Mouse TLR7 Reporter HEK293 Cells (NF-κB)

NF-κB–SEAP reporter HEK293 cells expressing mouse TLR7

Trafficking & signaling in HEK-Blue™ mTLR7 cells
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HEK-Blue™ mTLR7 cells were engineered from the human embryonic kidney HEK293 cell line to study the mouse Toll-like receptor 7 (TLR7)-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].

 More details

Description

HEK-Blue™ mTLR7 cells express the murine TLR7 gene as well as an NF-κB/AP-1-inducible SEAP reporter gene. SEAP (secreted embryonic alkaline phosphatase) levels produced upon TLR7 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 mTLR7, HEK-Blue™ mTLR7 cells strongly and stably respond to TLR7-specific ligands (see figures).

Interestingly, while HEK-Blue™ hTLR7 cells do not respond to TLR8 agonists (e.g. TL8-506), HEK-Blue™ mTLR7 cells do respond to these ligands (see figures). This may be explained by the strong homology between mTLR7 and mTLR8, and the possibility that these murine TLRs, but not their human counterparts, have evolved to detect a broad overlapping range of ligands. 

As HEK293 cells express endogenous levels of TLR3, TLR5, and NOD1, HEK-Blue™ mTLR7 cells will respond to their cognate ligands (see figures). 

Key Features

• Verified expression of murine (m)TLR7
• Strong and stable response to TLR7-specific ligands
• Distinct monitoring of NF-κB by assessing the SEAP activities

Applications

• Defining the distinct role of TLR7 in the NF-κB-dependent pathway
• Comparing human and murine TLR7-responses using the HEK-Blue™ hTLR7 cell line
• Screening for TLR7-specific agonists or inhibitors in comparison with their parental cell line HEK-Blue™ Null2-k 

References:

1. Georg P. & Sander L.E., 2019. Innate sensors that regulate vaccine responses. Curr. Op. Immunol. 59:31.

Figures

Dose-response of HEK-Blue™ mTLR7 cells to synthetic base analogs. HEK-Blue™ mTLR7 cells were cultured in HEK-Blue™ Detection medium with increasing concentrations of TLR7 agonists (CL264, Imiquimod, Gardiquimod), TLR7/8 agonists (R848, CL097, CL075), or a TLR8 agonist (TL8-506). After 24h incubation, the TLR7-induced NF-κB/AP-1 responses were assessed by measuring SEAP levels in the supernatant by reading the optical density (OD) at 650 nm. OD fold increase over non-induced cells is shown.

Species-driven TLR7 differential responses. HEK-Blue™ hTLR7 or mTLR7 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). Note: The HEK-Blue™ hTLR7 cell line used in this experiment is a different clone than the one on sale.

Response of HEK-Blue™ mTLR7 cells to various PRR agonists and cytokines. Cells were cultured in HEK-Blue™ Detection medium and incubated for 24 hours with cytokines and various TLR agonists: Human TNF-α (NF-κB-positive control, 1 ng/ml), Pam3CSK4 (TLR2 ligand, 1 µg/ml), Poly(I:C) HMW (TLR3 ligand, 1 µg/ml), LPS-EK Ultrapure (UP) (TLR4 ligand, 10 µg/ml), FLA-ST UP (TLR5 ligand, 10 µg/ml), C264 (TLR7 ligand, 1 µg/ml), R848 (TLR7/8 ligand, 1 µg/ml), ODN 2006 (TLR9 ligand, 10 µg/ml), or Tri-DAP (NOD1 ligand, 1 µg/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 murine TLR7 gene has been confirmed by RT-PCR.
• The activation of NF-κB/AP1 upon TLR7 stimulation has been verified using functional assays.
• The stability for 10 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™ mTLR7 cells is HEK-Blue™ Null2-k 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)

Details

Toll-Like Receptor 7

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].

TLR7 signaling

Upon viral infection, TLR7 translocates from the endoplasmic reticulum via the Golgi into the endosomes. Subsequently, it undergoes proteolytic cleavage and dimerization [1,3]. Once activated, it 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, TLR7-mediated signaling induces IFN-α and IFN-regulated cytokines or T helper 17 (Th17) polarizing cytokines, such as interleukin (IL)-1β and IL-23 [4].

TLR7 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 [4]. TLR7 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) [1-4]. Understanding the fundamental differences between these two related receptors could potentially be harnessed to discover novel drugs and improve vaccine efficacy/safety [4].

​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. de Marcken M, et al., 2019. TLR7 and TLR8 activate distinct pathways in monocytes during RNA virus infection. Sci Signal.;12(605):eaaw1347.  

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