Cytoskeletal Prestress Regulates RIG-I-Mediated Innate Immunity

Innate immunity is the body's first line of defense for mounting robust antiviral signaling. However, the role of cytoskeletal prestress, a hallmark of cellular mechanotransduction, in regulating innate immune pathways such as retinoic acid-inducible gene I (RIG-I) signaling remains poorly understood. Herein, we show that cells on soft vs. rigid substrates elicit cytoskeletal prestress-dependent activation of RIG-I signaling, leading to differential type-I interferon (IFN) gene expression. Cells were cultured on soft (0.6 kPa) and stiff (8.5 kPa) substrates to modulate cellular traction and prestress, followed by transfection of Poly(I:C), a synthetic viral dsRNA mimic, to measure the RIG-I-mediated innate immune response. Cells on soft substrates show minimal activation of RIG-I signaling, resulting in low expression of IFN-β1 and other IFN-stimulated genes (ISGs), compared to cells on stiff substrates. We further demonstrate that activation of TANK Binding Kinase 1 (TBK1), a downstream effector of the RIG-I pathway, is inhibited in cells on soft substrates due to the cytoplasmic sequestration of the Yes-associated protein (YAP), a HIPPO pathway effector protein. In contrast, cells on stiffer substrates experienced decreased TBK1 inhibition due to the nuclear localization of YAP and exhibited elevated TBK1 activation and heightened IFN and ISG expressions. Together, we demonstrate that cytoskeletal prestress represents a key biophysical regulator of innate immune signaling.