RNA Interference Mediated Interleukin-1 β Silencing in Inflamed Chondrocytes Decreases Target and Downstream Catabolic Responses

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 PGE 2 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 . PGE 2 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.