Reagents and antibodies

All reagents were purchased from Sigma-Aldrich unless otherwise stated. The following antibodies were obtained from Abcam, MMP13 (39012), osterix (22552), VEGF (46154) and nestin (18102). DRAQ5, NFκB p65 (4764) and nestin (47607) antibodies were purchased from Cell Signalling Technology Inc. WDR33 (374466) and ADAMTS5 (83186) antibodies were purchased from Santa Cruz Biotech. CPSF4 antibody was obtained from Protein Tech. PCNA (M0879) and CD68 (M0814) antibodies were obtained from Dako. Alkaline phosphatase and peroxidase kits as well as secondary antibodies were obtained from Vector Labs. MCSF was obtained from R&D Systems. RANKL was obtained from Peprotech.

Rodent models of OA

Studies were in accordance with UK Home Office Animals (Scientific Procedures) Act (1986) and the International Association for the Study of Pain guidelines and were approved by ethical review board at the University of Nottingham. Data are presented in line with the ARRIVE guidelines. All animal studies were conducted in a manner that minimised animal distress, and euthanization of the animal occurred via an appropriate S1 technique (as listed by the UK Home Office). Animals were anesthetised with isoflurane (2.5–3%) in 100% oxygen (1 L per min) prior to surgeries and intra-articular injections. Tissues including synovia and joints were collected for molecular biology, histological and immunohistochemistry studies.

Male Sprague Dawley rats (n = 10/group) weighing 180–200 g were given intra-articular injection of monosodium iodoacetate (MIA) (1 mg/50 μl) in saline at day 0 into their left knee32. Control rats received intra-articular injection of 50 µl of saline. For the therapeutic MIA study, at Day 14, cordycepin (4 mg/kg, 8 mg/kg or 16 mg/kg) or vehicle (1 ml distilled water) were mixed with 1 g of wet mash and administered every other day until day 28. For the pre-emptive MIA study, cordycepin (8 mg/kg) was given at day 0 (prior to intra-articular injection) for a period of 2 weeks, until day 14. Rats were food restricted for 2 hrs prior to being given cordycepin. Pain behaviour was measured twice weekly following model induction.

Eight to nine weeks old male C57BL/6 mice (at least n = 15/group) underwent surgery on their left knee joint at week 0 to displace the medial meniscus as described previously33. A small longitudinal incision was made over the joint and using blunt dissection the underlying medial meniscotibial ligament (MMTL), which anchors the medial meniscus to the tibial plateau was transected, destabilising the medial meniscus (DMM). The wound was sutured and mice observed until they regained consciousness. Control group included those having sham surgery, in which the ligament was visualised but not transected. From week 14 to 16 mice were orally gavaged every other day with 200 µl cordycepin (8 mg/kg) or vehicle (23% propylene glycol [PPG] in distilled water)34. Pain behaviour was measured once weekly following model induction and then twice weekly following cordycepin treatment, until week 16.

Pain behaviour was quantified as a change in hindlimb weight-distribution and hindpaw mechanical withdrawal thresholds, as previously described32.

Human osteoarthritic and post-mortem joint tissues

The joint tissue repository of the Arthritis Research UK Pain Centre, which contains samples from >1,700 subjects, was screened to select tissues obtained at the time of total knee replacement (TKR) for OA and tissues obtained post-mortem from age-matched subjects who had not sought medical attention for knee pain during the last year of life (non-OA control group). The tissue samples (n = 10 per group) were split into three distinct groups, OA group having high grade inflammation (median age [IQR]; 58 [57–73], 78% were male), OA group having low grade inflammation (median age [IQR]; 61 [58–70], 67% were male) and non-OA control group (median age [IQR]; 60 [50–70], 67% were male). Synovial inflammation was graded on a scale of 0–3 (where 0 = normal and 3 = severe [high grade] inflammation) by assessing the degree of synovial lining hyperplasia, inflammatory cell infiltrate, and cellularity35. Patients undergoing TKR fulfilled the American College of Rheumatology classification criteria for OA at the time of surgery36. Subjects from whom samples were obtained postmortem were recently deceased, had no history of rheumatoid arthritis or pseudogout, and had not previously sought help for knee pain during the last year of life, as determined by interviews with the relatives and review of case notes. Exclusion criteria for non-OA controls consisted of a history of OA, Heberden’s nodes identified on clinical examination, macroscopic chondropathy lesions of grade 3 or 4 in the medial tibiofemoral compartment, or osteophytes on direct visualization of the dissected knee. Informed consent was obtained from the TKR patients and from the next of kin of the postmortem subjects. All study protocols were performed in accordance with the relevant guidelines and regulations indicated by the UK National Research Ethics Service (Nottingham Research Ethics Committee 1 [05/Q2403/24] and Derby Research Ethics Committee 1 [11/H0405/2]).

Tissue processing

Rat synovia with patellae were dissected, embedded in OCT and snap frozen in isopentane. Tibiofemoral joints were fixed for 48 hrs in 4% paraformaldehyde (PFA), then decalcified in 10% ethylenediaminetetraacetic acid (EDTA) in 10 mM Tris buffer (pH 6.95) for 4 weeks on a shaker at room temperature (RT). Coronal sections of trimmed joint tissues were mounted in paraffin wax. Mice synovia with patellae and tibiofemoral joints were either frozen on dry ice or the whole knee joints were fixed in 4% paraformaldehyde for 24 hrs before being decalcified in EDTA for 6 days on a shaker at RT. Sagittal sections of trimmed joint tissues were mounted in paraffin wax. For human tissue samples, midcoronal sections of the middle one-third of the medial tibial plateau were fixed in neutral-buffered formalin and then decalcified in 10% EDTA in 10 mM Tris buffer (pH 6.95; at 4 °C) prior to embedding in wax. Surgeons and technicians were instructed to collect synovium from the medial joint line. Synovial tissues were fixed in formalin and embedded in wax without decalcification.

Joint histology

All sections for histology were cut at 5 μm and visualised using a 20 × objective lens unless otherwise indicated. All histomorphometry analysis was done on haematoxylin and eosin or Safranin-O/fast green-stained sections by at least two observers blinded to the treatment groups.

In the rat MIA model, cartilage damage, matrix proteoglycan and osteophytes were assessed as previously described37. The integrity of the osteochondral junction (OCJ) was measured as the number of channels (and those that were nestin positive) crossing the OCJ into the cartilage of the whole section of medial tibial plateau37. Synovial inflammation was graded as previously described38 on a scale from 0 (lining cell layer 1–2 cells thick) to 3 (lining cell layer >9 cells thick and/or severe increase in cellularity).

In the mice DMM model, joint pathology was assessed based on previously published scoring criteria39,40. Briefly, cartilage surface integrity was scored from 0 (normal) to 6 (vertical clefts/erosions to the calcified cartilage extending to >75% of the articular surface). Cartilage proteoglycan loss was scored from 0 (normal staining of non-calcified cartilage) to 5 (complete loss of safranin-o/fast green staining in the non-calcified cartilage extending to ≥75% of the articular surface). Chondrocyte hypertrophy score ranged from 0 (no chondrocyte hypertrophy) to 1 (enlarged chondrocyte lacunae with lack of safranin-o/fast green stain). Osteophyte size was scored from 0 (no osteophyte) to 3 (large osteophyte, greater than 3 x the thickness of the adjacent cartilage. Osteophyte maturity scores ranged from 0 (no osteophyte) to 3 (predominantly bone). Subchondral bone thickening score ranged from 0 (normal trabecular bone with greater than 50% marrow space) to 3 (solid bone spanning greater than two thirds of the width of the epiphysis). Synovial inflammation was graded on a scale of 0 (no inflammation: lining cell layer 1–2 cells thick) to 3 (severe inflammation: greater than 6 cells thick lining). In the human synovial sections inflammation was graded on a scale of 0–3 (where 0 = normal and 3 = severe inflammation) by assessing the degree of synovial lining hyperplasia, inflammatory cell infiltrate, and cellularity35.

Immunohistochemistry and immunofluorescence

Synovial inflammation was measured as CD68 (clone ED1) positive macrophages as previously described41. Proliferating cell nuclear antigen (PCNA) positive cells and PCNA-immunoreactive CD31-positive cells were used to identify proliferating cells and proliferating endothelial cells respectively, as measures of the extent of synovial proliferation and angiogenesis41. To detect ADAMTS5, MMP13, nestin, VEGF, PCNA, CD68, NF-ĸB and CPSF4 immunoreactivity in paraffin embedded tissue sections, the sections were first deparaffinised and rehydrated in graded ethanol and water, followed by antigen unmasking (10 mM sodium citrate buffer, pH 6) at 80–85 °C for 20 mins. Sections were cooled for 10 mins at RT followed by permeabilisation (0.1% Triton X-100) and blocking (5% serum) steps. Primary antibodies were incubated overnight at 4 °C and secondary antibodies for 45 mins at RT. Vectastain ABC-AP alkaline peroxidase with Fast Diaminobenzidene (DAB) was used to visualise ADAMTS-5, MMP13, nestin, VEGF, CPSF4 and PCNA staining. Preparations were mounted in DePeX. To detect NF-ĸB, WDR33, CPSF4 and CD68 immunofluorescence in tissue sections and cell cultures, Alexa Fluor 488 and 568 secondary antibodies were used. Cell cultures were fixed for 15 mins in 4% PFA before proceeding to the immunofluorescence protocol as described above. DRAQ5 was used as nuclear stain and sections were mounted in aqueous mounting media.

Osteoclast number

Tissue sections were dewaxed and recalcified before tartrate-resistant acid phosphatase (TRAP) staining. The number of TRAP-positive multinucleated osteoclasts were quantified within the subchondral bone area comprising the area between the cartilage/bone junction and the growth plate as described previously42.

In-vitro model of human macrophage and osteoclast differentiation

This study was approved by the Nottingham University Medical School Research Ethics Committee. Monocytes were isolated from peripheral blood of healthy human donors and either differentiated into macrophages or osteoclasts as previously described43. For osteoclast differentiation, monocytes were isolated from buffy coats by gradient centrifugation and seeded onto glass coverslips within a 24-well culture plates, and cultured in growth media supplemented with human macrophage colony stimulating factor (MCSF) and human receptor activator of NF-ĸB ligand (RANKL), unless otherwise stated. Cells were incubated at 37 °C, 7% CO2 for 2 hrs, and the medium replaced. Growth media containing cordycepin (20 µM) was then added to the cells. After 14 days, cells were washed and fixed with 4% PFA. Differentiated osteoclasts were identified by TRAP staining. For quantification of TRAP positive cells five random fields of view were counted per coverslip using four coverslips per condition. Cells that stained positive for TRAP and had three or more nuclei were counted. For macrophage differentiation, the monocytes were grown in RPMI 1640 supplemented with 5% foetal bovine serum (FBS) in the presence of MCSF for 5 days. Adherent cells were washed, replated onto coverslips in a 24-well plate and cultured for a further 24 h in 3% FBS before stimulation with LPS with and without cordycepin. Cells were fixed in 4% PFA before proceeding to the immunofluorescence protocol. RAW264.7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS in a humidified atmosphere of 5% CO 2 and 95% air at 37 °C. Twenty four hours before experimentation, the cells were washed with PBS and supplemented with 0.5% serum. The cells were then treated with cordycepin (20 µM) either 1 hr before or 10 mins after LPS stimulation (1 µg/ml). After which the cells were either processed for protein/RNA extraction or immunofluorescence protocol. RNA isolation for tissue culture cells was done using the Promega Reliaprep system.

Western blot analysis

RAW264.7 cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (0.5% Igepal, 0.5% deoxycholate, 0.05% sodium dodecyl sulfate, 1 mM β-glycerophosphate, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride) containing protease/phosphatase inhibitors to extract total cell protein content. Protein concentration was determined by Bradford Assay. Approximately 30 µg of protein was subjected to SDS-PAGE and transferred to nitrocellulose membrane. To block non-specific binding of proteins, membranes were treated in TBST with 5% skimmed milk for 1 hr at RT, and were incubated overnight with primary antibodies against IĸB and vinculin at 4 °C, followed by secondary antibody incubation for 1 hr at RT. Immunoreactivity was detected by chemiluminescence. Western blot images were not reassembled after cuts between the vertical lanes. The blots were cut horizontally so to make it easier to probe for various antibodies simultaneously on the same blot. Each continuous image represents a single exposure.

RNA isolation from tissues

At the end of the pain behavioural studies conducted in the DMM-model of OA, fresh-frozen synovial tissue and knee joints were collected and stored at −80 °C. Tissues were homogenized using the bullet blender. Total RNA was extracted from the tissues and RAW264.7 cells using TRIzol.

Quantitative real-time polymerase chain reaction (qRT-PCR)

500 ng of RNA was reversed transcribed to cDNA and diluted 5 fold with sterile distilled water before being subjected to qPCR using the GoTaq qPCR Master Mix containing the relevant forward and reverse primer sets (Supplementary Table 1). Primers for CD68, IL1β (spliced and unspliced), nestin, VEGF, PCNA, MYC, osterix, RUNX1, RUNX2 and TNF (spliced and unspliced) were designed with Primer Express 3.0 software. All qRT-PCR experiments were performed in triplicates, data were normalised to relative expression using the Qiagen Rotor-Gene Q software. All values were normalised to Ribosomal Protein L28 (RPL28) using the 2−∆∆ct method.

siRNA Transfection Protocol

RAW264.7 cells were transfected for 24 hrs with lipofectamine diluted in opti-MEM containing 5 nM siRNA for either WDR33 (Dharmacon SMARTpool ON-TARGETplus L-051645-01-0005) or CPSF4 (Dharmacon SMARTpool ON-TARGETplus L-052851-01-0005). Media was changed the next day and cells transfected again and incubated for a period of 24 hrs before being processed for either immunofluorescence or RNA extraction.

Microarray analysis

High-throughput analysis was conducted on RNA extracted from RAW 264.7 cells treated with either 20 μM cordycepin or vehicle control for 1 hr before being stimulated with LPS (1 μg/ml) or vehicle control for a further 1 hr, to reveal the genome wide changes brought about by cordycepin. Biological replicates (n = 4, 16 RNA samples in total) were then analysed on a mouse GE 8 × 60 K microarray (Agilent, cat no G4852A). This was followed by cluster analysis on the lists of RNAs whose levels were changed by cordycepin. This gene ontology analysis was done using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 software on the list of genes most strongly downregulated in the LPS with cordycepin treatment group compared to LPS alone group.

Image analysis and quantification

Synovial macrophage fractional area was the percentage of synovial section area positive for CD68, and synovial angiogenesis was measured as endothelial cell proliferation index, defined as the percentage of endothelial nuclei positive for PCNA, derived using four fields per section and one section per case as described previously2,44. Cartilage cellularity was quantified by counting the chondrocytes in three microscopic fields (355 μm x 265 μm) per section, at central, medial and lateral side of the medial tibial plateau (MTP) taken under 40 × magnification. Total number of chondrocytes and number of positively stained chondrocytes were counted in each section. Results were expressed as the percentage positive cells. At least 3 images per section and 3 sections per block were analysed45. Nestin and VEGF expression in the subchondral bone was analysed using imageJ software as the area (µm2) covered by nestin/VEGF immunoreactivity. PCNA expression in human synovial tissues was analysed on deconvoluted DAB and haematoxylin images in ImageJ as percentage of PCNA positive cells. NF-ĸB immunofluorescence was analysed on ImageJ as nuclear versus cytoplasmic intensity expression or intensity per µm2. All histology and immunohistochemistry image analyses were performed using a Zeiss Axioskop 50 microscope and a KS300 image analysis system. Immunofluorescence images were captured using a Leica confocal microscope.

Statistical analysis

Data were analyzed with GraphPad Prism version 6 and are presented as either the mean ± SEM or mean ± SD. For all comparisons, p < 0.05 was taken to indicate statistical significance. Parametric data were analysed using analysis of variance (ANOVA) with post hoc Dunnett’s test. Univariate comparisons were made against controls using the Student t test. Non-parametric data were analysed using the Kruskal–Wallis test followed by the Mann–Whitney test with Bonferroni correction.