Hyaluronan oligosaccharides in part mediate LPS-induced inflammation in human chondrocytes by up-regulating JAGGED1-NOTCH pathway

Introduction: Notch signalling is involved in the control of a number of processes and has recently also been linked to inflammatory diseases, including rheumatoid arthritis [1]. Notch can be activated by a wide array of inflammatory stimuli, including TLR ligands [2]. After stimulation, TLR4 leads to NF-kB and MAPK activation that increases JAG1 expression, responsible, in turn, of NOTCH receptorsactivation. The consequent up-regulation of IRF8, together with NF-kB, leads to the transcription of pro-inflammatory mediators [3]. In order to clarify the role of NOTCH signalling in pathologies involving cartilage injury, our study aimed to investigate its involvement in an experimental model of LPS and 6-mer HA-induced inflammatory response in human cultured chondrocytes. Methods: Changes in TLR-4, MAPK, JAGGED1, NOTCH1/3, RBP-JK, IRF8, HES1, IL-1beta and MMP-13 mRNA expression of LPS and 6-mer HA-stimulated chondrocytes were examined by qPCR, JAGGED1, IRF8, NOTCH1/3, IL-1beta and MMP-13 protein levels by western blot and ELISA. The NF-kB activation was also evaluated as well as experiments using siRNA JAGGED1 was also performed. Results:Treatment of chondrocytes with LPS produced NF-kB activation, as well as a significant increment in TLR-4, MAPK, JAGGED1, NOTCH1/3, RBP-JK, IRF8, HES1, IL-1beta and MMP-13 expression. 6-mer HA produced a similar effect but with less intensity. The exposition of cells to both LPS and 6-mer HA resulted in an additive effect on the considered parameters. Specific siRNAs blocking JAGGED1 confirmed the involvement of this pathway. Using a specific NF-kB inhibitor we also found that NF-kB pathway seems to be more effective than NOTCH pathway in the in transcription of inflammatory mediators. Discussion: Taken together, our results suggest that the activation of TLR4 induced by both LPS and HA oligosaccharides leads to the acute inflammatory response mediated both through the direct NFkB/ MAPK pathway and indirectly by JAGGED1/NOTCHs pathway.