En Macrophage reporter celle analyse til at undersøge Toll-lignende Receptor-medieret NF-kB/AP-1 signalering på Adsorbede protein lag på polymere overflader
Summary
Denne protokol giver forskerne en hurtig, indirekte metode til måling af TLR-afhængige NF-кb/AP-1 transkriptionsfaktor aktivitet i en murine makrofag cellelinje som reaktion på en række polymere overflader og adsorbede protein lag, der modellere biomaterialet implantat mikromiljø.
Abstract
Den vedholdende inflammatoriske vært respons på en implanteret biomaterial, kendt som den udenlandske krop reaktion, er en betydelig udfordring i udviklingen og gennemførelsen af biomedicinsk udstyr og vævs ingeniør konstruktioner. Makrofager, en medfødte immuncelle, er centrale aktører i den udenlandske krop reaktion, fordi de forbliver på implantatet stedet for levetiden af enheden, og er almindeligt undersøgt for at få en forståelse af denne skadelige vært svar. Mange biomaterialer forskere har vist, at adsorbede protein lag på implanterede materialer påvirke makrofag adfærd, og efterfølgende påvirke værten respons. Metoderne i dette papir beskriver en in vitro-model ved hjælp af adsorberede proteinlag indeholdende cellulære skade molekyler på polymer biomaterialet overflader til at vurdere makrofag respons. En NF-кb/AP-1 reporter makrofag cellelinje og den tilhørende kolorimetriske alkalisk fosfataseanalyse blev anvendt som en hurtig metode til indirekte at undersøge NF-кb/AP-1 transkriptionsfaktor aktivitet som reaktion på komplekse adsorptions proteinlag, der indeholder blodproteiner og skade relaterede molekylære mønstre, som en model af de komplekse adsorbede protein lag dannet på biomaterialet overflader in vivo.
Introduction
Den udenlandske krop reaktion (FBR) er en kronisk vært respons, der kan have en negativ indvirkning på ydeevnen af et implanteret materiale eller enhed (f. eks, Drug levering udstyr, biosensorer), gennem vedvarende frigivelse af inflammatoriske mediatorer og ved at hæmme integrationen mellem det implanterede materiale og det omgivende væv1. Dette medfødte immunrespons initieres af implantationsproceduren og er karakteriseret ved langvarig tilstedeværelse af medfødte immunceller og fibrøs kapsel dannelse omkring implantatet1. I forbindelse med materiale værts respons har makrofag-materiale interaktioner en betydelig indvirkning på udviklingen af værts respons og udvikling af en FBR1. Makrofager er en forskelligartet medfødte immuncelle population, rekrutteret til implantationsstedet enten fra væv-residente makrofag populationer eller fra blodet som monocyte-afledte makrofager. De begynder at akkumulere på implantationsstedet kort efter implantation, og inden for dage bliver den fremherskende cellepopulation i implantat mikromiljøet. Materiale-klædende makrofager, sammen med fremmedlegeme gigant celler (fbgc) dannet gennem makrofag fusion, kan fortsætte på den materielle overflade for levetiden af implantatet2,3. Derfor, makrofager anses for at være centrale aktører i den udenlandske krop respons på grund af deres roller orkestrering de karakteristiske trin af FBR: akut inflammatorisk respons, væv Remodeling, og dannelse af fibrotisk væv1.
Bompenge lignende receptorer (TLRs) er en familie af mønster genkendelses receptorer, der udtrykkes af mange immunceller, herunder makrofager, og har vist sig at spille en væsentlig rolle i inflammation og sårheling. Ud over det patogen afledte ligands er TLRs i stand til at binde endogene molekyler, kendt som skade relaterede molekylære mønstre (DAMPs), som frigives under celle nekrose og aktivere inflammatoriske signalerings veje, der resulterer i produktion af proinflammatoriske cytokiner4. Vi og andre har foreslået, at skader opstået under bløddels biomateriale implantations procedurer frigiver damps, som derefter adsorcerer til biomaterialet overflader ud over blodproteiner og modurer efterfølgende celle-materiale interaktioner5,6. Når makrofager interagerer med det adsorbede protein lag på et implantat, kan deres overflade-TLRs genkende adsorptions DAMPs og aktivere proinflammatoriske signalering kaskader, hvilket fører til NF-κB og AP-1 transkriptionsfaktor aktivering og produktion af proinflammatoriske cytokiner. Vi har tidligere vist, at murine makrofager har øget NF-κB/AP-1 aktivitet og tumor nekrose faktor α (TNF-α, proinflammatorisk cytokin) sekretion som reaktion på fugt-holdige adsorbede protein lag på en række polymere overflader sammenlignet med overflader med adsorbede serum eller plasma kun (dvs., ingen DAMPs til stede), og at dette svar er i vid udstrækning medieret af TLR2, mens TLR4 spiller en mindre rolle5.
NF-κb/AP-1 reporter makrofag Cell line (tabel over materialer), der anvendes i denne protokol er en bekvem metode til at måle relative NF-κb og AP-1 aktivitet i makrofager5,7,8. I kombination med TLR pathway hæmmere, denne cellelinje er et nyttigt redskab til at undersøge TLR aktivering og dens rolle i inflammation som reaktion på en række stimuli5,7,8. Den reporter celler er en modificeret mus makrofag-lignende cellelinje, der kan stabilt producere udskillet embryonale alkalisk fosfatase (SEAP) på NF-κB og AP-1 transkriptionen faktor aktivering9. Den kolorimetriske enzymatiske alkalisk fosfataseanalyse (tabel over materialer) kan derefter anvendes til at kvantificere relative mængder af SEAP-udtryk som et indirekte mål for NF-κb/AP-1-aktiviteten. Da NF-κB og AP-1 er nedstrøms for mange celle signalerings veje, kan neutraliserende antistoffer og hæmmere, der er rettet mod specifikke Tlr’er (f. eks. TLR2) eller TLR-adapter molekyler (f. eks. MyD88), bruges til at verificere rollen for en bestemt vej. Den metode, der er beskrevet i denne artikel, giver en enkel og hurtig tilgang til vurdering af bidraget fra TLR signalering i murine makrofag svar til en række polymere overflader med adsorberede proteinlag, der indeholder både blodproteiner og damps som en in vitro model af implanterede biomaterialer.
Protocol
Representative Results
Discussion
Et primært fokus i vores laboratorium er værts reaktionen på solide biomateriale bløddels implantater, og især hvordan de cellulære skader, der påløber under implantationsproceduren, påvirker værts reaktionen. Det arbejde, der præsenteres her beskriver indledende eksperimenter ved hjælp af en reporter makrofag cellelinje og in vitro-genereret fugtig-holdige cellulære lysat, at undersøge indflydelsen af molekyler frigivet under cellulære skader (dvs., fra implantatet kirurgi) på makrofag reaktioner på bio…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Forfatterne taknemmeligt anerkender operationel finansiering fra canadiske institutter for sundhedsforskning projekt (PTJ 162251), Queen’s University Senatet rådgivende forskningsudvalg og infrastruktur støtte fra det canadiske fundament for innovation John Evans Leadership Fund (projekt 34137) og ministeriet for forskning og innovation Ontario forskningsfond (projekt 34137). L.A.M. blev støttet af en Queen’s University R. Samuel McLaughlin Fellowship, et naturvidenskab og ingeniørvidenskab forskning Rådet af Canada Canadian Graduate Scholarship Master’s Award og en Ontario Graduate Scholarship. Forfatterne vil gerne takke Dr. Myron Vis for hans generøse gave af NF-κb/AP-1 reporter makrofag Cell line og DRS. Michael blennerhassett og Sandra lourenssen for brugen af deres gel Imaging System og plade læser.
Materials
| Cell culture reagents | |||
| anti-mouse/human CD282 (TLR2) | Biolegend | 121802 | |
| CLI-095 (TLR4 inhibitor) | Invivogen | TLRL-CLI95 | |
| C57 complement plasma K2 EDTA 10ml, innovative grade US origin | InnovativeResearch | IGMSC57-K2 EDTA-Compl-10ml | Mouse plasma |
| Dulbecco's modified eagle medium (DMEM) | Sigma Aldrich | D6429-500ML | |
| Dulbecco's phosphate buffered saline (DPBS) | Fisher Scientific | 14190250 | No calcium, no magnesium |
| Fetal bovine serum (FBS), research grade | Wisent | 98150 | |
| LPS-EK | Invivogen | TLRL-EKLPS | Lipopolysaccharide from Escherichia coli K12 |
| NIH/3T3 fibroblasts | ATCC | CRL-1658 | |
| Pam3CSK4 | Invivogen | tlrl-pms | Synthetic triacylated lipopeptide – TLR1/2 ligand |
| Penicillin/streptomycin | Sigma Aldrich | P4333-100ML | |
| Plasmocin | Invivogen | ANT-MPP | Mycoplasma elimination reagent |
| RAW-Blue cells | Invivogen | raw-sp | NF-κB/AP-1 reporter macrophage cell line |
| Trypan blue solution, 0.4% | Fisher Scientific | 15250061 | |
| TrypLE express enzyme (1X) | Fisher Scientific | 12604021 | animal origin-free recombinant cell dissociation enzyme |
| Zeocin | Invivogen | ANT-ZN-1 | |
| Kits and assays | |||
| ELISA precoated plates, mouse IL-6 | Biolegend | B213022 | |
| ELISA precoated plates, mouse TNF-α | Biolegend | B220233 | |
| Endotoxin (Escherichia coli) – Control standard endotoxin (CSE) | Associates of Cape Cope Inc. | E0005-5 | Endotoxin for standard curve in chromogenic endotoxin assay |
| LAL water, 100 mL | Associates of Cape Cope Inc. | WP1001 | Used with chromogenic endotoxin assay |
| Micro BCA protein assay | Fisher Scientific | PI23235 | |
| Limulus amebocyte lysate (LAL) Pyrochrome endotoxin test kit | Associates of Cape Cope Inc. | C1500-5 | Chromogenic endotoxin assay reagent |
| QUANTI-Blue alkaline phosphatase detection medium | Invivogen | rep-qb2 | Alkaline phosphatase assay to indirectly measure NF-κB/AP-1 activity |
| Polymeric coating reagents | |||
| Chloroform, anhydrous | Sigma Aldrich | 288306-1L | |
| Ethyl alcohol anhydrous | Commercial Alcohols | P006EAAN | Sigma: Reagent alcohol, anhydrous, 676829-1L |
| Straight tapered fine tip forceps | Fisher Scientific | 16-100-113 | |
| Fluorinert FC-40 solvent | Sigma Aldrich | F9755-100ML | Fluorinated solvent for fPTFE |
| Cell culture grade water (endotoxin-free) | Fisher Scientific | SH30529LS | |
| Poly(methyl methacrylate) (PMMA) | Sigma Aldrich | 182230-25G | |
| Sylgard 184 elastomer kit | Fisher Scientific | 50822180 | |
| Teflon-AF (fPTFE) | Sigma Aldrich | 469610-1G | Poly[4,5-difluoro-2,2-bis(trifluoromethyl)-1,3-dioxole-co-tetrafluoroethylene] |
| Consumables | |||
| Adhesive plate seals | Fisher Scientific | AB-0580 | |
| Axygen microtubes, 1.5 mL | Fisher Scientific | 14-222-155 | |
| Borosilicate glass scintillation vials, with white polypropylene caps | Fisher Scientific | 03-337-4 | |
| Clear PS 48-well plate | Fisher Scientific | 08-772-52 | |
| Clear TCPS 96-well plate | Fisher Scientific | 08-772-2C | |
| Clear TCPS 48-well plate | Fisher Scientific | 08-772-1C | |
| Cover glasses, circles | Fisher Scientific | 12-545-81 | |
| Falcon tissue culture treated flasks, T25 | Fisher Scientific | 10-126-10 | |
| sticky-Slide 8 Well | Ibidi | 80828 | |
| Superfrost microscope slides | Fisher Scientific | 12-550-15 | |
| Tissue culture treated flasks, T150 | Fisher Scientific | 08-772-48 | |
References
- Anderson, J. M., Rodriguez, A., Chang, D. T. Foreign body reaction to biomaterials. Seminars in Immunology. 20 (2), 86-100 (2008).
- Anderson, J. M., Miller, K. M. Biomaterial biocompatibility and the macrophage. Biomaterials. 5 (1), 5-10 (1984).
- Collier, T. O., Anderson, J. M. Protein and surface effects on monocyte and macrophage adhesion, maturation, and survival. Journal of Biomedical Materials Research. 60 (3), 487-496 (2002).
- Bianchi, M. E. DAMPs, PAMPs and alarmins: all we need to know about danger. Journal of Leukocyte Biology. 81 (1), 1-5 (2007).
- McKiel, L. A., Fitzpatrick, L. E. Toll-like Receptor 2-Dependent NF-κB/AP-1 Activation by Damage-Associated Molecular Patterns Adsorbed on Polymeric Surfaces. ACS Biomaterials Science & Engineering. 4 (11), 3792-3801 (2018).
- Babensee, J. E. Interaction of dendritic cells with biomaterials. Seminars in Immunology. 20 (2), 101-108 (2008).
- Sintes, J., Romero, X., de Salort, J., Terhorst, C., Engel, P. Mouse CD84 is a pan-leukocyte cell-surface molecule that modulates LPS-induced cytokine secretion by macrophages. Journal of Leukocyte Biology. 88 (4), 687-697 (2010).
- Tom, J. K., Mancini, R. J., Esser-Kahn, A. P. Covalent modification of cell surfaces with TLR agonists improves and directs immune stimulation. Chemical Communications. 49 (83), 9618-9620 (2013).
- Abdulkhalek, S., et al. Neu1 sialidase and matrix metalloproteinase-9 cross-talk is essential for toll-like receptor activation and cellular signaling. Journal of Biological Chemistry. 286 (42), 36532-36549 (2011).
- Gorbet, M. B., Sefton, M. V. Endotoxin: The uninvited guest. Biomaterials. 26 (34), 6811-6817 (2005).
- Xing, Z., Pabst, M. J., Hasty, K. A., Smith, R. A. Accumulation of LPS by polyethylene particles decreases bone attachment to implants. Journal of Orthopaedic Research. 24 (5), 959-966 (2006).
- Ding, H., et al. Comparison of the cytotoxic and inflammatory responses of titanium particles with different methods for endotoxin removal in RAW264.7 macrophages. Journal of Materials Science: Materials in Medicine. 23 (4), 1055-1062 (2012).
- Hoogenboom, R., Becer, C. R., Guerrero-Sanchez, C., Hoeppener, S., Schubert, U. S. Solubility and thermoresponsiveness of PMMA in alcohol-water solvent mixtures. Australian Journal of Chemistry. 63 (8), 1173-1178 (2010).
- Efimenko, K., Wallace, W. E., Genzer, J. Surface modification of Sylgard-184 poly(dimethyl siloxane) networks by ultraviolet and ultraviolet/ozone treatment. Journal of Colloid and Interface Science. 254 (2), 306-315 (2002).
- Godek, M. L., Sampson, J. A., Duchsherer, N. L., McElwee, Q., Grainger, D. W. Rho GTPase protein expression and activation in murine monocytes/macrophages is not modulated by model biomaterial surfaces in serum-containing in vitro cultures. Journal of Biomaterials Science. Polymer Edition. 17 (10), 1141-1158 (2006).
- Park, J. S., et al. Involvement of Toll-like Receptors 2 and 4 in Cellular Activation by High Mobility Group Box 1 Protein. Journal of Biological Chemistry. 279 (9), 7370-7377 (2004).
- Ohashi, K., Burkart, V., Flohé, S., Kolb, H. Cutting Edge: Heat Shock Protein 60 Is a Putative Endogenous Ligand of the Toll-Like Receptor-4 Complex. The Journal of Immunology. 164 (2), 558-561 (2000).
- Wong, T., McGrath, J. A., Navsaria, H. The role of fibroblasts in tissue engineering and regeneration. British Journal of Dermatology. 156 (6), 1149-1155 (2007).
- van Wachem, P. B., et al. The influence of protein adsorption on interactions of cultured human endothelial cells with polymers. Journal of Biomedical Materials Research. 21 (6), 701-718 (1987).
- Miller, K. M., Anderson, J. M. Human monocyte/macrophage activation and interleukin 1 generation by biomedical polymers. Journal of Biomedical Materials Research. 22 (8), 713-731 (1988).
- Bonfield, T. L., Colton, E., Anderson, J. M. Plasma protein adsorbed biomedical polymers: Activation of human monocytes and induction of interleukin 1. Journal of Biomedical Materials Research. 23 (6), 535-548 (1989).
- González, O., Smith, R. L., Goodman, S. B. Effect of size, concentration, surface area, and volume of polymethylmethacrylate particles on human macrophages in vitro. Journal of Biomedical Materials Research. 30 (4), 463-473 (1996).
- Anderson, J. M., et al. Protein adsorption and macrophage activation on polydimethylsiloxane and silicone rubber. Journal of Biomaterials Science. Polymer Edition. 7 (2), 159-169 (1995).
- Lord, M. S., Foss, M., Besenbacher, F. Influence of nanoscale surface topography on protein adsorption and cellular response. Nano Today. 5 (1), 66-78 (2010).
- Chen, S., et al. Characterization of topographical effects on macrophage behavior in a foreign body response model. Biomaterials. 31 (13), 3479-3491 (2010).
- Shen, M., Horbett, T. A. The effects of surface chemistry and adsorbed proteins on monocyte/macrophage adhesion to chemically modified polystyrene surfaces. Journal of Biomedical Materials Research. 57 (3), 336-345 (2001).
- Love, R. J., Jones, K. S. The recognition of biomaterials: Pattern recognition of medical polymers and their adsorbed biomolecules. Journal of Biomedical Materials Research Part A. 101 (9), 2740-2752 (2013).
- McNally, A. K., Anderson, J. M. Phenotypic expression in human monocyte-derived interleukin-4-induced foreign body giant cells and macrophages in vitro: Dependence on material surface properties. Journal of Biomedical Materials Research Part A. 103 (4), 1380-1390 (2015).
- Gambhir, V., et al. The TLR2 agonists lipoteichoic acid and Pam3CSK4 induce greater pro-inflammatory responses than inactivated Mycobacterium butyricum. Cellular Immunology. 280 (1), 101-107 (2012).
- Suzuki, O., Yagishita, H., Yamazaki, M., Aoba, T. Adsorption of Bovine Serum Albumin onto Octacalcium Phosphate and its Hydrolyzates. Cells and Materials. 5 (1), 45-54 (1995).
- Johnston, R. L., Spalton, D. J., Hussain, A., Marshall, J. In vitro protein adsorption to 2 intraocular lens materials. Journal of Cataract and Refractive Surgery. 25 (8), 1109-1115 (1999).
- Jin, J., Jiang, W., Yin, J., Ji, X., Stagnaro, P. Plasma proteins adsorption mechanism on polyethylene-grafted poly(ethylene glycol) surface by quartz crystal microbalance with dissipation. Langmuir. 29 (22), 6624-6633 (2013).
- Swartzlander, M. D., et al. Linking the foreign body response and protein adsorption to PEG-based hydrogels using proteomics. Biomaterials. 41, 26-36 (2015).
- Chamberlain, M. D., et al. Unbiased phosphoproteomic method identifies the initial effects of a methacrylic acid copolymer on macrophages. Proceedings of the National Academy of Sciences. 112 (34), 10673-10678 (2015).
- Dillman, W. J., Miller, I. F. On the adsorption of serum proteins on polymer membrane surfaces. Journal of Colloid And Interface Science. 44 (2), 221-241 (1973).
- Ishihara, K., Ziats, N. P., Tierney, B. P., Nakabayashi, N., Anderson, J. M. Protein adsorption from human plasma is reduced on phospholipid polymers. Journal of Biomedical Materials Research. 25 (11), 1397-1407 (1991).
- Warkentin, P., Wälivaara, B., Lundström, I., Tengvall, P. Differential surface binding of albumin, immunoglobulin G and fibrinogen. Biomaterials. 15 (10), 786-795 (1994).
- Berghaus, L. J., et al. Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors 2, 3, and 4. Comparative Immunology, Microbiology and Infectious Diseases. 33 (5), 443-454 (2010).
- Zhang, Y., Karki, R., Igwe, O. J. Toll-like receptor 4 signaling: A common pathway for interactions between prooxidants and extracellular disulfide high mobility group box 1 (HMGB1) protein-coupled activation. Biochemical Pharmacology. 98 (1), 132-143 (2015).
- Mizel, S. B., Honko, A. N., Moors, M. A., Smith, P. S., West, A. P. Induction of macrophage nitric oxide production by Gram-negative flagellin involves signaling via heteromeric Toll-like receptor 5/Toll-like receptor 4 complexes. Journal of Immunology. 170 (12), 6217-6223 (2003).
- Das, N., et al. HMGB1 Activates Proinflammatory Signaling via TLR5 Leading to Allodynia. Cell Reports. 17 (4), 1128-1140 (2016).
- Pelegrin, P., Barroso-Gutierrez, C., Surprenant, A. P2X7 Receptor Differentially Couples to Distinct Release Pathways for IL-1β in Mouse Macrophage. The Journal of Immunology. 180 (11), 7147-7157 (2008).
- Tak, P. P., Firestein, G. S. NF-κB: A key role in inflammatory diseases. Journal of Clinical Investigation. 107 (1), 7-11 (2001).
- Ashkenazi, A., Dixit, V. M. Death receptors: signaling and modulation. Science. 281 (5381), 1305-1308 (1998).
- Erridge, C. Endogenous ligands of TLR2 and TLR4: agonists or assistants. Journal of Leukocyte Biology. 87 (6), 989-999 (2010).
- Feng, Y., et al. A macrophage-activating, injectable hydrogel to sequester endogenous growth factors for in situ angiogenesis. Biomaterials. 134, 128-142 (2017).
- Lonez, C., et al. Cationic lipid nanocarriers activate Toll-like receptor 2 and NLRP3 inflammasome pathways. Nanomedicine: Nanotechnology, Biology, and Medicine. 10 (4), 775-782 (2014).
