Human TLR8 Reporter THP-1 Cells

TLR8 overexpressing or knockout (KO) dual reporter monocytes

InvivoGen expands its collection of human monocytic reporter cell lines designed to monitor the intertwined NF-κB and IRF responses of the human Toll-Like Receptor 8 (TLR8).

Trafficking and signaling in THP1-Dual™ TLR7/8 cells

THP1-Dual™ hTLR8 cells and THP1-Dual™ KO-TLR8 cells were generated from the THP1-Dual™ cell line harboring two inducible reporter genes, allowing the simultaneous monitoring of the NF-κB and IRF pathway, by monitoring the SEAP and Lucia luciferase activities, respectively. They feature either a stable expression of the human TLR8 or a knockout (KO) of the endogenous TLR8 gene.
 

These cells are derived from human leukemia monocytic THP-1 cells, a common model to study monocyte/macrophage functions, mechanisms, and signaling pathways. They express several pattern recognition receptors (PRRs) and co-adaptors, including TLR8 and UNC93B1, but not TLR7 [1-2].

Indeed, THP1-Dual™ cells do not respond to TLR7-specific ligands (see figures). To enhance the response to TLR8-specific ligands, the human TLR8 was overexpressed in THP1-Dual™ hTLR8 cells. This feature strongly increases the sensitivity to various TLR8 ligands. As expected, the biallelic KO in THP1-Dual™ KO-TLR8 cells abrogates these responses (see figures).

Both TLR7 and TLR8 recognize viral single-stranded RNA (ssRNA) as well as small synthetic molecules. Despite their structural homology and similar signaling pathway, TLR7 and TLR8 display different ligand specificities and tissue expression patterns [4].

 More details

Key Features

• Stable overexpression (OE) or biallelic KO of human (h)TLR8
• Increased and reproducible response to TLR8-specific ligands
• Distinct monitoring of NF-κB or IRF activation by assessing the SEAP and Lucia luciferase activities

Applications

• Defining the distinct role of TLR8 in the IRF and NF-κB-dependent pathway
• Highlighting possible overlap between TLR7 and TLR8 signaling pathways 
• Screening for TLR7- and TLR8-specific agonists or inhibitors 

Read our review on TLR7 and TLR8: same, same... but different!

​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. Majer, O., et al. 2019. UNC93B1 recruits syntenin-1 to dampen TLR7 signalling and prevent autoimmunity. Nature 575, 366–370.
3. Wang J, et al., 2006. The functional effects of physical interactions among Toll-like receptors 7, 8, and 9. J Biol Chem.; 281(49):37427-34. 
4. Georg P. & Sander L.E., 2019. Innate sensors that regulate vaccine responses. Curr. Op. Immunol. 59:31.

Figures

NF-κB responses comparing TLR8 overexpressing (OE) and TLR8-KO THP1-Dual™ -derived cells. THP1-Dual™, THP1-Dual™ hTLR8 cells and THP1-Dual™ KO-TLR8 cells were incubated for 24 hours with various TLR agonists: Pam3CSK4 (TLR2 ligand, 10 ng/ml), Poly(I:C) HMW (TLR3 ligand, 10 µg/ml), LPS-EK Ultrapure (UP) (TLR4 ligand, 1 ng/ml), FLA-ST UP (TLR5 ligand, 1 µg/ml), CL264 (TLR7 ligand, 1 µg/ml), CL307 (TLR7 ligand, 1 µg/ml), Imiquimod (TLR7 ligand, 10 µg/ml), R848 (TLR7/8 ligand, 1 µg/ml), CL097 (TLR7/8 ligand, 1 µg/ml), TL8-506 (TLR8 ligand, 1 µg/ml), and ODN 2006 (TLR9 ligand, 10 µg/ml). Human TNF-α (1 ng/ml) and hIFN-β (1000 U/ml) were used as an NF-kB-and IRF-positive control, respectively. After 24h incubation, the NF-kB-induced SEAP activity was assessed using QUANTI-Blue™. Data are shown as optical density (OD) at 630 nm (mean ± SEM).

IRF responses comparing TLR8-OE and TLR8-KO THP1-Dual™ -derived cells. THP1-Dual™, THP1-Dual™ hTLR8 cells and  THP1-Dual™ KO-TLR8 cells  were incubated for 24 hours with various TLR agonists: Pam3CSK4 (TLR2 ligand, 10 ng/ml), Poly(I:C) HMW (TLR3 ligand, 10 µg/ml), LPS-EK Ultrapure (UP) (TLR4 ligand, 1 ng/ml), FLA-ST UP (TLR5 ligand, 1 µg/ml), CL264 (TLR7 ligand, 1 µg/ml), CL307 (TLR7 ligand, 1 µg/ml), Imiquimod (TLR7 ligand, 10 µg/ml), R848 (TLR7/8 ligand, 1 µg/ml), CL097 (TLR7/8 ligand, 1 µg/ml), TL8-506 (TLR8 ligand, 1 µg/ml), and ODN 2006 (TLR9 ligand, 10 µg/ml). Human TNF-α (1 ng/ml) and hIFN-β (1000 U/ml) were used as an NF-kB-and IRF-positive control, respectively. 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

Growth medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml Normocin™

Antibiotic resistance: Blasticidin and Zeocin®

Quality Control:

• Human TLR8 overexpression has been verified by RT-qPCR and functional assays.
• Biallelic TLR8 knockout has been verified by PCR, DNA sequencing, Western blot, and functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free. 

Contents

Please note: Each cell line is sold separately. See TDS for the exact contents of each cell line. 

• 3-7 x 10⁶ THP1-Dual™ hTLR8 cells OR THP1-Dual™ KO-TLR8 cells in a cryovial or shipping flask
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
• 1 ml of Zeocin® (100 mg/ml)
• 1 ml of Blasticidin (10 mg/ml)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 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)

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

FAQ Cell Lines

Any questions about our cell lines ? Visit our frequently asked questions page

Details

Toll-Like Receptors 7 and 8

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 and TLR8 trafficking and signaling

Upon viral infection, the chaperone protein UNC93B1 interacts with TLR7 and TLR8, thereby facilitating their trafficking from the endoplasmic reticulum via the Golgi into the endosomes [4]. Subsequently, TLR7 and TLR8 undergo proteolytic cleavage and dimer rearrangement [1,3]. While TLR7 dimerizes upon ligand binding, TLR8, which exists as an unliganded inactive dimer, performs structural reorganization after ligand recognition [5]. Once activated, both TLR7 and TLR8 recruit the adaptor protein MyD88 to trigger IRF, AP-1, and NF-κB 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, whereas Th1-type cytokine (IL-12) production depends on TLR8 [6].

TLR7 and TLR8 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 [6]. TLR7/8 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) [1-6]. Understanding the fundamental differences between these two related receptors could potentially be harnessed to discover novel drugs and improve vaccine efficacy/safety [6].

​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. Majer, O., et al. 2019. UNC93B1 recruits syntenin-1 to dampen TLR7 signalling and prevent autoimmunity. Nature 575, 366–370.
5. Asami J, Shimizu T. 2021. Structural and functional understanding of the toll-like receptors. Protein Sci. (4):761-772.
6. de Marcken M, et al., 2019. TLR7 and TLR8 activate distinct pathways in monocytes during RNA virus infection. Sci Signal.;12(605):eaaw1347.