    Martín Pablo Cancela Sehabiague & Henrique Bunselmeyer Ferreira

  8. Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center, GD 3015, Rotterdam, The Netherlands

    Fabiany da Costa Gonçalves

  9. Serviço de Reumatologia, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350; 6º Andar, Porto Alegre, RS, 90035-903, Brazil

    Ricardo Machado Xavier

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  1. Suelen Pizzolatto Dalmolin
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Contributions

FCG, MF and RMX designed the experiments. ECFP, FCG, JMSS, MF, MPC, RTP, MLG, SPD and THR performed the experiments. ECFP, FCG, MF, RCES, RTP and SPD analyzed the data. FCG, MF and SPD wrote the manuscript. JMSS, RCES, RMX, RTP and MLG reviewed the manuscript. AHP, HBF and RMX contributed to the discussion. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Fabiany da Costa Gonçalves or Ricardo Machado Xavier.

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Collection of synovial liquid and in vitro procedures were approved by the Research Ethics Committee (CEP) of the Hospital de Clínicas de Porto Alegre (project identification: 18–0350, Plataforma Brasil CAAE: 89044918.8.0000.5327, approval date: 26/07/2018) and written informed consent was obtained from each patient, complying with all methods in accordance with CEP guidelines and regulations. Moreover, all experimental procedures in murines 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: 17–0310, approval date: 26/07/2017), complying with all methods in accordance with CEUA guidelines and regulations.

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Additional file 1

. Supplementary table S1. SD = standard deviation; ACPA = anti-citrullinated protein/peptide antibody; DAS-28 = Disease Activity Score 28; ESR = Erythrocyte Sedimentation Rate; DMARD = disease-modifying anti-rheumatic drug.

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Dalmolin, S.P., Pedó, R.T., da Rosa, T.H. et al. Fasciola hepatica extract suppresses fibroblast-like synoviocytes in vitro and alleviates experimental arthritis. Adv Rheumatol 62, 43 (2022). https://doi.org/10.1186/s42358-022-00275-y

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Advances in Rheumatology

ISSN: 2523-3106