Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that affects over one million people in the United States. SLE is characterized by the presence of anti-nuclear antibodies (ANA) directed against naked DNA and entire nucleosomes. It is thought that the resulting immune complexes accumulate in vessel walls, glomeruli and joints and cause a hypersensitivity reaction type III, which manifests as glomerulonephritis, arthritis and general vasculitis. The aetiology of SLE is unknown, but several studies suggest that increased liberation or disturbed clearance of nuclear DNA-protein complexes after cell death may initiate and propagate the disease. Consequently, Dnase1, which is the major nuclease present in serum, urine and secreta, may be responsible for the removal of DNA from nuclear antigens at sites of high cell turnover and thus for the prevention of SLE (refs 7-11). To test this hypothesis, we have generated Dnase1-deficient mice by gene targeting. We report here that these animals show the classical symptoms of SLE, namely the presence of ANA, the deposition of immune complexes in glomeruli and full-blown glomerulonephritis in a Dnase1-dose-dependent manner. Moreover, in agreement with earlier reports, we found Dnase1 activities in serum to be lower in SLE patients than in normal subjects. Our findings suggest that lack or reduction of Dnase1 is a critical factor in the initiation of human SLE.

Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.

Key Points• NET formation is required for neutrophil recruitment during sterile inflammation.• Platelet-induced NET formation requires stimulation of neutrophils by platelet chemokines and outside-in signaling via the integrin Mac-1.There is emerging evidence that neutrophil extracellular traps (NETs) play important roles in inflammatory processes. Here we report that neutrophils have to be simultaneously activated by integrin-mediated outside-in-and G-protein-coupled receptor (GPCR) signaling to induce NET formation in acute lung injury (ALI), which is associated with a high mortality rate in critically ill patients. NETs consist of decondensed chromatin decorated with granular and cytosolic proteins and they can trap extracellular pathogens. The prerequisite for NET formation is the activation of neutrophils and the release of their DNA. In a neutrophil-and platelet-dependent mouse model of ventilator-induced lung injury (VILI), NETs were found in the lung microvasculature, and circulating NET components increased in the plasma. In this model, blocking integrin-mediated outside-in or either GPCR-signaling or heteromerization of platelet chemokines decreased NET formation and lung injury. Targeting NET components by DNAse1 application or neutrophil elastase-deficient mice protected mice from ALI, whereas DNase1 2/2 /Trap1 m/m mice had an aggravated ALI, suggesting that NETs directly influence the severity of ALI. These data suggest that NETs form in the lungs during VILI, contribute to the disease process, and thus may be a promising new direction for the treatment of ALI. (Blood. 2014; 123(16):2573-2584

DNase1 is regarded as the major serum nuclease; however, a systematic investigation into the presence of additional serum nuclease activities is lacking. We have demonstrated directly that serum contains DNase1‐like 3 (DNase1l3) in addition to DNase1 by an improved denaturing SDS‐PAGE zymography method and anti‐murine DNase1l3 immunoblotting. Using DNA degradation assays, we compared the activities of recombinant murine DNase1 and DNase1l3 (rmDNase1, rmDNase1l3) with the serum of wild‐type and DNase1 knockout mice. Serum and rmDNase1 degrade chromatin effectively only in cooperation with serine proteases, such as plasmin or thrombin, which remove DNA‐bound proteins. This can be mimicked by the addition of heparin, which displaces histones from chromatin. In contrast, serum and rmDNase1l3 degrade chromatin without proteolytic help and are directly inhibited by heparin and proteolysis by plasmin. In previous studies, serum DNase1l3 escaped detection because of its sensitivity to proteolysis by plasmin after activation of the plasminogen system in the DNA degradation assays. In contrast, DNase1 is resistant to plasmin, probably as a result of its di‐N‐glycosylation, which is lacking in DNase1l3. Our data demonstrate that secreted rmDNase1 and murine parotid DNase1 are mixtures of three different di‐N‐glycosylated molecules containing two high‐mannose, two complex N‐glycans or one high‐mannose and one complex N‐glycan moiety. In summary, serum contains two nucleases, DNase1 and DNase1l3, which may substitute or cooperate with each other during DNA degradation, providing effective clearance after exposure or release from dying cells.

Objective. Dnase1-deficient mice with the 129 ؋ C57BL/6 genetic background develop symptoms of systemic lupus erythematosus, such as high titers of antinuclear autoantibodies directed against nucleosomes. In this study we analyzed a potential molecular pathomechanism leading to this autoimmunity, by exploring the influence of extracellular Dnase1 present in serum on the breakdown of chromatin in necrotic cells in vitro.Methods. Human breast adenocarcinoma cells (MCF-7) and other cell lines were subjected to necrosis induced by hydrogen peroxide, streptolysin O, or freezethawing. Subsequently, the influence of sera from Dnase1-deficient and wild-type mice as well as the influence of purified enzymes present in the culture medium on the process of necrotic chromatin breakdown was investigated.Results. Necrotic chromatin breakdown resembled apoptotic DNA laddering and was catalyzed by serum Dnase1 in conjunction with plasmin. During necrosis, Dnase1 and plasminogen penetrated the cell and accumulated in the cytoplasm and nucleus. Plasminogen bound to the cytoskeleton and nuclear structures, was activated to plasmin by either tissue-type or urokinase-type plasminogen activator, and degraded histone H1, thereby facilitating internucleosomal DNA cleavage by Dnase1.Conclusion. Our results suggest that serum Dnase1 in cooperation with the plasminogen system guarantees a fast and effective breakdown of chromatin during necrosis by the combined cleavage of DNA as well as of DNA binding proteins. The failure of such a clearance mechanism might lead to antinuclear autoimmunity similar to that observed in the Dnase1-deficient mouse.

Cisplatin is commonly used for chemotherapy in a wide variety of tumors; however, its use is limited by kidney toxicity. Although the exact mechanism of cisplatin-induced nephrotoxicity is not understood, several studies showed that it is associated with DNA fragmentation induced by an unknown endonuclease. It was demonstrated previously that deoxyribonuclease I (DNase I) is a highly active renal endonuclease, and its silencing by antisense is cytoprotective against the in vitro hypoxia injury of kidney tubular epithelial cells. This study used recently developed DNase1 knockout (KO) mice to determine the role of this endonuclease in cisplatin-induced nephrotoxicity. The data showed that DNase I represents approximately 80% of the total endonuclease activity in the kidney and cultured primary renal tubular epithelial cells. In vitro, primary renal tubular epithelial cells isolated from KO animals were resistant to cisplatin (8 M) injury. DNase I KO mice were also markedly protected against the toxic injury induced by a single injection of cisplatin (20 mg/kg), by both functional (blood urea nitrogen and serum creatinine) and histologic criteria (tubular necrosis and in situ DNA fragmentation assessed by the terminal deoxynucleotidyl transferase nick end-labeling). These data provide direct evidence that DNase I is essential for kidney injury induced by cisplatin.

Deoxyribonuclease 1 (DNASE1, DNase I) and deoxyribonuclease 1-like 3 (DNASE1L3, DNase gamma, DNase Y, LS-DNase) are members of a DNASE1 protein family that is defined by similar biochemical properties such as Ca2+/Mg2+-dependency and an optimal pH of about 7.0 as well as by a high similarity in their nucleic acid and amino acid sequences. In the present study we describe the recombinant expression of rat Dnase1 and murine Dnase1l3 as fusion proteins tagged by their C-terminus to green fluorescent protein in NIH-3T3 fibroblasts and bovine lens epithelial cells. Both enzymes were translocated into the rough endoplasmic reticulum, transported along the entire secretory pathway and finally secreted into the cell culture medium. No nuclear occurrence of the nucleases was detectable. However, deletion of the N-terminal signal peptide of both nucleases resulted in a cytoplasmic and nuclear distribution of both fusion proteins. Dnase1 preferentially hydrolysed 'naked' plasmid DNA, whereas Dnase1l3 cleaved nuclear DNA with high activity. Dnase1l3 was able to cleave chromatin in an internucleosomal manner without proteolytic help. By contrast, Dnase1 was only able to achieve this cleavage pattern in the presence of proteases that hydrolysed chromatin-bound proteins. Detailed analysis of murine sera derived from Dnase1 knockout mice revealed that serum contains, besides the major serum nuclease Dnase1, an additional Dnase1l3-like nucleolytic activity, which, in co-operation with Dnase1, might help to suppress anti-DNA autoimmunity by degrading nuclear chromatin released from dying cells.

Summary Background Acute thrombotic microangiopathies (TMAs) are characterized by excessive microvascular thrombosis and are associated with markers of neutrophil extracellular traps (NETs) in plasma. NETs are composed of DNA fibers and promote thrombus formation through the activation of platelets and clotting factors. Objective The efficient removal of NETs may be required to prevent excessive thrombosis such as in TMAs. To test this hypothesis, we investigated whether TMAs are associated with a defect in the degradation of NETs. Methods and Results We show that NETs generated in vitro were efficiently degraded by plasma from healthy donors. However, NETs remained stable after exposure to plasma from TMA patients. The inability to degrade NETs was linked to a reduced DNase activity in TMA plasma. Plasma DNase1 was required for efficient NET degradation and TMA plasma showed decreased levels of this enzyme. Supplementation of TMA plasma with recombinant human DNase1 restored NET‐degradation activity. Conclusions Our data indicate that DNase1‐mediated degradation of NETs is impaired in patients with TMAs. The role of plasma DNases in thrombosis is, as of yet, poorly understood. Reduced plasma DNase1 activity may cause the persistence of pro‐thrombotic NETs and thus promote microvascular thrombosis in TMA patients.