DNASE1L3 Research Articles

Targeting NETs using dual-active DNase1 variants.

Neutrophil Extracellular Traps (NETs) are key mediators of immunothrombotic mechanisms and defective clearance of NETs from the circulation underlies an array of thrombotic, inflammatory, infectious, and autoimmune diseases. Efficient NET degradation depends on the combined activity of two distinct DNases, DNase1 and DNase1-like 3 (DNase1L3) that preferentially digest double-stranded DNA (dsDNA) and chromatin, respectively. Here, we engineered a dual-active DNase with combined DNase1 and DNase1L3 activities and characterized the enzyme for its NET degrading potential in vitro. Furthermore, we produced a mouse model with transgenic expression of the dual-active DNase and analyzed body fluids of these animals for DNase1 and DNase 1L3 activities. We systematically substituted 20 amino acid stretches in DNase1 that were not conserved among DNase1 and DNase1L3 with homologous DNase1L3 sequences. We found that the ability of DNase1L3 to degrade chromatin is embedded into three discrete areas of the enzyme's core body, not the C-terminal domain as suggested by the state-of-the-art. Further, combined transfer of the aforementioned areas of DNase1L3 to DNase1 generated a dual-active DNase1 enzyme with additional chromatin degrading activity. The dual-active DNase1 mutant was superior to native DNase1 and DNase1L3 in degrading dsDNA and chromatin, respectively. Transgenic expression of the dual-active DNase1 mutant in hepatocytes of mice lacking endogenous DNases revealed that the engineered enzyme was stable in the circulation, released into serum and filtered to the bile but not into the urine. Therefore, the dual-active DNase1 mutant is a promising tool for neutralization of DNA and NETs with potential therapeutic applications for interference with thromboinflammatory disease states.

Breaking bad.

DNASE1 (D1) and DNASE1L3 (D1L3) synergistically reduce the severity of systemic infections caused by Staphylococcus aureus. In this issue of JEM, Lacey et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20221086) develop D1-/-, D1L3-/-, and D1-/-D1L3-/- mice to show that exogenous addition of the DNase formulation Dornase alfa can facilitate removal of biofilms.

Investigating Dnase1L3's role in inflammation and redesigning the enzyme to be used as a therapeutic.

Systemic lupus erythematosus (SLE) affects 1.5 million Americans. Lupus is a debilitating autoimmune disorder in which patients form antibodies that react to self-antigens. Anti-dsDNA antibodies bind to DNA, forming immune complexes (ICs) and deposit on organs. Over time accumulation of the ICs can lead to lupus nephritis, which will ultimately require a kidney transplant. Removing and breaking down the immune complexes from circulation and deposits would be beneficial in preventing further damage to organs. Some patients with SLE have a deficiency in Dnase1L3 (Dnase1-Like 3) enzyme caused by a pathogenic mutation in the Dnase1L3 gene. Dnase1L3 digests extracellular DNA in the peripheral circulation. In contrast to Dnase1, Dnase1L3 possesses the unique activity of digesting DNA complexed with other biological molecules in the form of microparticles and immune complexes; this activity is essential in preventing the creation of antibodies against self-antigens. Dnase1L3 degrades antigenic DNA complexes and is a potential therapeutic for SLE. Dnase1L3 has also been implicated in having a role in releasing inflammatory cytokines, but specific inhibitors block this action; it is still unclear what Dnase1L3 function is inhibited by these inhibitors. We have developed a unique bacterial expression system for Dnase1L3 and solved the X-ray crystal structure of the core enzyme without the C-terminal domain. I plan to determine the structure of the full-length enzyme bound to DNA to better understand the mechanism of action of Dnase1L3. To minimize any potential off-target side effects from a Dnase1L3 therapeutic, I am also investigating the inhibitors to elucidate the Dnase1L3 role that is blocked in the inflammatory pathway. The long-term goal of this project is to produce the most effective therapeutic for lupus nephritis.

Neutralizing Anti‒DNase 1 and ‒DNase 1L3 Antibodies Impair Neutrophil Extracellular Traps Degradation in Hidradenitis Suppurativa

Hidradenitis suppurativa (HS) is a debilitating inflammatory skin disorder characterized by abscess-like nodules and boils resulting in fistulas and tissue scarring. We previously reported evidence of an autoimmune signature in HS, characterized by enhanced neutrophil extracellular trap (NET) infiltration in HS skin lesions and dysregulation of the adaptive immune system characterized by the presence of autoantibodies. Timely removal of NETs is critical for tissue homeostasis to prevent a dysregulated generation of modified autoantigens and tissue damage. DNases 1 and 1L3 play important roles in proper NET removal. We tested the hypothesis that NETs in patients with HS are not effectively cleared owing to the presence of antibodies against DNase 1 and DNase 1L3. We report that HS serum poorly degraded NETs. Addition of exogenous DNase 1 restored NET degradation capabilities in a subset of HS samples. DNase 1 activity was significantly decreased in HS sera. Anti‒DNase 1 and ‒DNase 1L3 antibodies were detected in serum samples and skin lesions from patients with HS. Purified IgGs from HS decreased DNase 1 activity and NET degradation. Taken together, this identification of neutralizing antibodies against nucleases in HS expands the understanding of the pathogenesis of this disease to support an autoimmune mechanism in its underlying pathogenesis.

IRF5 risk variants contribute to pre-symptomatic SLE by enhancing the levels of circulating NET antigens

Abstract SLE is a complex multifactorial autoimmune disease characterized by high levels of autoantibodies that impact many organs. Neutrophil extracellular traps (NETs) are a potential source of antigen leading to the production of autoantibodies, as antibodies against NET components are detected in SLE patients. Higher level of NETs and decreased clearance of NETs are associated with SLE and other autoimmune diseases. Neutrophils play an key role in pediatric lupus and NETs from pediatric lupus patients activate plasmacytoid dendritic cells (pDCs) to produce high levels of IFN-α. Polymorphisms within and around the IRF5 gene associate with risk of developing SLE. We previously reported that healthy donor neutrophils from IRF5 homozygous risk carriers underwent increased spontaneous NETosis, as compared to non-risk donors, that resulted in increased IFN-α production, plasma cell differentiation and autoantibody production. Whether the observed increase in neutrophil-mediated ETosis is a systemic signature of IRF5-driven pre-symptomatic SLE is not known, nor is it known whether these IRF5 risk carriers have defects in NET clearance. We thus developed and optimized new assays to detect small quantities of NET remnants in plasma samples using a combined MPO-CitH3 antibody cocktail followed by detection of DNA by ELISA. Further, we designed a novel immunofluorescence technique to visualize and quantify plasma NETs in healthy donor risk and non-risk carriers, as compared to pediatric and adult SLE patients. In addition, plasma DNase 1 and DNase 1L3 levels were measured. This study will report on the mechanisms by which circulating NETs are increased in pediatric and adult SLE, as well as the contribution of IRF5 genetic risk to NET clearance. Supported by Lupus Research Alliance Department of Defense CDMRP LRP W81XWH-18-1-0674

A sputum 6-gene signature predicts airway inflammation endotypes and exacerbation frequency in chronic obstructive pulmonary disease.

Aim: To validate a sputum 6-gene signature (6GS), comprising of CLC, CPA, DNASE1L3, IL-1B, ALPL and CXCR2, for identifying different endotypes in chronic obstructive pulmonary disease. Methodology & results: Sputum cell CLC, CPA3 and DNASE1L3 gene expression correlated with eosinophil percentage, while IL-1B, ALPL and CXCR2 correlated with neutrophil percentage. Hierarchical cluster analyses of IL-1B, ALPL and CXCR2, and CLC, CPA3 and DNASE1L3, identified patient groups that differed in their sputum neutrophil and eosinophil levels, respectively. Multiple logistic regressions demonstrated that the 6GS could distinguish between eosinophilHigh and eosinophilLow patients, as well as neutrophilHigh and neutrophilLow, and could also predict exacerbation history. Conclusion: The 6GS may have applications in clinical practice or for stratifying patients for clinical trials.

Plasma DNA Profile Associated with DNASE1L3 Gene Mutations: Clinical Observations, Relationships to Nuclease Substrate Preference, and In Vivo Correction

SummaryPlasma DNA fragmentomics is an emerging area in cell-free DNA diagnostics and research. In murine models, it has been shown that the extracellular DNase, DNASE1L3, plays a role in the fragmentation of plasma DNA. In humans, DNASE1L3 deficiency causes familial monogenic systemic lupus erythematosus with childhood onset and anti-dsDNA reactivity. In this study, we found that human patients with DNASE1L3 disease-associated gene variations showed aberrations in size and a reduction of a “CC” end motif of plasma DNA. Furthermore, we demonstrated that DNA from DNASE1L3-digested cell nuclei showed a median length of 153 bp with CC motif frequencies resembling plasma DNA from healthy individuals. Adeno-associated virus-based transduction of Dnase1l3 into Dnase1l3-deficient mice restored the end motif profiles to those seen in the plasma DNA of wild-type mice. Our findings demonstrate that DNASE1L3 is an important player in the fragmentation of plasma DNA, which appears to act in a cell-extrinsic manner to regulate plasma DNA size and motif frequency.

Association of the DNASE1L3 rs35677470 polymorphism with systemic lupus erythematosus, rheumatoid arthritis and systemic sclerosis: Structural biological insights.

Although genome-wide association studies (GWAS) have identified hundreds of autoimmune disease-associated loci, much of the genetics underlying these diseases remains unknown. In an attempt to identify potential causal variants, previous studies have determined that the rs35677470 missense variant of the Deoxyribonuclease I-like 3 (DNASE1L3) gene was associated with the development of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and systemic sclerosis (SSc). DNase1L3 is a member of the human DNase I family, representing a nuclease that cleaves double-stranded DNA during apoptosis and serving a role in the development of autoimmune diseases. The present study aimed to determine the role of the rs35677470 variant at the DNASE1L3 gene leading to the R206C mutation in SLE, RA and SSc. The underlying mechanism potentially affecting protein structure loss of function was also assessed. DNASE1L3 evolution was investigated to define conservation elements in the protein sequence. Additionally, 3D homology modeling and in silico mutagenesis was performed to localize the polymorphism under investigation. Evolutionary analysis revealed heavily conserved sequence elements among species, indicating structural/functional importance. In silico mutagenesis and 3D protein structural analysis also demonstrated the potentially varied impact of the DNASE1L3 (rs35677470) single nucleotide polymorphism (SNP), providing an explanation for its effect on the R206C variant. Structural analysis demonstrated that the rs35677470 SNP encodes a non-conservative amino acid variation, R206C, which disrupted the conserved electrostatic network holding secondary protein structure elements in position. Specifically, the R206 to E170 interaction forming part of a salt bridge network stabilizing two α-helices was interrupted, thereby affecting the molecular architecture. Previous studies on the effect of this SNP in Caucasian populations demonstrated lower DNAse1L3 activity levels, which is consistent with the current results. The present study comprehensively evaluated the shared autoimmune locus of DNASE1L3 (rs35677470), which produced an inactive form of DNaseIL3. Furthermore, structural analysis explained the potential role of the produced mutation by modifying the placement of structural elements and consequently introducing disorder in protein folding, affecting biological function.

Detection and characterization of jagged ends of double-stranded DNA in plasma.

Cell-free DNA in plasma has been used for noninvasive prenatal testing and cancer liquid biopsy. The physical properties of cell-free DNA fragments in plasma, such as fragment sizes and ends, have attracted much recent interest, leading to the emerging field of cell-free DNA fragmentomics. However, one aspect of plasma DNA fragmentomics as to whether double-stranded plasma molecules might carry single-stranded ends, termed a jagged end in this study, remains underexplored. We have developed two approaches for investigating the presence of jagged ends in a plasma DNA pool. These approaches utilized DNA end repair to introduce differential methylation signals between the original sequence and the jagged ends, depending on whether unmethylated or methylated cytosines were used in the DNA end-repair procedure. The majority of plasma DNA molecules (87.8%) were found to bear jagged ends. The jaggedness varied according to plasma DNA fragment sizes and appeared to be in association with nucleosomal patterns. In the plasma of pregnant women, the jaggedness of fetal DNA molecules was higher than that of the maternal counterparts. The jaggedness of plasma DNA correlated with the fetal DNA fraction. Similarly, in the plasma of cancer patients, tumor-derived DNA molecules in patients with hepatocellular carcinoma showed an elevated jaggedness compared with nontumoral DNA. In mouse models, knocking out of the Dnase1 gene reduced jaggedness, whereas knocking out of the Dnase1l3 gene enhanced jaggedness. Hence, plasma DNA jagged ends represent an intrinsic property of plasma DNA and provide a link between nuclease activities and the fragmentation of plasma DNA.

Abstract IA01: Plasma DNA-based molecular diagnostics: Fragments, circles and beyond

Abstract My group has a longstanding interest in the diagnostic applications of cell-free DNA in plasma. For example, we have pioneered noninvasive prenatal testing (NIPT) and the use of plasma DNA for cancer screening. We believe that understanding the fundamental biology of circulating DNA is essential to further advance the field. In this regard, we have been particularly interested in the fragmentation patterns of cell-free DNA in plasma. We have shown that cell-free fetal DNA molecules in the plasma of pregnant women are shorter than those of maternal origin. The differential size profiles between circulating fetal and maternal DNA have enabled the use of plasma DNA size as an independent metric for NIPT. In the area of cancer liquid biopsy, we have used circulating Epstein-Barr virus (EBV) DNA as an example of tumor-derived DNA in plasma. Hence, circulating EBV DNA molecules are also short fragments of DNA. In another example, we have used hepatocellular carcinoma as a model system and have found that circulating tumor-derived DNA molecules have a shorter size profile than those of nontumoral origin. We are exploring enzymes that might be involved in the fragmentation of plasma DNA. We have shown that mice in which both copies of the Dnase1l3 gene have been knocked out have a larger size profile of circulating DNA. We have also developed a plasma DNA end motif metric to measure the effect of disruption of this nuclease system. In addition to the fragmentation of linear DNA in plasma, my group is also interested in exploring the presence of nonlinear DNA in plasma. Hence, we have used mitochondrial DNA (mtDNA) as a model system as mtDNA exists as a circular molecule inside mitochondria. Interestingly, we have observed the existence of both linear and circular mtDNA in plasma. Using liver and bone marrow transplantation models, we have shown that the circular form is predominantly of hematopoietic origin while the linear form is predominantly from the liver. Plasma mtDNA topologics thus holds information on the origin of circulating DNA. These studies therefore demonstrate some of the exciting findings that can be revealed through the study of plasma DNA fragmentomics and topologics. We believe that such new biologic understandings will drive new diagnostic applications. Citation Format: Yuk Ming Dennis Lo. Plasma DNA-based molecular diagnostics: Fragments, circles and beyond [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr IA01.

Loss-of-function variant in DNASE1L3 causes a familial form of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a complex autoimmune disease that causes substantial morbidity. As is typical for many other multifactorial disorders, much of the heritability of SLE remains unknown. We identified a rare autosomal recessive form of SLE, in which autozygome analysis revealed a null mutation in the DNASE1L3 gene. The DNASE1L3-related SLE we describe was always pediatric in onset and correlated with a high frequency of lupus nephritis. Our findings confirm the critical role of impaired clearance of degraded DNA in SLE pathogenesis.

Global genetic analysis of all single nucleotide polymorphisms in exons of the human deoxyribonuclease I‐like 3 gene and their effect on its catalytic activity

Deoxyribonucleases (DNases) have been suggested to be implicated in the pathophysiology of autoimmune diseases. In the DNASE1L3 gene encoding human DNase I-like 3 (DNase 1L3), a member of the DNase I family, only two non-synonymous (R178 H and R206C) single nucleotide polymorphisms (SNPs) have been examined [Ueki et al., Clin. Chim. Acta 2009, 407, 20-24]. Three other non-synonymous (G82R, K96N, and I243M) and four synonymous (S17S, T84T, R92R, and A181A) SNPs, in addition to R206C and R178H, have been identified in DNASE1L3. We investigated the distribution of all these SNPs in exons of the gene in eight Asian, three African, and three Caucasian populations worldwide using newly devised genotyping methods. SNP T84T showed polymorphism in all the populations, and R92R was polymorphic in the three African and three Caucasian populations; R206C was distributed only in Caucasian populations. In contrast, no minor allele was found in five SNPs (S17S, G82R, K96N, A181A, and I243M) in DNASE1L3. Generally, the DNase 1L3 gene shows relatively low genetic diversity with regard to exonic SNPs. When the effect of amino acid/nucleotide substitutions resulting from the SNPs on DNase 1L3 activity was examined, none of the synonymous SNPs had any effect on the DNase 1L3 activity, whereas among non-synonymous SNPs, SNP G82R diminished the activity of the enzyme, being similar to R206C. These findings permit us to assume that, although only R206 exhibits polymorphisms in a Caucasian-specific manner, at least SNPs G82R and R206C in DNASE1L3 might be potential risk factors for autoimmune disease.