Mechanical Stress Protects Against Chondrocyte Pyroptosis through Lipoxin A4 via Synovial Macrophage M2 Subtype Polarization in an Osteoarthritis Model

doi:10.21203/rs.3.rs-1411791/v1

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Research Article

Mechanical Stress Protects Against Chondrocyte Pyroptosis through Lipoxin A4 via Synovial Macrophage M2 Subtype Polarization in an Osteoarthritis Model

https://doi.org/10.21203/rs.3.rs-1411791/v1

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posted 08 Mar, 2022

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Our previous study found that lipoxin A₄ (LXA₄) exerted therapeutic effects on osteoarthritis (OA). In this study, we evaluated the effects of LXA₄ via synovial macrophage M1/M2 subtype polarization on chondrocyte pyroptosis and mechanisms during mechanical stimulation. Synovial macrophages were subjected to various LXA₄ concentrations and cyclic tensile strain (CTS) conditions to determine optimal co-culture conditions. The effects of LXA₄ on chondrocyte pyroptosis, as represented by macrophage M1/M2 subtype polarization, were detected by western blot, immunofluorescence, and flow cytometry analyses. Forty male Sprague-Dawley rats were randomly divided into four groups (n = 10): control group (CG), OA group (OAG), OA with moderate-intensity treadmill exercise (OAE), and OAE + BOC-2 (an LXA₄ antagonist). All rats were evaluated using histology, enzyme-linked immunosorbent assay (ELISA), quantitative PCR, and western blot analyses. We found that LXA₄ was downregulated in articular fluid and that CD 86/Arg 1 was up-regulated in the synovium of patients with increasing Kellgren-Lawrence grade. In vitro, CTS and LXA₄ both promoted M2 subtype polarization of synovial macrophages, inhibiting the nuclear translocation of NF-κB p65 and the NLRP3 formation in chondrocytes. In vivo, the OAE treatment exerted protective effects on articular cartilage and facilitated M2 polarization of synovial macrophages. These effects were suppressed by BOC-2 treatment. We concluded that moderate CTS enhances the therapeutic effects of LXA₄ by inhibiting the nuclear translocation of NF-κB p65 and NLRP3. Furthermore, the therapeutic effects of LXA₄ during treadmill exercise in monoiodoacetate-induced OA were driven by promotion of synovial macrophage M2 subtype polarization.

mechanical stimulation

lipoxin A4

synovial macrophages

osteoarthritis

pyroptosis

Osteoarthritis (OA) is a prevalent chronic joint disease characterized by structural alterations in the articular cartilage, subchondral bone, ligaments, capsules, synovial tissue, and periarticular muscles [1]; its main clinical features are pain and loss of function [2, 3]. Globally, OA incidence is rising due to increasingly aged populations and obesity [4].

Acute inflammatory response is an immune mechanism that produces multiple mediators, including cytokines and chemokines [5, 6]. However, in the case of OA, overactive immune response or chronic inflammation can lead to tissue damage and fibrosis [7, 8]. Lipoxin A4 (LXA₄) is an eicosanoid acid produced in the capillaries and infrapatellar fat pad during cell-cell interactions, and is an end signal of inflammation [9, 10]. In our previous studies, we found that LXA₄ was rapidly produced and metabolized during exercise therapy for OA, suggesting an important role in the efficacy of this treatment [11, 12]. This suggests that LXA₄ serves as an indicator for the curative effects of exercise, allowing for development of optimized therapeutic methods. Therefore, it is important to clarify how LXA₄ regulates inflammation during exercise therapy for OA.

Increasing evidence suggests that synovitis is related to OA progression [13]. Synovitis generates inflammatory mediators (TNF-α and IL-1β), which are released into the synovial cavity and play a key role in OA pathogenesis and cartilage degeneration. However, the cellular and molecular processes that occur in the OA synovium are poorly understood, and further research is required to develop novel therapeutics [14]. The synovium of the knee joint is divided into two layers: the intima and the subintima. The intima comprises synovial macrophages and fibroblasts, which are important to the knee joint microenvironment and affect the joint through the interaction of articular chondrocytes [15, 16]. Chondrocytes, which comprise articular cartilage, synthesize extracellular matrix and depend on intracellular signals generated in response to the microenvironment [8, 17]. Synovial macrophages are tissue-resident immune cells with critical roles in maintaining homeostasis and fighting inflammation. Locally-renewed and protective tight junction–mediated macrophage barrier functions are important for our general understanding of macrophage roles in health and disease [18]. Macrophages exert strong plasticity in response to a variety of microenvironmental stimuli, and can polarize into either of two functional states to generate a spectrum of functional phenotypes. Classical, or M1, macrophage polarization executes kill functions, whereas alternative, or M2, polarization is responsible for repair [19–21]. Recent studies have found that macrophage polarization is closely related to OA pathogenesis [22], and is regulated by a variety of signal pathways. Pyroptosis is a pro-inflammatory type of programmed cell death caused by inflammation, and includes the activation of Nod-like receptor protein 3 (NLRP3)/ caspase-1. This process induces cell rupture and release of contents in a manner similar to that of necrosis, but is caspase-driven, more like apoptosis [23, 24]

Appropriate exercise regimens are critical aspects of OA treatment plans [25–27]. Bodily tissues and cells are continuously exposed to complex mechanical cues from the environment. Mechanical stimulation affects cell proliferation, differentiation, and migration, as well as determining tissue homeostasis and repair [28]. Although synovial macrophages experience mechanical stress during exercise therapy, little is known about how these mechanical processes regulate immune responses.

Here, we show that cyclic tensile stress (CTS), similar to that experienced in the joint, initiates an inflammatory response via synovial macrophage polarization. The mechano-transduction signaling pathways of synovial macrophages are complex, and the mechanisms by which they convert mechanical stimuli into biochemical signals requires further investigation.

Several studies have emphasized the importance of exercise therapy to OA. However, most have focused on single cells, such as chondrocytes, synovial fibroblasts, or bone cells [29, 30]. Little is known regarding the complexity of how this treatment functions in living organisms. Thus, the effects of mechanical stimulation on chondrocyte behavior in multi-population systems is unclear.

In our preliminary experiment of this study, M2 polarization markers were upregulated, and LXA₄ was down-regulated in patients with high Kellgren-Lawrence grades. This finding was explored through in vivo and in vitro experiments. In vitro, the indirect effects of synovial macrophages on chondrocytes were studied by establishing a co-culture system using transwell chambers. Synovial macrophages were subjected to variable LXA₄ and CTS conditions. M2 polarization was found to inhibit the nuclear translocation of NF-κB p65 and the formation of NLRP3 in chondrocytes. In vivo, we used BOC-2, an antagonist of the LXA₄ receptor, to evaluate the effects of LXA₄ on synovial macrophage M2 polarization during moderate-intensity treadmill exercise in a rat model of monoiodoacetate (MIA)-induced OA [29]. These data provide new insights into the role of synovial macrophages in OA pathogenesis, and suggest that LXA₄ is suitable for assessing the effects of treadmill exercise on OA treatment and progression. Our findings offer to improve the development of exercise programs for patients with OA.

2.1 Human OA synovium, articular fluid, and clinicopathological features

This study was approved by the ethics committee of the Shengjing Hospital of China Medical University (No. 2017PS237K). All patients consented to their inclusion in this study. The synovium, articular fluid, and clinicopathological features of patients with different Kellgren-Lawrence grades were collected from January 2019 to August 2021, based on data from 184 patients treated by arthroscope in the Fourth Department of Orthopedic Surgery in our hospital. The inclusion criteria were: 1) epiphyseal closure, 2) sign informed consent, 3) no definite history of trauma within three months, and 4) no drug therapy within three months. The exclusion criteria were: 1) tumor and/or infection of knee tissue, 2) lack of obtained tissue samples, or 3) lack of imaging examination. (Table 1, Fig. 1A). Cartilage specimens from the tibial plateau, as well as medial and lateral femur condyles, removed after total knee arthroplasty were selected. The synovial tissue around the diseased cartilage was also collected.

Table 1

Baseline characteristics of patients
Basic information		Kellgren-Lawrence grade 0	Kellgren-Lawrence grade 1	Kellgren-Lawrence grade 2	Kellgren-Lawrence grade 3	Kellgren-Lawrence grade 4	Statistic	P value
Biological Sex	Male	13	15	12	15	12	χ² = 5.501	0.240
	Female	15	18	19	28	37
Age (years)	༜65	28	33	16	8	5	χ² = 108.99	< 0.001
	≥ 65	0	0	15	35	44
Obesity gradation	Under weight^a	2	1	0	1	2	χ² = 52.113	< 0.001
	Normal weight	22	27	21	24	10
	Overweight	4	4	8	15	23
	Obesity	0	1	2	3	14
^aUnderweight: BMI < 18.5; Normal weight: 18.5 ≤ BMI < 24.9; Overweight: 25 ≤ BMI < 30; Obesity: BMI ≥ 30.

2.2 Western blotting

Synovial tissue was washed twice in ice-cold potassium-buffered saline (PBS). Cytoplasm protein levels were measured using a bicinchoninic acid assay kit (Beyotime, Shanghai,China) according to manufacturer instructions. Equal protein concentrations (40 µg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 1% bovine serum albumin (BSA) in tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) at room temperature for 2 h. They were then incubated overnight at 4°C with the following primary antibodies: rabbit monoclonal anti-vascular endothelial growth factor (VEGF) receptor 1 (ab32152, 1:1000; Abcam; molecular weight 151 kDa); rabbit monoclonal anti-NLRP3 (ab210491, 1:1,000; Abcam; 118 kDa); rabbit monoclonal anti-MMP13 (ab51072, 1:1000; Abcam; 54 kDa); rabbit monoclonal anti-caspase-1 (ab108362, 1:5,000; Abcam; 45 kDa); rabbit monoclonal anti-pro caspase-1 (ab179515, 1:1,000; Abcam; 45 kDa, 42 kDa); rabbit monoclonal anti- TGF β1 (ab215715, 1:1000; Abcam 44 kDa); mouse monoclonal anti-β-actin (60008-1-lg, 1:5,000, Proteintech Group; 42 kDa); rabbit polyclonal anti-CD86 (ab220188, 1 µg/mL, Abcam; 38 kDa); rabbit polyclonal anti-Arg 1 (16001-1-AP, 1:20000; Proteintech 36 kDa); rabbit monoclonal anti-pro IL-1β antibody (ab216995, 1:1000; Abcam; 29 kDa, 35 kDa); rabbit monoclonal anti-IL-10 (ab133575, 1:1000; Abcam; 18 kDa). After washing three times with TBST, membranes were incubated with IgG-horseradish peroxidase–conjugated secondary antibodies (1:10000, Canlife) at room temperature for 2 h. After washing in TBST immunoreactivity was detected using enhanced chemiluminescence and proteins were quantified using Quantity ONE (Bio-Rad, Hercules, CA, USA) software. β-actin was used as an internal control.

2.3 Enzyme-linked immunoassay (ELISA) of LXA₄, tumor necrosis factor-α (TNF- α), and IL-1β

LXA₄ in articular fluid of patients was analyzed using ELISA kits (Shanghai Kemin Biotechnology Co. Ltd., Shanghai, China) according to manufacturer instructions. TNF-α and IL-1β levels in the knee intra-articular lavage fluid (IALF) and serum were also determined using ELISA kits (Tongwei) according to manufacturer instructions. Protein levels in IALF were measured to ensure that the dilution ratio was equal.

2.4 Isolation and culture of synovial macrophages and chondrocytes from patients with OA

Synovium and articular cartilage at the site of the lesion were collected from two males and four females who underwent total knee replacement, aged 58–71 years. Synovial macrophages and chondrocytes were derived from the synovium and articular cartilage of knee joints. Macrophages and chondrocytes we isolated from patients with a Kellgren-Lawrence grade of 4, ensuring consistent cellular responses to CTS and/or LXA₄ [31, 32]. Tissues were collected into sterile PBS.

Fat and connective tissue were removed and tissue was digested in 1 mg/mL collagenase (Sigma Aldrich, St. Louis, MO, USA) for 2–3 h at 37°C. Cells were separated from undigested tissue using a 70 µm cell strainer (BD, Franklin Lakes, NJ, USA) and cultured in 25 cm² cell culture flasks in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco BRL, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco BRL) and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin, and 0.1 mg/mL fungizone) in a humid 5% CO₂ atmosphere at 37°C. The cells were transferred into 25 cm² culture flasks at a density of 5 × 10⁶ cells/flask. At approximately 80–90% confluence, cells were detached using 0.25% trypsin and split into a 1:3 ratio. For all experiments, first and second cell passages were used. Synovial macrophages were identified by immunofluorescence staining with anti-CD 14 antibody (ab183322, 1:100; Abcam) (Fig. 2A, left).

Articular cartilage sections were sequentially digested in pronase (2 mg/mL) and collagenase D (1 mg/mL; Roche, Basel, Switzerland). Cells were cultured as previously described [33] and identified by immunohistochemical staining with anti-COLII antibody (ab34712, 1:100; Abcam) (Fig. 2A, right).

2.5 Exposure of synovial macrophages to CTS and LXA₄

Synovial macrophages were grown on fibronectin-coated Bioflex six-well culture plates (Flexcell International, Hillsborough, NC, USA) to 80–90% confluence. CTS experiments were conducted using the FX-5000 Flexcell system (Flexcell International, McKeesport, PA, USA). To provide uniform radial and circumferential strain on the membrane, plates were placed in a loading station (in a 5% CO₂ incubator), such that when a vacuum was applied, the membrane deformed across the post face, creating a uniform bi-axial strain. Synovial macrophages were subjected to CTS (10%, 0.5 Hz) of different durations (0, 6, 12, 24, 48, 72, or 96 h), and LXA₄ concentrations (0, 0.5, 1, 2, or 4 nM) for 24 h, following the methods of our previous study [11]. After these steps, we selected the optimal conditions for further study (Fig. 2B).

2.6 Determination of reactive oxygen species (ROS)

Synovial macrophages were exposed to CTS (10%, 0.5 Hz, 24 h) or LXA₄ (1 nM), and chondrocytes were harvested at 24 h. ROS production was measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (S0033, Beyotime), which is directly oxidized by ROS (e.g. superoxide ion, hydrogen peroxide, hydroxyl ions). Chondrocytes were incubated with 10 µM DCFH-DA for 45 min at 37°C in the dark. After this, they were washed three times in PBS. Fluorescence was detected by fluorescent microscopy and measured using the BD FACSCalibur instrument (BD Bioscience; 488 nm excitation, 525 nm emission).

2.7 Co-culture

Synovial macrophages were placed in the bottom compartment of a transwell system, and chondrocytes placed in the top. Cells were cultured with 0.4 µm inserts. Both cell types were mixed at a ratio of 3:1. Growth conditions were selected as previously described. Cell viability was > 95% before study commencement. Experiments were repeated at least three times with cells from different donors (one patient per experimental trial). Appropriate LXA₄ concentrations (1 nM) and CTS conditions (10%, 0.5 Hz, 24 h) were applied to synovial macrophages. After 24 h incubation, chondrocytes were collected for further study.

2.8 Immunofluorescence of chondrocytes

After washing in PBS and fixing in 4% paraformaldehyde for 20 min at room temperature, chondrocytes were permeabilized with 0.5% Triton X-100 for 30 min and incubated with blocking solution (5% BSA) for 30 min at room temperature. Rabbit polyclonal anti-NF-κB p65 antibody (AB21014, 1:50; Abcam) was then added, and cells were incubated overnight at 4 ºC. Cells were then stained with Alexa Fluor® 488 conjugated anti-rabbit antibody for 60 min at room temperature in the dark. Cytoskeleton structures were stained with phalloidin for 60 min at 37 ºC. Nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) for 2 min. After washing, cells adhering to Bioflex membranes were mounted in PBS with 20% glycerol. Chondrocytes were observed under a confocal microscope (Olympus, Tokyo, Japan).

2.9 Transmission electron microscopy (TEM)

After washing with PBS, the chondrocytes were harvested and centrifuged for 2 min at 2500 rpm before being fixed in cold 2.5% glutaraldehyde for 30 min. Samples were fixed again in 1% OsO₄ for 1 h, dehydrated, and embedded in epoxy resin. Ultrathin tissue sections (60 nm) were stained with uranyl acetate and lead citrate. The sections were examined via TEM (Hitachi 800, Tokyo, Japan) to observe cellular morphology.

2.10 Study animals

This study was approved by the ethics committee of Shengjing Hospital of China Medical University (No. 2017PS237K). Forty male Sprague-Dawley rats (230 g ± 10 g; specific pathogen–free) were obtained from HFK Bioscience Co. Ltd. (Beijing, China). Rat maintenance and care followed committee guidelines as previously described [12, 29]. Rats were adapted to laboratory conditions for one week prior to experimental procedures. All rats were familiarized with the ZH-PT treadmill exercise machine (Zhongshidichuang Science & Technology Development Co. Ltd., Beijing, China) for one week at 10 m/min for 10 min/day to reduce stress. All rats successfully adapted to the treadmill exercise [13]

2.11 OA model and treadmill protocols

After adaption to treadmill exercises, rats were randomly divided into four study groups (n = 10). Prior to treatment, all rats were anesthetized with 1.5% pentobarbital sodium (0.2 mL/100 g, intraperitoneal injection). The treatments were as follows: 1) Control group (CG), intra-articular injection of 50 µL sterile saline per cavity; 2) OA group (OAG), knee joint inflammation induced by intra-articular injection of MIA (1 mg per cavity in 50 µL sterile saline) through the lateral part of the inferior patellar ligament and into the bilateral knee joint cavity; 3) OAE, OAG conditions with moderate treadmill exercise (19.3 m/min with a 5° inclination, 60 min/day, 5 d/week for four weeks with appropriate photo-, acoustic-, and electric stimulation); 4) OAE + BOC-2, OAE conditions plus BOC-2 (an antagonist of LXA₄; Absin, Shanghai, China). BOC-2 (50 µg/kg) was administered by intraperitoneal injection before every treadmill exercise (10) (Fig. 5A). Because we demonstrated in previous studies that moderate exercise intensity showed an optimal therapeutic effect on osteoarthritis, we did not include low- or high-intensity exercise in this study [11, 34].

2.12 Sampling, tissue preparation, and histology

All rats were euthanized by cervical dislocation. The left knee joints were dissected and fixed in 4% paraformaldehyde solution. Skin and muscles along the patellar ligament were removed to open the capsula articularis, with care to avoid damage to the cartilage and synovium. Next, using a scalpel or rongeur, articular cartilage specimens were removed from the weight-bearing areas of the right femur and tibia condyles to analyze through western blot and quantitative PCR (qPCR). Synovium specimens were also removed. All tissue was stored at -80°C. Left knee joint tissue samples were stored in 4% paraformaldehyde for 7 d, then washed in water for 5 h and transferred to a 20% EDTA solution (Jianglai Reagent Co., Ltd, Shanghai, China) to decalcify for 21 d. The solution was changed every 3 d. Decalcified samples were dehydrated in an ethanol gradient series and embedded in paraffin. Serial 5 µm sagittal sections were removed from tibiofemoral joints for histological examination. Sections were stained in hematoxylin & eosin (HE) and toluidine blue to observe cartilage. Next, sections were observed with a ScanScope (APERIO CS2, Leica). Injury to the articular cartilage in the femur and tibia was assessed using the Modified Mankin score [35] on a 0–14 point scale, and using the Osteoarthritis Research Society International (OARSI) scale on a 0–24 point scale [36]. Because both tibial and femoral cartilage of left knee joints was evaluated, the maximum Mankin score was 28 and the maximum OARSI score was 48. In addition, we used microscopic scoring of synovium [37] to evaluate the histological changes of synovium. Two experienced observers (Yue Yang and Peng Shen) blindly scored samples.

2.13 Gross imaging and computed tomography

Knee joint images were captured by stereomicroscope (XTL-165-CB; Phenix), SkyScan 1276-CT (Bruker, Kontich, Belgium), and NRecon version 1.6 software (Bruker). Joint space narrowing, joint surface calcification, and articular cartilage damage were assessed. The degree of osteoarthritis was determined based on macro scores of surface roughness and erosion, according to the gross imaging scoring system described in previous literature; both the tibial and femoral joints were evaluated, with a maximum score of 10 [36, 38]. Based on the OsteoArthritis Computed Tomography-Score (OACT-Score), joint space narrowing, osteophytosis, and subchondral cysts were used to quantify the degree of osteoarthritis detected by computed tomography. The tibiofemoral joint score was evaluated based on a range from 0 to 7 [39].

2.14 Immunohistochemistry

After tissue section deparaffinization and rehydration, serial sections were stained for collagen II (COLII) and matrix metalloproteinase (MMP)-13 using a two-step method following manufacturer instructions. Sections were incubated overnight at 4°C with rabbit polyclonal anti-COLII antibody (ab34712, 1:100; Abcam) and rabbit polyclonal anti-MMP-13 antibody (ab219620, 1:50; Abcam). The slides were then washed three times in PBS, incubated for 20 min at 37°C with an anti-mouse/rabbit IgG detection system (PV-9000, Zhongshan Goldenbridge Biotechnology Co., China), and stained with diaminobenzidine (DAB). Nuclei were counterstained with hematoxylin for 5 min. The optical density of stained slides was measured using image analysis software (Nikon H600L Microscope and Image Analysis System, Japan). COLII was quantified by relative intensity. MMP-13 was quantified by the percentage of positive cells.

2.15 Quantitative real-time polymerase chain reaction (qPCR)

The qPCR protocol was conducted according to a previous study [22]. Expression levels were calculated using the 2^-△△CT method [40], with β-actin as the reference gene. Primer pair sequences were specific for rat CD86 (F: 5′-TCCTCCAGCAGTGGGAAACA-3′; R: 5′-TTTGTAGGTTTCGGGTATCCTTGC-3′), Arg1 (F: 5′-TTGATGTTGATGGACTGGAC-3′; R: 5′-TCTCTGGCTTATGATTACCTTC-3′), and β-actin (F: 5′-GGAGATTACTGCCCTGGCTCCTA-3′; R: 5′-GACTCATCGTACTCCTGCTTGCTG-3′).

2.16 Statistical analysis

Data were analyzed using SPSS statistical software version 16 (SSPS, Inc., Chicago, IL, USA) and expressed as means with 95% confidence intervals (CIs). Shapiro-Wilk's and Levene's tests were used to evaluate data normality and homogeneity, respectively. For variables that exhibited a normal distribution, independent sample t-tests and one-way ANOVA were used for statistical significance. A P value < 0.05 was accepted as statistically significant.

3.1 LXA₄ is downregulated in articular fluid and CD 86/Arg 1 is upregulated in synovial tissue of patients with increasing Kellgren-Lawrence grade.

To evaluate the potential role of LXA₄ and synovial macrophage M1/M2 subtype polarization in OA, we first detected the articular fluid and synovium samples divided into different Kellgren-Lawrence grades from 40 patients (Table 1). Western blots were evaluated for expression differences in CD86 (M1 macrophage marker) and Arg1 (M2 macrophage marker) [41] in patients with different of Kellgren-Lawrence grades. ELISA was evaluated for articular fluid expression differences for LXA₄ in patients with different of Kellgren-Lawrence grades. We observed that CD 86/Arg 1 was upregulated in the synovial tissue (Fig. 1B), whereas LXA₄ was downregulated in the articular fluid (Fig. 1C) of patients with increasing Kellgren-Lawrence grade.

3.2 CTS (10%, 0.5 Hz, 24 h) and 1 nM LXA₄ promote synovial macrophage M2 subtype polarization.

To identify the best condition for further study, we treated synovial macrophage with different durations of CTS (10%, 0.5 Hz) and concentrations of LXA₄ (Fig. 2B). At 24 and 48 h CTS, the percentages of CD-14-positive synovial macrophages and COL II-positive chondrocytes in the cultured cells exceeded 95% (Fig. 2A). We observed a significant decrease in CD86 and increase in Arg 1 levels, but no significant changes were observed between 24 and 48 h CTS (Fig. 2C). Similarly, we observed a significant decrease in CD86 and increase in Arg1 expression with different LXA₄ concentrations, with significant changes between 0.5 nM and 1 nM LXA₄. However, no significant change was seen in CD86 and Arg1 levels at 1, 2, and 4 nM LXA₄ (Fig. 2D). Thus, we selected 24 h CTS (10%, 0.5 Hz) and 1 nM LXA₄ for subsequent analyses.

As shown in Fig. 3A, MMP-13 and CD86 levels were significantly decreased, and IL-10, TGF-β1, VEGF receptor 1, and Arg 1 levels significantly increased in CTS (24 h, 10%, 0.5 Hz)- or LXA₄ (1 nM) -induced synovial macrophages when compared to the control group. Moreover, combined CTS and LXA₄ (1 nM) exhibited synergism on synovial macrophages (Fig. 3A). ROS production in synovial macrophages was assessed by fluorescence microscopy and flow cytometry (Fig. 3B). CTS and LXA₄ treatment resulted in significantly reduced ROS levels compared to the control (relative ROS intensity: Control = 1.00, 95% CI 0.94–1.06; CTS (24 h) = 0.88, 95% CI 0.82–0.95; LXA₄ (1 nM) = 0.64, 95% CI 0.61–0.68; CTS + LXA₄ = 0.45, 95% CI 0.38–0.51.). Additionally, synovial macrophages treated with CTS and LXA₄ exhibited significantly reduced apoptosis levels compared to those of the control (Fig. 3C).

3.3 CTS (10%, 0.5 Hz, 24 h) and 1 nM LXA₄ inhibit chondrocyte pyroptosis via synovial macrophage M2 subtype polarization.

We next explored the effects of CTS and LXA₄ on chondrocytes in co-culture with synovial macrophages. Pro-IL-1β, pro-caspase-1, caspase-1, and NLRP3 levels in chondrocytes decreased under CTS and LXA₄ treatment of synovial macrophage (Fig. 4B).

A considerable accumulation of NF-κB p65 occurred in chondrocyte nuclei in the control group. CTS and LXA₄ suppressed this nuclear translocation under co-culture with synovial macrophages (Fig. 4C). As shown in Fig. 4D, TEM images show that chondrocytes from patients with OA exhibited a tendency for pyroptosis, including severe cytoplasmic edema, swelling of the cell membrane, decreased overall electron density of the cell matrix, and serious cavitation. However, LXA₄ + CTS treatment of synovial macrophages inhibited the process of pyroptosis in the co-culture.

3.4 Treadmill exercise suppresses OA-related degeneration in MIA-induced rats

40 male SD rats were divided into four groups (Fig. 5A). Gross imaging macroscopic score (Fig. 5B), histological assessments (Mankin and OARSI scores)(Fig. 5C and 5D), and computed tomography (OACT-Score) (Fig. 5E), histological changes of synovium (microscopic score) (Fig. 5F) indicated that cartilage and synovium damage in the OAG group was more severe than that in the CG group. OAE treatment appeared to exert therapeutic effects on tibiofemoral joint cartilage when compared with that of the OAG group. Therapeutic effects in the OAE group were suppressed by BOC-2 treatment (Fig. 5C, 5F). Immunohistochemical staining (COL II and MMP-13) revealed similar Mankin and OARSI scores (Fig. 5D).

TNF-α and IL-1β serum concentrations in the OAG were higher than those in the CG. The OAE group exhibited decreased TNF-α and IL-1β serum concentrations when compared with the OAG. However, the decrease in TNF-α and IL-1β concentrations was attenuated by BOC-2 treatment. Changes in TNF-α and IL-1β concentrations in the IALF were similar to those in the serum levels. (Fig. 6B) Our data showed that the decrease of CD86 and the increase of Arg1 in synovial tissue were similar between the OAE group and the OAG. However, OAE effects were suppressed by BOC-2 (Fig. 6C). The relative expression levels of CD86 and Arg1 in synovial tissue as analyzed by qPCR were similar to those suggested by the western blot data (Fig. 6D).

In this study, we evaluated the effects of LXA₄ on chondrocyte pyroptosis via synovial macrophage M2 subtype polarization and identified the mechanisms by which it supports exercise therapy for OA.

Many studies have emphasized the importance of exercise therapy for OA [25, 26, 42]. However, most studies have focused on single cells, such as chondrocytes, synovial fibroblasts, or bone cells [29, 30], and little is known about multi-cell co-culture in organisms. In our previous study, we demonstrated that LXA₄ played an important role in exercise therapy for OA [11, 12] and that moderate-intensity exercise had the best therapeutic effect [34, 43]. Furthermore, we previously demonstrated that exercise could act on cartilage through mechanical stress [33] and altering the microenvironment around cartilage, such as cytokine LXA₄ in synovial fluid and synovial macrophages [44]. Therefore, it was important to understand mechanisms underlying mechanical stimulation and LXA₄ function on synovial macrophages, and similarly, understand the molecular communications between synovial macrophages and chondrocytes.

In our preliminary clinical experiment of the present study, we found that LXA₄ levels in articular fluid decreased and CD86/ Arg1 levels in synovial tissue increased with increasing Kellgren-Lawrence grade. In view of this finding, we predicted that LXA₄ may affect the pathogenesis of osteoarthritis through macrophage polarization. Next, we verified our conjecture through in vitro experiments. According to previous experiments, we identified LXA₄ as an exercise metabolite and an important factor in the exercise treatment of OA [11, 12], and we used CTS to simulate the microenvironment of exercise on chondrocytes and macrophages [33, 45]. We found that CTS supported by LXA₄ treatment inhibited chondrocyte pyroptosis via synovial macrophage M2 subtype polarization. In our in vivo experiments, we established treadmill exercise protocol to validate the data collected in vitro. Our data showed that CD86 levels decreased and Arg1 levels increased in a manner consistent with that seen in the human clinical data. Based on these results, we concluded that LXA₄ administration can support CTS therapy via synovial macrophage M2 subtype polarization, offering a new potential treatment for osteoarthritis.

Intra-articular injection of MIA in rats, which caused histological changes, such as cartilage surface erosion, matrix loss, and synovitis, is considered an accurate model of OA [38]. Our findings showed that therapeutic effects of moderate-intensity treadmill exercise were suppressed by BOC-2, an antagonist of the LXA₄ receptor which confirmed the results of our previous study [11]. The M2 polarization of macrophages is considered significant in treating various diseases. Although the positive effects of M2 polarization on synovial macrophages in OA have been reported [46], molecular mechanistic outputs from exercise therapy on synovial macrophages are unclear. We studied CD86, a marker of M1 macrophage, and Arg1, a marker of M2 macrophage [41] expression in the synovium of different experimental groups. Both western blotting and qPCR data showed decreased CD86 and increased Arg1 expression in synovium in the OAE group when compared with that of the OAG. These effects were suppressed by BOC-2 treatment.

The mechanisms underlying the conversion of biomechanical signals into intercellular events have become a major focus in OA research. Although synovial macrophages undergo mechanical stimulation during exercise therapy, little is known about how this stimulation regulates the immune response. Accordingly, mechanical stress may play a role in macrophage recruitment or polarization [47]. We showed that CTS initiated an inflammatory response similar to that induced in the joints during exercise, via the M2 polarization of synovial macrophages. Our data (Fig. 5C and 5D) indicated that CTS (10%, 0.5 Hz) for 24 h and LXA₄ administration (1 nM) decreased CD86 expression and increased Arg1 expression, causing M2 polarization of synovial macrophages. However, CTS (10%, 0.5 Hz) over 48 h exerted the opposite effects. Thus, we selected CTS (10%, 0.5Hz, 24 h) and LXA₄ (1 nM) for subsequent experiments. Our results showed these parameters significantly decreased ROS, MMP-13, and CD86 expression in synovial macrophages. Taken together, these findings demonstrated that CTS (10%, 0.5Hz, 24 h) and LXA₄ (1 nM) stimulated multiple chemokines, such as IL-10, TGF-β1, VEGF receptor 1 and Arg1, to facilitate macrophage M2 polarization. Equally, LXA4 and CTS stimulation also reduced apoptosis in synovial macrophages via M2 polarization. There is much evidence supporting physical and mechanical regulation of macrophage phenotype polarization and function.

Biophysical cues can function synergistically with soluble factors to mediate chondrocyte behavior [48]. The links between force distribution, tissue damage, morphogen gradients, and cell responses have garnered substantial research interest. We discovered the co-culture of synovial macrophages and chondrocytes by transwell chamber. Mechanical stimulation can induce alternative activation of macrophages, which have functional roles in chondrocyte regeneration. We further revealed that IL-10, TGF β1, VEGF receptor 1, and Arg 1, which are produced by M2 subtype macrophages, were functional mediators in OA exercise therapy. Our work suggested that CTS and LXA₄ are parameters of synovial macrophage M1/M2 polarization, and revealed an unexpected complexity in the co-culture of synovial macrophages and chondrocytes. Moderate CTS and LXA₄ treatment showed a synergistic effect on promoting M2 polarization in synovial macrophages, which inhibited the nuclear translocation of NF-κB p65, forming NLRP3 in chondrocytes.

Our previous studies showed that LXA₄ was rapidly produced and metabolized [11, 12], suggesting that LXA₄ plays an important role in exercise therapy for OA. Because there is no LXA₄ receptor (ALX/FPR2) in chondrocytes, LXA₄ cannot directly act on these cells. In the knee joint, LXA₄ combines with synovial macrophages, fibroblasts, and neutrophils to reduce chronic inflammation, generating therapeutic effects for OA [49, 50]. The effects of exercise on OA may be divided into systemic effects on the whole body and local effects on the knee joint. Appropriate exercise produces a hypoxic environment in blood vessels which promote platelet aggregation, lipoxygenase activation, and LXA₄ generation [9]. It is proposed that LXA₄ not only prevents chronic inflammation and limits neutrophil infiltration, but also relieves pain, reduces pain sensitivity, and inhibits peripheral nerve fiber sensitivity in systemic system of whole body [51, 52]. Additionally, appropriate exercise may generate synovial fluid and lipids in the infrapatellar fat pad, producing LXA₄ in the knee joint. LXA₄ may act on synovial macrophages to promote M2 polarization, and act on synovial fibroblasts to reduce MMPs and a disintegrin and metalloproteinase with thrombospondin motifs [11], thus relieving synovitis. Thus, our study identified a strategy whereby chondrocyte regeneration via synovial macrophage M2 subtype polarization can be encouraged via exercise therapy and LXA₄ treatment (Fig. 7).

Our study has several limitations that should be noted. First, due to ethical limitations, we were able to sample cartilage from OA patients with different Kellgren-Lawrence grades. Secondly, the MIA-induced OA rat model may not have fully represented all aspects of OA. This model involved measurements as a single time point, presenting a chance of random effects. We plan to study other OA models and analyze them in combination with the MIA model. Thirdly, we used BOC-2 as an antagonist of the LXA₄ receptor. It is well known that chemical inhibitors can produce off-target effects. Our research group has begun to conduct gene knockdown experiments to further study this problem. Fourth, in our previous studies, we demonstrated that moderate-intensity treadmill exercise alleviated MIA-induced OA in rats [11, 12], and that moderate CTS (10%, 0.5 Hz, 4 hr) ameliorated the degeneration of cartilage and chondrocytes [33, 45]. Because the main effect of exercise on articular cartilage is mechanical stimulation [53], we aim to study the similarities between CTS and treadmill exercise in animals in subsequent experiments, but the moderate CTS cannot be directly transformed into activities or common exercises OA patients may participate in. Finally, western blotting of whole synovial tissue is a very crude measure of CD86/Arg1, which could be expressed by numerous cell types in addition to macrophages and be influenced by other cells present in the synovial tissue. In this paper, we focused on the influence of synovial macrophages, and aim to study the role of other cells in this process in future experiments.

In summary, our work characterized mechanical stimulation and M2 polarization of the synovial macrophage-chondrocyte regeneration axis in response to LXA₄, and demonstrated how organs can regenerate in response to exercise therapy. We used treadmill exercises of MIA-induced OA rats, CTS and LXA₄ responses on synovial macrophages, and the co-culture of synovial macrophages and chondrocytes to explore their effects on OA. Our findings indicated that CTS and LXA₄ inhibited the nuclear translocation of NF-κB p65 and formation of NLRP3 in chondrocytes via M2 polarization of synovial macrophages. Our results identified the effects of mechanical stress and LXA₄ on synovial macrophages, and provided evidence of molecular signaling between synovial macrophages and chondrocytes during exercise therapy.

Statements and Declarations

FUNDING

This study was supported by the National Natural Science Foundation of China (Grant No. 82102613), and the Youth Science and Technology Talent Project of Liaoning Provincial Education Department (Grant No. QNZR2020014), and the 345 Talent Project of Shengjing Hospital of China Medical University.

CONFLICTS OF INTEREST

The authors declare that they have no competing interests.

AUTHORS’ CONTRIBUTIONS

Yue Yang and Peng Shen and Shuangshuo Jia contributed to study conception and design. Yue Yang, Yang Wang, Xiaonan Zhou, and Delong Zhang conducted treadmill exercise experiments. Yue Yang, Shuangshuo Jia, Yang Wang, Zhuangzhuang Jin, Ziyuan Wang, and Donghao Liu conducted cell culture and cyclic tensile strain experiments. All authors contributed to data acquisition and analysis. Yue Yang, Yang Wang, Lunhao Bai, Peng Shen and Shuangshuo Jia conducted statistical analysis and prepared the manuscript. Yue Yang, Peng Shen and Shuangshuo Jia approved of the final draft. Peng Shen and Shuangshuo Jia are co-first authors of the article. All authors contributed to critical manuscript revision for intellectual content and approved the manuscript for publication.

data availability statement

All data generated or analysed during this study are included in this published article.

ETHICS STATEMENT

This study was carried out under the approval and in accordance with the guidelines of the Ethics Committee of Shengjing Hospital of China Medical University.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

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Table 1. Baseline characteristics of patients

Basic information		Kellgren-Lawrence grade 0	Kellgren-Lawrence grade 1	Kellgren-Lawrence grade 2	Kellgren-Lawrence grade 3	Kellgren-Lawrence grade 4	Statistic	P value
Biological Sex	Male	13	15	12	15	12	c²=5.501	0.240
	Female	15	18	19	28	37
Age (years)	＜65	28	33	16	8	5	c²=108.99	<0.001
	≥ 65	0	0	15	35	44
Obesity gradation	Under weight^a	2	1	0	1	2	c²=52.113	<0.001
	Normal weight	22	27	21	24	10
	Overweight	4	4	8	15	23
	Obesity	0	1	2	3	14

^aUnderweight: BMI < 18.5; Normal weight: 18.5 ≤ BMI < 24.9; Overweight: 25 ≤ BMI < 30; Obesity: BMI ≥ 30.

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Mechanical Stress Protects Against Chondrocyte Pyroptosis through Lipoxin A4 via Synovial Macrophage M2 Subtype Polarization in an Osteoarthritis Model

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Human OA synovium, articular fluid, and clinicopathological features

2.2 Western blotting

2.3 Enzyme-linked immunoassay (ELISA) of LXA₄, tumor necrosis factor-α (TNF- α), and IL-1β

2.4 Isolation and culture of synovial macrophages and chondrocytes from patients with OA

2.5 Exposure of synovial macrophages to CTS and LXA₄

2.6 Determination of reactive oxygen species (ROS)

2.7 Co-culture

2.8 Immunofluorescence of chondrocytes

2.9 Transmission electron microscopy (TEM)

2.10 Study animals

3. Results

3.4 Treadmill exercise suppresses OA-related degeneration in MIA-induced rats

4. Discussion

Declarations

References

Table 1

Supplementary Files

Status:

Version 1

Mechanical Stress Protects Against Chondrocyte Pyroptosis through Lipoxin A4 via Synovial Macrophage M2 Subtype Polarization in an Osteoarthritis Model

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Human OA synovium, articular fluid, and clinicopathological features

2.2 Western blotting

2.3 Enzyme-linked immunoassay (ELISA) of LXA4, tumor necrosis factor-α (TNF- α), and IL-1β

2.4 Isolation and culture of synovial macrophages and chondrocytes from patients with OA

2.5 Exposure of synovial macrophages to CTS and LXA4

2.6 Determination of reactive oxygen species (ROS)

2.7 Co-culture

2.8 Immunofluorescence of chondrocytes

2.9 Transmission electron microscopy (TEM)

2.10 Study animals

3. Results

3.4 Treadmill exercise suppresses OA-related degeneration in MIA-induced rats

4. Discussion

Declarations

References

Table 1

Supplementary Files

Status:

Version 1

2.3 Enzyme-linked immunoassay (ELISA) of LXA₄, tumor necrosis factor-α (TNF- α), and IL-1β

2.5 Exposure of synovial macrophages to CTS and LXA₄