Knockdown adiponectin receptor 1 expression in synovial tissue alleviate the progression of collagen-induced arthritis

Background : Previous studies have shown that adiponectin (APN) is involved in the pathogenesis of rheumatoid arthritis (RA). The proinflammatory effect of APN is mainly mediated adiponectin receptor 1 (AdipoR1). The high expression of AdipoR1 have been suggested in RA synovial tissue. This study was aimed to investigate the effects of AdipoR1 in inflammation and bone erosion in collagen-induced arthritis (CIA) mice, and to further explore the underlying mechanisms. Methods : The expression of APN and AdipoR1 in synovial tissue of RA and osteoarthritis (OA) patient was tested by qPCR and western blot. RA synovial fibroblasts (RASFs) were stimulated with APN, IL-6 or TNF-α respectively. The expression of AdipoR1 on RASFs were tested by flow cytometry. To prove the pathogenic role, AdipoR1 was silenced in a human rheumatoid arthritis synovial fibroblast cell line (MH7A) and local joint of CIA mice by specific short hairpin RNAs (shRNAs) using a lentiviral delivery system. The levels of proliferation, apoptosis and inflammatory factors on MH7A were assessed in vitro . Local AdipoR1 knockdown on CIA mice were further estimated by arthritis clinical scores, inflammatory cytokine expression, micro-CT, H&E staining and receptor activator of nuclear factor к B ligand (RANKL) / osteoprotegerin (OPG) in vivo . Results : We found that the levels of APN and AdipoR1 expression were significantly higher in RASFs and the expression of AdipoR1 was upregulated by APN in RASFs. Silencing AdipoR1 could effectively reduce lipopolysaccharides (LPS) induced proliferation of MH7A cells, promote their apoptosis, and reduce the release of inflammatory factors. In CIA mice, local silencing AdipoR1 in arthritis markedly reduced joint inflammation and alleviated bone erosion and osteoporosis in vivo . Furthermore, local silencing AdipoR1 inhibited receptor activator of nuclear factor к B ligand (RANKL) expression and decreased RANKL / osteoprotegerin (OPG) ratio in knees and ankles of CIA mice. Conclusions : This study suggests that AdipoR1 plays a key role in the development of RA and silencing AdipoR1 might be a new target for the clinical treatment of RA. In present study, we intended to reveal relationships among APN, AdipoR1 and the inflammatory factors in synovial tissue of RA patients. Lentivirus-mediated technology was used to detect whether AdipoR1 knockdown could decrease the inflammatory responses in MH7A in vitro and delay the disease progression in collagen-induced arthritis (CIA) mice in vivo . This study aims to get insights into the pathogenic mechanism of AdipoR1 and provide the potential therapeutic targets for the treatments of RA.


Background
Rheumatoid arthritis (RA) is a chronic inflammatory disorder and irreversible destructive arthropathy. The inflammation is mainly driven by the overproduction of pivotal proinflammatory cytokines, including tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 [1]. These cytokines mediate long-term cartilage degradation and bone erosion, resulting in joint pain and dysfunction [2]. Notably, evidence suggests that rheumatoid arthritis synovial fibroblasts (RASFs) are key integrators of inflammatory signals in the inflamed rheumatoid synovium [3]. One of the most significant features of RASFs is destroying cartilage, and RASFs are considered as the primary cells responsible for marginal cartilage destruction by secretion of matrix-degrading enzymes and inflammatory cytokines [4].
Accumulating evidences demonstrated that APN could promote the secretion of IL-1β, IL-6, vascular cell adhesion molecule (VCAM)-1 and exacerbate erosive joint destruction in RA [5][6][7]. Recently, our group found that APN induced joint destruction through promoting osteopontin (OPN) expression in RASFs [8], and the overexpression of APN could promote the differentiation of T helper cell 17 (Th17 cell) and enhance the expression of RANKL thus exacerbating symptoms of collagen-induced arthritis (CIA) model [9]. It indicated that APN might be involved in the pathogenesis of RA. There are two main types of APN receptors, AdipoR1 and AdipoR2, that mediated its biological effects [10]. We found a high concentrations of adiponectin (APN) and the expression levels of adiponectin receptor 1 (AdipoR1) in RA patients [11], indicating that AdipoR1 might be the effector receptor.
Considering the dynamic effects of APN in vivo, directly knockdown its effector receptors might be an ideal strategy to intervene the local inflammation in the joint.
In present study, we intended to reveal relationships among APN, AdipoR1 and the inflammatory factors in synovial tissue of RA patients. Lentivirus-mediated technology was used to detect whether AdipoR1 knockdown could decrease the inflammatory responses in MH7A in vitro and delay the disease progression in collagen-induced arthritis (CIA) mice in vivo. This study aims to get insights into the pathogenic mechanism of AdipoR1 and provide the potential therapeutic targets for the treatments of RA.

Patients
RA and Osteoarthritis (OA) patients and the healthy controls (HC) were recruited randomly from the First Affiliated Hospital of Nanjing Medical University as our previously described [8]. In short, the synovial tissues were obtained from 3 patients with RA and 3 patients with OA that fulfilled the American College of Rheumatology Criteria for RA or OA.
This study was approved by the Ethics Committee at the First Affiliated Hospital of Nanjing Medical University, and all donors signed informed consent forms.

RNA extraction and Quantitative real-time PCR
RNA samples were isolated from collected cells using Trizol reagent (Invitrogen). RNA was converted to cDNA using Prime Script TM RT regent Kit (Takara) according to the manufacturer's instructions. PCR amplification and analysis were achieved using an ABI 7900 system (ABI), and the cycling parameters were performed an initial denaturation step at 95℃ for 10 minutes, followed by 40 cycles at 95℃ for 15 seconds and extension 60℃ for 1 minute. PCR primers used for real-time PCR were presented in Supplement Table S1.
Relative gene expression was determined by 2 -△△Ct method.

Protein extraction and western blotting analysis
Cells were solubilized with RIPA lysis buffer (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions and protein concentration was detected using a BCA kit (Thermo Fisher Scientific, Inc.). Approximately 50 μg of protein from each sample was separated by SDS-PAGE gels and electrophoretically transferred to PVDF membrane (Bio-Rad, USA). Then, the PVDF membrane were blocked with 5% skim fat dry milk in Tris-buffered saline (TBS) with 0.1% Tween-20 for 2 h at room temperature to block the non-specific sites on blots. The membranes were incubated with primary antibodies including

Cell counting kit 8 (CCK-8) assay
MH7A cells were plated in 96-well plates with the density of 5,000 cells/well for 72 h transfected with shAdipoR1 or corresponding controls (shNC) for 24 h. Cells were treated with lipopolysaccharide (LPS) at room temperature to construct a model of rheumatoid arthritis. The control group were treated with normal RPMI 1640 medium. CCK-8 solution (Invitrogen; Thermo Fisher Scientific, Inc.) was added to the wells after 12h, 24h, 48h, separately and samples were incubated at 37˚C for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader.

Flow cytometric analysis of apoptosis
Cells were seeded in the 6-well plate at a concentration of 1×10 6 /ml, treated with LPS and were harvested after transfection with AdipoR1 for 48 h. Cells were washed twice with cold PBS and resuspended in 100 μl binding buffer. Annexin V and propidium iodide (PI) double-staining was carried out using an Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, San Jose, CA, USA) according to manufacturer's instructions. Apoptosis analysis was performed using the BD LSRII Flow Cytometer System.

Induction of collagen-induced arthritis (CIA) mice and treatment
All animal experimental protocols were approved by the animal care and use committee of the First Affiliated Hospital of Nanjing Medical University and we confirmed that all methods were performed in accordance with the relevant guidelines and regulations. Male DBA/1 J mice (aged 8-12 weeks) were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Science. Experiments divided in four groups:NC, CIA mice, shAdipoR1 treated CIA mice and shNC treated CIA mice. CIA mice were induced as following described.
In Brief, 100 μg of bovine collagen type II (CII; Chondrex, Redmond, WA, USA) that was dissolved in 0.05 M acetic acid was emulsified with an equal volume of Freund's complete adjuvant (Difco, Detroit, MI, USA) and administered intradermally at the base of tail into DBA/1 J mice. On day 21 after the initial immunization, a booster immunization was administered intradermally near the primary injection site. Simultaneously, the lentivirus-mediated shAdipoR1(10 7 TU) was intraarticularly injected into the knee joints of shAdipoR1 treated CIA mice and the lentivirus-mediated shNC(10 7 TU) was injected into the knee joints of shNC treated CIA mice, and the other knees were treated with an equivalent volume of PBS as controls. Mice were examined by two blinded observers daily after the 2nd CII immunization for the signs of arthritis. The arthritis score was evaluated by grading each paw from 0 (no inflammation) to 4 based on erythema, swelling, and flexion of the joint and the clinical score was constructed by adding the scores from the 4 individual paws of each CIA mouse.

Haematoxylin & eosin (H&E) staining
The ankles or paws were removed from euthanized mice for histopathological examination after routine fixation, decalcification, and paraffin embedding of the tissue. The tissues were then sectioned into 3-μm slices, deparaffinized in xylene, rehydrated using a series of ethanol concentrations and stained with haematoxylin and eosin(H&E). The slides were evaluated for synovial hyperplasia, inflammatory infiltration pannus formation, and cartilage/subchondral bone destruction by two blinded observers. Each joint was scored from 0 to 4. Briefly, grade 0, no signs of inflammation; grade 1, mild inflammation with hyperplasia of the synovial lining without cartilage destruction; grades 2 to 4, increasing degrees of inflammatory cell infiltrate and cartilage/bone destruction.

Micro-computed tomography (Micro-CT) analysis
On day 45 post the 1st immunization, mice were sacrificed and knee joints and ankles were removed and fixed in 4% paraformaldehyde. Quantitative analysis of knee joints and ankles was performed using a Micro-CT Scan SkyScan1176S scanner at a resolution of 9 μ m.
Computed tomographic images of the knee joints and ankles of the mice in all four groups (n=3) were acquired. After scanning, cross-sectional slices were reconstructed.
Three-dimensional models of the knee joints and paws were performed to verify the bone destruction, using SkyScan CT Analyzer version 1.8. Cortical bone mineral density, bone mass, bone surface/volume ratio, trabecularbone mineral density, percent bone volume and structural model index were performed using scanner software (Skyscan CTAn).

Statistical analysis
For statistical evaluation, results are expressed as Mean ± SD. Student's t test was performed to assess the statistic difference between two groups. The statistic difference among three or more groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test. All results were considered statistically significant if the p value is < 0.05.

The expression of AdipoR1 was upregulated by APN in RASFs
Firstly, we detected the expression of APN and AdipoR1 in synovial tissue of RA patients. The result showed that the levels of APN and AdipoR1 mRNA expression were significantly higher in RA group, compared with OA group (Figure 1A, P＜0.05, n=3). These differences were also observed in protein levels (Figure 1B, P＜0.05, n=3). Furthermore, to find the reason of high expressed of AdipoR1 in RASF, primary RA synovial fibroblasts (RASFs) was stimulated with APN (10 μg/ml) or IL-6 (50 ng/ml) or TNF-α (50 ng/ml) respectively. Flow cytometry showed that the frequency of AdipoR1 was significantly higher in APN stimulated RASFs group (1.73±0.20 vs 1.00±0.07, P＜0.01). There was no significant difference among IL-6, TNF-α and NC groups ( Figure 1C). These results indicated that the expression of AdipoR1 was significantly higher in RASFs, and it was upregulated by the treatment of APN. AdipoR1 might function as a downstream signaling molecule of APN and play an important role in the micro-environmental changes of RA joints.

AdipoR1 knockdown prevented MH7A responses
To further determine the pathogenic role of AdipoR1 in RA, the expression of adipoR1 in MH7A was knocked down by shRNA/GFP lentivirus infection. Green fluorescent protein (GFP) expression in MH7A infected by the lentivirus vectors for 72h showed over 80% infection efficiency ( Figure S1A). The gene and protein expression levels also proved that AdipoR1 was successful knockdown (n=6, P＜0.01, Figure S1B and C). The result showed that the proliferation of MH7A was significantly decreased and the apoptosis of MH7A was increased after AdipoR1 knockdown ( P＜0.05, Figure 2A, B and C). In addition, AdipoR1 knockdown substantially suppressed the expression of IL-1β, IL-6 and TNF-α in MH7A induced by LPS ( Figure 2D). Therefore, these results suggested that APN-AdipoR1 pathway might be involved in RASFs proliferation, apoptosis, and pro-inflammatory release in RA.

AdipoR1 knockdown alleviated the progression of collagen-induced arthritis (CIA) mice
To further assess the effects of AdipoR1 on the process of inflammation in vivo, the lentivirus-mediated shAdipoR1 was injected into the knee joints of CIA mice on 21st day post the first immunization. AdipoR1 knockdown delayed onsets of arthritis and exhibited lower clinical scores CIA mice treated with shNC ( Figure 3A). Both the gene and protein expression of AdipoR1 in synovial tissue were significantly decreased in shAdipoR1 treated group  Figure 4A). Compared to shNC group, the cortical bone mineral density and bone volume were ascending, while the ratios of bone surface / volume were descending both in knee and ankle (Figure 4B, C). Besides, local inhibition of AdipoR1 pathway raised the trabecular bone density and percent bone volume and degraded the structure model index ( Figure 4D). The activation of RANKL have been proved tightly associated with osteoclast development [12]. Expectedly, shAdipoR1 group had both lower RANKL expression level and RANKL / osteoprotegerin (OPG) ratio compared to shNC group ( Figure 4E, F). It suggested that AdipoR1 knockdown treatment decreased joint erosion through suppressed RANKL/OPG axis via inhibiting the expression of RANKL. Taken together, our results confirmed that local AdipoR1 knockdown decrease synovial inflammation, joint erosion and osteoporosis progression in CIA mice.

Discussion
RA is characterized as the presence of an inflammatory synovitis accompanied by destruction of joint cartilage and bone. Although the underlying mechanism stay unknown, several studies find that multiple proinflammatory cytokines play critical roles in the pathophysiology of RA, including IL-1β, IL-6, TNF-α and so on [13]. However, the initiating factors of these inflammatory responses are still unclear.
APN was reported to be rather detrimental in RA and involved in disease progression, by inducing the secretion of pro-inflammatory molecules, chemokines and matrix-degrading enzymes [6,14,15]. There are two main types of APN receptors, AdipoR1 and AdipoR2, that mediated the biological effects [10]. In present study, AdipoR1 was found high-expressed in synovial tissue of RA patients. Moreover, the expression of AdipoR1 was upregulated after APN stimulation in primary RASFs. It suggested that AdipoR1 mediated the downstream changes, and it might be a therapeutic target in the treatment of RA.
RASFs mediate most relevant pathways of joint destruction. Molecular insights into these cells constitute an important target for novel therapeutic approaches that inhibit the destruction of cartilage and bone in RA [16]. When activated by LPS, RASFs could elaborate a broad repertoire of inflammatory mediators, justifying their classification as immune sentinel cells in RA [17]. In MH7A, AdipoR1 knockdown significantly suppressed the abnormal proliferation stimulated by LPS. Besides, evidence suggests that the expansion of fibroblast populations in the joint results primarily from inhibition of pro-apoptotic pathways, rather than large-scale proliferation [18]. In present study, AdipoR1 knockdown promoted the apoptosis and inhibited the inflammatory factors secretion of MH7A, suggesting a therapeutic effect in vitro.
In previously studies, we reported APN exacerbate disease progression, by enhancing Th17 response, prompting RANKL expression and osteopontin production [8,9]. Since RASFs play a central role in the progression of RA, lentivirus mediated shAdipoR1 was injected into the joints of CIA mice to achieve local AdipoR1 knockdown. The outcomes indicated that it significantly improved the pathological process, inflammatory reactions and bone erosion. In RA, bone erosions result from osteoclastic bone resorption at the sites of synovitis. RANKL plays a critical role in the bone homeostasis, which could be elevated by several cytokines [19]. RANKL/OPG axis determines the degree of proliferation and activity of the osteoclasts. Interestingly, local AdipoR1 knockdown could prevent CIA mouse from bone erosion by repressing the expression of RANKL without affecting the expression of OPG. It reveals a distinct mechanism of cartilage destruction and bone erosion in CIA mouse, and it provides us a novel way to block bone erosion in arthritis.
Current pharmacologic therapies have made a significant progress toward achieving disease remission without joint deformity. Despite this, a significant proportion of RA patients do not effectively respond to the current therapies and thus new drugs are urgently required [18]. Biological disease-modifying anti-rheumatic drugs (DMARDs) had revolutionized RA treatment. Current DMARDs targets a variety of receptors, such as Infliximab targets TNF-α [20], Anakinra targets IL-1 [4], Tocilizumab targets IL-6 [21], Ixekizumab targets RANKL [22] and so on. APN is widely distributed and plays systemically protective effects on human, including metabolic homeostasis, preventing cardiovascular diseases and so on [23]. Paradoxically, adiponectin has been proved to have both positive and negative influences in the inflammation mechanism of RA. In mice, systemic APN knock out aggravated the condition of arthritis [24]. Furthermore, APN significantly exacerbated the symptoms of RA in local joints [9]. Thereby, local AdipoR1 suppression might be an ideal target for the discovery of biological DMARDs in future.

Conclusions
In summary, we demonstrated that AdipoR1 knockdown treatment could decrease the inflammatory responses, improve the phenotype of RASFs and suppress the progression of RA through inhibiting RANKL expression. Our study reveals a new sight into the pathophysiological process of RA and provides a potential therapeutic target in the treatment of RA.