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. 2023 Apr 20;18(4):e0281834. doi: 10.1371/journal.pone.0281834

Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interaction

Hae-Ri Lee 1,#, Young-Jin Jeong 1,#, Joong-Woon Lee 1, JooYeon Jhun 2,3,4, Hyun Sik Na 2,3,4, Keun-Hyung Cho 2,3,4, Seok Jung Kim 5, Mi-La Cho 2,3,6, Tae-Hwe Heo 1,*
Editor: Hassan Zmerly7
PMCID: PMC10118179  PMID: 37079558

Abstract

Interleukin-1β (IL-1β) is one of the most potent pro-inflammatory cytokines implicated in a wide range of autoinflammatory, autoimmune, infectious, and degenerative diseases. Therefore, many researchers have focused on developing therapeutic molecules that inhibit IL-1β-IL-1 receptor 1 (IL-1R1) interaction for the treatment of IL-1-related diseases. Among IL-1-related diseases, osteoarthritis (OA), is characterized by progressive cartilage destruction, chondrocyte inflammation, and extracellular matrix (ECM) degradation. Tannic acid (TA) has been proposed to have multiple beneficial effects, including anti-inflammatory, anti-oxidant, and anti-tumor activities. However, it is unclear whether TA plays a role in anti-IL-1β activity by blocking IL-1β-IL-1R1 interaction in OA. In this study, we report the anti-IL-1β activity of TA in the progression of OA in both in vitro human OA chondrocytes and in vivo rat OA models. Herein, using-ELISA-based screening, natural compound candidates capable of inhibiting the IL-1β-IL-1R1 interaction were identified. Among selected candidates, TA showed hindering IL-1β-IL-1R1 interaction by direct binding to IL-1β using surface plasmon resonance (SPR) assay. In addition, TA inhibited IL-1β bioactivity in HEK-Blue IL-1-dependent reporter cell line. TA also inhibited IL-1β-induced expression of inducible nitric oxide synthase (NOS2), cyclooxygenase-2 (COX-2), IL-6, tumor necrosis factor-alpha (TNF-α), nitric oxide (NO), and prostaglandin E2 (PGE2) in human OA chondrocytes. Moreover, TA downregulated IL-1β-stimulated matrix metalloproteinase (MMP)3, MMP13, ADAM metallopeptidase with thrombospondin type 1 motif (ADAMTS)4, and ADAMTS5, while upregulating collagen type II (COL2A1) and aggrecan (ACAN). Mechanistically, we confirmed that TA suppressed IL-1β-induced MAPK and NF-κB activation. The protective effects of TA were also observed in a monosodium iodoacetamide (MIA)-induced rat OA model by reducing pain and cartilage degradation and inhibiting IL-1β-mediated inflammation. Collectively, our results provide evidence that TA plays a potential role in OA and IL-1β-related diseases by hindering IL-1β-IL-1R1 interaction and suppressing IL-1β bioactivity.

1. Introduction

Interleukin-1β (IL-1β) is a potent proinflammatory cytokine capable of inducing various inflammatory responses in various cell types [1, 2]. Binding of IL-1β to the cellular IL-1 receptor permits the association of the IL-1 receptor accessory protein (IL-1RAcP) to initiate a signaling cascade and induce the expression of inflammatory mediators involved in disease progression [3]. IL-1 receptors (IL-1R) are expressed in nearly all cells and tissues; thus, overproduction of IL-1β is a critical pathogenic mediator of various IL-1-related autoinflammatory, autoimmune, infectious, and degenerative diseases. Thus, many researchers have considered inhibiting the interaction between IL-1β and IL-1R1 as a therapeutic target for the treatment of various IL-1-related diseases. For instance, anakinra binds to IL-1R1 and inhibits its interaction with IL-1β. The anti-IL-1β activity of anakinra exhibit reduced mortality via anti-hyperinflammatory efficacy in patients with severe COVID-19 pneumonia [4]. It also showed anti-hyperinflammatory efficacy in other hyperinflammatory situations, including the cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome observed during antitumoral chimeric antigen receptor (CAR) T-cell therapy [5, 6]. In addition, there are several different approaches being tried to target the interaction between IL-1β and IL-1R1, and many studies have reported the therapeutic efficacies of IL-1 blockers in IL-1-related diseases [7, 8].

To date, three drugs targeting the IL-1β signaling pathway have been approved for clinical treatment. Anakinra is a recombinant IL-1 receptor antagonist; rilonacept, a dimeric fusion protein consisting of human IL-1R and IL-1RAcP; and canakinumab, an IL-1β neutralizing antibody [9]. In recent years, a number of studies have verified the efficacy of these approved drugs for various IL-1-related diseases, including autoimmune diseases such as osteoarthritis, gout, juvenile idiopathic arthritis, and diabetes [9, 10]. Therefore, the trend of these research reports is to focus on the possibility of its application as a therapeutic agent for various diseases related to IL-1 through the development of drugs that block the interaction between IL-1β and IL-1R1.

Among IL-1-related diseases, osteoarthritis (OA), the most prevalent form of arthritis, is characterized by progressive cartilage destruction, chondrocyte inflammation, and extracellular matrix (ECM) degradation [11]. Persistent chronic inflammation has a significant impact on the onset and progression of osteoarthritis. Inflammatory mediators, particularly cytokines, contribute to accelerated joint tissue degradation and pain [12]. Since accumulating evidence has demonstrated that IL-1β-mediated inflammation is important in the progression of OA, blocking IL-1β signaling may be used as an additional therapeutic strategy in drugs for OA. Therefore, it would be a good strategy to use IL-1β-mediated OA progression as an in vivo research model to evaluate the therapeutic efficacy of compounds with anti-IL-1β activity.

Tannins are naturally occurring plant phenolic compounds that are classified into hydrolyzed and condensed tannins. Among them, tannic acid (TA), a hydrolyzed tannin, is found not only in drinks such as black tea, coffee, and red wine, but also in fruits and various vegetables [13]. TA exhibits multiple beneficial effects including anti-inflammatory, anti-oxidant, anti-fungal, and anti-tumor activities [14–17]. In addition, TA also inhibits NLRP3 inflammasome-mediated IL-1β production via blocking NF-κB activation in macrophages [18]. However, it is unclear whether TA shows the anti-IL-1β activity by blocking the interaction between IL-1β and IL-1R1.

Therefore, in this study, we aimed to unveil the anti-IL-1β property of TA, which blocks the interaction between IL-1β and IL-1R1, and to determine the applicability of TA as an anti-IL-1β therapeutic agent using human OA chondrocytes and a rat OA model.

2. Materials and methods

2.1. Screening the library to discover IL-1β-blocking candidates

ELISA-based binding assays were performed for IL-1β-blocking candidates to block the interaction between IL-1β and IL-1R1 using 2303 natural compound libraries (TargetMol L-6000, Wellesley Hills, MA, USA; Selleckhem L-1400, Matsonford Road Radnor, PA, USA). In brief, ELISA plates were coated with 100 ng/well of recombinant human IL-1β (R&D Systems, Minneapolis, MN, USA) in PBS (Sigma-Aldrich, Burlington, MA, USA) at 4°C overnight. After coating, the plates were washed with PBST [PBS containing 0.05% Tween-20 (Sigma)] and blocked with PBSA [PBS containing 1% BSA (Bovogen Biologicals, East Keilor, Australia)] for 1 h at room temperature (RT). After an additional wash with PBST, each natural compound was diluted to 20 μM in PBSAT (PBS containing 1% BSA and 0.05% Tween-20), added to each well, and incubated for 2 h at RT. After three washes with PBST, recombinant human IL-1R1 protein (125 ng/ml, Abcam, Cambridge, UK) was added and incubated for 2 h. Subsequently, horseradish peroxidase (HRP)-conjugated anti-human IgG Fc (1:2000, Bethyl Laboratories, Montgomery, TX, USA) was added and incubated for 1 h. After washing, the TMB solution (Surmodics, Inc., Eden Prairie, MN, USA) was added to each well and incubated. Optical density (OD) at 450 nm was determined using an Epoch microplate spectrophotometer (BioTek Instruments, Santa Clara, CA, USA). Data are presented as the mean of triplicate samples. Dose-dependency tests of selected natural compounds for blocking recombinant human IL-1β and IL-1R1 interactions. Every step was identical to the primary screening except for the concentrations of the selected natural compounds (10, 40, or 160 μM) and washing buffer (PBS containing 0.05% Tween-20 and 0.01% Triton X-100). Binding of IL-1β and IL-1R1 without any single compound was used as a positive control (P.C.), and nonspecific binding of IL-1β and HRP-conjugated anti-human IgG secondary antibody without any single compound and IL-1R1 was used as a negative control (N.C.).

2.2. Surface plasmon resonance (SPR) analysis

Surface plasmon resonance (SPR) analysis was performed using a Biacore T200 instrument (GE Healthcare, Arlington Heights, IL, USA) with a CM5 sensor chip (GE Healthcare) at 25°C. To test TA (Sigma-Aldrich) binding of IL-1β, recombinant human IL-1β was immobilized on a CM5 sensor chip using the amine-coupling method, according to standard protocols. Various concentrations of TA were injected into the flow system at a flow rate of 20 μL/min for 300 s and allowed to dissociate for 600 s. The KD values of TA against IL-1β protein were obtained using the T200 BIA evaluation software (GE Healthcare).

2.3. Neutralizing bioassay with IL-1β-dependent HEK-Blue IL-1β cell

To examine IL-1β neutralizing activity of TA, HEK-Blue IL-1β cells (InvivoGen, San Diego, CA, USA) were used. Cells were cultured in DMEM (WELGENE, Gyeongsangbuk-do, Korea) containing 10% fetal bovine serum (FBS, Corning, Corning, NY, USA), 100 units/ml penicillin, 100 mg/ml streptomycin (Invitrogen), 100 μg/ml Normocin (InvivoGen), and 100 μg/ml Zeocin (InvivoGen), and cultured at 37°C in 5% CO2 humidified incubator. Cells (5×104 cells/well) were seeded in a 96-well plate and stimulated with human recombinant IL-1β (10 ng/ml) with or without various concentrations of TA (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, or 100 μM). After a 24 h incubation, SEAP activity was detected using QUANTI-Blue™ (Invitrogen) and the OD at 620 nm. Cell viability was measured by analyzing the OD at 450 nm using D-Plus CCK (DONGIN LS, Gyeonggi-do, Korea).

2.4. Human OA chondrocytes and stimulation

All protocols were approved by the institutional review board of Seoul St. Mary’s Hospital (KC21SISI0337) and were performed in accordance with the Declaration of Helsinki. Human articular cartilage was acquired from patients for replacement arthroplasty or joint replacement surgery, and the chondrocytes were obtained from the cartilage and maintained in DMEM (WELGENE) containing 10% FBS (Corning), 100 units/mL of penicillin, and 100 mg/mL of streptomycin (Invitrogen), and cultured at 37°C in 5% CO2 humidified incubator. Human OA chondrocytes were seeded at 3×105 cells/well or 2×105 cells/well into 6-well or 12-well plates, respectively. Following 24 h of starvation with insulin-transferrin-selenium (ITS-G, Thermo Fisher, Waltham, MA, USA), cells were stimulated with 10 ng/ml IL-1β with or without various concentrations of TA (0.5, 1, or 2 μM), recombinant human IL-1R1 Fc (1 μg/ml, Abcam), or anti-IL-1β neutralizing antibody (1 μg/ml, Abcam) in serum-free DMEM for 1, 3, or 48 h. Recombinant human IL-1R1 Fc or anti-IL-1β neutralizing antibody were used as a positive control and medium only group was used as a negative control. The supernatant and cells were collected for further analysis.

2.5. Enzyme-linked immunosorbent assay (ELISA)

The concentrations of IL-6, TNF, and PGE2 in the cell supernatants were measured using commercial human IL-6, TNF (R&D Systems), and PGE2 ELISA kits (R&D Systems), following the manufacturer’s protocol. As an indicator of NO production, nitrite was measured in the culture medium using the Griess Reagent System (Promega, Madison, WI, USA), following the manufacturer’s protocol. Optical density at 450 nm was determined using an Epoch microplate spectrophotometer (BioTek Instruments).

2.6. Western blot analysis

Total lysates of human OA chondrocytes were prepared using RIPA buffer (Biosesang, Gyeonggi-do, Korea). Primary antibodies against MMP3, MMP13 (1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA), COL2A1 (1:1000 dilution, Abcam), ACAN (1:500 dilution, Invitrogen), p38, p-p38, ERK, p-ERK, JNK, p-JNK, p65, p-p65, IκBα, p-IκBα, or β-actin (1:1000 dilution, Cell Signaling Technology) and appropriate HRP-conjugated secondary antibodies (1:2000 dilution, Bethyl) were used. Enhanced chemiluminescence (ECL) detection kit (Thermo Fisher Scientific) was used to detect for specific bands. Chemiluminescent signals were analyzed on a ChemiDoc XRS gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA), and intensity of specific bands was quantified using Image J software (NIH, MD, USA).

2.7. Reverse transcription–quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from chondrocytes using TRIzol® reagent (Ambion, Carlsbad, CA, USA), and 1 μg of total RNA was used for cDNA synthesis using a reverse transcription reagent kit (Meridian Bioscience, Cincinnati, OH, USA), according to the manufacturer’s protocol. RT-qPCR was performed using the SYBR® Green real-time PCR kit (Mbiotech, Seoul, Korea) on a Bio-Rad Real-Time PCR system (Bio-Rad Laboratories). Primers used for RT-qPCR reactions are listed in S1 Table. The relative expression levels of each gene were normalized to the internal reference gene GAPDH.

2.8. Animals

Male Wistar rats (Six-week-old, 230–280 g) were purchased from Central Lab Animal Inc. (Seoul, Korea). The animal experiments in this study were performed followed ethical guidelines for animal use and approved (Permit Number:2019-0302-01) by the Laboratory Animals Welfare Act (Korea), the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experiments of the Institutional Animal Care and Use Committee (IACUC) of the School of Medicine, Catholic University of Korea.

2.9. Monosodium iodoacetate model of OA and treatment with TA

Osteoarthritis rat model was induced by a single intra-articular injection of monosodium iodoacetate (MIA; Sigma-Aldrich). After anesthetization, the rats were injected with MIA (1 mg) into the intra-articular space of the right knee. Three days after MIA injection, the rats were received daily oral administration of TA (50 mg/kg) or celecoxib (50 mg/kg, positive control) during 21 days and were sacrificed using isoflurane anesthesia for further analysis.

2.10. Assessment of pain and weight-bearing measurement

Nociception tests in MIA-induced OA rats were performed by placing the rats on a metal mesh in the chamber and using a dynamic plantar sensory meter (Ugo Basile, Gemonio, Italy). When a microscopic plastic monofilament touched the paw and the monofilament’s force was gradually increased and stimulated, it caused pain in the rat, which was indicated by the rat’s paw withdrawal. The force used to produce the foot withdrawal reflex was measured. Rats acclimatized in an acrylic holder for a certain period of time fixed their feet on the pad and measured the weight balance of both feet for 5 seconds. The weights on the unguided and guided legs were determined. The percentage weight balance was obtained by comparing legs with and without OA.

2.11. Histopathological analyses

Three weeks after MIA injection, joint tissues were sectioned at 5 μm thickness and stained with safranin O and hematoxylin and eosin (H&E). Stained tissue samples were analyzed using the Osteoarthritis Research Society International (OARSI) and Mankin scoring systems [19].

2.12. Immunohistochemistry

Paraffin-embedded samples were incubated with the specific primary antibodies against IL-1β (1:500 dilution, Abcam, UK), IL-6 (1:500 dilution, Novus Biologicals, Centennial, CO, USA), and MCP-1 (1:500 dilution, Abcam). The tissue samples were then incubated with HRP-conjugated secondary antibody for 30 min. Visualization was conducted using the 3, 3-diaminobenzidine chromogen (Dako, Carpinteria, CA, USA). The percentage of positive cells was analyzed with HDAB (Hematoxylin & DBA) using the image J program (NIH, MD, USA).

2.13. Statistical analyses

Statistical analyses were performed using the nonparametric Mann–Whitney U-test for comparisons between two groups and one-way ANOVA with the Bonferroni post-hoc test for multiple comparisons. All graphs were constructed using GraphPad Prism, version 6 (GraphPad Software, San Diego, CA, USA). Data are presented as the mean ± standard deviation (SD). For all comparisons, p-value of < 0.05 was considered statistically significant.

3. Results

3.1. Screening and affinity determination of IL-1β-blocking candidate

Natural compound libraries were screened using an ELISA-based binding assay to discover candidate compounds that block IL-1β–IL-1R1 interaction. In the primary screening, TA inhibited the IL-1β-IL-1R1 interaction to 58.88 ± 0.89% compared to the control group of IL-1β-IL-1R1 interaction without any natural compounds (Fig 1A, orange bar; TA). Secondary screening was performed to confirm whether TA could inhibit the interaction between IL-1β and IL-1R1 under harsher conditions. Thus, every step of the ELISA-based binding assay was identical to the primary screening except for the concentrations of TA (10, 40, or 160 μM) and washing buffer (PBS containing 0.05% Tween-20 and 0.01% Triton X-100). As shown in Fig 1B, the interaction between IL-1β and IL-1R1 was inhibited by TA in a dose-dependent manner under harsh conditions. To verify the anti-IL-1β bioactivity of TA, we used the HEK-Blue IL-1β reporter cell line, which was engineered to detect IL-1β-induced activation of NF-κB and AP-1 through a secreted embryonic alkaline phosphatase (SEAP) reporter gene. TA reduced IL-1β-induced SEAP secretion (Fig 1C left panel, IC50 = 13.31 μM) and anti-IL-1β activity of TA showed a dose-dependent manner (Fig 1C, right panel). In addition, the cytotoxic effect of TA did not show a significant difference up to 100 μM compared to the medium alone group (Fig 1D). The ELISA-based binding assay and HEK-Blue IL-1β cell bioassay showed that TA blocked the IL-1β-IL-1R1 interaction and IL-1β-induced bioactivity. Therefore, an SPR assay was performed to investigate whether TA could directly bind to IL-1β. We first examined the kinetics of the interaction and binding affinity between TA and IL-1β. As shown in Fig 1E, TA bound to IL-1β in a dose-dependent and saturable manner, and the equilibrium dissociation constant KD was 8.224 × 10−7 M. Taken together, it can be inferred that TA is capable of binding to IL-1β to block IL-1β-induced bioactivity by inhibiting IL-1β-IL-1R1 interaction.

Fig 1. Screening and affinity determination of small molecule inhibitor of IL-1β.

Fig 1

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(A) 2303 natural compound libraries were tested for their ability to inhibit the interaction between recombinant human IL-1β and IL1 Receptor 1. Microtiter 96-well plates were coated with hIL-1β (100 ng/well) overnight and the blocking buffer without any single compound was used as a positive control. Diluted single compounds (20 μM) were added to each well and incubated for 2 h. After washing, recombinant human IL-1R1 (125 ng/ml) was added and incubated for 2 h. Subsequently, HRP-conjugated Anti-Human IgG Fc (1:2000) was added and incubated for 1 h. An OD450 was obtained following the TMB reaction. Data indicate mean ± SD (n = 3). (B) Dose-dependency test of selected natural compound for blocking the interaction between recombinant human IL-1β and IL-1R1. Every step was identical to primary screening except for concentrations of selected natural compound (10, 40, or 160 μM) and washing buffer (PBS containing 0.05% Tween-20 and 0.01% Triton X-100). * p <0.05 compared to the control group of IL-1β and IL1 Receptor 1 interaction without any natural compounds. (C) IL-1β-dependent HEK-Blue IL-1β cells (5×104 cell/well) were seeded onto a 96-well plate and treated with pre-incubation (20 min) of human IL-1β (10 ng/ml) with various concentrations of TA (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, or 100 μM). After a 24 h incubation, SEAP activity was assessed using QUANTI-Blue™ and the optical ensity (OD) at 620 nm. Cell viability was measured by analyzing the OD at 450 nm using D-Plus CCK. The IC50 value of tannic acid was determined using the GraphPadPrism 10 software. Data indicate mean ± SD (n = 3). (D) Surface plasmon resonance (SPR) assay was used to analyze the direct binding of tannic acid to human IL-1β. Human IL-1β protein (50 μg/ml) was immobilized on a CM5 sensor chip and various concentrations of TA (1.56, 3.125, 6.25, 12.5, 25, 37.5, 50, 62.5, 75, 87.5, or 100 μΜ) were injected into the flow system with a flow rate 20 μl/min for 300 s and allowed to dissociate for 600 s. The KD values of the tannic acid against human IL-1β were obtained using the T200 BIA evaluation software.

3.2. Effect of TA on NO, PGE2, IL-6, and TNF production in IL-1β-stimulated human OA chondrocytes

To confirm the inhibitory effects of TA on nitric oxide (NO), PGE2, IL-6, and TNF in IL-1β-stimulated human OA chondrocytes were evaluated using RT-qPCR, Griess reagent, and ELISA kits. As shown in Fig 2A, the mRNA expression levels of NOS2, COX-2, IL-6, and TNF increased after IL-1β treatment compared to the control, while IL-1β-induced NOS2, COX-2, IL-6, and TNF expression was significantly decreased by TA compared to the IL-1β treatment group. Similarly, the levels of NO, PGE2, IL-6, and TNF production in the culture supernatants were also clearly increased by IL-1β induction; however, TA also significantly reduced NO, PGE2, IL-6, and TNF release in response to IL-1β (Fig 2B). These results indicate that TA inhibited IL-1β-induced inflammatory mediators in human OA chondrocytes.

Fig 2. Effects of TA on the levels of inflammatory mediators in IL-1β-stimulated human OA chondrocytes.

Fig 2

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Human articular chondrocytes from OA patients were seeded onto 12-well plates (2×105 cells/well) and serum-starved cells were co-treated with various concentrations of TA (0.5, 1, or 2 μM) or IL-1R1 (1 μg/ml) and IL-1β (10 ng/ml) for 48 h. (A) The mRNA expression levels of iNOS, COX-2, IL-6, and TNF were measured using qRT-PCR. The relative quantity of each gene expression was normalized to the relative quantity of human GADPH. (B) Griess reaction was used to measure the NO levels in the culture supernatants and PGE2, IL-6, and TNF levels in the culture supernatants were evaluated using ELISA. # p < 0.05 compared with medium only control group and * p <0.05 compared with IL-1β-treated group.

3.3. Effect of TA on MMPs, ADAMTSs, COL2A1, and ACAN expression in IL-1β-stimulated human OA chondrocytes

We further investigated the effects of TA on IL-1β-stimulated MMP3, MMP13, ADAMTS4, ADAMTS5, COL2A1, and ACAN expression in human OA chondrocytes. The mRNA expression levels of MMP3, MMP13, ADAMTS4, and ADAMTS5 were clearly increased by IL-1β induction compared with the untreated control. Conversely, TA significantly suppressed IL-1β-induced MMP3, MMP13, ADAMTS4, and ADAMTS5 expression compared to the IL-1β treatment group (Fig 3A). In addition, the mRNA expression of COL2A1 and ACAN was downregulated by IL-1β induction compared to that in the untreated control. In contrast, the downregulated expression of COL2A1 and ACAN induced by IL-1β was increased by TA treatment (Fig 3A). Consistent with the RT-qPCR analysis, western blot analysis showed that the expression of MMP3 and MMP13 increased in response to IL-1β compared to the untreated control, and TA remarkably inhibited their expression (Fig 3B). Additionally, the protein expression of COL2A1 and ACAN was significantly decreased in response to IL-1β, and the application of TA attenuated the IL-1β-induced degradation of COL2A1 and ACAN. Collectively, these results suggest that TA could exert chondroprotective effects in the cartilage via the suppression of IL-1β-induced cartilage-degrading enzymes in human OA chondrocytes.

Fig 3. Effects of TA on the expression of MMPs, ADAMTSs, collagen type II and aggrecan in IL-1β-stimulated human OA chondrocytes.

Fig 3

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Human articular chondrocytes from OA patients were seeded onto 6-well plates (3×105 cells/well) and serum-starved cells were co-treated with various concentrations of TA (2 μM), IL-1R1 (1 μg/ml), or anti-IL-1 neutralizing antibody (1 μg/ml) and IL-1β (10 ng/ml) for 48 h. mRNA and protein expressions of MMPs, ADAMTSs, collagen type II, and aggrecan were detected by qRT-PCR (A) and Western blot analysis (B). The relative quantity of each gene expression was normalized to the relative quantity of human GADPH and β-actin was used as a loading control. # p < 0.05 compared with medium only control group and * p <0.05 compared with IL-1β-treated group.

3.4. Effect of TA on the MAPK and NF-κB signaling pathways in IL-1β-stimulated human OA chondrocytes

MAPKs and NF-κB regulate IL-1β-induced inflammatory activity during OA progression. Therefore, we evaluated whether the suppressive effect of TA on IL-1β-induced catabolic factors is regulated by MAPKs and NF-κB in human OA chondrocytes. As shown in Fig 4A, IL-1β significantly enhanced the phosphorylation of p38, ERK, and JNK compared with the untreated control. In contrast, TA showed a remarkable inhibitory effect on the IL-1β-induced phosphorylation of p38, ERK, and JNK. In addition, human OA chondrocytes stimulated with IL-1β induced the phosphorylation of p65 and IκBα, and treatment with TA markedly inhibited the IL-1β-induced phosphorylation of p65 and IκBα (Fig 4B). These results suggest that the inhibitory effect of TA on IL-1β-induced MAPK and NF-κB activation is related to its chondroprotective effect of TA on IL-1β-stimulated human OA chondrocytes.

Fig 4. Effects of TA on IL-1β-induced MAPK and NF-κB activation in human OA chondrocytes.

Fig 4

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Human articular chondrocytes from OA patients were seeded onto 6-well plates (3×105 cells/well), and serum-starved cells were co-treated with various concentrations of TA (2 μM), or anti-IL-1 neutralizing antibody (1 μg/ml) and IL-1β (10 ng/ml) for 1 h (A) or 3 h (B). Protein expressions of phosphorylated and non-phosphorylated forms of p38, ERK, JNK, p65, and IκBα were detected by Western blot analysis. β-actin was used as a loading control. Band intensity was analyzed using Image J program. # p < 0.01 compared with medium only control group and * p < 0.01 compared with IL-1β-treated group.

3.5. TA suppresses pain and cartilage destruction in MIA-induced OA rat model

To explore the anti-IL-1β efficacy of TA in in vivo animal experiments, we used a rat model of one of the IL-1β-related diseases, OA. Weight bearing, paw withdrawal threshold (PWT), and paw withdrawal latency (PWL) were examined to investigate whether TA can reduce pain in the MIA-induced OA rat model. After MIA injection, we observed that the PWT and PWL were significantly increased by TA compared to the vehicle (Fig 5A). These results indicate that oral administration of TA reduces pain. In addition, TA also showed a slight improvement in weight balance, similar to the celecoxib-treated positive control (Fig 5A). These results indicate that TA improved weight bearing, PWT, and PWL in rats with MIA-induced OA. Then, we collected the knee joints and used H&E and safranin O staining to evaluate the degree of cartilage destruction. As shown in Fig 5B, both the total Mankin score and the OARSI score in the vehicle group were markedly higher than those in the TA-treated group. In addition, TA significantly reduced articular cartilage damage and proteoglycan destruction compared with vehicle treatment in MIA-induced OA rats. Taken together, these data indicate that TA can inhibit the development of OA in MIA-induced OA rats. To further investigate the effect of TA on the expression of inflammatory mediators, IL-1β, IL-6, and MCP-1, in cartilage from the MIA-induced OA rat model was evaluated via immunohistochemistry. As shown in Fig 5B, IL-1β, IL-6, and MCP-1 were highly expressed in the vehicle group. In contrast, the expression levels of IL-1β and IL-6 were significantly decreased by TA, while the expression level of MCP-1 showed a decreasing trend in the TA-treated group. Therefore, the current results demonstrate that TA exerts a protective effect against OA development by inhibiting cartilage destruction and synovial inflammation in MIA-induced OA rats.

Fig 5. Therapeutic effect of TA in MIA-induced OA rat model.

Fig 5

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(A) Rats were injected with 1 mg of MIA in the right knee. TA (50 mg/kg) was administered orally daily for 3 days after MIA injection. Behavioral tests of mechanical hyperalgesia were evaluated using a dynamic plantar esthesiometer (n = 5). Paw withdrawal latency (PWL) and paw withdrawal threshold (PWT) were conducted right before the administration of TA. The weight balance of MIA-treated rats was analyzed using a capacitance meter. Celecoxib (50 mg/kg) was used as a positive control. Data are presented as the mean ± SD of three independent experiments. * p < 0.05 compared with vehicle group. (B) The knee joints from the OA rats treated with either TA, celecoxib, or vehicle control were stained with HE and Safranin O. The joint lesions were graded on a scale of 0–13 using the modified Mankin scoring system, giving a combined score for cartilage structure, cellular abnormalities, and matrix staining. Immunohistochemical analysis was performed to identify the expression of IL-1β, IL-6, and MCP-1 in the articular cartilage. The data are expressed as mean ± SEM for five animals per group. * p < 0.05, **p < 0.01, and ***p < 0.001 compared with the MIA-induced OA group.

4. Discussion

IL-1β is an important regulator in inflammatory immune responses and plays an essential role in a wide range of diseases, such as cancer, rheumatoid arthritis, and atherosclerosis [20, 21]. Therefore, a method of inhibiting IL-1β signaling by blocking the IL-1β:IL-1R1 interaction is considered a target for the development of therapeutic agents for IL-1β-related diseases [22–24]. Currently, there are three FDA-approved blockers that inhibit interleukin-1β signaling and are used safely in the clinic. These drugs include IL-1R antagonist (anakinra), IL-1 soluble receptor (rilonacept), and neutralizing IL-1β antibody (canakinumab). Despite the efficient use of these drugs in the clinic, all of them are biologics; thus, they have the potential for resistance, requirement of parenteral administration, and high treatment cost; hence, many studies have focused on developing small-molecule inhibitors [9, 25]. Therefore, the aim of present study was to identify small-molecule inhibitors that target IL-1β and to determine their anti-IL-1β efficacy by inhibiting the interaction between IL-1β and IL-1R1. In addition, its applicability as an anti-IL-1β therapeutic agent should be confirmed using an in vivo IL-1β related disease animal model.

Here, we conducted ELISA-based screening to identify natural compounds that could inhibit the interaction between IL-1β and IL-1R1. Among the 2303 compounds, we found that TA could directly bind to IL-1β and inhibit the IL-1β-induced activation of NF-κB and AP-1 in HEK-Blue IL-1β reporter cells. TA showed an anti-IL-1β bioactivity against expression of catabolic and anabolic factors in IL-1β-stimulated human OA chondrocytes via inhibition of IL-1β-induced MAPK and NF-κB activation. In addition, TA exerted anti-IL-1β activity in a MIA-induced rat OA model, and the protective effects of TA were confirmed by measuring pain control, cartilage destruction, and IL-1β-induced inflammatory responses. These results indicate that TA attenuated IL-1β-mediated inflammation by hindering the IL-1β:IL-1R1 interaction to diminish signaling downstream of IL-1β in in vitro and in vivo OA models.

TA is a natural hydrolyzable polyphenol that has been reported to have various efficacies in various research fields of biomedical for many years owing to its unique biochemical properties. In addition, a growing body of research about TA have demonstrated that TA has several therapeutic effects, such as antibacterial, anti-oxidant, antiviral, anti-inflammation, and anticancer activities [26]. In particular, it has been also reported that TA has the effect of inhibiting NLRP3 inflammasome-mediated IL-1β production by blocking NF-κB activation in macrophages [18]. However, no studies have shown that TA directly inhibits IL-1β–IL-1R1 interaction. In this study, we found that TA reduced IL-1β-induced SEAP secretion in a dose-dependent manner in a HEK-Blue IL-1β reporter cell line (Fig 1C). Moreover, we observed that TA showed binding activity to IL-1β in a dose-dependent and saturable manner (Fig 1D). Thus, we inferred that TA is capable of binding to IL-1β to block IL-1β-induced bioactivity by inhibiting the IL-1β–IL-1R1 interaction. Most studies on IL-1β signaling inhibition have reported that inhibition of IL-1β signaling has therapeutic effects in various inflammatory diseases such as rheumatoid arthritis, osteoarthritis, and systemic inflammatory conditions [7, 12, 27]. Further experiments were performed to determine whether TA, which directly binds to IL-1β and inhibits IL-1β signal transduction, exhibits therapeutic efficacy in IL-1β-related diseases.

Chondrocytes and the extracellular matrix are essential for the structure and function of articular cartilage, and IL-1β stimulates the production of various inflammatory mediators, including cytokines, NO, and PGE2 [28, 29]. IL-1β also stimulates chondrocytes to induce the secretion of MMPs and ADAMTSs, which degrade the extracellular matrix, such as COL2A1 and ACAN [30, 31]. Thus, upon stimulation of chondrocytes by IL-1β, the expression of factors related to OA pathology is regulated through NF-kB and MAPK signaling, and thus, NF-kB and MAPKs are major pathways involved in OA disease progression [32, 33]. Here, we found that TA reduced IL-1β-induced expression of inflammatory mediators, MMPs, and ADAMTSs, thereby inhibiting the degradation of COL2A1 and ACAN (Figs 2 and 3). It was also observed that the anti-catabolic and anti-inflammatory activities of TA were regulated by the NF-kB and MAPKs signaling pathways (Fig 4). Our observation of the anti-IL-1β activity of TA in human osteoarthritis chondrocytes prompted us to further test the anti-osteoarthritis therapeutic effect in the MIA-induced OA rat model. As expected, the anti-IL-1β activity of TA not only relieved pain in the MIA-induced OA rat model, but also significantly lowered the OARSI/MANKIN score and decreased the expression of IL-1β in the tissue, showing a therapeutic effect on OA (Fig 5).

It is well known that TA has beneficial effects in relieving inflammatory pain, and TA creams have therapeutic effects in OA of the knee in clinical trials. Furthermore, many studies have reported that TA and TA derivatives have therapeutic efficacy against OA in in vivo and in vitro OA models. Therefore, our results are consistent with those of previous reports on the suppression of OA-related factors by TA and various tannin derivatives [34–38]. Recently, the enthusiasm for IL-1 as a target in OA is rapidly dwindling due to in vivo models, showing a conflicting role for IL-1 molecule and some large double-blind randomized controlled clinical studies targeting IL-1 have failed [39]. Although the present study reports that IL-1β-direct targeting of TA showed anti-IL-1β activity by hindering the interaction between IL-1β and IL-1R1, further investigations are still needed to confirm whether TA can be applied as a therapeutic agent for other IL-1β-related diseases. In addition, the identification of the binding site of TA to IL-1β and IL-1R1, using molecular docking models, is also worthy of investigation. Nevertheless, this study is the first proves that TA can block the IL-1β–IL-1R1 interaction by binding to IL-1β and consequently inhibiting the activation of the IL-1β signaling pathway and IL-1β-related disease progression.

Supporting information

S1 Table. RT-qPCR primer sequences used in the present study.

(DOC)

Click here for additional data file. (44KB, doc)
S1 Raw images

(PDF)

Click here for additional data file. (3.3MB, pdf)

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (Grant number: NRF-2018R1A6A1A03025108 and 2021R1A2C2009782). This study was supported by the Research Fund, 2023 of The Catholic University of Korea.

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Decision Letter 0

Hassan Zmerly

8 Sep 2022

PONE-D-22-20982Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interactionPLOS ONE

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Reviewer #1: Dear Authors,

Thank you for submitting the manuscript entitled "Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interaction."

In this paper, authors investigated the role of tannic acid (TA) in the inhibition of the interaction between IL-1 β and IL-1R1, confirming the chondroprotective effects of TA due the suppression of MAPK and NF-κB signaling in IL-1β-induced OA rat model.

The paper has some strengths, but there are some critical points that need clarification and improvement especially concerning gene expression analyses.

Abstract

As reported in the submission guidelines, the abstract should not exceed 300 words. Please reduce its length trying to (i) describe the main objective of the study, (ii) explain how the study was done without methodological detail, and (iii) summarize the most important results and their significance.

Introduction

In the Introduction section (lines 85-86), authors assert that “many researchers have considered inhibiting the interaction between IL-1β and IL-1R1 as a therapeutic target for the treatment of various IL-1-related diseases.”

Please, list some examples of IL-1-related diseases, also adding appropriate recent references. For instance, since it is well-established that the most common side effects of CAR T cells therapy are cytokine release syndrome, developing new treatment options for CAR T cell-mediated toxicities based on IL-1 blockade has become an important part of immuno-oncological research.

Materials and Methods

Throughout the manuscript, please refer to the following reference for the correct nomenclature to use for human genes, especially for the use of italics (as also reported in the submission guidelines):

Bruford, E. A., Braschi, B., Denny, P., Jones, T., Seal, R. L., & Tweedie, S. (2020). Guidelines for human gene nomenclature. Nature genetics, 52(8), 754–758. https://doi.org/10.1038/s41588-020-0669-3.

2.7. Real-time polymerase chain reaction (RT-PCR)

This reviewer strongly recommends following the MIQE guidelines (The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009 Apr;55(4):611-22. doi: 10.1373/clinchem.2008.112797), not only to use the correct nomenclature in the text, but also to enable other investigators to reproduce results. In this respect, please replace abbreviation qPCR with RT-qPCR if you performed reverse transcription–qPCR.

Moreover, although the use of a reference gene an internal control is the most common method for normalizing cellular mRNA data, the chosen reference gene should be stably expressed between treatment groups.

Hence, normalization against a single reference gene (e.g., GAPDH), as reported by the authors, is not acceptable unless the investigators present clear evidence that confirms its invariant expression under the experimental conditions described.

To this regard, has the M-value (the measure of expression stability) of your reference gene been calculated?

Have the authors purchased certified primers? In this case, please add information regarding the Company. On the other hand, if primers were custom designed, please add the length of amplicons in an appropriate Table.

2.12. Histopathological Analyses

Since histopathological analyses were performed on tissues of OA rat model, this reviewer suggests referring to the work of Gerwin et al. for a more appropriate use of the OARSI score: Gerwin, N.; Bendele, A.M.; Glasson, S.; Carlson, C.S. The OARSI histopathology initiative—Recommendations for histological assessments of osteoarthritis in the rat. Osteoarthr. Cartil. 2010, 18, S24–S34.

Results

3.1. Screening and affinity determination of IL-1β-blocking candidate

Line 270: what is the meaning of #397 after Fig. 1A within the brackets?

Concerning Figure 1A, please enhance the scale of the y-axis to show all the SD bars.

3.4. Effect of TA on the MAPK and NF-κB signaling pathways in IL-1β-stimulated human OA chondrocytes

Since TA showed a remarkable inhibitory effect on the IL-1β-induced phosphorylation of p38, ERK, JNK, p65, and IκBα, have authors tested specific inhibitors of MAPK and NF-κB signaling pathways, as negative controls?

Discussion

In the Discussion section, authors should comment on the fact which the enthusiasm for IL-1 as a target in OA is rapidly dwindling. In this regard, as reported by Prof. TL Vincent (doi: 10.12688/f1000research.18831.1), in vivo models show a conflicting role for IL-1 molecule; early studies using therapeutic approaches in large animal models show a benefit, but most murine studies fail to demonstrate protection where the ligands (IL-1α/β), the cytokine activator (IL-1–converting enzyme), or the receptor (IL-1R) have been knocked out. Recently, some large double-blind randomised controlled clinical studies targeting IL-1 have failed.

Reviewer #2: The manuscript is technically sound and well written. However, several points need be tackled, especially in the statistical analysis part and in the corresponding conclusions, in order to improve the manuscript. Additionally, some ambiguities exist and they need clarification. Below are my suggestions and concerns:

Introduction

1.Lines 101 – 105: the sentences are ambiguous. Can the authors please reformulate these sentences?

Materials & Methods

2.The authors are advised to check section 2.5 for possible errors in listing the performed ELISA assays. Did the author perform ELISA assay for MMP-3 & MMP13 or PGE2?

3.Section 2.1: Can the authors please define what did they use as positive and negative controls?

4.Lines 233-234: the sentence is ambiguous. Can the authors please reformulate this sentence?

Results

5.Authors are advised to check the abbreviations and signs used in the figures as many of these are not defined in the legends (For ex: +/-; NC; PC; NT…).

6.Figure 1A: The authors included only few of the 2303 tested molecules in the figure. Obviously, it would be impossible to include all the tested molecules, but why the authors chose to represent specifically the results of these compounds among others? And what are these compounds? As in the codes used for these compounds are not defined in the legend.

7.Lines 274-275: “the interaction between IL-1β and IL-1R1 was inhibited by TA in a dose dependent manner under harsh conditions”. The p-value presented in the figure 1B is for the comparison between the different TA concentrations and the negative control. If the authors are testing the dose-dependent effect, the p-values for the comparisons between the different concentrations of TA should be calculated and used to make conclusions.

8.Lines 277-279: the authors concluded that “TA reduced Il-1β induced SEAP secretion in a dose dependent manner” and that TA showed no cytotoxic effect at 100μΜ. It is not clear what the obtained p-values for these analyses are. And which comparison do they represent?

9.Figure 2, 3 & 4: Authors should identify which groups serve as positive and negative controls (in the materials and methods section).

10.Figure 3: It seems like the legend attributed to figure A corresponds to figure B and vice-versa. Can the authors please checks figure 3 legend?

11.Figure 4: Why did the authors studied the effect of TA on MAPK pathway with a single TA concentration of 2 μΜ while the TA effect on NF-κB pathway was studied with 1 & 2 μΜ of TA?

12.Figure 5: The authors compared the therapeutic effect of TA and vehicule as well as Celecobix and vehicule. It would be interesting to compare also the effect of TA to Celecobix to see if the effect of TA on the studied OA features is similar to Celecoxib or inferior/superior

13.Line 626: Can the author please check the number of rats in each group as in figure 5B it looks as there is 15 rats/group but in the text it is mentioned that there are 5 rats/group.

Discussion

14.Lines 376-378: the sentence is ambiguous. Can the authors please reformulate this sentence?

15.Lines 378-379: “We confirmed that the reduction in IL-1β signaling by TA was similar to that observed following treatment with soluble IL-1R1 or an anti-IL-1β neutralizing antibody”. However, in figures 2 & 3, the authors didn’t compare the effect of TA to the effect of soluble IL-1R1 or anti-IL-1β neutralizing antibody. The p-values represented in the figures are for the comparison between different concentrations of TA & IL-1β treated group.

16.Lines 388 -389: lack references.

**********

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PLoS One. 2023 Apr 20;18(4):e0281834. doi: 10.1371/journal.pone.0281834.r002

Author response to Decision Letter 0

18 Oct 2022

PLoS One

Responses to the reviewer’s comments:

We are grateful to the reviewers for their valuable feedback and insightful comments that helped us to improve the quality of our manuscript. We have carefully considered all the comments and revised the manuscript accordingly. Our point-wise responses to the reviewers’ comments are given below. We hope that the response meets the reviewers’ expectations. We are ready to continue working with the editor and reviewers to bring this paper closer to publication.

Our responses are detailed below:

Reviewer 1:

Abstract

As reported in the submission guidelines, the abstract should not exceed 300 words. Please reduce its length trying to (i) describe the main objective of the study, (ii) explain how the study was done without methodological detail, and (iii) summarize the most important results and their significance.

Response: Thank you for the comments. We have revised the abstract accordingly. The new version of abstract is included in the revised manuscript (Revised lanes 37-60).

Introduction

In the Introduction section (lines 85-86), authors assert that “many researchers have considered inhibiting the interaction between IL-1β and IL-1R1 as a therapeutic target for the treatment of various IL-1-related diseases.”

Please, list some examples of IL-1-related diseases, also adding appropriate recent references. For instance, since it is well-established that the most common side effects of CAR T cells therapy are cytokine release syndrome, developing new treatment options for CAR T cell-mediated toxicities based on IL-1 blockade has become an important part of immuno-oncological research.

Response: We completely agree with your suggestion. We have included this information in the revised Introduction section. (Revised lines 83-88):” For instance, anakinra bind to IL-1R1 and inhibit its interaction with IL-1β. The anti-IL-1β activity of anakinra was shown to reduce mortality via anti-hyperinflammatory efficacy in patients with severe COVID-19 pneumonia [4]. Anakinra also showed anti-hyperinflammatory efficacy in other hyperinflammatory situations, including the cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome observed during antitumoral chimeric antigen receptor (CAR) T-cell therapy [5, 6].”

Materials and Methods

Throughout the manuscript, please refer to the following reference for the correct nomenclature to use for human genes, especially for the use of italics (as also reported in the submission guidelines):

Bruford, E. A., Braschi, B., Denny, P., Jones, T., Seal, R. L., & Tweedie, S. (2020). Guidelines for human gene nomenclature. Nature genetics, 52(8), 754–758. https://doi.org/10.1038/s41588-020-0669-3

Response: Thank you for your comments. We have corrected the nomenclature throughout the entire manuscript.

2.7. Real-time polymerase chain reaction (RT-PCR)

This reviewer strongly recommends following the MIQE guidelines (The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009 Apr;55(4):611-22. doi: 10.1373/clinchem.2008.112797), not only to use the correct nomenclature in the text, but also to enable other investigators to reproduce results. In this respect, please replace abbreviation qPCR with RT-qPCR if you performed reverse transcription–qPCR.

Moreover, although the use of a reference gene an internal control is the most common method for normalizing cellular mRNA data, the chosen reference gene should be stably expressed between treatment groups.

Hence, normalization against a single reference gene (e.g., GAPDH), as reported by the authors, is not acceptable unless the investigators present clear evidence that confirms its invariant expression under the experimental conditions described.

To this regard, has the M-value (the measure of expression stability) of your reference gene been calculated?

Response: Thank you for your valuable comment. We completely agree with your suggestion. Although we did not further evaluate the M-value of GAPDH as a reference gene, it was reported that GAPDH showed stable gene expression in IL-1β-treated human chondrocytes [1]. Thus, GAPDH was chosen as the reference housekeeping gene based on the majority of previous studies on chondrocyte gene expression.

In addition, using two other reference genes, RPL13a and YWHAZ [2], which are the most stable expressed genes for reference in human chondrocytes, we tried to normalize some of the tested gene expressions in Figure 2A and 2B in the manuscript. As shown below, the normalization results using two additional reference genes, RPL13a(A) and YWHAZ(B), were consistent with the existing normalization results using GAPDH as a reference gene.

[1] Toegel S et. al. Selection of reliable reference genes for qPCR studies on chondroprotective action. BMC Mol Biol. 2007; 8:13. DOI: 10.1186/1471-2199-8-13.

[2] Akira Ito et. al. Evaluation of reference genes for human chondrocytes cultured in several different thermal environments. Int J Hyperthermia. 2014;30(3):210-6. DOI:10.3109/02656736.2014.906048.

Have the authors purchased certified primers? In this case, please add information regarding the Company. On the other hand, if primers were custom designed, please add the length of amplicons in an appropriate Table.

Response: Thank you for the comment. We have added the information of primer sequences in Table S1. Please see methods section and supplementary information file.

2.12. Histopathological Analyses

Since histopathological analyses were performed on tissues of OA rat model, this reviewer suggests referring to the work of Gerwin et al. for a more appropriate use of the OARSI score: Gerwin, N.; Bendele, A.M.; Glasson, S.; Carlson, C.S. The OARSI histopathology initiative—Recommendations for histological assessments of osteoarthritis in the rat. Osteoarthr. Cartil. 2010, 18, S24–S34.

Response: We appreciate your valuable comments. However, this reference is suitable for the DMM or ACLT surgery model. In case of the MIA-induced osteoarthritis model, it shows severe bone destruction and cartilage damage. Therefore, the chemically induced model requires a scoring analysis focusing on cell death, bone destruction, and cartilage damage (Osteoarthritis and Cartilage (2006) 14, 13e29 Table III).

Results

3.1. Screening and affinity determination of IL-1β-blocking candidate

Line 270: what is the meaning of #397 after Fig. 1A within the brackets?

Response: We apologize for the error. We replaced the labels of Figure 1A #397 to Tannic acid. Please see revised manuscript and Fig. 1A.

Concerning Figure 1A, please enhance the scale of the y-axis to show all the SD bars.

Response: We have replaced the Figure 1A to improve clarity.

3.4. Effect of TA on the MAPK and NF-κB signaling pathways in IL-1β-stimulated human OA chondrocytes

Since TA showed a remarkable inhibitory effect on the IL-1β-induced phosphorylation of p38, ERK, JNK, p65, and IκBα, have authors tested specific inhibitors of MAPK and NF-κB signaling pathways, as negative controls?

Response: Thank you for your reasonable comments. Although we did not include negative control related data in previous manuscript, we have also compared the effects of TA with specific inhibitors of MAPK and NF-kB. IL-1β-induced phosphorylations of p38, ERK, JNK, p65, and IkBα were inhibited by TA as shown in Figures 4A and 4B; however, its inhibitory effects were lower than inhibition by MAPK or NF-kB specific inhibitors (SB203580; p38 inhibitor, U0126; ERK inhibitor, SP600125; JNK inhibitor, and Bay11-7082; NF-kB inhibitor).

Discussion

In the Discussion section, authors should comment on the fact which the enthusiasm for IL-1 as a target in OA is rapidly dwindling. In this regard, as reported by Prof. TL Vincent (doi: 10.12688/f1000research.18831.1), in vivo models show a conflicting role for IL-1 molecule; early studies using therapeutic approaches in large animal models show a benefit, but most murine studies fail to demonstrate protection where the ligands (IL-1α/β), the cytokine activator (IL-1–converting enzyme), or the receptor (IL-1R) have been knocked out. Recently, some large double-blind randomised controlled clinical studies targeting IL-1 have failed.

Response: We agree with your suggestion. We have included this information in Discussion (Revised lines 398-400). “Recently, the enthusiasm for IL-1 as a target in OA is rapidly dwindling due to in vivo models, showing a conflicting role for IL-1 molecule and some large double-blind randomized controlled clinical studies targeting IL-1 have failed [39].”

Reviewer #2:

Introduction

1.Lines 101 – 105: the sentences are ambiguous. Can the authors please reformulate these sentences?

Response: We have rewritten these sentences in the revised manuscript (Revised lines 103-107). “Since accumulating evidence has demonstrated that IL-1β-mediated inflammation is important in the progression of OA, blocking IL-1β signaling may be used as an additional therapeutic strategy in drugs for OA. Therefore, it would be a good strategy to use IL-1β-mediated OA progression as an in vivo research model to evaluate the therapeutic efficacy of compounds with anti-IL-1β activity.”

Materials & Methods

2.The authors are advised to check section 2.5 for possible errors in listing the performed ELISA assays. Did the author perform ELISA assay for MMP-3 & MMP13 or PGE2?

Response: We have corrected relevant information regarding ELISA assay in the revised manuscript section 2.5.

3.Section 2.1: Can the authors please define what did they use as positive and negative controls?

Response: Thank you for your comment. We have defined the positive and negative control in the revised manuscript.

4.Lines 233-234: the sentence is ambiguous. Can the authors please reformulate this sentence?

Response: We have rewritten the sentence. Please see revised manuscript (Revised lines 222-223). “The weights on the unguided and guided legs were determined. The percentage weight balance was obtained by comparing legs with and without OA.”

Results

5.Authors are advised to check the abbreviations and signs used in the figures as many of these are not defined in the legends (For ex: +/-; NC; PC; NT…).

Response: Thank you for the comments. We have corrected the abbreviations and signs in the revised figure legend (Revised lines 562-565, 592, 603, and 611). “Binding of IL-1β and IL-1R1 without any single compound was used as a positive control (P.C.), and nonspecific binding of IL-1β and HRP-conjugated anti-human IgG secondary antibody without any single compound and IL-1R1 was used as a negative control (N.C.). ** p <0.01 compared to the positive control group.” and ““+” indicates treated and “-” indicates un-treated.”

6.Figure 1A: The authors included only few of the 2303 tested molecules in the figure. Obviously, it would be impossible to include all the tested molecules, but why the authors chose to represent specifically the results of these compounds among others? And what are these compounds? As in the codes used for these compounds are not defined in the legend.

Response: Since we screened 2303 natural compound libraries, it would be impossible to include all the results of tested molecules. Therefore, we selected the anterior and posterior compound portions, including TA, from among the compound results within the library, and presented the results in Figure 1A. As suggested by the reviewer’s comment, we added the information of additional tested compounds in Figure legend (Revised lines 565-567). “Among 2303 natural compound, only some compounds were used for screening results, and the numbers 316 to 443 mean serial numbers represent the natural compounds in the TargetMol L-6000 library.”

7.Lines 274-275: “the interaction between IL-1β and IL-1R1 was inhibited by TA in a dose dependent manner under harsh conditions”. The p-value presented in the figure 1B is for the comparison between the different TA concentrations and the negative control. If the authors are testing the dose-dependent effect, the p-values for the comparisons between the different concentrations of TA should be calculated and used to make conclusions.

Response: Thank you for pointing this out. As suggested by the reviewer’s comment, comparisons between the different TA concentrations were performed against each other and the new information was included in the revised Figure 1B legend (Revised line 570-571). “## p <0.01 compared to the positive control group. ** p <0.01 compared to treatment groups with different TA concentrations (10 or 40 μM).”

8.Lines 277-279: the authors concluded that “TA reduced IL-1β induced SEAP secretion in a dose dependent manner” and that TA showed no cytotoxic effect at 100 μΜ. It is not clear what the obtained p-values for these analyses are. And which comparison do they represent?

Response: The comparisons between the different TA concentrations were performed against each other and we have added new information in the revised manuscript (Revised lines 258-261), Figure 1C and Figure legend 1. “TA reduced IL-1β-induced SEAP secretion (Fig. 1C left panel, IC50 = 13.31 µM) and anti-IL-1β activity of TA showed a dose-dependent manner (Fig. 1C, right panel). In addition, the cytotoxic effect of TA did not show a significant difference up to 100 μM compared to the medium alone group (Fig. 1D).”

9.Figure 2, 3 & 4: Authors should identify which groups serve as positive and negative controls (in the materials and methods section).

Response: We have defined positive and negative control in the revised Material and Methods section (Revised line 175-176). “Recombinant human IL-1R1 Fc or anti-IL-1β neutralizing antibody were used as a positive control and medium only group was used as a negative control.”

10.Figure 3: It seems like the legend attributed to figure A corresponds to figure B and vice-versa. Can the authors please checks figure 3 legend?

Response: We have corrected legend of Figure 3 in the revised manuscript (Revised line 598-602). “(A) mRNA expressions of MMPs, ADAMTSs, COL2A1, and ACAN were detected using RT-qPCR and the relative quantity of each gene expression was normalized to the internal reference gene GAPDH. (B) Protein expressions of MMPs, ADAMTSs, COL2A1, and ACAN were detected using western blot analysis and β-actin was used as a loading control. # p < 0.05 and ## p < 0.01 compared with medium only control group.”

11.Figure 4: Why did the authors studied the effect of TA on MAPK pathway with a single TA concentration of 2 μΜ while the TA effect on NF-κB pathway was studied with 1 & 2 μΜ of TA?

Response: Since the NF-κB pathway plays an important role in the pathogenesis of OA, the TA effect experiment on the NF-κB pathway was first conducted according to the TA concentration. Consequently, 2 μM of TA showed a more dramatic inhibitory efficacy, thus the MAPK pathway proceeded only with a single TA concentration of 2 μΜ.

We have modified the results for the NF-κB pathway to match MAPK pathway tested with a single concentration of 2 μM of TA to improve clarity.

12.Figure 5: The authors compared the therapeutic effect of TA and vehicle as well as Celecoxib and vehicle. It would be interesting to compare also the effect of TA to Celecoxib to see if the effect of TA on the studied OA features is similar to Celecoxib or inferior/superior

Response: Comparisons of the therapeutic effects between the TA and Celecoxib-treated group were performed. The therapeutic efficacy of TA showed a similarity to the celecoxib-treated positive control, and the p-value between the TA and Celecoxib-treated group was not significant.

13.Line 626: Can the author please check the number of rats in each group as in figure 5B it looks as there is 15 rats/group but in the text it is mentioned that there are 5 rats/group.

Response: To objectively evaluate histological scoring, each of the three evaluators who did not know the experimental group performed histological analysis, so the meaning of 15 dots refers to the results of each of the three evaluators for 5 rat samples per group. To avoid confusion, the results of the three evaluators were averaged and corrected for bar graphs. And we replaced the Fig. 5B. Please see revised Fig. 5B.

Discussion

14.Lines 376-378: the sentence is ambiguous. Can the authors please reformulate this sentence?

Response: We have rewritten the sentence. Please see revised manuscript (Revised lines 358-360). “TA showed an anti-IL-1β bioactivity against expression of catabolic and anabolic factors in IL-1β-stimulated human OA chondrocytes via inhibition of IL-1β-induced MAPK and NF-κB activation.”

15.Lines 378-379: “We confirmed that the reduction in IL-1β signaling by TA was similar to that observed following treatment with soluble IL-1R1 or an anti-IL-1β neutralizing antibody”. However, in figures 2 & 3, the authors didn’t compare the effect of TA to the effect of soluble IL-1R1 or anti-IL-1β neutralizing antibody. The p-values represented in the figures are for the comparison between different concentrations of TA & IL-1β treated group.

Response: Thank you for your pointing this out. As suggested by the reviewer’s comment, we have corrected Discussion section in the revised manuscript.

16.Lines 388 -389: lack references.

Response: We have added the reference information in the revised manuscript.

Attachment

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Decision Letter 1

Hassan Zmerly

2 Feb 2023

Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interaction

PONE-D-22-20982R1

Dear Dr. Tae-Hwe Heo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Hassan Zmerly, MD PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

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Reviewer #1: All comments have been addressed

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

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Reviewer #1: Yes

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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Reviewer #1: No

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Acceptance letter

Hassan Zmerly

11 Apr 2023

PONE-D-22-20982R1

Tannic acid, an IL-1β-direct binding compound, ameliorates IL-1β-induced inflammation and cartilage degradation by hindering IL-1β-IL-1R1 interaction

Dear Dr. Heo:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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    S1 Table. RT-qPCR primer sequences used in the present study.

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