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. 2022 Jan 11:12:817604.
doi: 10.3389/fimmu.2021.817604. eCollection 2021.

A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands

Katharina Radakovics  1 , Claire Battin  1 , Judith Leitner  1 , Sabine Geiselhart  2 , Wolfgang Paster  3 , Johannes Stöckl  4 , Karin Hoffmann-Sommergruber  2 , Peter Steinberger  1
Affiliations

A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands

Katharina Radakovics et al. Front Immunol. .

Abstract

Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.

Keywords: TLR; bacterial contamination; biosensor; reporter cell; toll-like receptor.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Jurkat-NF-κB::eGFP reporter cells detect the TLR5 ligand flagellin with high sensitivity and specificity. (A) eGFP expression in Jurkat JE6-1 cells stably transfected with an NF-κB::eGFP reporter gene treated with different TLR ligands and PMA/Ionomycin as a positive control for 24 h (n = 3 independent experiments performed in duplicates). (B) Representative histograms of eGFP expression in JE6-1 reporter cells stimulated with flagellin at the indicated concentrations (experiment shown is representative for three experiments independently performed). (C) Concentration-response curves upon stimulation of Jurkat-NF-κB::eGFP and THP-1-NF-κB::eGFP reporter cells with flagellin are shown (n = 3 for Jurkat and n = 2 for THP-1 independent experiments were performed in duplicates).
Figure 2
Figure 2
Generation of TLR reporter cells. Scheme for the generation of JE6-1-TLR reporter cell lines (created with BioRender.com) and exemplary histograms representative of at least three independent experiments showing the NF-κB::eGFP expression (standard logarithmic scale) after 24 h incubation with the typical TLR ligands Pam3CSK4 (violet, TLR2/1), Fsl-1 (light blue, TLR2/6), LPS (pink, TLR4) and flagellin (green, TLR5), as well as medium (grey, negative control) and PMA/Ionomycin (dark violet, positive control).
Figure 3
Figure 3
Sensitivity and specificity of TLR reporter cells. (A) Titration curves for typical ligands of each TLR reporter cell, with a dashed line at fold induction of 1.5 representing the limit of detection; summary of representative experiments performed in duplicates (n = 2 for TLR2/1/6, TLR2/1, TLR2/6, TLR4 reporter, n = 3 for TLR5 reporter). (B) Reactivity of each reporter cell line to the typical ligands of TLR2/1 (Pam3CSK4, 100 ng/mL), TLR2/6 (Fsl-1, 100 ng/mL), TLR4 (LPS-B5 ultrapure, 10 ng/mL) and TLR5 (Fla-ST ultrapure, 10 ng/mL) (n = 6, each experiment was performed in duplicates).
Figure 4
Figure 4
Comparison of LPS-detection systems. (A) Fold induction of eGFP expression in JE6-1-NF-κB::eGFP TLR4 reporter cells compared to fold induction of SEAP activity in HEK-Blue™-hTLR4 cells after incubation with different LPS concentrations overnight. Mean and standard deviation of two experiments performed in duplicates are shown. (B) Fold induction of eGFP expression in JE6-1-NF-κB::eGFP TLR4 reporter cells and THP-1-NF-κB::eGFP TLR4 reporter cells after 24 h incubation with different concentrations of LPS. Mean and standard deviation of two experiments (JE6-1) or one representative experiment (THP-1) performed in duplicates are shown. (C) Reactivity of the typical TLR ligands LPS (LPS-B5 ultrapure, TLR4), Pam3CSK4 (TLR2/1), Fsl-1 (TLR2/6), LTA-SA (TLR2) and MPLA-SM (TLR4) in the recombinant Factor C assay EndoZyme®II. The fold change of RFU in the recombinant Factor C assay was compared with the fold induction of gMFI (eGFP) in Jurkat-NF-κB::eGFP TLR4 reporter cells after 24 h incubation with the same ligands (n = 2 independent experiments performed in duplicates or triplicates).
Figure 5
Figure 5
TLR2 stimulatory capacity of different cell wall components. (A) Activation of indicated reporter cells by the microbial components peptidoglycan (PGN), isolated from the gram-positive B. subtilis (PGN-BS) or S. aureus (PGN-SA) or the gram-negative E. coli K12 (PGN-EK), purified lipoteichoic acid from S. aureus (LTA-SA) and the synthetic ligands Diprovocim-1 (TLR2/1), Pam3CSK4 (TLR2/1) and Fsl-1 (TLR2/6) (n = 8, each experiment performed in duplicates). Exemplary histograms of eGFP expression are shown for TLR2/1/6, TLR2/1 and TLR2/6 reporter cells. (B) TLR2 surface expression on different TLR reporter cell lines was compared by staining with an AF647-coupled anti-TLR2 antibody.
Figure 6
Figure 6
Detection of bacterial contamination. (A) Flagellin (Fla) preparations of different purity (std. – standard, 10% purity; u.p. – ultrapure, > 95% purity; rec. – recombinant) from S. typhimurium (-ST) or B. subtilis (-BS) at 10 ng/mL were compared regarding their capacity to activate TLR5 as well as TLR4 reporter cells. n = 7 independent experiments performed in duplicates and exemplary histograms. ns = p > 0.1; ** = p < 0.01; **** = p < 0.0001. (B) Activation of different TLR reporter cells by E. coli LPS extracts (10 ng/mL) of different purity (std. – standard, 10% purity; u.p. – ultrapure, > 95% purity) from E. coli 055:B5 (-B5), E. coli K12 (-EK) or E. coli 0127:B8 (-B8), as well as MPLA-SM (100 ng/mL), an LPS component of S. minnesota. n = 6 independent experiments performed in duplicates and exemplary histograms. (C) Exemplary histograms of eGFP expression in Jurkat-TLR2/1/6, -TLR2/1 and -TLR2/6 reporter cells and THP-1-NF-κB::eGFP reporter cells after overnight incubation with medium or cell culture supernatant containing mycoplasma (1:1 diluted). (D) Exemplary histograms of eGFP expression in different TLR reporter cells after overnight incubation with protein preparations of the C4d protein, either expressed in E. coli BL21 or ClearColi™ (10 μg/mL) or PMA(100 ng/mL)/Ionomycin(100 nM) as positive control.
Figure 7
Figure 7
Detection of TLR Ligands in allergen extracts. Exemplary histograms with gMFI of eGFP expression in TLR reporter cells after overnight incubation with allergenic extracts (15 μg/mL) are shown. HDM(1) - HDM(4): house dust mite extracts from different sources.
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