ADAP Y571 phosphorylation is required to prime STAT3 for activation in TLR4-stimulated macrophages

Adhesion and degranulation–promoting adapter protein (ADAP), originally identified as an essential adaptor molecule in TCR signaling and T cell adhesion, has emerged as a critical regulator in innate immune cells such as macrophages; however, its role in macrophage polarization and inflammatory responses remains unknown. In this study, we show that ADAP plays an essential role in TLR4-mediated mouse macrophage polarization via modulation of STAT3 activity. Macrophages from ADAP-deficient mice exhibit enhanced M1 polarization, expression of proinflammatory cytokines and capacity in inducing Th1 responses, but decreased levels of anti-inflammatory cytokines in response to TLR4 activation by LPS. Furthermore, overexpression of ADAP enhances, whereas loss of ADAP reduces, the LPS-mediated phosphorylation and activity of STAT3, suggesting ADAP acts as a coactivator of STAT3 activity and function. Furthermore, the coactivator function of ADAP mostly depends on the tyrosine phosphorylation at Y571 in the motif YDSL induced by LPS. Mutation of Y571 to F severely impairs the stimulating effect of ADAP on STAT3 activity and the ability of ADAP to inhibit M1-like polarization in TLR4-activated mouse macrophages. Moreover, ADAP interacts with STAT3, and loss of ADAP renders mouse macrophages less sensitive to IL-6 stimulation for STAT3 phosphorylation. Collectively, our findings revealed an additional layer of regulation of TLR4-mediated mouse macrophage plasticity whereby ADAP phosphorylation on Y571 is required to prime STAT3 for activation in TLR4-stimulated mouse macrophages.