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Toll-like receptor 2-mediated NF-κB activation by damage-associated molecular patterns on biomaterial surfaces Lindsay E. Fitzpatrick, Laura A. McKiel Department of Chemical Engineering, Queen’s University, Kingston, Canada Increased NF-κB-dependent SEAP activity was maintained on surfaces treated with lysate-containing protein mixtures, down to 0.1% total protein from lysate. Figure 1. Relative SEAP activity and cytokine secretion of RAW-Blue macrophages on lysate-adsorbed surfaces. TCPS *** 10 5 0 15 Control Lysate Pam3CSK4 (d) PMMA 10 *** *** 5 0 Control Lysate Untreated cells Pam3CSK4 Relative SEAP Activity (fold-change) TLR4 Inhibitor (c) *** Relative SEAP Activity (fold-change) (b) 15 Relative SEAP Activity (fold-change) Relative SEAP Activity (fold-change) TLR2 Inhibitor (a) 15 TCPS *** *** Lysate LPS ** *** 10 5 0 15 Control PMMA 10 5 0 Control Lysate LPS Untreated cells Anti-TLR2 CLI-095 Figure 2. Inhibition of TLR2 and TLR4 reduced NF-κB activity observed on lysate-coated surfaces. **** Relative SEAP Activity (fold-change) Statement of Purpose: 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. Methods: Tissue culture polystyrene (TCPS) and spincoated poly(methyl methacrylate) (PMMA) 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 RAWBlue macrophages was measured indirectly as NF-κBinducible 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 lysateadsorbed PMMA and TCPS surfaces for 20 hours had increased NF-κB/AP-1 activity, compared to cells cultured on FBS-adsorbed surfaces and the negative control (Fig. 1a), and increased secretion of proinflammatory cytokines (IL-6, TNF-α), compared to the negative control (Fig 1b-c). The increased NF-κBdependent SEAP activity on lysate-conditioned TCPS and PMMA surfaces was attenuated by treating RAW-Blue cells with a TLR2 neutralizing antibody (Fig 2a-b). Inhibiting TLR4 signalling in RAW-Blues with CLI-095 decreased SEAP activity on lysate-conditioned surfaces (Fig 2c-d), but to a lesser extent than the TLR2 neutralizing antibody. A competitive adsorption of DAMPs in the presence of serum proteins was performed to mimic competitive protein adsorption between DAMPs and serum proteins to a material surface in vivo. Solutions of lysate diluted in FBS (Fig 3; 1500 µg total protein per well, in 48 well plate) were adsorbed on TCPS surfaces. 10 8 6 * 4 ** 2 0 100% 50% 10% 5% 1% 0.5% 0.1% 0.05% 0.01% 0.005% % Lysate in FBS Total protein = 1500 µg per well 0% Media (100% FBS) Figure 3. Competitive adsorption of lysate- and serumderived proteins. Conclusions: Our work shows that: (1) lysate-derived DAMPs competed with serum proteins for biomaterial surface interactions in vitro; (2) 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; (3) lysate-derived DAMPs adsorbed on PMMA and TCPS surfaces activated NF-κB primarily through TLR2; and (4) 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 are capable of inducing a potent proinflammatory 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 include generating MyD88-knockout RAW-Blue line to investigate MyD88-dependent TLR signalling, and proteomic analysis of DAMPs adsorbed on polymer surfaces by mass spectrometry.