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

Summary

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.

Abstract

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-κB 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.

Introduction

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 transduction^1,2. 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 pathways^5,6. One is dependent upon the adaptor protein myeloid differentiation factor 88 (MyD88) — the MyD88-dependent pathway. All TLRs except TLR3 utilize this pathway to activate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-associated protein kinases (MAPKs), leading to the expression of pro-inflammatory mediators such as TNF-α, IL-6 and IL-8. The second pathway utilizes TIR-domain-containing adaptor-inducing interferon-β (TRIF) — the TRIF-dependent or MyD88-independent pathway — to activate interferon (IFN) regulatory factors (IRFs) and NF-κB, 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 'double-edged sword' and excessive, uncontrolled TLR activation is harmful. Overactive TLR responses contribute to the pathogenesis in many acute and chronic human inflammatory diseases^8,9. 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 pathophysiology^10,11,12. In addition, TLR5 has been found to contribute to chronic lung inflammation of patients with cystic fibrosis^13,14. 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 signaling^9,17,18. 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 properties^19,20,23. The nanoscale size allows these nano-therapeutics to have better bio-distribution and sustained circulation^24,25,26. They can be further functionalized to meet the desired pharmacodynamic and pharmacokinetic profiles^27,28,29. 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. 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-activities^30,31,32,33. This makes them a special class of drug (or "nano-drug") 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 cells^32,33. 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-κB 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-κB/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.

Protocol

1. Preparation of Cell Culture Media and Reagents

1. 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.
   1. Prepare the selection culture medium R10-Z by adding the antibiotics Zeocin (200 μg/mL) to R10 for maintaining the expression of SEAP under the control of NF-κB/AP-1 activation. To select for cells expressing both SEAP and luciferase reporter genes, add both Zeocin (100 μg/mL) and blasticidin (10 μg/mL) to R10 (as R10-ZB).
2. 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.
   1. Swirl the solution gently and incubate it at 37 °C for 1 h to ensure the complete dissolution of the substrates.
   2. Filter the solution using a 0.2 μm membrane to ensure its sterility (optional), and store it at 4 °C up to 2 weeks prior to use.
3. 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.
   1. After dissolving the powder completely, use the solution immediately. Alternatively, store the solution at 4 °C (up to a week) or at -20 °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.
4. Prepare the stock solution of phorbol 12-myristate 13-acetate (PMA) in molecular grade dimethyl sulfoxide (DMSO) to have a concentration of 500 μg/mL. Make aliquots of the stock solution (10 μL in a 500 μL tube) and store them at -20 °C.
5. 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 °C. Prepare a working LPS solution by diluting the stock LPS into phosphate buffered saline (PBS) at a concentration of 100 μg/mL and store at -20 °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.

2. Culture of THP-1 reporter cell-derived macrophages

1. 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-κB/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.
   1. 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.
   2. 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.
   3. 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 >98%. Keep a record of the passage number as the cells may behave differently after many passages (> 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.
2. 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.
   1. 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.
   2. Transfer 100 μL of the cell suspensions into each well of the 96-well plate using a multi-channel pipette. Incubate the plate at 37 °C (in a cell culture incubator) for 24 h.
   3. 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 μL/well) twice; add 100 μL 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.

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-κB/AP-1 activation while THP-1-Dual cells are for IRF activation from the TRIF-dependent signal transduction.

1. Identify the optimal LPS dose by generating a dose-response curve prior to the screening.
   1. 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 μL of solution after dilution.
   2. Gently remove the culture medium from the cell-seeded plate (from 2.2.3) using a vacuum aspirator.
   3. 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 °C (in a cell culture incubator) for 24 h.
   4. At 24 h, carefully transfer the supernatants (80 μL/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 °C (several hours) or -20 °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-κB/AP-1 activation as the Dual cells have a relatively high background of NF-κB/AP-1 activation after being differentiated into macrophages. However, it is feasible to use the Dual cells for reporting both NF-κB/AP-1 and IRF activation.
2. Develop the colorimetric assay for the NF-κB/AP-1 activation and the luciferase luminescence assay for IRF activation.
   1. To assess the NF-κB/AP-1 activation, transfer 20 μL of supernatant of each sample into a new 96-well flat-bottom culture plate; and add 180 μL of pre-warmed SEAP substrate solution into each well. Incubate the plate at 37 °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 >3).
   2. For the analysis of IRF activation, transfer 10 μL of supernatant of each sample into a 96-well clear flat-bottom white plate. Add the luciferase solution (50 μL) 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  incubation time and reading set-up for each well.
3. 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.
   1. 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.
   2. 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.
   3. Remove the culture medium from the cultured plate (2.2.3) and add 100 μL 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).
   4. After 24 h incubation at 37 °C, transfer the medium of each well into a tube and centrifuge the tubes at 18,000 x g, 4 °C for 30 min. Collect the supernatants (50-80 μL/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.

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-κB/AP-1 and IRF3 signals via immunoblotting.

1. 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.
2. For the immunoblotting approach, please follow the standard experimental procedures.
   1. 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.
   2. 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.
   3. Probe the signals of IκBα, phosphorylated p65, and phosphorylated IRF3 to examine the inhibitory effect of the lead hybrids on the activation of NF-κB and IRF3. Probe the β-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.

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.

1. Test various concentrations (1 ng/mL to 25 μg/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.
2. 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.

Results

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-κB/AP-1 and IRFs, respectively. The activation of NF-κB/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 ...

Discussion

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 use^35,36. Similarly, only a limited number of compounds have progressed to c...

Materials

List of materials used in this article

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