Toll-Like Receptor 2 and the Early Chronic Inflammatory and Fibrotic Macrophage Response to Adsorbed Damage Associated Molecular Patterns

Abstract

The foreign body reaction (FBR) is an aberrant wound healing process that occurs in response to an implanted biomaterial, which can lead to the implant failing prematurely. Recently, damage-associated-molecular-patterns (DAMPs) were identified as potential contributing factors to the onset and progression of the FBR. DAMPs, released as a result of tissue damage, can stimulate an inflammatory response via interaction with pattern recognition receptors, such as Toll-like receptors (TLRs), on the surface of immune cells. Recently, our research group demonstrated that macrophages stimulated by DAMP-adsorbed biomaterials have increased NF-κB/AP-1 activity and cytokine expression at 20 hours, with TLR2 inhibition reducing these effects. This thesis examined the effects of adsorbed DAMP-induced TLR2 signaling on pro-inflammatory and profibrotic signaling in macrophages and its downstream effects on fibroblasts to better understand the early chronic inflammatory response associated with biomaterials. After 72 hours, macrophages cultured on DAMP-adsorbed TeflonTM AF surfaces had increased NF-κB/AP-1 activity and pro-inflammatory and profibrotic cytokine expression compared to serum-adsorbed TeflonTM AF, which was reduced in the presence of a TLR2 neutralizing antibody. After 120 hours, NF-κB/AP-1 activity and profibrotic cytokine expression remained elevated for macrophages cultured on DAMP-adsorbed TeflonTM AF relative to the negative control, and again TLR2 inhibition reduced these effects. In addition, fibroblasts cultured in conditioned medium from macrophages cultured on DAMP-adsorbed TeflonTM AF surfaces for 72 or 120 hours had increased gene and protein expression of α smooth muscle actin (αSMA) compared to fibroblasts in conditioned medium from serum-adsorbed TeflonTM AF surfaces. This αSMA expression was reduced for the conditioned medium of DAMP-stimulated macrophages treated with a TLR2 neutralizing antibody. These results suggest that macrophages are still significantly activated by surface-adsorbed DAMPs at 72 and 120 hours and conditioned media from these cells could induce myofibroblast differentiation in fibroblasts. In addition, a single treatment with a TLR2 inhibitor for macrophages was sufficient to decrease the effects of adsorbed DAMPs at both 72 and 120 hours. However, the effects of both the adsorbed DAMPs and TLR2 inhibitor decreased over time in the macrophages, potentially due to paracrine signaling within the cultures overpowering the effects of the DAMP-signaling.