MyD88-dependent Toll-like receptor 2 signaling modulates macrophage activation on lysate-adsorbed Teflon™ AF surfaces in an in vitro biomaterial host response model

Abstract

The adsorbed protein layer on an implanted biomaterial surface is known to mediate downstream cell-material interactions that drive the host response. While the adsorption of plasma-derived proteins has been studied extensively, the adsorption of damage-associated molecular patterns (DAMPs) derived from damaged cells and matrix surrounding the implant remains poorly understood. Previously, our group developed a DAMP-adsorption model in which 3T3 fibroblast lysates were used as a complex source of cell-derived DAMPs and we demonstrated that biomaterials with adsorbed lysate potently activated RAW-Blue macrophages via Toll-like receptor 2 (TLR2). In the present study, we characterized the response of mouse bone marrow derived macrophages (BMDM) from wildtype (WT), TLR2-/- and MyD88-/- mice on Teflon™ AF surfaces pre-adsorbed with 10% plasma or lysate-spiked plasma (10% w/w total protein from 3T3 fibroblast lysate) for 24 hours. WT BMDM cultured on adsorbates derived from 10% lysate in plasma had significantly higher gene and protein expression of IL-1β, IL-6, TNF-α, IL-10, RANTES/CCL5 and CXCL1/KC, compared to 10% plasma-adsorbed surfaces. Furthermore, the upregulation of pro-inflammatory cytokine and chemokine expression in the 10% lysate in plasma condition was attenuated in TLR2-/- and MyD88-/- BMDM. Proteomic analysis of the adsorbed protein layers showed that even this relatively small addition of lysate-derived proteins within plasma (10% w/w) caused a significant change to the adsorbed protein profile. The 10% plasma condition had fibrinogen, albumin, apolipoproteins, complement, and fibronectin among the top 25 most abundant proteins. While proteins layers generated from 10% lysate in plasma retained fibrinogen and fibronectin among the top 25 proteins, there was a disproportionate increase in intracellular proteins, including histones, tubulins, actins, and vimentin. Furthermore, we identified 7 DAMPs or DAMP-related proteins enriched in the 10% plasma condition (fibrinogen, apolipoproteins), compared to 39 DAMPs enriched in the 10% lysate in plasma condition, including high mobility group box 1 and histones. Together, these findings indicate that DAMPs and other intracellular proteins readily adsorb to biomaterial surfaces in competition with plasma proteins, and that adsorbed DAMPs induce an inflammatory response in adherent macrophages that is mediated by the MyD88-dependent TLR2 signaling pathway.

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 . − …”

“…A key difference between this study and the McKiel study was that their study examined strongly adsorbed proteins, while this study included loosely adsorbed proteins, suggesting that these proteins were strongly adsorbed. In their study, the authors reported that adsorbed cell lysate and serum proteins generated a greater pro-inflammatory response than plasma alone in primary bone marrow-derived macrophages, similar to the findings in this study. One possible mechanism is through mutual transcriptional regulation, whereby TLR signaling can upregulate NOD mRNAs, while NOD signaling can upregulate MyD88 expression, an adapter protein involved in TLR signaling .…”

“…While we identified proteins in the whole cell lysate, we did not perform proteomics to identify which proteins adsorbed to TCPS or silicone in vitro . However, McKiel et al performed proteomics on strongly adsorbed proteins to a Teflon-coated surface and identified over 2550 unique proteins from a plasma and cell lysate solution compared to 321 unique proteins from a plasma-only solution, indicating that a large number of cell-derived proteins can adsorb to biomaterials. The in vitro studies used fetal bovine serum (FBS) as the source for preadsorbed serum proteins.…”

“…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. NOD and TLR signaling have also been shown to interact and cause a more potent inflammatory response, which has been hypothesized to occur through several mechanisms. − McKiel et al also found HGMB1, as well as heat shock proteins, on Teflon-coated surfaces, which had serum and fibroblast cell lysate preadsorbed. A key difference between this study and the McKiel study was that their study examined strongly adsorbed proteins, while this study included loosely adsorbed proteins, suggesting that these proteins were strongly adsorbed.…”

“…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. In a follow-up study from the same group, McKiel et al reported that plasma proteins when spiked with cell lysate and then preadsorbed onto a biomaterial (Teflon AF model surface) led to elevated pro-inflammatory cytokine production by macrophages at 24 h. Both studies identified TLR2 as the primary mediator of the cell-lysate-induced inflammatory response. These key findings provide evidence that DAMPs derived from injured cells induce an inflammatory response in macrophages and therefore may be an important contributor to the FBR.…”