Toll-like receptor 2-mediated NF-kB activation by damage-associated molecular patterns on biomaterial surfaces

McKiel, Laura A. (Department of Chemical Engineering, Queen's University, Kingston, Canada)
Fitzpatrick, Lindsay E. (Department of Chemical Engineering, Queen's University, Kingston, Canada)

Introduction

The innate immune response is the body’s primary defense against infection and injury, and is facilitated largely by white blood cells, such as macrophages. Toll-like receptors (TLRs) play a critical role in innate immunity by recognizing evolutionarily conserved pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively). DAMPs are released upon tissue injury and cell death, and are known to bind TLRs and initiate inflammatory responses via activation of NF-κB transcription factors, primarily through the MyD88 signalling pathway. We hypothesize that TLRs contribute to biomaterial-induced inflammation by binding DAMPs created at the implant site and adsorbed to the surface of the biomaterial. The aim of our research is to identify potential molecular targets for modulating inflammatory responses to biomaterials.

Materials and Methods

Tissue culture polystyrene (TCPS), spin-coated poly(methyl methacrylate) (PMMA), and polydimethylsiloxane (PDMS)-coated surfaces were exposed to NIH3T3 cell lysate (complex source of DAMPs) or FBS for 30 min, rinsed with PBS, then seeded with RAW-Blue™ (Invivogen, San Diego, CA) macrophage reporter cells. The NF-κB activity in RAW-Blue macrophages was measured indirectly as NF-κB-inducible secreted embryonic alkaline phosphate (SEAP) activity using an alkaline phosphatase assay. The concentration of IL-6 and TNF-α in the cell supernatant was analyzed using ELISA kits (BioLegend, San Diego, CA). TLR2 and TLR4 signalling was inhibited using a TLR2 neutralizing antibody (BioLegend) (50 µg/mL, 30 min) and TLR4 inhibitor CLI-095 (Invivogen) (1 µg/mL, 60 min), respectively. TLR2 and TLR4 agonists (PAM3CSK4 and LPS, respectively) were used as positive controls for TLR signaling. Cells cultured in 10% heat inactivated (HI)-FBS on untreated substrates were the negative control (“media”). Statistical analysis was performed by ANOVA with Tukey’s post hoc (α = 0.05).

Results

RAW-Blue macrophages cultured on lysate-adsorbed TCPS, PMMA, and PDMS surfaces for 20 hours had increased NF-κB/AP-1 activity, compared to cells cultured on FBS-adsorbed surfaces and the negative control. The increased NF-κB-dependent SEAP activity on lysate-conditioned TCPS, PMMA, and PDMS surfaces was attenuated by treating RAW-Blue cells with a TLR2 neutralizing antibody (Fig 1a). Inhibiting TLR4 signalling in RAW-Blues with CLI-095 decreased SEAP activity on lysate-conditioned surfaces (Fig 1b), but to a lesser extent than the TLR2 neutralizing antibody. RAW-Blues secreted the proinflammatory cytokines TNF-α and IL-6 in response to DAMP-coated PMMA and PDMS surfaces. TNF-α secretion was significantly reduced following anti-TLR2 treatment on both surfaces (Fig 2a), however IL-6 secretion only significantly decreased on PMMA following TLR2 neutralization (Fig 2b). Adsorption of DAMPs in the presence of serum proteins was performed to determine if the lysate-derived molecules were capable of stimulating NF-κB/AP-1 activity at very low concentrations. Solutions of lysate diluted in FBS (Fig 3; 300 μg total protein per well, in 48 well plate) were adsorbed on TCPS surfaces. Increased NF-κB-dependent SEAP activity was maintained on surfaces treated with lysate-containing protein mixtures, down to 0.1% total protein from lysate. 

Discussion and Conclusion

Our work shows that: (1) DAMP-adsorbed surfaces activated NF-κB transcription factors more strongly than serum proteins, and this is maintained down to 0.1% (total protein) lysate in serum; (2) lysate-derived DAMPs adsorbed on PMMA and TCPS surfaces activated NF-κB primarily through TLR2; and (3) exposure of RAW-Blue macrophages to DAMP-adsorbed surfaces strongly induced the production of pro-inflammatory cytokines IL-6 and TNF-α. These results suggest that adsorbed DAMPs, even in very small amounts, are capable of inducing a potent pro-inflammatory response in macrophages through TLRs, and that TLR signalling pathways should be investigated further as potential therapeutic targets for modulating host responses to implanted biomaterials. Future work will investigate the effect of adsorbed DAMPs on gene and protein expression in primary mouse bone marrow derived macrophages and proteomic analysis of DAMPs adsorbed on polymer surfaces by mass spectrometry.


Figure 1. Results of neutralizing TLR2 signalling in RAW-Blues on polymer surfaces (a). Results of inhibiting TLR4 signalling in RAW-Blues on polymer surfaces (b). ** p<0.01, *** p<0.001.

Figure 2. TNF-α (a) and IL-6 (b) concentration in the supernatant of RAW-Blue macrophages cultured for 20 hours on PMMA and PDMS, with and without TLR2 inhibition. *** p < 0.001, comparing TLR2 inhibition conditions. ` p < 0.05 and ```` p < 0.0001, compared to material-specific 10% FBS.

Figure 3. Dilution series of lysate in FBS based on total protein content to mimic competitive protein adsorption between DAMPs and serum proteins to a biomaterial surface. ** p<0.01.

Acknowledgements

We acknowledge support received from CFI John R. Evans Leaders Fund, Ontario Research Foundation Small Infrastructure Fund, NSERC Canada Graduate Scholarship – Masters, Ontario Graduate Scholarship, and Queen's University Principal's Development Fund and Senate Advisory Research Committee Annual Competition.

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