The extended effect of adsorbed damage-associated molecular patterns and Toll-like receptor 2 signaling on macrophage-material interactions

Abstract

Implanted biomaterials elicit an immune-mediated foreign body reaction (FBR) that results in the fibrous encapsulation of the implant and can critically impact the performance of some implants. Consequently, understanding the molecular mechanisms that underpin cell-materials interactions that initiate biomaterial-induced inflammation and fibrosis is critical to improving the performance of biomaterial implants negatively impacted by the FBR. Damage-associated molecular patterns (DAMPs) are endogenous mediators of inflammation that are released upon tissue injury and induce sterile inflammation via Toll-like receptors (TLRs). However, the prevalence of DAMPs within the adsorbed protein layer on material surfaces and their role mediating cell-material interactions is unclear. Previously, our group demonstrated that molecules in fibroblast lysates adsorbed to various biomaterials and induced a potent TLR2-dependent inflammatory response in macrophages at 24 h. In this study, we examined the extended response of RAW-Blue reporter macrophages on lysate or serum-adsorbed Teflon™ AF surfaces to understand the potential role of adsorbed DAMPs in macrophage-material interactions at later time points. Lysate-conditioned surfaces maintained increased nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1) transcription factor activity and increased expression Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES/CCL5) at 72 h and 120 h, compared to FBS-conditioned surfaces. In contrast, monocyte chemoattractant protein 1 (MCP-1/CCL2) was only elevated at 72 h in lysate conditions. Transforming growth factor beta 1 (TGF-β1) secretion was significantly increased on lysate-conditioned surfaces, while conditioned media from macrophages on lysate-conditioned surfaces induced alpha smooth muscle actin (αSMA) expression in 3T3 fibroblasts. TLR2 neutralizing antibody treatment significantly decreased NF-κB/AP-1 activity and attenuated TGF-β1 expression at both time points, and MCP-1 and RANTES at 72 h. Finally, multinucleated cells were observed on lysate-conditioned surfaces at 72 h, indicating adsorbed DAMPs induced a fusion permissive environment for adherent macrophages. This study demonstrates that adsorbed DAMPs continue to influence macrophage-material responses beyond the initial 24-h period and maintain a pro-inflammatory and fibrotic response that models aspects of the early FBR. Furthermore, the transient inhibition of TLR2 continued to exert an effect at these later time points, suggesting TLR2 may be a target for therapeutic interventions in FBR.

Cell- and Serum-Derived Proteins Act as DAMPs to Activate RAW 264.7 Macrophage-like Cells on Silicone Implants

“…The RAW 264.7 macrophage-like cell line was chosen as a model to study the DAMP-mediated cellular response. Berghaus et al reported that RAW 264.7 cells showed a similar phenotype and function to bone marrow-derived macrophages in response to TLR agonists, which is consistent with studies from our group and others. , LPS was chosen as a model DAMP, which binds TLR4, to represent damaged extracellular matrix that can act as a danger signal to recruit inflammatory cells in vivo . − …”

“…Preadsorbed serum-derived proteins invoked a stronger response than cell-derived proteins, but when both were combined, the effect was enhanced. Many DAMPs signal through TLRs, specifically TLR2 or TLR4 depending on the chemistry of the DAMP. ,,, Activation of cell surface receptors beyond TLRs that recognize cell-derived DAMPs could contribute to TLR signaling. For example, NOD1 and NOD2 have been shown to recognize DAMPs from ER stress, and RAGE has been shown to recognize several of the same DAMPs as TLRs, including HMGB1.…”

“…Berghaus et al 30 reported that RAW 264.7 cells showed a similar phenotype and function to bone marrowderived macrophages in response to TLR agonists, which is consistent with studies from our group 31 and others. 28,29 LPS was chosen as a model DAMP, which binds TLR4, to represent damaged extracellular matrix that can act as a danger signal to recruit inflammatory cells in vivo. 32−34 Our first objective was to establish a chemically defined, serum-free culture medium that would maintain RAW 264.7 cells and retain their activation capabilities by DAMPs.…”

“…24 In the FBR, possible sources of DAMPs include unfolded proteins from otherwise noninflammatory proteins, proteins released from injured cells, and degraded ECM fragments. 12,25,26 Studies from the Fitzpatrick group 27,28 evaluated the response of RAW 264.7-Blue murine macrophage-like cells to proteins derived from a NIH/3T3 cell lysate preadsorbed onto several biomaterials (poly(methyl methacrylate), poly-(dimethylsiloxane), and Teflon). The authors studied the effect of strongly adsorbed molecules and reported the upregulation of the inflammatory transcription factors NF-κB/AP-1 in response to cell lysate.…”

“…DAMPs bind to pattern recognition receptors on cells, and it has been demonstrated that DAMPs are capable of signaling through toll-like receptors (TLRs), to induce sterile inflammation. − Moreover, TLRs have been linked to the FBR in vivo . , If DAMP signaling is prolonged, chronic inflammation ensues, which is characteristic of the FBR . In the FBR, possible sources of DAMPs include unfolded proteins from otherwise noninflammatory proteins, proteins released from injured cells, and degraded ECM fragments. ,, Studies from the Fitzpatrick group , evaluated the response of RAW 264.7-Blue murine macrophage-like cells to proteins derived from a NIH/3T3 cell lysate preadsorbed onto several biomaterials (poly­(methyl methacrylate), poly­(dimethylsiloxane), and Teflon). The authors studied the effect of strongly adsorbed molecules and reported the upregulation of the inflammatory transcription factors NF-κB/AP-1 in response to cell lysate.…”