Neutrophil extracellular traps (NETs) are implicated in autoimmunity but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes, inducers of NETosis, require mitochondrial ROS for maximal NET stimulation. During this process, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro and, when injected into mice, stimulates type-I interferon (IFN) signaling through a pathway dependent on the DNA sensor, STING. Mitochondrial ROS is also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus (SLE). This was also observed in individuals with chronic granulomatous disease (CGD), which lack NADPH-oxidase activity, but still develop autoimmunity and type I-IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type-I IFN responses in a mouse model of lupus. These findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.

An abnormal neutrophil subset has been identified in the PBMC fractions from lupus patients. We have proposed that these “low density granulocytes” (LDGs) play an important role in lupus pathogenesis by damaging endothelial cells and synthesizing increased levels of proinflammatory cytokines and type I interferons. To directly establish LDGs as a distinct neutrophil subset, their gene array profiles were compared to those of autologous normal density neutrophils and control neutrophils. LDGs significantly overexpress mRNA of various immunostimulatory bactericidal proteins and alarmins, relative to lupus and control neutrophils. In contrast, gene profiles of lupus normal density neutrophils do not differ from those of controls. LDGs have heightened capacity to synthesize extracellular traps (NETs) which display increased externalization of bactericidal, immunostimulatory proteins and autoantigens, including LL-37, IL-17, and double-stranded DNA (dsDNA). Through NETosis, LDGs have increased capacity to kill endothelial cells and to stimulate IFN-α synthesis by pDCs. Affected skin and kidneys from lupus patients are infiltrated by netting neutrophils, which expose LL-37 and ds-DNA. Tissue NETosis is associated with increased anti-dsDNA in sera. These results expand the potential pathogenic roles of aberrant lupus neutrophils and suggest that dysregulation of NET formation and its subsequent responses may play a prominent deleterious role.

The early events leading to the development of rheumatoid arthritis (RA) remain unclear but formation of autoantibodies to citrullinated antigens (ACPA) is considered a key pathogenic phenomenon. Neutrophils isolated from patients with various autoimmune diseases display enhanced extracellular trap formation (NETs), a phenomenon that externalizes autoantigens and immunostimulatory molecules. We investigated whether aberrant NETosis occurs in RA, determined its triggers and examined its deleterious inflammatory consequences. Enhanced NETosis was observed in circulating and synovial fluid RA neutrophils, compared to neutrophils from healthy controls and from patients with osteoarthritis. Further, netting neutrophils infiltrated RA synovial tissue, rheumatoid nodules and skin. NETosis correlated with ACPA presence and levels and with systemic inflammatory markers. RA sera and immunoglobulin fractions from RA patients with high levels of ACPA and/or rheumatoid factor significantly enhanced NETosis, and the NETs induced by these autoantibodies displayed distinct protein content. During NETosis, neutrophils externalized citrullinated autoantigens implicated in RA pathogenesis, whereas anti-citrullinated vimentin antibodies potently induced NET formation. The inflammatory cytokines IL-17A and TNF-α induced NETosis in RA neutrophils. In turn, NETs significantly augmented inflammatory responses in RA and OA synovial fibroblasts, including induction of IL-6, IL-8, chemokines and adhesion molecules. These observations implicate accelerated NETosis in RA pathogenesis, through externalization of citrullinated autoantigens and immunostimulatory molecules that may promote aberrant adaptive and innate immune responses in the joint and in the periphery, and perpetuate pathogenic mechanisms in this disease.

BACKGROUNDAdult-onset inflammatory syndromes often manifest with overlapping clinical features. Variants in ubiquitin-related genes, previously implicated in autoinflammatory disease, may define new disorders. METHODSWe analyzed peripheral-blood exome sequence data independent of clinical phenotype and inheritance pattern to identify deleterious mutations in ubiquitin-related genes. Sanger sequencing, immunoblotting, immunohistochemical testing, flow cytometry, and transcriptome and cytokine profiling were performed. CRISPR-Cas9edited zebrafish were used as an in vivo model to assess gene function. RESULTSWe identified 25 men with somatic mutations affecting methionine-41 (p.Met41) in UBA1, the major E1 enzyme that initiates ubiquitylation. (The gene UBA1 lies on the X chromosome.) In such patients, an often fatal, treatment-refractory inflammatory syndrome develops in late adulthood, with fevers, cytopenias, characteristic vacuoles in myeloid and erythroid precursor cells, dysplastic bone marrow, neutrophilic cutaneous and pulmonary inflammation, chondritis, and vasculitis. Most of these 25 patients met clinical criteria for an inflammatory syndrome (relapsing polychondritis, Sweet's syndrome, polyarteritis nodosa, or giant-cell arteritis) or a hematologic condition (myelodysplastic syndrome or multiple myeloma) or both. Mutations were found in more than half the hematopoietic stem cells, including peripheral-blood myeloid cells but not lymphocytes or fibroblasts. Mutations affecting p.Met41 resulted in loss of the canonical cytoplasmic isoform of UBA1 and in expression of a novel, catalytically impaired isoform initiated at p.Met67. Mutant peripheral-blood cells showed decreased ubiquitylation and activated innate immune pathways. Knockout of the cytoplasmic UBA1 isoform homologue in zebrafish caused systemic inflammation. CONCLUSIONSUsing a genotype-driven approach, we identified a disorder that connects seemingly unrelated adult-onset inflammatory syndromes. We named this disorder the VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome.