of May 13, 2018.This information is current as

Plasmacytoid Dendritic Cellsof SiglecH Encompasses More Than

ControlDiphtheria Toxin Receptor under the Cell Depletion in Mice That Express

Christiane Ruedl, Giorgio Trinchieri and Marco ColonnaKim, Amiran Dzutsev, Susan Gilfillan, William Vermi, Melissa Swiecki, Yaming Wang, Elena Riboldi, Alfred H. J.

ol.1303135http://www.jimmunol.org/content/early/2014/03/28/jimmun

published online 28 March 2014J Immunol 

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The Journal of Immunology

Cell Depletion in Mice That Express Diphtheria ToxinReceptor under the Control of SiglecH Encompasses MoreThan 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

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

distribution or burden, and that cell depletion in SiglecH-DTR–Tg mice encompasses more than pDC. The Journal of Immu-

nology, 2014, 192: 000–000.

Plasmacytoid dendritic cells (pDC) are bone marrow–de-rived cells that are able to secrete large amounts of IFN-Iand, thus, are considered important mediators of antiviral

responses (1, 2). There is a great deal of controversy in the fieldregarding pDC as a major source of IFN-I and as APCs in vivo.Our previous work showed that pDC are an early, transient sourceof IFN-I and have a modest impact on virus-specific CD8 T cellresponses (3–5). In contrast, another study (6) demonstrated that

pDC have a more dramatic role in IFN-I production and promotingT cell–mediated immunity to viral infection.In our model, pDC express the diphtheria toxin receptor (DTR)

under the control of the CLEC4C promoter (CLEC4C-DTR trans-genic [Tg]) and can be eliminated with diphtheria toxin (DT) ad-ministration (3). CLEC4C, also known as blood dendritic cell (DC)Ag 2, is a type II C-type lectin that is specifically expressed byhuman pDC (7, 8). In the second model, DTR was inserted into theSiglecH locus (SiglecHDTR/DTR) (6). SiglecHDTR/DTR mice lackSiglecH expression and can be depleted of pDC with DT. SiglecH isa member of the sialic acid–binding Ig-like lectin family that iscommonly used to discriminate pDC from other cell types in mice,and it appears to have an immunomodulatory role during viralinfections (6, 9–11). Because the results and conclusions obtainedfrom these two inducible pDC-depletion models differ in magni-tude, it is important to investigate the reasons for the discrepancies.In this study, we evaluated the impact of SiglecH deficiency on

cytokine responses and the expression pattern of SiglecH invivo usingwild-type (WT) mice, previously generated SiglecH-eGFP knockinmice (3), CLEC4C-DTR–Tg mice, and newly generated SiglecH-DTR–Tg mice. We found that purified pDC from SiglecHeGFP/eGFP

(SiglecH-deficient) and WT mice produced similar amounts ofIFN-a after stimulation with CpGA or murine CMV (MCMV) exvivo. In vivo, infection with MCMV, but not CpGA administration,resulted in increased serum IFN-a levels in SiglecHeGFP/eGFP mice.These data suggested that lack of SiglecH might influence cytokineresponses by pDC or perhaps other cell types in vivo during anactive viral infection. To investigate this, we bred heterozygousSiglecHeGFP/+ and homozygous SiglecHeGFP/eGFP mice to CLEC4C-DTR–Tg mice to distinguish the effect of SiglecH deletion from pDCdepletion. Ablation of pDC in CLEC4C-DTR–Tg3 SiglecHeGFP/eGFP

mice revealed that lack of SiglecH enhanced IFN-I responses toviral infection in the presence and absence of pDC.

*Department of Pathology and Immunology, Washington University School of Med-icine, St. Louis, MO 63110; †Laboratory of Experimental Immunology, Cancer andInflammation Program, Center for Cancer Research, National Cancer Institute, Na-tional Institutes of Health, Frederick, MD 21702; ‡Department of PharmaceuticalSciences, University of Piemonte Orientale “A. Avagadro,” Novara, Italy 28100;xDivision of Rheumatology, Department of Internal Medicine, Washington UniversitySchool of Medicine, St. Louis, MO 63110; {Department of Molecular and Transla-tional Medicine, Section of Pathology, University of Brescia School of Medicine,Brescia, Italy 25123; and ‖Division of Molecular and Cell Biology, School of Bio-logical Sciences, Nanyang Technological University, Singapore 637551

Received for publication November 21, 2013. Accepted for publication February 27,2014.

M.S. was supported by Grant 1K01DK095972-01A1 from the National Institute ofDiabetes and Digestive and Kidney Diseases, Y.W. was supported by Pulmonary andCritical Care Training Grant 2T32HL007317-31 from the National Heart, Lung, andBlood Institute, W.V. was supported by Associazione Italiana per la ricerca sulcancro, 2010, IG 11924, and C.R. was supported by National Medical ResearchCouncil Grant NMMR/1253/2010.

Address correspondence and reprint requests to Prof. Marco Colonna, BJC Instituteof Health at Washington University, 425 South Euclid Avenue, Campus Box 8118,St. Louis, MO 63110. E-mail address: mcolonna@pathology.wustl.edu

Abbreviations used in this article: BAC, bacterial artificial chromosome; BM, bonemarrow; cDC, classical dendritic cell; DC, dendritic cell; DT, diphtheria toxin; DTR,diphtheria toxin receptor; LM-OVA, Listeria monocytogenes expressing OVA; LN,lymph node; MCMV, murine CMV; MM, metallophilic macrophage; MZM, marginalzone macrophage; pDC, plasmacytoid dendritic cell; p.i., postinfection; prepDC,precursor of plasmacytoid dendritic cell; qPCR, quantitative PCR; RT, room temper-ature; Tg, transgenic; WT, wild-type.

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Given that SiglecH deficiency may affect cytokine responses bycells other than pDC, we decided to evaluate the expression patternof SiglecH in vivo using anti-SiglecH mAb and heterozygousSiglecHeGFP/+ mice. We found that SiglecH was expressed by pDCand specialized macrophage subsets, such as marginal zone macro-phages (MZM), lymph node (LN) medullary macrophages, and micro-glia. SiglecH also was expressed by immediate precursors of pDC(prepDC) in the bone marrow (BM), which have the plasticity todifferentiate into pDC and classical DC (cDC) (12, 13). Analysis ofSiglecH-DTR–Tg and CLEC4-DTR–Tg mice indicated that MZM andprepDC were eliminated in DT-treated SiglecH-DTR–Tg mice but notin CLEC4C-DTR–Tg mice. Therefore, administration of DT to micethat express DTR under the control of the SiglecH promoter mightaffect a large number of APCs. Indeed, using two bacterial infectionmodels, we found that SiglecH-DTR–Tg mice injected with DT hadaltered bacterial uptake and were more susceptible to lethal Listeriamonocytogenes infection than were DT-treated CLEC4C-DTR–Tgmice. Thus, we envision that the broad expression pattern of SiglecHpotentially explains why data derived from inducible pDC-ablationmodels may be different.

Materials and MethodsMice, treatments, and infections

Animal studies were approved by the Washington University Animal StudiesCommittee. SiglecH-eGFP knockin mice and CLEC4C-DTR–Tg mice, bothon a C57BL/6 background, were bred in-house (3). SiglecH-DTR–Tg micewere generated and bred at the National Institutes of Health (C57BL/6) or atNanyang Technological University (BALB/c). CLEC4C-DTR–Tg mice andSiglecH-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 withDT in some experiments. CpGA 2216 (Operon; 6 mg/mouse) was complexedwith 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. mono-cytogenes expressing OVA (LM-OVA) (14) was injected i.p. at 2.5 3 107

CFU. Alexa Fluor 647–labeled, heat-killed Streptococcus pneumoniae R36Awas 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 bacte-rial artificial chromosome (BAC) recombineering. The BAC clone encodingthe complete SiglecH gene locus (RPA24-163A12) was obtained from theBACPAC Resources Center at Children’s Hospital Oakland Research Insti-tute (Oakland, CA). The BAC clone was modified by recombination usinga shuttle vector containing a bicistronic cassette consisting of the cDNAsequences encoding for human DTR and eGFP. The cassette was flanked bytwo 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 offertilized C57BL/6NCr oocytes at the Laboratory Animal Science Programfacility (National Cancer Institute). Single-cell embryos were implanted inpseudogravid females, and litters were screened to select Tg mouse founders.Two Tg mouse lines with high transgene expression were established. Theplasmid containing the human DTR sequence used in the shuttle vectorpreparation was a generous gift of Dr. T. Walzer (Universite de Lyon,Marseille, France). SiglecH-DTR–Tg mice on a BALB/c background weregenerated via BALB/c embryonic stem cells transfected with recombineeredBAC clones (Siglec-H: RP24-265E12) carrying insertions of human DTRsequence, with its pA site in the initiation codons replacing the first codingexon of the SiglecH gene (15).

Generation of SiglecH-DTR–Tg BM chimeras

BM from C57BL/6 SiglecH-DTR–Tg mice was prepared from tibias andfemurs. RBCs were lysed with RBC lysis buffer (Sigma-Aldrich). BM cellswere injected i.v. (5–10 million cells/mouse) into irradiated age/gender-matched C57BL/6 mice purchased from The Jackson Laboratory 8–10 hafter irradiation. Chimeric mice were used in experiments 4–5 mo later.

Cell preparations

Spleens were processed as previously described (3). BM was harvested fromtibias and femurs. Microglia were isolated as described (16). pDC were

enriched from BMby negative selection using the Plasmacytoid Dendritic CellIsolation Kit II (Miltenyi Biotec) and stimulated with CpGA (3 or 6 mg/ml).Purity was ∼50% after enrichment. FACS-sorted pDC (purity . 98%)were stimulated with CpGA (6 mg/ml) or MCMV tissue culture stock(multiplicity of infection 10:1). Cells were cultured in 96-well flat bottomplates at 0.25–1 3 105 cells/well with CpGA or MCMV. Sorted prepDC(1 3 105 cells/well) were cultured in 96-well flat-bottom plates in completemedium with GM-CSF (1 ng/ml) or Flt3L (10 ng/ml) (PeproTech) for 3 d.

Ab, flow cytometry, and cell sorting

Ab were purchased from BioLegend, eBioscience, or BD Biosciences. Thefollowing clones were used: SiglecH (551 or 440c), CD11c (HL3), Ly6C (AL-21), CCR9 (eBioCW-1.2), B220 (RA3-6B2), CD11b (M1/70), CD45 (30-F11),CD4 (GK1.5), CD8a (53-6.7), Gr-1 (RB6-8C5), F4/80 (BM8), and IA/IE (M5/114.15.2). Flow cytometry was conducted on a FACSCalibur or FACSCanto(BD Biosciences) and analyzed with FlowJo software (TreeStar). Cell sortingwas performed on a FACSAria II (BD Biosciences). Mature pDC andprepDC were defined/sorted as SiglecH+B220+CCR9+ and SiglecH+B220lo

CCR9lo/2 cells, respectively. cDC were sorted from spleens into CD11chi

MHCII+CD8a+ and CD11chiMHCII+CD8a2 subsets.

Cytokine analysis and quantitative PCR

IFN-a levels were determined by ELISA (PBL Interferon Source). Cytokineswere measured by Cytometric Bead Array (BD Biosciences). Expressionlevels of SiglecH and E2-2 were measured by quantitative PCR (qPCR) andnormalized to GAPDH. Primers used for qPCR included SiglecH forwardprimers: 59-ATT TCT GTG AGG AAA GGATC-39 and 59-AAT TCA CAGAAC TCC ACA GC-39; SiglecH reverse primers: 59-TAG GAC GAC CAAGCT CCA GT-39 and 59-GAT CCC AAG AAG CAG GAA TT-39; E2-2forward primers: 59-TGA GAT CAA ATC CGA CGA-39 and 59-ACAACG GAG CGA TGG GTA G-39; E2-2 reverse primers: 59-CGT TAT TGCTAG ATC TTG ACC T-39 and 59-GCA GGA GAG AAT GGC TGC CT-39.

FIGURE 1. Analysis of WT and SiglecH-eGFP reporter mice. (A)

SiglecHeGFP/+ mice were infected or not with HSV-1, and spleens were

analyzed 8 and 24 h later for pDC (Ly6C+SiglecH+eGFP+). (B) Body

weights and levels of blood urea nitrogen, creatinine, and total protein in

blood from WT and SiglecHeGFP/eGFP mice. Data are from two independent

experiments. (C) Representative light microscopy images of H&E-stained

kidney sections revealed no pathologic abnormalities in SiglecH-deficient

mice (original magnification 340).

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Blood work and kidney histology

Whole blood was collected by cardiac puncture into EDTA tubes for completeblood counts or in serum-collection tubes for measurement of blood ureanitrogen, creatinine, and total protein. Tests were performed by the Division ofComparative Medicine at Washington University School of Medicine. ForH&E staining, kidneys were fixed with 10% buffered formalin solution andembedded in paraffin; 5-mm sections were prepared and stained with H&E.

Immunohistochemistry and immunofluorescence

For immunohistochemistry, 5-mm frozen tissue sections were used forimmunohistochemical staining to visualize SiglecH+ cells in spleens and

LN. Digital images were taken using an Olympus BX60 microscope, cap-tured using a DP-70 Olympus digital camera, and processed using AnalysisImage Processing software (Olympus). For immunofluorescence, 8-mmfrozen spleen sections were fixed in acetone for 5 min at room temperature(RT) and stored at 280˚C. Frozen sections were blocked with 10% horseserum for 20 min at RT. Primary Ab to MARCO, Gr-1, SIGN-R1, andSialoadhesin were applied to tissue sections for 30 min at RT. After washingwith PBS, fluorescent-conjugated anti-rat secondary Ab were added to slidesfor 30 min at RT. Slides were mounted with Fluoromount-G and imaged ona Zeiss LSM 510 META confocal laser scanning microscope. Immunoflu-orescence images were adjusted globally for brightness and contrast usingAdobe Photoshop CS6.

Statistical analysis

Statistical significance was analyzed with an unpaired, two-tailed Studentt test or Mann–Whitney U test. The p values , 0.05 were considered sta-tistically significant. For susceptibility studies, p values were determined bythe log-rank test.

ResultsSiglecH effectively identifies pDC in steady-state and duringinfection

SiglecH is a DAP12-associated receptor used to discriminate pDCfrom other cell types in mice (9, 10). Our previous work (17) showedthat pDC numbers are reduced in spleens of infected mice, whichappeared to be a consequence of cell death. A recent study (11)indicated that, following stimulation with CpGA or infection withMCMV, SiglecH is downregulated in a TLR9/MyD88-dependentmanner. To determine whether reduced pDC numbers during

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

CpGA for 24 h. (A and B) IFN-a was measured in supernatants by ELISA.

(C) WT and SiglecHeGFP/eGFP mice were injected i.v. with CpGA, and serum

IFN-a levels were measured 6 h later. (D) pDC were sort-purified from WT

and SiglecHeGFP/eGFP mice and stimulated for 24 h with MCMV. IFN-a was

measured in supernatants by ELISA. (E) WTand SiglecHeGFP/eGFP mice were

infected i.p. with MCMV, and serum IFN-a levels were measured 48 h p.i.

Data are from two (A, B, D, and E) or three (C) experiments.

FIGURE 3. Lack of SiglecH impacts innate immune responses inde-

pendently of pDC in vivo. WT and SiglecHeGFP/eGFP mice were infected

i.v. with HSV-1. Serum IFN-a (A), TNF-a (B), and IL-6 (C) were mea-

sured 6 h p.i. (D) CLEC4C-DTR–Tg mice were bred to SiglecHeGFP/+ and

SiglecHeGFP/eGFP mice. Mice were injected with PBS or DT 24 h before

infection with HSV-1. Serum IFN-a levels were measured 6 h p.i. Data are

from two or three independent experiments, with at least three mice/group

in each experiment.

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infection are due to reduced detection of SiglecH expression, wecompared SiglecH and eGFP expression in pDC from spleens ofSiglecHeGFP/+ mice that were infected or not with HSV-1. Usingmarkers to identify pDC, such as Ly6C and CD11c, we found thatfrequencies of Ly6C+eGFP+ and Ly6C+SiglecH+ cells werecomparable and reduced in spleen to a similar extent 8 and 24 hpostinfection (p.i.) (Fig. 1A, data not shown), indicating that anti-SiglecH Ab are effective at identifying pDC in the steady-stateand during viral infection.

SiglecHeGFP/eGFP mice are healthy in steady-state

A recent report (18) suggested that an independently derivedSiglecH-deficient mouse strain had abnormal kidney pathology andfunction. Therefore, we evaluated body weight and performed bloodwork analyses on WT and SiglecHeGFP/eGFP mice (Fig. 1). WT andage/gender-matched SiglecHeGFP/eGFP mice had comparable bodyweights, percentages of segmented neutrophils and lymphocytes incirculation, and levels of blood urea nitrogen, creatinine, and totalprotein (Fig. 1B, data not shown). Consistent with the absence ofbiochemical abnormalities in renal function, no pathologic changeswere observed by light microscopy in the glomeruli or tubules ofSiglecHeGFP/eGFP mice (Fig. 1C). Thus, SiglecHeGFP/eGFP mice ap-pear to be healthy and have normal kidney function in steady-state.

SiglecH deficiency does not impact IFN-I production by pDCex vivo

It was shown that SiglecH has an immunomodulatory role duringinflammation and viral infections (6, 9, 11). To determine whetherSiglecH deficiency altered cytokine production by pDC, we mea-sured IFN-a levels in supernatants from enriched or sort-purifiedpDC from WT and SiglecHeGFP/eGFP mice stimulated ex vivo withCpGA. CpGA is a synthetic TLR ligand that induces IFN-I produc-tion by pDC through TLR9 (19). pDC fromWTand SiglecHeGFP/eGFP

mice produced comparable amounts of IFN-a in response to CpGA(Fig. 2A, 2B), suggesting that lack of SiglecH does not stronglyalter IFN-I production by pDC ex vivo. We next evaluated IFN-Iresponses to CpGA in vivo. WT and SiglecHeGFP/eGFP mice wereinjected i.v. with CpGA complexed with DOTAP, and serum IFN-awas measured 6 h later (Fig. 2C). Analyses of several mice revealedno significant differences in serum IFN-a levels between WT andSiglecHeGFP/eGFP mice. IFN-a was not detectable in serum fromnaive 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 issensed by pDC through TLR9 (20). Moreover, it was reported thatpDC are an important and early source of IFN-I during MCMVinfection (3, 20–23). Thus, we sort-purified pDC from WT andSiglecHeGFP/eGFP mice and cultured them with MCMV. pDC fromboth groups of mice produced similar levels of IFN-a (Fig. 2D),indicating that SiglecH deficiency did not affect IFN-I productionby virus-stimulated pDC ex vivo. We next evaluated whether micelacking SiglecH had altered IFN-I responses in vivo during MCMVinfection. We found that SiglecHeGFP/eGFP mice had increased levelsof systemic IFN-a compared with WT mice at 48 h p.i. (Fig. 2E),similar to a recent study (11). Taken together, these data suggestthat SiglecH deficiency does not affect IFN-I secretion by pDC inresponse to a synthetic TLR9 ligand or virus ex vivo, but it mayinfluence IFN-I production by pDC during viral infection in vivo.

Enhanced cytokine responses to HSV-1 in SiglecHeGFP/eGFP

mice occur in the presence and absence of pDC

SiglecHDTR/DTR mice were reported to have increased cytokineresponses to systemic HSV-1 infection (6). Corroborating thesefindings, SiglecHeGFP/eGFP mice also had slightly elevated levels of

systemic IFN-a and proinflammatory cytokines compared withWT mice after HSV-1 infection (Fig. 3A–C). These results areconsistent with an immunomodulatory role for SiglecH in antiviralresponses (6, 9, 11). To determine whether this effect was dueto pDC, we bred CLEC4C-DTR–Tg mice to SiglecHeGFP/+ andSiglecHeGFP/eGFP mice and infected them with HSV-1 in the pres-ence or absence of pDC. We found that SiglecHeGFP/eGFP micedepleted of pDC produced more IFN-a than did pDC-depletedSiglecHeGFP/+ mice or pDC-depleted CLEC4C-DTR–Tg mice(Fig. 3D). In all three lines of depleted mice, there was an ∼2-ngreduction in serum IFN-a levels relative to their undepleted coun-terparts. These findings suggest that lack of SiglecH may affectcytokine secretion by cells other than pDC during viral infection orthat viral burden and/or distribution is altered in the absence ofSiglecH.

SiglecH is expressed by MZM, medullary macrophages,microglia, and progenitors of cDC and pDC

Although SiglecH expression is mainly confined to pDC in cellsuspensions from primary and secondary lymphoid organs, it wasobserved by microscopy that specialized macrophage subsets in thespleen and LN express SiglecH (10). Corroborating this, we foundthat MZM in spleen (Fig. 4A) and medullary macrophages in LN(data not shown) were SiglecH+ by immunohistochemistry. In ad-dition, we observed that SiglecH was expressed by CD45intCD11b+

microglia in brain (Fig. 4B), in agreement with a recent study (24).We reported previously that cDC are eGFP+ in SiglecHeGFP/+

mice (3). Closer examination revealed that both CD4+ and CD42

cDC in spleen were eGFP+ (Fig. 5A); however, they did not ex-press SiglecH on the surface or at the transcript level (Fig. 5B,

FIGURE 4. SiglecH is expressed by specialized macrophages. (A)

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

magnification 3100, upper panels; 3400, lower panels). Scale bars, 200

mm (upper panels), 50 mm (lower panels). (B) Microglia were isolated

from WT mice and stained with CD45, CD11b, and SiglecH. Plots show

CD45intCD11b+ microglia (left panel), and SiglecH expression (right

panel). Data are representative of two independent experiments, with three

to five mice/experiment.

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data not shown). These findings indicated that the SiglecH pro-moter was active in DC progenitors during development, consistentwith a recent study (25), and that eGFP persists in differentiated ormature cDC. In the BM, there is a subset of SiglecH+ cells that hasbeen defined as prepDC (Fig. 5C), which can differentiate into pDCand cDC in vitro and in vivo (Fig. 5D) (12, 13). Both mature pDCand prepDC expressed the pDC-specific transcription factor Tcf4/E2-2 (Fig. 5E) (12, 26) and were eGFP+ in SiglecHeGFP/+ mice (datanot shown). Taken together, these data indicate that SiglecH isexpressed by mature pDC, specialized macrophages, and progenitorsof cDC and pDC.

PrepDC and MZM are depleted in SiglecH-DTR–Tg mice

Given that SiglecH expression is not restricted to mature pDC, wehypothesized that prepDC and MZM may also be depleted in micethat express DTR under the control of the SiglecH promoter. To testthis hypothesis, we used newly generatedC57BL/6 SiglecH-DTR–Tgmice that express DTR under the control of the SiglecH promoter.After DT injection, both mature pDC and prepDC were ablated inSiglecH-DTR–Tg mice (Fig. 5F). In contrast, only mature pDC wereeliminated in CLEC4C-DTR–Tg mice (Fig. 5F). Furthermore, whenCLEC4C-DTR–Tg mice were bred to SiglecHeGFP/+ mice, eGFP+

cDC were not depleted after DT administration (3).We next asked whether MZM were depleted in SiglecH-DTR–Tg

mice after DT treatment. To address this, we generated C57BL/6SiglecH-DTR–Tg BM chimeras, because normal C57BL/6 SiglecH-

DTR–Tg mice do not tolerate DT very well. After 4–5 mo of rest,we injected CLEC4C-DTR–Tg mice and SiglecH-DTR–Tg chimeraswith PBS or DT and harvested spleens 24–36 h later. Spleen sectionswere stained with Ab against the scavenger receptor MARCO, whichis expressed by MZM (Fig. 6A) (27, 28). Results indicated that MZMwere intact in CLEC4C-DTR–Tg mice treated with either PBS or DT.In contrast, very few MZM could be identified in spleen sections fromSiglecH-DTR–Tg chimeras injected with DT. The depletion of MZMin SiglecH-DTR–Tg mice was validated in a second SiglecH-DTR–Tg line that was generated on a BALB/c background (15). Spleensections stained for both MZM and metallophilic macrophages (MM)revealed that MZM, but not MM, were reduced in BALB/c SiglecH-DTR–Tg mice injected with DT (Fig. 6B).

Altered bacterial uptake and increased susceptibility toLM-OVA infection in depleted SiglecH-DTR–Tg mice

MZM are important for the clearance of apoptotic cells (29–31) andfor the uptake of bacteria, such as S. pneumoniae (32–34). Micelacking SIGN-R1, a receptor expressed by MZM, are more suscep-tible to systemic S. pneumoniae infection and show signs of alteredbacterial distribution in the spleen upon infection (33, 34). Therefore,we hypothesized that SiglecH-DTR–Tg mice, which appear to lackMZM after DT treatment, also would exhibit altered bacterial dis-tribution and perhaps be more susceptible to infection. To test this, weinjected control or depleted CLEC4C-DTR–Tg mice and SiglecH-DTR–Tg chimeras with fluorescent-labeled S. pneumoniae and

FIGURE 5. SiglecH expression in progeni-

tors 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 sub-

set. (D) Sorted prepDC from BM differentiate

into cDC or pDC after culture in GM-CSF or

Flt3L, respectively. (E) E2-2 expression in pDC,

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.

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examined spleens 1 h p.i. by flow cytometry. We first confirmeddepletion in mice treated with DT by staining for pDC (Fig. 7A,top panels). Next, using a variety of Ab, we evaluated which cellsfrom each group of mice were associated with fluorescent bacte-ria. In all groups of mice, bacteria were mainly found amongCD11b+Gr-1+F4/802 cells but not pDC (Fig. 7A, bottom panelsand data not shown). However, we noted that, in SiglecH-DTR–Tgchimeras treated with DT, there was a 3-fold increase in the fre-quency of Gr-1+ cells, presumably neutrophils, which were asso-ciated with bacteria. These findings suggested that the absence ofMZM in SiglecH-DTR–Tg mice resulted in increased bacterialburden in the red pulp. To confirm this, we analyzed spleens bymicroscopy 1 h after injection of fluorescent-labeled S. pneumo-niae (Fig. 7B). Very few fluorescent bacteria could be detected inCLEC4C-DTR–Tg mice injected with PBS or DT or in SiglecH-DTR–Tg chimeras injected with PBS. In contrast, SiglecH-DTR–Tg chimeras treated with DT had many bacteria located in the redpulp, as visualized by Gr-1 staining, confirming altered bacterialdistribution and impaired clearance in the absence of MZM.Given the differences in bacterial uptake and clearance between

CLEC4C-DTR–Tg and SiglecH-DTR–Tg chimeras, we sought todetermine whether SiglecH-DTR–Tg mice were more or less sus-ceptible to bacterial infection after DT administration. A study byTakagi et al. (6) found that SiglecHDTR/DTR mice treated with DT

were more resistant to lethal infection with LM-OVA than werenondepleted mice. Thus, we performed a similar experiment in ourCLEC4C-DTR–Tg mice and SiglecH-DTR–Tg chimeras. SiglecH-DTR–Tg chimeras treated with DT succumbed to LM-OVA infectionfaster than did mice in the other three groups (Fig. 8A). Moreover, itdid not appear that lack of pDC was responsible for this phenotype,because CLEC4C-DTR–Tg mice, depleted or not of pDC, exhibitedidentical rates of survival and died later than did DT-treated SiglecH-DTR–Tg chimeras (Fig. 8A). Analysis of serum cytokine levelsrevealed that DT-treated SiglecH-DTR–Tg chimeras produced verylittle IL-12p70 and had exaggerated levels of TNF-a and IL-6 in their

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

(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.

FIGURE 7. Distribution and clearance of bacteria are altered in depleted

SiglecH-DTR–Tg mice. PBS- or DT-treated CLEC4C-DTR–Tg mice and

C57BL/6 SiglecH-DTR–Tg chimeras were injected with Alexa Fluor 647–

labeled S. pneumoniae R36A. One hour after injection, spleens were har-

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-

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

numbers of bacteria in the red pulp of DT-treated SiglecH-DTR–Tg chimeras

(original magnification 310). Data are representative of three independent

experiments.

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serum compared with the other groups of mice (Fig. 8B). Thus, theincreased susceptibility and cytokine storm in DT-treated SiglecH-DTR–Tg mice most likely was a consequence of altered bacterialdistribution in the absence of SiglecH+ cells that were not pDC.

DiscussionIn this study, we found that SiglecH deficiency resulted in increasedcytokine responses during HSV-1 infection and other viral infections,which may be pDC independent. Indeed, SiglecH was expressed byMZM, which were reported to be major producers of IFN-I duringsystemic HSV-1 infection (35). SiglecH also was expressed by LNmedullary macrophages, microglia, and prepDC in the BM, whichcan differentiate into pDC and cDC (10, 12, 13, 24). Although cDCdid not express SiglecH on their surface or at the transcript level, theywere eGFP+ in SiglecH-eGFP–knockin mice (3), suggesting that theSiglecH promoter was active in a common DC progenitor during DCdevelopment (25). In addition, it was shown recently, using newlygenerated SiglecH-RFP reporter mice, that a fraction of B, T, NK,and NKT cells are RFP+, indicating SiglecH promoter activity ina common lymphoid progenitor (11). Thus, lack of SiglecH mayaffect cytokine production by a variety of cell types during a viralinfection, perhaps by altering viral burden and/or distribution.The semipromiscuous expression of SiglecH potentially explains

data inconsistencies between inducible pDC-ablation models. It ispossible that certain macrophage subsets and DC progenitors aredepleted in SiglecH-DTR-knockin or Tg mice, thus yielding strongerphenotypes than observed in CLEC4C-DTR–Tg mice. Indeed, usingtwo SiglecH-DTR–Tg mouse lines, we found that prepDC and MZMwere reduced after DT treatment in contrast to CLEC4C-DTR–Tgmice. Furthermore, depleted SiglecH-DTR–Tg mice showed alteredbacterial distribution and impaired clearance after injection ofS. pneumoniae compared with control mice and DT-treated CLEC4C-DTR–Tg mice. These results are consistent with a role for MZM inresponses to S. pneumoniae (32–34). Moreover, we found that de-pleted SiglecH-DTR–Tg mice were highly susceptible to lethal LM-

OVA infection. Previous studies showed that, early p.i., Listeria canbe found associated with SIGN-R1+ cells in the spleen (36, 37) andthat mice depleted of MZM and MM are impaired in their ability tocontrol and survive Listeria infection (38). We envision that lack ofMZM in DT-treated SiglecH-DTR–Tg mice may account for thecytokine 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 reportthat depleted pDC in SiglecHDTR/DTR mice. First, our depletedSiglecH-DTR–Tg mice were more susceptible to infection, whereasdepleted 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 in-fection, because PBS- and DT-treated mice had identical survivalrates and similar levels of systemic cytokines. It should be notedthat SiglecHDTR/DTR mice lack SiglecH expression (6). Therefore,the findings reported by Takagi et al. (6) do not discriminate effectsof SiglecH deficiency from pDC depletion. Although it has beensuggested that pDC may be detrimental during Listeria infectionbecause of their ability to produce IFN-I, studies showed that my-eloid cells and CD8a+ DC are important in pathogenesis as eitherIFN-I–producing cells or initial cellular entry points that establishproductive infection (39, 40). Taken together, we conclude that thebroad expression pattern of SiglecH should be considered whenusing SiglecH-DTR mice to evaluate pDC functions in vivo.

AcknowledgmentsWe thank C.A. Stewart (National Cancer Institute) for advice on BAC

recombineering, D. Leib (Geisel School of Medicine at Dartmouth) for

HSV-1, A. French (Washington University School of Medicine) for MCMV,

C. Rossini for technical support, J.F. Kearney and L. Jia (University of Ala-

bama at Birmingham) for R36A, and S. Srivatsan and E. Lantelme (Wash-

ington University School of Medicine) for help with BM chimeras and cell

sorting.

DisclosuresThe authors have no financial conflicts of interest.

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