Apoptotic extracellular vesicle formation via local phosphatidylserine exposure drives efficient cell extrusion.

Cell extrusion is a universal mode of cell removal from tissues, and it plays an important role in regulating cell numbers and eliminating unwanted cells. However, the underlying mechanisms of cell delamination from the cell layer are unclear. Here, we report a conserved execution mechanism of apoptotic cell extrusion. We found extracellular vesicle (EV) formation in extruding mammalian and Drosophila cells at a site opposite to the extrusion direction. Lipid-scramblase-mediated local exposure of phosphatidylserine is responsible for EV formation and is crucial for executing cell extrusion. Inhibition of this process disrupts prompt cell delamination and tissue homeostasis. Although the EV has hallmarks of an apoptotic body, its formation is governed by the mechanism of microvesicle formation. Experimental and mathematical modeling analysis illustrated that EV formation promotes neighboring cells' invasion. This study showed that membrane dynamics play a crucial role in cell exit by connecting the actions of the extruding cell and neighboring cells.

ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis.

The intestinal epithelium continually self-renews and can rapidly regenerate after damage. Dysregulation of intestinal epithelial homeostasis leads to severe inflammatory bowel disease. Additionally, aberrant signaling by the secreted protein angiopoietin-like protein 2 (ANGPTL2) causes chronic inflammation in a variety of diseases. However, little is known about the physiologic role of ANGPTL2 in normal tissue homeostasis and during wound repair following injury. Here, we assessed ANGPTL2 function in intestinal physiology and disease in vivo Although intestinal development proceeded normally in Angptl2-deficient mice, expression levels of the intestinal stem cell (ISC) marker gene Lgr5 decreased, which was associated with decreased transcriptional activity of ß-catenin in Angptl2-deficient mice. Epithelial regeneration after injury was significantly impaired in Angptl2-deficient relative to wild-type mice. ANGPTL2 was expressed and functioned within the mesenchymal compartment cells known as intestinal subepithelial myofibroblasts (ISEMFs). ANGPTL2 derived from ISEMFs maintained the intestinal stem cell niche by modulating levels of competing signaling between bone morphogenetic protein (BMP) and ß-catenin. These results support the importance of ANGPTL2 in the stem cell niche in regulating stemness and epithelial wound healing in the intestine.

DNase II-dependent DNA digestion is required for DNA sensing by TLR9.

DNase II digests DNA in endolysosomes. In the absence of DNase II, undigested DNA activates cytoplasmic DNA-sensing pathways. Little is known, however, about the role of DNase II in endolysosomal DNA sensing by TLR9. Here we show that DNase II is required for TLR9. We test two types of TLR9 ligands, CpG-A and CpG-B, and show that only CpG-A response is impaired in DNase II-deficient dendritic cells (DCs). Enzymatically inactive DNase II mutants cannot rescue CpG-A responses. DNase II cleaves CpG-A from 20-mer to 11-12-mer. The 3'11-mer CpG-A fragment activates DNase II-deficient DCs. CpG-A shows higher co-localization with LAMP-2(+) lysosomes than CpG-B and induces DNase II localization in LAMP-2(+) lysosomes. Moreover, we demonstrate that DNase II is required for TLR9 activation by bacterial genomic DNA. Taken together, these results demonstrate that TLR9 responds to DNA fragments generated by DNase II.

DNA degradation and its defects.

DNA is one of the most essential molecules in organisms, containing all the information necessary for organisms to live. It replicates and provides a mechanism for heredity and evolution. Various events cause the degradation of DNA into nucleotides. DNA also has a darker side that has only recently been recognized; DNA that is not properly degraded causes various diseases. In this review, we discuss four deoxyribonucleases that function in the nucleus, cytosol, and lysosomes, and how undigested DNA causes such diseases as cancer, cataract, and autoinflammation. Studies on the biochemical and physiological functions of deoxyribonucleases should continue to increase our understanding of cellular functions and human diseases.

Autoinflammation by endogenous DNA.

In various mammalian developmental processes such as programmed cell death, erythropoiesis, and lens-cell differentiation, chromosomal DNA is degraded into nucleotides by a set of specific nucleases. If this process does not proceed smoothly, the undigested DNA causes various problems. For example, when chromosomal DNA is not degraded in the lens cells, cataracts form. In other cases, undigested DNA in macrophages activates the innate immune system, like a DNA virus, and causes strong inflammation, resulting in anemia, arthritis, and lymphopenia. Here, we discuss when, where, and how DNA is degraded to maintain mammalian homeostasis.

Cytokine-dependent but acquired immunity-independent arthritis caused by DNA escaped from degradation.

DNase II digests the chromosomal DNA in macrophages after apoptotic cells and nuclei from erythroid precursors are engulfed. The DNase II-null mice develop a polyarthritis that resembles rheumatoid arthritis. Here, we showed that when bone marrow cells from the DNase II-deficient mice were transferred to the wild-type mice, they developed arthritis. A deficiency of Rag2 or a lack of lymphocytes accelerated arthritis of the DNase II-null mice, suggesting that the DNase II(-/-) macrophages were responsible for triggering arthritis, and their lymphocytes worked protectively. A high level of TNFα, IL-1ß, and IL-6 was found in the affected joints of the DNase II-null mice, suggesting an inflammatory-skewed cytokine storm was established in the joints. A lack of TNFα, IL-1ß, or IL-6 gene blocked the expression of the other cytokine genes as well and inhibited the development of arthritis. Neutralization of TNFα, IL-1ß, or IL-6 had a therapeutic effect on the developed arthritis of the DNase II-null mice, indicating that the cytokine storm was essential for the maintenance of arthritis in the DNase II-deficient mice. Methotrexate, an antimetabolite that is often used to treat patients with rheumatoid arthritis, had a therapeutic effect with the DNase II-null mice. These properties of arthritis in the DNase II-null mice were similar to those found in human systemic-onset juvenile idiopathic arthritis or Still's disease, indicating that the DNase II-null mice are a good animal model of this type of arthritis.

Interferon-induced TRAIL-independent cell death in DNase II-/- embryos.

The chromosomal DNA of apoptotic cells and the nuclear DNA expelled from erythroid precursors is cleaved by DNase II in lysosomes after the cells or nuclei are engulfed by macrophages. DNase II(-/-) embryos suffer from lethal anemia due to IFN-beta produced in the macrophages carrying undigested DNA. Here, we show that Type I IFN induced a caspase-dependent cell death in human epithelial cells that were transformed to express a high level of IFN type I receptor. During this death process, a set of genes was strongly activated, one of which encoded TRAIL, a death ligand. A high level of TRAIL mRNA was also found in the fetal liver of the lethally anemic DNase II(-/-) embryos, and a lack of IFN type I receptor in the DNase II(-/-) IFN-IR(-/-) embryos blocked the expression of TRAIL mRNA. However, a null mutation in TRAIL did not rescue the lethal anemia of the DNase II(-/-) embryos, indicating that TRAIL is dispensable for inducing the apoptosis of erythroid cells in DNase II(-/-) embryos, and therefore, that there is a TRAIL-independent mechanism for the IFN-induced apoptosis.

Protective targeting of high mobility group box chromosomal protein 1 in a spontaneous arthritis model.

OBJECTIVE: High mobility group box chromosomal protein 1 (HMGB-1) is a DNA binding nuclear protein that can be released from dying cells and activated myeloid cells. Extracellularly, HMGB-1 promotes inflammation. Clinical and experimental studies demonstrate that HMGB-1 is a pathogenic factor in chronic arthritis. Mice with combined gene deficiency for DNase II and IFNRI spontaneously develop chronic, destructive polyarthritis with many features shared with rheumatoid arthritis. DNase II is needed for macrophage degradation of engulfed DNA. The aim of this study was to evaluate a potential pathogenic role of HMGB-1 in this novel murine model. METHODS: The course of arthritis, assessed by clinical scoring and histology, was studied in DNase II(-/-) × IFNRI(-/-) mice, in comparison with heterozygous and wild-type mice. Synovial HMGB-1 expression was analyzed by immunohistochemistry. Serum levels of HMGB-1 were determined by Western immunoblotting and enzyme-linked immunosorbent assay (ELISA), and anti-HMGB-1 autoantibodies were detected by ELISA. Macrophage activation was studied by immunostaining for intracellular interleukin-1ß and HMGB-1. HMGB-1 was targeted with truncated HMGB-1-derived BoxA protein, acting as a competitive antagonist, with intraperitoneal injections every second day for 5 weeks. RESULTS: DNase II(-/-) × IFNRI(-/-) mice developed symmetric polyarthritis with strong aberrant cytosolic and extracellular HMGB-1 expression in synovial tissue, in contrast to that observed in control animals. Increased serum levels of HMGB-1 and HMGB-1 autoantibodies were recorded in DNase II(-/-) × IFNRI(-/-) mice, both prior to and during the establishment of disease. Systemic HMGB-1-specific blockade significantly ameliorated the clinical disease course, and a protective effect on joint destruction was demonstrated by histologic evaluation. CONCLUSION: HMGB-1 is involved in the pathogenesis of this spontaneous polyarthritis, and intervention with an HMGB-1 antagonist can mediate beneficial effects.

Autoimmunity and the clearance of dead cells.

To maintain organismal homeostasis, phagocytes engulf dead cells, which are recognized as dead by virtue of a characteristic "eat me" signal exposed on their surface. The dead cells are then transferred to lysosomes, where their cellular components are degraded for reuse. Inefficient engulfment of dead cells activates the immune system, causing disease such as systemic lupus erythematosus, and if the DNA of the dead cells is not properly degraded, the innate immune response becomes activated, leading to severe anemia and chronic arthritis. Here, we discuss how the endogenous components of dead cells activate the immune system through both extracellular and intracellular pathways.

IFN regulatory factor (IRF) 3/7-dependent and -independent gene induction by mammalian DNA that escapes degradation.

DNase II in macrophages cleaves the DNA of engulfed apoptotic cells and of nuclei expelled from erythroid precursor cells. Macrophages in DNase II-deficient mice accumulate undigested DNA and constitutively produce IFN-beta as well as TNF-alpha. The IFN-beta causes severe anemia in the DNase II(-/-) embryos, which die prenatally. On the other hand, when the DNase II gene is inactivated postnatally, mice develop polyarthritis owing to the TNF-alpha produced by macrophages. Here, we showed that the IFN-beta gene activation in DNase II(-/-) mice is dependent on IFN regulatory factor (IRF) 3 and 7. Accordingly, DNase II(-/-)IRF3(-/-)IRF7(-/-) mice do not suffer from anemia, but they still produce TNF-alpha, and age-dependently develop chronic polyarthritis. A microarray analysis of the gene expression in the fetal liver revealed a set of genes that is induced in DNase II(-/-) mice in an IRF3/IRF7-dependent manner, and another set that is induced independent of these factors. These results indicate that the mammalian chromosomal DNA that accumulates in macrophages due to inefficient degradation activates genes in both IRF3/IRF7-dependent and -independent manners.

Nucleases in programmed cell death.

DNA degradation is one of the hallmarks of programmed cell death, or apoptosis. Recent analyses of this process revealed that apoptotic DNA degradation is mediated by two independent mechanisms. First, the caspase-activated DNase (CAD) cell autonomously cleaves DNA into nucleosomal units in dying cells. Then, after the apoptotic cells are engulfed by macrophages, the fragmented DNA is further degraded by DNase II in the lysosomes of the macrophages. This chapter describes assay procedures for CAD and DNase II. It includes biochemical methods for quantifying DNase activity and cell culture systems to follow cell-autonomous and noncell-autonomous DNA degradation. These techniques are useful for studying DNases that are involved in programmed cell death and for following the engulfment of apoptotic cells by phagocytes.

Degradation of nuclear DNA by DNase II-like acid DNase in cortical fiber cells of mouse eye lens.

The eye lens is composed of fiber cells that differentiate from epithelial cells on its anterior surface. In concert with this differentiation, a set of proteins essential for lens function is synthesized, and the cellular organelles are degraded. DNase II-like acid DNase, also called DNase IIbeta, is specifically expressed in the lens, and degrades the DNA in the lens fiber cells. Here we report that DNase II-like acid DNase is synthesized as a precursor with a signal sequence, and is localized to lysosomes. DNase II-like acid DNase mRNA was found in cortical fiber cells but not epithelial cells, indicating that its expression is induced during the differentiation of epithelial cells into fiber cells. Immunohistochemical and immunocytochemical analyses indicated that DNase II-like acid DNase was colocalized with Lamp-1 in the lysosomes of fiber cells in a relatively narrow region bordering the organelle-free zone, and was often found in degenerating nuclei. A comparison by microarray analysis of the gene expression profiles between epithelial and cortical fiber cells of young mouse lens indicated that some genes for lysosomal enzymes (cathepsins and lipases) were strongly expressed in the fiber cells. These results suggest that the lysosomal system plays a role in the degradation of cellular organelles during lens cell differentiation.

Chronic polyarthritis caused by mammalian DNA that escapes from degradation in macrophages.

A large amount of chromosomal DNA is degraded during programmed cell death and definitive erythropoiesis. DNase II is an enzyme that digests the chromosomal DNA of apoptotic cells and nuclei expelled from erythroid precursor cells after macrophages have engulfed them. Here we show that DNase II-/-IFN-IR-/- mice and mice with an induced deletion of the DNase II gene develop a chronic polyarthritis resembling human rheumatoid arthritis. A set of cytokine genes was strongly activated in the affected joints of these mice, and their serum contained high levels of anti-cyclic citrullinated peptide antibody, rheumatoid factor and matrix metalloproteinase-3. Early in the pathogenesis, expression of the gene encoding tumour necrosis factor (TNF)-alpha was upregulated in the bone marrow, and administration of anti-TNF-alpha antibody prevented the development of arthritis. These results indicate that if macrophages cannot degrade mammalian DNA from erythroid precursors and apoptotic cells, they produce TNF-alpha, which activates synovial cells to produce various cytokines, leading to the development of chronic polyarthritis.

DNase II and the Chk2 DNA damage pathway form a genetic barrier blocking replication of horizontally transferred DNA.

We have previously shown that DNA from dying tumor cells may be transferred to living cells via the uptake of apoptotic bodies and may contribute to tumor progression. DNA encoding H-ras(V12) and c-myc oncogenes may be transferred to the nucleus of the phagocyte but will only integrate and propagate in p53- and p21-deficient mouse embryonic fibroblasts, whereas normal cells are resistant to transformation. Here, we show that this protective mechanism (activation of p53 and p21 after uptake of apoptotic bodies) is dependent on DNA fragmentation, where inhibition of the caspase-activated DNase in the apoptotic cells, in conjunction with genetic ablation of lysosomal DNase II in the phagocytes, completely blocks p53 activation and consequently allows DNA replication of transferred DNA. We, therefore, suggest that there is a causal relationship between DNA degradation during apoptosis and p53 activation. In addition, we could further show that Chk2-/- cells were capable of replicating the hyg(R) gene taken up from engulfed apoptotic cells, suggesting involvement of the DNA damage response. These data show that the phagocytosing cell is sensing the degraded DNA within the apoptotic cell, hence preventing these genes from being replicated, probably through activation of the DNA damage response. We, therefore, hypothesize that DNase II together with the Chk2, p53, and p21 pathway form a genetic barrier blocking the replication of potentially harmful DNA introduced via apoptotic bodies, thereby preventing transformation and malignant development.

Toll-like receptor-independent gene induction program activated by mammalian DNA escaped from apoptotic DNA degradation.

Deoxyribonuclease (DNase) II in macrophages cleaves the DNA of engulfed apoptotic cells and of nuclei expelled from erythroid precursor cells. DNase II-deficient mouse embryos accumulate undigested DNA in macrophages, and die in feto because of the activation of the interferon beta (IFNbeta) gene. Here, we found that the F4/80-positive macrophages in DNase II(-/-) fetal liver specifically produce a set of cytokines such as IFNbeta, TNFalpha, and CXCL10. Whereas, IFN-inducible genes (2'5'-oligo(A) synthetase, IRF7, and ISG15) were expressed not only in macrophages but also in other F4/80-negative cells. When DNase II(-/-) macrophages or embryonal fibroblasts engulfed apoptotic cells, they expressed the IFNbeta and CXCL10 genes. The ablation of Toll-like receptor (TLR) 3 and 9, or their adaptor molecules (MyD88 and TRIF), had no effect on the lethality of the DNase II(-/-) mice. These results indicate that there is a TLR-independent sensing mechanism to activate the innate immunity for the endogenous DNA escaping lysosomal degradation.

Phosphatidylserine-dependent engulfment by macrophages of nuclei from erythroid precursor cells.

Definitive erythropoiesis usually occurs in the bone marrow or fetal liver, where erythroblasts are associated with a central macrophage in anatomical units called 'blood islands'. Late in erythropoiesis, nuclei are expelled from the erythroid precursor cells and engulfed by the macrophages in the blood island. Here we show that the nuclei are engulfed by macrophages only after they are disconnected from reticulocytes, and that phosphatidylserine, which is often used as an 'eat me' signal for apoptotic cells, is also used for the engulfment of nuclei expelled from erythroblasts. We investigated the mechanism behind the enucleation and engulfment processes by isolating late-stage erythroblasts from the spleens of phlebotomized mice. When these erythroblasts were cultured, the nuclei protruded spontaneously from the erythroblasts. A weak physical force could disconnect the nuclei from the reticulocytes. The released nuclei contained an undetectable level of ATP, and quickly exposed phosphatidylserine on their surface. Fetal liver macrophages efficiently engulfed the nuclei; masking the phosphatidylserine on the nuclei with the dominant-negative form of milk-fat-globule EGF8 (MFG-E8) prevented this engulfment.

Lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested DNA.

The livers of DNase II-deficient mouse embryos contain many macrophages carrying undigested DNA, and the embryos die in utero. Here we report that erythroid precursor cells underwent apoptosis in the livers of DNase II-deficient embryos and that in the liver, interferon-beta mRNA was expressed by the resident macrophages. When the DNase II-deficient mice were crossed with mice deficient in type I interferon receptor, the resultant 'double-mutant' mice were born healthy. The double-mutant embryos expressed interferon-beta mRNA, but the expression of a subset of the interferon-responsive genes dysregulated in DNase II-deficient embryos was restored to normal. These results indicate that the inability to degrade DNA derived from erythroid precursors results in interferon-beta production that induces expression of a specific set of interferon-responsive genes associated with embryonic lethality in DNase II-deficient mice.

Nuclear cataract caused by a lack of DNA degradation in the mouse eye lens.

The eye lens is composed of fibre cells, which develop from the epithelial cells on the anterior surface of the lens. Differentiation into a lens fibre cell is accompanied by changes in cell shape, the expression of crystallins and the degradation of cellular organelles. The loss of organelles is believed to ensure the transparency of the lens, but the molecular mechanism behind this process is not known. Here we show that DLAD ('DNase II-like acid DNase', also called DNase IIbeta) is expressed in human and murine lens cells, and that mice deficient in the DLAD gene are incapable of degrading DNA during lens cell differentiation--the undigested DNA accumulates in the fibre cells. The DLAD-/- mice develop cataracts of the nucleus lentis, and their response to light on electroretinograms is severely reduced. These results indicate that DLAD is responsible for the degradation of nuclear DNA during lens cell differentiation, and that if DNA is left undigested in the lens, it causes cataracts of the nucleus lentis, blocking the light path.

Impaired thymic development in mouse embryos deficient in apoptotic DNA degradation.

Apoptosis is often accompanied by the degradation of chromosomal DNA. Caspase-activated DNase (CAD) is an endonuclease that is activated in dying cells, whereas DNase II is present in the lysosomes of macrophages. Here, we show that CAD(-/-) thymocytes did not undergo apoptotic DNA degradation. But, when apoptotic cells were phagocytosed by macrophages, their DNA was degraded by DNase II. The thymus of DNase II(-/-)CAD(-/-) embryos contained many foci carrying undigested DNA and the cellularity was severely reduced due to a block in T cell development. The interferon-beta gene was strongly up-regulated in the thymus of DNase II(-/-)CAD(-/-) embryos, suggesting that when the DNA of apoptotic cells is left undigested, it can activate innate immunity leading to defects in thymic development.