Name: screening bioactive nanoparticles in phagocytic immune cells for inhibitors of toll-like receptor signaling
Uploaded: 2017-07-26T14:00:00
Description: Watch this Scientific Journal Video about Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling at JoVE.com

Forfatterne vil gerne anerkende støtten fra programmet for professor i Special Appointment (Eastern Scholar) ved Shanghai Higher Learning (HY), startfonden fra Shanghai First People&#39;s Hospital (HY), Gaofeng Clinical Medicine Grant-støtte fra Shanghai Jiaotong University School of Medicine (HY), og finansieringen fra Crohns og Colitis Foundation of Canada (CCFC) (SET og HY).

1. Fremstilling af cellekulturmedier og reagenser <ol><li> Forbered det fuldstændige cellekulturmedium R10 ved at tilsætte supplementerne af 10% føtalt bovint serum (FBS), 2 mM L-glutamin og 1 mM natriumpyruvat i RPMI-1640-mediumet. <ol><li> Forbered udvælgelseskulturmediet R10-Z ved at tilsætte antibiotika Zeocin (200 μg / ml) til R10 for at opretholde ekspressionen af ​​SEAP under kontrol af NF-KB / AP-1-aktivering. For at udvælge celler, der udtrykker både SEAP- og luciferase-reporterge...

<ol>
	<li>Akira, S., Takeda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+signalling.">Toll-like receptor signalling.</a> Nat Rev Immunol. 4 (7), 499-511 (2004).</li><li>Beutler, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLRs+and+innate+immunity.">TLRs and innate immunity.</a> Blood. 113 (7), 1399-1407 (2009).</li><li>Gay, N. J., Symmons, M. F., Gangloff, M., Bryant, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+localization+of+Toll-like+receptor+signalling+complexes.">Assembly and localization of Toll-like receptor signalling complexes.</a> Nat Rev Immunol. 14 (8), 546-558 (2014).</li><li>Palm, N. W., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+recognition+receptors+and+control+of+adaptive+immunity.">Pattern recognition receptors and control of adaptive immunity.</a> Immunol Rev. 227 (1), 221-233 (2009).</li><li>Kawasaki, T., Kawai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+signaling+pathways.">Toll-like receptor signaling pathways.</a> Front Immunol. 5, 461 (2014).</li><li>Uematsu, S., Akira, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+and+Type+I+interferons.">Toll-like receptors and Type I interferons.</a> J Biol Chem. 282 (21), 15319-15323 (2007).</li><li>Maglione, P. J., Simchoni, N., Cunningham-Rundles, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+signaling+in+primary+immune+deficiencies.">Toll-like receptor signaling in primary immune deficiencies.</a> Ann N Y Acad Sci. 1356, 1-21 (2015).</li><li>Drexler, S. K., Foxwell, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+toll-like+receptors+in+chronic+inflammation.">The role of toll-like receptors in chronic inflammation.</a> Int J Biochem Cell Biol. 42 (4), 506-518 (2010).</li><li>O'Neill, L. A., Bryant, C. E., Doyle, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+targeting+of+Toll-like+receptors+for+infectious+and+inflammatory+diseases+and+cancer.">Therapeutic targeting of Toll-like receptors for infectious and inflammatory diseases and cancer.</a> Pharmacol Rev. 61 (2), 177-197 (2009).</li><li>Harter, L., Mica, L., Stocker, R., Trentz, O., Keel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+toll-like+receptor-2+and+-4+on+leukocytes+from+patients+with+sepsis.">Increased expression of toll-like receptor-2 and -4 on leukocytes from patients with sepsis.</a> Shock. 22 (5), 403-409 (2004).</li><li>Roger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+lethal+gram-negative+bacterial+sepsis+by+targeting+Toll-like+receptor+4.">Protection from lethal gram-negative bacterial sepsis by targeting Toll-like receptor 4.</a> Proc Natl Acad Sci U S A. 106 (7), 2348-2352 (2009).</li><li>Tsujimoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Toll-like+receptors+in+the+development+of+sepsis.">Role of Toll-like receptors in the development of sepsis.</a> Shock. 29 (3), 315-321 (2008).</li><li>Blohmke, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR5+as+an+anti-inflammatory+target+and+modifier+gene+in+cystic+fibrosis.">TLR5 as an anti-inflammatory target and modifier gene in cystic fibrosis.</a> J Immunol. 185 (12), 7731-7738 (2010).</li><li>Hartl, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immunity+in+cystic+fibrosis+lung+disease.">Innate immunity in cystic fibrosis lung disease.</a> J Cyst Fibros. 11 (5), 363-382 (2012).</li><li>Celhar, T., Fairhurst, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptors+in+systemic+lupus+erythematosus:+potential+for+personalized+treatment.">Toll-like receptors in systemic lupus erythematosus: potential for personalized treatment.</a> Front Pharmacol. 5, 265 (2014).</li><li>Chen, J. Q., Szodoray, P., Zeher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-Like+Receptor+Pathways+in+Autoimmune+Diseases.">Toll-Like Receptor Pathways in Autoimmune Diseases.</a> Clin Rev Allergy Immunol. 50 (1), 1-17 (2016).</li><li>Hennessy, E. J., Parker, A. E., O'Neill, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Toll-like+receptors:+emerging+therapeutics.">Targeting Toll-like receptors: emerging therapeutics.</a> Nat Rev Drug Discov. 9 (4), 293-307 (2010).</li><li>Hedayat, M., Netea, M. G., Rezaei, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+Toll-like+receptors:+a+decade+of+progress+in+combating+infectious+diseases.">Targeting of Toll-like receptors: a decade of progress in combating infectious diseases.</a> Lancet Infect Dis. 11 (9), 702-712 (2011).</li><li>Huang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+DNA+nanoparticles+as+immunomodulatory+reagents+that+activate+regulatory+T+cells.">Engineering DNA nanoparticles as immunomodulatory reagents that activate regulatory T cells.</a> J Immunol. 188 (10), 4913-4920 (2012).</li><li>Lemos, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+STING+adaptor+attenuates+experimental+autoimmune+encephalitis.">Activation of the STING adaptor attenuates experimental autoimmune encephalitis.</a> J Immunol. 192 (12), 5571-5578 (2014).</li><li>Hess, K. L., Andorko, J. I., Tostanoski, L. H., Jewell, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyplexes+assembled+from+self-peptides+and+regulatory+nucleic+acids+blunt+toll-like+receptor+signaling+to+combat+autoimmunity.">Polyplexes assembled from self-peptides and regulatory nucleic acids blunt toll-like receptor signaling to combat autoimmunity.</a> Biomaterials. 118, 51-62 (2017).</li><li>Tostanoski, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+Polyelectrolyte+Multilayers+to+Promote+Immunological+Tolerance.">Design of Polyelectrolyte Multilayers to Promote Immunological Tolerance.</a> ACS Nano. , (2016).</li><li>Foit, L., Thaxton, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+high-density+lipoprotein-like+nanoparticles+potently+inhibit+cell+signaling+and+production+of+inflammatory+mediators+induced+by+lipopolysaccharide+binding+Toll-like+receptor+4.">Synthetic high-density lipoprotein-like nanoparticles potently inhibit cell signaling and production of inflammatory mediators induced by lipopolysaccharide binding Toll-like receptor 4.</a> Biomaterials. 100, 67-75 (2016).</li><li>He, C., Hu, Y., Yin, L., Tang, C., Yin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+particle+size+and+surface+charge+on+cellular+uptake+and+biodistribution+of+polymeric+nanoparticles.">Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles.</a> Biomaterials. 31 (13), 3657-3666 (2010).</li><li>Hirn, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+size-dependent+and+surface+charge-dependent+biodistribution+of+gold+nanoparticles+after+intravenous+administration.">Particle size-dependent and surface charge-dependent biodistribution of gold nanoparticles after intravenous administration.</a> Eur J Pharm Biopharm. 77 (3), 407-416 (2011).</li><li>Blanco, E., Shen, H., Ferrari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+nanoparticle+design+for+overcoming+biological+barriers+to+drug+delivery.">Principles of nanoparticle design for overcoming biological barriers to drug delivery.</a> Nat Biotechnol. 33 (9), 941-951 (2015).</li><li>Arvizo, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+pharmacokinetics,+tumor+uptake+and+biodistribution+by+engineered+nanoparticles.">Modulating pharmacokinetics, tumor uptake and biodistribution by engineered nanoparticles.</a> PLoS One. 6 (9), e24374 (2011).</li><li>Ernsting, M. J., Murakami, M., Roy, A., Li, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+controlling+the+pharmacokinetics,+biodistribution+and+intratumoral+penetration+of+nanoparticles.">Factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles.</a> J Control Release. 172 (3), 782-794 (2013).</li><li>Lin, P. J., Tam, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+the+pharmacokinetic/pharmacodynamic+properties+of+therapeutic+nucleotides+using+lipid+nanoparticle+systems.">Enhancing the pharmacokinetic/pharmacodynamic properties of therapeutic nucleotides using lipid nanoparticle systems.</a> Future Med Chem. 7 (13), 1751-1769 (2015).</li><li>Yang, H., Fung, S. Y., Liu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+the+cellular+uptake+of+physiologically+stable+peptide-gold+nanoparticle+hybrids+with+single+amino+acids.">Programming the cellular uptake of physiologically stable peptide-gold nanoparticle hybrids with single amino acids.</a> Angew Chem Int Ed Engl. 50 (41), 9643-9646 (2011).</li><li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+Acid+Structure+Determines+the+Immune+Responses+Generated+by+Peptide-Gold+Nanoparticle+Hybrids.">Amino Acid Structure Determines the Immune Responses Generated by Peptide-Gold Nanoparticle Hybrids.</a> Particle &amp; Particle Systems Characterization. 30 (12), 1039-1043 (2013).</li><li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+Acid-Dependent+Attenuation+of+Toll-like+Receptor+Signaling+by+Peptide-Gold+Nanoparticle+Hybrids.">Amino Acid-Dependent Attenuation of Toll-like Receptor Signaling by Peptide-Gold Nanoparticle Hybrids.</a> ACS Nano. 9 (7), 6774-6784 (2015).</li><li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosomal+pH+modulation+by+peptide-gold+nanoparticle+hybrids+enables+potent+anti-inflammatory+activity+in+phagocytic+immune+cells.">Endosomal pH modulation by peptide-gold nanoparticle hybrids enables potent anti-inflammatory activity in phagocytic immune cells.</a> Biomaterials. 111, 90-102 (2016).</li><li>Ospelt, C., Gay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLRs+and+chronic+inflammation.">TLRs and chronic inflammation.</a> Int J Biochem Cell Biol. 42 (4), 495-505 (2010).</li><li>Kuznik, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+endosomal+TLR+inhibition+by+antimalarial+drugs+and+imidazoquinolines.">Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines.</a> J Immunol. 186 (8), 4794-4804 (2011).</li><li>Lee, S. J., Silverman, E., Bargman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+antimalarial+agents+in+the+treatment+of+SLE+and+lupus+nephritis.">The role of antimalarial agents in the treatment of SLE and lupus nephritis.</a> Nat Rev Nephrol. 7 (12), 718-729 (2011).</li><li>Mullarkey, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+endotoxin+response+by+e5564,+a+novel+Toll-like+receptor+4-directed+endotoxin+antagonist.">Inhibition of endotoxin response by e5564, a novel Toll-like receptor 4-directed endotoxin antagonist.</a> J Pharmacol Exp Ther. 304 (3), 1093-1102 (2003).</li><li>Barochia, A., Solomon, S., Cui, X., Natanson, C., Eichacker, P. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eritoran+tetrasodium+(E5564)+treatment+for+sepsis:+review+of+preclinical+and+clinical+studies.">Eritoran tetrasodium (E5564) treatment for sepsis: review of preclinical and clinical studies.</a> Expert Opin Drug Metab Toxicol. 7 (4), 479-494 (2011).</li><li>Rossignol, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety,+pharmacokinetics,+pharmacodynamics,+and+plasma+lipoprotein+distribution+of+eritoran+(E5564)+during+continuous+intravenous+infusion+into+healthy+volunteers.">Safety, pharmacokinetics, pharmacodynamics, and plasma lipoprotein distribution of eritoran (E5564) during continuous intravenous infusion into healthy volunteers.</a> Antimicrob Agents Chemother. 48 (9), 3233-3240 (2004).</li><li>Rossignol, D. P., Wong, N., Noveck, R., Lynn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+pharmacodynamic+activity+of+eritoran+tetrasodium,+a+TLR4+antagonist,+during+intermittent+intravenous+infusion+into+normal+volunteers.">Continuous pharmacodynamic activity of eritoran tetrasodium, a TLR4 antagonist, during intermittent intravenous infusion into normal volunteers.</a> Innate Immun. 14 (6), 383-394 (2008).</li><li>Tidswell, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+2+trial+of+eritoran+tetrasodium+(E5564),+a+toll-like+receptor+4+antagonist,+in+patients+with+severe+sepsis.">Phase 2 trial of eritoran tetrasodium (E5564), a toll-like receptor 4 antagonist, in patients with severe sepsis.</a> Crit Care Med. 38 (1), 72-83 (2010).</li><li>Opal, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+eritoran,+an+antagonist+of+MD2-TLR4,+on+mortality+in+patients+with+severe+sepsis:+the+ACCESS+randomized+trial.">Effect of eritoran, an antagonist of MD2-TLR4, on mortality in patients with severe sepsis: the ACCESS randomized trial.</a> JAMA. 309 (11), 1154-1162 (2013).</li><li>Fung, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+screening+of+marine+sponge+extracts+for+anti-inflammatory+agents+combined+with+chemical+genomics+identifies+girolline+as+an+inhibitor+of+protein+synthesis.">Unbiased screening of marine sponge extracts for anti-inflammatory agents combined with chemical genomics identifies girolline as an inhibitor of protein synthesis.</a> ACS Chem Biol. 9 (1), 247-257 (2014).</li></ol>

Da TLR er involveret i patogenesen af ​​mange inflammatoriske sygdomme, er de fremkommet som terapeutiske mål for moduleringen af ​​immunresponser og inflammatoriske tilstande. Den kliniske udvikling af terapeutiske midler til at inhibere TLR-signalveje har imidlertid haft begrænset succes til dato. Det antimalariale lægemiddel hydroxychloroquin, der hæmmer TLR7 og TLR9, er i klinisk anvendelse 35 , 36 . Tilsvarende har kun et begrænset antal forbin...

Toll-lignende receptorer (TLR&#39;er) er et af hovedelementerne i det medfødte immunsystem, der bidrager til den første forsvarslinje mod infektioner. TLR&#39;er er ansvarlige for at registrere invaderende patogener ved at genkende et repertoire af patogenassocierede molekylære mønstre (eller PAMP&#39;er) og montering af forsvarsreaktioner gennem en kaskade af signaltransduktion 1 , 2 . Der er identificeret 10 menneskelige TLR&#39;er; Bortset fra TLR10, for hvilken liganden (e) forbliver uklare, kan hver TLR genkende en særskilt konserveret gruppe af PAMP&#39;er. For eksempel kan TLR2 og TLR4, primært placeret på celleoverfladen, detektere lipoproteiner og glycolipider fra henholdsvis Gram-positiv og Gram-negativ bakterier; Mens TLR3, TLR7 / 8 og TLR9, der hovedsagelig er til stede i endosomale rum, kan mærke RNA og DNA-produkter fra virus og bakterier 3 . Når stimuleret af PAMP&#39;er udløser TLR&#39;er væsentlige immunresponser ved at frigive pro-infLammatoriske mediatorer, rekrutterings- og aktiverende effektorimmunceller og koordinering af efterfølgende adaptive immunhændelser 4 . TLR-signaltransduktionen kan simpelthen kategoriseres i to hovedveje 5 , 6 . Den ene er afhængig af adapter-myeloid differentieringsfaktor 88 (MyD88) - den MyD88-afhængige vej. Alle TLR&#39;er undtagen TLR3 anvender denne vej til aktivering af kaktakaktiver kappa-lette kædeforstærkere af aktiverede B-celler (NF-KB) og mitogenassocierede proteinkinaser (MAPK&#39;er), hvilket fører til ekspression af proinflammatoriske mediatorer, såsom TNF- A, IL-6 og IL-8. Den anden vej benytter TIR-domæne-indeholdende adapter-inducerende interferon-β (TRIF) - den TRIF-afhængige eller MyD88-uafhængige vej - for at aktivere interferon (IFN) regulatoriske faktorer (IRF&#39;er) og NF-KB, hvilket resulterer i produktion af Type I IFN&#39;er. Intact TLR signaleringEr afgørende for vores daglige beskyttelse mod mikrobielle og virale infektioner; Defekter i TLR signalveje kan føre til immunsvigt og er ofte skadelige for menneskers sundhed. 7 TLR-signalering er imidlertid et &quot;dobbeltkantet sværd&quot; og overdreven, ukontrolleret TLR-aktivering er skadelig. Overaktive TLR-reaktioner bidrager til patogenesen i mange akutte og kroniske humane inflammatoriske sygdomme 8 , 9 . F.eks. Skyldes sepsis, som er karakteriseret ved systemisk inflammation og multiorganskader, primært akutte, overvældende immunresponser mod infektioner, idet TLR2 og TLR4 spiller en afgørende rolle i sepsispatofysiologien 10 , 11 , 12 . Derudover har TLR5 vist sig at bidrage til kronisk lungebetændelse hos patienter med cystisk fibrose 13, 14 . Desuden er dysreguleret endosomal TLR-signalering (f.eks. TLR7 og TLR9) stærkt forbundet med udvikling og progression af flere autoimmune sygdomme, herunder systemisk lupus erythematosus (SLE) og reumatoid arthritis (RA) 15 , 16 . Disse konvergerende bevislinier identificerer TLR-signalering som et potentielt terapeutisk mål for mange inflammatoriske sygdomme 17 . Selv om farmakologisk regulering af TLR-responser forventes at være gavnlig under mange inflammatoriske tilstande, er der desværre i øjeblikket meget få forbindelser klinisk tilgængelige til at hæmme TLR-signalering 9 , 17 , 18 . Dette skyldes dels kompleksiteten og redundansen af ​​TLR-veje involveret i immune homeostasen og sygdomspatologien. Derfor søger efter roman, potenTerapeutiske midler til målretning af flere TLR-signalveje kunne bygge bro over et grundlæggende hul og overvinde udfordringen ved at fremme TLR-hæmmere ind i klinikken. I lyset af de hurtige fremskridt inden for nanovidenskab og nanoteknologi fremkommer nanodevices som næste generation TLR modulatorer på grund af deres unikke egenskaber 19 , 20 , 23 . Nanoskala-størrelsen gør det muligt for disse nano-terapeutiske stoffer at få en bedre biofordeling og vedvarende omsætning 24 , 25 , 26 . De kan yderligere funktionaliseres for at opfylde de ønskede farmakodynamiske og farmakokinetiske profiler 27 , 28 , 29 . Mere spændende opstår biaktiviteten af ​​disse nye nanodevices fra deres egenskaber, som kan skræddersyes tilSpecifikke medicinske anvendelser, snarere end blot at virke som et leveringsmiddel til et terapeutisk middel. For eksempel blev et HDD-lignende nanopartikel med høj densitet designet til at hæmme TLR4-signalering ved fjernelse af TLR4-liganden LPS 23 . Derudover har vi udviklet et peptid-guld nanopartikel hybrid system, hvor de dekorerede peptider kan ændre overfladeegenskaberne af guld nanopartikler, og tillade dem at have forskellige bioaktiviteter 30 , 31 , 32 , 33 . Dette gør dem til en speciel klasse af stof (eller &quot;nanoteknologi&quot;) som næste generations nano-terapeutik. I denne protokol præsenterer vi en tilgang til at identificere en ny klasse af peptid-guld nanopartikler (peptid-GNP) hybrider, der potentielt kan inhibere flere TLR signalveje i fagocytiske immunceller 32 , </suP&gt; 33 . Tilgangen er baseret på kommercielt tilgængelige THP-1 reportercellelinier. Reportercellerne består af to stabile, inducerbare reporterkonstruktioner: den ene bærer et sekretiseret embryonalt alkalisk phosphatase- (SEAP) -gen under kontrol af en promotor inducerbar af transkriptionsfaktorerne NF-KB og aktivatorprotein 1 (AP-1); Den anden indeholder et secerneret luciferase reportergen under kontrol af promotorer inducerbare af interferon regulatoriske faktorer (IRF&#39;er). Ved TLR-stimulering fører signaltransduktionen til aktiveringen af ​​NF-KB / AP-1 og / eller IRF&#39;er, som tænder reportergenene til hemmeligt SEAP og / eller luciferase; Sådanne hændelser kan let registreres ved anvendelse af deres tilsvarende substratreagenser med et spektrofotometer eller luminometer. Ved hjælp af denne fremgangsmåde til screening af vores tidligere etablerede bibliotek af peptid-GNP-hybrider, identificerede vi blykandidater, som potentielt kan hæmme TLR4-signalveje. Den inhibitoriske aktivitet af blypeptid-BNP-hybrider blev derefter valideret ved anvendelse af en anden biokemisk tilgang til immunoblotting og evalueret på andre TLR-veje. Denne fremgangsmåde muliggør hurtig og effektiv screening af nye agenter, der retter sig mod TLR-signalveje.

<table>
	<tbody>
		<tr>
			<td>THP-1-XBlue reporter cell</td>
			<td>InvivoGen</td>
			<td>thpx-sp</td>
			<td>keep cell culture passage under 20</td>
		</tr>
		<tr>
			<td>THP-1-Dual repoter cell</td>
			<td>InvivoGen</td>
			<td>thpd-nfis</td>
			<td>keep cell culture passage under 20</td>
		</tr>
		<tr>
			<td>RPMI-1640 (no L-glutamine)</td>
			<td>GE Health Care</td>
			<td>SH30096.02</td>
			<td>Warm up to 37 &#176;C before use; add supplements to make a complete medium R10</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (qualified)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12484028</td>
			<td>Heat inactivated; 10% in RPMI-1640</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30034.02</td>
			<td>2 mM in the complete medium R10</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Thermo Fisher Scientific</td>
			<td>11360-070</td>
			<td>1 mM in the complete medium R10</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline, 1X, without calcium, magnesium</td>
			<td>GE Health Care</td>
			<td>SH30028.02</td>
			<td>Use for cell washing and reagent preparation</td>
		</tr>
		<tr>
			<td>QUANTI-Blue</td>
			<td>InvivoGen</td>
			<td>rep-qb1</td>
			<td>SEAP substrate</td>
		</tr>
		<tr>
			<td>QUANTI-Luc</td>
			<td>InvivoGen</td>
			<td>rep-qlc2</td>
			<td>Luciferase substrate</td>
		</tr>
		<tr>
			<td>Zeocin</td>
			<td>InvivoGen</td>
			<td>ant-zn-1</td>
			<td>Selection antibiotics for reporter cells</td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>InvivoGen</td>
			<td>anti-bl-1</td>
			<td>Selection antibiotics for reporter cells</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO) for molecular biology</td>
			<td>Sigmal-Aldrich</td>
			<td>D8418-100ML</td>
			<td>Use for reagent preparation</td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA) for molecular biology</td>
			<td>Sigmal-Aldrich</td>
			<td>P1585-1MG</td>
			<td>Use for cell differentiation</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS) from E. coli K12</td>
			<td>InvivoGen</td>
			<td>tlrl-eklps</td>
			<td>TLR4 ligand</td>
		</tr>
		<tr>
			<td>Pam3CSK4</td>
			<td>InvivoGen</td>
			<td>tlrl-pms</td>
			<td>TLR2/1 ligand</td>
		</tr>
		<tr>
			<td>Poly (I:C) HMW</td>
			<td>InvivoGen</td>
			<td>tlrl-pic</td>
			<td>TLR3 ligand</td>
		</tr>
		<tr>
			<td>Flagellin from S. Typhimurium (FLA-ST), ultrapure</td>
			<td>InvivoGen</td>
			<td>tlrl-epstfla</td>
			<td>TLR5 ligand</td>
		</tr>
		<tr>
			<td>SpectraMax Plus 384 microplate reader</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>Read colorimetric assay</td>
		</tr>
		<tr>
			<td>Infinite M200 Pro multimode microplate reader with injectors</td>
			<td>Tecan</td>
			<td>N/A</td>
			<td>Read luminiscience</td>
		</tr>
		<tr>
			<td>Microfuge 22R centrifuge</td>
			<td>Beckman Coulter</td>
			<td>N/A</td>
			<td>Temperature controlled micro-centrifugator (up to 18,000 g)</td>
		</tr>
		<tr>
			<td>Allegra X-15R centrifuge</td>
			<td>Beckman Coulter</td>
			<td>N/A</td>
			<td>Temperature controlled general purpose centrifugator (for cell culture use)</td>
		</tr>
		<tr>
			<td>Costar assay plate, 96-well white with clear flat bottom, tissue culure treated</td>
			<td>Corning Costar</td>
			<td>3903</td>
			<td>Used for luminiscence assay</td>
		</tr>
	</tbody>
</table>

screening bioactive nanoparticles in phagocytic immune cells for inhibitors of toll-like receptor signaling

Farmakologisk regulering af Toll-lignende receptor (TLR) reaktioner har stort løfte i behandlingen af ​​mange inflammatoriske sygdomme. Der har imidlertid været begrænsede forbindelser hidtil til at dæmpe TLR-signalering, og der har ikke været klinisk godkendte TLR-hæmmere (undtagen det anti-malariale lægemiddelhydroxychloroquin) i klinisk anvendelse. I lyset af de hurtige fremskridt inden for nanoteknologi kan manipulation af immunrespons ved hjælp af nanoapparater give en ny strategi til behandling af disse sygdomme. Her præsenterer vi en high-throughput screeningsmetode til hurtigt at identificere nye bioaktive nanopartikler, der hæmmer TLR-signalering i fagocytiske immunceller. Denne screeningsplatform er bygget på THP-1-cellebaserede reporterceller med kolorimetriske og luciferaseanalyser. Reportercellerne er konstrueret fra den humane THP-1 monocytiske cellelinie ved stabil integration af to inducerbare reporterkonstruktioner. Den ene udtrykker et udskilt embryonalt alkalisk phosphatase (SEAP) genUnder kontrol af en promotor inducerbar af transkriptionsfaktorerne NF-KB og AP-1, og den anden udtrykker et udskilt luciferase-reportergen under styring af promotorer inducerbare af interferonregulatoriske faktorer (IRF&#39;er). På grund af TLR-stimulering aktiverer reportercellerne Transkriptionsfaktorer og producerer efterfølgende SEAP og / eller luciferase, som kan detekteres under anvendelse af deres tilsvarende substratreagenser. Ved anvendelse af et bibliotek af hybrider af peptid-guld nanopartikel (GNP), der blev etableret i vores tidligere undersøgelser som eksempel, identificerede vi en peptid-GNP-hybrid, som effektivt kunne hæmme de to arme af TLR4-signaleringskaskade udløst af dets prototypiske ligand, lipopolysaccharid (LPS). Resultaterne blev valideret ved standard biokemiske teknikker, herunder immunoblotting. Yderligere analyse fastslog, at denne bly hybrid havde et bredt inhibitorisk spektrum, der virker på flere TLR-veje, herunder TLR2, 3, 4 og 5. Denne eksperimentelle tilgang gør det muligt hurtigt at vurdere hvemRa nanopartikel (eller andre terapeutiske forbindelser) kan modulere specifik TLR-signalering i fagocytiske immunceller.

Den samlede eksperimentelle tilgang er illustreret i figur 1 . De to THP-1-reportercellelinier, THP-1-XBlue og THP-1-Dual, anvendes til hurtig screening af TLR-responserne ved at undersøge aktiveringen af ​​henholdsvis NF-KB / AP-1 og IRF&#39;er. Aktiveringen af ​​NF-KB / AP-1 kan påvises ved hjælp af SEAP-kolorimetrisk analyse, mens IRF-aktivering overvåges ved luciferase luminescens. De monocytiske THP-1-celler kan let afledes i makrofager for ...

Watch this Scientific Journal Video about Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling at JoVE.com

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Toll-lignende receptor (TLR) signalering spiller en vigtig rolle i patofysiologien af ​​mange humane inflammatoriske sygdomme, og regulering af TLR-responser af bioaktive nanopartikler forventes at være gavnlig under mange inflammatoriske tilstande. THP-1-cellebaserede reporterceller tilvejebringer en alsidig og robust screeningsplatform til identifikation af nye inhibitorer af TLR-signalering.

Screening af bioaktive nanopartikler i phagocytiske immunceller til inhibitorer af Toll-lignende Receptor Signalering

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Research

Education

JoVE Journal

JoVE Core

Biology

Medicine

Cellular Processes

Cell Signaling

SCIENTISTS IN ACTION

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

High Content Screening in Neurodegenerative Diseases

Højt indhold Screening i neurodegenerative sygdomme

The functional annotation of genomes, construction of molecular networks and novel drug target identification, are important challenges that need to be ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Screening for Melanoma Modifiers anvendelse af et zebrafisk Autokton tumormodel

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

En Multiplexed Luciferase-baseret Screening Platform for forhører Kræft-associeret signaltransduktion i dyrkede celler

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Præklinisk evaluering af tyrosinkinasehæmmere for Behandling af akut leukæmi

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

High-throughput Flowcytometri cellebaseret assay til påvisning af antistoffer til N-methyl-D-aspartat-receptor eller dopamin-2-receptoren i humant serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Profilering af østrogen-regulerede MicroRNA i brystkræftceller

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Udnyttelse af blød agar Colony Formation Assay til at identificere inhibitorer for tumorgenicitet for Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...