ASC-particle-induced Peritonitis

Abstract

In response to pathogen infection and tissue damage, inflammasome sensors such as NLRP3 and AIM2 are activated, which triggers PYRIN domain (PYD)-mediated ASC nucleation, followed by self-perpetuating ASC polymerization, which ultimately culminates in caspase-1 activation, interleukin (IL)-1β and IL-18 processing and release and pyroptosis (Ratsimandresy et al., 2013; Cai et al., 2014). Inflammasomes release not only cytokines, but also the polymeric ASC danger particles (pASC) by pyroptosis, which perpetuate and propagate inflammasome responses to bystander cells to engage cell intrinsic ASC and caspase-1 (Baroja-Mazo et al., 2014; Franklin et al., 2014). In this protocol we describe intraperitoneal injection of polymeric ASC particles as a danger signal and measure neutrophil infiltration and levels of the pro-inflammatory cytokine IL-1β by ELISA in the peritoneal lavage (de Almeida et al., 2015).

Figure 1. Total cell lysates from stable ASC-GFP-expressing HEK293 cells were GFP-sorted by flow cytometry after inducing ASC polymerization and control (Ctrl) and ASC-GFP (pASC)-containing fractions analyzed by fluorescence microscopy

HEK293 cell lysates were used as a negative control.

Video 1. Peritoneal lavage

The video shows how to wash peritoneal cavity after injection of pASC-GFP particles to measure IL-1β by ELISA.