Nucleotide-binding oligomerization domain (NOD) signaling defects and cell death susceptibility cannot be uncoupled in X-linked inhibitor of apoptosis (XIAP)-driven inflammatory disease

Abstract

The X-linked inhibitor of apoptosis (XIAP) protein has been identified as a key genetic driver of two distinct inflammatory disorders, X-linked lymphoproliferative syndrome 2 (XLP-2) and very-early-onset inflammatory bowel disease (VEO-IBD). Molecularly, the role of XIAP mutations in the pathogenesis of these disorders is unclear. Recent work has consistently shown XIAP to be critical for signaling downstream of the Crohn's disease susceptibility protein nucleotide-binding oligomerization domain-containing 2 (NOD2); however, the reported effects of XLP-2 and VEO-IBD XIAP mutations on cell death have been inconsistent. In this manuscript, we describe a CRISPR-mediated genetic system for cells of the myeloid lineage in which XIAP alleles can be replaced with disease-associated XIAP variants expressed at endogenous levels to simultaneously study inflammation-related cell death and NOD2 signaling. We show that, consistent with previous studies, NOD2 signaling is critically dependent on the BIR2 domain of XIAP. We further used this system to reconcile the aforementioned inconsistent XIAP cell death data to show that XLP-2 and VEO-IBD XIAP mutations that exhibit a loss-of-function NOD2 phenotype also lower the threshold for inflammatory cell death. Last, we identified and studied three novel patient XIAP mutations and used this system to characterize NOD2 and cell death phenotypes driven by XIAP. The results of this work support the role of XIAP in mediating NOD2 signaling while reconciling the role of XLP-2 and VEO-IBD XIAP mutations in inflammatory cell death and provide a set of tools and framework to rapidly test newly discovered XIAP variants.

Small molecule inhibitors reveal an indispensable scaffolding role of RIPK 2 in NOD 2 signaling

“…Kinase inhibitors antagonize RIPK2-XIAP interaction to inhibit NOD2 signaling XIAP and cIAPs ubiquitinate RIPK2 following NOD2 stimulation (Damgaard et al, 2012), and the interaction between the XIAP BIR2 domain and the RIPK2 kinase domain is needed for NOD2 signaling Chirieleison et al, 2017). Small molecule IAP antagonists such as Compound A (CpA) can block NOD2 signaling by interfering with the interaction between XIAP and RIPK2 and thereby prevent the ubiquitination of RIPK2 by XIAP ( Fig 5A; Krieg et al, 2009;Damgaard et al, 2013;Hrdinka et al, 2016).…”

Live-cell visualization of gasdermin D-driven pyroptotic cell death

“…To study GSDMD in macrophages, Gsdmd was genetically deleted in immortalized bone marrow-derived macrophages (iBMDM) through the use of CRISPR-Cas9 as previously described ( Fig. 1A) (16,18). The p30 fragment of murine GSDMD (AA 1-276) has been shown to be cytotoxic via formation of the pyroptotic pore, whereas the cleaved p20 fragment (AA 277-487) is thought to be located diffusely in the cytosol following caspase cleavage (12)(13)(14)(15).…”

NOD2 and inflammation: current insights

“… 34 , 35 Interestingly, loss of Pellino 3 decreases RIP2 ubiquitination and activation of NF-κB and mitogen-activated protein kinases (MAPKs), while genetic X-linked inhibitor of apoptosis protein loss causes a blunted NOD2 response. 34 − 36 Tripartite Motif Containing 27 negatively regulates NOD2 by ubiquitination, while the E3-ubiquitin ligase ZNRF4, degrading RIP2, is a negative regulator of NOD2-induced NF-κB activity as well. 37 , 38 Differently, the ovarian tumor family deubiquitinase OTULIN was shown to dampen NOD2 signaling by increasing NF-κB transcription.…”