Neutrophil elastase escapes azurophilic granules, translocates to the nucleus, and degrades histones to promote chromatin decondensation necessary for NET formation.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which patients develop autoantibodies to DNA, histones, and often to neutrophil proteins. These form immune complexes that are pathogenic and may cause lupus nephritis. In SLE patients, infections can initiate flares and are a major cause of mortality. Neutrophils respond to infections and release extracellular traps (NETs), which are antimicrobial and are made of DNA, histones, and neutrophil proteins. The timely removal of NETs may be crucial for tissue homeostasis to avoid presentation of self-antigens. We tested the hypothesis that SLE patients cannot clear NETs, contributing to the pathogenesis of lupus nephritis. Here we show that serum endonuclease DNase1 is essential for disassembly of NETs. Interestingly, a subset of SLE patients’ sera degraded NETs poorly. Two mechanisms caused this impaired NET degradation: ( i ) the presence of DNase1 inhibitors or ( ii ) anti-NET antibodies prevented DNase1 access to NETs. Impairment of DNase1 function and failure to dismantle NETs correlated with kidney involvement. Hence, identification of SLE patients who cannot dismantle NETs might be a useful indicator of renal involvement. Moreover, NETs might represent a therapeutic target in SLE.

The signaling mechanisms leading to the formation of neutrophil extracellular traps (NETs), relevant in infections, sepsis and autoimmune diseases, are poorly understood. Neutrophils are not amenable to studies with conventional genetic techniques. Using a new chemical genetic analysis we show that the Raf-MEK-ERK pathway is involved in NET formation through activation of NADPH oxidase and upregulation of antiapoptotic proteins. We identify potential targets for drugs addressing NET-associated diseases.

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IntroductionActivated neutrophils kill microbes intracellularly after phagocytosis and by extracellular mechanisms, including neutrophil extracellular traps (NETs), which are composed of chromatin decorated with granular proteins. 1 NETs bind bacteria 1 and fungi 2 and expose antimicrobial molecules. Generation of NETs requires reactive oxygen species produced by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. 3 Chronic granulomatous disease (CGD) is caused by mutations in genes encoding NADPH oxidase subunits. CGD patients do not produce reactive oxygen species, kill microbes poorly, and are susceptible to recurrent life-threatening infections. 4 Aspergillus spp infections cause pneumonia and disseminated disease and are the leading cause of death in these patients. [4][5][6] It is unclear how Aspergillus infections are controlled in healthy persons. [7][8][9][10][11][12][13] In CGD patients, these infections are frequently refractory to antifungal therapy, treatment with interferon-␥, or granulocyte transfusions. 5 Here we show that the recently discovered NADPH oxidase-dependent microbicidal pathway through NETs 1-3 is efficient against Aspergillus nidulans conidia and hyphae in vitro and that restoration of NET formation by GT of X-CGD aided clearing severe invasive A nidulans infection in vivo. Methods Gene therapyWe treated an 8.5-year-old boy with X-linked gp91 phox -deficient CGD and therapy refractory A nidulans lung infection with a monocistronic long terminal repeat-driven gamma-retroviral SF71gp91 phox vector (see supplemental data, available on the Blood website; see the Supplemental Materials link at the top of the online article). The protocol for the patient's treatment was approved by the ethics review board of the University Children's Hospital Zurich and the Swiss Expert Committee for Bio-Safety, after written informed consent from his parents in accordance with the Declaration of Helsinki. For follow-up monitoring, gp91 phox expression was measured by fluorescence-activated cell sorter (FACS) on peripheral neutrophils after 30 minutes of staining at room temperature with 10 g/mL gp91 phox -fluorescein isothiocyanate (FITC) antibody (Anti-Flavocytochrome b558, clone 7D5, MBL). NADPH oxidase activity was measured by standard dihydrorhodamine and nitroblue tetrazolium tests (supplemental materials). Bone marrow colony assays and determination of proviral gp91 phox sequences in genomic DNA were performed as described. 14 NET inductionNET formation was visualized as described (supplemental materials) and quantified after stimulation of 5 ϫ 10 4 neutrophils for 3 hours with 40 nM phorbol 12-myristate 13-acetate (PMA) and staining the NET-DNA with 1 M Sytox green (Invitrogen) in a black 96-well plate (BD Biosciences). The plates were read in a fluorescence microplate reader (Victor, 3 PerkinElmer Life and Analytical Sciences) with a filter setting of 485 nm/535 nm (excitation/emission). NET antifungal activityThe A nidulans strain used was isolated from bronchoalveolar lavage fluid ...