A genome‐wide screen identifies IRF2 as a key regulator of caspase‐4 in human cells

Abstract

Caspase‐4, the cytosolic LPS sensor, and gasdermin D, its downstream effector, constitute the non‐canonical inflammasome, which drives inflammatory responses during Gram‐negative bacterial infections. It remains unclear whether other proteins regulate cytosolic LPS sensing, particularly in human cells. Here, we conduct a genome‐wide CRISPR/Cas9 screen in a human monocyte cell line to identify genes controlling cytosolic LPS‐mediated pyroptosis. We find that the transcription factor, IRF2, is required for pyroptosis following cytosolic LPS delivery and functions by directly regulating caspase‐4 levels in human monocytes and iPSC‐derived monocytes. CASP4, GSDMD, and IRF2 are the only genes identified with high significance in this screen highlighting the simplicity of the non‐canonical inflammasome. Upon IFN‐γ priming, IRF1 induction compensates IRF2 deficiency, leading to robust caspase‐4 expression. Deficiency in IRF2 results in dampened inflammasome responses upon infection with Gram‐negative bacteria. This study emphasizes the central role of IRF family members as specific regulators of the non‐canonical inflammasome.

Pyrin dephosphorylation is sufficient to trigger inflammasome activation in familial Mediterranean fever patients

“…UCN‐01 is a PKC superfamily inhibitor and thereby not a specific inhibitor of PKN1/2. We were unable to invalidate PKN2 by CRISPR/Cas9 despite multiple assays and more than 50 other human genes successfully invalidated in the meantime in the same cellular system (Benaoudia et al , ). PKN2 ‐edited clones were recovered but contained small deletions/insertions not affecting the open‐reading frames (see one example in ), suggesting that PKN2 is necessary for cell growth/survival of U937 cells.…”

“…Four clones are shown. Clones 3 and 6 were selected. BAbsolute transcript level of the indicated genes was quantified by RNAseq in U937 cells (Benaoudia et al , ) and expressed as count per million (cpm). PYCARD encodes ASC and is shown as a reference. C PKN2 transcript levels were assessed in U937 cells at 24 h post‐electroporation with the indicated non‐targeting (NT) siRNA or three different siRNAs targeting PKN2 . D–FU937 cells with the indicated Pyrin variant were treated (+Dox, plain lines) or not (No Dox, dotted lines) with doxycycline (Dox) at 24 h post‐electroporation with the indicated siRNA.…”

IRF 1 and IRF 2 regulate the non‐canonical inflammasome

“…Understanding the mechanism and regulation of this system is of great interest, given its central role in mouse models of bacterial septic shock. In this issue of EMBO Reports, Benaoudia and colleagues sought to discover extra players in the human non-canonical inflammasome using a CRISPR library screen; the only strongly positive hit apart from the known components caspase-4 and gasdermin D was interferon regulatory factor-2 (IRF2) [1]. IRF2 was found to be a transcriptional activator of caspase-4, and in its absence, induction of IRF1 could substitute to maintain caspase-4 expression.EMBO Reports (2019) 20: e48891…”

Vying for the control of inflammasomes: The cytosolic frontier of enteric bacterial pathogen–host interactions

“…The ‘priming’ signals (signal 1) activate the transcription and/or post‐translational regulation of inflammasome‐associated genes/proteins and are best understood for the activation of the NLRP3 inflammasome in macrophages. These include: (i) the transcription of pro‐IL‐1β, (ii) the transcriptional and translational licensing of NLRP3 via toll‐like receptors (TLRs) and NF‐κB (Bauernfeind et al, ; Fernandes‐Alnemri et al, ; Lin et al, ), (iii) interferon‐dependent up‐regulation of murine caspase‐11 (Rathinam et al, ; Benaoudia et al, ) and mouse and human GBPs (Kim et al, ) and (iv) IRF2‐driven expression of human caspase‐4 (Benaoudia et al, ) and mouse GSDMD (Kayagaki et al, ). Importantly, bacterial ligands such as cell wall components and nucleic acids can serve as signal 1, and pathogen‐associated virulence factors and/or activities, as discussed below, serve as a second signal specific to the NLR/ALR/PYRIN inflammasome sensors.…”

“…The 'priming' signals (signal 1) activate the transcription and/or posttranslational regulation of inflammasome-associated genes/proteins and are best understood for the activation of the NLRP3 inflammasome in macrophages. These include: (i) the transcription of pro-IL-1β, (ii) the transcriptional and translational licensing of NLRP3 via toll-like receptors (TLRs) and NF-κB Fernandes-Alnemri et al, 2013;Lin et al, 2014), (iii) interferondependent up-regulation of murine caspase-11 Benaoudia et al, 2019) and mouse and human GBPs and (iv) IRF2-driven expression of human caspase-4 (Benaoudia et al, 2019) and mouse GSDMD (Kayagaki et al, 2019).…”