Comprehensive effects of ibuprofen on gene expression in chondrocytes as determined by RNA-Seq

Purpose: Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat pain associated with osteoarthritis (OA). In addition to their well-known analgesic effects, there is also some data about their potential disease-modifying effects in OA. These effects are controversial, as both potential benefits (e.g. inhibition of harmful low-grade inflammation in the joint, reduction of cartilage catabolism) and harms (e.g. impairment of cartilage anabolism, accelerated radiographic joint destruction) have been reported in different studies. We carried out a genome-wide expression analysis on the effects of the NSAID ibuprofen to gene expression in OA chondrocytes using RNA-Seq, with or without stimulation by the central proinflammatory cytokine interleukin 1 (IL-1). Methods: Cartilage samples were obtained from ten OA patients undergoing knee replacement surgery, and chondrocytes were isolated by enzyme digestion. The cells were cultured 1) alone, 2) with ibuprofen, 3) with IL-1 or 4) with IL-1 and ibuprofen, for 24 hours. Total mRNA was sequenced using Illumina HiSeq2500 system and aligned to a reference genome. Differentially expressed genes were identified with edgeR using pairwise comparisons. Functional analysis was performed using Ingenuity Pathway Analysis (IPA), and interactions between the protein products of differentially expressed genes were studied with STRING. Results: In the absence of exogenous cytokines, no differentially expressed genes were identified when ibuprofen-treated chondrocytes were compared to those cultured without ibuprofen. When comparing chondrocytes treated with IL1 and ibuprofen to those treated with IL1 alone, 60 genes were up- and 48 downregulated in a statistically significant manner with a fold change > 1.5 into either direction. The upregulated genes included several anti-inflammatory factors such as peroxisome proliferator-activated receptor gamma (PPARG) and its coactivator PPARGC1B, interleukin 10 receptor alpha subunit (IL10RA), but also genes linked to increased cartilage catabolism, including phosphodiesterase 5A (PDE5A) and C-X-C motif chemokine receptor 3 (CXCR3). On the other hand, several mediators of inflammation (such as IL-23A and IL-6) were among the downregulated genes. HAS1 and STC1, previously shown to be upregulated in inflamed OA synovium compared to non-inflamed (Lambert et al. Arthritis & Rheumatology 2014), were downregulated by ibuprofen. In the pathway analysis (IPA), many canonical pathways were identified. Significantly upregulated pathways include several associated with cell adhesion and inflammation, such as integrin, ERK/MAPK and VEGF signaling pathways, as well as cAMP-mediated signaling, while PTEN signaling was downregulated. In the analysis of diseases and bio functions, cellular migration and movement were found to be upregulated, while inflammation and degradation of connective tissue were downregulated. Also, genes promoting cell viability were upregulated. In the STRING analysis (see figure), IL-6 (which was downregulated by ibuprofen) was identified as a central node in the interaction network. Other genes occupying central places in the network include peroxisome proliferator-activated receptor gamma, granulocyte-macrophage colony-stimulating factor and selectin E (PPARG, GM-CSF and SELE respectively, all upregulated by ibuprofen). Conclusions: In conclusion, ibuprofen alone did not have any significant effect on gene expression in primary OA chondrocytes. In cells treated with IL-1, ibuprofen increased the expression several genes associated with regulation of inflammation and cell movement. On the other hand, inflammatory mediators and catabolic factors were downregulated. Interestingly, genes downregulated by ibuprofen include those shown to be upregulated in inflamed OA tissue compared to non-inflamed tissue, suggesting that ibuprofen can, to some extent, “normalize” the phenotype of OA tissue under inflammatory conditions. No findings pointing to catabolic effects of NSAIDs were identified at the level of genome-wide expression analysis.