Dura Role Of TNF In Inducing Osteoclast Differentiation

Background TNF is the major cytokine driving inflammation in rheumatoid arthritis(RA), a chronic inflammatory disease affecting about 1% of the world's population and characterized by synovial inflammation and joint destruction, leading to severe morbidity and premature mortality. Transgenic mice over-expressing TNF(TNF-Tg mice) develop a form of arthritis that isvery similar to human RA. Although anti-TNF therapies have significantly reduced the morbidity and joint destruction in RA, they are expensive, and only about60% of patients have a good response to these agents. In non-responding patients, TNF inhibitors typically are administered for several months before a decision is made to switch to an alternative treatment,which is often another TNF inhibitor that also may be ineffective. Thus, there is a need to better understand how TNF induces joint inflammation and destruction.Purpose We aim to explore the mechanism of TNF role in osteoclast,for TNF induces bone loss in common bone diseases by promoting osteoclast formation directly and indirectly, and it can also inhibit osteoclast differentiation through expressing NF-k B p100, we use the TNF expression in different OCPs to induce the joint inflammation and destruction to acquire the dura role of the TNF in inducing osteoclast differentiation..Methods To characterize TNF-induced OCPs, we examined expression of the cell surface markers CD11 b,F4/80, Ly6 C,CD11c and Gr1,Freshly isolated bone marrow cells(BMCs) from 3-month-old C57Bl6 mice were cultured with M-CSF,M-CSF~+TNF or M-CSF ~+RANKL to recruit OCPs, cells attached to the dishes were collected and stained with antibodies to analyze expression of cell surface markers by flowcytometry; Then We sorted each of these OCPs into four populations were stained with the fluorescent-labeled, Ly6C~+Gr1~-and Ly6C~-Gr1~- populations from CD11b~+F4/ 80~+ cells were sorted by flowcytometry to observe the ability of TNF-induced macrophages express M1 markers;subsequently to investigate the role of RelB in TNF induced OC formation, M-,R-,and T-OCPs generated were treated with PBS,RANKL,TNF or RANKL~+TNF by which time mature Ocs had formed,Cell lysates were subjected to Western blot analysis of RelB and ?-actin.we next to study the biphasic effect of RelB on OC formation,BMCs from C57Bl6 mice were cultured with M-CSF by treatment of ?volume of p MX-GFP or p MX-GFP-RelB retroviral supernatant,GFP~+F4/80~+ cells were analyzed by flow cytometry and RelB protein levels in GFP or RelB expressing cells that had been treated with P,R or T were tested by Western blot,and m RNA expression of NFATc1 normalized to ?-actin was tested by real-time PCR.Results1. TNF switches the differentiation of M-CSF-induced Ly6C~-Gr1~- M2 to Ly6C~+Gr1~-CD11c~+ and Ly6C~-Gr1~-CD11c~+ inflammatory M1 macrophages.2. TNF-induced CD11b~+F4/80~+Ly6C~+Gr1~- and CD11b~+F4/80~+Ly6C~-Gr1~-CD11c~+ macrophages have higher OC forming potentialthan M-CSF-induced macrophages.3. Ly6C~+Gr1~- cells express M1 macrophage markers and Ly Ly6C~-Gr1~- cells from T-OCPs are also polarized to M1 macrophages.4. TNF induction of RelB promotes the differentiation of Ly6C~+Gr1~- and Ly6C~-Gr1~-macrophages.5. TNF inhibits RANKL-induced osteoclastogenesis from OCPs primed by M-CSF alone, but not by RANKL and TNF.6. Biphasic effect of RelB on OC formationConclusions1.TNF induction of RelB directly mediates terminal osteoclast differentiation independent of NFATc1 and limits RANKL-induced osteoclastogenesis by inhibiting NFATc1 activation.2.The dominant role of TNF is to expand the OCP pool by switching the differentiation of M-CSF-induced M2 to M1 macrophages with enhanced osteoclast forming potential. Strategies to degrade RelB could prevent TNF-induced M2/M1 switching and reduce osteoclast formation.