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

The authors would like to acknowledge the support from the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (H.Y.), the starting fund from Shanghai First People&#39;s Hospital (H.Y.), Gaofeng Clinical Medicine Grant support from Shanghai Jiaotong University School of Medicine (H.Y.), and the funding from the Crohn&#39;s and Colitis Foundation of Canada (CCFC) (S.E.T. and H.Y.).

1. Preparation of Cell Culture Media and Reagents
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	<li>Prepare the complete cell culture medium R10 by adding the supplements of 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1 mM sodium pyruvate into the RPMI-1640 medium.
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		<li>Prepare the selection culture medium R10-Z by adding the antibiotics Zeocin (200 &#956;g/mL) to R10 for maintaining the expression of SEAP under the control of NF-&#954;B/AP-1 activation. To select for cells expressing both SEAP and luciferase reporter genes, ad...

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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. 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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. 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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. 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Since TLRs are involved in the pathogenesis of many inflammatory diseases, they have emerged as therapeutic targets for the modulation of immune responses and inflammatory conditions. However, the clinical development of therapeutics to inhibit TLR signaling pathways has had limited success to date. The antimalarial drug hydroxychloroquine which inhibits TLR7 and TLR9 is in clinical use35,36.&#160;Similarly, only a limited number of compounds have progressed to c...

Toll-like receptors (TLRs) are one of the key elements in the innate immune system contributing to the first line of defense against infections. TLRs are responsible for sensing invading pathogens by recognizing a repertoire of pathogen-associated molecular patterns (or PAMPs) and mounting defense reactions through a cascade of signal transduction1,2. There are 10 human TLRs identified; except TLR10 for which the ligand(s) remain unclear, each TLR can recognized a distinct, conserved group of PAMPs. For example, TLR2 and TLR4, primarily located on the cell surface, can detect lipoproteins and glycolipids from Gram-positive and Gram-negative bacteria, respectively; while TLR3, TLR7/8 and TLR9, mainly present in the endosomal compartments, can sense RNA and DNA products from viruses and bacteria3. When stimulated by PAMPs, TLRs trigger essential immune responses by releasing pro-inflammatory mediators, recruiting and activating effector immune cells, and coordinating subsequent adaptive immune events4.
The TLR signaling transduction can be simply categorized into two main pathways5,6. One is dependent upon the adaptor protein myeloid differentiation factor 88 (MyD88) &#8212; the MyD88-dependent pathway. All TLRs except TLR3 utilize this pathway to activate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) and mitogen-associated protein kinases (MAPKs), leading to the expression of pro-inflammatory mediators such as TNF-&#945;, IL-6 and IL-8. The second pathway utilizes TIR-domain-containing adaptor-inducing interferon-&#946; (TRIF) &#8212; the TRIF-dependent or MyD88-independent pathway &#8212; to activate interferon (IFN) regulatory factors (IRFs) and NF-&#954;B, resulting in the production of type I IFNs. Intact TLR signaling is critical to our daily protection from microbial and viral infections; defects in TLR signaling pathways can lead to immunodeficiency and are often detrimental to human health.7
However, TLR signaling is a &#39;double-edged sword&#39; and excessive, uncontrolled TLR activation is harmful. Overactive TLR responses contribute to the pathogenesis in many acute and chronic human inflammatory diseases8,9. For instance, sepsis which is characterized by systemic inflammation and multi-organ injury, is primarily due to acute, overwhelming immune responses toward infections, with TLR2 and TLR4 playing a crucial role in the sepsis pathophysiology10,11,12. In addition, TLR5 has been found to contribute to chronic lung inflammation of patients with cystic fibrosis13,14. Moreover, dysregulated endosomal TLR signaling (e.g., TLR7 and TLR9) is strongly associated with the development and progression of several autoimmune diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA)15,16. These converging lines of evidence identify TLR signaling as a potential therapeutic target for many inflammatory diseases17.
Although pharmacological regulation of TLR responses is anticipated to be beneficial in many inflammatory conditions, unfortunately, there are currently very few compounds clinically available to inhibit TLR signaling9,17,18. This is partly due to the complexity and redundancy of the TLR pathways involved in the immune homeostasis and disease pathology. Therefore, searching for novel, potent therapeutic agents to target multiple TLR signaling pathways could bridge a fundamental gap, and overcome the challenge of advancing TLR inhibitors into the clinic.
In light of the rapid advances in nanoscience and nanotechnology, nanodevices are emerging as the next generation TLR modulators owing to their unique properties19,20,23. The nanoscale size allows these nano-therapeutics to have better bio-distribution and sustained circulation24,25,26. They can be further functionalized to meet the desired pharmacodynamic and pharmacokinetic profiles27,28,29. More excitingly, the bio-activity of these novel nanodevices arises from their intrinsic properties, which can be tailored for specific medical applications, rather than simply acting as a delivery vehicle for a therapeutic agent. For example, a high-density lipoprotein (HDL)-like nanoparticle was designed to inhibiting TLR4 signaling by scavenging the TLR4 ligand LPS23.&#160;In addition, we have developed a peptide-gold nanoparticle hybrid system, where the decorated peptides can alter the surface properties of the gold nanoparticles, and allow them to have various bio-activities30,31,32,33. This makes them a special class of drug (or &#34;nano-drug&#34;) as the next generation nano-therapeutics.
In this protocol, we present an approach to identify a novel class of peptide-gold nanoparticle (peptide-GNP) hybrids that can potently inhibit multiple TLR signaling pathways in phagocytic immune cells32,33. The approach is based on commercially available THP-1 reporter cell lines. The reporter cells consist of two stable, inducible reporter constructs: one carries a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and activator protein 1 (AP-1); the other contains a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs). Upon TLR stimulation, the signal transduction leads to the activation of NF-&#954;B/AP-1 and/or IRFs, which turns on the reporter genes to secret SEAP and/or luciferase; such events can be easily detected using their corresponding substrate reagents with a spectrophotometer or luminometer. Using this approach to screen our previously established library of peptide-GNP hybrids, we identified lead candidates that can potently inhibit TLR4 signaling pathways. The inhibitory activity of the lead peptide-GNP hybrids was then validated using another biochemical approach of immunoblotting, and evaluated on other TLR pathways. This approach allows for fast, effective screening of novel agents targeting TLR signaling pathways.

<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

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

The overall experimental approach is illustrated in Figure 1. The two THP-1 reporter cell lines, THP-1-XBlue and THP-1-Dual, are used to fast screen the TLR responses by probing the activation of NF-&#954;B/AP-1 and IRFs, respectively. The activation of NF-&#954;B/AP-1 can be detected by the SEAP colorimetric assay, whereas IRF activation is monitored by luciferase luminescence. The monocytic THP-1 cells can be easily derived into macrophages to screen the na...

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-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

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

The overall goal of this screening procedure is to identify novel bioactive nanoparticles that can inhibit toll-like receptor signaling. This method can help you physically identify novel nanoinhibitors targeting toll-like receptor signaling in innate immune cells. The main advantage of this technique is that it is fast and robust and easy to perform using reporter cell systems.The applications of this technique extend toward the therapy of many inflammatory diseases because toll-like receptor signaling is found to be involved in the pathogenesis of these diseases. Though this method can provided high screening on and gold nanoparticles, it can also be applied to other nanoparticle systems as well as therapeutic small molecules and the biologies. To begin this procedure, prepare the culture mediums as outlined in the text protocol.Next, add 100 milliliters of ultra-pure endotoxin-free water to a clean 125 milliliter glass flask. Add one pouch of secreted embryonic alkaline phosphatase substrate powder, and swirl the solution gently until it is fully dissolved. Incubate at 37 degrees Celsius for one hour to ensure complete dissolution of the substrates.Using a 0.2 micrometer membrane, filter the solution. Store at four degrees Celsius until ready to use. Next, add one pouch of luciferase substrate powder to a 50 milliliter centrifuge tube containing 25 milliliters of ultra pure endotoxin-free water.After the powder has dissolved completely, store at four degrees Celsius for up to a week until ready to use. Then, prepare the stock solutions of all other reagents as outlined in the text protocol. After culturing and preparing the cells, transfer them from the culture flask to a centrifuge tube.Spin down the cells at 300 times G for five minutes. Then, resuspend them in the R-10 medium at a concentration of 1 X 10 to the 6 cells per milliliter. Add aliquots of the PMA solution to the cell suspension such that the final concentration is 50 nanograms per milliliter.Pour the cell suspension into a sterile reagent reservoir. Using a multi-channel pipette, transfer 100 microliters of the cell suspension into each well of a 96-well flat bottom culture plate. Incubate in a cell culture incubator for 24 hours at 37 degrees Celsius.After this, use a vacuum aspirator to carefully remove the culture medium. Using an inverted microscope, check the cells to ensure that the derived macrophages are adhered at the bottom of the well. Gently wash the cells twice using 100 microliters of PBS per well for each wash.Then, add 100 microliters of fresh R-10 medium to each well. Let the cells rest for two days in an incubator at 37 degress Celsius before conducting the reporter assay. First, determine the optimal LPS dose as outlined in the text protocol.Next, add 180 microliters of pre-warmed Quanti-Blue solution to each well of a new 96-well flat bottom plate. Then, transfer 20 microliters of supernatant from each sample into the plate. Incubate at 37 degrees Celsius for one to two hours to allow for the color to develop from pink to dark blue.After the optical density is above one, use a plate reader to determine the absorption at 655 nanometers. To begin analysis of IRF activation, transfer 10 microliters of fresh supernatant from each sample into a fresh 96-well clear flat bottom white plate. Using auto-injection, add 50 microliters of the luciferase solution to each well.Then immediately collect the luminescence well by well to produce a dose response curve to determine the optimal LPS concentration for nanoparticle screening. Next, centrifuge 20 volumes of the peptide-GMP hybrid solution in 1.5 milliliter Eppendorf tubes at 18, 000 times G for 30 minutes. Carefully discard the supernatants.Transfer the hybrids to a single fresh tube and wash them twice with one milliliter of PBS. After this, resuspend the washed hybrids in one volume of R-10 medium. Mix equal volumes of the concentrated hybrids and the LPS containing R-10 medium such that the final concentration of the hybrids and LPS are 100 nanomolar and 10 nanograms per milliliter, respectively.Next, remove the culture medium from the macrophage culture plate. Add 100 microliters of the hybrid LPS mixture into each well, with three replicates for each condition. Include a negative control and an LPS control.Incubate at 37 degrees Celsius for 24 hours. Then, transfer the medium of each well to a separate centrifuge tube. Centrifuge the tubes at 18, 000 times G and four degrees Celsius for 30 minutes.Transfer the supernatants into a fresh 96-well round bottom plate. To begin the reporter assay on these samples, add 180 microliters of pre-warmed Quanti-Blue solution to each well of a new 96-well flat bottom plate. Then, transfer 20 microliters of the supernatants from each sample into this plate.Incubate at 37 degrees Celsius for one to two hours until the dark blue color develops to an optical density over one. After this, use a plate reader to record the absorption at 655 nanometers. Next, transfer 10 microliters of fresh supernatant from each sample into a 96-well clear flat bottom white plate.Using an automatic injector, add 50 microliters of the luciferase solution to each well, and immediately record the luminescence well by well. Then, validate the inhibitory effect of the potential candidates, and evaluate the TLR specificity as outlined in the text protocol. In this study, the activation of NF-Kappa B AP1 is detected by the SEAP colorimetric assay, in which TLR-4 activation by LPS results in the activation of NF-Kappa B AP1 and the production of SEAP.The released SEAP converts the substrate, leading to a color change that is proportional to the amount of SEAP released upon stimulation. This is quantified by measuring the absorbance at 655 nanometers. Next, LPS-mediated activation of IRF led to the expression of luciferase, catalyzing the substrate to produce luminescence.An optimal LPS concentration of 10 nanograms per milliliter is determined from these dose responses to screen a previously established library of peptide-GMP hybrids. From this, a group of hybrids is identified for their potent ability to inhibit LPS-triggered activation for both NF-Kappa B AP1 and IRF. Validation of this inhibitory activity shows that as the concentration of LPS increases, the inhibitory effects of the hybrid reduce, as expected.Immunoblotting is then conducted to confirm inhibition activity. The lead hybrid is seen to reduce p65 phosphorylation, inhibit I-Kappa B alpha degradation, and delay IRF3 phosphorylation, confirming that the lead hybrid, P12, is able to inhibit LPS-mediating TLR-4 signaling. Additional investigation reveals that P12 also reduces TLR-2 and TLR-5-mediated NF-Kappa B AP1 signaling, as well as TLR-3-mediated IRF activation, suggesting that P12 has potent inhibitory activity on multiple TLR pathways.Following this procedure, other measures like immunoblotting and the cytotoxicity assay is to be performed in order to validate the screening results and to avoid false positive discovery. After its development, this technique paved the way for other researchers in the field of nanomedicine to explore nanoinhibitors in toll-like receptor signaling as next-generation and informatory therapeutics.

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