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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.

The prolonged persistence of extracellular chromatin and DNA is a salient feature of diseases like cystic fibrosis, systemic lupus erythematosus and COVID-19 associated microangiopathy. Since deoxyribonuclease I (DNase1) is a major endonuclease involved in DNA-related waste disposal, recombinant DNase1 is an important therapeutic biologic. Recently we described the production of recombinant murine DNase1 (rmDNase1) in Pichia pastoris by employing the α-mating factor prepro signal peptide (αMF-SP) a method, which we now applied to express recombinant human DNASE1 (rhDNASE1). In addition to an impaired cleavage of the αMF pro-peptide, which we also detected previously for mDNase1, expression of hDNASE1 resulted in a 70–80 times lower yield although both orthologues share a high structural and functional homology. Using mDNase1 expression as a guideline, we were able to increase the yield of hDNASE1 fourfold by optimizing parameters like nutrients, cultivation temperature, methanol supply, and codon usage. In addition, post-translational import into the rough endoplasmic reticulum (rER) was changed to co-translational import by employing the signal peptide (SP) of the α-subunit of the Oligosaccharyltransferase complex (Ost1) from Saccharomyces cerevisiae . These improvements resulted in the purification of ~ 8 mg pure mature rmDNase1 and ~ 0.4 mg rhDNASE1 per Liter expression medium of a culture with a cell density of OD 600 = 40 in 24 hours. As a main cause for the expression difference, we assume varying folding abilities to reach a native conformation, which induce an elevated unproductive unfolded protein response within the rER during hDNASE1 expression. Concerning functionality, rhDNASE1 expressed in P. pastoris is comparable to Pulmozyme®, i.e. rhDNASE1 produced in Chinese hamster ovary (CHO) cells by Roche - Genentech. With respect to the biochemical effectivity, rmDNase1 is superior to rhDNASE1 due to its higher specific activity in the presence of Ca 2 + /Mg 2 + and the lower inhibition by monomeric actin.

Soluble nucleases of the deoxyribonuclease 1 (DNase1) family facilitate DNA and chromatin disposal (chromatinolysis) during certain forms of cell differentiation and death and participate in the suppression of anti-nuclear autoimmunity as well as thrombotic microangiopathies caused by aggregated neutrophil extracellular traps. Since a systematic and direct comparison of the specific activities and properties of the secretory DNase1 family members is still missing, we expressed and purified recombinant murine DNase1 (rmDNase1), DNase1-like 2 (rmDNase1L2) and DNase1-like 3 (rmDNase1L3) using Pichia pastoris . Employing different strategies for optimizing culture and purification conditions, we achieved yields of pure protein between ~3 mg/l (rmDNase1L2 and rmDNase1L3) and ~9 mg/l (rmDNase1) expression medium. Furthermore, we established a procedure for post-expressional maturation of pre-mature DNase still bound to an unprocessed tri-N-glycosylated pro-peptide of the yeast α-mating factor. We analyzed glycosylation profiles and determined specific DNase activities by the hyperchromicity assay. Additionally, we evaluated substrate specificities under various conditions at equimolar DNase isoform concentrations by lambda DNA and chromatin digestion assays in the presence and absence of heparin and monomeric skeletal muscle α-actin. Our results suggest that due to its biochemical properties mDNase1L2 can be regarded as an evolutionary intermediate isoform of mDNase1 and mDNase1L3. Consequently, our data show that the secretory DNase1 family members complement each other to achieve optimal DNA degradation and chromatinolysis under a broad spectrum of biological conditions.

The stratum corneum of the epidermis constitutes the mammalian skin barrier to the environment. It is formed by cornification of keratinocytes, a process which involves the removal of nuclear DNA. Here, we investigated the mechanism of cornification-associated DNA degradation by generating mouse models deficient of candidate DNA-degrading enzymes and characterizing their epidermal phenotypes. In contrast to Dnase1l2 −/− mice and keratinocyte-specific DNase2 knockout mice ( Dnase2 Δep ), Dnase1l2 −/− Dnase2 Δep mice aberrantly retained nuclear DNA in the stratum corneum, a phenomenon commonly referred to as parakeratosis. The DNA within DNase1L2/DNase2-deficient corneocytes was partially degraded in a DNase1-independent manner. Isolation of corneocytes, i.e. the cornified cell components of the stratum corneum, and labelling of DNA demonstrated that corneocytes of Dnase1l2 −/− Dnase2 Δep mice contained DNA in a nucleus-shaped compartment that also contained nucleosomal histones but lacked the nuclear intermediate filament protein lamin A/C. Parakeratosis was not associated with altered corneocyte resistance to mechanical stress, changes in transepidermal water loss, or inflammatory infiltrates in Dnase1l2 −/− Dnase2 Δep mice. The results of this study suggest that cornification of epidermal keratinocytes depends on the cooperation of DNase1L2 and DNase2 and indicate that parakeratosis per se does not suffice to cause skin pathologies.

The murine basic helix-loop-helix transcription (bHLH) factor mouse atonal homolog 6 (Math6) is expressed in numerous organs and supposed to be involved in several developmental processes. However, so far neither all aspects nor the molecular mechanisms of Math6 function have been explored exhaustively. To analyze the in vivo function of Math6 in detail, we generated a constitutive knockout (KO) mouse ( Math6 −/− ) and performed an initial histological and molecular biological investigation of its main phenotype. Pregnant Math6 −/− females suffer from a disturbed early placental development leading to the death of the majority of embryos independent of the embryonic Math6 genotype. A few placentas and fetuses survive the severe uterine hemorrhagic events at late mid-gestation (E13.5) and subsequently develop regularly. However, these fetuses could not be born due to obstructions within the gravid uterus, which hinder the birth process. Characterization of the endogenous spatiotemporal Math6 expression during placenta development reveals that Math6 is essential for an ordered decidualization and an important regulator of the maternal-fetal endocrine crosstalk regulating endometrial trophoblast invasion and differentiation. The strongly disturbed vascularization observed in the maternal placenta appears as an additional consequence of the altered endocrine status and as the main cause for the general hemorrhagic crisis.

Increased concentrations of circulating chromatin, especially oligo-nucleosomes, are observed in sepsis, cancer and some inflammatory autoimmune diseases like systemic lupus erythematosus (SLE). In SLE, circulating nucleosomes mainly result from increased apoptosis and decreased clearance of apoptotic cells. Once released, nucleosomes behave both as an autoantigen and as a damage-associated molecular pattern (DAMP) by activating several immune cells, especially pro-inflammatory cells. Deoxyribonuclease 1 (DNase1) is a major serum nuclease whose activity is decreased in mouse and human lupus. Likewise, the mitochondrial chaperone tumor necrosis factor (TNF) receptor-associated protein-1 (Trap1) protects against oxidative stress, which is increased in SLE. Here, using wild type, DNase1-deficient and DNase1/Trap1-deficient mice, we demonstrate that DNase1 is a major serum nuclease involved in chromatin degradation, especially when the plasminogen system is activated. In vitro degradation assays show that chromatin digestion is strongly impaired in serum from DNase1/Trap1-deficient mice as compared to wild type mice. In vivo , after injection of purified chromatin, clearance of circulating chromatin is delayed in DNase1/Trap1-deficient mice in comparison to wild type mice. Since defective chromatin clearance may lead to chromatin deposition in tissues and subsequent immune cell activation, spleen cells were stimulated in vitro with chromatin. Splenocytes were activated by chromatin, as shown by interleukin (IL)-12 secretion and CD69 up-regulation. Moreover, cell activation was exacerbated when Trap1 is deficient. Importantly, we also show that cytokines involved in lupus pathogenesis down-regulate Trap1 expression in splenocytes. Therefore, combined low activities of both DNase1 and Trap1 lead to an impaired degradation of chromatin in vitro , delayed chromatin clearance in vivo and enhanced activation of immune cells. This situation may be encountered especially, but not exclusively, in SLE by the negative action of cytokines on Trap1 expression.

The basic helix-loop-helix transcription factor Math6 was shown to have important regulatory functions during many developmental events. However, a systematic description of Math6 expression during mouse embryonic development is up to now still lacking. We carried out this study to show Math6 expression at different stages of mouse embryonic development aiming to provide a wide insight into the regulatory functions during the mouse organogenesis. Using immunohistochemistry, we could show that Math6 expression is activated in the inner cell mass at the blastocyst stage and in the neural tube as well as somatic and splanchnic mesoderm at stage E8.5. At stages E8.5 and E10.5, Math6 transcripts were detected in the myotome, neural tube, pharyngeal arches, foregut and heart. At stages E11.5 and E12.5, Math6 transcripts were accumulated in the developing brain, heart, limb buds and liver. The heterozygous transgenic mouse embryos carrying EGFP–Cre under the Math6 promoter were used to analyze Math6 expression at later stages by means of immunohistochemistry against EGFP protein. EGFP was observed in the neural tube, heart, lung, skeletal muscle, skin, cartilage, trachea and aorta. We have observed Math6 expression in various organs at early and late stages of mouse development, which illustrates the involvement of Math6 in multiple developmental events.