Human TLR9 Reporter THP-1 Cells

Reporter systems in THP1-Dual™ hTLR9 cells

Dual reporter monocytes for TLR9 pathway studies

InvivoGen offers a human monocyte-derived cell line, specifically designed for the study of human TLR9 (Toll-Like Receptor 9) signaling pathways:

• THP1-Dual™ hTLR9 cells

THP1-Dual™ hTLR9 cells overexpress the human TLR9 (hTLR9) gene. They also 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.
Unlike their parental cells, THP1-Dual™,  which weakly express TLR9, THP1-Dual™ hTLR9 cells are responsive to stimulation with TLR9 agonists, such as oligonucleotides containing CpG motifs (CpG ODNs). As expected, hTLR9 overexpression in THP1-Dual™ cells allows potent NF-κB and IRF responses upon incubation with CpG-ODNs of class A (ODN 2216), class B (ODN 2006, ODN 1826), and class C (ODN 2395). Of note, THP1-Dual™ hTLR9 cells are more responsive to the class B, human-preferred, ODN 2006  (see Figures).

Background:

Toll-Like Receptor 9 (TLR9) is one of the most studied pattern recognition receptors (PRRs) for nucleic acids. It is an endosomal receptor that triggers NF-κB- and IRF-mediated pro-inflammatory responses upon the recognition of unmethylated cytosine-phosphorothioate-guanosine (CpG) forms of DNA [1-3]. TLR9 agonists can be mimicked by synthetic oligonucleotides containing CpG motifs (CpG ODNs) [1,3].

 More details

Key features:

• Verified overexpression of TLR9 gene by qRT-PCR
• Functionally validated with a selection of TLR9 agonists
• Readily assessable Lucia luciferase and SEAP reporter activities

Applications:

• Study of IRF and NF-κB-dependent TLR9 signaling pathways
• Screening of novel specific activators or inhibitors of the TLR9 signaling pathways

References

1. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
2. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
3. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.

Figures

Human TLR9 expression in THP1-Dual™ hTLR9 cells.
Total mRNA was extracted from ~1x10⁶ THP1-Dual™ (parental, blue) and THP1-Dual™ hTLR9 (red) cells. Human TLR9 (hTLR9) mRNA was amplified using quantitative RT-qPCR. Data are represented as the log₂ fold change comparing hTLR9 relative expression between THP1-Dual™ and THP1-Dual™ hTLR9 cells.

NF-κB and IRF responses in THP1-Dual™-derived cells.
THP1-Dual™ and THP1-Dual™ hTLR9 cells were incubated with 40 nM ODN 2216 (class A, human TLR9-preferred), ODN 2006 (class B, human TLR9-preferred), ODN 1826 (class B, mouse TLR9-preferred), or ODN 2395 (class C, human/mouse TLR9-preferred), and 1 ng/ml human TNF-α (hTNF-α), 300 ng/ml Tri-DAP, 104 U/ml human IFN-β (hIFN-β), or 3 μg/ml 2'3'-cGAMP as controls. After overnight incubation, the NF-κB response was assessed by measuring the SEAP activity in the supernatant using QUANTI-Blue™ Solution. Data are shown as optical density (OD) at 630 nm (mean ± SEM) (A).
The IRF response was assessed by measuring Lucia luciferase activity in the supernatant using QUANTI-Luc™. Data are shown as a fold increase (mean ± SEM) over non-induced cells (B).

NF-κB and IRF responses induced by TLR9 agonist ODNs.
THP1-Dual™ hTLR9 cells were incubated with increasing concentrations of ODN 2216 (class A, human TLR9-preferred), ODN 2006 (class B, human TLR9-preferred), ODN 1826 (class B, mouse TLR9-preferred), or ODN 2395 (class C, human/mouse TLR9-preferred). After overnight incubation, the NF-κB response was assessed by measuring the SEAP and IRF activity in the supernatant using QUANTI-Blue™ Solution (A), or QUANTI-Luc™ (B), respectively. Data are shown as a fold increase (mean ± SEM) over non‑induced cells.

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, Zeocin®, and Puromycin

Quality Control:

• Human TLR9 expression has been verified by qRT-PCR and functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free. 

Contents

• 3-7 x 10⁶ THP1-Dual™ hTLR9 cells in a cryovial or shipping flask
• 1 ml of Blasticidin (10 mg/ml)
• 1 ml of Zeocin® (100 mg/ml)
• 1 ml of Puromycin (10 mg/ml)
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)
• 1 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)

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

Details

Toll-Like Receptor 9 (TLR9) is an endosomal receptor that triggers NF-κB- and IRF-mediated pro-inflammatory responses upon the recognition of unmethylated cytosine-phosphorothioate-guanosine (CpG) forms of DNA [1-3]. Unmethylated CpG dinucleotides are a hallmark of microbial (bacterial, viral, fungal, and parasite) DNA, as well as mitochondrial self-DNA [3,4]. These TLR9 agonists can be mimicked by synthetic oligonucleotides containing CpG motifs (CpG ODNs), which have been extensively studied to improve adaptive immune responses in the context of vaccination [1,3].

TLR9 is mainly expressed in subsets of Dendritic Cells and in B cells of all mammals. In rodents, but not in humans, TLR9 is also expressed in monocytes and macrophages [3]. The structure of the receptor varies by 24% between human TLR9 (hTLR9) and mouse TLR9 (mTLR9) [3]. They recognize different CpG motifs, the optimal sequences being GTCGTT and GACGTT for hTLR9 and mTLR9, respectively [5].
 

Get more information about CpG-ODNs Classes.

References

1. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
2. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
3. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.
4. Kumar V., 2021. The trinity of cGAS, TLR9, and ALRs: guardians of the cellular galaxy against host-derived self-DNA. Front. Immunol. 11:624597.
5. Bauer S. et al., 2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA, 98(16):9237-42.

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