Caspase-1 in inflammmasome responses:

Rapid responses to infections and tissue damages are largely mediated by the inflammasomes. The formation of these cytosolic signaling platforms occurs according to a consensual two-step model. The priming step induces the transcription of pro-IL-1β. The activation step triggers the multimerization of the activated inflammasome sensor with the ASC adaptor and pro-caspase-1. This assembly permits caspase-1 self-activation, which in turn induces the maturation of IL-1β and IL-18 cytokines. Activated caspase-1 also cleaves the N-terminal fragment of gasdermin D (GSDMD), which accumulates to form pores at the cell membrane. These pores allow the unconventional secretion of IL-1β and IL-18, and in the absence of membrane repair, their accumulation leads to pyroptosis.

Application

THP1-defCASP1 cells are designed to study the signals involved in inflammasome activation.

To become susceptible to inflammasome inducers, these cells must be induced by stimuli commonly used for induction in model systems, such as lipopolysaccharide (LPS) and phorbol 12-myristate acetate (PMA). Stimulation by LPS or differentiation with PMA induces the production of pro-IL-1β, the immature form of IL-1β. Subsequent stimulation with inflammasome inducers, such as ATP and alum crystals, leads to caspase-1 activation and IL-1β maturation and secretion. Mature IL-1β can be detected by Western blot, ELISA, or a cell-based assay.

InvivoGen has developed a new method to detect bioactive IL-1β, based on HEK293 cells specifically engineered to selectively respond to IL-1β, named HEK-Blue™ IL-1β cells. These cells feature the SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of an NF-kB-inducible promoter. They naturally express the IL-1β receptor (IL-1R), and all the proteins involved in the MyD88-dependent IL-1R signaling pathway that leads to NF-kB activation. Thus upon IL-1β binding to IL-1R, a signaling cascade is initiated triggering NF-kB activation and the subsequent production of SEAP. Detection of SEAP in the supernatant of HEK-Blue™ IL-1β cells can be readily assessed using QUANTI-Blue™, a SEAP detection medium. QUANTI-Blue™ turns blue in the presence of SEAP which can be easily quantified using a spectrophotometer.

Detection of IL-1β in THP-1 supernatants

Detection of IL-1beta in THP-1 supernatants

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