of December 11, 2018. This information is current as Infection Leishmania Following Development of Regulatory Dendritic Cells Cooperate To Support Increased Stromal Cell-Derived CXCL12 and CCL8 Aziz, Paul M. Kaye and Mattias Svensson Anh Thu Nguyen Hoang, Hao Liu, Julius Juaréz, Naveed ol.0903673 http://www.jimmunol.org/content/early/2010/07/12/jimmun published online 12 July 2010 J Immunol Material Supplementary 3.DC1 http://www.jimmunol.org/content/suppl/2010/07/12/jimmunol.090367 average * 4 weeks from acceptance to publication Fast Publication! • Every submission reviewed by practicing scientists No Triage! • from submission to initial decision Rapid Reviews! 30 days* • Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. All rights reserved. 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on December 11, 2018 http://www.jimmunol.org/ Downloaded from by guest on December 11, 2018 http://www.jimmunol.org/ Downloaded from
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of December 11, 2018.This information is current as
InfectionLeishmaniaFollowing Development of Regulatory Dendritic CellsCooperate To Support Increased Stromal Cell-Derived CXCL12 and CCL8
Aziz, Paul M. Kaye and Mattias SvenssonAnh Thu Nguyen Hoang, Hao Liu, Julius Juaréz, Naveed
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Print ISSN: 0022-1767 Online ISSN: 1550-6606. All rights reserved.1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,
is published twice each month byThe Journal of Immunology
Stromal Cell-Derived CXCL12 and CCL8 Cooperate ToSupport Increased Development of Regulatory Dendritic CellsFollowing Leishmania Infection
Anh Thu Nguyen Hoang,* Hao Liu,† Julius Juarez,* Naveed Aziz,‡ Paul M. Kaye,† and
Mattias Svensson*
In the immune system, stromal cells provide specialized niches that control hematopoiesis by coordinating the production of
chemokines, adhesion molecules, and growth factors. Stromal cells also have anti-inflammatory effects, including support for the
differentiation of hematopoietic progenitors into dendritic cells (DCs) with immune regulatory properties. Together, these obser-
vations suggest that the alterations in hematopoiesis commonly seen in infectious disease models, such as experimental visceral
leishmaniasis in mice, might result from altered stromal cell function. We report in this study that the stromal cell-derived chemo-
kines CXCL12 and CCL8 cooperate to attract hematopoietic progenitors with the potential to differentiate into regulatory DCs. We
also show that infection of murine bone marrow stromal cells by Leishmania donovani enhanced their capacity to support the
development of regulatory DCs, as well as their capacity to produce CCL8. Likewise, in experimental visceral leishmaniasis, CCL8
production was induced in splenic stromal cells, leading to an enhanced capacity to attract hematopoietic progenitor cells. Thus,
intracellular parasitism of stromal cells modifies their capacity to recruit and support hematopoietic progenitor differentiation into
regulatory DCs, and aberrant expression of CCL8 by diseased stromal tissue may be involved in the switch from resolving to
persistent infection. The Journal of Immunology, 2010, 185: 000–000.
Stromal cells, suchasfibroblasts, endothelial cells, andstromalmacrophages, are important in regulating tissue homeostasisand supporting the differentiation of hematopoietic stem and
progenitor cells (HSPCs) into terminally differentiated blood cells.Under homeostatic conditions,HSPCs aremost abundant in the bonemarrow (BM), where they reside with stromal cells in specializedniches that control HSPC homing, migration, survival, proliferation,differentiation, and self-renewal (1). Homing and migration ofHSPCs involves the action of locally produced chemokines (2, 3).The chemokine CXCL12 is constitutively expressed at high levelsby stromal cells in the BM and acts as the major chemoattractant of
HSPCs (4). Inactivation of CXCL12 and its receptor CXCR4 leadsto mobilization of HSPCs and impaired hematopoiesis (5–8). Al-though these data suggest an important role for CXCR4/CXCL12 inBM retention of HSPCs, the relative contribution to progenitor re-tention by other stromal cell-derived chemokines is poorly defined.Furthermore, under homeostatic conditions, HSPCs in the BM arenot entirely sessile but contain a population of highlymigratory cells,and some HSPCs recirculate constantly between BM and blood (9).Blood HSPC numbers can be dramatically modified during inflam-mation and following stimulation by several cytokines, includingCSF-2 and CSF-3 (10, 11). Moreover, studies in mice revealed thatBM is not the exclusive destination of blood-borne HSPCs, becauseHSPCs have also been recovered from extramedullary sites, includ-ing liver (12), spleen (3), and muscles (13). Accordingly, diversetissue stromal cells provide specialized local niches that controlHSPC migration and survival, and that may have the capacity tosupport local differentiation of HSPCs into diverse populations ofimmune cells.Dendritic cells (DCs) are a heterogeneous family of hematopoietic
CD11c+ and MHC-II+ cells with specialized Ag-presenting capaci-ties (14, 15). Differentiation of DCs mainly proceeds from HSPCswithin theBM,and several developmental intermediates ofDCshavebeen isolated from blood and various tissues (15–18). In a previousstudy, it was reported that migratory HSPCs also proliferate anddifferentiate within extramedullary tissues and give rise to tissue-resident DCs (9). As a heterogeneous population, DCs contributeto the functional differentiation of effector and regulatory T cells;therefore, DCs play a dual role in inducing adaptive immuneresponses to foreign Ags and in maintaining tolerance to self (19,20). Notably, regulatoryT cell differentiation is controlled by subsetsof DCs (regulatory DCs), which, in turn, arise from tissue-specificand stromal cell-directed differentiation of HSPCs (21). We andother investigators showed that murine splenic stromal cells playan active role in supporting the differentiation of BM lineagenegative (BMLin2) CD117+ HSPCs into regulatory DCs that
*Department of Medicine, Center for Infectious Medicine, Karolinska Institutet,Stockholm, Sweden; and †Centre for Immunology and Infection, Hull York MedicalSchool and ‡Department of Biology, Technology Facility, University of York, York,United Kingdom
Received for publication November 17, 2009. Accepted for publication June 10,2010.
Work in the authors’ laboratories was supported by grants from the British MedicalResearch Council and Wellcome Trust (to P.K.), the Swedish Research Council, theSwedish Foundation for Strategic Research, the Erik och Edit Fernstroms Foundation,and the Karolinska Institutet (to M.S.). A.T.N.H. was a recipient of a Karolinska post-graduate studentship. H.L. was a recipient of a Medical Research Center Dorothy Hodg-kins Research Studentship. J.J. was a recipient of a Marie Curie postdoctoral fellowship.
The sequences in this article have been submitted to the European BioinformaticsInstitute ArrayExpress database (www.ebi.ac.uk/microarray-as/ae/) under accessionnumber E-MEXP-2554.
Address correspondence and reprint requests to Dr. Mattias Svensson, Center forInfectious Medicine, F59, Department of Medicine, Karolinska Institutet, KarolinskaUniversity Hospital, Huddinge, 141 86 Stockholm, Sweden. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: BM, bone marrow; BSS, balanced salt solution; CT,cycle threshold; DC, dendritic cell; EVL, experimental visceral leishmaniasis; HSPC,hematopoietic stem and progenitor cell; LAMP, lysosomal membrane protein-1;MHC-II, MHC class II; MOI, multiplicity of infection; n.d., not detectable; NGS,normal goat serum; PTX, pertussis toxin; RT, room temperature.
Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903673
Published July 12, 2010, doi:10.4049/jimmunol.0903673 by guest on D
produce IL-10 and have a CD11cloCD11bhi phenotype (21). Stromalcell-dependent regulatoryDCswere capable of inhibiting T cell pro-liferation via NO production (22) or IL-10–dependent mecha-nisms (22, 23) and caused IL-10–producing CD4+ regulatoryT cells to be able to transfer Ag-specific tolerance (23). More re-cently, Xia et al. (24) reported that liver stromal cells also supportBMLin2CD117+ HSPC differentiation into CD11cloCD11bhi
MHC-IIlo regulatory DCs that have the capacity to alleviateexperimental autoimmune hepatitis. Significantly, natural subsetsof regulatory DCs resembling those developing on stromal cellsin vitro have been isolated from various tissues, including spleen(22, 25, 26), lung (27, 28), and liver (24).Tissue stromal cells are important in many pathological pro-
cesses. For example, at the time of tissue injury, fibroblasts par-ticipate in wound healing by producing extracellular matrixproteins, as well as responding to and synthesizing cytokines,chemokines, and other mediators of inflammation. Stromal cells areclearly not fixed in terms of their capacity to support hematopoieticactivity (29). In chronic inflammation resulting from infection withLeishmania donovani, considerable remodeling of the splenicmicroarchitecture coincides with altered splenic stromal cellfunctions and increased hematopoietic activity (30, 31). Aug-mented accumulation of mRNA for CSF-1, CSF-2, and CSF-3 isobserved, indicative of enhanced hematopoietic activity (31).Moreover, this infection also promotes the capacity of splenicstromal cells to support regulatory DC differentiation (23). Similarchanges may also occur in experimental malaria infections (32).However, the precise cellular components and niche-derived fac-tors allowing HSPC differentiation into regulatory DCs within var-ious vascular niches at steady state and in the course of chronicinfectious diseases remain unclear.In this study, we sought to identify whether stromal cell-derived
chemokines play a role in the differentiation of HSPCs into regula-tory DCs and whether altered chemokine expression might, there-fore, underpin the observed increase in this activity evident instromal cells isolated from mice infected with L. donovani (23).Our data demonstrate that stromal cell-guided differentiation ofBMLin2CD117+ HSPCs into regulatory DCs involves the stromalcell-derived chemokines CXCL12 and CCL8. Furthermore, ourdata also indicate that direct infection of stromal cells with L.donovani enhances their capacity to support regulatory DC differ-entiation and that this parasite selectively modulates expression ofthose chemokines involved in stromal cell-guided HSPC differen-tiation into regulatory DCs in vitro and in vivo.
Materials and MethodsMice and infection
C57BL/6 (B6) and BALB/c mice were obtained from Charles River Lab-oratories (Margate, U.K.) or bred at the Department of Microbiology andTumor Biology, Karolinska Institutet. Micewere used at 6–10 wk of age andhoused under conventional conditions with food and water ad libitum.Parasites of the Ethiopian strain of L. donovani (LV9) were maintained byserial passage in Syrian hamsters or B6.RAG12/2 mice. Amastigotes wereisolated from infected spleens and used for infections of mice and cell lines.Mice were infected with 2 3 107 L. donovani amastigotes i.v. in 100 mlRPMI 1640 (Invitrogen, Paisley, U.K.) and killed by cervical dislocation, asrequired. All animal care and experimental procedures were approved by theUniversity of York Animal Procedures and Ethics Committee and per-formed under U.K. Home Office License and were also approved by theAnimal Ethics Committee of Stockholm in Sweden.
Primary stromal cells and cell lines
Stromal cells from naive or infected B6 mice were obtained as previouslydescribed (23). The BM-derived stromal cell line MBA-1 (a gift fromDr. D. Zipori, Weizmann Institute of Science, Reovot, Israel) was maintainedin completeDMEM(50mM2-ME,100U/mlpenicillin, 100g/ml streptomycin,
10 mMHEPES, nonessential amino acids; all from Invitrogen), 1 mM sodiumpyruvate, and 2 mM L-glutamine, supplemented with 10% heat-inactivatedFBS (all from Sigma-Aldrich, Poole, U.K.). The RAW264.7 macrophage cellline (American TypeCulture Collection, Teddington, U.K.) wasmaintained incomplete DMEM containing 10% heat-inactivated FBS. When cell lineshad reached 80–90% confluency, cells were removed by incubation inenzyme-free cell-dissociation buffer (Invitrogen) at 37˚C for 30 min andby using a cell lifter (Corning, Amsterdam, The Netherlands). Collectedcells were washed twice in serum-free DMEM and were incubated in0.5 mg/ml collagenase for 45 min at room temperature (RT). Collagen-digested cells were washed twice in complete DMEM. Pelleted cells wereresuspended in complete DMEM supplemented with 5% FBS, and 2 3 103,104, 105, or 106 cells were seeded in 24-well plates or T25 tissue culture flasks,or 2 3 103 cells were seeded onto coverslips in 24-well plates. Cells wereincubated for 48 h in a humidified, 5% CO2 incubator at 37˚C; supernatantswere collected, and cells were used for infections, cocultures with HSPCs,chemotaxis assays, or gene-expression analyses.
In vitro infections of cell lines
Freshly isolated L. donovani amastigotes were added to the adherent cells intissue culture plates or flasks at various multiplicities of infection, rangingfrom 5 to 20:1. Tissue cultures were incubated in a humidified, 5% CO2
incubator at 37˚C for 1 h. Noninternalized parasites were removed by threewashes with complete DMEM. The efficiency of infection (parasite-containing cells/100 cells) was determined from triplicate samples ofGiemsa-stained coverslip preparations with 104 cells or coverslip prepara-tions stained for immunofluorescence analysis after incubation in completeDMEM with 5% heat-inactivated FBS for up to 48 h in a humidified, 5%CO2 incubator at 37˚C. Alternatively, infected cells were used for mRNAextraction, gene-expression analysis, or cocultures with progenitors.
Purification and culture of HSPCs
BMLin2CD117+ HSPCs were purified using a cell-lineage depletion kit(Miltenyi Biotec, Bergisch Gladbach, Germany), as previously described(23). A total of 5 3 104 or 2 3 105 BMLin2CD117+ cells were culturedtogether with MBA-1 or RAW264.7 cells in 24-well plates, or BMLin2
CD117+ cells were cultured in Transwell inserts (0.4, 3.0, or 5.0 mm pore-size, Corning Glass) with stromal cells or stromal cell-derived supernatant inthe lower chamber. Alternatively, BMLin2 cells were cultured in 10% culturesupernatant from a myeloma cell line transfected with murine CSF-2 cDNA.As required, LPS (1 mg/ml) was added for the final 48 h of culture. Allcultures containing BMLin2CD117+ cells used complete DMEM supple-mented with 5% FCS in humidified, 5% CO2 incubators at 37˚C.
Flow cytometry
For flow cytometry, cells were incubated with 10 mg/ml 2.4G2 anti-Fcreceptor mAb (BD Pharmingen, San Diego, CA), followed by labelingwith directly conjugated mAb. Cells were labeled with FITC-conjugatedanti–Sca-1 (clone D7), anti-H2Kb (clone AF6-88.5), anti-CD86 (cloneGL-1), anti-F4/80 (clone CI:A3-1), and anti-GR1 (clone RB6-8C5); PE-conjugated anti-CD117 (clone 2B8), anti-H2Ab (clone M5/114.15.2), andanti-CD11b (clone M1/70); PerCp-conjugated anti-CD45 (clone 30-F11);and allophycocyanin-conjugated CD11c (clone HL3) mAbs (all fromBD Pharmingen, except for F4/80, which was from Serotec, Oxford,U.K.). Minimal background staining was observed using control FITC-conjugated, PE-conjugated, PerCp-conjugated rat IgG2a and IgG2b Absand allophycocyanin-conjugated hamster IgG Ab (all from BD Pharmin-gen). All labeling was performed on ice for 30 min in PBS containing 2%FCS, 5 mM EDTA, and 0.01% sodium azide. Flow-cytometry analysiswas performed with a FACSCalibur (BD Biosciences, San Jose, CA) on50,000 cells and analyzed using CellQuest software (BD Biosciences).
Immunohistochemistry and immunofluorescence staining
Splenic tissue for cryosectioning was prepared from infected and naiveanimals by embedding the tissue in OCT compound on a cork block. Blockswere snap-frozen in liquid nitrogen, stored at280˚C until required, and then8-mm cryosections were cut onto diagnostic microscope glass slides(Thermo Scientific, Waltham, MA) using a MICROM cryostat HM 560MV (Zeiss, Jena, Germany) and fixed in 2% freshly prepared formal-dehyde in PBS for 15 min at RT. For immunohistochemistry, cryostat sec-tions were blocked with 10% FCS in Earl’s balanced salt solution (BSS)with 0.1% saponin for 30 min at RT, followed by additional blocking with2% H2O2 in BSS-saponin and an avidin and biotin blocking reagent (VectorLaboratories, Burlingame, CA). Primary monoclonal rat anti-mouse CCL8(15 mg/ml, clone 146123; R&D Systems, Minneapolis, MN) and ER-TR7(BMA Biomedicals, Augst, Switzerland) Abs were diluted in BSS solution
containing saponin and incubated overnight at RT. After incubation, tissuesections were washed and blocked with 1% normal rabbit serum in BSS-saponin before the addition of biotinylated rabbit anti-rat IgG (diluted 1:200;DakoCytomation, Glostrup, Denmark) diluted in 1% normal rabbit serum inBSS-saponin. Avidin-peroxidase solution was added (Vectastain-Elite; VectorLaboratories), and the color reaction developed by the addition of 3,3-diaminobenzidine (Vector Laboratories), followed by counterstaining with he-matoxylin. Tissue sections were visualized using a Leica DMR microscope.
For immunofluorescence labeling of splenic tissue, cryostat sections wereblocked with an avidin and biotin blocking reagent, followed by additionalblocking with 0.1% BSA-c (Aurion, Wageningen, The Netherlands) in PBSwith 0.1% saponin for 30min at RT. Sectionswere incubated overnight at RTwith amixtureof the twoprimaryAbs: rat anti-mouseCCL8 (15mg/ml, clone146123; R&D Systems) and serum from Leishmania-infected hamsters.After incubation, tissue sections were washed and incubated with the bio-tinylated rabbit anti-rat IgG (diluted 1:200; DakoCytomation) for 30 min atRT; after washing, sections were incubated with Alexa Fluor 546-conjugated goat anti-hamster IgG (1:500; Molecular Probes. Eugene, OR)for 30 min at RT. Sections were mounted in SlowFade Gold Antifade withDAPI (Molecular Probes). Tissue sections were visualized using a LeicaTCS SP2 confocal microscope.
For immunofluorescence labeling, cells derived from freshly isolatedsplenic tissue or in vitro cultures were grown on coverslips or spun onto glassslides by cytospin, followed by fixation in 4% paraformaldehyde in PBS for15 min RT. After fixing, samples were blocked and permeabilized by in-cubation with 1.5% normal goat serum (NGS) in 0.2% saponin in PBS for 45min at RT. Sampleswere incubatedwithAbs diluted in PBS containing 1.5%NGS and 0.2% saponin for 45 min at RT. Serum from Leishmania-infectedhamsters and the following primary reagents were used: hamster anti-CD11c (clone N418; Serotec), rat anti-H2Ab (clone 2G9), anti-CD107a(lysosomal associated membrane protein-1 [LAMP-1]; clone 1D4B),anti–ER-TR7 (BMA Biomedicals), and anti-CXCL12 (clone 79014; R&DSystems). After washing cells four times in 1.5% NGS in 0.2% saponin inPBS, specific staining was detected by Alexa Fluor 488-conjugated goatanti-rat IgG (Molecular Probes), Alexa Fluor 488-conjugated goat antimouse IgG (Molecular Probes), Alexa Fluor 546-conjugated goat anti-rat,and Alexa Fluor 546-conjugated goat anti-hamster IgG (Molecular Probes).BODIPY-650 phalloidin was used to reveal F-actin (Molecular Probes), andDAPI was used to reveal cell nuclei. Coverslips were mounted in antifade(Molecular Probes) and visualized with a confocal microscope (Leica TCSSP2 AOBS, LeicaMicrosystems,Wetzlar, Germany or Zeiss LSM 510MetaAxioplan 2 system; Zeiss). Images shown are single optical slices (0.8 to1.0 mm), or projections were made from five 1-mm-thick optical sections.
Allogeneic MLRs
Splenic BALB/c CD4+ T cells were isolated, as previously described (23).Primary MLRs were set up in flat-bottom 96-well plates (BD Biosciences)with 1.53 105 responderBALB/cCD4+ T cells perwell and variable numbersof allogeneic stimulator cells. All stimulator cells were irradiated with 2000rad.MLRswere incubated for 96 h in humidified, 5%CO2 incubators at 37˚C.Eight hours before termination, 0.5 mCi [3H]thymidine was added per well.The culture medium was RPMI 1640, with sodium pyruvate and L-glutamineand supplemented with 50 mM 2-ME, 100 U/ml penicillin, 100 g/ml strepto-mycin, and 10% FBS (all from Invitrogen).
Migration assay
Migration of progenitor cells was assayed using Transwell cell-cultureinserts (BD Biosciences). Stromal cells or RAW264.7 macrophages dilutedin 500 ml complete DMEM were placed into 24-well culture plates (Costar,Cambridge, MA) and allowed to adhere and condition the culture mediumfor 48 h prior to initiating the assay. Alternatively, 500 ml culture supernatantfrom MBA-1 cells, freshly isolated stromal cells, or rCXCL12 and rCCL8diluted in 500 ml complete DMEM were placed into the 24-well cultureplates. Transwell culture inserts with a pore size of 3.0 or 5.0 mm thatallow cellular transmigration were placed into 24-well culture plates, and105 BMLin2CD117+ HSPCs in 100 ml complete DMEM were seeded in theculture inserts. Alternatively, inserts, with a pore size of 0.4 mm, that onlyallow diffusion of soluble products were used. Prior to seeding progenitors,Matrigel (BD Biosciences) was added to the inserts to form a thin matrixlayer. Cells were allowed to migrate for 3 or 16 h at 37˚C in 5% CO2, afterwhich inserts were removed or kept for the total coculture period of 6 d.Migrated cells were enumerated and analyzed by flow cytometry, and theamount of migrated cells relative to the total BMLin2CD117+ HSPCs ini-tially seeded in the upper chamber was calculated. Alternatively, chemotaxiswas quantified by flow cytometry following the addition of an internallabeled standard and expressed as a percentage of the total input cells inthe upper chamber.
Microarray analysis
To analyze gene-expression levels in MBA-1 and RAW264.7 cells, we per-formed a genome-wide expression analysis using the GeneChip Mouse Ge-nome 430 2.0 array (Affymetrix, Santa Clara, CA), which consists of.45,000 probe sets representing.34,000mouse genes. A total of 106 controlor infected MBA-1 and RAW264.7 cells were incubated for 12 and 48 h.Samples were prepared in triplicate on three separate occasions. RNA wasextracted from the cell lines using TRIzol reagent, according to the manufac-turer’s protocol (Sigma-Aldrich). One of the triplicate samples from each ofthe three experiments was used for microarray analysis. Extracted RNA qual-ity was assessed by Agilent Model 2100 Bioanalyzer (Agilent, Palo Alto,CA). RNAwas processed for the microarray using the Affymetrix GeneChipone-cycle target-labeling kit (Affymetrix), according to the manufacturer’srecommended protocols and hybridized to individual arrays. Raw data pro-cessing was performed using Affymetrix GCOS 1.2 software. After hybrid-ization and scanning, probe cell intensities were calculated and summarizedfor the respective probe sets by means of the MAS5 algorithm. For eachexperiment and time points, data sets from infected and noninfectedMBA-1 cells were compared with infected and noninfected RAW264.7 cells.MAS5-normalized data were collected and analyzed using the ArrayAssistExpression software, Version 5.5 (Stratagene, La Jolla, CA). Cluster analysison the genes was performed, and differentially expressed genes were identi-fied using a two-class t test, and the significance level was set at p , 0.05.Genes thatwere up- or downregulated.2-fold between groups were selected.Data from these studies have been deposited in the European BioinformaticsInstitute ArrayExpress database (www.ebi.ac.uk/microarray-as/ae/; accessionnumber: E-MEXP-2554).
Real-time RT-PCR
Total RNA was extracted from splenic tissue, spleen-derived stromalcells, or cell lines using an RNeasy Mini kit (Qiagen, Valencia, CA),following the manufacturer’s protocol. RNA was converted into cDNAusing a cDNA reverse-transcription kit (Applied Biosystems, Foster City,CA), according to the manufacturer’s instructions. Amplification of cDNAwas performed using the Applied Biosystems 7500 Fast Real-Time PCRSystem. The primers and probes for CXCL12 (Mm00445552_m1), CCL8(Mm01297183_m1), and 18S rRNA (4310893E) were purchased as Pre-Developed TaqMan Gene Expression Assays (Applied Biosystems). The18S rRNA served as an endogenous control to normalize the amount ofsample cDNA. The TaqMan Gene Expression Master Mix (Applied Biosys-tems) was used for the reaction mixtures of 10 ml, as recommended by themanufacturer. Relative amounts of the two chemokines were calculated usingthe comparative threshold cycle (CT) method (33). The threshold correlateswith the cycle number where there is sufficient amplified product to givea detectable reading; if the threshold is not attained after 40 cycles, the mRNAis considered undetectable. Data are presented as relative expression of che-mokine mRNA in MBA-1 cells compared with the chemokine mRNA inRAW264.7 cells. Data are also presented as fold changes in the mRNA ofinfected samples compared with the mRNA of naive control samples.
Chemokine measurement
A sandwich ELISA construction kit to detect CXCL12 and CCl8 was usedaccording to the manufacturer’s protocols (Antigenix, Huntington Station,NY). CXCL12 was detected using 1 mg/ml capture Ab and 0.25 mg/mldetection Ab. CCL8 was detected using 0.5 mg/ml capture Ab and 0.25mg/ml detection Ab. Assays were developed by adding streptavidin-peroxidaseand 3,39,5,59-tetramethyl-benzidine as the substrate. Absorbance was read at450 nm using a Microplate Manager 6 reader (Bio-Rad, Hemel Hempstead,U.K.). rCXCL12 and rCCL8 were used as standards, and the concentration ofchemokines in the test samples was calculated using the linear part of a “four-parameter fit” standard curve run in parallel with the samples.
Statistical analysis
Statistical analyses to detect significant differences between groups wereperformed using the unpaired Student t test or the Mann–Whitney U test, asindicated in the figure legends. Statistical analyses were performed usingPrism v.5 software (GraphPad, San Diego, CA).
ResultsMBA-1 BM stromal cells support HSPC differentiation intoregulatory DCs
Freshly isolated stromal cells have the capacity to support HSPCdifferentiation into regulatoryDCs (23). In this study,we investigatedwhetherMBA-1 (34), a fibroblast-like BM stromal cell line shown to
have hematopoietic colony-stimulating activity and the ability toreduce rejection of a transplanted MHC-mismatched allograft (35),could also support HSPC differentiation into DCs with immuneregulatory properties. In cocultures of BMLin2CD117+ HSPCs(Supplemental Fig. 1) and MBA-1 cells in the absence of exogenousgrowth factors, cells with DC morphology that express CD11c andMHC class II (MHC-II) emerged at day 6 (Fig. 1A, 1B). Most ofthese DCs were weakly associated with the MBA-1 cells and couldbe harvested as a nonadherent population consisting of CD45+ cellsthat stained positive for MHC-II but had very low expression ofCD86 (Supplemental Fig. 2). Furthermore, DCs developing onMBA-1 cells expressed a relatively heterogeneous level of CD11cand somewhat higher levels of CD11b compared with BMLin2
CD117+ HSPCs that had been differentiated into DCs for the sameperiod of time using CSF-2 (Fig. 1C). The surface Ags F4/80 andGr-1 could not be detected on DCs developing in cocultures (Sup-plemental Fig. 2). To determine whether CD11c-purified DCs de-rived on MBA-1 cells (Fig. 1D) had regulatory capacity, we testedtheir capacity to inhibit an MLR stimulated by CSF-2–derived DCs.DCs developing on stromal cells could suppress theMLR induced byCSF-2–derived DCs by almost 90% when admixed at a 1:1 ratio(Fig. 1E). In addition, we found that the CD11c2 fraction alsosuppressed the MLR induced by CSF-2–derived DC by ∼70%when admixed at a 1:1 ratio (Fig. 1F ). This result indicates that
the CD11c2 hematopoietic population developing on stromal cellsalso encompasses some immune regulatory function (Fig. 1F).Nevertheless, these data indicate that MBA-1 stromal cells share,with splenic (22, 23) and liver stroma (24), the capacity to supportHSPC differentiation into regulatory DCs.
Stromal cell-derived chemokines regulate functionalhematopoietic progenitor differentiation into regulatory DCs
Stromal cell-derived chemokines direct and coordinate HSPC mi-gration toward their preferred niches. However, the role of chemo-kines in stromal cell-guideddifferentiation ofHSPCs into regulatoryDCs has not been reported. Therefore, we determined the impor-tance of chemokine-directed migration of HSPCs for their differen-tiation into regulatory DCs. For this purpose, we establisheda Transwell system in which MBA-1 cells were seeded in the lowerchamber, and BMLin2CD117+ HSPCs were seeded in the upperchamber, followed by coculture periods of up to 6 d. First, weexamined whether HSPCs migrated to the lower chamber usinga permeable membrane that allows cells to transmigrate. CD45+
H2Kb+ cells were detected in the lower chamber after 3 h (Fig. 2A)and 16 h (Fig. 2B) of coculture, indicating that HSPCs had migratedtoward the MBA-1 cells contained in the lower chamber. Next, weexamined whether migrated HSPCs differentiated into CD11c+
DCs within the Transwell system. As shown in Fig. 2C, CD11c+
FIGURE 1. MBA-1 BM stromal cells support HSPC differentiation into regulatory DCs. To establish whether the BM stromal cell line MBA-1 could
support differentiation of HSPCs into DCs with regulatory properties, HSPCs and MBA-1 cells were cocultured for 6 d in the absence of exogenous growth
factors. A, Cocultures of MBA-1 cells and BMLin2CD117+ HSPCs were examined on day 6, and cells with DC morphology were detected (arrowhead).
Original magnification 3800. B, Outgrowing cells were fixed, permeabilized, and examined for CD11c (red) and MHC-II (green) expression using
immunofluorescence confocal microscopy (original magnification 3800). C, Dot plots show CD11b and CD11c expression on DCs generated in MBA-1–
HSPC cocultures (left panel) or CSF-2–derived DCs (right panel). DCs generated in MBA-1–HSPC cocultures were also stained with isotype control Abs
(middle panel). D, Expression of CD11c on the positive (thin line) and negative (thick line) fraction following CD11c enrichment. E, CD4+ T cells from
BALB/cmicewere stimulated with 13 103 CSF-2–derived LPS-stimulatedDCs fromB6mice in the absence or presence of the indicated numbers of CD11c+
DCs that had developed onMBA-1 cells. F, CD4+ T cells fromBALB/c micewere stimulated with 13 103 CSF-2–derived LPS-stimulated DCs from B6mice
in the absence or presence of the indicated numbers of CD11c2 cells that had developed on MBA-1 cells. Thymidine incorporation was determined on day 4.
Value for level of suppression is shown as the percentage reduction in maximumT cell proliferation. All results are representative of at least three independent
experiments. Data in E and F are mean values of T cell proliferation in triplicate from one representative experiment (6 SD).
and CD11c2 cells expressing CD11b could be collected from thelower chamber of the Transwell system after 6 d of coculture, in-dicating that migrated HSPCs differentiated into CD11c+CD11b+
DCs, as well as CD11c2CD11b+ cells. In contrast, HSPCs remain-ing in the insert differentiated only into CD11c2CD11b+ cells.Together, this suggested that soluble mediators from MBA-1 cellsare sufficient to support differentiation of CD11c2CD11b+ cells,whereas migration of HSPCs, followed by contact with stromalcells, is required for differentiation into CD11c+CD11b+ DCs. Toconfirm that DCs derived from rapidly migrating HSPCs had reg-ulatory properties, we isolated CD11c+ cells from 6-d cocultures inwhich the inserts had been removed 3 h after seeding of the HSPCs.Such DCs also suppressed the MLR induced by CSF-2–derived
DCs (Fig. 2D). Thus, rapidly migrating HSPCs have the capacityto differentiate into regulatory DCs.Chemokines signal via receptors coupled to G proteins (36),
including the Gi/o subtypes (37). Therefore, we used pertussis toxin(PTX), an inhibitor of Gi/o proteins, to determine whether migra-tion of HSPCs was a chemokine-directed event. Pretreatment ofHSPCs with PTX reduced HSPC migration by .95% at 16 h ofcoculture (0.5 6 0.4% versus 11 6 1% of cells migrated in thepresence or absence of PTX, respectively), and it almost com-pletely abolished their differentiation into CD11c+ cells (Fig.2E). Conversely, HSPC differentiation into CD11c2CD11b+ cellswithin the upper chamber was unaffected when progenitors werepretreated with PTX (Fig. 2E). Thus, Gi protein signaling was
FIGURE 2. MBA-1 bone marrow stromal cells support the development of regulatory DCs by mechanisms involving progenitor migration and Gi protein
signaling. To determine the importance of chemokines in stromal cell-mediated development of regulatory DCs, BMLin2CD117+ HSPCs were subjected to
transmigration assays. A, B, Dot plots indicate the amount of BMLin2CD117+ HSPCs that migrated in Transwells in response to MBA-1 cells (104).
Migrated progenitors were stained with anti-H2Kb and CD45 and analyzed by flow-cytometry analysis. Cells collected from the lower chamber at 3 h (A) or
16 h (B) after seeding BMLin2CD117+ HSPCs in an insert (upper chamber) with a 3.0-mm membrane that allowed cells to transmigrate. Number indicates
the percentage of migrated cells at 3 and 16 h among total (5 3 104) BMLin2CD117+ HSPCs initially seeded in the upper chamber. C, Dot plots indicate
CD11b and CD11c expression on cells developing on MBA-1 cells (23 103) in the lower (left panel) or upper (right panel) chamber after 6 d of coculture.
D, CD4+ T cells from BALB/c mice were stimulated with 1 3 103 CSF-2 derived LPS-stimulated DCs from B6 mice alone or in the presence of 1 3 103
CD11c-enriched DCs developing on MBA-1 cells in the lower chamber of the Transwell cocultures in which the inserts were removed 3 h after seeding the
HSPCs in the insert. Thymidine incorporation was determined on day 4. E, Bar graphs indicate the numbers of CD11c+ DCs (filled bars) or CD11c2 cells
(open bars) developing in Transwell cocultures after BMLin2CD117+ HSPCs were treated with PTX. Cells developing on MBA-1 cells (104) in the lower
chamber (left panel) or in the upper chamber (right panel) were collected 6 d after seeding the HSPCs in the insert of the Transwell coculture. HSPCs were
treated with 1 mg/ml PTX for 30 min prior to adding them to the insert. F, Bar graphs indicate the numbers of CD11c+ DCs (filled bars) or CD11c2 cells
(open bars) developing in Transwell cocultures after replating migrated HSPCs on fresh stromal cells or in wells with MBA-1 cells and inserts with
a 0.4-mm membrane that allowed diffusion of soluble products but not cellular migration. G, Bar graphs indicate the numbers of CD11c+ DCs (filled bars)
or CD11c2 cells (open bars) developing in Transwell cocultures after replating migrated HSPCs in wells with varying numbers of MBA-1 cells and inserts
with a 0.4-mm membrane. All results are representative of two or three independent experiments. Data presented in D–G are mean values of T cell
proliferation in triplicates or numbers of cells collected per condition in triplicates from one representative experiment (6 SD).
essential for the migration required to differentiate HSPCs intoregulatory DCs. To further test whether chemokines were sufficientto support HSPC differentiation into regulatory DCs, we replatedHSPCs that had migrated directly onto fresh MBA-1 cells or intoinserts containing 0.4-mm membranes that allow diffusion ofsoluble mediators but not cellular transmigration. As shown inFig. 2F, migrated HSPCs recovered at 16 h and then replated onfresh MBA-1 cells differentiated into CD11c+CD11b+ DCs andCD11c2CD11b+ cells, whereas migrated HSPCs exposed only toMBA-1 cell-conditioned medium (i.e., in the 0.4-mm insert) did notdifferentiate into CD11c+CD11b+ DCs. In assays in which varyingnumbers of MBA-1 cells were used to condition the medium, mi-grated and replated (in the 0.4-mm insert) HSPCs still did notdifferentiate into CD11c+CD11b+ DCs, whereas the output num-ber of CD11c2CD11b+ cells increased with increasing numbersof MBA-1 cells. Together, these findings indicate that stromal cell-derived chemokines attract HSPCs and that sustained (.16 h) con-tact with stromal cells is required for HSPC differentiation intoregulatory DCs.
Stromal cell-guided HSPC differentiation into regulatory DCsis modulated in response to infection with L. donovani
In previous studies, it was demonstrated that the protozoan parasiteL. donovani could infect BM (29) and splenic stromal cells (23),inducing growth factors and a range of chemokines (29, 31, 38) and,that during L. donovani infection, splenic stromal cells acquired anenhanced capacity to support HSPC differentiation into regulatoryDCs (23). However, in the latter study, the relative importance ofdirect infection with L. donovani compared with the influence ofinflammatory cytokines was not ascertained. Therefore, to de-termine whether L. donovani directly affects stromal cell function,in the absence of other cellular sources of inflammatory cytokines,we infected MBA-1 cells (and a well-established macrophage cellline RAW264.7) with L. donovani amastigotes (Fig. 3A). Two hourspostinfection, intracellular parasites were readily detected inboth cell populations, as assessed by Giemsa staining (data notshown) or double staining with Leishmania- and LAMP-1–specific Abs (Fig. 3A). The ability to infect MBA-1 (44 6 5%)and RAW264.7 (36 6 8%) cells at comparable levels (by alteringthe multiplicity of infection [MOI]) allowed comparative analysisof the functional consequences of infection. We first determined
whether infection with L. donovani altered the capacity to supportHSPC differentiation. As shown in Fig. 3B, infection of theMBA-1 cell line promotes enhanced HSPC differentiation intoCD11c+ DCs. In direct contrast, HSPC differentiation was unde-tectable in cocultures with uninfected or L. donovani-infectedRAW264.7 cells (Fig. 3B). This finding was consistent with ourobservation that RAW264.7 cells also failed to attract HSPCs inthe Transwell system (data not shown). DCs derived on infectedMBA-1 cells also had regulatory capacity (Fig. 3C). Together, thesedata confirm that direct infection of stromal cells, but not macro-phages, modulates their ability to support HSPC differentiation intoregulatory DCs.
Stromal cells express a unique chemokine profile modulated byinfection with L. donovani
Next, we sought to identify chemokines expressed in MBA-1cells, but not in RAW264.7 cells, in the absence and presence ofL. donovani infection. We performed genome-wide mRNA expres-sion profiling of uninfected and infected MBA-1 and RAW264.7cells. This revealed eight chemokine genes (CCL7, CCL8, CCL27,CXCL1, CXCL3, CXCL5, CXCL12, and CXCL15) differentiallyexpressed inMBA-1 cells compared with RAW264.7 cells (Fig. 4A,Supplemental Fig. 3). Our microarray analyses also revealed thatCCL8 was reproducibly increased (2.3-fold increase; p, 0.005) inMBA-1 cells in response to L. donovani at 48 h postinfection.Despars et al. (39) also identified CCL8 gene expression bya splenic stromal cell line as potentially relevant to the regulationof DC development. Thus, on the basis of these observations andthe known role of CXCL12 in regulating HSPC homing and mobi-lization (4–8, 40), we focused further investigations on CXCL12and CCL8. First, we validated the microarray data by real-timeRT-PCR analysis. Consistent with the microarray analysis, real-time RT-PCR analysis revealed that CXCL12 (average CT value =16.46 0.3) and CCL8 (average CT value = 18.76 0.2) mRNAwasconstitutively expressed at much higher levels in MBA-1 comparedwith RAW264.7 cells (average CT values ranged from 39–40 forboth of the chemokines; Fig. 4B, 4C). That CXCL12 and CCL8were produced by MBA-1 cells but not by RAW 264.7 cells wasverified using ELISA (Fig. 4D, 4E). In addition, real-time RT-PCRanalysis of infected MBA-1 cells revealed that the expression ofCXCL12 (Fig. 4F) was downmodulated (p , 0.05), whereas
FIGURE 3. Infection of stromal cells with L. donovani promotes enhanced HSPC differentiation into regulatory DCs. A, MBA-1 cells and RAW264.7
macrophages were stained for L. donovani amastigotes and LAMP-1 using anti-L. donovani Ab (red) and LAMP-1 Ab (green) and analyzed by immunoflu-
orescence confocal microscopy. MBA-1 cells were infected with amastigotes at MOI of 10:1 and macrophages at MOI of 5:1 for 1 h. B, Numbers of CD11c+
DCs collected on day 6 from cocultures of HSPCs with uninfected or L. donovani-infected MBA-1 cells (open bars) or RAW.264.7 cells (filled bar). C, CD4+
T cells from BALB/c mice were stimulated with 1 3 103 CSF-2–derived LPS-stimulated DCs from B6 mice alone or in the presence of 1 3 103 CD11c+ DCs
developing on control MBA-1 cells (open bar) or 1 3 103 CD11c+ cells developing on infected MBA-1 cells (hatched bar). Thymidine incorporation was
determined on day 4. All results are representatives of two or three independent experiments. Data in B and C are mean values of T cell proliferation in
triplicates or numbers of cells collected per condition in triplicates from one representative experiment (6 SD). p , 0.05; ppp , 0.01.
the expression of CCL8 (Fig. 4G) was increased (p , 0.05), inresponse to L. donovani infection 48 h postinfection. Together, thesedata demonstrate that stromal cells have a unique chemokine
expression profile and that infection with L. donovani has thepotential to selectively modulate chemokine expression in stromalcells.
FIGURE 4. Chemokine expression by stromal cells is modulated in response to L. donovani infection. A, Heat map of chemokine genes differentially
expressed in MBA-1 cells compared with RAW264.7 cells at 12 and 48 h. Genome-wide mRNA expression in control cells or cells infected with
L. donovani was performed using Affymetrix microarrays. Total mRNA was extracted from control cells or cells infected at MOI of 10:1 (MBA-1) or
5:1 (RAW264.7) for 12 and 48 h. Red indicates high expression, and blue indicates low expression. Where multiple probes were present, an average of all
probes was calculated to be included in the heat map. Pooled data from three independent experiments are shown. The relative expression of CXCL12 (B)
and CCL8 (C) in MBA-1 and RAW264.7 cells was analyzed by real-time RT-PCR. Data represent normalized expression levels relative to the RAW264.7
cell sample. The concentration of CXCL12 (D) and CCL8 (E) in medium conditioned for 48 h with MBA-1 (104) or RAW264.7 (104) cells was determined
by ELISA. The relative expression of CXCL12 (F) and CCL8 (G) in control MBA-1 cells or MBA-1 cells infected with L. donovani was analyzed by real-
time RT-PCR at 12 and 48 h postinfection. In B–G, pooled data from at least three independent experiments are shown as the mean values of RNA
expression or protein concentration (6 SD). pp , 0.05; Mann–Whitney test. n.d., non detectable.
CCL8 expression is induced in splenic stromal cells duringL. donovani infection
During L. donovani infection, the expression of CXCL12 or CCL8and the potential cellular sources of these chemokines have not beenreported. First, we determined whether CXCL12 and CCL8 mRNAexpression could be detected in splenic tissue from naive mice usingreal time RT-PCR. In agreement with previous studies (41, 42),CXCL12 mRNA was abundantly expressed (average CT value =25.4 6 0.8), whereas CCL8 mRNA was expressed at low levels(average CT value = 34.9 6 0.49), in naive spleen. Next, we ana-lyzed the accumulation of CXCL12 and CCL8 mRNA in splenictissue from L. donovani-infected mice at day 21 postinfection. Al-though CXCL12 expression was unaltered or minimally reduced inresponse to infection (Fig. 5A), CCL8 mRNA accumulation in-creased by 100–1000 times (p , 0.05; Fig. 5A). To confirm thatsplenic stromal cells increase their expression of CCL8 duringL. donovani infection, we performed real time RT-PCR analysis ofex vivo enriched splenic stromal cells. In mice infected withL. donovani, CCL8 mRNA accumulation increased by∼10,000 fold(p , 0.01), reflecting a .10-fold enrichment of signal by stromal-cell purification compared with that seen in whole spleen (Fig. 5A).Conversely, CXCL12 mRNA expression was moderately decreased(p, 0.01; Fig. 5A). Consistent with the real-time RT-PCR analysis,immunohistology analysis revealed that CCL8 was abundantly pro-duced in splenic tissue from L. donovani-infected mice, whereas noCCL8protein could bedetected in naive spleen (Fig. 5B). ThatCCL8was abundantly produced by splenic stromal cells in infected micewas confirmed by the detection of CCL8 protein in medium con-ditionedwith freshly isolated splenic stromal cells fromL. donovani-infectedmice (Fig. 5C). Furthermore, the immunohistology analysisrevealed that production of CCL8 in response to infection (Fig. 5B)was predominantly associated with areas of tissue that stained pos-itive for the fibroblast-specific marker ER-TR7 (Fig. 5D). In addi-tion, staining for CCL8 and parasites in combination indicated thatsome CCL8+ cells also seemed to be infected with L. donovaniamastigotes (Fig. 5E). CXCL12 was also still evident in enrichedsplenic stromal cells from naive and L. donovani-infectedmice (Fig.5F). These data demonstrate that during visceral leishmaniasis,stromal cells in the spleen regulate their expression of chemokines,most significantly that of CCL8.
L. donovani-induced CCL8 is associated with increasedstromal cell-guided recruitment of HSPCs
Because MBA-1 cells constitutively accumulate relatively high levelsof CXCL12 and CCL8 mRNA, and infection with L. donovani in-creased CCL8 production in splenic stromal cells, we first tested thecontribution that these two chemokines make to the chemotaxis ofHSPCs induced byMBA-1 cells, by performingmigration assayswithneutralizing Abs. As shown in Fig. 6, neutralizing Ab to CXCL12(Fig. 6A) was as efficient as PTX (Fig. 6B) in blocking the migrationof HSPCs induced by MBA-1 cells. Thus, CXCL12 production isnecessary for the induced migration of HSPCs, confirming previousstudies in other systems (4, 40). Interestingly, neutralizing Ab toCCL8 also blocked the migration of HSPCs induced byMBA-1 cells, although only partially (Fig. 6C). To further investigatethe role of CXCL12 and CCL8 in the migration of HSPCs, wesubstituted MBA-1 cells for MBA-1 cell-conditioned medium inthe Transwell assay. By using conditioned medium, rather thanMBA-1 cells, we sought to avoid involving the contribution of chemo-kines that may be induced following contact between HSPCs andMBA-1 cells. As shown in Fig. 6D, this conditioned medium inducedgreater migration ofHSPCs thanwas achievedwith a concentration ofrCXCL12 similar to that detected in MBA-1–conditioned medium,thus supporting the role for other chemokines in augmenting
CXCL12-dependent migration induced by MBA-1 cells. As alsoshown in Fig. 6D, the addition of neutralizing Ab to CCL8 reducedthe recruitment of HSPCs induced by MBA-1 cell-conditioned me-dium to the level seen with rCXCL12. Furthermore, as shown in Fig.6E, although CXCL12 alone was able to induce migration of HSPCs,CCL8 failed to do so; however, in combination, CXCL12 and CCL8induced migration to levels equivalent to those seen with MBA-1–conditioned medium.Having established a role for stromal dell-derived CCL8 in aug-
menting CXCL12-dependent recruitment of HSPCs (Fig. 6E), trans-migration assays with splenic stromal cells from uninfected andL. donovani-infected mice were used to determine whether the in-duction of CCL8 in experimental visceral leishmaniasis (EVL) alsoaffects HSPC recruitment. We first determined whether infectionwith L. donovani altered the capacity to support HSPC recruitment.As shown in Fig. 6F, splenic stromal cells from infected mice in-duced greater migration of HSPCs than was achieved with splenicstromal cells from naive mice. To determine the contribution thatincreased production of CCL8makes to the enhanced chemotaxis ofHSPCs induced by stromal cells from infected mice, we substitutedsplenic stromal cells for splenic stromal cell-conditioned medium inthe Transwell assay. As shown in Fig. 6G, neutralizing Ab to CCL8reduced the recruitment of HSPCs induced by medium conditionedwith stromal cells from infected mice to the level seen with mediumconditionedwith stromal cells from naivemice. Furthermore, as alsoshown in Fig. 6G, neutralizingAb toCXCL12 blocked themigrationof HSPCs induced by medium conditioned with stromal cells frominfected mice to the level seen with medium only. Thus, CXCL12production is also necessary for splenic stromal cell-induced migra-tion of HSPCs, and CCL8 cooperates with CXCL12 to maximizeHSPC migration toward stromal cells from infected mice. Thus, ourdata demonstrate that stromal cell-controlled differentiation ofHSPCs involves both of these chemokines and that infection withL. donovani modulates the stromal cell niche in ways that lead tomore efficient recruitment ofHSPCswith the potential of developinginto regulatory DCs.
DiscussionThe stromal cell niche is increasingly recognized as influencinga broad range of events involved in inflammation and the hostresponse to infection. In this study, we assessed the requirementsfor stromal cell-derived chemokines in the development of regula-tory DCs from HSPCs. We first showed that a BM-derived stromal-cell line, MBA-1, induced chemotaxis in a population of HSPCs,with the potential to differentiate into regulatory DCs. The recruit-ment of HSPCs was mediated by stromal cell-derived CXCL12 andCCL8 that cooperate to induce maximal chemotaxis. We thenshowed that infection of MBA-1 cells with the intracellular proto-zoan parasite L. donovani modulates the expression of CXCL12and CCL8 and, that during EVL, splenic stromal cells selectively in-creased their capacity to produce CCL8 compared with CXCL12.Thus, regulation of CCL8 may represent a key checkpoint respon-sible for the enhanced recruitment of HSPCs to the spleen (31) andthe augmented output of regulatory DCs associated with L. dono-vani infection (23). During infection, a defining characteristic of theimmune system is the dramatic changes in trafficking, activation,expansion, and contraction of many of its constituent cells. Suchimmune regulation requires highly orchestrated interactions be-tween diverse cell populations with specialized functions. Whathas only recently been recognized is the role of stromal cells inmany aspects of immune regulation, including cellular traffickingorchestrated by the coordinated expression of chemokines. How-ever, few studies have examined how stromal cell-derived chemo-kines are influenced in the context of chronic infections and whether
targeting of stromal cells by pathogens can influence their chemo-kine production.We and other investigators have demonstrated that splenic stroma
can support, in the absence of exogenous growth factors, the dif-ferentiation of regulatory DCs from HSPCs, as well as by inducingfurther differentiation of mature DCs (22, 23, 26). This function is
not restricted to splenic stromal cells; it has also been observed withliver and pulmonary stromal cells (24, 28), the BM-derived fibro-blast line MBA-1, and a stromal macrophage cell line (14M1.4;M. Svensson and P. M. Kaye, unpublished observations), althoughnot with long-term lymph node-derived stromal cells (39) or withconventional macrophage cells lines, such as RAW264.7. Thus,
FIGURE 5. EVL is associated with an induction of CCL8 in splenic tissue stromal cells. To determine whether CXCL12 and CCL8 expression is modulated
in splenic tissue during L. donovani infection, spleens were collected from naive C57BL/6 mice and mice infected for 21 d with L. donovani. A, CXCL12 and
CCL8 mRNA accumulation was determined in total splenocytes (left panel) and ex vivo-enriched stromal cells (right panel) from naive and infected mice.
CXCL12 andCCL8mRNAexpression in infected samples was comparedwith noninfected samples. The horizontal line represents a value of 1; a fold change. 1
indicates increased expression, whereas a fold change , 1 indicates decreased expression. Data are derived from two experiments, with mRNA obtained from
two to four mice per experiment. B, The presence of CCL8 (brown) in the spleens of infected mice was visualized by immunohistochemistry (original
magnification3200). Splenic sections from naive and infected mice were stained with an anti-CCL8 Ab or control Ab. C, CCL8 content in medium conditioned
for 48 hwith freshly isolated splenic stromal cells fromnaive or L. donovani-infectedmice. CCL8 content was determined by ELISA.D, The association of CCL8
with areas rich in stromal cells was verified in a parallel section to that used in B, by staining for ER-TR7 (original magnification3200). E, The distribution of
CCL8 (green) and L. donovani amastigotes (red, arrowheads) in spleens of infected mice was visualized by immunofluorescence (original magnification3800).
Splenic sections were stained with an anti-CCL8 in combination with serum from Leishmania-infected hamsters. F, The association of CXCL12 with stromal
cells enriched from naive and infectedmicewas verified by immunofluorescence (original magnification3800). Stromal cells were stained with an anti-CXCL12
Ab (green) in combination with an anti ER-TR7 Ab (red), and nuclei (including parasite nuclei) were counterstained with DAPI (blue). All results are
representative of two or three independent experiments. Data in C are mean values of protein concentration in triplicates from one representative experiment
a broad, but tissue-specific, range of stromal cells is able to indi-rectly exert immune regulatory function via their influence on localHSPC differentiation.Coordinated expression of chemokines by stromal cells is essen-
tial in controlling the migration, homing, and differentiation ofHSPCs into functionally diverse populations of immune cells. Ourfinding that the stromal cell-guided HSPC differentiation into regu-latory DCs was abolished by PTX blockade of G protein signalingsupported the hypothesis that a G protein-coupled pathway, suchas chemokine receptor signaling, was involved in the developmentof regulatory DCs. CXCL12 is a well-defined chemoattractant forHSPCs (4) and the involvement of CXCL12 in orchestrating themigration of HSPCs that is required for regulatory DC development
was confirmed by expression studies in MBA-1 and splenicstromal cells and the use of rCXCL12 and neutralizing mAbs inin vitro migration assays. However, CXCL12 was less potent thanMBA-1–conditioned medium in promoting HSPC migration,suggesting other chemokines might contribute to this process. In astudy by O’Neil and colleagues (39), gene-expression profiling ona stromal cell line (STX3) that supported DC development and onethat did not (2RL22) identified a number of chemokine genes poten-tially involved in stromal cell-supported DC development. By com-parison with chemokines expressed in MBA-1 cells, we identifiedCCL5, CCL8, and CXCL10 as being of further interest. However,CCL5 andCXCL10were also expressed inRAW264.7macrophagesthat do not supportmigration and differentiation ofHSPCs. Thus, we
FIGURE 6. Stromal cell-guided recruitment of HSPCs involves CXCL12 and CCL8. To determine the contribution that CXCL12 and CCL8 make to the
chemotaxis of HSPCs induced by MBA-1 cells, migration assays with neutralizing Abs were performed. Migrated progenitors were enumerated, stained
with anti-H2Kb and CD45, and analyzed by flow cytometry. A, Dot plots indicate the number of HSPCs that migrated in Transwells in response to MBA-1
cells (104) in the presence of control Abs (left panel) or 1 mg/ml anti-CXCL12 Abs (right panel). Cells collected from the lower chambers 3 h after seeding
HSPCs in an upper chamber with a 5.0-mm membrane that allowed cells to transmigrate are shown. B, Dot plots indicate the number of HSPCs that
migrated over 3 h in response to MBA-1 cells (104). HSPCs were not treated (left panel) or pretreated (right panel) with PTX (1 mg/ml). C, Dot plots
indicate the number of HSPCs that migrated in Transwells in response to MBA-1 cells (104) in the presence of control Abs (left panel) or 1 mg/ml anti-
CCL8 Abs (right panel). Cells collected from the lower chambers 3 h after seeding HSPCs in an upper chamber with a 5.0-mm membrane that allowed cells
to transmigrate are shown. D, Chemotaxis of HSPCs in response to stromal cell supernatant in the absence or presence of anti-CXCL12 Ab (1 mg/ml) or
CCL8 Ab (1 mg/ml). Chemotaxis induced by supernatant in the presence of control IgG Abs was 40.7 6 3.4%, which was not significantly different from
that induced by supernatant alone. Chemotaxis in response to rCXCL12 (2 ng/ml) is also shown. E, Three-hour chemotactic response of HSPCs exposed to
stromal cell supernatant or CXCL12 (100 ng/ml) and/or CCL8 (100 ng/ml). Medium was conditioned for 48 h prior to protein quantification using ELISA.
F, Three-hour chemotactic response of HSPCs induced by splenic stromal cells from naive or L. donovani-infected mice. G, Three-hour chemotactic
response of HSPCs induced by medium conditioned with splenic stromal cells from naive mice, as well as medium conditioned with splenic stromal cells
from infected mice in the presence of control IgG, anti-CCL8 Ab (1 mg/ml), or 1 mg/ml anti-CXCL12 Abs. Migrated cells were quantified by flow
cytometry as described in Materials and Methods. Numbers in A–C indicate the percentage of migrated cells among total (5 3 104) BMLin2CD117+
HSPCs initially seeded in the upper chamber. All results are representative of two or three independent experiments. Data in D–G are mean values of
chemotaxis per condition in triplicates from one representative experiment (6 SD). pp , 0.05; ppp , 0.01; Student t test.
focused our subsequent investigations on the role of CXCL12 andCCL8. Our experiments demonstrated that CCL8, in cooperationwith CXCL12, enhanced the number of HSPCs that migrate underthe guidance of MBA-1 cells, highlighting for the first time cooper-ation between these two chemokines to drive maximal HSPC mig-ration. Further studies will be required to determine the exactmechanisms bywhich these two chemokines induce receptor signal-ing that may cooperate in regulating enhanced cell migration. Nev-ertheless, it is reasonable to propose that the regulated expression ofCCL8 may provide a checkpoint to control enhanced HSPC recruit-ment and differentiation during this, and perhaps other, situationsinvolving chronic inflammation and extramedullary hematopoiesis.In leishmaniasis, chemokine production represents one of the
earliest detected responses to infection in vitro and invivo (43). Thisincludes production of CCL2, CCL3, and CXCL10 among others.Although Leishmania infection was shown to modulate a number ofchemokines in diverse cells types, the accumulation of CCL8mRNA in response to infection has not previously been noted. ThatCCL8 regulation in response to L. donovani infection has pre-viously gone unnoticed may reflect that CCL8 production is re-stricted to stromal cells rather than the macrophage and otherleukocyte populations that are most commonly studied duringLeishmania infection. Indeed, our data underscore the significanceof studying tissue-specific nonhematopoietic cells when examiningmechanisms of pathogenesis. Importantly, our data do not argueagainst a role for CXCL12 in the regulation of hematopoietic dif-ferentiation of HSPCs. Rather, our finding that CXCL12 and CCL8cooperate in regulating hematopoiesis provides evidence for addi-tional pathways regulating hematopoietic activity that can be tar-geted by intracellular pathogens. However, a causal link betweenCCL8 expression and the development and/or maintenance ofchronic infection will be dependent upon the future developmentof tools for the specific in vivo blockade of CCL8 and/or the gen-eration of conditional CCL8 knockout mice.Although different DC subpopulations arise mainly from variably
committed DC precursors that are selectively recruited from BM tothe peripheral tissue (15–18), tissue-specific differentiation of DCsprovides complementary routes for establishing DC heterogeneityin diverse tissues (9, 44, 45). In the steady state, this model of DCdifferentiation would involve tissue-resident HSPCs and/or theirdescendents; during inflammation, this might be supplemented bythe recruitment of additional HSPCs. In addition, HSPC differentia-tion can be amplified upon exposure to TLR agonists (9). Thus, thelocal microenvironment has multiple opportunities for shaping theDC repertoire. The advantage of stromal cell plasticity to the organ-ism is that it provides a mechanism able to meet the changing re-quirements for hematopoietic cells of a given lineage. In the contextof infections, such as leishmaniasis (23, 29, 31), tuberculosis (46),and malaria (32, 47), alterations in local stromal cells, as well as theenhanced capacity to support HSPC renewal and recruitment thatpromotes the differentiation of DCs and T cells with regulatory func-tion,might be commonhost responses to several pathogens to preventimmune-mediated pathology, although also promoting pathogen per-sistence. Similarly, an accumulation of DCs and other myeloid cellswith immune-suppressive functions has been noted in cancers asso-ciated with altered stromal cell function (48). By further understand-ing themechanisms bywhich stromal cells control the differentiationand function of regulatory DCs, we might uncover potential targetsfor manipulating the DC repertoire in a therapeutic setting.
AcknowledgmentsWe thank Dr. D. Zipori for providing the MBA-1 cell line and the staff of
the Biological Services Facilities at York and at the Karolinska Institut for
animal husbandry.
DisclosuresThe authors have no financial conflicts of interest.
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