Notification: Reference #thpd-mctlr4-av can only be ordered together with reference #thpd-mctlr4.
Reference #thpd-mckotlr4-av can only be ordered together with reference #thpd-mckotlr4.

CD14 and MD-2 expressing dual reporter monocytes for LPS studies

InvivoGen expands its collection of human monocytic reporter cell lines designed to monitor the TLR4 (Toll-like receptor 4)-dependent NF-kB and IRF responses upon stimulation with rough and smooth LPS (lipopolysaccharide):

Rough and smooth LPS-induced signaling

— THP1-Dual™ MD2-CD14-TLR4 cells

— THP1-Dual™ MD2-CD14 KO-TLR4 (control) cells

These cells stably express MD-2 (myeloid differentiation factor 2) and CD14 (cluster of differentiation 14), two co-adaptors important for LPS-induced TLR4 signaling. Additionally, THP1-Dual™ MD2-CD14-TLR4 cells feature TLR4 overexpression and their control THP1-Dual™ MD2-CD14 KO-TLR4, a biallelic TLR4 knockout (KO). They were generated from the THP1-Dual™ cell line harboring two inducible reporter genes for SEAP (secreted embryonic alkaline phosphatase) and Lucia luciferase. This allows the simultaneous monitoring of the MyD88-dependent NF-κB and TRIF-dependent IRF pathway, respectively.

Due to the stable expression of CD14 and MD-2, the TRIF-dependent IRF response to LPS is completely restored in these cells. The MyD88-dependent NF-kB activation is strongly increased upon stimulation using smooth or rough LPS. The additional overexpression of hTLR4 enhances the sensitivity to various TLR4 ligands. As expected, the control cells THP1-Dual™ MD2-CD14 KO-TLR4 do not respond to any type of LPS (see figures below). Moreover, this KO cell line can be used to distinguish between TLR4- and other TLRs-mediated responses.

THP1-Dual™ MD2-CD14-TLR4 and their control derive from the human leukemia monocytic THP-1 cells. This cell line has become a common model to study monocyte/macrophage functions, mechanisms and signaling pathways. It expresses several pattern recognition receptors (PRRs), including TLR4, which upon sensing of their cognate PAMPs (pathogen-associated molecular patterns) trigger the activation of the NF-kB and/or IRF transcription factors [1]. TLR4-induced IRF-activation in THP-1-derived cells (e.g. THP1-Dual™) requires their differentiation into macrophages using PMA (Phorbol 12-myristate 13-acetate). Overexpression of MD-2/CD14 in THP1-Dual™ MD2-CD14-TLR4 and THP1-Dual™ MD2-CD14 KO-TLR4 (control) cells eliminates this requirement, thus TLR4-dependent IRF activation can be studied without PMA treatment.

The adaptor proteins, MD-2 and CD14, are essential for TLR4-dependent sensing of rough or smooth LPS. The wildtype, smooth (s)LPS requires CD14 co-expression, whereas the truncated form of LPS (rough LPS) can also interact with TLR4 in a CD14-independent manner. Their recognition lead to NF-κB and IRF activation and the production of proinflammatory cytokines and interferons, respectively [2-3]. Thus, the LPS-induced TLR4 responses are highly inflammatory, which makes it a remarkable therapeutic target [3].

More details

Bypass PMA Differentiation Bias: THP1-Dual™ MD2-CD14-TLR4 cells (& control) do not require PMA treatment prior to IRF-stimulation (see figures).

Key Features

Stable overexpression of CD14 and MD-2
Verified overexpression or biallelic KO of the hTLR4 gene
Increased and reproducible response to rLPS and sLPS
Measurable IRF response without PMA-differentiation
Distinct monitoring of NF-κB or IRF activation by assessing the SEAP and Lucia luciferase activities

Applications

Detecting sample contamination with rLPS and sLPS
Defining the role of TLR4 in LPS-induced signaling pathways
Screening for TLR4 agonists or antagonists
Highlighting possible overlap between TLR4 and other TLR-related signaling pathways (e.g. TLR2)

References:

1. Hornung V. et al. 2002. Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol.; 168(9):4531-7.
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.

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