Plasmacytoid Dendritic Cells of Siglech Encompasses More Than Control Diphtheria Toxin Receptor Under the Cell Depletion in Mice

Cell Depletion in Mice That Express Diphtheria Toxin Receptor under the Control of SiglecH Encompasses More Than Plasmacytoid Dendritic Cells This information is current as of September 29, 2021. Melissa Swiecki, Yaming Wang, Elena Riboldi, Alfred H. J. Kim, Amiran Dzutsev, Susan Gilfillan, William Vermi, Christiane Ruedl, Giorgio Trinchieri and Marco Colonna J Immunol 2014; 192:4409-4416; Prepublished online 28 March 2014; Downloaded from doi: 10.4049/jimmunol.1303135 http://www.jimmunol.org/content/192/9/4409 http://www.jimmunol.org/ References This article cites 40 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/192/9/4409.full#ref-list-1

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Cell Depletion in Mice That Express Diphtheria Toxin Receptor under the Control of SiglecH Encompasses More Than Plasmacytoid Dendritic Cells

Melissa Swiecki,* Yaming Wang,* Elena Riboldi,†,‡ Alfred H. J. Kim,x Amiran Dzutsev,† Susan Gilﬁllan,* William Vermi,*,{ Christiane Ruedl,‖ Giorgio Trinchieri,† and Marco Colonna*

Plasmacytoid dendritic cells (pDC) produce IFN-I in response to viruses and are routinely identified in mice by SiglecH expression. SiglecH is a sialic acid–binding Ig-like lectin that has an immunomodulatory role during viral infections. In this study, we evaluated the impact of SiglecH deficiency on cytokine responses in the presence and absence of pDC. We found that lack of SiglecH enhanced IFN-I responses to viral infection, regardless of whether pDC were depleted. We also examined the expression Downloaded from pattern of SiglecH and observed that it was expressed by specialized macrophages and progenitors of classical dendritic cells and pDC. Accordingly, marginal zone macrophages and pDC precursors were eliminated in newly generated SiglecH–diphtheria toxin receptor (DTR)–transgenic (Tg) mice but not in CLEC4C-DTR–Tg mice after diphtheria toxin (DT) treatment. Using two bacterial models, we found that SiglecH-DTR–Tg mice injected with DT had altered bacterial uptake and were more susceptible to lethal Listeria monocytogenes infection than were DT-treated CLEC4C-DTR–Tg mice. Taken together, our findings suggest that lack of SiglecH may affect cytokine responses by cell types other than pDC during viral infections, perhaps by altering viral http://www.jimmunol.org/ distribution or burden, and that cell depletion in SiglecH-DTR–Tg mice encompasses more than pDC. The Journal of Immu- nology, 2014, 192: 4409–4416.

lasmacytoid dendritic cells (pDC) are bone marrow–de- pDC have a more dramatic role in IFN-I production and promoting rived cells that are able to secrete large amounts of IFN-I T cell–mediated immunity to viral infection. P and, thus, are considered important mediators of antiviral In our model, pDC express the diphtheria toxin receptor (DTR) responses (1, 2). There is a great deal of controversy in the field under the control of the CLEC4C promoter (CLEC4C-DTR trans- regarding pDC as a major source of IFN-I and as APCs in vivo. genic [Tg]) and can be eliminated with diphtheria toxin (DT) ad- by guest on September 29, 2021 Our previous work showed that pDC are an early, transient source ministration (3). CLEC4C, also known as blood dendritic cell (DC) of IFN-I and have a modest impact on virus-specific CD8 T cell Ag 2, is a type II C-type lectin that is specifically expressed by responses (3–5). In contrast, another study (6) demonstrated that human pDC (7, 8). In the second model, DTR was inserted into the SiglecH locus (SiglecHDTR/DTR) (6). SiglecHDTR/DTR mice lack SiglecH expression and can be depleted of pDC with DT. SiglecH is a member of the sialic acid–binding Ig-like lectin family that is *Department of Pathology and Immunology, Washington University School of Med- commonly used to discriminate pDC from other cell types in mice, † icine, St. Louis, MO 63110; Laboratory of Experimental Immunology, Cancer and and it appears to have an immunomodulatory role during viral Inflammation Program, Center for Cancer Research, National Cancer Institute, Na- tional Institutes of Health, Frederick, MD 21702; ‡Department of Pharmaceutical infections (6, 9–11). Because the results and conclusions obtained Sciences, University of Piemonte Orientale “A. Avagadro,” Novara, Italy 28100; from these two inducible pDC-depletion models differ in magni- xDivision of Rheumatology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110; {Department of Molecular and Transla- tude, it is important to investigate the reasons for the discrepancies. tional Medicine, Section of Pathology, University of Brescia School of Medicine, In this study, we evaluated the impact of SiglecH deficiency on ‖ Brescia, Italy 25123; and Division of Molecular and Cell Biology, School of Bio- cytokine responses and the expression pattern of SiglecH in vivo using logical Sciences, Nanyang Technological University, Singapore 637551 wild-type (WT) mice, previously generated SiglecH-eGFP knockin Received for publication November 21, 2013. Accepted for publication February 27, 2014. mice (3), CLEC4C-DTR–Tg mice, and newly generated SiglecH- DTR–Tg mice. We found that purified pDC from SiglecHeGFP/eGFP M.S. was supported by Grant 1K01DK095972-01A1 from the National Institute of Diabetes and Digestive and Kidney Diseases, Y.W. was supported by Pulmonary and (SiglecH-deficient) and WT mice produced similar amounts of Critical Care Training Grant 2T32HL007317-31 from the National Heart, Lung, and IFN-a after stimulation with CpGA or murine CMV (MCMV) ex Blood Institute, W.V. was supported by Associazione Italiana per la ricerca sul cancro, 2010, IG 11924, and C.R. was supported by National Medical Research vivo. In vivo, infection with MCMV, but not CpGA administration, eGFP/eGFP Council Grant NMMR/1253/2010. resulted in increased serum IFN-a levels in SiglecH mice. Address correspondence and reprint requests to Prof. Marco Colonna, BJC Institute These data suggested that lack of SiglecH might influence cytokine of Health at Washington University, 425 South Euclid Avenue, Campus Box 8118, responses by pDC or perhaps other cell types in vivo during an St. Louis, MO 63110. E-mail address: [email protected] active viral infection. To investigate this, we bred heterozygous Abbreviations used in this article: BAC, bacterial artificial chromosome; BM, bone SiglecHeGFP/+ and homozygous SiglecHeGFP/eGFP mice to CLEC4C- marrow; cDC, classical dendritic cell; DC, dendritic cell; DT, diphtheria toxin; DTR, DT receptor; LM-OVA, Listeria monocytogenes expressing OVA; LN, DTR–Tg mice to distinguish the effect of SiglecH deletion from pDC lymph node; MCMV, murine CMV; MM, metallophilic macrophage; MZM, mar- depletion. Ablation of pDC in CLEC4C-DTR–Tg 3 SiglecHeGFP/eGFP ginal zone macrophage; pDC, plasmacytoid DC; p.i., postinfection; prepDC, precursor of pDC; qPCR, quantitative PCR; RT, room temperature; Tg, trans- mice revealed that lack of SiglecH enhanced IFN-I responses to genic; WT, wild-type. viral infection in the presence and absence of pDC. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303135 4410 FUNCTION AND EXPRESSION OF SiglecH

Given that SiglecH deficiency may affect cytokine responses by enriched from BM by negative selection using the Plasmacytoid Dendritic Cell cells other than pDC, we decided to evaluate the expression pattern Isolation Kit II (Miltenyi Biotec) and stimulated with CpGA (3 or 6 mg/ml). Purity was ∼50% after enrichment. FACS-sorted pDC (purity . 98%) of SiglecH in vivo using anti-SiglecH mAb and heterozygous m eGFP/+ were stimulated with CpGA (6 g/ml) or MCMV tissue culture stock SiglecH mice. We found that SiglecH was expressed by pDC (multiplicity of infection 10:1). Cells were cultured in 96-well flat bottom and specialized macrophage subsets, such as marginal zone macro- plates at 0.25–1 3 105 cells/well with CpGA or MCMV. Sorted prepDC phages (MZM), lymph node (LN) medullary macrophages, and micro- (1 3 105 cells/well) were cultured in 96-well flat-bottom plates in complete glia. SiglecH also was expressed by immediate precursors of pDC medium with GM-CSF (1 ng/ml) or Flt3L (10 ng/ml) (PeproTech) for 3 d. (prepDC) in the bone marrow (BM), which have the plasticity to Ab, flow cytometry, and cell sorting differentiate into pDC and classical DC (cDC) (12, 13). Analysis of Ab were purchased from BioLegend, eBioscience, or BD Biosciences. The SiglecH-DTR–Tg and CLEC4-DTR–Tg mice indicated that MZM and following clones were used: SiglecH (551 or 440c), CD11c (HL3), Ly6C (AL- prepDC were eliminated in DT-treated SiglecH-DTR–Tg mice but not 21), CCR9 (eBioCW-1.2), B220 (RA3-6B2), CD11b (M1/70), CD45 (30-F11), in CLEC4C-DTR–Tg mice. Therefore, administration of DT to mice CD4 (GK1.5), CD8a (53-6.7), Gr-1 (RB6-8C5), F4/80 (BM8), and IA/IE (M5/ that express DTR under the control of the SiglecH promoter might 114.15.2). Flow cytometry was conducted on a FACSCalibur or FACSCanto affect a large number of APCs. Indeed, using two bacterial infection (BD Biosciences) and analyzed with FlowJo software (TreeStar). Cell sorting was performed on a FACSAria II (BD Biosciences). Mature pDC and models, we found that SiglecH-DTR–Tg mice injected with DT had prepDC were defined/sorted as SiglecH+B220+CCR9+ and SiglecH+B220lo altered bacterial uptake and were more susceptible to lethal Listeria CCR9lo/2 cells, respectively. cDC were sorted from spleens into CD11chi 2 monocytogenes infection than were DT-treated CLEC4C-DTR–Tg MHCII+CD8a+ and CD11chiMHCII+CD8a subsets. mice. Thus, we envision that the broad expression pattern of SiglecH Cytokine analysis and quantitative PCR potentially explains why data derived from inducible pDC-ablation Downloaded from a models may be different. IFN- levels were determined by ELISA (PBL Interferon Source). Cytokines were measured by Cytometric Bead Array (BD Biosciences). Expression levels of SiglecH and E2-2 were measured by quantitative PCR (qPCR) and Materials and Methods normalized to GAPDH. Primers used for qPCR included SiglecH forward Mice, treatments, and infections primers: 59-ATT TCT GTG AGG AAA GGA TC-39 and 59-AAT TCA CAG AAC TCC ACA GC-39; SiglecH reverse primers: 59-TAG GAC GAC CAA Animal studies were approved by the Washington University Animal Studies GCT CCA GT-39 and 59-GAT CCC AAG AAG CAG GAA TT-39;E2-2 Committee. SiglecH-eGFP knockin mice and CLEC4C-DTR–Tg mice, both forward primers: 59-TGA GAT CAA ATC CGA CGA-39 and 59-ACA http://www.jimmunol.org/ on a C57BL/6 background, were bred in-house (3). SiglecH-DTR–Tg mice ACG GAG CGA TGG GTA G-39; E2-2 reverse primers: 59-CGT TAT TGC were generated and bred at the National Institutes of Health (C57BL/6) or at TAG ATC TTG ACC T-39 and 59-GCA GGA GAG AAT GGC TGC CT-39. Nanyang Technological University (BALB/c). CLEC4C-DTR–Tg mice and SiglecH-DTR–Tg mice were injected i.p. with 100–200 ng or 200–500 ng DT (Sigma-Aldrich), respectively. Non-Tg control mice also were injected with DT in some experiments. CpGA 2216 (Operon; 6 mg/mouse) was complexed with DOTAP and injected i.v. HSV-1 KOS strain was injected i.v. at 1 3 107 PFU. MCMV Smith strain was injected i.p. at 5 3 104 PFU. L. monocytogenes expressing OVA (LM-OVA) (14) was injected i.p. at 2.5 3 107 CFU. Alexa Fluor 647–labeled, heat-killed Streptococcus pneumoniae R36A by guest on September 29, 2021 was a generous gift from J.F. Kearney (University of Alabama at Birming- ham, Birmingham, AL) and was injected i.v. at ∼1 3 108 CFU/mouse. Generation of SiglecH-DTR–Tg mice C57BL/6-Tg(SiglecH-hDTR-EGFP)NCr–Tg mice were generated by bacterial artificial chromosome (BAC) recombineering. The BAC clone encoding the complete SiglecH gene locus (RPA24-163A12) was obtained from the BACPAC Resources Center at Children’s Hospital Oakland Research Insti- tute (Oakland, CA). The BAC clone was modified by recombination using a shuttle vector containing a bicistronic cassette consisting of the cDNA sequences encoding for human DTR and eGFP. The cassette was flanked by two homologous regions targeting the transgenes to the desired site of in- sertion (SiglecH exon I, after the second triplet of the open reading frame). The modified BAC clone was linearized and injected into the pronuclei of fertilized C57BL/6NCr oocytes at the Laboratory Animal Science Program facility (National Cancer Institute). Single-cell embryos were implanted in pseudogravid females, and litters were screened to select Tg mouse founders. Two Tg mouse lines with high transgene expression were established. The plasmidcontainingthehumanDTRsequenceusedintheshuttlevector preparation was a generous gift of Dr. T. Walzer (Universite´ de Lyon, Marseille, France). SiglecH-DTR–Tg mice on a BALB/c background were generated via BALB/c embryonic stem cells transfected with recombineered BAC clones (Siglec-H: RP24-265E12) carrying insertions of human DTR sequence, with its pA site in the initiation codons replacing the first coding exon of the SiglecH gene (15). Generation of SiglecH-DTR–Tg BM chimeras BM from C57BL/6 SiglecH-DTR–Tg mice was prepared from tibias and A femurs. RBCs were lysed with RBC lysis buffer (Sigma-Aldrich). BM cells FIGURE 1. Analysis of WT and SiglecH-eGFP reporter mice. ( ) eGFP/+ were injected i.v. (5–10 million cells/mouse) into irradiated age/gender- SiglecH mice were infected or not with HSV-1, and spleens were matched C57BL/6 mice purchased from The Jackson Laboratory 8–10 h analyzed 8 and 24 h later for pDC (Ly6C+SiglecH+eGFP+). (B)Body after irradiation. Chimeric mice were used in experiments 4–5 mo later. weights and levels of blood urea nitrogen, creatinine, and total protein in blood from WT and SiglecHeGFP/eGFP mice. Data are from two independent Cell preparations experiments. (C) Representative light microscopy images of H&E-stained Spleens were processed as previously described (3). BM was harvested from kidney sections revealed no pathologic abnormalities in SiglecH-deficient tibias and femurs. Microglia were isolated as described (16). pDC were mice (original magnification 340). The Journal of Immunology 4411

Blood work and kidney histology LN. Digital images were taken using an Olympus BX60 microscope, cap- tured using a DP-70 Olympus digital camera, and processed using Analysis Whole blood was collected by cardiac puncture into EDTA tubes for complete Image Processing software (Olympus). For immunofluorescence, 8-mm blood counts or in serum-collection tubes for measurement of blood urea frozen spleen sections were fixed in acetone for 5 min at room temperature nitrogen, creatinine, and total protein. Tests were performed by the Division of (RT) and stored at 280˚C. Frozen sections were blocked with 10% horse Comparative Medicine at Washington University School of Medicine. For serum for 20 min at RT. Primary Ab to MARCO, Gr-1, SIGN-R1, and H&E staining, kidneys were fixed with 10% buffered formalin solution and Sialoadhesin were applied to tissue sections for 30 min at RT. After washing m embedded in paraffin; 5- m sections were prepared and stained with H&E. with PBS, fluorescent-conjugated anti-rat secondary Ab were added to slides Immunohistochemistry and immunofluorescence for 30 min at RT. Slides were mounted with Fluoromount-G and imaged on a Zeiss LSM 510 META confocal laser scanning microscope. Immunoflu- For immunohistochemistry, 5-mm frozen tissue sections were used for orescence images were adjusted globally for brightness and contrast using immunohistochemical staining to visualize SiglecH+ cells in spleens and Adobe Photoshop CS6. Statistical analysis Statistical significance was analyzed with an unpaired, two-tailed Student t test or Mann–Whitney U test. The p values , 0.05 were considered sta- tistically significant. For susceptibility studies, p values were determined by the log-rank test.

Results SiglecH effectively identiﬁes pDC in steady-state and during infection Downloaded from SiglecH is a DAP12-associated receptor used to discriminate pDC from other cell types in mice (9, 10). Our previous work (17) showed that pDC numbers are reduced in spleens of infected mice, which appeared to be a consequence of cell death. A recent study (11) indicated that, following stimulation with CpGA or infection with http://www.jimmunol.org/ MCMV, SiglecH is downregulated in a TLR9/MyD88-dependent manner. To determine whether reduced pDC numbers during by guest on September 29, 2021

FIGURE 2. Effect of SiglecH deficiency on pDC responses to CpGA and MCMV. (A) pDC from WT and SiglecHeGFP/eGFP mice were enriched from BM and stimulated with CpGA for 48 h. (B) pDC from WT and SiglecHeGFP/eGFP mice were sort-purified from BM and stimulated with FIGURE 3. Lack of SiglecH impacts innate immune responses inde- CpGA for 24 h. (A and B)IFN-a was measured in supernatants by ELISA. pendently of pDC in vivo. WT and SiglecHeGFP/eGFP mice were infected (C) WT and SiglecHeGFP/eGFP mice were injected i.v. with CpGA, and serum i.v. with HSV-1. Serum IFN-a (A), TNF-a (B), and IL-6 (C) were mea- IFN-a levels were measured 6 h later. (D) pDC were sort-purified from WT sured 6 h p.i. (D) CLEC4C-DTR–Tg mice were bred to SiglecHeGFP/+ and and SiglecHeGFP/eGFP mice and stimulated for 24 h with MCMV. IFN-a was SiglecHeGFP/eGFP mice. Mice were injected with PBS or DT 24 h before measured in supernatants by ELISA. (E)WTandSiglecHeGFP/eGFP mice were infection with HSV-1. Serum IFN-a levels were measured 6 h p.i. Data are infected i.p. with MCMV, and serum IFN-a levels were measured 48 h p.i. from two or three independent experiments, with at least three mice/group Data are from two (A, B, D,andE)orthree(C) experiments. in each experiment. 4412 FUNCTION AND EXPRESSION OF SiglecH infection are due to reduced detection of SiglecH expression, we systemic IFN-a and proinflammatory cytokines compared with compared SiglecH and eGFP expression in pDC from spleens of WT mice after HSV-1 infection (Fig. 3A–C). These results are SiglecHeGFP/+ mice that were infected or not with HSV-1. Using consistent with an immunomodulatory role for SiglecH in antiviral markers to identify pDC, such as Ly6C and CD11c, we found that responses (6, 9, 11). To determine whether this effect was due frequencies of Ly6C+eGFP+ and Ly6C+SiglecH+ cells were to pDC, we bred CLEC4C-DTR–Tg mice to SiglecHeGFP/+ and comparable and reduced in spleen to a similar extent 8 and 24 h SiglecHeGFP/eGFP mice and infected them with HSV-1 in the pres- postinfection (p.i.) (Fig. 1A, data not shown), indicating that anti- ence or absence of pDC. We found that SiglecHeGFP/eGFP mice SiglecH Ab are effective at identifying pDC in the steady-state depleted of pDC produced more IFN-a than did pDC-depleted andduringviralinfection. SiglecHeGFP/+ mice or pDC-depleted CLEC4C-DTR–Tg mice ∼ eGFP/eGFP (Fig. 3D). In all three lines of depleted mice, there was an 2-ng SiglecH mice are healthy in steady-state reduction in serum IFN-a levels relative to their undepleted coun- A recent report (18) suggested that an independently derived terparts. These findings suggest that lack of SiglecH may affect SiglecH-deficient mouse strain had abnormal kidney pathology and cytokine secretion by cells other than pDC during viral infection or function. Therefore, we evaluated body weight and performed blood that viral burden and/or distribution is altered in the absence of work analyses on WT and SiglecHeGFP/eGFP mice (Fig. 1). WT and SiglecH. age/gender-matched SiglecHeGFP/eGFP mice had comparable body SiglecH is expressed by MZM, medullary macrophages, weights, percentages of segmented neutrophils and lymphocytes in microglia, and progenitors of cDC and pDC circulation, and levels of blood urea nitrogen, creatinine, and total protein (Fig. 1B, data not shown). Consistent with the absence of Although SiglecH expression is mainly confined to pDC in cell Downloaded from biochemical abnormalities in renal function, no pathologic changes suspensions from primary and secondary lymphoid organs, it was were observed by light microscopy in the glomeruli or tubules of observed by microscopy that specialized macrophage subsets in the SiglecHeGFP/eGFP mice (Fig. 1C). Thus, SiglecHeGFP/eGFP mice ap- spleen and LN express SiglecH (10). Corroborating this, we found pear to be healthy and have normal kidney function in steady-state. that MZM in spleen (Fig. 4A) and medullary macrophages in LN (data not shown) were SiglecH+ by immunohistochemistry. In ad- SiglecH deficiency does not impact IFN-I production by pDC int +

dition, we observed that SiglecH was expressed by CD45 CD11b http://www.jimmunol.org/ ex vivo microglia in brain (Fig. 4B), in agreement with a recent study (24). + eGFP/+ It was shown that SiglecH has an immunomodulatory role during We reported previously that cDC are eGFP in SiglecH + 2 inflammation and viral infections (6,9,11).Todeterminewhether mice (3). Closer examination revealed that both CD4 and CD4 + SiglecH deficiency altered cytokine production by pDC, we mea- cDC in spleen were eGFP (Fig. 5A); however, they did not ex- sured IFN-a levels in supernatants from enriched or sort-purified press SiglecH on the surface or at the transcript level (Fig. 5B, pDC from WT and SiglecHeGFP/eGFP mice stimulated ex vivo with CpGA. CpGA is a synthetic TLR ligand that induces IFN-I production by pDC through TLR9 (19). pDC from WT and SiglecHeGFP/eGFP mice produced comparable amounts of IFN-a in response to CpGA by guest on September 29, 2021 (Fig. 2A, 2B), suggesting that lack of SiglecH does not strongly alter IFN-I production by pDC ex vivo. We next evaluated IFN-I responses to CpGA in vivo. WT and SiglecHeGFP/eGFP mice were injected i.v. with CpGA complexed with DOTAP, and serum IFN-a was measured 6 h later (Fig. 2C). Analyses of several mice revealed no significant differences in serum IFN-a levels between WT and SiglecHeGFP/eGFP mice. IFN-a was not detectable in serum from naive WT or SiglecHeGFP/eGFP mice (data not shown). We next evaluated whether SiglecH deficiency influenced IFN-I production by pDC after exposure to a live virus. MCMV is sensed by pDC through TLR9 (20). Moreover, it was reported that pDC are an important and early source of IFN-I during MCMV infection (3, 20–23). Thus, we sort-purified pDC from WT and SiglecHeGFP/eGFP mice and cultured them with MCMV. pDC from both groups of mice produced similar levels of IFN-a (Fig. 2D), indicating that SiglecH deficiency did not affect IFN-I production by virus-stimulated pDC ex vivo. We next evaluated whether mice lacking SiglecH had altered IFN-I responses in vivo during MCMV infection. We found that SiglecHeGFP/eGFP mice had increased levels of systemic IFN-a compared with WT mice at 48 h p.i. (Fig. 2E), similar to a recent study (11). Taken together, these data suggest that SiglecH deficiency does not affect IFN-I secretion by pDC in response to a synthetic TLR9 ligand or virus ex vivo, but it may FIGURE 4. SiglecH is expressed by specialized macrophages. (A) influence IFN-I production by pDC during viral infection in vivo. SiglecH is expressed by pDC and MZM in spleen. Black arrowheads de- note pDC in the T cell area, and red arrowheads indicate MZM (original Enhanced cytokine responses to HSV-1 in SiglecHeGFP/eGFP magnification 3100, upper panels; 3400, lower panels). Scale bars, 200 mice occur in the presence and absence of pDC mm(upper panels), 50 mm (lower panels). (B) Microglia were isolated from WT mice and stained with CD45, CD11b, and SiglecH. Plots show DTR/DTR SiglecH mice were reported to have increased cytokine CD45intCD11b+ microglia (left panel), and SiglecH expression (right responses to systemic HSV-1 infection (6). Corroborating these panel). Data are representative of two independent experiments, with three findings, SiglecHeGFP/eGFP mice also had slightly elevated levels of to five mice/experiment. The Journal of Immunology 4413 data not shown). These findings indicated that the SiglecH pro- DTR–Tg mice do not tolerate DT very well. After 4–5 mo of rest, moter was active in DC progenitors during development, consistent we injected CLEC4C-DTR–Tg mice and SiglecH-DTR–Tg chimeras with a recent study (25), and that eGFP persists in differentiated or with PBS or DT and harvested spleens 24–36 h later. Spleen sections mature cDC. In the BM, there is a subset of SiglecH+ cells that has were stained with Ab against the scavenger receptor MARCO, which been defined as prepDC (Fig. 5C), which can differentiate into pDC is expressed by MZM (Fig. 6A) (27, 28). Results indicated that MZM and cDC in vitro and in vivo (Fig. 5D) (12, 13). Both mature pDC were intact in CLEC4C-DTR–Tg mice treated with either PBS or DT. and prepDC expressed the pDC-specific transcription factor Tcf4/ In contrast, very few MZM could be identified in spleen sections from E2-2 (Fig. 5E) (12, 26) and were eGFP+ in SiglecHeGFP/+ mice (data SiglecH-DTR–Tg chimeras injected with DT. The depletion of MZM not shown). Taken together, these data indicate that SiglecH is in SiglecH-DTR–Tg mice was validated in a second SiglecH-DTR– expressed by mature pDC, specialized macrophages, and progenitors Tg line that was generated on a BALB/c background (15). Spleen of cDC and pDC. sections stained for both MZM and metallophilic macrophages (MM) revealed that MZM, but not MM, were reduced in BALB/c SiglecH- PrepDC and MZM are depleted in SiglecH-DTR–Tg mice DTR–Tg mice injected with DT (Fig. 6B). Given that SiglecH expression is not restricted to mature pDC, we hypothesized that prepDC and MZM may also be depleted in mice Altered bacterial uptake and increased susceptibility to that express DTR under the control of the SiglecH promoter. To test LM-OVA infection in depleted SiglecH-DTR–Tg mice this hypothesis, we used newly generated C57BL/6 SiglecH-DTR–Tg MZM are important for the clearance of apoptotic cells (29–31) and mice that express DTR under the control of the SiglecH promoter. for the uptake of bacteria, such as S. pneumoniae (32–34). Mice

After DT injection, both mature pDC and prepDC were ablated in lacking SIGN-R1, a receptor expressed by MZM, are more suscep- Downloaded from SiglecH-DTR–Tg mice (Fig. 5F). In contrast, only mature pDC were tible to systemic S. pneumoniae infection and show signs of altered eliminated in CLEC4C-DTR–Tg mice (Fig. 5F). Furthermore, when bacterial distribution in the spleen upon infection (33, 34). Therefore, CLEC4C-DTR–Tg mice were bred to SiglecHeGFP/+ mice, eGFP+ we hypothesized that SiglecH-DTR–Tg mice, which appear to lack cDC were not depleted after DT administration (3). MZM after DT treatment, also would exhibit altered bacterial dis- We next asked whether MZM were depleted in SiglecH-DTR–Tg tribution and perhaps be more susceptible to infection. To test this, we

mice after DT treatment. To address this, we generated C57BL/6 injected control or depleted CLEC4C-DTR–Tg mice and SiglecH- http://www.jimmunol.org/ SiglecH-DTR–Tg BM chimeras, because normal C57BL/6 SiglecH- DTR–Tg chimeras with ﬂuorescent-labeled S. pneumoniae and by guest on September 29, 2021

FIGURE 5. SiglecH expression in progenitors of cDC and pDC. (A) CD11chi cells in SiglecHeGFP/+ mice express eGFP. The dot plot shows live spleen cells stained with CD4 and CD11c (left panel). Histograms show eGFP expression in CD4+CD11chi DC and CD42 CD11chi DC (right panel). (B)pDC,CD8a+ DC, and CD8a2 DC were sorted from spleens, and SiglecH expression was measured by qPCR. (C) PrepDC in BM express SiglecH. The dot plot shows B220hiSiglecH+ pDC and B220loSiglecH+ prepDC, and histograms show expression of CCR9, Ly6C, CD11c, and MHCII on each subset. (D) Sorted prepDC from BM differentiate into cDC or pDC after culture in GM-CSF or Flt3L, respectively. (E)E2-2expressioninpDC, CD8a+ DC, and CD8a2 DC sorted from spleen and mature pDC and prepDC sorted from BM. (F) SiglecH-DTR–Tg mice, CLEC4C-DTR Tg mice, and their littermates (DTR2) were injected with DT, and BM was analyzed for mature pDC and prepDC 48 h later. Plots in (A), (C), and (F) are representative of 5–10 mice. In (B), (D), and (E), cells were sorted from ﬁve or six mice for qPCR or in vitro differentiation. 4414 FUNCTION AND EXPRESSION OF SiglecH Downloaded from http://www.jimmunol.org/

FIGURE 6. MZM are depleted in SiglecH-DTR–Tg mice. (A) Spleen sections from PBS- or DT-treated CLEC4C-DTR–Tg mice and C57BL/6 SiglecH-DTR–Tg chimeras were stained for MARCO to identify MZM by guest on September 29, 2021 (original magnification 310). (B) Spleen sections from BALB/c SiglecH- DTR–Tg mice and their non-Tg littermates (WT) injected with DT were stained with Abs to SIGN-R1 and Sialoadhesin (Sn) to detect MZM and MM, respectively (original magnification 320). Data are representative of two or three independent experiments. examined spleens 1 h p.i. by flow cytometry. We first confirmed depletion in mice treated with DT by staining for pDC (Fig. 7A, FIGURE 7. Distribution and clearance of bacteria are altered in depleted top panels). Next, using a variety of Ab, we evaluated which cells SiglecH-DTR–Tg mice. PBS- or DT-treated CLEC4C-DTR–Tg mice and from each group of mice were associated with fluorescent bacte- C57BL/6 SiglecH-DTR–Tg chimeras were injected with Alexa Fluor 647– ria. In all groups of mice, bacteria were mainly found among labeled S. pneumoniae R36A. One hour after injection, spleens were har- CD11b+Gr-1+F4/802 cells but not pDC (Fig. 7A, bottom panels vested for flow cytometry and microscopy. (A) pDC frequencies in PBS- and DT-treated mice (top panels). Frequencies of live, Gr-1+ cells (middle pan- and data not shown). However, we noted that, in SiglecH-DTR–Tg + + chimeras treated with DT, there was a 3-fold increase in the fre- els). Frequencies of Alexa Fluor 647 cells among live, Gr-1 cells (bottom panels). (B) Spleen sections were stained with Gr-1 and show increased quency of Gr-1+ cells, presumably neutrophils, which were asso- numbers of bacteria in the red pulp of DT-treated SiglecH-DTR–Tg chimeras ciated with bacteria. These findings suggested that the absence of (original magnification 310). Data are representative of three independent MZM in SiglecH-DTR–Tg mice resulted in increased bacterial experiments. burden in the red pulp. To confirm this, we analyzed spleens by microscopy 1 h after injection of fluorescent-labeled S. pneumoniae (Fig. 7B). Very few fluorescent bacteria could be detected in were more resistant to lethal infection with LM-OVA than were CLEC4C-DTR–Tg mice injected with PBS or DT or in SiglecH- nondepleted mice. Thus, we performed a similar experiment in our DTR–Tg chimeras injected with PBS. In contrast, SiglecH-DTR– CLEC4C-DTR–Tg mice and SiglecH-DTR–Tg chimeras. SiglecH- Tg chimeras treated with DT had many bacteria located in the red DTR–Tg chimeras treated with DT succumbed to LM-OVA infection pulp, as visualized by Gr-1 staining, confirming altered bacterial faster than did mice in the other three groups (Fig. 8A). Moreover, it distribution and impaired clearance in the absence of MZM. did not appear that lack of pDC was responsible for this phenotype, Given the differences in bacterial uptake and clearance between because CLEC4C-DTR–Tg mice, depleted or not of pDC, exhibited CLEC4C-DTR–Tg and SiglecH-DTR–Tg chimeras, we sought to identical rates of survival and died later than did DT-treated SiglecH- determine whether SiglecH-DTR–Tg mice were more or less sus- DTR–Tg chimeras (Fig. 8A). Analysis of serum cytokine levels ceptible to bacterial infection after DT administration. A study by revealed that DT-treated SiglecH-DTR–Tg chimeras produced very Takagi et al. (6) found that SiglecHDTR/DTR mice treated with DT little IL-12p70 and had exaggerated levels of TNF-a andIL-6intheir The Journal of Immunology 4415

OVA infection. Previous studies showed that, early p.i., Listeria can be found associated with SIGN-R1+ cells in the spleen (36, 37) and that mice depleted of MZM and MM are impaired in their ability to control and survive Listeria infection (38). We envision that lack of MZM in DT-treated SiglecH-DTR–Tg mice may account for the cytokine storm and rapid death following LM-OVA infection. The results that we obtained in our LM-OVA experiments using SiglecH-DTR–Tg mice differ in two ways from an earlier report that depleted pDC in SiglecHDTR/DTR mice. First, our depleted SiglecH-DTR–Tg mice were more susceptible to infection, whereas depleted SiglecHDTR/DTR mice appeared to be more resistant (6). Second, our model of specific pDC depletion using CLEC4C-DTR– Tg mice revealed no major role for pDC in lethal LM-OVA infection, because PBS- and DT-treated mice had identical survival rates and similar levels of systemic cytokines. It should be noted that SiglecHDTR/DTR mice lack SiglecH expression (6). Therefore, the findings reported by Takagi et al. (6) do not discriminate effects of SiglecH deficiency from pDC depletion. Although it has been

suggested that pDC may be detrimental during Listeria infection Downloaded from because of their ability to produce IFN-I, studies showed that my- eloid cells and CD8a+ DC are important in pathogenesis as either IFN-I–producing cells or initial cellular entry points that establish FIGURE 8. Susceptibility of CLEC4C-DTR–Tg mice and SiglecH- productive infection (39, 40). Taken together, we conclude that the DTR–Tg mice to LM-OVA. Groups of ﬁve mice (PBS- or DT-treated broad expression pattern of SiglecH should be considered when

CLEC4C-DTR–Tg mice and SiglecH-DTR–Tg chimeras) were infected using SiglecH-DTR mice to evaluate pDC functions in vivo. http://www.jimmunol.org/ i.p. with LM-OVA. Survival of mice was monitored every 12 h (A), and serum was collected from three mice in each group 24 h p.i. for cytokine Acknowledgments B analysis ( ). We thank C.A. Stewart (National Cancer Institute) for advice on BAC recombineering, D. Leib (Geisel School of Medicine at Dartmouth) for serum compared with the other groups of mice (Fig. 8B). Thus, the HSV-1, A. French (Washington University School of Medicine) for MCMV, increased susceptibility and cytokine storm in DT-treated SiglecH- C. Rossini for technical support, J.F. Kearney and L. Jia (University of Ala- DTR–Tg mice most likely was a consequence of altered bacterial bama at Birmingham) for R36A, and S. Srivatsan and E. Lantelme (Wash- distribution in the absence of SiglecH+ cells that were not pDC. ington University School of Medicine) for help with BM chimeras and cell

sorting. by guest on September 29, 2021 Discussion Disclosures In this study, we found that SiglecH deficiency resulted in increased The authors have no financial conflicts of interest. cytokine responses during HSV-1 infection and other viral infections, which may be pDC independent. Indeed, SiglecH was expressed by MZM, which were reported to be major producers of IFN-I during References systemic HSV-1 infection (35). SiglecH also was expressed by LN 1. Gilliet, M., W. Cao, and Y. J. Liu. 2008. Plasmacytoid dendritic cells: sensing medullary macrophages, microglia, and prepDC in the BM, which nucleic acids in viral infection and autoimmune diseases. Nat. Rev. Immunol. 8: 594–606. can differentiate into pDC and cDC (10, 12, 13, 24). Although cDC 2. Trinchieri, G. 2010. Type I interferon: friend or foe? J. Exp. Med. 207: 2053– did not express SiglecH on their surface or at the transcript level, they 2063. were eGFP+ in SiglecH-eGFP–knockin mice (3), suggesting that the 3. Swiecki, M., S. Gilfillan, W. Vermi, Y. Wang, and M. Colonna. 2010. Plasma- cytoid dendritic cell ablation impacts early interferon responses and antiviral NK SiglecH promoter was active in a common DC progenitor during DC and CD8(+) T cell accrual. Immunity 33: 955–966. development (25). In addition, it was shown recently, using newly 4. Wang, Y., M. Swiecki, M. Cella, G. Alber, R. D. Schreiber, S. Gilfillan, and M. Colonna. 2012. Timing and magnitude of type I interferon responses by generated SiglecH-RFP reporter mice, that a fraction of B, T, NK, distinct sensors impact CD8 T cell exhaustion and chronic viral infection. Cell + and NKT cells are RFP , indicating SiglecH promoter activity in Host Microbe 11: 631–642. a common lymphoid progenitor (11). Thus, lack of SiglecH may 5. Swiecki, M., Y. Wang, S. Gilfillan, and M. Colonna. 2013. Plasmacytoid dendritic cells contribute to systemic but not local antiviral responses to HSV affect cytokine production by a variety of cell types during a viral infections. PLoS Pathog. 9: e1003728. infection, perhaps by altering viral burden and/or distribution. 6. Takagi, H., T. Fukaya, K. Eizumi, Y. Sato, K. Sato, A. Shibazaki, H. Otsuka, The semipromiscuous expression of SiglecH potentially explains A. Hijikata, T. Watanabe, O. Ohara, et al. 2011. Plasmacytoid dendritic cells are crucial for the initiation of inflammation and T cell immunity in vivo. Immunity data inconsistencies between inducible pDC-ablation models. It is 35: 958–971. possible that certain macrophage subsets and DC progenitors are 7. Dzionek, A., A. Fuchs, P. Schmidt, S. Cremer, M. Zysk, S. Miltenyi, D. W. Buck, and J. Schmitz. 2000. BDCA-2, BDCA-3, and BDCA-4: three markers for distinct depleted in SiglecH-DTR-knockin or Tg mice, thus yielding stronger subsets of dendritic cells in human peripheral blood. J. Immunol. 165: 6037–6046. phenotypes than observed in CLEC4C-DTR–Tg mice. Indeed, using 8. Dzionek, A., Y. Sohma, J. Nagafune, M. Cella, M. Colonna, F. Facchetti, two SiglecH-DTR–Tg mouse lines, we found that prepDC and MZM G. Gunther,€ I. Johnston, A. Lanzavecchia, T. Nagasaka, et al. 2001. BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates an- were reduced after DT treatment in contrast to CLEC4C-DTR–Tg tigen capture and is a potent inhibitor of interferon alpha/beta induction. J. Exp. mice. Furthermore, depleted SiglecH-DTR–Tg mice showed altered Med. 194: 1823–1834. bacterial distribution and impaired clearance after injection of 9. Blasius, A. L., M. Cella, J. Maldonado, T. Takai, and M. Colonna. 2006. Siglec-H is an IPC-specific receptor that modulates type I IFN secretion through DAP12. S. pneumoniae compared with control mice and DT-treated CLEC4C- Blood 107: 2474–2476. DTR–Tg mice. These results are consistent with a role for MZM in 10. Zhang, J., A. Raper, N. Sugita, R. Hingorani, M. Salio, M. J. Palmowski, V. Cerundolo, and P. R. Crocker. 2006. Characterization of Siglec-H as a novel responses to S. pneumoniae (32–34). Moreover, we found that de- endocytic receptor expressed on murine plasmacytoid dendritic cell precursors. pleted SiglecH-DTR–Tg mice were highly susceptible to lethal LM- Blood 107: 3600–3608. 4416 FUNCTION AND EXPRESSION OF SiglecH

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