Toll-Like Receptor Signaling In Macrophages Is Regulated By Extracellular Substrate Stiffness And Rho-Associated Coiled-Coil Kinase (Rock1/2)

Macrophages participate in immunity, tissue repair and tissue homeostasis. Activation of Toll-like receptors (TLRs) by conserved exogenous or endogenous structures initiates signaling cascades that result in the release of cytokines such as tumor necrosis factor alpha (TNF alpha). Extracellular substrate stiffness is known to regulate functions of non-immune cells through a process called mechanotransduction, yet less is known about how physical cues affect macrophage function or TLR signaling. To investigate this question, we cultured murine primary bone marrow-derived macrophages (BMMs) and RAW264.7 cells on fibronectin-coated polyacrylamide (PA) gels of defined stiffnesses (1, 20 and 150 kPa) that approximate the physical properties of physiologic tissues. BMMs on all gels were smaller and more circular than those on rigid glass. Macrophages on intermediate stiffness 20 kPa PA gels were slightly larger and less circular than those on either 1 or 150 kPa. Secretion of the pro-inflammatory cytokine,TNF alpha, in response to stimulation of TLR4 and TLR9 was increased in macrophages grown on soft gels versus more rigid gels, particularly for BMMs. Inhibition of the rho-associated coiled-coil kinase 1/2 (ROCK1/2), key mediators in cell contractility and mechanotransduction, enhanced release of TNF alpha in response to stimulation of TLR4. ROCK1/2 inhibition enhanced phosphorylation of the TLR downstream signaling molecules, p38, ERK1/2 and NF kappa B. Our data indicate that physical cues from the extracellular environment regulate macrophage morphology and TLR signaling. These findings have important implications in the regulation of macrophage function in diseased tissues and offer a novel pharmacological target for the manipulation of macrophage function in vivo.