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Abstract
Non-steroidal anti-inflammatory drugs are a widely used symptomatic treatment in osteoarthritis (OA), but their effects on cartilage remain controversial. We studied the effects of ibuprofen on gene expression in chondrocytes from patients with OA using RNA-Seq. Chondrocytes were isolated from cartilage samples of patients with OA undergoing knee replacement surgery, cultured with ibuprofen, and total mRNA was sequenced. Differentially expressed genes were identified with edgeR using pairwise comparisons. Functional analysis was performed using ingenuity pathway analysis (IPA). Ibuprofen did not induce statistically significant changes in chondrocyte transcriptome when the cells were cultured in the absence of added cytokines. In inflammatory conditions (when the cells were exposed to the OA-related cytokine interleukin (IL)-1β), 51 genes were upregulated and 42 downregulated by ibuprofen with fold change >1.5 in either direction. The upregulated genes included anti-inflammatory factors and genes associated with cell adhesion, while several mediators of inflammation were among the downregulated genes. IPA analysis revealed ibuprofen having modulating effects on inflammation-related pathways such as integrin, IL-8, ERK/MAPK and cAMP-mediated signalling pathways. In conclusion, the effects of ibuprofen on primary OA chondrocyte transcriptome appear to be neutral in normal conditions, but ibuprofen may shift chondrocyte transcriptome towards anti-inflammatory phenotype in inflammatory environments.
- chondrocytes
- osteoarthritis
- anti-inflammatory agents
- non-steroidal
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Key messages
The current evidence about the effects of non-steroidal anti-inflammatory drugs (NSAIDs) on osteoarthritis (OA) cartilage is conflicting.
We investigated the effects of ibuprofen on gene expression in OA chondrocytes by using RNA-Seq.
In neutral conditions (in the absence of added inflammatory factors), ibuprofen had no statistically significant effects on gene expression in OA chondrocytes.
In inflammatory conditions mimicked by the presence of interleukin (IL)-1β, ibuprofen upregulated several anti-inflammatory factors while downregulating inflammatory mediators such as IL-6 and IL-23. Ibuprofen also inhibited phosphatase and tensin homolog (PTEN) signalling.
The findings support the assumption that NSAIDs are safe for cartilage when treating OA pain. They also may shift chondrocyte transcriptome towards an anti-inflammatory phenotype in OA exacerbations.
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat osteoarthritis (OA) pain but there are some concerns on their effects on chondrocyte biology.1
OA is characterised by constant low-grade joint inflammation and transient inflammatory exacerbations. The inflammatory nature of the disease is evidenced by the increased production of proinflammatory cytokines, particularly interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor α (TNFα). They drive the production of catabolic enzymes such as matrix metalloproteinases (MMPs), accelerating joint destruction.2
NSAIDs exert their effects by inhibiting the synthesis of prostanoids, particularly prostaglandin E2 (PGE2) by cyclo-oxygenase (COX) enzymes. By altering the balance of proinflammatory and anti-inflammatory mediators in the joint, they have been hypothesised to affect OA pathogenesis. These effects, if any, are however controversial, as both potential benefits (eg, alleviation of joint inflammation and reduction of cartilage catabolism) and harms (eg, impairment of cartilage anabolism and accelerated radiographic joint destruction) have been reported.1 3
We carried out a genome-wide expression analysis on the effects of the NSAID ibuprofen on gene expression in OA chondrocytes in normal and inflammatory conditions in vitro by using RNA-Seq.
Methods
Cartilage samples were obtained from 10 patients with OA (mean age 67 years (SEM 3.8 years), 8 females, Kellgren-Lawrence grade 3.7 (SEM 0.15)) undergoing knee replacement surgery in Coxa Hospital for Joint Replacement, Tampere, Finland.
Chondrocytes were isolated by enzyme digestion and seeded on 24-well plates for 24 hours. Thereafter the experiments were started, and the cells were cultured either alone, with ibuprofen (10 µM), with IL-1β (100 pg/mL), or with a combination of ibuprofen and IL-1β for 24 hours. Cell culture, RNA sequencing, RT-PCR and data analysis are described in online supplemental data S1.
Supplemental material
Results
The effects of ibuprofen on OA chondrocytes in neutral conditions
In the absence of exogenous cytokines, no genes were found to be differentially expressed between chondrocytes cultured with or without ibuprofen when the results were adjusted by false discovery rate.
The effects of ibuprofen on OA chondrocytes in inflammatory conditions
In inflammatory conditions (ie, in the presence of the OA-related cytokine IL-1β), ibuprofen induced the upregulation of 51 genes while 42 were downregulated in a statistically significant manner with a fold change >1.5 into either direction (table 1). All differentially expressed genes are listed in online supplemental tables S2 and S3.
Supplemental material
Supplemental material
All genes upregulated or downregulated by ibuprofen in the presence of IL-1β with FC ≥1.5 in either direction
The upregulated genes included anti-inflammatory factors such as peroxisome proliferator-activated receptor gamma (PPARG) and its coactivator PPARGC1B as well as IL-10 receptor subunit alpha. In addition, some genes associated with inflammation, including C-X-C motif chemokine receptor 3 (CXCR3), selectin E (SELE) and granulocyte-macrophage colony stimulating factor (CSF2/GM-CSF) were also upregulated (table 1).
On the other hand, several mediators of inflammation (such as IL23A, IL6 and NAMPT (nicotinamide phosphoribosyltransferase aka visfatin)) were downregulated, as was the catabolic enzyme ADAMTS6 (ADAM metallopeptidase with thrombospondin type 1 motif 6). Insulin-like growth factor-binding protein 4 (IGFBP4), which sequesters IGF and regulates chondrocyte proliferation,4 was also downregulated. Hyaluronan synthase 1 (HAS1) and stanniocalcin-1 (STC1), previously shown to be upregulated in inflamed OA synovium,5 were also downregulated by ibuprofen (table 1).
Differential expression of selected inflammation and cartilage-related genes (PPARG, PPARGC1B, CSF2, IL23, HAS1, IGFBP4, ADAMTS6 and IL6) was confirmed with RT-PCR using chondrocytes from a different set of 10 patients (online supplemental figure S4). As expected, IL-1β was shown to strongly increase the synthesis of prostanoids, and this increase was inhibited by ibuprofen (online supplemental figure S5).
Supplemental material
Supplemental material
When all genes affected by ibuprofen in a statistically significant manner in the presence of IL-1β were analysed with ingenuity pathway analysis (IPA), activated canonical pathways included several associated with inflammation and cell adhesion such as IL-8, integrin, ERK/MAPK and cAMP-mediated signalling pathways (table 2). Conversely, phosphatase and tensin homolog (PTEN) signalling was inhibited (table 2). Differentially expressed genes included in the significantly activated/inhibited pathways are listed in online supplemental table S6.
Supplemental material
Canonical IPA pathways significantly upregulated or downregulated (z-score ≥2.5 or ≤−2.5) by ibuprofen in the presence of IL-1β
Among the genes with FC >1.5 in either direction, STRING analysis identified IL6 (which was downregulated by ibuprofen) as a central node in the interaction network (figure 1). Other genes occupying central places include PPARG, granulocyte-macrophage colony-stimulating factor and selectin E (PPARG, CSF2 and SELE respectively, all upregulated by ibuprofen).
Interactions among the genes that were upregulated or downregulated by ibuprofen with an FC 1.5 or greater (in either direction) in IL-1β-treated cells. Genes with no identified interactions are excluded from the graph. Colours of the edges: green=activation, blue=binding, black=chemical reaction, red=inhibition, violet=catalysis, pink=posttranslational modification, yellow=transcriptional regulation, grey=other interaction.
Discussion
Ibuprofen did not have any significant effects on gene expression in primary OA chondrocytes cultured in the absence of added cytokines. This implies that ibuprofen has a neutral effect on chondrocyte transcriptome in non-inflamed joints. In cells treated with IL-1β, ibuprofen regulated the expression of both proinflammatory and anti-inflammatory factors and seemed to shift the balance to favour the latter.
Ibuprofen is a widely used non-selective NSAID. Like other NSAIDs, it exerts its effects by inhibiting prostanoid, particularly PGE2, synthesis by COX-1 and COX-2 enzymes. In addition to their role as mediators of a pain, prostanoids such as PGE2 mediate various inflammatory responses. Prostanoids have also been implicated in the pathogenesis OA by affecting cartilage matrix integrity and proteoglycan degradation as well as chondrocyte dedifferentiation and apoptosis.1 6 Cellular effects of prostanoids are mediated through G-protein coupled receptors; many prostaglandin receptor subtypes, particularly DP1, EP2, EP4 and IP,7 activate adenylate cyclase leading to increased intracellular levels of the multifunctional second messenger cAMP. By activating protein kinase A and transcription factors such as cAMP response element-binding protein, cAMP also regulates the expression of a number of genes.8 This pathway offers a possible prostanoid-dependent mechanism for the changes in gene expression seen in the present study. In addition, the IPA analysis showed that ibuprofen regulates several other inflammatory pathways which may mediate its effects on chondrocyte transcriptome by prostanoid dependent or independent manner.
In our data, ibuprofen increased the expression of PPARG and its coactivator 1 beta (PPARGC1B). PPARG expression has been shown to be downregulated in OA cartilage,9 and PPARG may affect the pathogenesis of OA by suppressing joint inflammation, downregulating the production of catabolic enzymes and inhibiting chondrocyte apoptosis.10 Induction of some proinflammatory factors such as CSF2/GM-CSF by ibuprofen can be regarded as a potentially deleterious effect, as CM-CSF has been shown to promote OA development and pain.11 To our knowledge, this is the first study linking NSAIDs to GM-CSF production in chondrocytes.
IL6 and IL23A as well as ADAMTS6 (ADAM metallopeptidase with thrombospondin type 1 motif 6) are examples of proinflammatory/catabolic factors that were suppressed by ibuprofen. Ibuprofen downregulated also hyaluronan synthase 1 (HAS1) and stanniocalcin-1 (STC1) both of which have been shown to be upregulated in inflamed OA joints.5 These data suggest that ibuprofen can, to some extent, ‘normalise’ the phenotype of OA tissue under inflammatory conditions. Notably, IL23A was the most strongly downregulated gene in our data. The potential local roles of this proinflammatory cytokine in OA cartilage appear relatively understudied, but its serum levels in patients with OA have been found to be higher compared with controls.12 IL-6 is considered a central proinflammatory mediator in OA.13 HAS1 is one of the three principal enzymes participating in the synthesis of hyaluronan, a central extracellular matrix (ECM) component. It may also promote inflammation by producing pericellular, monocyte-attracting hyaluronan coats.14 STC1 is a calcium-regulating and phosphate-regulating protein whose effects on cartilage appear to be complex. It may inhibit cartilage development,15 but its expression in synovial cells has also been linked to slower OA progression.16
Integrin signalling was the IPA pathway most strongly activated by ibuprofen. This is interesting, as dysregulated integrin signalling has been implicated in OA pathogenesis.17 Other significantly upregulated pathways include several linked to inflammation (such as IL-8, NF-κB and MAPK/ERK signalling). Looking at the specific genes included in these pathways and affected by ibuprofen (online supplemental table S6) reveals that these can be mostly considered negative feedback genes rather than the major proinflammatory mediators/effectors of these pathways. Examples include several integrins (ITGAM, ITGAX, ITGB2, ITGB3 and ITGB5) in the IL-8 and NF-κB pathways, growth factors and their receptors (VEGFA, VEGFC, HBEGF and FGFR3) in IL-8 signalling as well as anti-inflammatory MAPK phosphatases and PPAR pathway constituents (DUSP1, DUSP2, DUSP4, PRKAR1A, PRKAR1B, PRKAR2B and PPARG) in MAPK/ERK signalling.
Intriguingly, PTEN signalling was inhibited by ibuprofen. PTEN is a modulator of phosphoinositide 3-kinase/Akt (PI3K/Akt) signalling with various potential effects including promotion of apoptosis, regulation of cell adhesion and inhibition of cell proliferation. PTEN is upregulated in OA chondrocytes, where it inhibits the production of ECM components,18 and interventions that inhibit PTEN slow the development of osteoarthritic changes in cartilage.19 To our knowledge, PTEN has not previously been linked to NSAIDs in cartilage.
Previous studies have investigated the effects of NSAIDs and COX-2 selective inhibitors on cartilage/synovial explants.6 20 Both prostaglandin-mediated and prostaglandin-independent effects have been observed; these include, for example, inhibition of chondrocyte apoptosis, reduction of nitric oxide synthesis as well as reduced production of catabolic MMPs on IL-1β stimulation.1 Our study expands these results by investigating the whole transcriptome of ibuprofen-treated OA chondrocytes and provides a starting point for future studies.
In conclusion, ibuprofen alone had no significant effects on gene expression in chondrocytes supporting cartilage safety of COX inhibitors in the treatment of OA pain. When used in a setting of joint inflammation, ibuprofen seems to shift chondrocyte transcriptome towards an anti-inflammatory phenotype.
Ethics statements
Patient consent for publication
Ethics approval
The study was approved by the Ethics Committee of Tampere University Hospital, Tampere, Finland (decision reference ETL R16076), and performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from the patients.
Acknowledgments
We thank research coordinator Heli Kupari for her assistance with the cartilage samples. We are also grateful to Ms. Meiju Kukkonen and Mrs. Salla Hietakangas for excellent technical assistance, as well as to Mrs. Heli Määttä for great secretarial help.
References
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
Contributors Conceptualisation, methodology: all authors. Formal analysis: AP, TM, EM. Investigation: AP, LT, TL, MH. Writing: AP, EM. Visualisation: AP. Supervision and funding: EM.
Funding This study was supported by grants from Finnish Society of Rheumatology and the Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital.
Disclaimer The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.