P078 Transforming growth factor-beta dampens pro-inflammatory interleukin-6 signaling in chondrocytes by decreasing the interleukin-6 receptor

Abstract

Career situation of first and presenting authorYoung investigatorIntroductionThe pro-inflammatory cytokine interleukin-6 (IL-6) is present in high levels in arthritic diseases and has been implicated in cartilage degeneration. In contrast, transforming growth factor-β (TGF-β) is a homeostatic regulator of cartilage that decreases the catabolic effects of pro-inflammatory cytokines in chondrocytes. In tissues other than cartilage, multiple mechanisms of cross-talk between TGF-β and IL-6 are reported.ObjectivesIn this study, we investigated whether TGF-β is able to control IL-6 signaling in chondrocytes.MethodsHuman primary chondrocytes and the human G6 chondrocyte cell line were stimulated with rhTGF-β1 (1.0 ng/ml). We determined mRNA expression levels of IL-6 and IL-6 receptor (IL-6R) using qPCR, measured IL-6 protein release using Luminex technology and IL-6R protein levels by Western Blot. rhIL-1β was used as a positive control for IL-6 induction. Induction of signal transducer and activator of transcription (STAT)3 phosphorylation by TGF-β was determined using Western Blot in presence of the IL-6R inhibitor Tocilizumab. Induction of suppressor of cytokine signaling (SOCS)3 gene expression was used as a read-out for IL-6/STAT3 mediated transcriptional activity.ResultsTGF-β increased both IL-6 mRNA expression and protein release in G6 and primary chondrocytes. Moreover, we observed activation of the main IL-6R downstream signaling protein STAT3 after TGF-β exposure, which could be completely prevented by blockade of the IL-6R, suggesting IL-6 dependency. Surprisingly however, TGF-β did not increase expression of the well known IL-6 target gene SOCS3, despite the increased levels of IL-6 and p-STAT3 we observed in these cells. Moreover, the effect of exogenously added rhIL-6 was markedly inhibited by TGF-β, as 6h pre-treatment prevented rhIL-6-mediated induction of both SOCS3 and p-STAT3. Interestingly, we observed decreased expression of the IL-6R in both primary and G6 chondrocytes after exposure to TGF-β, which might explain the decreased intracellular IL-6 signaling. This effect was not dependent on IL-6 itself, as IL-1β did not affect IL-6R expression, despite being a strong inducer of IL-6. Finally, we showed that rescuing IL-6R levels using soluble IL-6R dose-dependently restored induction of SOCS3 and p-STAT3 by rhIL-6 in the presence of TGF-β.ConclusionsOur study reveals a novel, protective effect of TGF-β that could help preserve cartilage homeostasis under inflammatory conditions by dampening IL-6 signaling via IL-6R down regulation. In arthritic diseases, the presence of soluble IL-6R might bypass this protective TGF-β effect and contribute to cartilage damage.Disclosure of InterestNone declared

The hUCMSCs Exerts Excellent Anti-inflammation Effect which is Different from TCZ IL-6 Antagonist by Inducing High IL-6 Secretion

“…4b). Based on the above experimental results, we drew the following conclusions: IL-6 is mainly secreted by hUCMSCs, while IL-6R is mainly expressed on the cell membrane of PBMCs, and the absence of either IL-6 or IL-6R hinders activation of IL-6-related "anti-in ammatory responses" [15].…”

“…The synthesis of IL-6 is regulated at both the transcriptional and post-transcriptional levels. Upon infection or injury, the biosynthesis of IL-6 is induced immediately as an emergency signal to activate host defenses and an acute immune response [15]. After the environmental stress diminishes or disappears, the concentration of IL-6 decreases and returns to baseline [2].…”

“…IL-6 exerts its biological activities mainly through IL-6R, which exists in two forms, a membrane-bound form (mIL-6R) that exerts its actions through cell membranes, and a soluble form (sIL-6R) that is secreted into the extracellular space and in body uids. The latter mainly binds to IL-6 and exerts its activity in extracellular environments [10,15,16]. Signals mediated by mIL-6R and sIL-6R are termed classical signaling and trans-signaling, respectively.…”

“…mIL-6R is the predominant form of IL-6R in human [17,18]. These different IL-6R form may be a key to the different anti-in ammatory pathways involving IL-6 [15].…”