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 to TLRs and initiate inflammatory responses via activation of NF-κB and AP-1 transcription factors[1].
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 objective of this research is to create an in vitro model of macrophage response to DAMP-adsorbed biomaterials.
Materials and Methods: Borosilicate coverslips (12 mm) and microscope slides (25 x 75 mm) were spin coated (3000 rpm, 2 min) with 20 mg/mL poly(methyl methacrylate) (PMMA) in chloroform, then UV sterilized for 30 min. 3T3 fibroblasts were lysed by freeze/thaw cycling to yield a lysate containing DAMPs. Fetal bovine serum (FBS) was used as a source of serum proteins.
RAW-Blue cells were seeded onto serum or lysate-adsorbed PMMA and/or TCPS surfaces. RAW-Blue cells are a macrophage reporter line that stably express an NF-κB/AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) that can be detected using QUANTI-Blue reagent. Prior to cell seeding, surfaces were exposed to lysate (30 min) or serum (HI-FBS or FBS, 20 hr) then rinsed with PBS. Lipopolysaccharide (LPS, 1.5 µg/mL), a known TLR agonist, was the positive control. HI-FBS-treated TCPS without cells was the negative control.
Results and Discussion: Water contact angles for PMMA-coated compared to uncoated glass increased (71° vs 62°, p<0.05), showing complete coverage of PMMA. 3D AFM images of the surfaces show the changes in surface morphology and height following protein adsorption (Figure 1).
Figure 1. 3D AFM images of PMMA films before and after protein adsorption. PMMA films alone (a) or following incubation in lysate (30 min) (b), or 10% HI-FBS (20 hr) (c).
Lysate yielded the highest NF-кB/AP-1 activation compared to the other conditions (p<0.01) (Figure 2). The results suggest that PMMA activates these factors more than TCPS (p<0.01). NF-кB/AP-1 activity in FBS is higher than HI-FBS due to alkaline phosphatases in serum.
Figure 2. NF-кB/AP-1-dependent SEAP activity in the supernatant of RAW-Blue cells cultured in HI-FBS or FBS on TCPS or PMMA surfaces. Relative SEAP activity was calculated as a fold-change relative to the control (TCPS with HI-FBS, no cells). (Mean ± SD, n=3; ** p<0.01, *** p<0.001)
Conclusion: Molecules in cell lysate adsorb to polymer surfaces and strongly activate NF-кB/AP-1 in macrophages that are grown on PMMA. Future research will include TLR-signaling inhibition assays to define if increased NF-кB/AP-1 activity is attributed to TLR activation.
Canada Foundation for Innovations (CFI) John R. Evans Leaders Fund (JELF); Senate Advisory Research Committee (SARC) grant; The R. Samuel McLaughlin Fellowship
References:
[1] Miyake, K, Sem Immunol, 2007, 19:3-10.