In addition, autophagy things generated products have been linked to innate and adaptive defenses. Although OBs have been shown to express NLRP 3 re quired for caspase 1 activation associated with OB death in response to Inhibitors,Modulators,Libraries infection, we find that MSU activates NLRP3 in human OBs with no production of pro IL 1B or IL 1B. We identified a new role for NLRP3 in MSU induced autophagy in these bone cells. In OBs, MSU upregulates NLRP3, which is a positive regulator of the formation of MSU autophagosomes. Phagocytosis of MSU by OBs is a prerequisite process to MSU induced autophagy. However, signaling pathways of phagocytosis by OBs are not similar to those of professional phago cytes. In addition, OBs stimulated by MSU reduce their proliferation rate without change of their viability, and MSU crystals remain intact inside OBs.
Together with the bone matrix irregularly calcified and the reduced number of OBs present on the osteoid close to MSU deposits, the present results indicate that MSU mi crocrystals, when phagocytized by the nonprofessional phagocyte OBs, activate NLRP3, which in turn upregu lates a nonproductive macroautophagy that fails to Inhibitors,Modulators,Libraries clear MSU. Reduced anabolic functions and increased cata bolic functions of OBs subsequent to MSU phagocytosis also suggest that MSU activated OBs can be responsible for reduction of calcified bone matrix and increase of matrix Inhibitors,Modulators,Libraries degradation. Moreover, inefficient phagocytosis and autophagy of these MSU microcrystals lead to their persistent presence in autophagosomes without degradation.
Methods Reagents The incubation media MEM, FBS, and penicillin streptomycin were purchased from Wisent Inc. Triclinic MSU microcrystals were kindly provided by Dr R. Inhibitors,Modulators,Libraries De Mdicis Inhibitors,Modulators,Libraries and were used under sterile pyrogen free conditions. The mean size of the MSU mi crocrystals used was 10 1. 25 um, as determined by scanning electron microscopy. MSU was suspended at 10 mg ml in MEM supplemented with 10% FBS. Accu tase was from eBioscience. Calcein AM, propidium iodide, cell tracker orange CMTMR, lipofectamine and Trizol were purchased from Invitrogen Canada. Colchicine, cytochala sin D, SB203580, PD98069, 3 methyladenine, spautin 1, dynasore, and alizarin red S were obtained from Sigma Chemical Co. Piceatannol, wortmannin, LY4294002, G6979, B glycerophosphate, and 4 amino 5 7 pyrazolo pyrimidine were from Calbiochem. GF109203X was from Biomol International Lp.
The IB antibodies were from Cell Signaling Technology. The rabbit polyclonal anti pro IL 1B antibody was from Santa Cruz Biotechnology. IL 1B was assessed by using the DuoSet ELISA Development kit. The mouse monoclo nal anti NLRP3 antibody and the rabbit polyclonal anti LC3B antibody were from Novus Biologicals. Cell preparation All volunteers signed a consent form inhibitor Nintedanib that included participation to the present study and publication of the results in accordance with the Declaration of Helsinki.