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
<ol>
	<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.
	<ol>
		<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, add both Zeocin (100 &#956;g/mL) and blasticidin (10 &#956;g/mL) to R10 (as R10-ZB).</li>
	</ol>
	</li>
	<li>Prepare the SEAP substrate solution by dissolving one pouch of the substrate powder (e.g., QUANTI-Blue) into 100 mL ultrapure, endotoxin free water in a clean 125 mL glass flask.
	<ol>
		<li>Swirl the solution gently and incubate it at 37 &#176;C for 1 h to ensure the complete dissolution of the substrates.</li>
		<li>Filter the solution using a 0.2 &#956;m membrane to ensure its sterility (optional), and store it at 4 &#176;C up to 2 weeks prior to use.</li>
	</ol>
	</li>
	<li>Prepare the luciferase substrate solution by dissolving one pouch of the substrate powder (e.g., QUANTI-Luc) into 25 mL ultrapure, endotoxin free water in a sterile 50 mL centrifuge tube.
	<ol>
		<li>After dissolving the powder completely, use the solution immediately. Alternatively, store the solution at 4 &#176;C (up to a week) or at -20 &#176;C (up to a month) prior to use. 
		CAUTION: Both substrate solutions are light sensitive, and should avoid light exposure whenever is possible. Multiple freeze-thaw cycles of the solution can cause instability of the substrate and shorten its shelf-life.</li>
	</ol>
	</li>
	<li>Prepare the stock solution of phorbol 12-myristate 13-acetate (PMA) in molecular grade dimethyl sulfoxide (DMSO) to have a concentration of 500 &#956;g/mL. Make aliquots of the stock solution (10 &#956;L in a 500 &#956;L tube) and store them at -20 &#176;C.</li>
	<li>Prepare LPS (E-coli K12) stock solution in sterile, endotoxin free water at a concentration of 5 mg/mL, and make aliquots for long-term storage at -20 &#176;C. Prepare a working LPS solution by diluting the stock LPS into phosphate buffered saline (PBS) at a concentration of 100 &#956;g/mL and store at -20 &#176;C prior to use. 
	CAUTION: The reagent preparation for culture uses should be carried out in a biosafety cabinet, and all the containers should be sterilized prior to use. Repeated freeze-thaw cycles of PMA and LPS solutions should be avoided.</li>
</ol>
2. Culture of THP-1 reporter cell-derived macrophages
<ol>
	<li>Use two THP-1 reporter cell lines: THP-1-XBlue and THP-1-Dual cells. The former has a SEAP reporter gene controlled by NF-&#954;B/AP-1, and the latter has a dual reporter gene system, the same SEAP reporter gene and a IRF controlled luciferase gene. Their culture procedures are identical except the selection culture media.
	<ol>
		<li>Thaw a working cell stock (~5 x 106 cells) in 10 mL of R10 medium, spin down the cells at 300 x g for 5 min. Resuspend cells in 10 mL of R10 medium, and transfer them into a T75 culture flask. Exchange the culture medium every 2-3 days until cells reach a density of 1 x 106 cells/mL.</li>
		<li>When cell growth reaches its capacity, passage cells to reduce the cell density to in the range of 2-5 x 105 cells/mL, so the cells can continue to grow.</li>
		<li>After at least 1 passage, start to culture the cells in the selection culture medium: R10-Z for THP-1-XBlue, and R10-ZB for THP-1-Dual. After culturing the cells with selection culture medium for at least 1 passage, cells are ready for the experiment. 
		CAUTION: Cell overgrowing can lead to significant cell death; the cell viability should maintain &#62;98%. Keep a record of the passage number as the cells may behave differently after many passages (&#62; 20 passages). 
		NOTE: Cell culture flasks can be reused several times to save costs; however, this practice may increase the risk of general contamination and cross-contamination among different flasks (if handling different cell lines at the same time). During media exchange and cell passage, the centrifugation is set at 300 x g for 5 min.</li>
	</ol>
	</li>
	<li>To perform reporter cell assay, seed cells into a 96-well flat-bottom cell culture plate and differentiate them into macrophages. The procedures are described in the following.
	<ol>
		<li>Transfer the cells from the flask to a centrifuge tube, spin down the cells at 300 x g for 5 min, and resuspend them in the R10 medium at a concentration of 1 x 106 cells/mL. Add aliquots of the PMA solution into the cell suspensions with a final concentration of 50 ng/mL.</li>
		<li>Transfer 100 &#956;L of the cell suspensions into each well of the 96-well plate using a multi-channel pipette. Incubate the plate at 37 &#176;C (in a cell culture incubator) for 24 h.</li>
		<li>After incubation, carefully remove the culture medium using a vacuum aspirator (or with a multi-channel pipette), and gently wash the cells with PBS (100 &#956;L/well) twice; add 100 &#956;L of fresh R10 medium in each well. Rest the cells for 2 days in an incubator before conducting the reporter assay. 
		CAUTION: The resting step is very important to allow cells to calm down after PMA stimulation to their normal quiet status. This can significantly reduce the background signals of the reporter genes under unstimulated conditions. 
		NOTE: After 24 stimulation with PMA, cells are differentiated into macrophage-like phenotype, with a characteristic of cell adhesion at the bottom of the well, and a morphological feature of pseudopodia.</li>
	</ol>
	</li>
</ol>
3. Screening for Potential TLR4 Nano-inhibitors Using the Reporter Cells
NOTE: Since TLR4 signaling utilizes both MyD88-dependent and TRIF-dependent pathways, it is selected as the primary target to encompass a wide range of TLR signaling pathways. THP-1-XBlue reporter cells are used to mainly examine the NF-&#954;B/AP-1 activation while THP-1-Dual cells are for IRF activation from the TRIF-dependent signal transduction.
<ol>
	<li>Identify the optimal LPS dose by generating a dose-response curve prior to the screening.
	<ol>
		<li>Dilute the working LPS solution to a final concentration of 0.01 - 100 ng/mL in R10 medium (make dilution in log10 scale). Layout the plate design and make dilution in a 96-well round-bottom culture plate. Make sure that each well has a minimum of 110 &#956;L of solution after dilution.</li>
		<li>Gently remove the culture medium from the cell-seeded plate (from 2.2.3) using a vacuum aspirator.</li>
		<li>Transfer the LPS containing R10 medium prepared in the dilution plate (3.1.1) into the culture plate according to the layout of the samples. Incubate the plate at 37 &#176;C (in a cell culture incubator) for 24 h.</li>
		<li>At 24 h, carefully transfer the supernatants (80 &#956;L/well) into a new 96-well round-bottom plate. Conduct the colorimetric and/or luciferase luminescence assay on these solutions immediately or store them at 4 &#176;C (several hours) or -20 &#176;C (days) prior to the assay development. 
		NOTE: The design of plate layout should be simple and clear for experiment and data analysis. For each condition, 2-4 replicates should be considered. Always include a negative control group (LPS null). It is recommended to use THP-1-XBlue cells for NF-&#954;B/AP-1 activation as the Dual cells have a relatively high background of NF-&#954;B/AP-1 activation after being differentiated into macrophages. However, it is feasible to use the Dual cells for reporting both NF-&#954;B/AP-1 and IRF activation.</li>
	</ol>
	</li>
	<li>Develop the colorimetric assay for the NF-&#954;B/AP-1 activation and the luciferase luminescence assay for IRF activation.
	<ol>
		<li>To assess the NF-&#954;B/AP-1 activation, transfer 20 &#956;L of supernatant of each sample into a new 96-well flat-bottom culture plate; and add 180 &#956;L of pre-warmed SEAP substrate solution into each well. Incubate the plate at 37 &#176;C for 1 - 2 h to allow the color development (pink to dark blue). Collect the absorption at 655 nm on a plate reader. 
		CAUTION: The incubation time can be varied based on the color development (30 min up to overnight incubation). It is recommended to wait until the optical density (O.D.) of the darkest color reaches above 1, while avoiding the saturation of the color development (O.D &#62;3).</li>
		<li>For the analysis of IRF activation, transfer 10 &#956;L of supernatant of each sample into a 96-well clear flat-bottom white plate. Add the luciferase solution (50 &#956;L) and immediately collect the luminescence well by well. 
		NOTE: It is highly recommended to use a luminescence plate reader with an auto-injection function to ensure the consistency in the luminescence reading from well to well and from plate to plate. If the substrate solutions are manually injected, please ensure a consistent &#160;incubation time and reading set-up for each well.</li>
	</ol>
	</li>
	<li>Conduct the screening assay on various peptide-GNP hybrids with 10 ng/mL LPS stimulation (based on 3.1). Follow the same procedure in 3.2 for the reporter assay development.
	<ol>
		<li>Concentrate the peptide-GNP hybrids to 200 nM in R10 medium using the centrifugation method. Centrifuge 20 volumes of the hybrid solution (10 nM) at 18,000 x g for 30 min, and carefully discard the supernatants. Collect the hybrids (at the bottom of the tube) into a tube, wash them with PBS twice, and re-suspend the hybrids in one volume of the R10 medium.</li>
		<li>Mix equal volumes of the concentrated hybrids and the LPS (20 ng/mL) containing R10 medium to have a final concentration of the hybrids and LPS to be 100 nM and 10 ng/mL, respectively.</li>
		<li>Remove the culture medium from the cultured plate (2.2.3) and add 100 &#956;L of the mixed solution into each well (3 replicates for each condition); include a negative control (medium only) and a LPS control (10 ng/mL LPS without hybrids).</li>
		<li>After 24 h incubation at 37 &#176;C, transfer the medium of each well into a tube and centrifuge the tubes at 18,000 x g, 4 &#176;C for 30 min. Collect the supernatants (50-80 &#956;L/tube) into a 96-well round-bottom plate, and perform the reporter assay as described in 3.2. 
		CAUTION: When discarding the supernatants after centrifugation, do not agitate the hybrids at the bottom of the tube. 
		NOTE: The centrifugation in the step 3.3.4 is very important to remove the non-internalized nanoparticle hybrids from the culture medium, and can avoid the interference of the hybrids with the colorimetric/luminescence readings. This is because the gold nanoparticles can absorb wide wavelengths of light depending on their size and agglomeration.</li>
	</ol>
	</li>
</ol>
4. Validating the Inhibitory Effect of the Potential Candidates
NOTE: To confirm the inhibitory effect of the potential candidates from the screening, two approaches are employed. One is to examine the dose responses of the stimulants (LPS) at a fixed hybrid concentration (or the other way around); the other is to directly look at the inhibition on the NF-&#954;B/AP-1 and IRF3 signals via immunoblotting.
<ol>
	<li>In approach one, perform the reporter assay with 100 nM of the lead hybrids and two LPS concentrations at 1 ng/mL and 10 ng/mL following the same procedures described in 3.3. Include an inactive hybrid (based on the screening results) as a hybrid control for comparison.</li>
	<li>For the immunoblotting approach, please follow the standard experimental procedures.
	<ol>
		<li>Culture THP-1 cells in R10 medium, seed the cells into a 12-well culture plate (2 x 106 cells/well), and differentiate them into macrophages by treating the cells with 50 ng/mL PMA for 24 h followed by resting for 2 days.</li>
		<li>After cell differentiation, stimulate cells with 10 ng/mL LPS with/without the hybrids (100 nM) over time (up to 4 h). At various time points (0, 5, 15, 30, 60, 120 and 240 min), prepare the cell lysates for immunoblotting. Include an inactive hybrid as a control.</li>
		<li>Probe the signals of I&#954;B&#945;, phosphorylated p65, and phosphorylated IRF3 to examine the inhibitory effect of the lead hybrids on the activation of NF-&#954;B and IRF3. Probe the &#946;-actin and total IRF3 signals as internal controls. 
		NOTE: The signal transduction often occurs much faster (in a few hours) then the expression of the reporter enzymes SEAP and luciferase (24 h). It is highly recommended to also perform a viability assay of the tested hybrids at 24 h as another validation method.</li>
	</ol>
	</li>
</ol>
5. Evaluating the TLR Specificity
NOTE: To investigate the TLR specificity of the lead peptide-GNP hybrid, other TLR signaling pathways are tested, including TLR2, TLR3 and TLR5. TLR7, 8 and 9 are excluded because the THP-1 derived macrophages do not respond well to the stimulation of these TLRs due to the lack of TLR7, 8 and 9 expression in macrophages34.
<ol>
	<li>Test various concentrations (1 ng/mL to 25&#160;&#956;g/mL) of the ligands specific to TLR2 (Pam3CSK4), TLR3 (poly I:C) and TLR5 (flagellin) on both reporter cells derived macrophages to obtain the optimal concentration following the same procedure in 3.1 and 3.2.</li>
	<li>Treat the cells with the mixtures of the lead hybrid (100 nM) and each TLR ligand at the concentration obtained from 5.1 to evaluate the inhibitory specificity of the lead hybrid according to the experimental procedure described in 3.3. Include the inactive hybrid as a control for comparison.</li>
</ol>

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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. 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>

The authors have nothing to disclose.

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 clinical trials including eritoran, a TLR4 antagonist, that exhibited potent inhibitory effects on LPS-mediated inflammatory responses in pre-clinical studies37,38, showed positive results in the phase I/II clinical trials39,40,41, but ultimately the phase III trial failed to reduce the mortality of patients with severe sepsis42. This failure has many possible reasons, one being that sepsis-associated inflammatory responses are often triggered through multiple TLR pathways, and thus blocking only TLR4 may not be sufficient to reduce the overwhelming inflammation. Therefore, developing novel, potent poly-TLR inhibitors could overcome such clinical challenges and become the next generation anti-inflammatory therapeutics. The screening strategy and protocol described here are expected to serve as an efficient experimental tool in studying TLR signaling, and more importantly as a drug discovery platform to accelerate the search for the next generation TLR inhibitors.
This screening approach provides several advantages in searching for new TLR inhibitors. First, the screening can be achieved in a high-throughput fashion using the reporter cell systems that are fast, sensitive and quantitative. Second, with both reporter cell lines, the screening can be done to cover a wide range of TLR signaling cascades, including the NF-&#954;B/AP-1 pathway and the type I interferon signaling (via IRFs); thus, they are ideal for the screening of multiple TLR pathways. Third, these reporter cells are genetically engineered from the human monocytic THP-1 cell line, which is a good model system to study interventions targeting the innate immune response. Fourth, the cell line is easily maintained, and particularly, can be differentiated into macrophages; and since macrophages play a key role in many disease-associated inflammatory conditions, they serve as an ideal target for the screening of immunotherapeutics targeting TLR signaling. Fifth, monocytes and macrophages have impressive phagocytic capacity, which allows for high cellular uptake of the nanoparticles, making them especially suitable for studying nanoscale therapeutic agents. Furthermore, this screening protocol can be applied to search for not only the nano-based therapeutic agents, but also other types of bio-active compounds.
Although this screening platform is very versatile and robust, some caution must be applied to avoid false discovery. The screening is primarily based on the reporter assay, which relies on the expression of the reporter gene under control of specific signaling events. Ideally, the reporter gene expression (SEAP and luciferase) is proportional to the intensity of the signal transduction pathway of interest, and the impact of the drug candidates is reflected in the assay readouts. However, in reality, any biological events occurring upstream of the reporter gene expression could affect the result, sometimes leading to a false positive discovery. For instance, low expression of SEAP could result from the inhibition of the protein synthesis process rather than from the down regulation of TLR signaling43. To avoid such a false discovery, the inhibitory activity of the identified candidates should always be validated, and the gold standard method is to directly look at the signaling pathways via immunoblotting. Another important aspect in screening nanoparticle-based therapeutic agents is the surface properties of these nanodevices. Based on the surface modifiers, the nanodevices can have various biological activity. However, they may also have non-specific binding capability to certain biomolecules. In the case of non-specific binding to SEAP or luciferase, the catalytic activity to the substrates could be compromised by these nanodevices, leading to potential false discovery. Including extra control groups (e.g., nanodevice only) in the screening reduces the risk of false discovery. Last but not the least, the cytotoxicity of the identified lead candidates must be examined to exclude cytotoxicity as a contributing factor to false discovery. This can be done simultaneously during the screening process using a standard viability assay (e.g., MTS or MTT), or in a separate experiment.

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 &amp;deg;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&#039;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 nanodevices for their immunomodulatory activity on the innate phagocytic immune cells. With the reporter system, the screening can be conducted in a high-throughput fashion; such an approach is versatile to the discovery of new immunotherapeutics, particularly targeting on innate immune signaling such as TLR signaling.
The screening procedure and representative results are shown in Figure 2. Briefly, the reporter cells are seeded into a 96-well plate and derived into macrophages (Figure 2A and 2B). Different concentrations of the TLR ligands (TLR4 as an example) are first tested to obtain the optimal concentration for the actual screening of the peptide-GNP hybrids. The TLR4 stimulation by LPS resulted in the activation of NF-&#954;B/AP-1 and the production of SEAP. The released SEAP converted the substrate and changed its photophysical property, which could be monitored by the shifting in the light absorption, leading to the solution color change (Figure 2C). Such a change is proportional to the amount of SEAP released upon stimulation, and can be quantified by measuring the absorbance at 655 nm on a spectrophotometer (Figure 2D). Similarly, the activation of IRFs (triggered by LPS) led to the expression of luciferase, which catalyzed the substrate to produce luminescence (Figure 2E). Based on these dose responses, an optimal concentration of LPS (10 ng/mL) was used to screen a small previously established library of peptide-GNP hybrids (Table 1). The fabrication of the hybrids and their physicochemical characteristics were described in our previous publications30,31,32. From the screening, a group of hybrids (P12 and its derivatives) were identified for their potent inhibitory activity on both NF-&#954;B/AP-1 and IRF activation triggered by LPS (Figure 2F); interestingly, the hybrid P13, just slightly different from P12 in the peptide coatings, did not have any inhibitory activity, which could be served as a hybrid control for comparison. The P13 derivatives showed various degree of mild inhibitory activity depending on the other decorated peptide on the surface.
After identifying the lead hybrid, it is important to validate the inhibitory activity. The inhibition was first confirmed by examining the different ratios of the hybrid to LPS to exclude potential false positive results due to technical artifacts. As the concentration of LPS increased, the inhibitory effect of the hybrid (at a fixed concentration) reduced as expected (Figure 3A and 3B). To further ensure that the observed inhibition from the reporter assays was indeed a result of down-regulating the NF-&#954;B and IRF signaling by the lead hybrid, the immunoblotting was conducted to directly assess the protein signal transduction over time. The activation of NF-&#954;B and IRF3 was examined by probing the phosphorylation of the NF-&#954;B subunit p65 and the degradation of the NF-&#954;B inhibitor I&#954;B&#945;, and phosphorylation of IRF3, respectively. As shown in Figure 3C, the lead hybrid P12 could reduce p65 phosphorylation, inhibit I&#954;B&#945; degradation, and delayed IRF3 phosphorylation, while the inactive hybrid P13 could not (data not shown). All these results confirmed that the identified lead hybrid was able to inhibit LPS-mediated TLR4 signaling by down-regulating both NF-&#954;B and IRF3 activation.
In addition to TLR4 signaling, the inhibitory activity of the lead hybrid was further evaluated on other TLR pathways including TLR2, TLR3 and TLR5 to address the TLR specificity. As shown in Figure 4, the lead hybrid P12 was able to reduce TLR2- and TLR5-mediated NF-&#954;B/AP-1 signaling, as well as TLR3-mediated IRF activation; again, the inactive hybrid P13 did not show any inhibitory activity. These results suggested that the identified lead hybrid has a potent inhibitory activity on multiple TLR pathways.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56075/56075fig1.jpg" /> 
Figure 1: Overall experimental approach of high-throughput screening of TLR inhibitors using the reporter cell assay. Two reporter cell lines are used: THP-1-XBlue and THP-1-Dual. The former has a SEAP reporter gene under the control of NF-&#954;B/AP-1 activation, whereas the Dual system has an additional luciferase reporter gene under the control of IRFs activation. These cells can be easily differentiated into macrophages for high-throughput screening of immune modulatory nanoparticles on innate immune signaling. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56075/56075fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56075/56075fig2.jpg" /> 
Figure 2: High-throughput screening of nano-inhibitors on TLR4 signaling. 
(A) A scheme of experimental procedures. (B) An optical microscopic image of differentiated macrophages (200x magnification). (C) A representative image of the solution color change from the SEAP reporter assay; the color of the substrate solution turned into purple or dark blue (from original pink) depending on the SEAP expression in the culture medium. (D) Quantitative analysis of the SEAP substrate absorption at 655 nm in response to LPS stimulation. (E) The luciferase luminescence from the IRF reporter system in proportion to LPS stimulation. (F) High-throughput screening identifying the lead peptide-GNP hybrid P12 and its derivatives in inhibiting both NF-&#954;B/AP-1 and IRF pathways of TLR4 signaling; the hybrid concentration = 100 nM; the LPS concentration = 10 ng/mL; the bar represents mean &#177; standard deviation; n = 2. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56075/56075fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56075/56075fig3.jpg" /> 
Figure 3: Validation of the inhibitory activity of the lead hybrid. Confirmation of the inhibitory effect of the lead hybrid with various concentrations of LPS on both (A) SEAP and (B) luciferase reporter systems. (C) Confirming the inhibition of the lead hybrid on the NF-&#954;B and IRF3 signaling via immunoblotting. The inactive hybrid P13 was used as a hybrid control for comparison. The hybrid concentration = 100 nM; the LPS concentration = 10 ng/mL; the bar represents mean &#177; standard deviation; n = 3; * and *** denote p &#60;0.05 and p &#60;0.001, respectively, using one way ANOVA analysis. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56075/56075fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56075/56075fig4.jpg" /> 
Figure 4: Inhibitory effect of the lead hybrid on other TLR signaling pathways. 
The inhibition of NF-&#954;B/AP-1 by the lead hybrid following the TLR2 stimulation (Pam3CSK4 = 1 ng/mL) (A) and TL5 stimulation (flagellin = 100 ng/mL) (B). (C) The reduction of both NF-&#954;B/AP-1 and IRF signaling by the lead hybrid following the TLR3 stimulation (poly I:C = 25 &#956;g/mL). The hybrid concentration = 100 nM; the bar represents mean &#177; standard deviation; n = 3; *, **, and&#160;*** denote p &#60;0.05, p &#60;0.01,&#160;and p &#60;0.001, respectively, using one way ANOVA analysis; ns: non-significant. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56075/56075fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/56075/56075table1.jpg" /> 
Table 1: A small library of peptide-GNP hybrids established in our early studies. 
The hybrids are made of a gold nanoparticle core (~13 nm in diameter) and various hexapeptide coatings on the surface. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56075/56075table1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

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12.3K Views.  Shanghai Jiaotong University School of Medicine. 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. 

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

Detection of  Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR G&alpha;i proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl &alpha;-2,3-linked &beta;-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells 6. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.

The sialidase assay is a simple technical approach that will elucidate novel molecular mechanism(s) of TLR sensors of microbial infections and involvement in inflammatory diseases at the receptor level on the cell surface of live macrophages.

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of ...

Immunology and Infection

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Bioengineering

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

Cancer Research

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay described here will aid in elucidating the role of microglia in neuroinflammation and neurodegenerative diseases. The use of microglia, resident immune cells of the brain, as a surrogate biosensor provides novel insight into how inflammatory responses mediate neuronal insults. Here, we utilize an immortalized murine microglial cell line, designated BV2, and describe a method for nanoparticle exposure using silver nanoparticles (AgNPs) as a standard. We describe how to expose microglia to nanoparticles, how to remove nanoparticles from supernatant, and how to use supernatant from activated microglia to determine toxicity, using hypothalamic cell survival as a measure. Following AgNP exposure, BV2 microglial activation was validated using a tumor necrosis factor alpha (TNF-&#945;) enzyme linked immunosorbent assay (ELISA). The supernatant was filtered to remove the AgNP and to allow cytokines and other secreted factors to remain in the conditioned media. Hypothalamic cells were then exposed to supernatant from AgNP activated microglia and survival of neurons was determined using a resazurin-based fluorescent assay. This technique is useful for utilizing microglia as a surrogate biomarker of neuroinflammation and determining the effect of neuroinflammation on other cell types.

Microglia (immune cells of the brain), are used as a surrogate biosensor to determine how nanoparticles influence neurotoxicity. We describe a series of experiments designed to assay microglial response to nanoparticles and exposure of hypothalamic neurons to supernatant from activated microglia to determine neurotoxicity.

Nanoparticles found in air pollutants can alter neurotransmitter profiles, increase neuroinflammation, and alter brain function. Therefore, the assay ...

Neuroscience

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

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

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

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Chapters in this video

Detection of Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1α Nanoparticles

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Microglia as a Surrogate Biosensor to Determine Nanoparticle Neurotoxicity

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

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo