TLR8 KO Dual Reporter THP-1 Cells

TLR8 knockout dual reporter monocytes

THP1-Dual™ KO-TLR8 cells were generated from the THP1-Dual™ cell line through the stable knockout of the TLR8 gene. They feature two inducible reporter genes, allowing the concomitant study of the IRF and NF-κB pathways, by monitoring the Lucia luciferase and SEAP (secreted embryonic alkaline phosphatase) activities, respectively. As expected, NF-κB- and IRF-mediated responses are abolished in THP1-Dual™ KO-TLR8 cells upon incubation with TLR8 and TLR7/8 agonists when compared to the THP1-Dual™ cells, with no notable difference for the other ligands tested (see Figures).

Toll-like receptor 8 (TLR8) is a crucial intracellular sensor involved in the detection of viral and bacterial pathogens [1,2]. Similar to its structural homolog TLR7, TLR8 is activated by single-stranded (ss)RNA, as well as synthetic imidazoquinoline compounds such as R848. TLR8 and its structural homolog, TLR7, have similar signaling pathways but display different ligand specificities and expression patterns [2]. TLR8 activation triggers NF-κB- and IRF-mediated production of pro-inflammatory cytokines and type-I IFNs, respectively.

More details

NF-κB and IRF signaling pathways in THP1-Dual™ KO-TLR8 cells

Key Features:

• Verified knockout of the TLR8 gene (PCR, DNA sequencing, and Western blot)
• Functionally validated with a selection of PRR ligands and cytokines
• Readily assessable Lucia luciferase and SEAP reporter activities

Applications:

• Defining the role of TLR8 in PRR-induced signaling, or other cell signaling pathways 
• Highlighting the possible overlap between TLR8 and other signaling pathways
• Screening specificity of novel TLR8 agonists for use in vaccine adjuvants

References

1. Moen, S.H. et al., 2019. Human Toll-like Receptor 8 (TLR8) is an Important Sensor of Pyogenic Bacteria and Is Attenuated by Cell Surface TLR Signaling. Front Immunol, 10: 1209.
2. Georg P. & Sander L.E., 2019. Innate sensors that regulate vaccine responses. Curr. Op. Immunol. 59:31

Figures

Validation of TLR8 KO. PMA-differentiated THP1-Dual™ (WT) and THP1-Dual™ KO‑TLR8 (KO) cells were treated with 20 ng/ml interferon(IFN)-γ to induce the expression of TLR8. Lysates from non-treated (-) and treated (+) WT and KO cells were analyzed using an anti‑human TLR8 antibody, followed by an HRP‑conjugated anti‑rabbit secondary antibody (WES assay). As expected, a band was detected in the IFN-γ treated WT cells only (green arrow). The predicted molecular weight of TLR8 is ~120 kDa.

NF-κB responses in THP1-Dual™-derived cells. THP1-Dual™ and THP1-Dual™ KO-TLR8 cells were incubated with 0.3 ng/ml human (h)TNF-α (NF-κB-SEAP positive control), 1 x 104 U/ml hIFN-β (IRF-Lucia positive control), 300 ng/ml 3p-hpRNA/LyoVec™ (RIG-I agonist), 1 μg/ml LPS-EK Ultrapure (UP; TLR4),0.1 ng/ml Pam3CSK4 (TLR2 agonist), 10 µg/ml R848 (TLR7/8 agonist), 10 µg/ml CL075 (TLR7/8 agonist), 5 µg/ml ORN06/LyoVec™ (TLR8 agonist), 5µg/ml ssRNA40/LyoVec™ (TLR8 agonist) and 10 μg/ml 2’3’-cGAMP (STING agonist). 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).

IRF responses in THP1-Dual™-derived cells. THP1-Dual™ and THP1-Dual™ KO-TLR8 cells were incubated with 0.3 ng/ml human (h)TNF-α (NF-κB-SEAP positive control), 1 x 104 U/ml hIFN-β (IRF-Lucia positive control), 300 ng/ml 3p-hpRNA/LyoVec™ (RIG-I agonist), 1 μg/ml LPS-EK Ultrapure (UP; TLR4),0.1 ng/ml Pam3CSK4 (TLR2 agonist), 10 µg/ml R848 (TLR7/8 agonist), 10 µg/ml CL075 (TLR7/8 agonist), 5 µg/ml ORN06/LyoVec™ (TLR8 agonist), 5µg/ml ssRNA40/LyoVec™ (TLR8 agonist) and 3 μg/ml 2’3’-cGAMP (STING agonist). After overnight incubation, the IRF response was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI-Luc™. Data are shown as a fold increase over non-induced cells (Lucia luciferase readout).

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:

• 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. 
   

InvivoGen's products are covered by a Limited Use License (See Terms and Conditions).

Contents

• 3-7 x 10⁶ 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 pouch of QUANTI-Luc™ (Lucia luciferase detection reagent)
• 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)

Details

TLR8 background

Toll-like receptor 8 (TLR8) is a crucial sensor involved in the detection of viral and bacterial pathogens within endosomes [1,2]. Similar to its structural homolog TLR7, TLR8 surveys phagocytic cargo for the presence of microbial single-stranded (ss) RNA. Despite having similar signaling pathways, TLR7 and TLR8 display different ligand specificities and expression patterns [2]. TLR7 is essentially expressed by plasmacytoid dendritic cells (pDCs), whereas TLR8 is highly expressed by myeloid cells [2]. This suggests that TLR7 and TLR8 have evolved to mediate distinct immune responses. TLR8 contains two distinct ligand interaction sites that facilitate its recognition of RNA degradation products and small synthetic nucleoside analogs such as resiquimod (R848) [2,3]. Ultimately, this interaction induces a conformational change and leads to the activation of NF-κB- and IRF-mediated production of pro-inflammatory cytokines (e.g. IL-1β and IL-12) and type I IFNs (IFN-β), respectively. There is growing evidence for TLR8 as a critical sensor in the essential humoral immune response needed to achieve optimal vaccine efficiencies with live attenuated vaccines [2, 4]. Therefore, there are ongoing efforts to develop more potent, specific, and less toxic TLR8 agonists for vaccine adjuvants. 
 

References:

1. Moen, S.H. et al., 2019. Human Toll-like Receptor 8 (TLR8) is an Important Sensor of Pyogenic Bacteria and Is Attenuated by Cell Surface TLR Signaling. Front Immunol, 10: 1209.
2. Georg P. & Sander L.E., 2019. Innate sensors that regulate vaccine responses. Curr. Op. Immunol. 59:31
3. Tanji H. et al., 2015. Toll-like receptor 8 senses degradation products of single-stranded RNA. Nat. Struct. Mol. Biol. 22:109.
4. Ugolini M. et al., 2018. Recognition of microbial viability via TLR8 drives TFH cell differentiation and vaccine responses. Nat. Immunol. 19:386.

FAQ

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