Blockade of 4-1BB and 4-1BBL Interaction Reduces Obesity-Induced Skeletal Muscle Inflammation
Figure 2
Inhibition of 4-1BB/4-1BBL interaction reduces inflammatory responses in myotube/macrophage coculture. C2C12 myotubes (Mb) were established for 3 days and then cocultured with Raw264.7 macrophages (M) (25%) in serum-free DMEM for 24 h. ((a) and (b)) Raw macrophages were directly seeded into the plates containing established myotubes (Contact) or were added to transwell inserts (Transwell). (a) Expression of 4-1BB, 4-1BBL, and inflammatory cytokine (TNF, IL-6, and MCP-1) mRNAs was analyzed by qRT-PCR. (b) TNF, IL-6, and MCP-1 proteins released in the culture media were measured by ELSA. ((c) and (d)) Myotubes and Raw264.7 macrophages in the contact cocultures (Contact-Mb/M) or the transwell cocultures (Transwell-Mb/M) (control) were separated using a CD11b MicroBeads system. Expression of 4-1BB, 4-1BBL, and inflammatory cytokine mRNAs in isolated myotubes (c) and in isolated-Raw macrophages (d) was analyzed by qRT-PCR. (e–g) Raw264.7 macrophages were seeded onto myotubes with or without pretreatment with neutralizing anti-4-1BBL antibody (TKS-1, 5 g/mL) or rat IgG (5 g/mL) in serum-free DMEM for 24 h. (e) TNF, IL-6, and MCP-1 proteins released in the culture media were measured by ELISA. ((f) and (g)) Myotubes and Raw264.7 macrophages in the TKS-1- or rat IgG-treated cocultures were separated using a CD11b MicroBeads system. Expression of inflammatory cytokine mRNAs in isolated myotubes (f) and in isolated-Raw macrophages (g) was analyzed by qRT-PCR. Data are means ± SEM of three independent triplicate experiments. *, **, and *** compared with controls.
