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 ....

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....

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