Quantitative single-molecule imaging of TNFR1 reveals zafirlukast as antagonist of TNFR1 clustering and TNFα-induced NF-&x138;B signaling

TNFR 1 is a crucial regulator of NF-& x138;B-mediated proinflammatory cell survival responses and programmed cell death (PCD). Deregulation of TNF alpha- and TNFR1-controlled NF-& x138;B signaling underlies major diseases, like cancer, inflammation, and autoimmune diseases. Therefore, although being routinely used, antagonists of TNF alpha might also affect TNFR2-mediated processes, so that alternative approaches to directly antagonize TNFR1 are beneficial. Here, we apply quantitative single-molecule localization microscopy (SMLM) of TNFR1 in physiologic cellular settings to validate and characterize TNFR1 inhibitory substances, exemplified by the recently described TNFR1 antagonist zafirlukast. Treatment of TNFR1-mEos2 reconstituted TNFR1/2 knockout mouse embryonic fibroblasts (MEFs) with zafirlukast inhibited both ligand-independent preligand assembly domain (PLAD)-mediated TNFR1 dimerization as well as TNF alpha-induced TNFR1 oligomerization. In addition, zafirlukast-mediated inhibition of TNFR1 clustering was accompanied by deregulation of acute and prolonged NF-& x138;B signaling in reconstituted TNFR1-mEos2 MEFs and human cervical carcinoma cells. These findings reveal the necessity of PLAD-mediated, ligand-independent TNFR1 dimerization for NF-& x138;B activation, highlight the PLAD as central regulator of TNF alpha-induced TNFR1 oligomerization, and demonstrate that TNFR1-mEos2 MEFs can be used to investigate TNFR1-antagonizing compounds employing single-molecule quantification and functional NF-& x138;B assays at physiologic conditions.

Automated summary

TNFR1 is a crucial regulator of proinflammatory cell survival and programmed cell death, and deregulation of TNFα and TNFR1 signaling underlies major diseases. Therefore, finding alternative approaches to directly antagonize TNFR1 is beneficial.