---
title: "DNASE1L3 enhances innate immunity to DNA viruses via the cGAS-STING pathway"
id: "frontiers-in-immunology-13-deoxyribonuclease-dnase1l3-promotes-innate-immunity-against-dna-virus-infection"
canonical_url: "https://medichelpline.com/clinical-feed/frontiers-in-immunology-13-deoxyribonuclease-dnase1l3-promotes-innate-immunity-against-dna-virus-infection"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "Frontiers in Immunology"
source_url: "https://www.frontiersin.org/articles/10.3389/fimmu.2026.1806552"
published_at: "2026-10-23T00:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# DNASE1L3 enhances innate immunity to DNA viruses via the cGAS-STING pathway
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/frontiers-in-immunology-13-deoxyribonuclease-dnase1l3-promotes-innate-immunity-against-dna-virus-infection
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** Frontiers in Immunology
- **Source URL:** [Original Journal Publication](https://www.frontiersin.org/articles/10.3389/fimmu.2026.1806552)
- **Published At:** 2026-10-23T00:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- Investigators reported that **DNASE1L3** promotes innate immune responses against **DNA virus** infection by enhancing the **cGAS-STING** signaling pathway, according to the Frontiers in Immunology article title. - The source material provided here did not include experimental design, model systems, dosing regimens, or quantitative results; those details were not reported in the supplied content. - The article type is Original Research, indicating primary data were likely generated and analyzed, but the supplied excerpt contains only site navigation and the article title; no figures, methods, or results were accessible. - Key mechanistic claim centers on modulation of the cytosolic DNA-sensing axis (cGAS-STING) by a deoxyribonuclease, implying a link between DNA processing and downstream innate signaling. - Unanswered questions in the provided content include which DNA viruses were studied, whether effects were demonstrated in vitro or in vivo, the cellular sources of **DNASE1L3**, and any measured impact on antiviral cytokine production or viral load. - The supplied excerpt did not report safety, tolerability, or translational implications; therefore clinical relevance and applicability remain unspecified in the available text. - For clinicians and researchers, the most immediate takeaways are the molecular target (**DNASE1L3**) and the pathway (**cGAS-STING**); further appraisal requires full-text access to evaluate methods, statistical robustness, and reproducibility.
## Clinical Analysis & Structured Key Points
Frontiers | Deoxyribonuclease DNASE1L3 promotes innate immunity against DNA virus infection by enhancing the cGAS-STING pathway ORIGINAL RESEARCH article Front. Immunol. , 23 October 2026 Sec. Molecular Innate Immunity Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1806552 Published in Frontiers in Immunology Molecular Innate Immunity 7 impact factor 11.3 citescore Editor & Reviewers Edited by T I Tomozumi Imamichi Reviewed by C A Ceri Alan Fielding T D Tianhao Duan Outline Figures and Tables Figure 1 View in article Figure 2 View in article Figure 3 View in article Figure 4 View in article Figure 5 View in article Figure 6 View in article Figure 7 View in article Figure 8 View in article ORIGINAL RESEARCH article Front. Immunol. , 23 October 2026 Sec. Molecular Innate Immunity Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1806552 Deoxyribonuclease DNASE1L3 promotes innate immunity against DNA virus infection by enhancing the cGAS-STING pathway M W Min Wang 1 † Y W Yilan Wang 1 † J N Jian Ni 2 † Y Z Yujie Zhan 3 M B Menglan Bao 1 X W Xiyang Wang 1 Q S Qingsong Shen 1 X Z Xuechun Zhang 1 K P Kailing Pan 4 X C Xianguo Chen 4 Z H Zan Huang 1 * +3 more M P Min Peng 3 * 1. College of Life Sciences, Hubei Key Laboratory of Cell Homeostasis, Department of Oncology of Renmin Hospital, Wuhan University, Wuhan, China 2. Department of Cardiology of Renmin Hospital, Wuhan University, Wuhan, China 3. Department of Oncology of Renmin Hospital, Wuhan University, Wuhan, China 4. Department of Cardiothoracic Surgery, Jinhua Municipal Central Hospital, Affiliated Jinhua Hospital of Zhejiang University School of Medicine, Jinhua, China See more Article metrics View details Abstract Deoxyribonucleases are known to scavenge self-DNA to dampen innate immune responses and prevent autoimmune diseases. Whether and how deoxyribonucleases respond to DNA virus infection are largely elusive. In this study, HSV-1 infection significantly upregulated and activated DNASE1L3. In vitro , DNASE1L3 overexpression upregulated the production of type I interferons and pro-inflammatory cytokines, enhanced IRF3 phosphorylation, and dampened viral replication whereas DNASE1L3 deficiency caused the opposite phenotypes. In vivo , Dnase1l3 knockout impaired innate immunity against HSV-1 and led to an early onset of death and low survival rate of mice. Mechanistically, DNASE1L3 enhanced the activation of cGAS-STING pathway through novel mechanisms. On one hand, DNASE1L3 bound to STING, enhanced its oligomerization and the recruitment of TBK1 independent of the nuclease activity. On the other hand, DNASE1L3 facilitated the release of HMGB1, and cGAS from chromatin dependent on the nuclease activity, which ultimately augmented cGAS oligomerization and cGAMP production. Our study revealed novel roles of DNASE1L3 and the underlying mechanisms coordinating innate immune responses to self-DNA and viral DNA. 1 Introduction Innate immune responses to DNA are tightly associated with multiple pathophysiological conditions including autoimmune diseases, DNA virus infection, and tumor immunity, and the activation of cGAS-STING pathway plays essential roles in these processes ( 1 , 2 ). Physiologically, self-DNA has to be cleared or tolerated whereas microbe DNA activates innate immune signaling to fight pathogenic infection. Chromatin-bound cGAS is inactive but reactivated by viral infection ( 3 ), which suggests a structural basis for differential innate immune responses to DNA in different biological processes. However, mechanisms coordinating distinct innate immune responses to DNA in different biological processes remain to be further clarified. DNA degradation by deoxyribonucleases is an effective way for the body to prevent autoimmune diseases ( 4 ). Among them, DNASE1, DNASE1L1, and DNASE1L3 are mainly responsible for clearing extracellular DNA, while DNASE1L2, TREX1, and PLD3/PLD4 are mainly responsible for clearing intracytoplasmic DNA ( 5 ). Mutations in these genes are closely correlated to systemic lupus erythematosus (SLE), systemic sclerosis, and rheumatoid arthritis ( 5 , 6 ). Deficiencies of these genes cause defects in clearing autologous DNA that leads to sustained activation of the STING signaling, endolysosome TLR7/9 pathway, or RIG-I-MAVS pathway ( 5 , 6 ). Of note, DNASE1L3 is structurally characterized by nuclear localization signals and a C-terminal domain, which confer DNASE1L3 of nuclear localization as well as the ability to cleave nucleosomal and liposomal DNA extracellularly and intracellularly ( 6 , 7 ). DNASE1L3 mutations or decreased activity associated with systemic lupus erythematosus and rheumatoid arthritis ( 8 , 9 ), and DNASE1L3 knockout mice mimic these phenotypes by inducing B cell differentiation and the expression of type I interferons in plasmacytoid dendritic cells via endosomal TLR7/9 ( 10 – 12 ). However, whether and how DNASE1L3 may regulate innate immunity in DNA virus infection remains unclear. In this study, we found that DNASE1L3 was upregulated and activated by herpes simplex virus 1 (HSV-1) that in turn dampened HSV-1 infection and promoted mice survival in vitro and in vivo . DNASE1L3 did so by promoting STING oligomerization in cytoplasm and cGAS reactivation in nucleus. Our study unveils unexpected roles of DNASE1L3 and the underlying mechanisms in innate immune response to DNA virus infection, which are opposite to its function in dampening innate immune response via endosomal TLR7/9 pathway to prevent self-DNA-induced autoimmune diseases. 2 Materials and methods 2.1 Mice Dnase1l3 −/− mice (C57BL/6 background) were generated using the CRISPR/Cas9 technique and Dnase1l3 gene knockout was confirmed as previously reported ( 13 ). All animal experiments were approved by the Animal Care and Use Committee of the College of Life Sciences of Wuhan University. Care was provided following the criteria outlined in the Guide for The Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health. 2.2 Cell culture THP-1 cells (ATCC, Cat#TIB-202) were cultured in complete RPMI 1640 medium (Gibco, Cat#31800022). L929 (ATCC, Cat#CCL-1), 143B (ATCC, Cat#CVCL-2270), and Vero (ATCC, Cat# CCL-81) cells were maintained in complete DMEM medium (Gibco, Cat#12100046). HEK293T cells were kindly provided by Dr. Bo Zhong from Wuhan University, which retain the ability to response to HSV-1 infection and DNA transfection for dual-luciferase assay ( 14 – 16 ). Primary mouse embryo fibroblasts (MEFs) were prepared from E14.5 embryos and cultured in complete DMEM medium. To culture bone marrow-derived macrophages (BMDMs), bone marrow cells were isolated from femurs of wildtype ( Wt ) and Dnase1l3 −/− ( KO ) mice and cultured in complete DMEM medium supplemented with 20% of L929-conditioned medium. All complete medium contained FBS (10%, vol/vol) (Cegrogen, Cat#A0500-3010) and streptomycin–penicillin (1%, vol/vol) (HyClone, Cat# SV30010). All cells were incubated at 37°C in a 5% CO 2 and 90% humidity atmosphere. For transient transfection of HEK293T cells, polyethyleneimine (Polyscience, Cat#24765) was used according to the manufacturer’s protocol. To establish stable overexpression or knockdown cells, THP-1 cells were infected with lentivirus in the presence of polybrene (10 μg/mL) and selected with puromycin (10 μg/mL) for 5 days as previously described ( 17 ). Sequences of sgRNAs specific for DNASE1L3 were listed in Supplementary Table S2 and the knockdown efficiency was verified by Western blotting. 2.3 Cell model and mouse model of herpes simplex virus infection Cells were infected with HSV-1 (5×10 6 PFU per 1×10 6 cells) for one hour. The supernatants were removed and cells were washed twice with pre-warmed PBS followed by culture in full medium for 16 hours. Viral replication was analyzed by quantitative RT-PCR. To infect mouse, age- and sex-matched Wt and Dnase1l3 −/− mice were injected with HSV-1 (1×10 7 PFU per mouse) through tail vein and the survival of animals was monitored every day as previously described ( 14 ). Mice were sacrificed and the liver, spleen, lung, brain or serum were collected for quantitative RT-PCR analysis, Western blotting analysis, or ELISA at the indicated time points post infection. 2.4 Plaque formation assay The supernatants of HSV-1-infected BMDMs or THP-1 cells were collected and used to infect monolayers of Vero cells. One hour after infection, the supernatants were removed and the infected Vero cells were washed with pre-warmed PBS twice followed by incubation with complete DMEM medium supplemented with 1% methylcellulose for 36–48 hours. Cells were fixed with 4% paraformaldehyde for 15 minutes and stained with 1% crystal violet for 15 minutes before counting the plaques. 2.5 Measurement of deoxyribonuclease activity THP-1 and HEK293T cells were washed and lysed in PBS containing 0.2% NP40. To examine the effect of PV6R on deoxyribonuclease activity, PV6R (100 μM) was added to the culture medium 6 hours prior to sample collection. The deoxyribonuclease activity in the cell lysates or cell culture supernatants was assayed by using genomic DNA or nucleosomal DNA from 143B human osteosarcoma cells, which do not express DNASE1L3 and possess stable chromatin structure, uniform nucleosome assembly, and abundant Alu repetitive sequences. Therefore, genomic DNA from 143B cells have been adopted as a standard substrate for studying DNase1, DNASE1L3, and CAD family nuclease due to such unique characters ( 8 , 18 , 19 ). In brief, 5 μL of the deoxyribonuclease sample was applied to the genomic DNA or nucleus solution (derived from 1×10 5 143B cells in a reaction buffer containing 25 mM HEPES, 1 mM MgCl 2 , 1 mM CaCl 2 , 100 μg/ml BSA with total reaction volume of 30 μL). After incubation for 30 minutes at room temperature, EDTA was added to a final concentration of 10 mM to stop the reaction. DNA fragmentation was assessed by agarose gel electrophoresis, and the Alu200/Alu76 ratio was measured by quantitative PCR. A lower ratio indicates greater DNA fragmentation and higher nuclease activity. 2.6 Subcellular fractionation of cytoplasmic, nuclear-soluble, and chromatin-bound constituents HEK293T and THP-1 cells (5×10 6 ) were collected. Cytoplasmic fraction, nuclear-soluble fraction, and chromatin-bound fraction were prepared as previously described ( 14 ) and used for Western blotting. Detailed protocol was provided in Supplemental information and data. 2.7 Dual luciferase reporter assay HEK293T cells were co-transfected with firefly luciferase-expressing reporter vectors and renilla luciferase-expressing inner control vector in combinations of vectors overexpressing different genes as indicated ( 14 ). After culture for 48 hours, cells were harvested and lysed in lysis buffer for 30 minutes at room temperature. A luciferase kit (Promega, Cat#E1980) and Promega Max20/20 was used to measure the firefly and renilla luciferases activities following manufactory manual. 2.8 Quantitative RT-PCR Total RNA was extracted with TRIzol (Vazyme, Cat#R411-01), reverse-transcribed with All-in-One RT SuperMix Perfect (Vazyme, #R333-01), and used for PCR with SYBR qPCR Master Mix (Vazyme, #Q311-02) following manufactory instructions. PCR was performed in the following conditions: 95 °C for 2 min followed by 95 °C for 15 s and 60 °C for 30 s for 40 cycles. The reactions were run in triplicates on the Quantagene q225 Real-Time PCR System (Kubo Tech). The relative quantitation of real-time PCR product was measured using the comparative ΔΔCT method and presented in bar graph format. The sequences of primer sets were listed in Supplementary Table S1 in Supplemental information and data. 2.9 Co-immunoprecipitation and Western blotting Co-immunoprecipitation and Western blotting were performed as previously described ( 17 , 20 ). Antibodies used in this study were listed in the Supplemental information and data. 2.10 Semi-denaturing detergent agarose gel electrophoresis SDD-AGE was performed as previously described ( 14 , 21 ). In brief, cells were collected and lysed for 30 minutes with lysis buffer (20 mM Tris–HCl, pH 7.4-7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40) containing inhibitors for proteases and phosphatases. After centrifugation for 10 minutes at 12,000×g, supernatants were collected and added with 1×Sample buffer (0.5×TBE, 10% glycerol, 2% SDS, and 0.0025% bromophenol blue) and loaded onto a vertical 1.5% agarose gel. After electrophoresis in the running buffer (1×TBE and 0.1% SDS) for 60 minutes with a constant voltage of 100 V at 4°C, the proteins were subjected to Western blotting analysis. 2.11 ELISA The IFN-β and IL-6 in serum were determined by ELISA kits (JONLBIO, Cat#JL20219, Cat#JL20268) following manufactory manual. 2.12 Immunofluorescence assay Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at room temperature. Cells then were blocked and permeabilized for 30 minutes in blocking buffer (PBS containing 1% bovine serum albumin (BSA) and 0.1% Triton X-100). Cells were then incubated with primary antibody at 4 °C overnight and secondary antibody for 1 hour at room temperature. Nuclei were labeled by staining with DAPI. Images were collected using a Leica SP8 confocal laser microscopy system (Leica Microsystems) and processed with LAS X (V3.7.4.23463). All images are representative of at least three independent experiments. 2.13 Detection of cGAMP by mass spectrometry Standard cGAMP was purchased from Topscience (Cat#T10065L). The cGAMP levels in samples were measured by liquid-mass spectrometry (Vanquish Core HPLC and Q Exactive HF-X MS Orbitrap from Thermo Fisher). Detailed protocol was provided in Supplemental information and data. 2.14 Statistical analysis All data were presented as the mean ± SEMs and analyzed by GraphPad software. Data are representative of ≥3 independent biological replicates. Differences between experimental and control groups were determined by Student’s t -test (where two groups of data were compared) or by one-way or two-way ANOVA analysis (where more than two groups of data were compared). The P value <0.05 was defined as statistical significance. For animal survival analysis, the Kaplan–Meier method was adopted to generate the survival curves and analyzed with log-rank. The statistical analysis method used in each experiment was specified in the figure legends. 3 Results 3.1 DNASE1L3 upregulation by HSV-1 infection enhances innate immunity in vitro Nucleases and nucleic acid editing enzymes play key roles in DNA sensing and autoimmune diseases ( 4 ). However, whether and how these enzymes may regulate DNA virus infection is largely unknown. In this study, cell lysates from HSV-1-infected THP-1 cells exhibited stronger nuclease activities to degrade naked genomic DNA isolated from 143B cells than that from mock-infected THP-1 cells evidenced by the reduction of Alu200/Alu76 ratio (the ratio of long 200-bp Alu sequence to short 76-bp Alu sequence amplified by quantitative PCR) ( Figure 1A ; the corresponding agarose gel electrophoresis is shown in Supplementary Figure S1A ). This result suggests that HSV-1 infection increases the total intracellular nuclease activity. Consistently, HSV-1 infection upregulated the expression of multiple nuclease genes in human cell lines and primary mouse cells, of which DNASE1L3 (D1L3) was consistently upregulated in all cells ( Supplementary Figures S1B–E ). To test how these genes might contribute to HSV-1-induced innate immune responses, HEK293T cells were employed to perform dual luciferase reporter assay, which retained modest response to HSV-1 infection and DNA transfection for dual-luciferase assay described in previous reports ( 14 – 16 ). TREX1 was shown to reduce the HSV-1-induced activity of IFNB gene promoter as previously reported ( 22 , 23 ). Interestingly, DNASE1L1 (D1L1), DNASE1L2 (D1L2), and DNASE1L3 (D1L3) promoted the activation of IFNB gene promoter, of which DNASE1L3 displayed the most significant effect and exerted function dose-dependently ( Figures 1B, C ). Indeed, cell lysates from HSV-1-infected THP-1 cells showed increased ability to digest the nucleosomal DNA isolated from 143B cells ( Figure 1D ; the corresponding agarose gel electrophoresis is shown in Supplementary Figure S1F ), which is an optimal substrate for DNASE1L3 but not for other nucleases as a unique character of DNASE1L3. Furthermore, DNASE1L3 enhanced the activity of ISRE and NF-κB reporters ( Figure 1E ). These observations strongly suggest that DNASE1L3 upregulation is tightly associated with HSV-1 infection and enhances innate immune response. The endogenous protein expression of DNASE1L3 in THP-1 and HEK293T cells was detected and multiple bands were observed as previously reported, which was further verified by CRISPR/Cas9-mediated DNASE1L3 knockout ( Supplementary Figure S1G, H ). DNASE1L3 overexpression showed lower molecular weight than the endogenous DNASE1L3 in HEK293T cells as expected ( 13 ). It has been speculated that the multiple bands of DNASE1L3 originate from cell-type-specific differences in post-translational modifications such as ADP-ribosylation, proteolytic cleavage, or secretory processing in these cell lines ( 13 , 24 – 28 ). Moreover, only low-molecular-weight DNASE1L3 was secreted outside the THP-1 cells and measurable low-molecular-weight DNASE1L3 was observed in cytoplasm and nucleus in DNASE1L3-overexpressing THP-1 cells upon HSV-1 infection ( Supplementary Figure S1I ). DNASE1L3 overexpression promoted HSV-1-induced IRF3 phosphorylation, enhanced the expression of IFNB , TNFA , and IL6 , and inhibited HSV-1 replication in THP-1 cells ( Figures 1F–I ). These results demonstrate that upregulation and activation of DNASE1L3 by HSV-1 infection in turn enhances innate immune response. Figure 1 DNASE1L3 upregulation by HSV-1 infection enhances innate immunity in vitro . (A) Genomic DNA isolated from 143B cells was treated with cell lysates from Mock or HSV-1-infected THP-1 cells for times as indicated. Quantitative PCR was performed to measure the repetitive sequence Alu and the ratio of the long fragment amplification product Alu200 to the short fragment amplification product Alu76 was calculated. (B) Analysis on the activity of IFNB gene promoter by dual luciferase reporter assay in HSV-1-infected HEK293T cells co-transfected with Ctrl, DNASE1L1- (D1L1), DNASE1L2- (D1L2), DNASE1L3- (D1L3), DNASE2B- (D2B), or TREX1-overexpressing plasmids. (C) Analysis on the activity of IFNB gene promoter by dual luciferase reporter assay in HEK293T cells transfected with different doses of DNASE1L3-expressing plasmids as indicated. (D) The nucleus isolated from 143B cells was treated with cell lysates from Mock or HSV-1-infected THP-1 cells. Quantitative PCR was performed and the Alu200/Alu76 was calculated. (E) Analysis on the activity of IFNB gene promoter, ISRE reporter, or NF-κB reporter in Mock or HSV-1-infected HEK293T cells transfected with vector (Ctrl) or DNASE1L3-expressing (D1L3) plasmids as indicated. (F) Cell lysates from Mock or HSV-1-infected control (Ctrl) or DNASE1L3-overexpressing (D1L3) THP-1 cells were used for Western blotting analysis as indicated. (G) Quantitative RT-PCR analysis on the expression of IFNB , TNFA , and IL6 in Mock- or HSV-1-infected Ctrl or D1L3 THP-1 cells. (H) Quantitative RT-PCR analysis on the expression of UL30 mRNA to measure HSV-1 replication in HSV-1-infected Ctrl and D1L3 THP-1 cells. (I) Plaque formation assay to measure HSV-1 replication using supernatants from HSV-1-infected Ctrl and D1L3 THP-1 cells. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, non-significance. Statistical significance was determined using two-tailed Student’s t tests in (D, H, I) or one-way ANOVA in (A–C) , two-way ANOVA in (E, G) . 3.2 DNASE1L3 deficiency inhibits innate immunity against HSV-1 in vitro We further tested the effe
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