Inhibition of toll-like receptor 4 protects against inflammation-induced mechanobiological alterations to intervertebral disc cells

Abstract

Intervertebral disc (IVD) degeneration is associated with elevated levels of inflammatory cytokines implicated in disease aetiology and matrix degradation. Toll-like receptor-4 (TLR4) has been shown to participate in the inflammatory responses of the nucleus pulposus (NP) and its levels are upregulated in disc degeneration. Activation of TLR4 in NP cells leads to significant, persistent changes in cell biophysical properties, including hydraulic permeability and osmotically active water content, as well as alterations to the actin cytoskeleton. The study hypothesis was that inflammation-induced changes to cellular biomechanical properties and actin cytoskeleton of NP cells could be prevented by inhibiting TLR4 signalling. Isolated NP cells from bovine discs were treated with lipopolysaccharide (LPS), the best studied TLR4 agonist, with or without treatment with the TLR4 inhibitor TAK-242. Cellular volume regulation responses to step osmotic loading were measured and the transient volume-response was captured by time-lapse microscopy. Volume-responses were analysed using mixture theory framework to investigate hydraulic permeability and osmotically active intracellular water content. Hydraulic permeability and cell radius were significantly increased with LPS treatment and these changes were blocked in cells treated with TAK-242. LPS-induced remodelling of cortical actin and IL-6 upregulation were also mitigated by TAK-242 treatment. These findings indicated that TLR4 signalling participated in NP cell biophysical regulation and may be an important target for mitigating altered cell responses observed in IVD inflammation and degeneration.