Homeostatic Milieu Induces Production of Deoxyribonuclease 1–Like 3 from Myeloid Cells

Homeostatic Milieu Induces Production of Deoxyribonuclease 1−like 3 from Myeloid Cells

This information is current as Shoichiro Inokuchi, Hiroki Mitoma, Shotaro Kawano, Shota of September 25, 2021. Nakano, Masahiro Ayano, Yasutaka Kimoto, Mitsuteru Akahoshi, Yojiro Arinobu, Hiroshi Tsukamoto, Koichi Akashi, Takahiko Horiuchi and Hiroaki Niiro J Immunol published online 18 March 2020

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 18, 2020, doi:10.4049/jimmunol.1901304 The Journal of Immunology

Homeostatic Milieu Induces Production of Deoxyribonuclease 1–like 3 from Myeloid Cells

Shoichiro Inokuchi,*,† Hiroki Mitoma,* Shotaro Kawano,* Shota Nakano,* Masahiro Ayano,* Yasutaka Kimoto,‡ Mitsuteru Akahoshi,* Yojiro Arinobu,* Hiroshi Tsukamoto,*,x Koichi Akashi,* Takahiko Horiuchi,‡ and Hiroaki Niiro{

DNase 1–like 3 (DNase1L3), which belongs to DNase1 family, was originally identified as one of apoptosis- and necrosis-related endonucleases that fragmentate intranucleosomal DNA. A loss-of-function mutation has been reported in murine models of systemic lupus erythematosus (SLE) and in familial SLE patients. These reports suggest DNase1L3 plays an important role in the prevention of developing SLE; however, expression and function of DNase1L3 in human immune systems have been largely unclarified. As previous reports showed DNase1L3 is expressed in hematopoietic organs, we first analyzed expression levels of DNase1L3 in each subset of human peripheral blood cells by quantitative real-time PCR. Plasmacytoid dendritic cells showed the Downloaded from highest expression levels of DNase1L3 mRNA among peripheral blood cells. IL-4 enhanced DNase1L3 expression in monocytes, monocyte-derived dendritic cells, and monocyte-derived macrophages (MDMs), but not in T cells, B cells, or plasmacytoid dendritic cells. Together with IL-4, all-trans retinoic acid and apoptotic cells efficiently upregulated expression of DNalse1L3 in MDMs. As a result of intracellular signaling analysis, Jak1-IRS2-ERK/PI3K pathway was essential for IL-4–induced DNase1L3 expression. IL-4–treated monocyte-derived dendritic cells and MDMs secreted active DNase1L3 protein that could degrade liposome–DNA complexes, which were resistant to DNase1. Our results indicate DNase1L3 is secreted by innate immune cells http://www.jimmunol.org/ and may play a critical role in the tissue homeostasis and on prevention of developing autoimmunity by degrading self-DNA. The Journal of Immunology, 2020, 204: 000–000.

eoxyribonuclease 1–like 3 (DNase1L3) belongs to DNase1 DNase1 because of the surrounding DNA-binding proteins, family (1). This nuclease originally identified as one such as histones. D of apoptosis- (2, 3) and necrosis-related endonucleases Disruption of the nuclease activity has been highlighted in the (4) that fragmentate intranucleosomal DNA (5, 6). Unlike DNase1, context of autoimmunity, notably, systemic lupus erythematosus DNase1L3 has positively charged amino acid sequences in its (SLE). One of the key features of SLE has been thought to be by guest on September 25, 2021 C-terminal region (7), and it is postulated that this feature charac- broken tolerance toward self-component, especially dead cells terizes the function of DNase1L3 to digest protein-associated DNA in (12, 13). The clearance of dead cells is impaired in SLE patients addition to naked DNA (8–11). Therefore, DNase1L3 can degrade from various causes, which leads to an accumulation of auto- nucleosomal DNA more efficiently than DNase1, which is resistant to antigens, followed by autoimmune responses like the emergence of autoreactive lymphocytes and the production of autoanti- bodies. Deletion or loss of function in some DNases was variably associated with the development of autoimmune diseases due to *Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka 812-8582, Japan; †Department of Rheumatol- impaired clearance of dead cells in mice and humans. DNase1- ogy, National Hospital Organization Fukuoka National Hospital, Fukuoka 811-1394, deficient mice develop SLE with classical findings, and poly- Japan; ‡Department of Internal Medicine, Kyushu University Beppu Hospital, Beppu x morphisms of DNase1 were reported in SLE patients (14–16). 874-0838, Japan; Department of Rheumatology, Shin-Kokura Hospital, Kitakyushu 803-8505, Japan; and {Department of Medical Education, Faculty of Medical Sci- DNase2-deficient mice show stimulator of IFN genes (STING)– ences, Kyushu University, Fukuoka 812-8582, Japan dependent type I IFN signature (17, 18), and patients having ORCIDs: 0000-0002-5501-8852 (S.I.); 0000-0002-2107-7459 (H.M.); 0000-0002-5221- loss-of-function mutations in DNASE2 demonstrate severe 1008 (S.K.); 0000-0002-2954-9676 (M. Ayano); 0000-0002-3556-4709 (M. Akahoshi). neonatal anemia, membranoproliferative glomerulonephritis, Received for publication October 29, 2019. Accepted for publication February 12, liver fibrosis, deforming arthropathy, and increased anti-DNA 2020. Abs. Mutation of three prime repair endonuclease (TREX1) was This work was supported by Japan Society for the Promotion of Science KAKENHI reportedtocauseAicardi–Goutie`res syndrome, which manifests Grant JP14535306. STING-dependent type I IFN response and SLE-like symptoms Address correspondence and reprint requests to Dr. Hiroki Mitoma, Department of Medicine and Biosystemic Sciences, Kyushu University Graduate School of Medical (19, 20). Polymorphism of TREX1 was also reported in SLE Sciences, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail address: patients (21). [email protected] As other deoxynucleases, loss of function of DNase1L3 has been The online version of this article contains supplemental material. reported to cause autoimmune phenotypes. Mutations of DNase1L3 Abbreviations used in this article: ATRA, all-trans retinoic acid; DNase1L3, DNase 1–like gene that cause loss of function have been reported in murine models 3; HA, hemagglutinin; IFN-MoDC, MoDC differentiated by type 1 IFN and GM-CSF; IL-4–MoDC, MoDC differentiated by IL-4 and GM-CSF; IRS2, insulin receptor of SLE (22). Familial SLE is caused by DNase1L3 mutations with substrate 2; MDM, monocyte-derived macrophage; MoDC, monocyte-derived den- an autosomal recessive pattern of inheritance in a very-high dritic cell; NET, neutrophil extracellular trap; pDC, plasmacytoid dendritic cell; qPCR, penetrance rate, which onset in childhood and show the typical quantitative PCR; shRNA, short hairpin RNA; SLE, systemic lupus erythematosus. phenotype of SLE, such as elevation of anti-dsDNA Abs, low Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 serum complement, and lupus nephritis (23). In addition, a recent

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901304 2 HOMEOSTASIS-ASSOCIATED MYELOID CELLS PRODUCE DNase1L3 report showed that DNase1L3-deficient mice also develop features medium was replaced by fresh medium with or without 20 ng/ml human of SLE (24). These mice presented impaired processing of the IL-4. After 24 h, indicated analyses were performed. chromatin in microparticles from apoptotic cells, and the authors Differentiation of human monocyte-derived cells suggested a deficiency of DNase1L3 led to a loss of self-tolerance toward apoptotic cells and contribute to SLE pathogenesis. RPMI 1640 supplemented with 10% FBS (Sigma-Aldrich), MEM Non- Essential Amino Acids Solution (FUJIFILM Wako Pure Chemical), Moreover, several genetic variants of DNase1L3, which have 50 nM of 2-ME (Sigma-Aldrich), and Penicillin-Streptomycin Solution less nuclease activity than wild type, were also associated with (FUJIFILM Wako Pure Chemical) were used for primary cell culture. disease susceptibility in systemic sclerosis (25–27), rheumatoid Monocytes were isolated from healthy subjects using RosetteSep Human arthritis (28), and hypocomplementemic urticarial vasculitis Monocyte Enrichment Cocktail (STEMCELL Technologies). For differentiation of monocyte-derived dendritic cells (MoDCs), 2.5 3 105 of syndrome (29). monocytes in 500 ml of medium with GM-CSF (20 ng/ml; PeproTech) and Accumulating evidences indicate that DNase1L3 plays an es- IL-4 (20 ng/ml; PeproTech) were seeded into 48-well tissue culture plates. sential role in processing self-DNA and is required for preventing Culture medium was changed every 3 d. After 5 d of differentiation, the development of autoimmunity. DNase1L3-deficient mice and indicated experiments were performed. In some experiments, 20 ng/ml humans normally express the other DNases; hence, DNase1L3 may of GM-CSF and Universal Type I IFN (1000 U/ml; R&D Systems) were added to the monocytes for the differentiation into the other have a unique function in degrading self-DNA in vivo. Expression form of MoDCs (MoDCs differentiated by type 1 IFN and GM-CSF and function of DNase1L3 in human immune systems have been [IFN-MoDCs]). In the case of IFN-MoDCs, indicated experiments were largely unknown; therefore, we aimed to reveal how the expression performed after 3 d of differentiation. To differentiate monocyte-derived 3 5 of DNase1L3 is regulated and how DNase1L3 degrades DNA in macrophages (MDMs), 1.5 10 of monocytes in 300 ml of medium with M-CSF (50 ng/ml; PeproTech) was seeded on 48-well tissue culture plates. human immune cells. Culture medium was changed every 3 d. After 7 d of differentiation, in- Downloaded from dicated experiments were performed. Materials and Methods Flow cytometry and Abs Cell lines Peripheral blood was collected from healthy donors, and PBMCs were The 293T, THP-1, and HUVEC were purchased from American Type separated by a density gradient method using Lymphoprep (Axis-Shield). Culture Collection, and fibroblast-like synoviocyte from rheumatoid For isolation of WBCs, whole blood was treated with RBC lysis buffer arthritis patients were purchased from Cell Applications. All cell lines (BioLegend) according to the manufacturer’s instrument. PBMCs or WBCs http://www.jimmunol.org/ were kept in culture for ,1 consecutive mo. The subcultures were done in were washed with PBS and resuspended with 2% FBS (Sigma-Aldrich) accordance with the manufacturer’s instruments. When differentiating with 2 mM EDTA (Thermo Fisher Scientific) in PBS. The cells were in- THP-1 toward macrophage, 0.5 3 106 cells per well of THP-1 in 48-well cubated with fluorescence-labeled Abs for 20 min at 4˚C. After washing plates were incubated with 300 nM PMA (Sigma-Aldrich) for 24 h, and the cells, dead cells were stained using propidium iodide (BioLegend), and

FIGURE 1. DNase1L3 expression in hu- by guest on September 25, 2021 man blood cells. (A) Data of DNase1L3 mRNA expressions in human peripheral blood cells were obtained from GSE24759. (B and C) Quantitative real-time PCR of DNase1L3 mRNA was performed in each immune cell subset from peripheral blood cells (B), and in monocytes (Mono) or Mono-derived cells (C). Relative expression levels of DNase1L3 were normalized to b-actin and compared with the values obtained for Mono in each assay. The sample of Mono was assigned the arbitrary value of 1. Data are representative data from two to three independent experiments with different donors, respectively. Analysis for each sample was carried out in triplicate. The error bars indicate mean 6 SD. *p , 0.05 versus Mono, Student t test. Baso, basophil; cm, central memory; CMP, common myeloid progenitor; DN-B, double negative B cell; em, effector memory; Eos, eosinophil; ERY, erythroid cell; Gran, granulocyte; HSC, hematopoietic stem cell; IFN-DC, MoDC differentiated with type I IFN and GM-CSF; IgM-Bmem, IgM memory B cell; imm-Mono, immature Mono; MDM, monocyte-derived macrophages; Mega, megakaryocyte; MEP, megakaryocyte/ erythroid progenitor; Swi-Bmem, switched memory B cell. The Journal of Immunology 3

Table I. Fluorescence-labeled Abs used in the study [v/v]) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). For fractionation of cytosolic protein and nu- Ag Fluorescence Clone Source clear protein, NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) was used. Protein were denaturated with CD3 FITC UCHT1 BioLegend 43 sample buffer (200 mM Tris HCl [pH 6.8], 12.5% SDS [w/v], 40% CD3 PerCP-Cy5.5 UCHT1 BioLegend glycerol [v/v], and 24% 2-ME [v/v]) at 100˚C for 5 min. Protein were CD4 Allophycocyanin RPA-T4 BioLegend resolved by SDS-PAGE and transferred to PVDF membranes. The mem- CD8 Allophycocyanin-Cy7 HIT8a BioLegend brane was blocked in 3% BSA (w/v) and 0.05% Tween 20 (v/v) in TBS CD11c Allophycocyanin B-ly6 BD Biosciences and incubated with mouse anti-hemagglutinin (HA) Ab (1:10,000, CD14 FITC M5E2 BD Biosciences TANA2; Medical & Biological Laboratories), mouse anti-GAPDH– CD14 PerCP-Cy5.5 63D3 BioLegend peroxidase Ab (1:10,000, GAPDH-71.1; Sigma-Aldrich), mouse anti– CD16 PE 3G8 Beckman Coulter histone deacetylase Ab (1:2000, 2E10; MilliporeSigma). Anti-rabbit IgG CD16 PerCP-Cy5.5 3G8 BioLegend HRP-linked Ab or anti-mouse IgG HRP-linked Ab (GE Healthcare) were CD19 PE-Cy7 HIB19 BD Biosciences used for the detection of primary rabbit or mouse Ab, respectively. CD19 PerCP-Cy5.5 HIB19 BioLegend When detecting DNase1L3, capillary electrophoresis immunoassay using CD56 PE HCD56 BioLegend the Wes machine (ProteinSimple) was applied in accordance with the CD56 PerCP-Cy5.5 HCD56 BioLegend manufacturer’s protocols. In brief, 5 ml of denatured lysates were loaded CD123 PE 6H6 BioLegend on the plate and separated by size. DNase1L3 protein was labeled with polyclonal rabbit anti-DNase1L3 Ab (1:1000; Lifespan Biosciences) and detected by chemiluminescence using anti-rabbit IgG HRP-linked Ab live cell sorting was performed for each cell subset using FACSAria supplied from the manufacturer. cell sorter (BD Biosciences) according to the following gates: neutrophils (CD16+ cells in granulocyte gate), eosinophils (CD162 cells in Immunofluorescence and in situ hybridization Downloaded from + + + granulocyte gate), CD4 T cells (CD3 CD4 cells in lymphocyte gate), To detect DNase1L3 mRNA in immune cells in human spleen formalin- + + + 2 + CD8 T cells (CD3 CD8 in lymphocyte gate), NK cells (CD3 CD56 fixed, paraffin-embedded specimen (OriGene Technologies), ViewRNA + cells in lymphocyte gate), B cells (CD19 cells in lymphocyte gate), in situ hybridization method followed by immunohistochemistry was 2 2 + monocytes (CD3 CD94 CD14 cells), myeloid dendritic cells applied. Human DNase1L3 probe was obtained from Thermo Fisher Sci- (CD32CD192CD942CD142HLA-DRhighCD11c+CD1232 cells), and plas- 2 2 2 2 entific. Primary rabbit anti-CD11c mAb was obtained from Abcam. All macytoid dendritic cells (pDCs) (CD3 CD19 CD94 CD14 HLA- procedures were performed by Tokushima Molecular Pathology Institute. high 2 +

DR CD11c CD123 cells). Sorted cells were lysed in RLT buffer http://www.jimmunol.org/ and used for expression analysis (described below). Fluorescent- Overexpression of human DNase1L3 labeled Abs used in the assay were summarized in Table I. DNsae1L3 cDNA was cloned into HA-pCMV plasmid (Takara Bio) and Small chemical inhibitors and blocking Ab pcDNA 3.1/H5-His (Thermo Fisher Scientific). DNase1L3 expression vectors were transfected into 293T with Lipofectamine 3000 (Thermo SP600125 (JNK inhibitor) was purchased from Sigma-Aldrich. U0126 Fisher Scientific). The efficacy of overexpression was routinely checked by (ERK inhibitor) was obtained from Promega, and SB203580 (p38 inhibitor) qPCR. Forty eight hours after transfection, cells and supernatants were and LY294002 (PI3K inhibitor) were purchased from Merck. Anti–IL-4 collected and used for indicated assays. blocking Ab (MAB204) was purchased from R&D Systems. Each inhibitor was added at the indicated concentration at 30 min before stimulation. Lentiviral transduction

Quantitative real-time PCR Lentiviral vector pLKO.1-expressing scrambled sequences or short hairpin by guest on September 25, 2021 RNA (shRNA) sequences targeting insulin receptor substrate 2 (IRS2) Total RNA was extracted using RNeasy Mini Kit (QIAGEN) in accordance (TRCN0000061253 and TRCN0000061257) were purchased from Sigma- with the manufacturer’s protocol. cDNA was synthesized by iScript Aldrich. psPAX2 and pMD2.G were obtained from Addgene and trans- Reverse Transcriptase (Bio-Rad Laboratories). Quantitative PCR (qPCR) fected together with pLKO.1 into 293T to produce lentiviral particles. was performed using iTaq Universal SYBR Green Supermix (Bio-Rad THP-1 were infected with lentiviral particles under 4 mg/ml polybrene Laboratories). The primers used in the study were applied from previous (Sigma-Aldrich) for 48 h. Lentivirus-transduced cells were selected under studies (30, 31) or designed using Primer3 software. The primer sequences 5 mg/ml puromycin (InvivoGen) for 48 h. The efﬁcacy of IRS2 knockdown are shown in Table II. All reactions were normalized to b-actin to evaluate was determined by qPCR. the relative gene expression. Analysis of DNase activity Western blotting To evaluate DNA digestion, 500 ng DNA puriﬁed from PBMC using Whole cell lysate was extracted in ice-cold lysis buffer (1% Triton X-100 [v/v], NucleoSpin Tissue (Takara Bio) was incubated with supernatant from 50 mM Tris HCl [pH 7.5], 5 mM EDTA, 150 mM NaCl, and 10% glycerol DNase1L3-expressing, DNase1-expressing, or mock 293T for 30 min at

Table II. qPCR primers used in this studya

Primer Name Primer Sequence Ref ACTB forward 59-AGAGCTACGAGCTGCCTGAC-39 30 ACTB reverse 59-AGCACTGTGTTGGCGTACAG-39 30 DNASE1L3 forward 59-ATCTGCTCCTTCAACGTCAG-39 DNASE1L3 reverse 59-GTTGCTGTCCTTGATTTCCATC-39 IRS2 forward 59-ACCTACGCCAGCATTGACTT-39 31 IRS2 reverse 59-CATCCTGGTGATAAAGCCAGA-39 31 DNASE1L1 forward 59-CATTTGTGGCCCAGTTCTCT-39 DNASE1L1 reverse 59-GGGAGACCTCCAGAAACACA-39 DNASE1L2 forward 59-TTCGGTGACAGCAAAGTGTC-39 DNASE1L2 reverse 59-GCTGTTGATCTGCTCCATGA-39 DNASE1 forward 59-CCCTCGTCAGCTACATTGTG-39 DNASE1 reverse 59-TCTGGTGCATCCTGATTGAG-39 DNASE2 forward 59-ACCAGCTGGAAGGGATCTTT-39 DNASE2 reverse 59-GGCAAAGCTCTGGAAAACAG-39 aPrimers for qPCR were applied from previous studies or designed using Primer3 software. Ref, reference. 4 HOMEOSTASIS-ASSOCIATED MYELOID CELLS PRODUCE DNase1L3

37˚C. Degraded DNA was analyzed by agarose gel electrophoresis. For producing liposome–DNA complex, 1 ng of pCMV plasmid was complexed with Lipofectamine 2000 (Thermo Fisher Scientific) in Opti-MEM (Thermo Fisher Scientific). The supernatant from DNase1L3-expressing, DNase1-expressing, or mock 293T was incubated with liposome–DNA complex or 1 ng pCMV plasmid for 30 min at 37˚C. Residual DNA was measured by qPCR using plasmid-specific primer, and DNase activity was calculated. Evaluation of microarray datasets To assess DNase1L3 mRNA expression in human blood cells, we analyzed dataset GSE24759 (Broad Institute Differentiation Map Portal), in which 211 arrays for 38 types of purified normal human hematopoietic cells were included (32). We used average expression of DNase1L3 for all replicates of each cell type in the dataset from the Differentiation Map. When evaluating DNase1L3 expression in mice, we obtained microarray datasets from GSE15907 (ImmGen Consortium), which contain 653 arrays for isolated primary cells from multiple immune lineages (33). DNase1L3 expression in each cell subset was extracted, and the cell populations were categorized into groups based on tissue (thymus, spleen, bone marrow, liver, lung, lymph node, peritoneal, intestine, pancreas, skin, adipose, brain, synovial fluid, kidney, and blood) and cell types (dendritic cell, macrophage, monocyte, thymocyte, gdT cell, CD4-positive T cell, Downloaded from CD8-positive T cell, NK cell, stem/progenitor/precursor cell, granulocyte, fibroblast, stromal cell, epithelial cells, and endothelial cell). Statistical analysis Results were presented as mean 6 SD, and the statistical analyses were performed using R studio software, version 3.5.2 (www.r-project.org). Unpaired, two-tailed Student t test and two-way ANOVA were used for http://www.jimmunol.org/ the evaluation of statistical significance as indicated in the figure legends. The p values ,0.05 were considered statistically significant.

Results DNase1L3 mRNA is highly expressed in myeloid cells and monocyte-derived cells Hematopoietic organs are one of the major DNase1L3-expressing

tissues in human (34). We first investigated the expression levels of by guest on September 25, 2021 DNase1L3 in human blood cells and human tissues using published datasets. The GSE24759 dataset published by Novershtern et al. (32) contained various cell populations and has been cited in many scientific articles (35–37). Therefore, we searched DNase1L3 expression in human blood cells using this dataset and found myeloid cells, especially pDCs, have high expression levels of DNase1L3 mRNA (Fig. 1A). To validate expression levels of DNase1L3 in immune cells, we analyzed DNase1L3 mRNA expression via qPCR in each population of human peripheral blood cells purified by flow sorting (Tables I and II). Our results confirmed that peripheral blood cells, especially pDCs, expressed

(20 ng/ml), or TGF-b (5 ng/ml). (E) CD19-positive B cells were treated with veh, IL-4 (20 ng/ml) or IFN-g (20 ng/ml). (F) M-CSF–differentiated MDMs were treated with or without apoptotic lymphocytes (apop), in concomitant use of IL-4 (20 ng/ml) and/or ATRA (1 mM) or nonstimulation as indicated. (G) Prior to stimulation, M-CSF–differentiated MDMs were pretreated with (anti-IL-4) or without (ctrl) anti–IL-4 neu- tralization Ab (5 mg/ml) for 30 min. After that, IL-4 (20 ng/ml) plus ATRA (1 mM) or veh was added to wells. (H) M-CSF–differentiated MDMs were treated with indicated cytokines. Relative expression levels of DNase1L3 were normalized to b-actin and compared with the values obtained for veh in each assay. The experiments (A, B, and F) are representative of three FIGURE 2. IL-4 induces DNase1L3 expression in human myeloid cells. independent experiments with different donors. The experiment (C–E, G, (A–E) Quantitative real-time PCR of DNase1L3 was performed in human and H) is one of three independent experiments performed with blood cells immune cells treated with indicated cytokines and chemicals. (A) Mono- from a single donor. The sample of nonstimulation was assigned the ar- cytes were treated with vehicle (veh), IL-4 (20 ng/ml), or IFN-g (20 ng/ml). bitrary value of 1. Analysis for each sample was carried out in triplicate. (B) M-CSF–differentiated MDMs were treated with veh, IL-4 (20 ng/ml), The error bars indicate mean 6 SD. *p , 0.05, **p , 0.05, ***p , 0.05, or IFN-g (20 ng/ml). (C) pDCs were treated with veh or IL-4 (20 ng/ml). versus veh and by Student t test, by Student t test, and by two-way (D) CD4-positive T cells were treated with veh, IL-4 (20 ng/ml), IFN-g ANOVA, respectively. The Journal of Immunology 5

DNase1L3 mRNA (Fig. 1B). As myeloid cells are the potent repair. Retinoic acid is essential for the conversion of MDMs into populations expressing DNase1L3 mRNA, we further investigated tissue-resident macrophages (39, 40); therefore, we stimulated DNase1L3 mRNA expression in monocyte-derived cells. MoDCs MDMs with apoptotic cells and/or all-trans retinoic acid (ATRA) differentiated by IL-4 and GM-CSF (IL-4–MoDCs) had prominent with or without IL-4. Apoptotic cells and ATRA in combination DNase1L3 mRNA expression compared with monocytes. In with IL-4 induced extremely high expression of DNase1L3. To a contrast, IFN-MoDC did not show elevation of DNase1L3 mRNA lesser extent, stimulation of apoptotic cells or ATRA with IL-4 expression compared with monocytes (Fig. 1C). Therefore, also upregulated the expression of DNae1L3 mRNA (Fig. 2F). DNase1L3 mRNA was expressed in human peripheral blood cells, Taken together, the treatment of myeloid cells with IL-4 efﬁciently especially resting pDCs and IL-4–MoDCs. enhanced DNase1L3 expression. Moreover, factors associated with tissue-resident macrophages, apoptotic cells, and ATRA co- IL-4 efﬁciently induces DNase1L3 mRNA expression in ordinated with IL-4 to induce DNase1L3 expression. myeloid cells The result that IL-4–MoDCs expressed much higher DNase1L3 DNase1L3 is expressed in human spleen mRNA than IFN-MoDCs led us to further investigate the effect To investigate DNase1L3 expression in human tissue, we selected of IL-4 on DNase1L3 expression. IL-4–treated MDMs and spleen according to the following reasons: 1) global high monocytes showed markedly high expression levels of DNase1L3 DNase1L3 mRNA expression in spleen (34), 2) IL-4 expression mRNA, whereas IL-4–treated T cells, B cells, and pDCs did in lymphoid organs, and 3) presence of abundant apoptotic not show substantial upregulation of DNase1L3 expression lymphocytes. Because of the absence of available DNase1L3-

(Fig. 2A–E). Although other cytokines associated with macrophage specific Ab for tissue specimen, mRNA in situ hybridization Downloaded from polarization were investigated, any of them did not upregulate together with immunofluorescence was performed. As shown in DNase1L3 mRNA expression (Fig. 2H). To evaluate the effect of Fig. 3, CD11c-positive cells, which contain mainly monocytes/ IL-4 on DNase1L3 mRNA expression in nonhematopoietic cells, we macrophages, highly expressed DNase1L3 mRNA, whereas used human fibroblast-like synoviocytes from patients with rheuma- CD11c-negative cells also expressed DNase1L3 mRNA. toid arthritis and HUVECs. IL-4–induced upregulation of DNase1L3 To confirm DNase1L3 expression in tissue cells, we evalu-

mRNA was not observed in these cells (Supplemental Fig. 1). ated transcriptome of each cell subset from murine tissue and http://www.jimmunol.org/ IL-4 with apoptotic cells has been reported to polarize MDMs blood (GSE15907) (33). Macrophages and dendritic cells from toward tissue-repairing macrophages (38); hence, we hypothesized intestine, spleen, and lymph node showed remarkably high that DNase1L3 may work to degrade DNA in the process of tissue expression of DNase1L3 (Supplemental Fig. 2). The fact that by guest on September 25, 2021

FIGURE 3. DNase1L3 expression in human spleen. (A–C) DNase1L3 mRNA in 5-mm-thick sections of formalin-fixed, paraffin-embedded human spleen was visualized by in situ hybridization. Subsequently, immunofluorescence was performed to detect the colocalization of DNase1L3 mRNA and CD11c. Arrowheads de- note DNase1L3 mRNA expression in CD11c-positive cells. 6 HOMEOSTASIS-ASSOCIATED MYELOID CELLS PRODUCE DNase1L3

the highest expression was identiﬁed in intestinal macrophages supports our ﬁnding of ATRA-induced DNase1L3 expression, because intestinal tissue contain a high amount of retinoic acid, which contribute to maintaining intestinal homeostasis (41). These data indicate that myeloid cells are the potent DNa- se1L3 producer in peripheral blood and tissues and that the polarization toward tissue-resident macrophages via IL-4, apoptotic cells, and ATRA may be the important factor for up- regulating DNase1L3. IRS2 is required for DNase1L3 mRNA expression We next investigated intracellular signaling of IL-4 required for the upregulation of DNase1L3 expression. IL-13R shares IL-4R a-chain with IL-4R, and IL-13 is also able to polarize macrophages into M2 phenotype through STAT6-dependent signaling as with IL-4 (42). We analyzed the effect of IL-13 on DNase1L3 mRNA expression in MDMs. IL-13 did not upregulate DNa-

se1L3 mRNA (Fig. 4A). One of the differences between the Downloaded from signaling of IL-4 and IL-13 is the usage of IRS2, an adaptor protein associated only with the IL-4R, but not with the IL-13R. IL-4 signaling through IRS2 induces activation of ERK and PI3K, leading to IL-4–dependent gene expression in macrophages(43,44).ToclarifytheeffectsofSTAT6andIRS2on

DNase1L3 mRNA expression, small molecule inhibitors of each http://www.jimmunol.org/ molecule were used. As shown in Fig. 4B, NT157 (IRS2 inhibitor) inhibited the IL-4–induced DNase1L3 mRNA expression, but AS1517499 (STAT6 inhibitor) did not. To conﬁrm the involvement of IRS2, shRNA was performed in THP-1 monocytes. IRS2-knockdown cells showed signiﬁcantly reduced DNase1L3 mRNA expression compared with scramble controls after stimulation with IL-4 (Fig. 4C). IRS2 is phosphorylated by JAKs and recruit downstream signaling molecules (43, 44) To

identify which JAKs are responsible for DNase1L3 expression, by guest on September 25, 2021 effects of tofacitinib (JAK1/3 inhibitor) and baricitinib (JAK1/2 inhibitor) were examined. Both of these compounds completely inhibited IL-4–induced DNase1L3 expression; hence, JAK1 was proposed for the responsible kinase in this intracellular signaling pathway (Fig. 4D). We next examined the involvement of MAPKs and PI3K, which are downstream of IRS2, in the expression of DNase1L3 via IL-4 using speciﬁc chemical inhibitors for p38, JNK, ERK, and PI3K (Fig. 4E, 4F). Inhibition of MAPKs, especially ERK, and PI3K led to suppression of DNase1L3 in- duction via IL-4, indicating that MAPK and PI3K are required for DNase1L3–mRNA expression via IL-4. In summary, IRS2 acti- A F FIGURE 4. IRS-2 is essential for DNase1L3 expression in MDM. ( – ) vated by JAK1 is a key adaptor signaling adaptor molecule for the Quantitative real-time PCR of DNase1L3 mRNA was performed. (A) transcription of DNase1L3 via activating MAPK and PI3K. M-CSF–differentiated MDMs were stimulated with IL-4 (20 ng/ml) or IL-13 (20 ng/ml). (B) M-CSF–differentiated MDMs were pretreated with the in- DNase1L3 protein is secreted into the extracellular space dicated inhibitors or vehicle (veh [DMSO]) for 1 h and then stimulated with 20 ng/ml of IL-4. (C) IRS-2 was knocked down with shRNA using the DNase1L3 contains a signal peptide sequence in the N terminus lentiviral system (sh-IRS2-1 and sh-IRS2-2) in THP-1 cells. For control, and has been thought to be secreted into the extracellular space vectors carrying scrambled sequences (scr1 and scr2) were also included. (34). However, some other reports have documented that this THP-1 cells transduced with the indicated shRNA lentiviral particles, in- protein also bears potential nuclear localization sequences and cluding the puromycin-resistant gene, were treated with puromycin for se- could localize in nucleus. To clarify whether DNase1L3 protein lection. Selected cells were differentiated into macrophages with PMA and could be secreted into extracellular space or not, we used a 293T- treated with IL-4. (D–F) M-CSF–differentiated MDMs were pretreated with overexpression system. DNase1L3 cDNA was cloned into pCMV the indicated inhibitors or veh (DMSO) for 1 h and then stimulated with 20 plasmid, and DNase1L3-coding pCMV plasmid was transfected ng/ml of IL-4. Relative expression levels of DNase1L3 were normalized to into 293T cells. As shown in (Fig. 5A), DNase1L3, similarly b-actin and compared with the values obtained for veh in each assay. The with DNase1, was detected in culture supernatants. We next sample of veh was assigned the arbitrary value of 1. The experiments, except for (C), were performed with monocytes from a single donor, and data are evaluated intracellular localization of DNase1L3 in human THP-1 representative of at least three independent experiments. The error bars in- monocytes via subcellular fractionation. DNase1L3 existed in the dicate mean 6 SD. *p , 0.05, **p , 0.05, versus veh and by two-way cytosol, but not in the nucleus at steady-state (Fig. 5B, 5C). ANOVA, respectively. A157949, STAT6 inhibitor (200 nM); Bar, baricitinib Therefore, DNase1L3 is localized in the cytosol and could be (300 nM); NT-157, IRS-2 inhibitor (1 mM); Tof, tofacitinib (300 nM). secreted into the extracellular space. The Journal of Immunology 7

resistant to DNase1 (7, 24); therefore, we tested nuclease activities of secreted DNase1L3 against DNA-associated liposome, Lipofect- amine 2000. Secreted DNase1L3 from 293T cells overexpressing DNase1L3 could degrade DNA–liposome complex, whereas DN- ase1 could not (Fig. 6B). Nuclease activity toward neutrophil extracellular traps (NETs), which is net-like DNA structure ex- truded from neutrophils undergoing cell death called NETosis, was also examined in our system, because impairment of degradation of NETs have been reported in autoimmune disease like SLE (45–47). As shown in Fig. 6C, secreted DNase1L3 could degrade NETs to a similar extent to DNase1. Synergistic effects between DNase1 and DNase1L3 was not observed. We further evaluated nuclease activities of supernatants from myeloid cells. Because of the sensitivity, we applied a real-time qPCR-based quantiﬁcation system as previously described in primary cells (24). As a result, the enhanced DNase activity in supernatant from IL-4–MoDC was observed (Fig. 6D, 6E). Additionally, the supernatant from IL-4–treated MDMs had higher DNase activity

than supernatant from nontreated MDMs (Fig. 6F). Because Downloaded from IL-4 could not upregulate DNase-associated molecules other than DNase1L3 (Supplemental Fig. 3A–D), it is suggested that IL-4–induced nuclease activity in supernatant secreted from MDM was mediated by DNase1L3.

Discussion http://www.jimmunol.org/ In this study, we identified IL-4 as an important regulator of DNase1L3 expression, especially in myeloid cells. A recent report showed IL-4–induced DNase1L3 secretion from MoDC, which is consistent with our findings (48). As IL-4 is thought to be required for the tissue-repair response through alternative macrophage activation (42), DNase1L3 may play a role in tissue homeostasis after tissue injury. IL-4 is expressed in lymphoid organs at steady-state (49), and DNase1L3 was expressed in CD11c- positive and -negative cells in spleen. As an accumulation of by guest on September 25, 2021 dead cells can be occurred in spleen and in secondary lymphoid organs (50, 51), secreted DNase1L3 in these tissues may contribute to the clearance of dead cells. In the absence of DNase1L3, an accumulation of autoantigen followed by eliciting autoimmune responses could undergo and lead to the development of autoimmunity like SLE. The concept of the importance of DNase1L3 for tissue homeostasis is also supported by our result that ATRA and apoptotic cells in concert with IL-4 induce higher FIGURE 5. DNase1L3 protein is localized in the cytosol at a steady-state expression of DNase1L3 than IL-4 alone. ATRA can polarize and secreted into the extracellular space. (A) Human DNase1L3 (1L3) and human DNase1 (D1) were cloned into pCMV-HA vector. The constructs or macrophages into tissue-resident phenotype (39, 40), and apo- mock vector were transfected into 293T cells (mock, HA-D1, and HA-1L3) ptotic cells enhance the IL-4–induced expressions of genes asso- with Lipofectamine 3000. The cell lysates and supernatants were collected. ciated with tissue-repair responses (38). Immunoprecipitation using the supernatant was performed by anti-HA Ab. IL-4 and IL-13 have similar roles in terms of allergic, fibrotic, The samples were separated by SDS-PAGE and probed with anti-HA Ab. (B and tissue-repair response; however, the distribution of their re- and C) The cytosol and nucleus of THP-1 cells were separated by NE-PER ceptors among cell types and downstream intracellular signaling Nuclear and Cytoplasmic Extraction Reagents (THP1-Cyto and THP1-Nuc, are different (52). Furthermore, IL-4 and IL-13 stimulate macro- respectively). The cell lysate of THP1 cells and 293T cell lysates [same phages distinctively, and one of the critical differences between A B as ( )] were separated with Wes Simple Western System ( )orwith IL-4 and IL-13 signaling is the association of adaptor protein IRS2 SDS-PAGE (C) and then stained with anti-HA Ab, anti–histone deacetylase (43, 44). IRS2 is expressed in macrophages and associates with (HDAC) Ab (a marker for nucleus), or GAPDH Ab (a marker for cytosol). The arrowheads indicate the specific signals of DNase1L3. Each blot is one ERK, followed by PI3K activation in a JAK-dependent manner. representative of at least three independent experiments. Consequently, IL-4 can induce robust gene expression related to M2-macrophage phenotype compared with IL-13. In this report, we identified DNase1L3 expression is induced by IL-4–IRS-ERK/PI3K Secreted DNase1L3 proteins have the nuclease activity signaling in MDMs. The result that M-CSF–differentiated MDMs We next investigated the nuclease activities of secreted DNa- showed higher response to IL-4 than GM-CSF–differentiated se1L3. As indicated in Fig. 6A, DNase activities toward naked MDMs (data not shown) also supports DNase1L3 expression in DNA of secreted DNase1L3 and DNase1 from HEK-293T cells M2-macrophage. overexpressing DNase1L3 or DNase1 were confirmed. Previous Macrophages are highly heterogeneous populations, and various reports showed that DNase1L3 could degrade DNA associated stimuli could polarize macrophages into distinct cell types (42, 53). with liposome or lipid-like apoptotic microparticles, which is Apoptotic cells are recognized by receptors for dead cells such as 8 HOMEOSTASIS-ASSOCIATED MYELOID CELLS PRODUCE DNase1L3

TIM4, AXL, and MerTK on macrophages and promote homeostatic program associated with tissue-repair response, apoptotic cell clearance, and anti-inflammatory phenotype (54–59). Retinoic acid was the essential factor for differentiation from monocyte- derived to tissue-resident phenotype in peritoneal macrophage in mice (39, 40), indicating these stimuli may potentiate macrophage differentiation toward tissue-repairing and/or homeostatic macrophage. We analyzed the effect of apoptotic cells and ATRA for macrophages and identified these stimuli enhanced IL-4–induced DNase1L3 mRNA upregulation. Although we could not evaluate the effect of IL-4 and these stimuli for tissue-resident macrophages in human tissue, the fact that ATRA and apoptotic cells could enhance IL-4–induced DNase1L3 expression may suggest the role of DNase1L3 in context of tissue repairing and homeostasis. Our results identified DNase1L3 mRNA expression in CD11c- positive cells in the human spleen specimen. CD11c is expressed on many of dendritic cells, some macrophages, and a part of B cells

like age-associated B cells (60). The morphologies of CD11c- Downloaded from positive cells expressing DNase1L3 mRNA imply these cells were dendritic cells or macrophages. However, the possibility that CD11c-positive B cells expressed DNase1L3 mRNA cannot be ruled out, as the system used in our study did not work well for the other molecules such as CD3, CD19, and CD68. In con-

trast, CD11c-negative cells also expressed DNase1L3 mRNA. http://www.jimmunol.org/ Additionally, B1 cells and marginal zone B cells showed high expressions of DNase1L3 from GSE15907 dataset (33) (Supplemental Fig. 2, data not shown). Thus, innate-like B cells could be a potential DNase1L3-expressing population in human spleen. In addition, medullary thymic epithelial cells have an extremely high expression of DNase1L3 (Supplemental Fig. 2). Although upregulations of DNase1L3 mRNA in nonhematopoietic cells were not induced by IL-4 and/or ATRA in vitro, it is possible that other conditions promote DNase1L3 mRNA expression in by guest on September 25, 2021 nonhematopoietic cells. Further investigations are necessary to clarify the regulation of DNase1L3 expression. We showed secreted DNase1L3, as well as DNase1, could degrade nucleic acids in NETs. One recent report showed deficiencies of both of these nucleases, but not a single deficiency, led to vascular occlusion via intravascular NETs formation in mice (61), whereas loss of DNase1 or DNase1L3 developed lupus-like diseases (14, 15, 24). These facts may propose a redundant function for NETs degradation among DNase1 and DNase1L3. In contrast, DNase1L3 has a specialized role in some types of DNA, such as DNA packed in apoptotic microparticles; therefore, lack of DNase1L3 may lead to the development of SLE. The majority of familial SLE patients carrying a loss-of-function mutation of DNase1L3 had anti-neutrophil cytoplasmic Ab, which targets FIGURE 6. IL-4–stimulated macrophages or dendritic cells secrete NETs components (23). Moreover, several studies have shown that nuclease that is able to cleave DNA–liposome complex. (A and B)Five DNase1L3 had privileged functions for degrading DNA complexed A hundred nanograms of genomic DNA (gDNA) purified from PBMC ( )or with proteins, like histone, compared with DNase1 in vitro (10, 11). B gDNA complexed with Lipofectamine 2000 (L2K) ( ) was treated with the There might be complementary ability between DNase1 and supernatant of mock, DNase1, or DNase1L3-overexpressed 293T cells DNase1L3 to process DNA-associated autoantigen, which is re- with or without 2 mM EDTA. The degradation of DNA was analyzed by agarose gel electrophoresis. (C) Neutrophils from healthy donors were sponsible for autoimmune responses like productions of anti-DNA treated with 30 nM PMA for inductions of NETs. Plate-bound NETs Abs or anti-neutrophil cytoplasmic Ab. In DNase1L3-deficient were washed three times with PBS and supernatant of mock or DNase1L3- overexpressed 293T cells with or without the indicated concentration of rDNase1. The solubilized NETs were analyzed by agarose gel electrophoresis. (D–F) One nanogram of pCMV DNA plasmid with or without on the residual DNA. The experiment (A–D) is representative of at least L2K were treated with the supernatant of mock, DNase1, or DNase1L3- three independent experiments. (E) and (F) are performed in three inde- overexpressed 293T (D), with the supernatant of monocyte, IL-4-MoDC, pendent experiments using monocytes from one individual, and repre- MDM, or pDC (E) or with the supernatant of MDM stimulated with or sentative data are shown. The error bars indicate mean 6 SD. Analysis for without IL-4 (F). The residual DNA was estimated by qPCR using plasmid- each sample was carried out in triplicate. *p , 0.05 versus vehicle (veh) specific primers. DNase activity of each supernatant was calculated based sample, Student t test. The Journal of Immunology 9 mice, NET-like structures were detected in cultured splenic cells, 15. Napirei, M., H. Karsunky, B. Zevnik, H. Stephan, H. G. Mannherz, and T. Mo¨roÿ. 2000. Features of systemic lupus erythematosus in Dnase1-deficient indicating that DNase1L3 may contribute to processing and re- mice. Nat. Genet. 25: 177–181. moving NETs in spleens (9). As a specialized role of DNase1L3 in 16. Yasutomo, K., T. Horiuchi, S. Kagami, H. Tsukamoto, C. Hashimura, degrading NETs is controversial, whether DNase1L3 deficiency M. Urushihara, and Y. Kuroda. 2001. Mutation of DNASE1 in people with systemic lupus erythematosus. Nat. Genet. 28: 313–314. alone leads to an accumulation of NETs and NET-dependent 17. Kawane, K., M. Ohtani, K. Miwa, T. Kizawa, Y. Kanbara, Y. Yoshioka, autoantibody production in vivo should be determined by fur- H. Yoshikawa, and S. Nagata. 2006. Chronic polyarthritis caused by mammalian ther studies. DNA that escapes from degradation in macrophages. [Published erratum appears in 2007 Nature 446: 102.] Nature 443: 998–1002. In summary, our study proposed the roles of DNase1L3 on tissue 18. 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