Abstract
Receptor crosstalk, the interaction between different receptors to modulate signaling, is crucial for fine-tuning the inflammatory responses of innate immune cells. Although the synergistic crosstalk between Toll-like receptor (TLR)4 and Fc gamma receptor (FcγR) is well documented, the detailed mechanism underlying this synergy remains unclear. In this study, we addressed this knowledge gap by imaging the molecular organization of TLR4 and FcγR on the macrophage cell surface and correlating it with their synergistic co-activation using ligands functionalized on lipid bilayers. We confirmed that co-activation of TLR4 and FcγR enhances whole-cell pro-inflammatory responses and tyrosine phosphorylation at the receptor level. Super-resolution microscopy revealed that TLR4 and FcγR each form discrete nanoclusters after ligand stimulation, and their synergistic co-activation increases both the size and spatial overlap of these nanoclusters. Contrary to previous assumptions that TLR4 and FcγR form heterodimers during their crosstalk, our results emphasize the critical role of nanoscale spatial organization between distinct receptor clusters in modulating innate immune responses. Additionally, these findings align with similar receptor interaction mechanisms that we previously reported in other receptor pairs, such as Dectin-1/TLR2 and FcγR/TLR2, suggesting that nanocluster interactions may represent a predominant mechanism governing crosstalk between TLRs and ITAM-containing receptors.