Proteasome dysfunction can lead to autoinflammatory disease associated with elevated type I interferon (IFN-αβ) and NF-κB signaling; however, the innate immune pathway driving this is currently unknown. Here, we identified protein kinase R (PKR) as an innate immune sensor for proteotoxic stress. PKR activation was observed in cellular models of decreased proteasome function and in multiple cell types from patients with proteasome-associated autoinflammatory disease (PRAAS). Furthermore, genetic deletion or small-molecule inhibition of PKR in vitro ameliorated inflammation driven by proteasome deficiency. In vivo, proteasome inhibitor–induced inflammatory gene transcription was blunted in PKR-deficient mice compared with littermate controls. PKR also acted as a rheostat for proteotoxic stress by triggering phosphorylation of eIF2α, which can prevent the translation of new proteins to restore homeostasis. Although traditionally known as a sensor of RNA, under conditions of proteasome dysfunction, PKR sensed the cytoplasmic accumulation of a known interactor, interleukin-24 (IL-24). When misfolded IL-24 egress into the cytosol was blocked by inhibition of the endoplasmic reticulum–associated degradation pathway, PKR activation and subsequent inflammatory signaling were blunted. Cytokines such as IL-24 are normally secreted from cells; therefore, cytoplasmic accumulation of IL-24 represents an internal danger-associated molecular pattern. Thus, we have identified a mechanism by which proteotoxic stress is detected, causing inflammation observed in the disease PRAAS.

Biallelic loss of function mutations in the linear chain specific deubiquitinase (DUB) OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) result in OTULIN Related Autoinflammatory Syndrome (ORAS). To date all reported ORAS patients have had homozygous or compound heterozygous loss of function mutations, however we identified a patient with a monoallelic heterozygous mutation p.Cys129Ser. Consistent with the ORAS phenotype, we observed accumulation of linear ubiquitin chains, increased sensitivity to TNF induced cell death and dysregulation of inflammatory signalling in both patient cells and in vitro exogenous expression models. Levels of the mutant OTULIN protein were consistent with wild type OTULIN in patient cells and exogenous expression systems and maintained binding capacity to both LUBAC and linear ubiquitin chains. However, even in a heterozygous context this mutant DUB promoted the global accumulation of linear ubiquitin chains. Furthermore, it allowed accumulation of ubiquitin on the linear ubiquitin chain assembly complex (LUBAC). Altered ubiquitination of LUBAC leads to a dysregulation of NF-κB signalling and promotion of TNF induced cell death. By reporting the first dominant negative mutation driving ORAS this study expands our clinical understanding of Otulin mediated pathology.

Missense mutations in PLCG2 can cause autoinflammation with phospholipase C gamma 2-associated antibody deficiency and immune dysregulation (APLAID). Here, we generated a mouse model carrying an APLAID mutation (p.Ser707Tyr) and found that inflammatory infiltrates in the skin and lungs were only partially ameliorated by removing inflammasome function via the deletion of caspase-1. Also, deleting interleukin-6 or tumor necrosis factor did not fully prevent APLAID mutant mice from autoinflammation. Overall, these findings are in accordance with the poor response individuals with APLAID have to treatments that block interleukin-1, JAK1/2 or tumor necrosis factor. Cytokine analysis revealed increased granulocyte colony-stimulating factor (G-CSF) levels as the most distinct feature in mice and individuals with APLAID. Remarkably, treatment with a G-CSF antibody completely reversed established disease in APLAID mice. Furthermore, excessive myelopoiesis was normalized and lymphocyte numbers rebounded. APLAID mice were also fully rescued by bone marrow transplantation from healthy donors, associated with reduced G-CSF production, predominantly from non-hematopoietic cells. In summary, we identify APLAID as a G-CSF-driven autoinflammatory disease, for which targeted therapy is feasible.