In addition, whether inflammasome-dependent expulsion is an epithelial cell type-intrinsic response, or whether it involves other cell types, has not been addressed

In addition, whether inflammasome-dependent expulsion is an epithelial cell type-intrinsic response, or whether it involves other cell types, has not been addressed. In this study, we provide genetic evidence for a coordinated intestinal epithelial cell-intrinsic inflammasome response locus under Cre-inducible control (iNLRC4 mice, Fig. al., 2014; Romberg et al., 2014). studies of NLRC4-deficient animals demonstrated a role for NLRC4 in infection (Broz et al., 2010; Carvalho et al., 2012; Franchi et al., 2012; Lara-Tejero et al., 2006; Mariathasan et al., 2004; Miao et al., 2010) and inflammation-induced colon cancer (Hu et al., 2010), but whether NLRC4 has an IEC-intrinsic function in these scenarios has not been addressed. Studies utilizing intestinal epithelium-specific NAIP deficient mice demonstrated an epithelium intrinsic requirement for NAIPs in infection (Sellin et al., 2014) and colon SCH-1473759 tumorigenesis (Allam et al., 2015), but whether and/or how NLRC4 or Caspase-1 is involved remains unclear (Allam et al., 2015). A study using bone marrow transfer suggested NLRC4 in non-hematopoetic cells SCH-1473759 is protective in infection with the epithelial cell-adhering pathogen (Nordlander et al., 2013), but interpretation of bone-marrow chimeras is complicated by the possible presence of radioresistant hematopoietic cells. In macrophages, NAIP-NLRC4 activation results in Caspase-1 dependent processing and release of pro-inflammatory interleukins-1 and -18, and a lytic cell death called pyroptosis. By contrast, NAIP-NLRC4 activation in IECs was recently reported to result in their non-lytic expulsion into the gut lumen and protection from Salmonella invasion (Sellin et al., 2014). Why macrophages but not epithelial cells would undergo pyroptosis upon Caspase-1 activation is not clear. In addition, whether inflammasome-dependent expulsion is an epithelial cell type-intrinsic response, or whether it involves other cell types, has not been addressed. In this study, we provide genetic evidence for a coordinated intestinal epithelial cell-intrinsic inflammasome response locus under Cre-inducible control (iNLRC4 mice, Fig. 1A, Fig. S1A, S1B). In these mice, Cre excises a floxed transcriptional STOP cassette that is upstream of allows identification of cells in which the STOP cassette has been excised. Crossing iNLRC4 mice to a or transgene resulted in mice in which NLRC4 and GFP are expressed selectively in Villin+ IECs or LysM+ hematopoietic cells (monocytes, macrophages, granulocytes), respectively. To activate NLRC4 in the absence of potentially confounding toll-like receptor (TLR) stimulation SCH-1473759 that accompanies bacterial infections, we used FlaTox, a previously described reagent for delivery of the NAIP5 ligand flagellin to the cytosol of cells (Ballard et al., 1996; Kofoed and Vance, 2011; Moltke et al., 2012; Rauch et al., 2016; Zhao et al., 2011). As reported previously (Moltke et al., 2012), systemic NAIP5-NLRC4 activation by FlaTox treatment of wild-type mice caused Rabbit polyclonal to ACADM rapid hypothermia and vascular fluid loss, resulting in a marked increase in hematocrit, whereas locus after successful gene targeting with iNLRC4-IRES-GFP. Yellow triangles represent LoxP sites. (B, C) Mice were injected with 0.8g/g PA and 0.4g/g LFn-Fla and monitored for (B) body temperature and (C) hematocrit (30 minutes, n=3). (D, E) Mice were injected with 0.8g/g PA and 80ng/g LFn-Fla166 and (D) wet/dry ratio of intestinal content and (E) PGE2 amounts of intestinal tissue from mice treated for 30 minutes were determined (n=4C5). (F) Quantification of serum IL-18 using ELISA in mice treated as in B), 60 minute timepoint (n=3). (G) H&E staining of small intestinal tissue from mice treated as in B), 60 minutes post injection. Scale bar = 100m (H) SCH-1473759 CFU in cecum 18h after oral invasion To determine whether the epithelium-intrinsic inflammasome response was sufficient to protect against an invasive bacterial pathogen, we infected iNLRC4 mice with serovar Typhimurium (singly-deficient mice generated by CRISPR/Cas9; Fig. S4ACC). However, Caspase-1 and -11-deficient mice nevertheless exhibited kinetically delayed hypothermia and hemoconcentration after injection with FlaTox (Moltke SCH-1473759 et al., 2012). These effects were accompanied by significant epithelial cell expulsion (Fig. 3A). The intestines of in IECs, we generated inducible mice (iCasp1 mice, analogous to iNLRC4 mice, see Fig. S3ACD) and crossed these mice to the and transgenic lines. Upon FlaTox treatment, observations, when we treated intestinal organoids derived from ***p 0.001. Please see also Figure S3 Gasdermin D is required for IEC pyroptosis but not IEC expulsion Gasdermin D was recently identified as the main.