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.
Highlights
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)
Its mutation leads to two major distinct clinical presentations: XLP-2, a disease characterized by life-threatening exaggerated cytokine responses to Epstein-Barr virus, and VEO-IBD, a severe, difficult-to-treat form of inflammatory bowel disease [17, 18, 62, 63]
Given that loss-of-function nucleotide-binding oligomerization domain-containing 2 (NOD2) polymorphisms predispose to the development of Crohn’s disease, it is attractive to attribute the totality of the role of XIAP in VEO-IBD to NOD2 dysfunction
Summary
The investigation of XIAP loss in innate immune signaling pathways has been somewhat limited by the reagents available. As seen in the NF-B luciferase assays, loss of XIAP blunted the NOD2-driven expression of immuneregulated genes, evidenced by significantly lower MDP-induced expression of IRG1 in dendritic cells lacking XIAP (Fig. 1c) Taken together, these cell lines recapitulate signaling defects at innate receptors known to require XIAP activity and provide a platform to dissect XIAP function. In contrast to the DC2.4s, the TNF- or LPSstimulated iBMDM cell death was exclusively due to apoptosis rather than necroptosis, as evidenced by increased cleaved caspase-3 but absent phosphorylated MLKL at each time point (Fig. 2, f and h) These results demonstrate that XIAP is a critical regulator of both NOD2 signaling activity as well as TNF- and LPS-driven cell death in both dendritic cells and macrophages
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