Materials

The TNFα and IL-6 ELISA kits, MTT, and apoptosis ELISA detection kit were obtained from Sigma-Aldrich Chemicals (Bangalore, India). The PGE2 and LTB4 ELISA kits were procured from Enzo Life Sciences (New Delhi, India). The Lipofectamine RNAiMAX reagent, Lipofectamine-2000 reagent, human-specific LPCAT3 siRNA (product ID. 16915), mouse-specific LPCAT3 siRNA (product ID. 89055), human-specific MALT1 siRNA (product ID. 105148) and scrambled siRNAs (product ID. 8390844) were procured from Thermo Fisher Scientific (MA, USA). Mepazine Acetate was purchased from Merck, CA, USA. The inhibitors, KJPyr9, and EN4 were purchased from Sigma-Aldrich Chemicals (Bangalore, India). The LPCAT3 polyclonal antibody was purchased from Novus Biologicals, CO, USA (Product no: NBP3-10074). The human anti-LPCAT3 antibody was purchased from Abcam MA, USA (Product no: ab232958). MALT1 antibody was procured from Cell Signaling Technology, USA (Product no: 2494). Myc (Product no: sc-40) and GAPDH (Product no: sc-32243) antibodies were procured from Santa Cruz Biotechnology, Texas, USA.

MethodsIsolation of chondrocytes

OA human cartilage was obtained from OA patients ages 53–76 years diagnosed according to the International Cartilage Repair Society criteria undergoing total knee replacement. Cartilage samples were cut into small pieces and digested with trypsin–EDTA at 37°C for 20 min. The trypsin solution was washed and tissue slices wereincubated with 2 mg/ml type II collagenase (Sigma Aldrich, Bangalore, India) in DMEM (Himedia Laboratories, Mumbai, India) for 16h. Next, it was filtered using nylon mesh and washed with medium. The isolated chondrocytes were seeded in T75 culture flasks and incubated in DMEM containing 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 mg/ml) at 37°C in an atmosphere of 5% CO2. At confluence, the cells were detached and seeded in 24-well plates at a density of 1 × 105/well. Cell passages 1 to 2 were used in our study.

Transfection of plasmids or siRNA and immunoblotting

Human chondrocytes were seeded in a 24-well plate at a density of 1 × 105cells/well with DMEM and 10%FBS. The next day, transfection of MALT1 plasmids or siRNA was done using Lipofectamine. The chondrocytes were transfected with scrambled RNA (scRNA), LPCAT3 siRNA(200 nM),or MALT1 siRNA (200 nM) using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher, MA, USA) in 500μl RPMI medium for 8 h as described in our previous studies [22, 23, 65]. Then the medium was replaced with serum-richDMEM medium and cultured for 48h or as described in the figure legends. Similarly, 1 μg of pCMV6-XL4-MALT1 or pCMV-XL4 empty plasmids (Origene, MD, USA) were transfected using Lipofectamine 2000 (Thermo Fisher, MA, USA) in DMEM medium without antibiotics for 12h. Later the medium was replaced with fresh medium. 48 h post-transfection, the cells were stimulated with IL-1β as described in figure legends, and cell medium was collected for analysis of cytokines and eicosanoids. Alternatively, the cells were lysed in the SDS-PAGE sample buffer, and an equal amount of protein or volume was subjected to SDS-PAGE, followed by immunoblot analysis. The immunoblots were developed with chemiluminescence using HRP-substrate (Sigma). Densitometric analysis was performed using the Bio-Rad Chemi XRS system and Image J software.

RNA extraction and qRT-PCR

Total RNA from the chondrocytes or cartilage tissues was extracted using TRIzol reagent (Sigma Aldrich, Banglore, India). The total RNA was extracted, and cDNA was prepared by reverse transcription (iScript cDNA Synthesis Kit, Bio-Rad) and amplified by PCR (Bio-Rad, CA, USA), as described in our previous studies [65, 66]. The qRT-PCR was done in triplicates and the expression of target genes was normalized to GAPDH. The relative gene expression levels were calculated using the 2−ΔΔCT method and expressed as fold changes [65]. Sequences of the primers used in this study are presented in Supplementary Table 1.

Cell viability assay

Cell viability was assessed by MTT assay as described previously [67, 68]. Briefly, human chondrocytes were seeded in 96-well plates at a density of 1.0 × 104 cells/well in DMEM with 10% FBS. The next day, the cells were transfected with scRNA or siRNA targeting LPCAT3 or MALT1 (20 nM) for 12h hours in the serum-free medium using Lipofectamine RNAiMAX transfection reagent. Later, the medium was replaced with a complete medium. 48 h post-transfection, the cells were treated with IL-1β for 24 h. In the mepazine treated group, mepazine was added 2h prior toIL-1β treatment. Cell viability was measured by MTT assay. Briefly, fresh MTT (5 mg/ml in PBS) was prepared and 25 μl was added to each well. 4 h later, the supernatant was removed, and 100 μl of DMSO was added to each well. The absorbance of the samples was measured at 540 nm using a plate reader. Cell viability was measured as % absorbance of IL-1β-treated cells compared to the cells not treated with IL-1β.

Apoptosis assay by ELISA

The apoptosis assay was determined by a cell-death ELISA detection kit (Sigma-Aldrich, Bangalore, India) that measures histone-DNA complexes generated during DNA fragmentation caused by apoptosis [69, 70]. The assay was done using the manufacturer’s instructions and absorbance was read at 405 nm using a spectrophotometer.

Culture of cartilage explants

The Human cartilage explants were cultured as described previously [5]. Briefly, cartilage explants were cut into 4 × 5 mm volumes and they were cultured in DMEM containing 10% FBS. They were transfected with scRNA, human LPCAT3 siRNA, or MALT1 siRNA using Lipofectamine RNAiMax reagent in a serum-free medium for 10 h. Later, the medium was replaced with serum-rich DMEM and 10% FBS with antibiotics. 48 h post-transfection, they were stimulated with IL-1β (10 ng/ml) for 24 h. The cartilage explants were then collected for immunoblot or RT-PCR analysis. The medium was collected for analysis of sulfated glycosaminoglycans (sGAG) by 1, 9, dimethyl methylene blue (DMMB) (Sigma-Aldrich, Bangalore) assay using chondroitin sulfate as standard [71]. The PGE2, LTB4, and IL-6 were analyzed in the medium by ELISA.

Lipid extraction and fatty acid analysis in the cells

Total lipids in the cells were extracted [25], saponified with 0.5 M KOH and methylated with 40% boron trifluoride in methanol as described in our previous studies [24, 25]. The methyl esters of fatty acids were extracted with hexane. Fatty acid analysis was carried out as described in our previous studies [24, 25]. The arachidonic acid content in the cells was presented as a relative percentage peak area with respect to other fatty acids [24].

Synthesis of LPCAT3 siRNA nanoparticles

We synthesized liponanoparticles containing LPCAT3 siRNA, delivered to mice, and assessed its effects on DMM-induced OA. These nanoparticles have been designed for efficient, safe, and targeted transfection of siRNA [72, 73]. The mouse-specific LPCAT3 siRNA (product ID. 89055) and scrambled siRNAs (product ID. 8390844) were procured from Thermo Fisher Scientific. The lipids 1,2-dipalmitoyl-sn-glycero-3-phospho((ethyl-1′,2′,3′-triazole)triethylene glycol) (ammonium salt) (PEG-DPPE), and N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium (chloride salt) (DOTAP), cholesterol, lecithin, were purchased from Sigma Aldrich Chemicals, Bangalore, India.

The siRNA encapsulation was performed as described previously [74, 75]. Briefly, the DOTAP, lecithin, cholesterol, and PEG-DPPE were dissolved in ethanol at a molar ratio of 45:15:35:5 to a final concentration of 15 mM. The mice-specific LPCAT3 siRNA or scRNA dissolved at 2 mg/ml in 50 mmol/l citrate, pH 4.0 was added drop-wise to an ethanolic solution of lipid mixture and incubated at 31 °C for 30 min with constant stirring to a final siRNA/lipid weight ratio of 8/1, wt/wt. The ethanol was removed and the neutralization was performed by dialysis against phosphate-buffered saline (PBS), pH 7.4 overnight using a Spectra dialysis membrane. The synthesized LPCAT3-siRNA-lipid nanoparticles were used for mice OA studies.

Induction of OA by DMM-surgery in mice knee tissues

The DMM surgery was performed in the hind knee of 10–12 week-old mice following administration of general anesthesia (inhalation of isoflurane in oxygen) by transection of the anterior attachment of the medial meniscotibial ligament in the knee [76].The joint capsule was closed with a continuous 8–0 suture and the subcutaneous layer with 7–0 suture. The skin was closed by tissue adhesive. The skin of the knee joint was resected in the sham-operated group. Later, the buprenorphine (1.0 mg/kg) was given subcutaneously to alleviate pain after surgery.

Intraarticular delivery of mepazine or LPCAT3-siRNA nanoparticles

The DMM-operated mice were intraarticularly injected immediately with mepazine or vehicle and thereafter weekly twice post-DMM surgery at a dosage of 10 mg/kg body weight in 20 μl volume using an insulin syringe. The mice were sacrificed at 8-weeks post DMM and knee tissues were collected. The external muscle tissue was removed using scissors and the knee tissues were either fixed in formalin. The LPCAT3-siRNA lipid nanoparticles were administered after DMM surgery in mice, and also at 1, 2, 4, and 6 weeks after surgery (5 doses). The 20 μl of LPCAT3-siRNA lipid nanoparticle was injected IA using a 30-gauge insulin syringe which was equivalent to 0.5 mg/kg body weight of siRNA/mouse. The untreated sham mice received PBS and the scrambled RNA (scRNA) group received non-targeted scrambled RNA. The knee tissues were collected at 8 weeks post DMM and they were fixed in formalin. Alternatively, the synovial articular cartilage tissues were isolated by microdissection using a scapel and frozen immediately for the analysis of cytokines and eicosanoids.

Histological analysis of knee tissues

Eight weeks after DMM surgery, the mice were sacrificed and the knee tissues were harvested, fixed in buffered formalin for 72 h, and transferred to 70% alcohol until they were used. The joint tissues were decalcified by immersing them in a decalcifier (stat-lab) for 48 h and then embedded in paraffin for sectioning. The tissues were sectioned (6-μm thick) and selected sections were stained with safranin O-fast green staining [41, 77]. The cartilage damage was measured using the Osteoarthritis Research Society International (OARSI) scoring system [46, 78, 79]. In brief, grade 0 = healthy cartilage; 0.5 = loss of Safranin O staining with no structural changes; 1 = small fibrillation with no loss of cartilage; 2 = vertical clefts down to the layer below the superficial layer; 3–6 = vertical clefts and erosion of calcified cartilage in the articular surface with < 25%, 25%–50%, 50%–75% and > 75%, respectively. The OARSI scoring of cartilage damage was performed by 2 blinded observers. The mean values of scores were used for analysis. The synovitis score (score scale 0–6) was determined based on the thickness of the synovial lining cell layer, and cellular density in the synovial stroma. The mean values of individual scores for both parameters were used for analysis [46, 80, 81]. Briefly, the degree of synovitis in Safranin O stained sections were obtained using a scoring system that measured the thickness of the synovial lining cell layer on a scale of 0–3 (0 = 1–2 cells, 1 = 2–4 cells, 2 = 4–9 cells and 3 = 10 or more cells) and cellular density in the synovial stroma on a scale of 0–3 (0 = normal cellularity, 1 = slightly increased cellularity, 2 = moderately increased cellularity and 3 = greatly increased cellularity).

Immunohistochemistry

For immunohistochemistry, the antigen retrieval was done by boiling cartilage tissue sections for 15 min in sodium citrate buffer (10 mM, pH 6.0). The sections were incubated in 3% hydrogen peroxide for 20 min to quench endogenous peroxide activity. After blocking the tissue sections with an antibody diluent containing background reducing components (Thermofisher, MA, USA), they were incubated with control IgG, or primary antibody against LPCAT3 (5 µg/ml) overnight at 4°C. The sections were then incubated with biotin-labeled goat anti-rabbit IgG (1:500) and ultrasensitive streptavidin-HRP (1:500) (Sigma, Bangalore, India) and developed using DAB substrate kit (Sigma-Aldrich, Bangalore, India), mounted, and visualized with an Olympus BX41 microscope.

Measurement of cytokines

Chondrocytes were stimulated with IL-1β (10ng/ml) for 24 h or indicated time points as described in the figure legends. The TNFα, IL-6, LTB4, and PGE2 levels in the cell supernatants were estimated using ELISA kits according to the manufacturer’s instructions. The synovial cartilage tissues from mice were snap-frozen in liquid nitrogen, and the frozen tissue was pulverized into powder. The powder was suspended in RIPA buffer containing protease inhibitors and indomethacin. The lysate was briefly sonicated and centrifuged at 10,000 × g for 20 min at 4°C. The TNFα, IL-6, MCP1, LTB4, and PGE2 levels in tissue extracts were estimated using ELISA kits.

Data analysis

All experiments were conducted independently in triplicates. The data were shown as mean ± SD. Normality tests; Shapiro–Wilk was used for normal distribution of data. Most of the data passed the normality test and parametric tests were used to calculate statistical significance. In qRT-PCR experiments, data are expressed as relative fold changes. We analyzed the data for the two groups using unpaired two-tailed t test. One-way ANOVA with post-hoc Tukey–Kramer test was used to analyze the differences among more than two groups. p < 0.05 was considered statistically significant. GraphPad Prism version 8.0.2 was used for statistical analysis.