A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands
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...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-01-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2021.817604/full |
| _version_ | 1798035885898334208 |
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| author | Katharina Radakovics Claire Battin Judith Leitner Sabine Geiselhart Wolfgang Paster Johannes Stöckl Karin Hoffmann-Sommergruber Peter Steinberger |
| author_facet | Katharina Radakovics Claire Battin Judith Leitner Sabine Geiselhart Wolfgang Paster Johannes Stöckl Karin Hoffmann-Sommergruber Peter Steinberger |
| author_sort | Katharina Radakovics |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-11T21:04:33Z |
| format | Article |
| id | doaj.art-170cbda73b0842d78411fb0c103cf73e |
| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-04-11T21:04:33Z |
| publishDate | 2022-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Immunology |
| spelling | doaj.art-170cbda73b0842d78411fb0c103cf73e2022-12-22T04:03:23ZengFrontiers Media S.A.Frontiers in Immunology1664-32242022-01-011210.3389/fimmu.2021.817604817604A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR LigandsKatharina Radakovics0Claire Battin1Judith Leitner2Sabine Geiselhart3Wolfgang Paster4Johannes Stöckl5Karin Hoffmann-Sommergruber6Peter Steinberger7Division of Immune Receptors and T Cell Activation, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, AustriaDivision of Immune Receptors and T Cell Activation, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, AustriaDivision of Immune Receptors and T Cell Activation, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, AustriaDepartment of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, AustriaClinical Cell Biology and FACS Core Unit, St. Anna Children´s Cancer Research Institute (CCRI), Vienna, AustriaDivision Regulation of the Immune System, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, AustriaDepartment of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, AustriaDivision of Immune Receptors and T Cell Activation, Institute of Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, AustriaToll-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.https://www.frontiersin.org/articles/10.3389/fimmu.2021.817604/fulltoll-like receptorTLRbiosensorbacterial contaminationreporter cell |
| spellingShingle | Katharina Radakovics Claire Battin Judith Leitner Sabine Geiselhart Wolfgang Paster Johannes Stöckl Karin Hoffmann-Sommergruber Peter Steinberger A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands Frontiers in Immunology toll-like receptor TLR biosensor bacterial contamination reporter cell |
| title | A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands |
| title_full | A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands |
| title_fullStr | A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands |
| title_full_unstemmed | A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands |
| title_short | A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands |
| title_sort | highly sensitive cell based tlr reporter platform for the specific detection of bacterial tlr ligands |
| topic | toll-like receptor TLR biosensor bacterial contamination reporter cell |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2021.817604/full |
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