Human TLR3 Reporter HEK293 Cells (NF-κB and IRF)

NF-κB–SEAP and IRF–Lucia reporter HEK293 cells expressing human TLR3

Signaling pathways in HEK-Dual™ hTLR3 cells
(click to enlarge and see legend)

HEK-Dual™ hTLR3 cells were engineered from HEK-Dual™ cells, a human embryonic kidney HEK293-derived cell line, to study the human Toll-like receptor 3 (hTLR3)-dependent NF-κB and IRF responses. This important pattern recognition receptor (PRR) recognizes double-stranded (ds)RNA, a hallmark of viral replication, and triggers antiviral NF-κB and IRF immune responses [1].

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Description

HEK-Dual™ hTLR3 cells feature the stable expression of the TLR3 gene as well as two inducible reporter genes for SEAP (secreted embryonic alkaline phosphatase) and Lucia luciferase. As a result, these cells allow the simultaneous study of the NF-κB pathway, by monitoring the activity of SEAP, and the IRF pathway, by assessing the activity of the secreted Lucia luciferase. Upon TLR3 activation, both reporter proteins are readily measurable in the cell culture supernatant when using QUANTI-Blue™ Solution, a SEAP detection reagent, and QUANTI-Luc™ 4 Lucia/Gaussia, a Lucia luciferase detection reagent. 

HEK-Dual™ hTLR3 cells are highly responsive to synthetic analogs of dsRNA. They show potent NF-κB and IRF responses upon incubation with TLR3-specific ligands, such as Poly(I:C) (polyinosinic-polycytidylic acid) or Poly(A:U) (polyadenylic–polyuridylic acid), when compared to HEK-Blue™ hTLR3 cells and their parental cell line HEK-Dual™ (see figures). 

Of note, HEK293 cells express endogenous levels of various PRRs, including TLR3, TLR5, and NOD1, and therefore might respond to their cognate ligands (see figures). 

Key features

• Stable expression of human TLR3
• Strong response to dsRNA and analogs
• Distinct monitoring of TLR3-dependent NF-κB or IRF activation by assessing the SEAP and Lucia luciferase activities

Applications

• Defining the role of TLR3-dependent IRF and NF-κB signaling pathways
• Screening for novel TLR3 agonists and inhibitors

References

1. Chen Y, et al., 2021.  Toll-like receptor 3 (TLR3) regulation mechanisms and roles in antiviral innate immune responses. J Zhejiang Univ Sci B.;22(8):609-632. 

Figures

NF-κB responses of HEK-Blue™ hTLR3 vs. HEK-Dual™ hTLR3. HEK-Blue™ hTLR3 and HEK-Dual™ hTLR3 cells were stimulated with various TLR agonists and cytokines: TNF-α (10 ng/ml), Poly(I:C) HMW (TLR3 agonist; 100 ng/ml), Poly(I:C) LMW (TLR3 agonist; 100 ng/ml), and Poly(A:U) (TLR3 agonist; 100 ng/ml). After overnight incubation, the activation of NF-κB was assessed by measuring the activity of SEAP in the supernatant using QUANTI-Blue™ Solution. Data are shown as optical density (OD) at 630 nm (mean ± SEM).

NF-κB responses in HEK-Dual™ -derived cells. HEK-Dual™ and HEK-Dual™ hTLR3 cells were incubated for 24 hours with cytokines and various TLR agonists: Human TNF-α (NF-κB-positive control, 10 ng/ml), hIFN-β (IRF-positive control, 1000 U/ml), Pam3CSK4 (TLR2 ligand, 100 ng/ml), Poly(I:C) LMW and HMW (TLR3 ligands, 1 µg/ml), Poly(A:U) (TLR3 ligand, 10 µg/ml), LPS-EK Ultrapure (UP) (TLR4 ligand, 100 ng/ml), FLA-ST UP (TLR5 ligand, 100 ng/ml), R848 (TLR7/8 ligand, 10 µg/ml), and ODN 2006 (TLR9 ligand, 10 µg/ml). After 24h incubation, the NF-kB-induced SEAP activity was assessed using QUANTIBlue™. Data are shown as optical density (OD) at 630 nm (mean ± SEM).

IRF responses in HEK-Dual™ -derived cells. HEK-Dual™ and HEK-Dual™ hTLR3 cells were incubated for 24 hours with cytokines and various TLR agonists: Human TNF-α (NF-κB-positive control, 10 ng/ml), hIFN-β (IRF-positive control, 1000 U/ml), Pam3CSK4 (TLR2 ligand, 100 ng/ml), Poly(I:C) LMW and HMW (TLR3 ligands, 100 ng/ml), Poly(A:U) (TLR3 ligand, 10 µg/ml), LPS-EK Ultrapure (UP) (TLR4 ligand, 100 ng/ml), FLA-ST UP (TLR5 ligand, 100 ng/ml), R848 (TLR7/8 ligand, 10 µg/ml), and ODN 2006 (TLR9 ligand, 10 µg/ml). After 24h incubation, the IRF response was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI-Luc™. Data are shown in fold response over non-induced cells (mean ± SEM).

Specifications

Antibiotic resistance: Blasticidin, Hygromycin, 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:

• Human TLR3 expression has been verified by RT-qPCR and functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free.

All of these products are covered by a Limited Use License (See Terms and Conditions).

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 1 ml Blasticidin (10 mg/ml)
• 1 ml Hygromycin B Gold (100 mg/ml)
• 1 ml Normocin™ (50 mg/ml)
• 1 ml Zeocin® (100 mg/ml)
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

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

Details

Toll-Like Receptor 3

In humans, four Toll-Like Receptor (TLR) family members TLR3, TLR7, TLR8, and TLR9 are specialized in sensing viral-derived components and are mainly found in the endosome. Among these, TLR3 recognizes double-stranded (ds)RNA, a hallmark of viral replication, and triggers antiviral immune responses [1]. TLR3 is expressed in myeloid dendritic cells, macrophages, as well as non-immune cells [2].

TLR3 signaling

TLR3 activation upon viral infection involves several steps, including translocation of TLR3 from the ER (endoplasmic reticulum) via the Golgi to the endosome, proteolytic cleavage and dimerization of TLR3, and finally receptor-ligand binding [3]. In order to start the signaling cascade, activated TLR3 recruits the adaptor protein TRIF (TIR domain-containing adapter-inducing interferon-β). TRIF binds to TRAF3 (TNF receptor-associated factor 3), which then recruits TBK1 (TANK-binding kinase 1) and IKKε (IκB kinase ε), thus activating the transcription factor IRF3 (interferon regulatory factor 3) and stimulating the production of type I IFNs (interferons). Additionally, TRIF interacts with TRAF6 and RIP1 (kinase receptor-interacting protein 1). RIP1 in turn binds to TAK1 (transforming growth factor β-activated kinase 1) and IKK. TAK1 phosphorylates IKKα and IKKβ, leading to the phosphorylation of IκB, the NF-κB inhibitor. Ultimately, this leads to the release and translocation of NF-κB into the nucleus and the induction of pro-inflammatory cytokines [2,4]. 

Pathology

Given its important role in dsRNA recognition, TLR3 signaling has been intensively studied. Various TLR3-agonists, such as the synthetic dsRNA analog Poly(I:C) are being used in vaccine development and cancer therapy [4]. Yet, recent studies have indicated that TLR3 may act as a double-edged sword by showing both protective and damaging functions in the context of some human viral infections [3,5]. Moreover, rare mutations in TLR3 have been associated with viral susceptibility; specifically, infections with HSV-1 (herpes simplex virus 1), influenza, and SARS-Co-V2 have been linked to pathogenic germline variants in TLR3 pathway genes [2]. Understanding the TRIF-dependent TLR3 pathway may be essential for the establishment of specific therapeutic approaches to diminish TLR3-driven disease and exploit its protective functions [3].

References

1. Manuela Sironi, et al., 2012. A Common Polymorphism in TLR3 Confers Natural Resistance to HIV-1 Infection. J Immunol 15; 188 (2): 818–823. 
2. Aluri, J, et al., 2021. Toll-Like Receptor Signaling in the Establishment and Function of the Immune System. Cells, 10, 1374.
3. Chen Y, et al., 2021.  Toll-like receptor 3 (TLR3) regulation mechanisms and roles in antiviral innate immune responses. J Zhejiang Univ Sci B.;22(8):609-632.
4. Komal A, et al., 2021. TLR3 agonists: RGC100, ARNAX, and poly-IC: a comparative review. Immunol Res. 69(4):312-322. 
5. Perales-Linares R, Navas-Martin S. 2013. Toll-like receptor 3 in viral pathogenesis: friend or foe? Immunology.;140(2):153-67.

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