Gasdermin-D KO THP-1 Cells

Gasdermin D knockout in THP-1 cells

Inflammasome signaling in THP1-KO-GSDMD cells

Gasdermin D (GSDMD) is a cytoplasmic protein with a pore-forming ability that has been described as a major actor in early canonical and non-canonical inflammasome responses [1]. GSDMD pores are formed via its N-terminal domain following cleavage of the full-length GSDMD protein by caspase-1 or human CASP4/5 (CASP11 in mice). These pores allow the release of alarmins (e.g. HMGB1) and the secretion of mature IL-1β and IL-18 inflammatory cytokines.

More details

InvivoGen has developed THP1-KO-GSDMD cells, which were generated from the human monocytic THP-1 cell line THP1-Null2, through the stable knockout of the GSDMD gene.

• THP1-KO-GSDMD cells – Knockout (KO) of the GSDMD gene

These cells exhibit impaired early IL-1β secretion and pyroptosis responses upon canonical and non-canonical inflammasome activation. Although these cells are fully KO for the GSDMD protein, mature IL-1β secretion is detected upon prolonged incubation with some inflammasome inducers. This observation illustrates the reported IL-1β secretion upon GSDMD-independent cell death [2, 3].

Features of THP1-KO-GSDMD cells:

• Generated from the parental cell line THP1-Null2
• Verified biallelic knockout of the GSDMD gene (DNA sequencing, PCR, and Western blot)
• Altered IL-1β secretion and pyroptosis early upon canonical and non-canonical inflammasome activation

For detecting and quantifying the release of mature human (h)IL-1β, InvivoGen provides HEK-Blue™ IL-1β sensor cells, which express an NF-κB-inducible SEAP reporter gene. QUANTI-Blue™ Solution allows rapid colorimetric detection and measure of SEAP activity by reading the optical density at 630-650 nm.

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References:

1. Kovacs S.B. & Miao E.A. 2017. Gasdermins: effectors of pyroptosis. Trends Cell. Biol. 27:673.
2. Schneider K.S. et al.,2018. The Inflammasome Drives GSDMD-Independent Secondary Pyroptosis and IL-1 Release in the Absence of Caspase-1 Protease Activity. Cell Rep. 21:3846.
3. Zeng C-H. et al., 2019. ATP induces caspase-3/gasdermin E-mediated pyroptosis in NLRP3 pathway-blocked murine macrophages. Apoptosis.  24(9-10):703-717.

Figures

Validation of GSDMD KO in THP1-KO-GSDMD cells. (A) The targeted GSDMD region in THP1-Null2 (WT) and THP1-KO-GSDMD (KO) cells was amplified by PCR. THP1-KO-GSDMD cells feature a biallelic deletion (arrow). (B) Lysates from THP1-Null2 (WT) and THP1‑KO‑GSDMD (KO) cells were analyzed by Western blot (Wes™) using an anti-human GSDMD antibody and an HRP-conjugated anti-mouse secondary antibody. The arrow indicates the expected band for the GSDMD protein (48 kDa).

Altered mature IL-1β secretion by THP1-KO-GSDMD cells upon inflammasome activation. ~3x10⁵ THP1-Null2 (blue) and THP1-KO-GSDMD cells (red) were incubated for 3h at 37°C with LPS-EK (1 μg/ml) (priming) and then incubated (activation) with Nigericin (5 μM), Alum Hydroxide (150 μg/ml), transfected Poly(dA:dT) (1 μg/ml), or E. coli outer membrane vesicles (OMVs) (100 μg/ml). After 6h, the secretion of mature human (h)IL-1β was assessed in the culture supernatant using HEK-Blue™ IL-1β sensor cells, which express an NF-κB SEAP reporter gene. QUANTI‑Blue™ Solution was used to measure SEAP activity. Optical density (OD) was read at 630 nm. 

Altered pyroptosis by THP1-KO-GSDMD cells upon inflammasome activation. ~3x10⁵ THP1-Null2 (blue) and THP1-KO-GSDMD cells (red) were incubated for 3h at 37°C with LPS-EK (1 μg/ml) (priming) and then incubated (activation) with Nigericin (5 μM). After 6h, cell death was assessed using the lactate dehydrogenase (LDH) assay.

Specifications

Antibiotic resistance: Zeocin®

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

Quality Control:

• Biallelic GSDMD knockout has been verified by DNA sequencing, PCR, Western blot (Wes™), 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-KO-GSDMD cells in a cryovial or shipping flask
• 1 ml of Zeocin® (100 mg/ml). Store at 4 °C or at -20 °C.
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.

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

Details

GSDMD belongs to a family of six and ten gasdermins in humans and mice, respectively, which all have different expression patterns [1, 2]. GSDMD consists of two distinct domains, whereby the C-terminal domain exerts an auto-inhibitory function on the N-terminal domain.

GSDMD is cleaved by activated caspase-1 (CASP1) downstream of NLRP1, NLRP3, AIM2, NLRC4, or Pyrin canonical inflammasomes, or by activated CASP4/5 (human), CASP11 (mouse) non-canonical inflammasomes. The released GSDMD N-terminal domain oligomerizes to form 10-15 nm diameter pores at the cell membrane, thereby allowing the release of alarmins (e.g. HMGB1) and the secretion of mature IL-1β and IL-18 inflammatory cytokines. The accumulation of GSDMD pores in the membrane causes cell swelling and rupture, leading to an inflammatory cell death termed pyroptosis  [1, 3].

Importantly, GSDMD links the canonical and non-canonical inflammasome responses with the pore formation leading to stress signals, such as cytosolic ion concentration imbalances (i.e. K+ efflux) and ATP release. These signals induce the activation of NLRP3 and CASP1-mediated IL-1β/IL-18 maturation and secretion [2, 4].

1. Kovacs S.B. & Miao E.A. 2017. Gasdermins: effectors of pyroptosis. Trends Cell. Biol. 27:673.
2. Groslambert M. & Py B. 2018. Spotlight on the NLRP3 inflammasome pathway. J. Inflamm. Res. 11:359.
3. Feng S. et al., 2018. Mechanisms of Gasdermin family members in inflammasome signaling and cell death. J. Mol. Biol. 430:3068.
4. Mathur A. et al., 2017. Molecular mechanisms of inflammasome signaling. J. Leuk. Biol. 103:233.

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