Myeloid OTULIN deficiency couples RIPK3-dependent cell death to Nlrp3 inflammasome activation and IL-1ß secretion.

Loss-of-function mutations in the deubiquitinase OTULIN result in an inflammatory pathology termed "OTULIN-related autoinflammatory syndrome" (ORAS). Genetic mouse models revealed essential roles for OTULIN in inflammatory and cell death signaling, but the mechanisms by which OTULIN deficiency connects cell death to inflammation remain unclear. Here, we identify OTULIN deficiency as a cellular condition that licenses RIPK3-mediated cell death in murine macrophages, leading to Nlrp3 inflammasome activation and subsequent IL-1ß secretion. OTULIN deficiency uncoupled Nlrp3 inflammasome activation from gasdermin D-mediated pyroptosis, instead allowing RIPK3-dependent cell death to act as an Nlrp3 inflammasome activator and mechanism for IL-1ß release. Accordingly, elevated serum IL-1ß levels in myeloid-specific OTULIN-deficient mice were diminished by deleting either Ripk3 or Nlrp3. These findings identify OTULIN as an inhibitor of RIPK3-mediated IL-1ß release in mice.

OTULIN protects the intestinal epithelium from apoptosis during inflammation and infection.

The intestinal epithelium is a single cell layer that is constantly renewed and acts as a physical barrier that separates intestinal microbiota from underlying tissues. In inflammatory bowel disease (IBD) in humans, as well as in experimental mouse models of IBD, this barrier is impaired, causing microbial infiltration and inflammation. Deficiency in OTU deubiquitinase with linear linkage specificity (OTULIN) causes OTULIN-related autoinflammatory syndrome (ORAS), a severe inflammatory pathology affecting multiple organs including the intestine. We show that mice with intestinal epithelial cell (IEC)-specific OTULIN deficiency exhibit increased susceptibility to experimental colitis and are highly sensitive to TNF toxicity, due to excessive apoptosis of OTULIN deficient IECs. OTULIN deficiency also increases intestinal pathology in mice genetically engineered to secrete excess TNF, confirming that chronic exposure to TNF promotes epithelial cell death and inflammation in OTULIN deficient mice. Mechanistically we demonstrate that upon TNF stimulation, OTULIN deficiency impairs TNF receptor complex I formation and LUBAC recruitment, and promotes the formation of the cytosolic complex II inducing epithelial cell death. Finally, we show that OTULIN deficiency in IECs increases susceptibility to Salmonella infection, further confirming the importance of OTULIN for intestinal barrier integrity. Together, these results identify OTULIN as a major anti-apoptotic protein in the intestinal epithelium and provide mechanistic insights into how OTULIN deficiency drives gastrointestinal inflammation in ORAS patients.

OTULIN maintains skin homeostasis by controlling keratinocyte death and stem cell identity.

OTULIN is a deubiquitinase that specifically cleaves linear ubiquitin chains. Here we demonstrate that the ablation of Otulin selectively in keratinocytes causes inflammatory skin lesions that develop into verrucous carcinomas. Genetic deletion of Tnfr1, knockin expression of kinase-inactive Ripk1 or keratinocyte-specific deletion of Fadd and Mlkl completely rescues mice with OTULIN deficiency from dermatitis and tumorigenesis, thereby identifying keratinocyte cell death as the driving force for inflammation. Single-cell RNA-sequencing comparing non-lesional and lesional skin reveals changes in epidermal stem cell identity in OTULIN-deficient keratinocytes prior to substantial immune cell infiltration. Keratinocytes lacking OTULIN display a type-1 interferon and IL-1ß response signature, and genetic or pharmacologic inhibition of these cytokines partially inhibits skin inflammation. Finally, expression of a hypomorphic mutant Otulin allele, previously shown to cause OTULIN-related autoinflammatory syndrome in humans, induces a similar inflammatory phenotype, thus supporting the importance of OTULIN for restraining skin inflammation and maintaining immune homeostasis.

OTULIN in NF-κB signaling, cell death, and disease.

Tight control of inflammatory signaling pathways is an absolute requirement to avoid chronic inflammation and disease. One of the proteins responsible for such control is OTU deubiquitinase with linear linkage specificity (OTULIN), the only mammalian deubiquitinating enzyme (DUB) exclusively hydrolyzing linear ubiquitin chains from proteins modified by the linear ubiquitin chain assembly complex (LUBAC) described thus far. Recent findings show that loss-of-function mutations in OTULIN underlie a severe early-onset human autoinflammatory disease and severe pathology in experimental mouse models. Here, we review the molecular and cellular mechanisms by which OTULIN controls inflammation and discuss the involvement of OTULIN in inflammatory disease development. We also highlight several newly identified roles for OTULIN, including a ubiquitin-independent function.

OTULIN Prevents Liver Inflammation and Hepatocellular Carcinoma by Inhibiting FADD- and RIPK1 Kinase-Mediated Hepatocyte Apoptosis.

Inflammatory signaling pathways are tightly regulated to avoid chronic inflammation and the development of disease. OTULIN is a deubiquitinating enzyme that controls inflammation by cleaving linear ubiquitin chains generated by the linear ubiquitin chain assembly complex. Here, we show that ablation of OTULIN in liver parenchymal cells in mice causes severe liver disease which is characterized by liver inflammation, hepatocyte apoptosis, and compensatory hepatocyte proliferation, leading to steatohepatitis, fibrosis, and hepatocellular carcinoma (HCC). Genetic ablation of Fas-associated death domain (FADD) completely rescues and knockin expression of kinase inactive receptor-interacting protein kinase 1 (RIPK1) significantly protects mice from developing liver disease, demonstrating that apoptosis of OTULIN-deficient hepatocytes triggers disease pathogenesis in this model. Finally, we demonstrate that type I interferons contribute to disease in hepatocyte-specific OTULIN-deficient mice. Our study reveals the critical importance of OTULIN in protecting hepatocytes from death, thereby preventing the development of chronic liver inflammation and HCC.