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.

Objectives An imbalance between neutrophil extracellular trap (NET) formation and degradation has been described in systemic lupus erythematosus (SLE), potentially contributing to autoantigen externalisation, type I interferon synthesis and endothelial damage. We have demonstrated that peptidylarginine deiminase (PAD) inhibition reduces NET formation and protects against lupus-related vascular damage in the New Zealand Mixed model of lupus. However, another strategy for inhibiting NETs—knockout of NOX2—accelerates lupus in a different murine model, MRL/lpr. Here, we test the effects of PAD inhibition on MRL/lpr mice in order to clarify whether some NET inhibitory pathways may be consistently therapeutic across models of SLE. Methods NET formation and autoantibodies to NETs were characterised in lupus-prone MRL/lpr mice. MRL/lpr mice were also treated with two different PAD inhibitors, Cl-amidine and the newly described BB-Cl-amidine. NET formation, endothelial function, interferon signature, nephritis and skin disease were examined in treated mice. Results Neutrophils from MRL/lpr mice demonstrate accelerated NET formation compared with controls. MRL/lpr mice also form autoantibodies to NETs and have evidence of endothelial dysfunction. PAD inhibition markedly improves endothelial function, while downregulating the expression of type I interferon-regulated genes. PAD inhibition also reduces proteinuria and immune complex deposition in the kidneys, while protecting against skin disease. Conclusions PAD inhibition reduces NET formation, while protecting against lupus-related damage to the vasculature, kidneys and skin in various lupus models. The strategy by which NETs are inhibited will have to be carefully considered if human studies are to be undertaken.

Neutrophil extracellular traps (NETs) represent an important defense mechanism against microorganisms. Clearance of NETs is impaired in a subset of patients with systemic lupus erythematosus (SLE), while NETosis is increased in neutrophils and, particularly, in low-density granulocytes derived from lupus patients. NETs are toxic to the endothelium, expose immunostimulatory molecules, activate plasmacytoid dendritic cells and may participate in organ damage through incompletely characterized pathways. In order to better understand the role of NETs in fostering dysregulated inflammation, we examined inflammasome activation in response to NETs or to LL-37, an antibacterial protein externalized on the NETs. Both NETs and LL-37 activate caspase-1, the central enzyme of the inflammasome, in both human and murine macrophages, resulting in release of active IL-1β and IL-18. LL-37 activation of the NLRP3 inflammasome utilizes P2×7 receptor-mediated potassium efflux. NET and LL-37-mediated activation of the inflammasome is enhanced in macrophages derived from lupus patients. In turn, IL-18 is able to stimulate NETosis in human neutrophils. These results suggest that enhanced formation of NETs in lupus patients can lead to increased inflammasome activation in adjacent macrophages. This leads to release of inflammatory cytokines which further stimulate NETosis, resulting in a feed-forward inflammatory loop that could potentially lead to disease flares and/or organ damage.