Paquet et al.TNF gene targeting via TFO and arthritispage # 1
Supporting Information: Material and Methods
Isolation and cultivation of rat chondrocytes and synoviocytes
Synoviocytes wereisolated from synovial membranesof healthy Wistar male rats (6 weeks old, 150 to 175 g) (Charles River, Saint-Aubinles-Elbeuf, France), digested overnight by a mix ofcollagenase and dispase (Roche) (at 0.1 units/mL and 0.8 units/mLrespectively). Chondrocytes were isolated from femoral heads by sequential digestion with Pronase (2 mg/mL, 2 h) and collagenase B(1.5 mg/mL, overnight) (Roche, Meyland, France) as previously described [1]. Chondrocytes were used in the first and synoviocytes in the third passage to prevent dedifferentiation.
Transient transfections
Chondrocytes and synoviocytes were seeded on 24-well culture plates at 60,000 cells per well and grown to 70% confluence. Cells were transfected with anti-TNF alpha oligonucleotides (ranging from 1fM to 100nM in the final culture medium). Tranfections were performed for two hours with polyethylenimine reagent (Euromedex, Souffelweyersheim, France) for TFO and with JetSITM-ENDO (Polyplus-transfection, Illkirch, France) for siRNA, according to the manufacturers’ recommendations. At 24 hours after transfection, cells were stimulated either with recombinant rat IL-1β (10ng/mL, R&D Systems, Lille, France) or with LPS (1µg/mL) for four hours (mRNA expression analysis) and 18 hours (production of mediators in supernatants). Cells were then frozen and stored at -70°C until processing.
Scoring of arthritis
The disease course was monitored by the repeated assessment of knee joint width using a caliper (Kroeplin Längenmesstechnik, Schlüchtern, Germany). Raw data were used to estimate joint circumference using the geometric formula of ellipse circumference:
2π X (a= knee height and B= knee breadth).
The results were representative of joint swelling and expressed in millimeters as the difference between the joint size at a given day compared to the size measured just before arthritis induction (Day zero). Body-weight gain was also monitoreddaily.
Weight-bearing assessment protocol
Hindlimb weightbearing was determined using an incapacitance tester (Linton Instrumentation, Norfolk, UK) consisting of a dual channel weight averager. Weight distribution was measured between sensitized (intra-articular injection of mycobacterium walls and mBSA) and contralateral (saline injected) hindlimbs and used as an index of joint discomfort in the arthritic knee. Rats were carefully placed in an angled Plexiglas chamber positioned so that each hindpaw rested on a separate force plate. Care was taken to ensure that the animal weight was directed onto the force plates and not dissipated through the walls of the chamber. The force exerted by each hindlimb (measured in grams) was averaged over a period of 5 s.Each data point is the mean of three readings. The percentage of weight distributed onto the sensitized (arthritic) hindlimb was calculated by the following equation: [weight on ipsilateral hindlimb /(weight on ipsilateral + weight on contralateral)] x100 [2]. Weight distribution of arthritic rats was compared to that of control rats (injected bilaterally with saline into the knee joints).
Assessment of proteoglycan metabolism in patellar cartilage
Proteoglycan synthesis was studied by ex vivo incorporation of Na235SO4 into patellar cartilage as previously described [3]. Data are expressed as the percentage of variation from healthy controls, with a negative value representing a decrease in proteoglycan synthesis.
Histological analyses
Knee joints were collected at necropsy, fixed immediately for 24 hours in 4% paraformaldehyde, then decalcified in rapid bone decalcifier (RDO; Apex Engineering, Plainfield, IL, USA) at room temperaturefor 6 hours, dehydrated and further fixed in 4% paraformaldehyde before being embedded in paraffin. Sections (5 μm thick) were rehydrated in a graded ethanol series and stained with hematoxylin/eosin/saffron, toluidine blue, sirius red and safranin-O. The histological characteristics of articular cartilage, bone and periarticular soft tissues were scored (adapted from Mankin et al.) [4]independently by two observers who were blind to the samples. Cartilage degradation was graded from 0 to 3, where 0 = fully stained cartilage, 1 = loss of proteoglycan staining in the superficial layer, 2 = complete loss of proteoglycan staining, and 3 = complete loss of cartilage. The following morphological criteria were used for bone erosion: 0 = normal, 1 = mild loss of cortical bone at a few sites, 2 = moderate loss of cortical and trabecular bone, and 3 = marked loss of bone at many sites. Synovium from knee joint was graded using a scoring technique adapted from Rooney et al.[5]. Briefly, samples were evaluated on a scale from 0 to 4 (with 0 = normal and 4 = major changes) for hyperplasia of synovial fibroblasts (depth of lining layer), fibrosis (percentage replacement of loose connective tissue), angiogenesis (number of proliferating blood vessels), perivascular infiltrates of lymphocytes (percentage of vessels surrounded by lymphocytes) and tissue infiltration by lymphocytes (size of aggregates, percentage infiltrating cells).
Assessment of cytokine mRNA expression
Tissue sampling and preparation.Samples of knee joint synovial membrane and patella cartilage were obtained from rats sacrificed 24 hours after arthritis induction. At necropsy, tissue pieces were frozen and stored at -80°C until processing.
RNA extraction and real-time PCR analysis. Frozen tissues (synovial membranes and patella cartilage) were homogenized immediately after thawing using a dispersing system and mixed with 350µL RLT buffer added with β-mercapto-ethanol. Samples were then placed on QIAshredder columns (Qiagen, RNeasy Kit, Courtaboeuf, France) and mRNA was extracted with the RNeasy kit® following the manufacturer’s recommendations. A total of 0.5 µg of mRNA from each sample was then reverse-transcribed with 200 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen) and oligo(dT) primers (Eurogentec, Liège, Belgium), according to the supplier’s recommendations. Expression of TNF-α, IL-1β, IL-6, vEGF, iNos and MMP13 was quantified by real-time PCR with the Lightcycler® (Roche) technology and the SYBRgreen master mix system® (Qiagen, Courtaboeuf, France). For quantification of the changes in gene expression, the comparative Ct method was used to calculate the relative-fold changes normalized against the ribosomal RNA [6]. Data are presented as the mean of three samples and each sample was assayed in triplicate. The gene-specific primer pairs used were as follows:
TNF-α, sense 5'-AGCCCTGGTATGAGCCCATGTA-3',
antisense 5'- CCGGACTCCGTGATGTCTAAGT-3';
IL-1β, sense 5'-CTTCCCCAGGACATGCTAGG-3',
antisense 5'-CAAAGGCTTCCCCTGGAGAC-3';
IL-6, sense 5'-CCGGAGAGGAGACTTCACAG-3',
antisense 5'-ACAGTGCATCATCGCTGTTC-3';
COX2, sense 5’-TACAAGCAGTGGCAAAGGCC-3’,
antisense 5’-CAGTATTGAGGAGAACAGATGGG-3’;
MMP13,sense 5’-AGGCCTTCAGAAAAGCCTTC-3’,
antisense 5’-GAGCTGCTTGTCCAGGTTTC-3’;
vEGF, sense 5’-CACATCTGCAAGTACGTTCGTTTA-3’,
antisense 5’-CAGAGCGGAGAAAGCATTTGTT-3’;
iNos, sense 5’-AATGCGGAAGGTCATGGC-3’,
antisense 5’-CAGCTTTCCTGTCTCAGTAGCAAA-3’;
S29, sense 5'-AAGATGGGTCACCAGCAGCTCTACG-3',
Antisense 5'-AGACGCGGCAAGAGCGAGAA-3'.
Assessment of cytokine protein expression
Tissue sampling and preparation.For joint fluid sampling, at necropsy (48 hours after arthritis induction), the patellar ligament was cut and the articular cavity incised perpendicularly to the patella. The synovial fluid was then collected by impregnation of small pieces of filter paper (Schleicher & Schuell, GmbH, Germany) (4mm2). These paper pieces were left in 150µL of sterile salineat 4°Cfor 12 hours, and then frozen at -80°C until processing [7]. For in vitro studies, supernatants were collected after 18 hours of stimulation by IL-1β (10ng/mL, R&D Systems, Lille, France).
ELISA test and nitrite assay.The contents of the samples (cell culture supernatants and synovial fluid) were analyzed by both NO and TNF-α dosage. NO production was determined spectrophotometrically by measuring the accumulation of nitrites inculture supernatants by the Griess reaction [8].
The TNF-α concentrations were determined using a sandwich ELISA (R&D System) as previously described [9].
Multiplex immuno-assay. Levels of 24 cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p(70), IL-13, IL-17, IL-18, Leptin, GRO/KC, TNFα, IFNγ, GMCSF, RANTES, MCP-1, MIP-1α, G-CSF, IP-10, Eotaxin, vEGF) were determined in synovial fluid (sensitized and contralateral) at 7 and 24 hours and at Day7 with the LINCOplex KIT (LINCO Research, USA). Millipore multiscreen 96-well filter plates (Bedford, USA) for multiplex cytokine kits were used. Assays were run in triplicate according to the manufacturers’ protocol [10]. Data were collected using the Luminex-100 software Version 1.7 (Luminex, Austin, USA) and analysis was performed with the MasterPlex QT 1.0 system (MiraiBio, Alameda, CA). A five-parameter regression formula was used to calculate the sample concentrations from the standard curves.
Statistical analysis
All results are expressed as mean ± SEM. All analyses and editing of figures were performed using GraphPad Prism (release 4, GraphPad Software, Inc. San Diego, USA). Student’s t-test was used to compare a batch to its own control and ANOVA with post-hoc Bonferroni when required (i.e. when groups ≥ 3). The Mann-Whitney-Wilcoxon test was used for non-parametric data.
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