IRF5 KO Dual Reporter THP-1 Cells

IRF5 knockout dual reporter monocytes

THP1-Dual™ KO-IRF5 cells were generated from the THP1-Dual™ cell line through the stable knockout of the IRF5 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-mediated responses are significantly reduced in THP1-Dual™ KO-IRF5 cells upon incubation with TLR4 and TLR7/8 agonists  [1] when compared to THP1-Dual™ cells, with no notable difference for the other ligands tested (see Figures).

Interferon regulatory receptor 5 (IRF5) is a transcription factor that plays a central role in inflammation, mediating the induction of type I interferons and proinflammatory cytokines [1]. IRF5 has been implicated downstream of several pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) [2]. There is increasing evidence that IRF5 plays a key role in numerous inflammatory and autoimmune diseases, including rheumatoid arthritis (RA) and inflammatory bowel disease [1,3]. Importantly, because IRF5 acts in a cell-type and activity-specific manner, it is an attractive therapeutic target [1].

NF-κB and IRF signaling pathways in THP1-Dual™ KO-IRF5 cells

More details

Key Features:

• Verified knockout of the IRF5 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 IRF5 in PRR-induced signaling, or other cell signaling pathways 
• Highlighting the possible overlap between IRF5 and other signaling pathways
• Developing novel IRF5-targeting therapeutics

References

1. Almuttaqi, H. & Udalova, I.A. 2019. Advances and challenges in targeting IRF5, a key regulator of inflammation. FEBS J 286, 1624-1637.
2. Zhao, G.N. et al. 2015. Interferon regulatory factors: at the crossroads of immunity, metabolism, and disease. Biochim Biophys Acta. 2015 Feb;1852(2):365-78.
3. Kaur, A. et al. 2018. IRF5-mediated immune responses and its implications in immunological disorders. Int Rev Immunol 37, 229-248.

Figures

Validation of IRF5 KO. (A) The targeted IRF5 region in THP1-Dual™ (WT; blue arrow) parental cells and THP1‑Dual™ KO-IRF5 (KO; red arrows) cells was amplified by PCR. THP1-Dual™ KO-IRF5 cells feature two different frameshift deletions for each allele, causing an early stop codon and inactivation of IRF5. (B) Lysates from THP1-Dual™ (WT) and THP1-Dual™ KO-IRF5 (KO) cells were analyzed using an anti-human IRF5 antibody (green arrow), followed by an HRP‑conjugated anti‑rabbit secondary antibody (WES assay). As expected a band was detected at ~56 kDa in the WT cells only.

NF-κB responses in THP1-Dual™-derived cells.
THP1-Dual™ and THP1-Dual™ KO-IRF5 cells were incubated with 1 ng/ml human (h)TNF-α (NF-κB-SEAP positive control), 1000 U/ml hIFN-β (IRF-Lucia positive control), 10 ng/ml LPS-EK Ultrapure (UP; TLR4 agonist), 3 μg/ml CL075 (TLR7/8 agonist), 3 μg/ml R848 (TLR7/8 agonist), 30 μg/ml 2’3’-cGAMP (STING agonist), and 300 ng/ml 3p-hpRNA complexed with LyoVec™ (LV; RIG-I 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 PMA-differentiated THP1-Dual™-derived cells.
THP1-Dual™ and THP1-Dual™ KO-IRF5 cells were pretreated with PMA (Phorbol 12-myristate 13-acetate; 10 ng/ml for 3 hours). After 3 days recovery, the cells were incubated with 1 ng/ml human (h)TNF-α (NF-κB-SEAP positive control), 1000 U/ml hIFN-α or hIFN-β (IRF-Lucia positive control), 10 ng/ml LPS-EK Ultrapure (UP; TLR4 agonist), 1 μg/ml R848 (TLR7/8 agonist), 300 ng/ml 3p-hpRNA complexed with LyoVec™ (LV; RIG-I agonist), and 10 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 IRF5 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

• 3-7 x 10⁶ THP1-Dual™ KO-IRF5 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)

Details

IRF5 background

Interferon regulatory factor 5 (IRF5) is a transcription factor that plays a central role in inflammation. It mediates the induction of type I interferons (IFNs) and proinflammatory cytokines through binding to ISRE and NF-κB motifs, respectively [1]. IRF5 has been implicated downstream of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) [2].  Depending on the PRR that is triggered, IRF5 is activated through distinct mechanisms. TLR stimulation elicits IRF5 activation downstream of the MyD88 or the TRIF adaptors. Nucleic acid sensing by RIG-I, MDA5, cGAS or STING, elicits IRF5 activation through TBK1 (TANK-binding kinase 1). IRF5 can also be activated by the IKKβ kinase downstream of the MAVS adaptor associated with RIG-I or MDA5 [2]. IRF5 plays a key role in numerous inflammatory and autoimmune diseases including rheumatoid arthritis (RA) and inflammatory bowel disease [1, 3]. Importantly, because IRF5 acts in a cell-type and activity-specific manner, it is an attractive therapeutic target [1].

1. Almuttaqi, H. & Udalova, I.A. 2019. Advances and challenges in targeting IRF5, a key regulator of inflammation. FEBS J 286, 1624-1637.
2. Zhao, G.N. et al. 2015. Interferon regulatory factors: at the crossroads of immunity, metabolism, and disease. Biochim Biophys Acta. 2015 Feb;1852(2):365-78.
3. Kaur, A. et al. 2018. IRF5-mediated immune responses and its implications in immunological disorders. Int Rev Immunol 37, 229-248.

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