Download 10:30 a.m. 287. Toll-like receptor 2-mediated NF

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
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15
Control
Lysate
Pam3CSK4
(d)
PMMA
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Control
Lysate
Untreated cells
Pam3CSK4
Relative SEAP Activity
(fold-change)
TLR4 Inhibitor
(c)
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Relative SEAP Activity
(fold-change)
(b)
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Relative SEAP Activity
(fold-change)
Relative SEAP Activity
(fold-change)
TLR2 Inhibitor
(a)
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TCPS
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Lysate
LPS
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Control
PMMA
10
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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.
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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.
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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.