Effect of high mobility group box-1 on receptor for advanced glycation endproducts (RAGE) and toll-like receptor-4 (TLR4) diffusion and signaling.

High mobility group box-1 (HMGB1) is a damage-associated molecular pattern and a ligand for multiple immune receptors, including receptors for advanced glycation endproducts (RAGE) and toll-like receptor 4 (TLR4). Binding of HMGB1 to these receptors initiates inflammatory signaling cascades implicated in diseases such as septic shock, chronic inflammation, and autoimmune disorders. The spatial and molecular mechanisms underlying HMGB1-mediated receptor signaling at the plasma membrane remain poorly understood. Here, single-particle tracking was used to investigate the effects of extracellular HMGB1 on RAGE's and TLR4's diffusion properties in human embryonic kidney 293 (HEK293) cells. HMGB1 treatment reduced the Brownian diffusion coefficients by 27% and 24% for RAGE (with and without TLR4 co-expression, respectively) and by 40% and 21% for TLR4 (with and without RAGE co-expression, respectively). HMGB1 treatment reduced the size of the confined domains the receptors diffuse into and increased the number of times the two receptors came in close proximity. In contrast, HMGB1 had no detectable effect on the diffusion properties of an S391A RAGE mutant, which lacks the ability to recruit adapter proteins. Lipid diffusion measurements revealed that HMGB1 treatment decreases lipid membrane fluidity in all cell lines studied. Similarly, phalloidin staining revealed no detectable HMGB1-induced changes in actin organization in RAGE expressing cells. Functionally, HMGB1 treatment activated the extracellular signal-regulated kinase (ERK) (1/2) signaling pathway through both RAGE and TLR4, whereas ERK (1/2) and p38 MAPK activation by HMGB1 were abolished in cells expressing S391A RAGE. Together, these findings show that HMGB1 reduced confined receptor diffusion and increased nanoscale proximity while promoting MAPK signaling. This work sheds new light on the nanoscale regulation of innate immune receptors by HMGB1 and emphasizes membrane biophysics as one critical component of inflammatory signaling outcomes.