Toll-like receptors (TLR) play a critical role in host defence by initiating sterile inflammatory responses to endogenous damage-associated molecular patterns (DAMPs), which are released following tissue injury. Upon ligand binding, TLRs induce inflammation via NF-κB activation and the expression of pro-inflammatory cytokines, including TNF-α and IL-6. Tissue injury accompanying biomaterial implantation likely results in the release of DAMPs. However, the role of DAMP-TLR signaling in host responses to solid polymer implants in unclear. The aim of our research is to understand the molecular mechanisms through which the innate immune system responds to materials, in order to identify potential molecular targets for modulating cell-material interactions. Our current focus is examining the molecular mechanism through which biomaterial-adsorbed DAMPs activate macrophages and mediate the inflammatory biomaterial microenvironment.
In this study, we used cell lysate generated from NIH3T3 fibroblasts via freeze-thaw cycling as a complex, in vitro source of DAMPs. Lysate or fetal bovine serum (FBS) was adsorbed to polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA) surfaces or tissue culture polystyrene (TCPS) surfaces for 30 min and rinsed with PBS. RAW-Blue™ (Invivogen) macrophages, an NF-κB reporter cell line, were seeded on the different polymer surfaces for 20 hours and the NF-κB activity was measured indirectly using an inducible-secreted embryonic alkaline phosphatase (SEAP) activity assay. The concentration of IL-6 and TNF-α in the cell supernatant was measured by ELISA. TLR2 and TLR4 signalling were inhibited using a TLR2 neutralizing antibody and TLR4 inhibitor, CLI-095. PAM3CSK4 and LPS were used as positive controls of TLR2 and TLR4 signalling, respectively.
Macrophages cultured on lysate-adsorbed PMMA, PDMS and TCPS surfaces had a greater than 6-fold increase in NF-κB activity, compared to FBS-adsorbed surfaces and the negative control (p < 0.001). Lysate-adsorbed surfaces also potently induced the secretion of TNF-α and IL-6, compared to the negative control (p < 0.001). The increased NF-κB activity and cytokine secretion on lysate-adsorbed surfaces were strongly attenuated by TLR2 neutralizing antibodies, whereas inhibiting TLR4 resulted in a modest reduction in NF-κB activity.
These data show that lysate-derived DAMPs adsorbed to polymer surfaces potently NF-κB transcription factors, to a greater extent than serum protein; DAMP-adsorbed polymer surfaces activated NF-κB primarily through TLR2; and exposure of RAW-Blue macrophages to DAMP-adsorbed surfaces strongly induced the production of pro-inflammatory cytokines in a TLR2-dependent manner. These results suggest that DAMPs, in their adsorbed conformation, are capable of inducing a potent pro-inflammatory response in macrophages through TLRs, and that TLR pathways should be investigated as potential therapeutic targets for modulating biomaterial host responses.