Human TLR8 Reporter HEK293 Cells (NF-κB)

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NF-κB–SEAP reporter HEK293 cells expressing human TLR8

Trafficking & signaling in HEK-Blue™ hTLR8 cells
(click to enlarge and see legend)

HEK-Blue™ hTLR8 cells were engineered from the human embryonic kidney HEK293 cell line to study the Toll-like receptor 8 (TLR8)-dependent NF-κB pathway. This important pattern recognition receptor (PRR) recognizes viral single-stranded RNA (ssRNA) structures and subsequently triggers NF-κB and IRF immune responses [1].

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Description

HEK-Blue™ hTLR8 cells express the human TLR8 gene as well as an NF-κB/AP-1-inducible SEAP reporter gene. SEAP (secreted embryonic alkaline phosphatase) levels produced upon TLR8 stimulation can be readily determined by performing the assay in HEK-Blue™ Detection, a cell culture medium that allows for real-time detection of SEAP. Alternatively, SEAP activity may be monitored using QUANTI-Blue™, a SEAP detection reagent.

Due to the expression of hTLR8, HEK-Blue™ hTLR8 cells strongly and stably respond to TLR8-specific ligands (see figures). Interestingly, while HEK-Blue™ murine (m)TLR8 cells do not respond to the TLR8 agonist ssRNA40, HEK-Blue™ hTLR8 cells respond to this ligand (see figures). Thus, findings regarding mouse TLR8 are not transposable to its human counterpart [1,2]. 

HEK-Blue™ hTLR8 cells do not respond to TLR7-specific ligands, since HEK293 cells do not express endogenous TLR7 (see figures). However, as they express endogenous levels of TLR3, TLR5, and NOD1 [in-house data], HEK-Blue™ hTLR8 cells may respond to their cognate ligands such as Poly(I:C), flagellin, or Tri-DAP (see figure).

As TLR8 activates both the NF-κB and IRF signaling pathways, InvivoGen also offers HEK-Dual™ hTLR8 cells; a cell line expressing a dual reporter system comprising an NF-κB-inducible SEAP and an IRF-inducible-Lucia luciferase (learn more here). 

Of note, HEK293 cells express endogenous levels of various PRRs, including TLR3 and TLR5. Therefore, they might respond to their cognate ligands (see figures).

Key Features

• Verified expression of human (h)TLR8
• Strong and stable response to TLR8-specific ligands
• Distinct monitoring of NF-κB by assessing the SEAP activities

Applications

• Defining the distinct role of TLR8 in the NF-κB-dependent pathway
• Comparing human and murine TLR8-responses using the HEK-Blue™ mTLR8 cell line
• Screening for novel TLR8-specific agonists or inhibitors in comparison with their parental cell line HEK-Blue™ Null1

References:

1. Heil F. et al., 2004. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science. 303:1526.
2. Eigenbrod T. & Dalpke A.H., 2015. Bacterial RNA: an underestimated stimulus for innate immune responses. J. Immunol. 195:411.
3. Georg P. & Sander L.E., 2019. Innate sensors that regulate vaccine responses. Curr. Op. Immunol. 59:31.

Figures

Dose-response of HEK-Blue™ hTLR8 cells to synthetic base analogs. HEK-Blue™ hTLR8 cells were cultured in HEK-Blue™ Detection medium with increasing concentrations of a TLR8 agonist (TL8-506), various TLR7/8 agonists (R848, CL097, CL075) or TLR7 agonists (CL264, Imiquimod, Gardiquimod). After 24h incubation, TLR8-induced NF-κB/AP1 responses were assessed by measuring SEAP levels in the supernatant and reading the optical density (OD) at 650 nm. OD fold increase over non-induced cells is shown.

Species-driven TLR8 differential responses. HEK-Blue™ hTLR8 or mTLR8 were cultured in HEK-Blue™ Detection medium with 1 μg/ml R848 (TLR7/8 agonist), 3 μg/ml Imiquimod (TLR7 agonist), 5 μg/ml ssRNA40/LyoVec™ (referred as human TLR8 agonist), or 1 μg/ml TL8-506 (TLR8 agonist, VTX-2337 analog). After 24h incubation, TLR8-induced NF-κB/AP1 responses were assessed by measuring SEAP levels in the supernatant and reading the OD at 650 nm. OD fold increase over non-induced cells is shown (mean ± SEM).

Response of HEK-Blue™ hTLR8 cells to various PRR agonists and cytokines. Cells were cultured in HEK-Blue™ Detection medium and stimulated for 24 hours with cytokines and various TLR agonists: Human TNF-α (NF-κB-positive control, 1 ng/ml), Pam3CSK4 (TLR2 ligand, 100 ng/ml), Poly(I:C) HMW (TLR3 ligand, 100 ng/ml), LPS-EK Ultrapure (UP) (TLR4 ligand, 100 ng/ml), FLA-ST UP (TLR5 ligand, 10 ng/ml), C264 (TLR7 ligand, 1 µg/ml), R848 (TLR7/8 ligand, 1 µg/ml), ODN 2006 (TLR9 ligand, 10 µg/ml), Tri-DAP (NOD1 ligand, 100 ng/ml), or MDP (NOD2 ligand, 100 ng/ml). After 24h incubation, TLR8-induced NF-κB/AP1-induced SEAP activity was determined by measuring the SEAP level in the supernatant. Data are shown as OD at 650 nm (mean ± SEM).

Specifications

Antibiotic resistance: Blasticidin, Zeocin®.

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™.

Quality Control:

• The expression of the human TLR8 gene has been confirmed by RT-PCR.
• The activation of NF-κB/AP1 upon TLR8 stimulation has been verified using functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free.

Note: HEK293 cells express endogenous levels of TLR3, TLR5, and NOD1. The appropriate parental cell line for HEK-Blue™ hTLR8 cells is HEK-Blue™ Null1 cells.

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 1 ml Blasticidin (10 mg/ml)
• 1 ml Zeocin® (100 mg/ml)
• 1 ml Normocin™ (50 mg/ml)
• 1 pouch of HEK-Blue™ Detection (cell culture medium for real-time detection of SEAP)

Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

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Details

Toll-Like Receptor 8

In humans, four Toll-Like Receptor (TLR) family members TLR3, TLR7, TLR8, and TLR9, mainly found in the endosome, are specialized in sensing viral-derived components. TLR7 and TLR8 recognize single-stranded (ss)RNA structures, such as viral ssRNA, miRNA, and various synthetic agonists [1]. Despite their similarities in PAMP (pathogen-associated molecular pattern) recognition, structure, and signaling partners, they highly differ in expression profiles and signaling responses, with TLR7 being more involved in the antiviral immune response and TLR8 mastering the production of proinflammatory cytokines [2]. TLR7 is mainly found in plasmacytoid dendritic cells (pDCs) and B cells, whereas TLR8 is highly expressed in monocytes, monocyte-derived DCs (mDCs), and macrophages [3].

TLR8 signaling

Upon viral infection, TLR8 translocates from the endoplasmic reticulum via the Golgi into the endosomes. Subsequently, it undergoes proteolytic cleavage in order to facilitate dimer rearrangement [1,3]. While TLR7 dimerizes upon ligand binding, TLR8, which exists as an unliganded inactive dimer, performs structural reorganization after ligand recognition [4]. Once activated, TLR8 recruits the adaptor protein MyD88 to trigger IRF, AP-1, and NF-kB responses via TRAF6 (TNF receptor-associated factor 6) [1,3]. Depending on the stimulus and cell type, TLR8-mediated signaling induces Th1-type cytokine (IL-12) production [5].

TLR8 therapeutic targeting

The involvement of nucleic acid-sensing mechanisms in the immune response against infections and other diseases makes them interesting targets for drug design [5]. TLR7/8 agonists are currently been tested as vaccine adjuvants and immunomodulatory therapeutics. They are extensively studied in the context of viral infection (e.g. SARS-CoV-2, Influenza, HIV), autoimmune (e.g. asthma, Lupus), and autoinflammatory diseases (e.g. cancer) [1-5]. Understanding the fundamental differences between these two related receptors could potentially be harnessed to discover novel drugs and improve vaccine efficacy/safety [5].

​References:

1. Martínez-Espinoza I & Guerrero-Plata A. 2022. The Relevance of TLR8 in Viral Infections. Pathogens. 11(2):134.
2. Salvi V, et al., 2021. SARS-CoV-2-associated ssRNAs activate inflammation and immunity via TLR7/8. JCI Insight.;6(18):e150542.
3. Georg P. & Sander L.E., 2019. Innate sensors that regulate vaccine responses. Curr. Op. Immunol. 59:31.
4. Asami J, Shimizu T. 2021. Structural and functional understanding of the toll-like receptors. Protein Sci. (4):761-772.
5. de Marcken M, et al., 2019. TLR7 and TLR8 activate distinct pathways in monocytes during RNA virus infection. Sci Signal.;12(605):eaaw1347.