Human TLR4 Reporter HEK293 Cells (NF-κB)

NF-κB–SEAP reporter HEK293 cells expressing human TLR4

Signaling pathways in HEK-Blue™ hTLR4 cells
(click to enlarge and see legend)

HEK-Blue™ hTLR4 cells were engineered from the human embryonic kidney HEK293 cell line to study the Toll-like receptor 4 (TLR4)-dependent NF-κB response upon stimulation with lipopolysaccharide (LPS). LPS recognition via TLR4 and its co-adaptors leads to NF-κB and IRF activation and the production of proinflammatory cytokines and interferons, respectively [1].

 More details

Description

HEK-Blue™ hTLR4 cells feature the stable expression of TLR4, as well as the adapter proteins MD-2 (myeloid differentiation factor 2) and CD14 (cluster of differentiation 14). These cells also express an inducible reporter gene for SEAP (secreted embryonic alkaline phosphatase). SEAP levels produced upon TLR4-dependent NF-κB activation 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.

HEK-Blue™ hTLR4 cells show a potent NF-κB response to both types of LPS, smooth and rough, compared to their parental cell line HEK-Blue™ Null2 (see figures). 

As TLR4 triggers both the NF-κB and IRF signaling pathways, InvivoGen also offers HEK-Dual™ hTLR4 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, TLR5, and RIG-I-like receptors, and therefore might respond to their cognate ligands (see figures). 

Key features

• Stable expression of human TLR4, MD2, and CD14
• Increased and reproducible response to rough and smooth LPS
• Distinct monitoring of TLR4-dependent NF-κB activation by assessing the SEAP activities

Applications

• Detecting sample contamination with LPS
• Defining the role of TLR4 in LPS-induced signaling pathways 
• Screening for TLR4 agonists or antagonists
• Highlighting the importance of TLR4 when used in combination with HEK-Blue™ hMD2-CD14 (no endogenous TLR4 expression)

1. Godowski, P., 2005. A smooth operator for LPS responses. Nat Immunol 6, 544–546.

Figures

Response of HEK-Blue™-derived cells to TLR4 agonists. HEK-Blue™ Null2, HEK-Blue™ hMD2-CD14, and HEK-Blue™ hTLR4 cells were cultured in HEK-Blue™ Detection reagent and stimulated for 24 hours with 10 ng/ml of the following TLR4 agonists: LPS-EB Ultrapure (UP) and LPS-EK UP. Human TNF-α (1 ng/ml) serves as an NF-κB-positive control. After 24h incubation, the NF-κB-induced SEAP activity was assessed by measuring the SEAP level in the supernatant. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

Dose-response of HEK-Blue™ hTLR4 cells to TLR4 agonists. Cells were cultured in HEK-Blue™ Detection reagent and stimulated with increasing concentrations of LPS-EB UP, LPS-EK UP, MPLAs (synthetic), and MPLA-SM* (MPLA from Salmonella minnesota R595) After 24 hour incubation, the NF‑kB‑induced SEAP activity was determined by reading the OD at 650 nm (mean ± SEM).

Response of HEK-Blue™ hTLR4 cells to various PRR agonists and cytokines. Cells were cultured in HEK-Blue™ Detection reagent and stimulated for 24 hours with cytokines and various PRR 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 UP (TLR4 ligand, 100 ng/ml), CRX-527 (synthetic TLR4 ligand, 10 ng/ml) FLA-ST UP (TLR5 ligand, 10 ng/ml), R848 (TLR7/8 ligand, 10 µg/ml), ODN 2006 (TLR9 ligand, 10 µg/ml), Tri-DAP (NOD1 ligand, 10 µg/ml), and MDP (NOD2 ligand, 10 µg/ml). After 24h incubation, the NF-κB-induced SEAP activity was assessed by measuring the SEAP level in the supernatant. Data are shown as OD at 650 nm (mean ± SEM).

NF-κB responses of HEK-Blue™ hTLR4 vs. HEK-Dual™ hTLR4​. HEK-Blue™ hTLR4 and HEK-Dual™ hTLR4 cells were stimulated with various TLR4 agonists: LPS-EB Ultrapure (UP) (1 ng/ml), LPS-EK UP (1 ng/ml), LPS-B5 UP (100 pg/ml), or synthetic MPLAs (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 650 nm (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:

• Stable expression of human (h)TLR4, MD-2, and CD14 has been verified by RT-qPCR and functional assays.
• The activation of NF-κB/AP1 upon TLR4 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™ hTLR4 cells is HEK-Blue™ Null2. 

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 2 ml 250X HEK-Blue™ Selection
• 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 4 signaling

The Toll-like receptor 4 (TLR4) was the first TLR identified and is an important pattern recognition receptor (PRR) in innate immunity and inflammation. It is found both on the cell surface and in endosomes of innate immune cells including monocytes and macrophages, as well as on intestinal epithelium and endothelial cells [1]. TLR4 can recognize pathogen- and damage-associated molecular patterns (PAMPs and DAMPs). However, it is primarily activated by lipopolysaccharide (LPS) and its toxic moiety Lipid A [2]. TLR4 does not directly interact with LPS but requires essential adaptor proteins [3]. The soluble LPS-binding protein (LBP) extracts monomeric LPS from the microbial membrane and transfers it to CD14 (cluster of differentiation 14). This membrane-bound protein then interacts with MD-2 (myeloid differentiation factor 2), constitutively associated with the TLR4 ectodomain. The ligand-loaded MD-2 subsequently binds to another TLR4/MD-2/LPS complex, leading to their dimerization [4]. Then, TLR4 triggers two distinct signaling cascades [5]:

• the MyD88-dependent activating NF-κB pathway (at the cell surface) 
• the TRIF-dependent activating IRF pathway (in endosomes) 

At the cell surface, activation of TLR4 initiates the TIRAP-MyD88-dependent pathway, ultimately leading to the activation of NF-κB and the production of a pro-inflammatory response. Also, the TLR4 complex can be endocytosed into endosomes in a CD14-mediated fashion. This results in the stimulation of IRF3 (interferon regulatory factor), which modulates the expression of type I IFN [3].

TLR4 signaling is crucial in both acute and chronic inflammatory disorders and thus, is an attractive target for novel treatments [1]. Stimulating drugs are useful for the development of vaccine adjuvants or cancer immunotherapeutics, whereas TLR4-inhibition is a therapeutic approach to treat septic shock or autoimmune inflammatory pathologies such as atherosclerosis [5].

Rough vs smooth LPS

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria. It comprises three covalently linked regions:

• the lipid A (endotoxin)
• the rough core oligosaccharide
• the O-antigenic side chain.

Wild-type LPS contains the O-side chain and is referred to as smooth (sLPS). Few bacterial strains (e.g. Salmonella Typhimurium, Brucella canis) lost the O-side chain. This mutated form is called rough (rLPS). Both sLPS and rLPS share the same receptor complex (TLR4-MD-2-CD14), but their mechanism of action differs. While CD14 is necessary for sLPS NF-κB and IRF signaling, it is dispensable for rLPS NF-κB signaling. It has been hypothesized that rLPS activates a broader range of cells (CD14 positive, low, and negative), accounting for higher toxicity [6]. 

Nevertheless, both LPS variations elicit potent innate immune responses. The resulting signaling triggers the release of pro‑inflammatory cytokines, which can lead to both acute and chronic inflammatory diseases. It is all about balance: small controlled amounts of LPS can be protective and large uncontrolled amounts can lead to disastrous outcomes, such as septic shock [7]. Despite its highly inflammatory nature, LPS has remarkable therapeutic potential and features many characteristics needed for an effective vaccine adjuvant [8]. 

References:

1. Ou, T. et al. 2018. The Pathologic Role of Toll-Like Receptor 4 in Prostate Cancer. Front Immunol 9, 1188.
2. Cochet, F. et al. 2017. The Role of Carbohydrates in the Lipopolysaccharide (LPS)/Toll-Like Receptor 4 (TLR4) Signalling. Int J Mol Sci 18
3. Kuzmich, N.N. et al. 2017. TLR4 Signaling Pathway Modulators as Potential Therapeutics in Inflammation and Sepsis. Vaccines (Basel) 5.
4.Tanimura N. et al. 2014. The attenuated inflammation of MPL is due to the lack of CD14-dependent tight dimerization of the TLR4/MD2 complex at the plasma membrane. Int Immunol.(6):307-14.
5. Romerio A, Peri F. 2020. Increasing the Chemical Variety of Small-Molecule-Based TLR4 Modulators: An Overview. Front Immunol.;11:1210.
6. Zanoni I,.et al., 2012. Similarities and differences of innate immune responses elicited by smooth and rough LPS. Immunol Lett. 2012 Feb 29;142(1-2):41-7.
7. Godowski, P., 2005. A smooth operator for LPS responses. Nat Immunol 6, 544–546.
8. McAleer, J.P. & Vella, A.T., 2010.  Educating CD4 T cells with vaccine adjuvants: lessons from lipopolysaccharide. Trends Immunol 31, 429-435.

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