The role of DNASE1L3 in systemic lupus erythematosus: from pathogenesis to clinical implications

Abstract

Systemic Lupus Erythematosus (SLE) is a chronic inflammatory autoimmune disease characterized by multi-organ damage, a complex interplay of immune dysregulation, and antibodies against nuclear macromolecules like DNA. Among these, anti-dsDNA antibodies are not only crucial diagnostic markers but also active participants in disease pathogenesis, forming immune complexes that deposit in various tissues and trigger inflammation. Emerging research has highlighted the critical role of Deoxyribonuclease 1-Like 3 (DNASE1L3), a secreted endonuclease, in maintaining homeostasis of cell-free DNA (cfDNA). Deficiencies or dysfunction of DNASE1L3, whether due to genetic mutations or neutralizing autoantibodies, lead to impaired clearance of cfDNA derived from apoptotic cells, microparticles, and Neutrophil Extracellular Traps (NETs). This accumulation of cfDNA plays an important role in SLE. This review explores the characteristics of DNASE1L3, the current status of research on the mechanisms through which its defects contribute to SLE pathogenesis, and its promising potential in the clinical diagnosis, monitoring, and treatment of this complex disease.

Introduction

Systemic Lupus Erythematosus is a chronic inflammatory disease characterized by multi-organ damage and autoimmune responses. Its diagnosis heavily relies on specific biomarkers, especially highly specific antinuclear antibodies (ANAs) binding to DNA, RNA, and other nucleic acid complexes [1–4]. Among these antibodies, anti-dsDNA antibodies, considered a part of the anti-chromatin antibody spectrum, exhibit a stronger correlation with disease activity and organ damage. These antibodies are not only key indicators in SLE classification criteria but also directly participate in disease pathogenesis. By binding to DNA, they form immune complexes that deposit in tissues such as the kidneys and skin, activating the complement system and recruiting inflammatory cells, ultimately leading to organ damage [5]. Notably, high levels of anti-dsDNA antibodies in SLE patients significantly correlate with disease activity and organ damage, suggesting that abnormal exposure of DNA is a crucial part of the pathological process [6, 7].

Under physiological conditions, dsDNA and nucleosomes from nuclear fragments released by apoptotic cells [8], including the produced microparticles [9], NETosis [10], and other cell death, are promptly degraded by nucleases and phagocytosed by macrophages, thus preventing autoimmune recognition. However, when DNA clearance mechanisms are imbalanced, these undegraded nuclear fragments can persist in circulation, becoming a critical pathogenic factor in SLE [11, 12]. The accumulated nuclear debris can be captured and processed by dendritic cells and B cells in vivo, leading to a breakdown of immune tolerance and activation of both innate and adaptive immune responses. This ultimately results in the production of high-titer autoantibodies. The immune complexes formed by these autoantibodies and nuclear debris deposit in sites like the glomerular basement membrane, triggering severe inflammatory reactions, such as lupus nephritis [2]. Therefore, the loss of DNA homeostasis, through the substrate effect of nuclear debris accumulation, directly promotes the formation of core SLE autoantigens and subsequent immune activation.

These fragments of extracellular DNA, referred to as cell-free DNA (cfDNA), can be detected in plasma or other body fluids. Such a condition is manifested by an increase in the concentration of cfDNA detected and changes in the distribution of fragments [13, 14] and is related to disease activity [15]. These accumulated cfDNA with varying sizes from the normal distribution peak exhibit stronger immunogenicity and have a greater correlation with disease [16, 17]. Studies have found that the secreted nuclease DNASE1L3 plays a crucial role in maintaining the normal distribution and concentration of cfDNA. DNASE1L3 efficiently cleaves DNA-histone complexes in chromatin and NETs, or DNA bound to lipids in microparticles. Its functional deficiency has been confirmed to be closely related to the production of autoantibodies and disease progression in SLE patients and animal models [18]. Therefore, an in-depth analysis of the regulatory mechanisms of DNASE1L3 in SLE not only provides new perspectives on the origin and accumulation of autoimmune DNA but also lays a theoretical foundation for developing targeted intervention strategies.

DNASE1L3: a specialized guardian of nucleic acid homeostasis

DNASE1L3, also known as DNase γ, DNAS1L3, or LS-DNase, is an essential member of the Deoxyribonuclease I (DNase I) superfamily of endonucleases. DNASE1L3 was first isolated in 1994 from nuclei of rat thymocytes [19], while sharing sequence homology and certain catalytic features with other DNase I family members [20]. DNASE1L3 has a critical function due to its distinct substrate preferences, cellular sources, and pivotal role in preventing autoimmunity. Its gene is located on human chromosome 3p14.3–p21.1 [21, 22]. The enzyme’s discovery and subsequent characterization have illuminated crucial aspects of how the body handles the potentially dangerous cargo of cfDNA [23].

Expression profile: cellular origin, synthesis, and systemic distribution

Expression of the DNASE1L3 gene was verified for the spleen, liver, thymus, lymph node, bone marrow, small intestine, and kidney. But the primary production sites of DNASE1L3 are mainly immune cells of myeloid lineage [24].

Specifically, compelling evidence indicates that DNASE1L3 is predominantly synthesized and secreted by monocyte-derived dendritic cells and macrophages. These cells are strategically positioned throughout the body, particularly in organs central to immune surveillance and clearance, such as Kupffer cells in the liver and red pulp macrophages and marginal zone macrophages in the spleen [25]. Studies have shown that the homeostatic milieu itself can induce the production of DNASE1L3 from these myeloid cells, suggesting a constitutive role in maintaining cfDNA balance [26]. Lower levels of expression have also been detected in other immune cell populations, including plasmacytoid dendritic cells, marginal zone B cells, and B-1a cells, although their relative contribution to systemic DNASE1L3 levels is likely less significant than that of conventional dendritic cells and macrophages.

Furthermore, immunohistochemical data, which is available from the Human Protein Atlas describe a different view. It confirms that DNASE1L3 protein is also notably abundant in key SLE-affected organs, including the kidney and the skin, correlating its cellular synthesis with its presence in sites of lupus pathology. However, there is currently no research clearly indicating the expression of DNASE1L3 in these tissues, and further studies are required.

DNASE1L3 is secreted via the classic endoplasmic reticulum-Golgi pathway into the blood, lymph, and body cavities, like joint cavities. In the circulatory system, DNASE1L3 exists in a free form, its activity dependent on Ca²⁺/Mg²⁺ ions [27]. When cells are stimulated or undergo apoptosis, the N-terminal signal peptide of DNASE1L3 is cleaved, and the C-terminal nuclear localization sequence plays a major role, translocating it from the endoplasmic reticulum to the nucleus to participate in the fragmentation of nuclear chromatin [28, 29].

Distinctive structural architecture: form dictating function

The unique functional capabilities of DNASE1L3, particularly its proficiency in degrading complex DNA substrates implicated in autoimmunity, are deeply rooted in its distinct three-dimensional structure. Comprehensive structural analyses have provided invaluable insights into the molecular architecture of DNASE1L3, revealing a bipartite organization comprising a conserved core catalytic domain and a unique C-terminal domain (CTD).

The core catalytic domain: a modified DNase I fold

The N-terminal portion of DNASE1L3, approximately amino acids 23–280 in the mature protein, constitutes the core catalytic domain.

This domain adopts a classic α-β sandwich fold, a structural motif characteristic of the Exonuclease/Endonuclease/Phosphatase superfamily of enzymes. It shares significant structural homology with the canonical DNase1, including the overall fold and the arrangement of key catalytic residues [30]. The active site, responsible for the hydrolysis of phosphodiester bonds in the DNA backbone, harbors two highly conserved histidine residues: His155 and His274 in DNASE1L3, corresponding to His134 and His252 in bovine DNase1 [31] (Fig. 1). These histidines function as a general acid and a general base, respectively, in the catalytic mechanism.

Fig. 1.

Structural comparison of DNASE1L3 and DNase1 highlighting conserved α–β sandwich fold, unique CTD, and key residue differences. (A) Crystal structures of DNASE1L3 (lacking the C-terminal domain; PDB ID: 7KIU) in blue and DNase1 (PDB ID: 4AWN) in yellow both exhibit the canonical α–β sandwich fold characteristic of DNase I-like endonucleases, with α-helices and β-sheets. (B) The unique C-terminal domain (CTD) of DNASE1L3, comprising residues 283–305, is rendered with a spectrum gradient to indicate residue position from the N-terminal to the C-terminal end of the CTD. This positively charged, flexible extension—absent in DNase1—is predicted to enhance DNA binding and facilitate clearance of extracellular chromatin. (C) Structural detail of Phe131 in DNASE1L3, replacing Ala114 in DNase1, and the loop region corresponding to the primary actin-binding α-helix in DNase1 (residues 67–75) is shown. The DNASE1L3 loop adopts a distinct conformation, potentially contributing to its reduced actin-binding capacity. All structural representations were prepared in PyMOL with cartoon rendering and ray tracing for clarity. (D) Structural representation of DNASE1L3, highlighting the Cys194–Cys231 disulfide bond and key variant sites. The DNASE1L3 protein backbone is shown in blue from the N-terminus to the C-terminus. The position of the Cys194–Cys231 disulfide bond is depicted as two black dots, illustrating its role in stabilizing the protein’s tertiary structure. Key sites affected by genetic variants are highlighted: Arg206Cys in a blue circle, p.Trp215Glyfs*2 and p.Asn191Ser in yellow circles, and p.Thr97Ilefs*2 in a red circle to illustrate the extent of their influence on the structure of DNASE1L3 from mild to severe. These mutations and the SNP can affect the spatial structure of this critical disulfide bond, leading to protein inactivation or secretion defects. The linear diagram of DNASE1L3 protein was created in https://BioRender.com/2qrvgf3

Despite the overall similarity to DNase1, the core domain of DNASE1L3 possesses subtle but functionally important differences.

A particularly salient feature of DNASE1L3’s core domain is its inherent resistance to inhibition by monomeric globular actin. DNase1 is potently inhibited by globular actin, which binds to a specific site on the nuclease, sterically hindering its access to DNA substrates. This inhibition is a significant regulatory mechanism for DNase1 activity [22, 32]. DNASE1L3, however, largely evades this inhibition [20, 33]. Structural comparisons and mutagenesis studies have revealed several key differences that account for this resistance (Table 1). One of the crucial substitutions in DNASE1L3 is Phe131, which replaces Ala114 found in DNase1 at a similar position within the actin-binding interface [32]. The bulkier phenylalanine side chain at this position is thought to create steric clashes that prevent the stable docking of actin onto the DNASE1L3 surface. On the other hand, the loop region corresponding to the primary actin-binding α-helix in DNase1, typically residues 67–75, exhibits differences in DNASE1L3 (Fig. 1). The original document mentions an insertion of two additional amino acids and a resultant charge reversal in this region. This dramatic shift in electrostatic properties would further disfavor the interaction with the predominantly negatively charged surface of actin [34].

Table 1.

DNA nucleases involved in SLE

	DNASE1L3	DNase1	DNASE2	DFFB	TREX1
Inhibitors	Zn2+, heparin	Zn2+, G-actin	various salts	DFFA	SPP
Optimal pH	Neutral	Neutral	Acid	Neutral	Neutral
Expression	high levels in lymphoid organs, liver, spleen	Pancreas,, salivary glands	widely distributed	widely distributed	widely distributed
Site of action	Intracellular and extracellular	Extracellular	Lysosomes	Intracellular	Intracellular
Typical DNA product size	Mononucleosomes	< Mononucleosomes	< Oligonucleotides	> Oligonucleosomes	-
Main function	Clearance of cfDNA complexed with lipids and proteins	Clearance of naked DNA	Clearance of nucleic acids generated during apoptosis	Cutting of DNA into internucleosomal fragments	Clearance of endogenous DNA debris

These structural modifications ensure that DNASE1L3 can remain catalytically active even in environments where actin might be released from damaged cells, such as sites of inflammation or extensive apoptosis, a condition highly relevant to SLE.

The C-Terminal domain: a unique appendage for enhanced substrate interaction

The most striking structural distinction between DNASE1L3 and other DNase I family members is its C-terminal domain (CTD), a segment of approximately 20–25 amino acids, residues 283–305 in human DNASE1L3, appended to the core catalytic domain. This relatively short extension is rich in positively charged amino acid residues, such as lysine and arginine.

Nuclear Magnetic Resonance and circular dichroism studies have demonstrated that the isolated CTD is intrinsically disordered in solution, lacking a stable, well-defined secondary or tertiary structure. Importantly, even when tethered to the core domain or when DNASE1L3 is bound to its DNA substrates, the CTD appears to retain a significant degree of flexibility and conformational heterogeneity. This intrinsic disorder allows the CTD to adopt multiple conformations and likely facilitates its interaction with diverse, structurally varied substrates (Fig. 1).

The primary function of CTD attribution is to enhance the interaction between enzymes and structurally complex DNA substrates and to improve their degradation capability, particularly for DNA substrates that are not easily accessible to conventional nucleases such as DNase1, including chromatin and immune complexes [33, 35–37]. This special structure drives its ability to degrade diverse DNA substrates through multifunctional interactions. Its positively charged clusters bind chromatin via the DNA phosphate backbone and nucleosomal histones, enhancing enzymatic targeting. The CTD also mediates hydrophobic/electrostatic interactions with lipid membranes, enabling access to encapsulated DNA, as shown by a 10-fold activity loss in ΔCTD mutants [33, 38]. In autoimmune contexts like SLE, the CTD anchors DNASE1L3 to antibody-DNA complexes, disrupting their structure to expose DNA for cleavage. By integrating electrostatic binding, membrane penetration, and macromolecular remodeling, the CTD equips DNASE1L3 to clear pathological DNA forms—chromatin, lipid-associated, or immune-complexed, which are critical for preventing DNA-driven inflammation and autoimmunity [33].

Functional characteristics: a specialized nuclease for complex DNA substrates

The distinct structural features of DNASE1L3 translate into a specialized functional profile that sets it apart from other endonucleases and underscores its critical role in maintaining immune tolerance. Its primary physiological function is the efficient degradation of potentially immunogenic self-DNA, particularly in forms that are poorly handled by other nucleases [11].

Substrate specificity: beyond naked DNA

While DNASE1L3 can also cleave naked DNA, its hallmark is its superior ability to degrade DNA bound to proteins or lipids, such as nuclear fragments, including chromatin, nucleosomes, and double-stranded DNA with histone cores [20]. The body employs a multi-tiered system of nucleases to manage DNA from various sources, and DNASE1L3 plays a specialized, largely non-redundant role within this network.

DNASE1L3 excels at cleaving DNA within chromatin, the physiological state of DNA in eukaryotic cells where it is complexed with histone proteins to form nucleosomes [28]. It can efficiently digest DNA in isolated nucleosomes and larger chromatin fragments, producing the characteristic internucleosomal ladder pattern seen during apoptosis. This activity is vital for processing the vast amounts of chromatin released from dying cells. Its preference for complex DNA, such as mononucleosomes, over free DNA is a key distinguishing feature from DNase1, which is less effective against chromatin structures and immunogenic DNA-protein complexes [22, 33]. This selectivity is strongly attributed to the CTD, whose basic residues interact favorably with the negatively charged chromatin, anchoring the enzyme to its preferred substrate.

NETs are complex structures composed of a scaffold of decondensed chromatin studded with various antimicrobial proteins [18]. DNASE1L3, along with DNase1, plays a crucial role in the timely degradation of NETs. Inefficient NET clearance is increasingly implicated in the pathogenesis of SLE and other autoimmune and thrombotic disorders. The ability of DNASE1L3 to access and fragment DNA within these dense, protein-rich NET structures is critical for their resolution [39].

Apoptotic cells shed membrane-bound vesicles known as apoptotic bodies or microparticles, which contain nuclear fragments, including chromatin and DNA [40]. DNASE1L3 is uniquely equipped to digest DNA on the surface of or within these microparticles [41]. As mentioned earlier, its CTD is crucial for interacting with and degrading lipid-associated DNA, which is a prominent feature of these microparticles. By neutralizing the DNA cargo of microparticles, DNASE1L3 prevents them from becoming potent sources of autoantigens (Fig. 2).

Fig. 2.

Sources and characteristics of DNASE1L3-cleaved cell-free DNA. cfDNA originates from various sources, including microparticles (MPs) released by apoptotic cells and neutrophil extracellular traps (NETs) formed during neutrophil activation. DNASE1L3 efficiently digests these complex DNA substrates, producing cfDNA fragments with a predominant length of approximately 166 bp, corresponding to mononucleosomal DNA. In addition, DNASE1L3 cleavage generates characteristic end-motif patterns, notably a cytosine (C) at the 5′ termini, reflecting its substrate specificity and enzymatic cleavage preferences. These features distinguish DNASE1L3-dependent cfDNA processing from that of other nucleases and are relevant for understanding its role in immune tolerance and systemic lupus erythematosus pathogenesis. The figure is created https://BioRender.com/jmc21ok

Atypical DNA structures, such as Z-DNA, are biologically relevant due to potential infectious triggers in SLE [42]. However, DNASE1L3’s specific activity against these atypical conformations remains to be defined.

Enzymatic properties: activity spectrum and optimal conditions

DNASE1L3 is an endonuclease that introduces single- and double-strand breaks in DNA, ultimately producing DNA fragments with 3’-hydroxyl and 5’-phosphate ends. Its activity is highly dependent on specific environmental conditions.

The first condition is divalent cations. Like other DNase I family members, DNASE1L3 requires divalent cations for its activity, primarily Mg²⁺ and Ca²⁺. These ions are involved in substrate binding, positioning of the attacking water molecule, and stabilization of the transition state during phosphodiester bond hydrolysis [27].

Secondly, DNASE1L3 exhibits optimal activity at physiological pH, typically around pH 7.4 (Table 1). Its activity significantly diminishes in acidic environments, which has implications for its function in different tissue microenvironments or within intracellular compartments like late endosomes or lysosomes if it were to be internalized [43]. For example, DNASE2 is primarily located within lysosomes. Its main function is to degrade DNA from phagocytosed material, such as apoptotic cells or pathogens, that has been delivered to the lysosomal compartment. DNASE2 operates optimally at the acidic pH found within lysosomes (Table 1).

DNA homeostasis: synergistic and non-redundant roles of nuclease

The body employs a multi-tiered system of nucleases to manage DNA from various sources, and DNASE1L3 plays a specialized, largely non-redundant role within this network.

In the extracellular space, DNASE1L3 synergizes with DNase1, which is primarily expressed in the pancreas and salivary glands, secreted into the digestive tract and circulation. Their main role is thought to be the degradation and the clearance of cf-DNA released into the bloodstream [20]. However, DNase1 is less efficient than DNASE1L3 in degrading chromatin and is also inhibited by actin.

Notably, DNASE1L3 generates cfDNA with distinctive fragmentomic signatures. Its cleavage preferentially yields ~ 166 bp mononucleosomal fragments and shows a strong enrichment of cytosine at the 5′ termini [44] (Fig. 2). These features distinguish DNASE1L3 activity from other nucleases and may serve as biomarkers of nuclease function and cfDNA clearance efficiency.

In the intracellular space, multiple nucleases participate in DNA. DFFB, located in the nucleus, this nuclease participates in DNA fragmentation with DNASE1L3 during apoptosis [45]. But this function with DNASE1L3 is redundant [44]. Other nucleases like DNASE2 and the cytosolic 3’→5’ exonuclease TREX1 also play important roles in clearing endogenous DNA debris [46].

Pathogenic mechanisms: causes of DNASE1L3 deficiency in SLE

Genetic alterations: mutations and single nucleotide polymorphisms

DNASE1L3 gene mutations mainly include frameshift mutations and point mutations. These different mutations lead to varying degrees of reduced DNASE1L3 enzyme activity, impaired function, or inability to secrete, ultimately resulting in impaired DNA degradation capacity, triggering immune complex accumulation and autoantibody production, primarily leading to early-onset monogenic lupus [47–52].

The first clinical link between DNASE1L3 deficiency caused by a mutation and SLE was established in 2011, when a consanguineous family from Saudi Arabia was reported to harbor a loss-of-function variant (c.643delT) in DNASE1L3 (Fig. 1). Affected children developed severe, early-onset SLE, often presenting within the first three years of life with lupus nephritis and profound [50]. Two years later, DNASE1L3 mutations were also identified in hypocomplementemic urticarial vasculitis syndrome (HUVS), where three families carried a homozygous frameshift mutation (c.289_290delAC) [53]. Notably, over 50% of HUVS patients progress to an SLE-like phenotype, underscoring a shared pathogenic axis [54]. Since HUVS is an immune complex–mediated disease, these findings suggest that DNASE1L3 loss-of-function facilitates immune complex accumulation and type I interferon–driven B cell dysregulation. Collectively, these studies provided the first clinical evidence connecting DNASE1L3 deficiency to lupus pathogenesis.

Single nucleotide polymorphisms (SNPs) of DNASE1L3 are related to susceptibility to various autoimmune diseases, including SLE [55]. The single nucleotide polymorphisms are exemplified by rs35677470 (p.Arg206Cys) (Fig. 1) [56]. The mutation of arginine to cysteine disrupts the catalytic domain integrity, reducing enzyme activity by 70% and conferring homozygous-biased risk for SLE, rheumatoid arthritis, and systemic sclerosis. Homozygous individuals have a significantly higher disease risk than heterozygous carriers [57, 58].

The sequence of DNASE1L3 is relatively conserved, and current research has not found copy number variations in DNASE1L3 [59].

Autoantibodies: the emergence of Anti-DNASE1L3

The role of circulating DNase and its respective autoantibodies has long been a focus in SLE. For instance, decreased DNase1 enzyme activity and the presence of inhibitory anti-DNase1 antibodies were early findings linked to the impaired clearance of DNA [36, 37, 60–62].

Although early research focused on congenital DNASE1L3 deficiency caused by gene mutations, subsequent studies also found patients with reduced serum DNASE1L3 activity who did not have gene mutations [63]. Recent research has revealed that anti-DNASE1L3 antibodies occur in 18%-30% of sporadic SLE patients (~ 43% with nephritis) and correlate with SLEDAI scores [17, 64].

Pathogenic mechanisms: consequences of DNASE1L3 deficiency in SLE

The deficiency or diminished activity of DNASE1L3 sets in motion a cascade of molecular events that ultimately leads to the breakdown of self-tolerance and the development of SLE. The pathogenic process is initiated by the inadequate clearance of cfDNA, primarily derived from apoptotic cells and NETs [39, 64], which then engages specific innate and adaptive immune pathways. Our understanding of this cascade has been meticulously built by combining clinical observations with insights from elegantly designed animal models that recapitulate key aspects of the human disease.

Main pathway: the central role of the type I interferon pathway

Evidence about the mutation mentioned above [53, 65] hints that the primary and most well-defined mechanism driving autoimmunity in DNASE1L3 deficiency is the aberrant activation of the type I interferon (Type I IFN) system. The essential function of DNASE1L3 is to digest chromatin within microparticles and other debris released from apoptotic cells. In its absence, this immunogenic self-DNA accumulates in the circulation. This core hypothesis was powerfully validated using engineered mouse models. The first complete Dnase1l3 knockout (KO) mice, generated by Sisirak et al., provided definitive proof of causality. These mice spontaneously developed a disease strikingly similar to human SLE, characterized by a rapid breach in B-cell tolerance, with high-titer anti-dsDNA and anti-nucleosome antibodies appearing as early as five weeks of age. This early seroconversion was followed by a much more protracted course leading to overt clinical disease, with fatal lupus nephritis—marked by glomerular immune complex deposition—developing much later, typically between 9 and 12 months [41]. This temporal disconnection between early autoantibody production and delayed end-organ damage observed in the KO mice provided a crucial window into a multi-step pathogenic process.

The link between the accumulated cfDNA and autoimmunity is the endosomal sensor, Toll-like receptor 9 (TLR9), and, in certain contexts, Toll-like receptor 7 (TLR7). These receptor, highly expressed in plasmacytoid dendritic cells (pDCs), B cells, and macrophages, recognizes unmethylated CpG motifs within the undigested DNA, which are abundant in microbial DNA but are typically methylated in mammalian DNA [66]. In SLE, the immunogenicity of self-DNA is further enhanced because mammalian DNA undergoes partial demethylation in T cells and B cells, which is associated with the downregulation of DNA methyltransferase 1 (DNMT1) protein levels in these immune cells. This reduction impairs the maintenance of DNA methylation patterns following cell division. The resulting hypomethylation of key promoter regions leads to the pathological overexpression of normally silenced T-cell and B-cell genes, pushing these lymphocytes toward an activated, autoreactive state [67]. At the same time, cfDNA originating from apoptotic cells or NETs can undergo modifications or become associated with proteins that facilitate its uptake and delivery to TLR7/9-containing endosomes. TLR7 mainly recognizes single-stranded RNA [68], but in SLE, DNA bound to autoantibodies can form DNA-RNA hybrids or DNA-protein complexes, exposing single-stranded regions that are then mistakenly recognized by TLR7 [69, 70]. Once inside, accumulated cfDNA activates endosomal TLR7/9 in pDCs/B cells, driving IFN-I production via MyD88-dependent signaling [41, 71] (Fig. 3). This mistaken recognition thus acts as an epigenetic defect in concert with promoting immune dysregulation and the loss of tolerance in SLE.

Fig. 3.

The pDC-TLR7/9-IFN axis in a DNASE1L3-deficient condition. With the deficiency or decrease of DNASE1L3, cfDNA accumulates in the circulation. These abnormal cfDNA could be recognized and engulfed by pDCs and B cells with TLR7/9. In pDCs, it promotes their nuclear translocation and the production of type I interferons, which can stimulate T-cell-dependent B-cell differentiation in the ExFO pathway. This figure shows the ExFO pathway activated by accumulated cfDNA in the spleen, which occurs mainly in the marginal zone and the T-B cell boundary. In B cells themselves, the TLR7/9 activation can also stimulate their differentiation into plasma cells. These cells secrete auto-antibodies that can bind to cfDNA itself and other molecules, ultimately creating organ damage. The figure is created in BioRender. Deng, S. (2025) https://BioRender.com/dfxq8rt

Notably, autoimmunity in these mice develops in a manner that does not depend on the cytosolic DNA sensor STING, suggesting that the pDC–TLR7/9–IFN axis is a major contributor to disease initiation in this model [66]. Other nucleic acid-sensing receptors, such as Interferon-induced helicase C domain-containing protein 1 (IFIH1), are also implicated as a key cytoplasmic sensor of double-stranded RNA and major drivers of the Type I IFN signature. Genetic evidence strongly supports its role in SLE: common risk SNPs in IFIH1 are associated with increased sensitivity to IFN-α, while rare, monogenic gain-of-function mutations in IFIH1 have been identified in patients with severe, early-onset SLE, often leading to constitutive, pathogenic Type I IFN production [72]. Whether these RNA receptors also play a role in disease pathogenesis remains to be determined. The Type I IFN produced is not merely a bystander but a key pathogenic driver. Further studies in these mice by Soni et al. revealed that the resulting Type I IFN storm promotes a T-cell-dependent, but spatially distinct, extrafollicular B cell response. In this pathway, autoreactive B cells rapidly differentiate into short-lived, antibody-secreting plasmablasts outside of traditional germinal centers, a process sustained by specialized extrafollicular helper T cells (Fig. 3). Blocking the Type I IFN receptor in Dnase1l3 KO mice was sufficient to prevent the expansion of these plasmablasts and ameliorate the disease, confirming Type I IFN as a master regulator of the autoantibody response [71]. These models also highlighted the importance of the genetic context in shaping this IFN-driven pathology; KO mice on a C57BL/6 genetic background develop significantly milder nephritis than those on a 129SvEvTac background, demonstrating that other modifier genes heavily influence the penetrance of the phenotype caused by this single-gene defect.

Additional pathways: other contributing mechanisms of pathogenesis

While macrophage-derived DNASE1L3 is crucial for systemic DNA clearance, its specific deletion only causes attenuated lupus, revealing redundancy (e.g., dendritic cells) and requiring near-total deficiency for fulminant disease [73]. DNASE1L3 deficiency also enables parallel B-cell activation: self-DNA complexes directly trigger TLR2, inducing IL-6 secretion and autoreactivity [74]. Intracellularly, DNASE1L3 regulates inflammasomes (promoting ASC speck formation), enabling IL-1β maturation and HMGB1 release; HMGB1 then chaperones DNA to TLR9, amplifying inflammation [75]. DNASE1L3 itself becomes an autoantigen, with antibodies evolving via epitope spreading to cross-react with dsDNA [76].

It is crucial, however, to contextualize these findings. While indispensable for dissecting these fundamental pathways, mouse models have limitations. The polygenic complexity [77] and diverse environmental triggers [78] of human SLE contrast with the monogenic nature and controlled environment of laboratory mice. Nonetheless, the clear pathogenic sequence established in these models—linking a specific molecular defect to aberrant innate sensing [79], IFN production, and a targeted autoimmune response—provides an invaluable and robust framework for understanding the human disease.

Clinical spectrum: Phenotypes, CfDNA Alterations, and immune signatures of DNASE1L3 deficiency

The detailed molecular and cellular understanding of DNASE1L3-driven autoimmunity, built upon this wealth of animal model data, has profound clinical implications. Firstly, the functional deficiency of DNASE1L3 is closely linked to the onset and clinical phenotype of SLE. Although this mutation is relatively rare, reports to date indicate that patients with DNASE1L3 gene deficiency exhibit early-onset SLE as other monogenic SLE [48], low age of onset, accompanied by high disease activity, kidney involvement, and hypocomplementemia. The positive rates for anti-dsDNA antibodies, ANA, and anti-cardiolipin antibodies are significantly elevated. In these patients, abnormal secretion or loss of DNASE1L3 enzyme activity leads to impaired cfDNA clearance. Undegraded DNA from chromatin fragments and NETs accumulates in the circulation, directly driving complement consumption and immune complex deposition. Similarly, patients with accumulated DNA and positive anti-DNASE1L3 antibodies are also associated with higher disease activity and incidence of nephritis, as well as anemia [76]. Immunological abnormalities in these patients include the coexistence of polyclonal autoantibodies (such as anti-dsDNA, anti-β2 glycoprotein I, and anti-Ro52 antibodies). Simultaneously, polynucleosomal DNA fragments are enriched in circulating microparticles, which exacerbate tissue damage by enhancing the affinity of anti-dsDNA antibodies.

Secondly, impaired cfDNA clearance due to DNASE1L3 functional deficiency is closely related to SLE pathogenesis and disease activity. SLE patient plasma cfDNA exhibits elevated concentrations and specific fragmentation patterns different from healthy individuals [14, 80]. Specifically, short cfDNA fragments (< 115 bp) are increased by about 40% in SLE patients, with a more significant increase in patients with high disease activity. Long cfDNA fragments (> 250 bp), such as MP-derived cfDNA, increase by more than twofold. The 166 bp mononucleosomal peak, which is most abundant in healthy individuals, is significantly decreased [14].DNASE1L3 mutations in humans also disrupt plasma DNA fragmentomic profiles [81], altering size distributions, end motifs [82], and methylation profiles [83]. These features correspond to the function of DNASE1L3 in recognizing specific sequences and cutting chromatin into mononucleosomes.

Finally, the interferon signature is a typical feature of SLE. Transcriptome analysis further reveals that interferon signaling pathway genes and neutrophil activation markers are significantly upregulated in the peripheral blood of antibody-positive patients, indicating abnormal immune activation [76, 84]. Furthermore, DNASE1L3-related SNPs increase SLE susceptibility by regulating gene expression or enzyme activity and are associated with anti-dsDNA antibody production and activation of the interferon pathway [85].

Clinical applications: a biomarker and therapeutic target in SLE

Biomarker utility: diagnosis and disease monitoring

The presence and characteristics of DNASE1L3 and its targeting antibodies show promise for diagnosing and monitoring SLE activity. Antibodies against DNASE1L3 are significantly associated with the clinical and immunological features of active SLE. Patients positive for both anti-DNASE1L3 antibodies and anti-dsDNA antibodies tend to exhibit higher disease activity, suggesting that monitoring these antibodies can provide insights into the disease state.

A critical subset includes dual-reactive antibodies—initially categorized as anti-dsDNA but also reactive with DNASE1L3. Identifying these populations could refine diagnostic and prognostic assessments, as they are associated with more pathogenic SLE features and are linked to specific gene expression patterns, such as the interferon signature and neutrophil activation [76].

Patients who test positive for anti-DNASE1L3 antibodies exhibit a phenotype similar to those with genetic deficiencies and are also associated with higher disease activity, lower complement levels, and an increased incidence of nephritis [17, 76].

Although the prevalence of anti-DNASE1L3 autoantibodies in SLE is relatively low (18%-30%) [17, 76], analyzing the clinical characteristics of this small patient subgroup may help to understand disease heterogeneity and potential mechanistic pathways. Meanwhile, DNASE1L3 single nucleotide polymorphisms also occur in patients with other autoimmune diseases [58], so their definitive clinical value, specificity, and correlation with disease activity still require further large-scale validation (Table 2).

Table 2.

Comparative performance table of SLE biomarkers

Marker	Prevalence	Specificity	PPV	NPV	AUC	Correlation with SLEDAIe
Anti-dsDNAa	39%-76%	63%-98.3%	43%-96.1%	60%-61.6%	0.70–0.85	R = 0.23–0.42
Anti-Sm	25%-30%	91.5%-98.9%	50.4%	93%-94.6%	-	P > 0.05
Anti-Chromatinb	63%-100%	80% − 91.5%	50.4%	81% − 94.6%	-	R = 0.43–0.58
Anti-DNASE1L3c	30%	≈ 98.4%	≈ 97.9%	≈ 35.6%	-	P = 0.002
cfDNAd	-	73.7%	-	-	0.743	R2 ≈ 0.3, P < 0.001

^a There are three main detection methods. Indirect immunofluorescence (IIF) is clinically favored for its high specificity (98.3%) and is often used as a confirmatory test. Digital liquid chip method (DLCM) and chemiluminescent immunoassay (CLIA) offer slightly higher sensitivities (43.65% and 41.57%, respectively), but with modestly lower specificity and PPV (e.g., DLCM specificity 96.7%, PPV 93.48%). All three methods yield similar NPV values, ranging from 60.05% to 61.59% [89]

^b Prevalence estimates in SLE vary significantly depending on the antigenic target (e.g., histones H1, H2B) and detection method, due to the complex composition of chromatin and nucleosome complexes. This methodological heterogeneity limits direct comparability across studies [90]

^c Currently, there is a lack of data on specificity in non-SLE autoimmune disease cohorts. Reported PPV, NPV, AUC, and correlation values are derived from calculations using data from SLE vs. healthy controls and should be interpreted as estimates rather than directly measured outcomes [76]

^d Plasma cfDNA levels are significantly elevated in SLE patients. Research has focused on its dynamic changes in relation to disease activity and treatment response. Its diagnostic performance (PPV/NPV) is highly dependent on assay thresholds. The cfDNA–SLEDAI association has been reported with a coefficient of determination around R2 [91]

^e While anti-Sm alone shows limited correlation with disease activity, the co-positivity of anti-Sm and anti-dsDNA correlates strongly with severe disease [92, 93]. Anti-DNASE1L3 positivity is significantly associated with higher SELENA-SLEDAI scores (median 3.4 vs. 1.8, P = 0.002) [76]. However, most studies treat anti-DNASE1L3 as a binary variable and do not report a Pearson correlation (r) between antibody titers and SLEDAI as continuous variables

The concentration and fragmentation patterns of cfDNA, which are influenced by DNASE1L3 activity, are currently being explored as biomarkers (Table 2). For example, studies in cervical cancer show that changes in cfDNA levels and specific end-motif profiles (patterns at the ends of cfDNA fragments) correlate with treatment response [86]. This indicates that cfDNA characteristics, partly shaped by DNASE1L3, could also potentially be used to monitor treatment efficacy in SLE. The p.Arg206Cys variant in the DNASE1L3 gene significantly affects cfDNA properties, including fragment size and concentration [87]. This genetic variation can impact cfDNA-based diagnostic tests, highlighting the importance of considering DNASE1L3 status in such assays.

Therapeutic opportunities: current and emerging treatments

Since DNASE1L3 deficiency, whether genetic or caused by neutralizing autoantibodies, is implicated as a driver of SLE, enzyme replacement therapy is a potential treatment strategy. Researchers have designed a long-acting enzyme biologic with dual DNase1/DNASE1L3 activity that is resistant to inhibitors of both enzymes. This engineered biologic has shown promising results in preclinical models of SLE in mice, capable of preventing the onset of autoimmunity and reducing organ damage and mortality in inducible lupus animal models. Importantly, the human isoform of this engineered enzyme was found to be non-reactive to neutralizing autoantibodies present in the plasma of SLE patients and effectively degraded various forms of cfDNA, including those implicated in SLE pathology, such as NETs [88]. However, enzyme replacement may lead to increased or new anti-DNASE1L3 antibodies in some patients. Moreover, the therapeutic work in mouse models has been entirely preventative, given before disease onset, which poses a significant challenge for translation to established human disease. Given that impaired DNASE1L3 function leads to the accumulation of immunogenic self-DNA and subsequent inflammation, therapies aimed at reducing this DNA load or dampening the subsequent immune activation can also be considered.

In summary, DNASE1L3 plays a crucial role in SLE by influencing self-DNA processing and the subsequent immune response. The involvement of DNASE1L3 offers promising avenues for the development of novel diagnostic tools to stratify patients better and monitor disease activity, as well as targeted therapies, such as enzyme replacement therapy, to address the underlying pathology of the disease.

Discussion and perspective

DNASE1L3 has firmly established itself as a pivotal enzyme in the intricate web of immune regulation, with its deficiency directly implicated in the pathogenesis of SLE. While significant progress has illuminated its function and clinical relevance, ongoing research efforts focused on addressing current limitations and pursuing novel diagnostic and therapeutic strategies hold immense promise.

The future of DNASE1L3 research lies in further unraveling its regulatory mechanisms, expanding genetic insights through diverse cohort sequencing, and developing integrated biomarker panels using advanced assays and machine learning to enhance SLE diagnosis and monitoring. Concurrently, therapeutic innovation—including engineered biologics, small-molecule modulators, and targeted clinical trials—must align with precision medicine approaches, while exploring DNASE1L3’s broader role in autoimmune and inflammatory diseases could unlock novel diagnostic and therapeutic avenues beyond SLE.

Abbreviations

ANA

Antinuclear antibody

APC

Antigen-presenting cell

cfDNA

Cell-free DNA

cGAS

Cyclic GMP–AMP synthase

CTD

C-terminal domain

DCs

Dendritic cells

dsDNA

Double-stranded DNA

DNase1

Deoxyribonuclease 1

DNase2

Deoxyribonuclease 2

DNASE1L3

Deoxyribonuclease 1-like 3

DNMT1

Downregulation of DNA methyltransferase 1

HUVS

Hypocomplementemic urticarial vasculitis syndrome

IFIH1

Interferon-induced helicase C domain-containing protein 1

IFNAR

Interferon α/β receptor

IRF

Interferon regulatory factor

KO

knockout

NETs

Neutrophil extracellular traps

pDCs

Plasmacytoid dendritic cells

SLE

Systemic lupus erythematosus

SNP

Single nucleotide polymorphism

ssRNA

Single-stranded RNA

STING

Stimulator of interferon genes

TLR7

Toll-like receptor 7

TLR9

Toll-like receptor 9

Type I IFN

Type I interferon

Author contributions

Xiaohan Liu the main manuscript text and prepared Figs. 1, 2 and 3. All authors reviewed the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No.32450786, No. 82271847, and No.32400583), Tongji Hospital Clinical Research Flagship Program Funding (No.2019CR206), and Tongji Hospital High-Quality Clinical Research Funding (No.2024TJCR008).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.

Ethics statement

This article is a review and does not contain any studies with human participants or animals performed by any of the authors.