A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

McKiel, Laura A.; Woodhouse, Kimberly A.; Fitzpatrick, Lindsay E.

doi:10.3791/60317

Cited by 5 publications

(11 citation statements)

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“…However, while both Teflon ™ AF and PTFE are fluoropolymers, they have different surface chemistries due to the oxygen content of the PDD comonomer in Teflon ™ AF. Teflon ™ AF can be easily incorporated into cell culture systems, as it is soluble in perfluorinated solvents and can be cast from solution (29,30). Furthermore, the amorphous structure of Teflon ™ AF imparts excellent optical clarity, which is beneficial when visualizing adherent cells.…”

Section: Teflon ™ Af Surface Preparationmentioning

confidence: 99%

“…Plates were dried in a vacuum oven (50 cmHg, 40 °C) for 48 hours to remove solvent. The wells were then cleaned with 70% (by volume) ethanol for one hour, followed by 30 minutes of ultraviolet (UV) sterilization (30,35). Endotoxin-free water washes were performed on the wells for 1 hour (three times), 12 hours, and 24 hours prior to use to remove any remaining solvent.…”

Section: Teflon ™ Af Surface Preparationmentioning

confidence: 99%

“…All batches of Teflon ™ AF-coated wells were tested indirectly for endotoxin (n = 3 per batch, plated in duplicate) with a LAL Pyrochrome kit (CapeCod and Associates, East Falmouth, MA), and endotoxin levels were consistently below 0.05 EU/mL. Details on the indirect endotoxin assay methods have been previously described (30).…”

Section: Teflon ™ Af Surface Preparationmentioning

confidence: 99%

“…This study builds upon previous work from our group demonstrating that DAMP-containing fibroblast lysates adsorb to polymeric surfaces in the presence of blood proteins and induce a pro-inflammatory and pro-fibrotic response in the RAW264.7 and RAW-Blue mouse macrophage cell lines over 120 hours, which mimics the cytokine secretion profile and macrophage fusion of in vivo macrophage-material interactions (28,29). Furthermore, this pro-inflammatory response was shown to occur primarily through Toll-like receptor 2 (TLR2) signaling (28)(29)(30). TLR2 is a cell surface TLR that, upon ligation, forms a heterodimer with either TLR1 or TLR6 and induces the myeloid differentiation primary-response gene 88 (MyD88)-dependent activation of nuclear factor-кB (NF-кB) transcription factors, and the production of pro-inflammatory cytokines, such as tumour necrosis factor a (TNF-a), interleukin 1 beta (IL-1b), and interleukin 6 (IL-6) (31).…”

Section: Introductionmentioning

confidence: 99%

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

McKiel,

Ballantyne,

Negri

et al. 2023

Front. Immunol.

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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.

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Section: Teflon ™ Af Surface Preparationmentioning

confidence: 99%

Section: Teflon ™ Af Surface Preparationmentioning

confidence: 99%

Section: Teflon ™ Af Surface Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

McKiel,

Ballantyne,

Negri

et al. 2023

Front. Immunol.

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“…As a kind of PRR, TLRs can participate in the signal transduction process in cells and play an extremely important role in the synthesis and release of proinflammatory cytokines [9,10]. Continued interactions with bacteria require the co-ordination of multiple PRR signaling pathways that dictate the outcome of microbial colonization [11].…”

Section: Introductionmentioning

confidence: 99%

DC-SIGN of Largemouth Bass (Micropterus salmoides) Mediates Immune Functions against Aeromonas hydrophila through Collaboration with the TLR Signaling Pathway

Huang,

Liu,

Yuan

et al. 2024

IJMS

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C-type lectins in organisms play an important role in the process of innate immunity. In this study, a C-type lectin belonging to the DC-SIGN class of Micropterus salmoides was identified. MsDC-SIGN is classified as a type II transmembrane protein. The extracellular segment of MsDC-SIGN possesses a coiled-coil region and a carbohydrate recognition domain (CRD). The key amino acid motifs of the extracellular CRD of MsDC-SIGN in Ca2+-binding site 2 were EPN (Glu-Pro-Asn) and WYD (Trp-Tyr-Asp). MsDC-SIGN-CRD can bind to four pathogen-associated molecular patterns (PAMPs), including lipopolysaccharide (LPS), glucan, peptidoglycan (PGN), and mannan. Moreover, it can also bind to Gram-positive, Gram-negative bacteria, and fungi. Its CRD can agglutinate microbes and displays D-mannose and D-galactose binding specificity. MsDC-SIGN was distributed in seven tissues of the largemouth bass, among which the highest expression was observed in the liver, followed by the spleen and intestine. Additionally, MsDC-SIGN was present on the membrane of M. salmoides leukocytes, thereby augmenting the phagocytic activity against bacteria. In a subsequent investigation, the expression patterns of the MsDC-SIGN gene and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) exhibited an up-regulated expression response to the stimulation of Aeromonas hydrophila. Furthermore, through RNA interference of MsDC-SIGN, the expression level of the DC-SIGN signaling pathway-related gene (RAF1) and key genes associated with the TLR signaling pathway (TLR4, NF-κB, and IL10) was decreased. Therefore, MsDC-SIGN plays a pivotal role in the immune defense against A. hydrophila by modulating the TLR signaling pathway.

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Macrophage plasticity: signaling pathways, tissue repair, and regeneration

Yan,

Wang,

Cai

et al. 2024

MedComm

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Macrophages are versatile immune cells with remarkable plasticity, enabling them to adapt to diverse tissue microenvironments and perform various functions. Traditionally categorized into classically activated (M1) and alternatively activated (M2) phenotypes, recent advances have revealed a spectrum of macrophage activation states that extend beyond this dichotomy. The complex interplay of signaling pathways, transcriptional regulators, and epigenetic modifications orchestrates macrophage polarization, allowing them to respond to various stimuli dynamically. Here, we provide a comprehensive overview of the signaling cascades governing macrophage plasticity, focusing on the roles of Toll‐like receptors, signal transducer and activator of transcription proteins, nuclear receptors, and microRNAs. We also discuss the emerging concepts of macrophage metabolic reprogramming and trained immunity, contributing to their functional adaptability. Macrophage plasticity plays a pivotal role in tissue repair and regeneration, with macrophages coordinating inflammation, angiogenesis, and matrix remodeling to restore tissue homeostasis. By harnessing the potential of macrophage plasticity, novel therapeutic strategies targeting macrophage polarization could be developed for various diseases, including chronic wounds, fibrotic disorders, and inflammatory conditions. Ultimately, a deeper understanding of the molecular mechanisms underpinning macrophage plasticity will pave the way for innovative regenerative medicine and tissue engineering approaches.

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A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

Cited by 5 publications

References 0 publications

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

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

DC-SIGN of Largemouth Bass (Micropterus salmoides) Mediates Immune Functions against Aeromonas hydrophila through Collaboration with the TLR Signaling Pathway

Macrophage plasticity: signaling pathways, tissue repair, and regeneration

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