A2M inhibits inflammatory mediators o by blocking IL-1β/NF-κB pathway

A hallmark of osteoarthritis (OA) is cartilage degeneration, which has been previously correlated with dramatic increases in inflammatory enzymes. Specifically, interleukin-1 beta (IL-1 beta) and subsequent upregulation of nuclear factor kappa B (NF-kappa B) is implicated as an important player in the development of posttraumatic osteoarthritis (PTOA). Alpha 2-macroglobulin (A2M) can inhibit this inflammatory pathway, making it a promising therapy for PTOA. Herein, we demonstrate that A2M binds and neutralizes IL-1 beta, blocking downstream NF-kappa B-induced catabolism seen in in vitro. Human chondrocytes (cell line C28) were incubated with A2M protein and then treated with IL-1 beta. A2M was labeled with VivoTag (TM) 680 to localize the protein postincubation. The degree of binding between A2M and IL-1 beta was evaluated through immunoprecipitation (IP). Catabolic proteins, including IL-1 beta and NF-kB, were detected by Western blot. Pro-inflammatory and chondrocyte-related gene expression was examined by qRT-PCR. VivoTag (TM) 680-labeled A2M was observed in the cytoplasm of C28 human chondrocytes by fluorescence microscopy. IP experiments demonstrated that A2M could bind IL-1 beta. Additionally, western blot analysis revealed that A2M neutralized IL-1 beta and NF-kappa B in a dose-dependent manner. Moreover, A2M decreased levels of MMPs and TNF-alpha and increased the expression of cartilage protective genes Col2, Type2, Smad4, and aggrecan. Mostly importantly, A2M was shown to directly neutralize IL-1 beta to downregulate the pro-inflammatory responses mediated by the NF-kB pathway. These results demonstrate a mechanism by which A2M reduces inflammatory catabolic activity and protects cartilage after joint injury. Further in vivo studies are needed to fully understand the potential of A2M as a novel PTOA therapy.