RNA Interference Mediated Interleukin-1β Silencing in Inflamed Chondrocytes Decreases Target and Downstream Catabolic Responses. (17th March 2016)

• Record Type: Journal Article
• Authors: Ortved, Kyla F.; Austin, Bethany S.; Scimeca, Michael S.; Nixon, Alan J.
• Other Names: van der Kraan Peter M. Academic Editor.
• Is Part Of: Arthritis. Volume 2016(2016)
• Publication Date: 2016-03-17
• Subjects: Arthritis -- Periodicals; 616.722005
• DOI: 10.1155/2016/3484961
• Languages: English
• ISSNs: 2090-1984

Abstract

Abstract : Posttraumatic activation of the catabolic cascade plays a major role in degradation of cartilage. Interleukin-1 β (IL-1 β ), a primary instigator in the catabolic axis, is upregulated in chondrocytes following injury. IL-1 β activates key degradative enzymes, including MMPs and aggrecanases, and other proinflammatory mediators such as PGE2 which contribute to ECM breakdown. Posttranscriptional silencing of IL-1 β by RNA interference (RNAi) may drive a reduction in IL-1 β . We hypothesized that transduction of chondrocytes using rAAV2 expressing a short hairpin RNAi motif targeting IL-1 β (shIL-1 β ) would significantly decrease IL-1 β expression and, in turn, decrease expression of other catabolic enzymes. Chondrocyte cultures were transduced with rAAV2-tdT-shIL-1 β in serum-free media. The fluorescent protein, tdTomato, was used to determine transduction efficiency via flow cytometry and fluorescent microscopy. Cells were stimulated with lipopolysaccharide (LPS) 48 hours following transduction. After 24-hour stimulation, supernatants were collected for cytokine analysis, and cells lysed for gene expression analysis. IL-1 β knockdown led to significantly decreased expression of IL-1β, TNF-α, and ADAMTS5 . PGE2 synthesis was also significantly downregulated. Overall, effective silencing of IL-1 β using rAAV2 vector expressing a short hairpin IL-1 β knockdown sequence was shown. Additionally, significant downstream effects were evident, including decreased expression of TNF-α and ADAMTS5 . Targeted silencing of catabolic cytokines may provide a promising treatment avenue for osteoarthritic (OA) joints.