Human MD2 and CD14 Reporter HEK293 Cells (NF-κB)

NF-κB–SEAP reporter HEK293 cells expressing human MD2 and CD14

Signaling pathways in HEK-Blue™ hMD2-CD14 cells

HEK-Blue™ hMD2-CD14 cells were engineered from the human embryonic kidney HEK293 cell line. These cells represent a great tool for studying different Toll-like receptor 4 (TLR4) mutations (gain-of-functions, loss-of-function) since they feature the important adaptor proteins MD2 (myeloid differentiation factor 2) and CD14 (cluster of differentiation 14) without the interference of endogenous TLR4 expression.
 

Description

HEK-Blue™ hMD2-CD14 cells stably express MD2 and CD14, two adaptor proteins crucial for TLR4 signaling. Moreover, these cells harbor an inducible reporter gene for SEAP (secreted embryonic alkaline phosphatase). SEAP levels produced upon TLR4 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.

HEK-Blue™ hMD2-CD14 cells do not express the Toll-like receptor 4 (TLR4). Therefore, they don't respond to any type of LPS, when compared to HEK-Blue™ hTLR4 - a cell line that stably expresses TLR4, MD2, and CD14. In addition, this cell line can be used to distinguish between TLR4- and other TLRs-mediated responses, as HEK293 cells express endogenous levels of various PRRs, including TLR3, TLR5, and RIG-I-like receptors. The parental cell line of HEK-Blue™ hMD2-CD14 cells is HEK-Blue™ Null2 cells.

The two adaptor proteins MD2 and CD14 are essential for TLR4-dependent sensing of lipopolysaccharide (LPS). LPS is a major constituent of the outer membrane of Gram-negative bacteria and the main microbial mediator implicated in the pathogenesis of septic shock [1].

 More details

Key features

• Stable expression of human MD2 and CD14
• No endogenous expression of human TLR4
• Distinct monitoring of NF-κB activation by assessing the SEAP activities

Applications

• Investigating different TLR4 variants (loss-of-function, gain-of-function mutations,...)
• Defining the role of MD2 and CD14 in LPS-induced signaling pathways 
• Screening for TLR4 agonists or antagonists
• Highlighting the importance of TLR4 when used in combination with HEK-Blue™ hTLR4 

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

Figures

Response of HEK-Blue™ hMD2-CD14 cells to TLR4 agonists after transient transfection of hTLR4. Cells were transiently transfected with a hTLR4A plasmid and stimulated with human TNF-α (NF-κB-positive control, 10 ng/ml), LPS-EB UP (1 µg/ml), and LPS-EK UP (1 µg/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).

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).

Response of HEK-Blue™ hMD2-CD14 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, 10 ng/ml), Pam3CSK4 (TLR2 ligand, 1 µg/ml), Poly(I:C) HMW (TLR3 ligand, 1 µg/ml), LPS-EK UP (TLR4 ligand, 1 µg/ml), FLA-ST UP (TLR5 ligand, 1 µg/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).

Specifications

Antibiotic resistance: 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™.

Guaranteed mycoplasma-free

Contents

• 1 vial containing 3-7 x 10⁶ cells
• 1 ml of Hygromycin B Gold (100 mg/ml)
• 1 ml of Zeocin® (100 mg/ml)
• 1 ml of 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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