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 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.
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) — 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 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. 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 "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 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-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.
1. Preparation of Cell Culture Media and Reagents
2. Culture of THP-1 reporter cell-derived macrophages
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.
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.
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.
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 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-κB/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-κB/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-κB and IRF signaling by the lead hybrid, the immunoblotting was conducted to directly assess the protein signal transduction over time. The activation of NF-κB and IRF3 was examined by probing the phosphorylation of the NF-κB subunit p65 and the degradation of the NF-κB inhibitor IκBα, and phosphorylation of IRF3, respectively. As shown in Figure 3C, the lead hybrid P12 could reduce p65 phosphorylation, inhibit IκBα 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-κB 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-κB/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.
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-κB/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. Please click here to view a larger version of this figure.
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-κB/AP-1 and IRF pathways of TLR4 signaling; the hybrid concentration = 100 nM; the LPS concentration = 10 ng/mL; the bar represents mean ± standard deviation; n = 2. Please click here to view a larger version of this figure.
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-κB 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 ± standard deviation; n = 3; * and *** denote p <0.05 and p <0.001, respectively, using one way ANOVA analysis. Please click here to view a larger version of this figure.
Figure 4: Inhibitory effect of the lead hybrid on other TLR signaling pathways.
The inhibition of NF-κB/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-κB/AP-1 and IRF signaling by the lead hybrid following the TLR3 stimulation (poly I:C = 25 μg/mL). The hybrid concentration = 100 nM; the bar represents mean ± standard deviation; n = 3; *, **, and *** denote p <0.05, p <0.01, and p <0.001, respectively, using one way ANOVA analysis; ns: non-significant. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. 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-κB/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.
The authors have nothing to disclose.
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's Hospital (H.Y.), Gaofeng Clinical Medicine Grant support from Shanghai Jiaotong University School of Medicine (H.Y.), and the funding from the Crohn's and Colitis Foundation of Canada (CCFC) (S.E.T. and H.Y.).
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 |