Phenotypic, Morphological, and Functional Heterogeneity of SplenicImmature Myeloid Cells in the Host Response to Tularemia

John W. Rasmussen,a* Jason W. Tam,a,c Nihal A. Okan,a* Patricio Mena,a Martha B. Furie,a,b,c David G. Thanassi,a,c Jorge L. Benach,a,b,c

and Adrianus W. M. van der Veldena,b,c

Center for Infectious Diseases,a Department of Pathology,b and Department of Molecular Genetics and Microbiology,c Stony Brook University, Stony Brook, New York, USA

Recent studies have linked accumulation of the Gr-1� CD11b� cell phenotype with functional immunosuppression in diversepathological conditions, including bacterial and parasitic infections and cancer. Gr-1� CD11b� cells were the largest populationof cells present in the spleens of mice infected with sublethal doses of the Francisella tularensis live vaccine strain (LVS). In con-trast, the number of T cells present in the spleens of these mice did not increase during early infection. There was a significantdelay in the kinetics of accumulation of Gr-1� CD11b� cells in the spleens of B-cell-deficient mice, indicating that B cells play arole in recruitment and maintenance of this population in the spleens of mice infected with F. tularensis. The splenic Gr-1�

CD11b� cells in tularemia were a heterogeneous population that could be further subdivided into monocytic (mononuclear) andgranulocytic (polymorphonuclear) cells using the Ly6C and Ly6G markers and differentiated into antigen-presenting cells fol-lowing ex vivo culture. Monocytic, CD11b� Ly6Chi Ly6G� cells but not granulocytic, CD11b� Ly6Cint Ly6G� cells purified fromthe spleens of mice infected with F. tularensis suppressed polyclonal T-cell proliferation via a nitric oxide-dependent pathway.Although the monocytic, CD11b� Ly6Chi Ly6G� cells were able to suppress the proliferation of T cells, the large presence ofGr-1� CD11b� cells in mice that survived F. tularensis infection also suggests a potential role for these cells in the protectivehost response to tularemia.

Francisella tularensis is a small, aerobic, nonmotile, Gram-neg-ative, pleomorphic coccobacillus. It is a facultative intracellu-

lar organism that replicates in macrophages and hepatocytes (4, 5,7, 14, 16, 27–29, 46, 47). Four subspecies have been identified. Themost virulent subspecies in humans is F. tularensis subsp. tularen-sis (also known as type A), and it is the predominant cause oftularemia in North America. F. tularensis subsp. holarctica (typeB) predominates in Eurasia and causes less severe human diseasethan does type A. F. tularensis subsp. novicida and F. tularensissubsp. mediasiatica are not important pathogens for humans. TheF. tularensis live vaccine strain (LVS) is an attenuated type B strainand is infectious and virulent in mice but not in humans. Thismurine infection model has served as a very useful surrogate forthe human illness (27). The clinical severity of tularemia, its pro-tean manifestations, and its lethality, particularly in type A infec-tions, are the main reasons for the inclusion of F. tularensis in thecategory A group of agents of bioterrorism (http://www.bt.cdc.gov/agent/agentlist.asp).

The basis for the virulence and clinical severity of infectionwith F. tularensis is not completely understood. The bacteremiaand hepatitis of tularemia are undoubtedly contributors to theclinical severity, but there is also evidence suggesting that earlydysfunction of the immune system could play a role. The immuneresponse to this bacterium is being scrutinized closely, but gapsremain in understanding the mechanisms that depress the adap-tive response (23). Immune suppression during infection with F.tularensis could delay the development of adaptive immunity andcontribute to high morbidity and mortality. The composition ofthe cellular immune response in the livers of infected mice hasprovided a potential clue to immune suppression. The histopa-thology of hepatic tularemia is characterized by the formation ofgranuloma-like lesions (13–15), and the role of gamma interferon(IFN-�) in their development has been demonstrated (6, 36, 71).We previously characterized the cellular composition of infected

livers using specific cell surface markers showing several types ofcells that express the myeloid cell marker CD11b (also known asMac-1) (55). The largest subpopulation of cells infiltrating theinfected livers expressed both Gr-1 and CD11b.

Recent studies have linked the accumulation of cells with theGr-1� CD11b� phenotype to functional immunosuppression inbacterial and parasitic infections, acute and chronic inflamma-tion, and cancer. Most attention has been focused on the role ofGr-1� CD11b� myeloid cells in cancer since they accumulate inlarge numbers in tumors in practically all tested experimentalmodels, as well as in patients with different types of cancer, andcause a global and profound immune suppression (2, 8–11, 42–45,57, 64). Gr-1� CD11b� cells are a heterogeneous population thathave been referred to as myeloid-derived suppressor cells (31). Werefer to this cell phenotype here as immature myeloid cells (IMC)to avoid a functional connotation.

Although there are some differences among the results and theexperiments that have been done in the context of IMC and infec-tion, the data are similar in their demonstration of immunosup-

Received 9 April 2012 Accepted 12 April 2012

Published ahead of print 23 April 2012

Editor: A. J. Bäumler

Address correspondence to Adrianus W. M. van der Velden, vandervelden@notes.cc.sunysb.edu.

* Present address: John W. Rasmussen, Department of Biological Sciences, BoiseState University, Boise, Idaho, USA, and Nihal A. Okan, Department ofMicrobiology and Immunobiology, Harvard Medical School, Boston,Massachusetts, USA.

J.W.R. and J.W.T. contributed equally to this article.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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pression associated with the Gr-1� CD11b� phenotype. Earlyobservations of precursor myeloid cells being involved in immu-nosuppression were made in a Salmonella infection model in1991. In this study, the appearance of macrophage precursors wasshown to play an important regulatory role in the immune re-sponse to these bacteria (3). Burns infected with Pseudomonasaeruginosa have large populations of Gr-1� CD11b� cells thatinhibit the production of antimicrobial peptides by keratinocytes(40). A recent study showed that Gr-1� CD11b� cells cause im-munosuppression in experimental sepsis after passive transfer(19). Gr-1� CD11b� cells have also been implicated in immunesuppression in protozoal (34) and fungal (50) infections.

The heterogeneous Gr-1� CD11b� cells can be subdividedinto functionally and morphologically distinct subpopulations.Murine monocytes are composed of two distinct subpopulationscharacterized by expression or absence of Gr-1 (32, 33). Gr-1 isexpressed on neutrophils, inflammatory monocytes, and somepopulations of dendritic cells (69), and monoclonal antibodies toGr-1 recognize both Ly6C and Ly6G isoforms (26). More recently,monoclonal antibodies have been used to separate Gr-1� sub-populations into neutrophil-like (CD11b� Ly6Cint Ly6G�) andinflammatory monocytic (CD11b� Ly6Chi Ly6G�) cells (18, 74).In fact, Gr-1� CD11b� cells form two separate myeloid lineageswith apparently opposing functions that develop as a result ofinfection with mycobacteria (20). In infections with Trypanosomagondii, Listeria monocytogenes, or Aspergillus fumigatus, the mono-cytic component is required for an effective host response (17, 21,35, 37, 54, 58, 59, 61, 62). Thus, the heterogeneous IMC play acomplex role in host-pathogen interactions.

Both monocytes/macrophages and neutrophils are importantin innate immunity to F. tularensis. F. tularensis replicates withinmacrophages and are thus shielded from the humoral immuneresponse (27). Mice that are depleted of neutrophils with antiserato Gr-1 succumb to sublethal doses of F. tularensis LVS (41, 67).However, these results are difficult to interpret as multiple celltypes express the Gr-1 surface marker.

Due to their potentially immunosuppressive nature and theiraccumulation in large numbers in the livers of mice infected withF. tularensis, we considered that Gr-1� CD11b� cells could play animportant role in the host response. In the present study, we dem-onstrate that Gr-1� CD11b� cells can be elicited in the spleens ofmice infected with sublethal doses of F. tularensis, purified by cellsorting, differentiated into antigen-presenting cells, and subdi-vided into granulocytic (CD11b� Ly6Cint Ly6C�) and monocytic(CD11b� Ly6Chi Ly6C�) subpopulations. The monocytic sub-population inhibited T-cell proliferation via a nitric oxide-depen-dent mechanism, suggesting that elicitation of these cells by F.tularensis may contribute to disease progression by suppressinghost immune function. However, the large presence of Gr-1�

CD11b� cells in surviving mice also suggests that these cells maycontribute to host protection. Thus, the Gr-1� CD11b� cellscould in fact have dual roles in infection, providing a balance ofimmunosuppressive and protective functions where the tipping ofthis balance may be an important factor influencing the outcomeof infection.

MATERIALS AND METHODSBacteria. F. tularensis LVS (29684; American Type Culture CollectionManassas, VA) was cultured in Mueller-Hinton (MH) broth (BD Biosci-ences, Sparks, MD) supplemented with 2% IsoVitaleX Enrichment (BD

Biosciences), 5.6 mM D-glucose, 625 �M CaCl2, 530 �M MgCl2, and 335�M ferric pyrophosphate and then incubated at 37°C and 5% CO2. Bac-teria were cultured as previously described (55). To enumerate the bacte-ria, organs from mice were homogenized in sterile Whirl-Paks (Nasco, Ft.Atkinson, WI). Whole blood was used for the detection of bacteremia.Serial dilutions of emulsified organs and blood were made in sterile phos-phate-buffered saline (PBS), plated on Chocolate II agar (BD Biosci-ences), and incubated at 37°C and 5% CO2 for 48 h before the CFU werecounted.

Mice. Female C3H/HeN, C57BL/6 and BALB/c mice were purchasedfrom Charles River Laboratories (Wilmington, MA) and used at 6 to 10weeks of age. B-cell-deficient mice in a C57BL/6 strain background(B6.129S2-Igh-6tmlCgn/J) were purchased from The Jackson Laboratory(Bar Harbor, ME). B6.129S2-Igh-6tmlCgn/J mice are deficient in mature Bcells because they lack the expression of membrane-bound IgM (39). Allmice were housed in microisolator cages with free access to food andwater. Mice received intradermal injections of 105 to 106 CFU of F. tula-rensis LVS. At various times postinoculation, mice were euthanized, andtheir blood and organs were used for determination of bacterial burdens.Mice were weighed immediately after euthanization to calculate a ratio ofspleen to body weight. The Institutional Animal Care and Use Committeeat Stony Brook University approved all animal procedures.

Flow cytometry. Excised spleens were teased apart to a single cellsuspension and collected in Dulbecco modified Eagle medium (DMEM;Invitrogen, Carlsbad, CA). Bone marrow cells were collected from mice byremoving the femur and tibiae of both legs and cutting the epiphysis toexpose the marrow. The cavities of the bones were flushed with DMEM tocollect the bone marrow cells. Both spleen and bone marrow cells weretreated with NH4Cl buffer (144 mM NH4Cl and 17 mM Tris [pH 7.4] inwater) to lyse red blood cells before resuspension in fluorescence-acti-vated cell sorter (FACS) buffer (0.2% bovine serum albumin [Sigma] and0.09% NaN3 [Sigma] in PBS). Cells (106) were then incubated with anti-Fc�R antibody (clone 2.4G2; BD Pharmingen, San Diego, CA) beforeappropriate amounts of conjugated antibodies or isotype-matched con-trol antibodies were added, followed by incubation in the dark for 30 minat 4°C. Stained cells were washed twice with FACS buffer and fixed in 1%formalin. At least 10,000 viable cells were acquired and analyzed using aFACSCalibur flow cytometer with CellQuest Pro Software (BD Biosci-ences, San Jose, CA). Additional analysis was performed using WinListsoftware (Verity Software House, Topsham, ME) and FlowJo software(Tree Star, Inc., Ashland, OR).

Microscopy. Spleens were fixed in 10% neutral buffered formalin,dehydrated in ethanol, embedded in Blue Ribbon paraffin (Surgipath,Richmond, IL), sectioned at 5 �m, stained with hematoxylin and eosin,and mounted with Acrymount (Statlab Medical Products, Lewisville, TX).For the detection of bacteria and cellular markers in the spleen, paraffin sec-tions and frozen tissue sections were stained with the antibodies listed below,as previously described (55). F. tularensis in paraffin sections was detectedwith polyclonal rabbit antisera, followed by alkaline phosphatase-conju-gated anti-rabbit IgG and Vulcan Fast Red chromogen (Biocarta, SanDiego, CA). For immunofluorescence assays, secondary fluorescein iso-thiocyanate (FITC) anti-rabbit IgG (Chemicon International, Temecula,CA) or Alexa Fluor 488 anti-rabbit IgG (Invitrogen) was used to detect F.tularensis. For frozen specimens, organs were embedded in Neg-50 freez-ing compound (Richard-Allan Scientific, Kalamazoo, MI), frozen in iso-pentane that had been cooled with liquid nitrogen, cut at 5 �m in thecryostat at �25°C, air dried, and fixed in acetone for 30 s. After applicationof the appropriate primary and secondary antibodies, slides were washedand mounted in Opti-Mount (Richard-Allan Scientific). Slides were ex-amined by phase-contrast and epifluorescence microscopy using a NikonEclipse E600 microscope, and images were captured using a Spot camera(Diagnostic Instruments, Inc., Sterling Heights, MI). Slides for confocalmicroscopy were analyzed using a Leica DM IRE2 confocal microscope.Images of the red, green, and blue emission signals were captured sepa-

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rately with the Leica LCS software package. Images were then processedwith Adobe Photoshop software.

Antibodies for flow cytometry and confocal microscopy. The follow-ing antibodies were used for flow cytometry and confocal microscopy:fluorescein isothiocyanate (FITC) anti-mouse CD11c (clone HL3), FITCanti-mouse CD49b/Pan NK cells (clone DX5), FITC anti-mouse CD21(clone 7G6), R-phycoerythrin (PE) anti-mouse CD3 (clone 17A2), PEanti-mouse I-A/I-E (major histocompatibility complex class II [MHC-II];clone M5/114.15.2), PE anti-mouse Ly-6G and Ly-6C (Gr-1; clone RB6-8C5), PE anti-mouse CD138 (Syndecan-1; clone 281-1), peridinin chlo-rophyll-a protein (PerCP) anti-mouse CD4 (clone RM4-5), PerCP-Cy5.5anti-mouse CD11b (Mac-1; clone M1/70), PerCP-Cy5.5 anti-mouse IgM(clone R6-60.2), allophycocyanin (APC) anti-mouse Ly-6G and Ly-6C(Gr-1; clone RB6-8C5), APC anti-mouse NK1.1 (clone PK136), and APCanti-mouse CD8 (clone 53-6.7) from BD Pharmingen; APC anti-mouseCD23 (clone 2G8) from Southern Biotech (Birmingham, AL); Alexa Fluor647 anti-mouse CD11b (Mac-1; clone M1/70) from Biolegend (San Di-ego, CA); and Alexa Fluor 488 anti-mouse F4/80 (clone CI:A3-1) fromSerotec (Raleigh, NC). Isotype-matched antibodies, as well as secondaryantibodies (all from BD Pharmingen), were used as a control for nonspe-cific binding in all experiments. The following antibodies were used forexperiments with T cells: PerCP anti-mouse CD11b (clone M1/70), PEanti-mouse Ly6G (clone 1A8), APC anti-mouse Ly6C (clone HK1.4),APC anti-mouse CD90.2 (clone 30-H12), anti-CD3 (clone 145-2C11),and anti-mouse CD28 (clone E18) from Biolegend.

Purification of splenocytes. Live cell sorting was performed fromboth uninfected and infected spleens to purify cells expressing Gr-1� andCD11b�. First, CD11b� cells were collected in magnetic cell sorting buf-fer (2 mM EDTA and 0.5% bovine serum albumin in PBS at pH 7.2) froma suspension of spleen cells by using magnetic beads specific for CD11baccording to the protocol of the manufacturer (Miltenyi Biotec, Auburn,CA). Magnetically purified CD11b� cells were stained with APC anti-mouse Ly-6G and Ly-6C (anti-Gr-1; clone RB6-8C5) and sorted by theFACSAria cell sorting system (BD Biosciences). Purity of sorted cell pop-ulations was verified by flow cytometry and was consistently �98%.Staining of cells with an annexin V and propidium iodide apoptosis de-tection kit (BD Pharmingen) was used to evaluate cell viability. Single cellsuspensions of sorted cell populations were either fixed in methanol forGiemsa stain or stained with DAPI (4=,6=-diamidino-2-phenylindole; In-vitrogen) and mounted with Vectashield (Vector Laboratories, Burlin-game, CA) for microscopy.

Ex vivo culture of Gr-1� CD11b� cells. Purified Gr1� CD11b� cellswere cultured ex vivo in RPMI 1640 medium (Invitrogen) supplementedwith 10% fetal bovine serum, 2 mM L-glutamine, 200 U of penicillin/ml,and 50 �g of streptomycin/ml at 37°C and 5% CO2 on 24-well cell cultureplates (BD Biosciences) at a density of 3 � 105 cells/well. For all ex vivoexperiments, aliquots of the sorted cells, as well as medium, were platedand cultured on Chocolate II agar for the detection of contaminatingresidual bacteria. Cells were stimulated with 10 ng of recombinant mousegranulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Sys-tems, Minneapolis, MN)/ml for 7 days after sorting, detached with por-cine pancreatic trypsin 1-300 (MP Biomedicals, Solon, OH) and EDTA,and analyzed for cell differentiation by flow cytometry.

Measurement of T-cell proliferation. C3H/HeN mice were infectedwith 105 CFU of F. tularensis LVS. After 10 days, the splenocytes wereharvested and stained with conjugated monoclonal antibodies againstCD11b, Ly6G, and Ly6C (Biolegend). CD11b� Ly6Cint Ly6G� granulo-cytic IMC and CD11b� Ly6Chi Ly6G� monocytic IMC were identified byflow cytometry. For experiments on T-cell proliferation, the splenocyteswere enriched for CD11b� cells using magnetic microbeads and separa-tion columns (Miltenyi Biotec). The resulting populations enriched forCD11b� cells were stained with antibodies against CD11b, Ly6G, andLy6C (Biolegend) and sorted by flow cytometry as described above. T cellswere enriched from spleens of uninfected, syngeneic mice using anti-CD90.2 magnetic microbeads and separation columns (Miltenyi Biotec)

and labeled with the fluorescent dye carboxyfluorescein diacetate succin-imidyl ester (CFSE; Invitrogen) at 5 �M, as described previously (48, 49).The CFSE-labeled T cells were then seeded into tissue culture plates coatedwith 3 �g of anti-CD3 (Biolegend)/ml and cultured with 5 �g of anti-CD28 (Biolegend)/ml. Where indicated, granulocytic or monocytic IMCwere added at a ratio of 10 IMC per T cell. In some samples, the nitricoxide (NO) synthase inhibitor 1400W (Sigma) was added to the culturesat a final concentration of 200 �M. Cultures were incubated for 96 h at37°C and 5% CO2. After 96 h, the cells were stained with antibodiesagainst CD90.2 and analyzed by flow cytometry, as described above. Di-lution of CFSE was used as a measure of cell proliferation.

Statistics. Statistical analysis was performed using unpaired one-wayanalysis of variance (ANOVA) with Dunnett’s multiple-comparison posttest. P values were calculated by comparing data from uninfected mice todata from infected mice, and the significance was determined using InStatsoftware (GraphPad, San Diego, CA). For experiments involving T cells,two-way ANOVA with the Bonferroni post test (to compare the two IMCpopulations) and one-way ANOVA with the Tukey post test (to comparethe levels of T-cell proliferation) were used (InStat software; GraphPad).

RESULTSGr-1� CD11b� cells constitute the majority of the cellular infil-trate of the spleens in mice infected with F. tularensis. The spleenenlarged during acute tularemia due to increased cellularity andunderwent changes in architecture and histopathology, includinglymphoid follicle disintegration and involution with associatedexpansion of the red pulp (Fig. 1A and B). Vulcan Fast Red stain-ing was used to detect the presence of bacteria in the spleen(Fig. 1C and D). The spleen/mouse weight ratio increased almost5-fold in the first 10 days after intradermal inoculation of a sub-lethal dose of F. tularensis (Fig. 1E and F). The composition ofspleen cells for the first 20 days of the infection showed that thegreatest increase was in CD11b� cells (Table 1). Some of these cellsexpressed Gr-1 but not F4/80 or MHC-II. The Gr-1� CD11b�

F4/80� MHC-II� population (referred to hereafter as Gr-1�

CD11b� cells) is known to include IMC. Another subpopulationof CD11b� cells expressed both F4/80 and MHC-II, all of whichare markers of monocytes/macrophages. Three- and two-fold in-creases in dendritic cells (CD11c� CD11b�) and NK cells (DX5�

NK1.1�), respectively, were also noted in the spleens of mice in-fected with F. tularensis (Table 1). By day 10 postinfection, T lym-phocytes reached numbers above those in uninfected spleens (Ta-ble 1). The T-lymphocytic compartment (CD3�) increased inboth the CD8� and CD4� subsets along the same ratio present inthe normal spleen (Table 1).

Gr-1� CD11b� cells were the most prominent population inthe spleens of infected mice by day 10. Their accumulation in thespleen was apparent from the first day after inoculation and pro-gressed rapidly to a peak in numbers by day 10 postinoculation(Table 1). After 20 days, the numbers of these cells were reducedfrom the levels at 10 days after inoculation. In addition, the pop-ulation of monocyte/macrophages (F4/80� MHC-II� Gr-1�

CD11b�) showed large increases (from 0.7 � 106 cells at day 1 to8.8 � 106 cells at day 5) in the infected spleens. Although thepresence of this antigen-presenting cell phenotype is likely impor-tant for host defense, these cells were not major contributors to theincrease in cellularity at 10 days postinoculation (Table 1).

The increase in the splenic Gr-1� CD11b� cell population overtime in F. tularensis-infected C3H/HeN mice is documented inFig. 2A. Although similar increases in splenic Gr-1� CD11b� cellpopulations were observed in F. tularensis-infected C57BL/6 (Fig.2B) and BALB/c (Fig. 2C) mice, the magnitudes of these responses

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were not as large as in C3H/HeN mice. To determine bacterialloads, organ burden assays were performed during the progres-sion of infection. At each time point, similar numbers of CFU of F.tularensis were recovered from the spleens of C3H/HeN, C57BL/6,and BALB/c mice (Fig. 2D). Therefore, we continued our subse-quent investigations using C3H/HeN mice, except for experi-ments that required genetically modified mice in the C57BL/6background (see below). Nonetheless, there are noted differencesin the responses of different strains of mice to infection with F.tularensis (30), and the role of Gr-1� CD11b� cells in this differ-ential response may be of importance.

We used several markers to document the location of F. tular-ensis relative to the Gr-1� CD11b� cells in the spleen. Because thehistological architecture of the spleen changed dramatically dur-ing infection, the bacteria appeared to be distributed throughoutthe organ and not specifically associated with either the red orthe white pulp (Fig. 1). Some bacteria colocalized with Gr-1�

CD11b� cells in the spleen (Fig. 2E, arrows), suggesting these cellsare infected, and the high numbers of both bacteria and cells ofthis phenotype were evident in spleens of mice infected 5 daysearlier. Similar observations were made for liver granuloma-likelesions containing large numbers of Gr-1� CD11b� cells withsuperimposed F. tularensis (55).

Significant increases in the percentages of Gr-1� CD11b� cellsin the bone marrow of mice infected with F. tularensis also oc-curred (Fig. 2F), paralleling the increases in the spleen (Table 1,Fig. 2) and liver (55). The presence of large numbers of Gr-1�

CD11b� cells in the bone marrow and the organs infected by F.tularensis suggests that these cells play an important role in infec-tion.

Shift in splenic B-cell populations during acute infectionwith F. tularensis. The number of B cells in the spleens of micewith tularemia showed an initial decrease until day 10, when allB-cell populations rebounded, along with the appearance ofplasmablasts (IgM� CD138�). Follicular (FO) B cells (IgM�

CD21�/� CD23�) were found to decrease significantly in num-bers until day 10 (Fig. 3A). Marginal zone (MZ) B cells (IgM�

CD21� CD23�) followed a similar trend (Fig. 3B). This loss innumbers of FO B cells and MZ B cells could have resulted fromtheir differentiation into plasmablasts, which increased in numbersignificantly by day 10 (Fig. 3C). Alternatively, the loss might havebeen due to their egress into the red pulp. The simultaneous de-creases of the FO and MZ B cells, together with the large accumu-lation of Gr-1� CD11b� cells, led us to the premise that theseopposing shifts might be associated. To investigate this premise,

FIG 1 Hematoxylin-and-eosin-stained sections of spleens of mice infectedwith sublethal doses of F. tularensis. (A) Normal spleen. (B) By 7 days postin-oculation, an influx of mononuclear cells in the red and white pulp disruptsnormal splenic architecture. (C) Detection with Vulcan Fast Red of vastgrowth of F. tularensis in the red pulp at 5 days after infection. (D) High-powerview as in panel C. Bars, 200 �m (A to C) and 50 �m (D). (E) Means andstandard deviations of spleen/mouse body weight ratios. The increase is due toboth an increase in spleen weight and a loss of mouse total body weight duringinfection. (F) Comparison of an age- and sex-matched spleen from a mousethat was not infected (NI) with a spleen from mouse infected 10 days earliershows splenomegaly of the latter. The data shown are from a single experimentusing three mice per experimental group. Asterisks indicate statistically signif-icant differences (*, P � 0.05; **, P � 0.01).

TABLE 1 Flow cytometric analysis of surface marker expression by splenocytes from mice that were either not infected or infected with F. tularensisat various days postinoculation

Cell surface marker(s)

Mean no. of splenocytes (106) � SDa

NI Day 1 Day 5 Day 10 Day 20

Gr-1� CD11b� MHC-II� 5.7 � 0.3 10.5 � 2.5 16.1 � 3.3 70.7 � 9.3** 14.0 � 2.0Gr-1� CD11b� F4/80� 5.4 � 0.5 9.9 � 2.6 18.2 � 7.2 78.4 � 10.8** 13.3 � 1.6F4/80� MHC II� Gr-1� CD11b� 0.4 � 0.0 0.7 � 0.1 8.8 � 4.4** 7.6 � 1.0** 1.2 � 0.3CD3� 27.8 � 2.5 17.1 � 3.5 35.6 � 3.1 40.2 � 9.0* 33.8 � 10.2CD3� CD4� 18.2 � 1.9 11.5 � 2.6 20.8 � 1.0 26.1 � 7.1* 20.1 � 5.7CD3� CD8� 7.5 � 0.8 4.9 � 0.9 9.0 � 0.2 14.8 � 4.2** 11.9 � 3.1CD11c� CD11b� 0.8 � 0.3 0.6 � 0.2 1.7 � 1.1 2.4 � 0.5* 1.2 � 0.3DX5� NK1.1� 0.6 � 0.2 0.3 � 0.1 0.9 � 0.6 1.3 � 0.2 0.5 � 0.2a Numbers are means from a single experiment using three C3H/HeN mice per group. NI. not infected. *, P � 0.05; **, P � 0.01.

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B6.129S2-Igh-6tmlCgn/J mice (B cell deficient) and the corre-sponding C57BL/6 wild-type (WT) mice were infected with dif-ferent doses of the LVS in order to establish a 50% lethal dose(LD50). The LD50s of the WT and B6.129S2-Igh-6tmlCgn/J micewere not different, which is consistent with earlier studies (22, 24).In agreement with these findings, WT and B-cell-deficient miceharbored similar levels of F. tularensis in spleen and bloodthroughout the experimental course of infection (Fig. 3D and datanot shown).

Splenocytes from WT and B-cell-deficient mice infected withF. tularensis were harvested and analyzed by flow cytometry forexpression of surface Gr-1 and CD11b. There was a difference inthe kinetics of splenic accumulation of Gr-1� CD11b� cells be-tween F. tularensis-infected WT and B-cell-deficient mice, with asignificant delay in the accumulation of these cells by day 5 in theanimals that lacked B cells (Fig. 3E). This delay could be due to (i)reduced splenic infiltration, (ii) reduced proliferation outside ofthe bone marrow, or (iii) the lower numbers of Gr1� cells presentin B-cell-deficient mice prior to infection. In contrast, Gr-1�

CD11b� cell numbers remained significantly elevated in B-cell-deficient mice by day 20, at a time when these cells had returned to

normal levels in the WT mice. Collectively, these results suggest arole for B cells in the control of the rate of Gr-1� CD11b� cellaccumulation in spleens of mice infected with F. tularensis.

Gr-1� CD11b� cells from both infected and uninfectedmouse spleens can differentiate into cells with macrophage- anddendritic cell-like phenotypes following ex vivo culture. UsingFACS, we were able to purify a morphologically heterogeneouspopulation of Gr-1� CD11b� cells from both infected and normalmouse spleens. Giemsa staining of the purified cells showed both amononuclear cell type and a neutrophil-like cell type with a ringnucleus but with scant granulation (Fig. 4A). Mature polymor-phonuclear cells were not seen in these purified preparations. Theheterogeneous nature of the Gr-1� CD11b� cells has been docu-mented before in infection (19, 20, 51) and in cancer (2, 9, 74).Flow cytometric analysis using forward scatter and side scattershowed that the sorting process yielded mostly live cells (Fig. 4B),and, with vital stains, 8 to 10% dead cells (Fig. 4C). Consistently,we reached high levels of purity of cells sorted using Gr-1 andCD11b markers (Fig. 4D to F).

For baseline comparisons, aliquots of the Gr-1� CD11b� cellspurified from infected mice at day 10 postinoculation were rean-

FIG 2 Numbers of Gr-1� CD11b� IMC in the spleens and bone marrow of mice infected with sublethal doses of F. tularensis. Spleens were removed on variousdays postinoculation from C3H/HeN mice (A), C57BL/6 mice (B), and BALB/c mice (C) or from mice of the same strain that were not infected (NI). Splenocyteswere collected, stained fluorescently for the cell surface markers Gr-1 and CD11b, and analyzed by flow cytometry. (D) Bacterial loads per gram of spleen tissueharvested from C3H/HeN mice (s), C57BL/6 mice (�) and BALB/c mice (�) infected with F. tularensis. (E) Immunofluorescent detection of F. tularensis, Gr-1,and CD11b in spleen tissue harvested from a C3H/HeN mouse infected 10 days earlier. The merged image shows Gr-1� cells (PE, red), CD11b� cells (Alexa Fluor647, blue) and F. tularensis (Alexa Fluor 488, green). Note the colocalization of F. tularensis with Gr-1� CD11b� cells (white arrows). Bar, 75 �m. (F) Means andstandard deviations of percentages of Gr-1� CD11b� cells in the bone marrow of infected mice or mice that were not infected (NI). Bone marrow from femursand tibiae was collected, stained for Gr-1 and CD11b, and analyzed by flow cytometry. Data shown are from a single experiment using three to five mice perexperimental group. Asterisks indicate statistically significant differences (*, P � 0.05; **, P � 0.01).

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alyzed immediately by flow cytometry for expression of markersassociated with monocyte/macrophages (F4/80� CD11b�) anddendritic cells (CD11c�), as well as for Gr-1. As expected, theseassays reflected the purity levels obtained after sorting with Gr-1and CD11b markers (Fig. 4D to F). The numbers of freshly puri-fied Gr-1� CD11b� cells expressing F4/80 or CD11c were lessthan 1% (Fig. 5B and C, D0 infected).

Aliquots of the Gr-1� CD11b� cells from uninfected and in-fected mice were cultured in RPMI 1640 medium supplementedwith 10 ng of GM-CSF/ml for 7 days to characterize the ability ofthese cells to differentiate ex vivo. As shown in Fig. 5A, the cellsincreased in size and granularity over the 7 days, comparably incells isolated from either infected or uninfected spleens. Flow cy-tometric analysis revealed that Gr-1� CD11b� cells from infectedmice differentiated into F4/80� CD11b� cells (Fig. 5B, D7 in-fected) and into CD11c� dendritic cells (Fig. 5C, D7 infected) andalmost totally lost Gr-1 expression (Fig. 5D, D7 infected). This

same differentiation pattern was seen for Gr-1� CD11b� cellsfrom uninfected mice, where monocyte/macrophage and den-dritic cell markers were acquired with a concomitant loss of Gr-1(Fig. 5B and D, D7 uninfected). Expression of CD11b was main-tained at high levels (95 to 99%) for the 7 days of ex vivo culturewith GM-CSF (Fig. 5B). Therefore, splenic Gr-1� CD11b� cellsfrom both infected and uninfected mice could differentiate intomonocyte/macrophages and dendritic cells in the presence of theappropriate growth factor. Although GM-CSF is essential for theex vivo culture of sorted Gr-1� CD11b� cells, we cannot excludethe possibility that GM-CSF induced the differentiation of theheterogeneous Gr-1� CD11b� cells, resulting in survival and en-richment of newly differentiated monocyte/macrophages anddendritic cells.

Monocytic IMC but not granulocytic IMC purified fromspleens of mice infected with F. tularensis can inhibit T-cell pro-liferation. CD11b� cells enriched from spleens of infected or un-

FIG 3 Response of B cells in mice infected with F. tularensis. Single-cell suspensions were prepared from spleens of mice infected with F. tularensis or mice thatwere not infected (NI). The cells were stained with antibodies to IgM, CD21, CD23, and CD138 as markers specific for B cells. The percentages of each cellsubpopulation were multiplied by the total number of splenocytes to determine absolute numbers in the various subpopulations. (A) Follicular B cells (IgM�

CD21�/� CD23�). (B) Marginal zone B cells (IgM� CD21� CD23�). (C) IgM plasmablasts (IgM� CD138�). (D) Bacterial loads per gram of spleen tissueharvested from B-cell-deficient mice (circles) and C57BL/6 wild-type (WT) mice (squares) infected with F. tularensis. (E) Kinetics of accumulation andmaintenance of splenic Gr-1� CD11b� IMC in B-cell-deficient mice (�) and C57BL/6 WT mice (s) infected with F. tularensis. The data shown are from a singleexperiment using three mice per experimental group. Asterisks indicate statistically significant differences (*, P � 0.05; **, P � 0.01).

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infected mice were stained with anti-mouse Ly6G and anti-mouseLy6C antibodies. The granulocytic IMC component (CD11b�

Ly6Cint Ly6G�) represented 15% � 1.2% (mean � the standarddeviation) of the total number of cells from infected spleens,whereas uninfected spleens had 5% � 1%. In contrast, the mono-cytic component (CD11b� Ly6Chi Ly6G�) represented 4% �0.2% of cells from infected spleens and 1% � 1% of cells fromuninfected organs (Fig. 6A).

Given that there is evidence to suggest that IMC can mediatethe suppression of T-cell responses in cancer, we used an in vitroassay (70) to determine whether IMC purified from spleens ofmice infected with F. tularensis could suppress the proliferation ofT cells. T cells enriched from spleens of uninfected mice werelabeled with CFSE and seeded in tissue culture plates coated withanti-CD3 and supplemented with anti-CD28 (both part of theT-cell immunological synapse and required for the proliferationof T cells in vitro). CFSE is a fluorescent dye that is distributedevenly among daughter cells with each round of cell division, pro-viding a measure of cell proliferation. Monocytic IMC or granu-locytic IMC purified from spleens of mice infected with F. tular-ensis were then added to the T cells. After 96 h, the cells wereharvested, stained, and analyzed by flow cytometry. As shown inFig. 6, T cells cultured with monocytic IMC but granulocytic IMCexhibited significantly reduced levels of proliferation in responseto anti-CD3/CD28 (Fig. 6B and C). The suppressive activity ofIMC is often associated with production of nitric oxide (NO). Todetermine whether F. tularensis-induced monocytic IMC inhibitproliferation of T cells via NO, a selective inhibitor of inducibleNO synthase, 1400W, was added to the cultures. The addition of

1400W blocked the suppression of T-cell proliferation by mono-cytic IMC purified from spleens of mice infected with F. tularensis(Fig. 6B and C). Collectively, these results suggest an importantrole for NO in mediating monocytic IMC-induced suppression ofT-cell responses during tularemia.

DISCUSSION

The splenomegaly of experimental infection with F. tularensis wascharacterized by lymphoid follicle disintegration and involution,along with expansion of the red pulp. Expansion of the Gr-1�

CD11b� population (Table 1 and Fig. 2) was a major contributorto splenomegaly. These cells were not at all prominent in unin-fected mice. Most Gr-1� CD11b� cells in the tularemia-infectedspleen were negative for the expression of MHC-II and F4/80,emphasizing their immature myeloid nature (Table 1). Simulta-neous analysis of the splenocytes for four myeloid markers (F4/80,MHC-II, Gr-1, and CD11b) provided further evidence that themajority of these cells had not developed into the monocyte/mac-rophage lineage. Moreover, typical granular polymorphonuclearcells were generally absent from the sorted Gr-1� CD11b� popu-lation isolated 10 days after infection, indicating that there werevery few mature neutrophils. The splenic responses to experimen-tal tularemia centered on the expansion of the Gr-1� CD11b�

population and, as such, parallel the expansion of these cells in thehepatic granulomas characteristic of this infection (55). Althoughcells of the myeloid lineage are derived from the bone marrow,extramedullary proliferation of these cells (i.e., proliferation ofthese cells outside of the bone marrow) in the spleen in response totumor-derived growth factors, proinflammatory proteins, and

FIG 4 Purification of Gr-1� CD11b� cells from mice infected with F. tularensis. Splenocytes were harvested from mice infected with F. tularensis 10 days earlierand incubated with anti-CD11b microbeads to collect CD11b� cells by magnetic separation. CD11b� cells were then stained with a fluorescent antibody to Gr-1and purified by FACS. (A) Giemsa staining of purified Gr-1� CD11b� IMC show a heterogeneous population with mononuclear (black arrows) or ring-shaped(white arrow) nuclei. Bar, 10 �m. (B and C) Forward-scatter versus side-scatter profile (B) and cell death profile of purified Gr-1� CD11b� IMC (C), as detectedby flow cytometry. (D to F) Three separate experiments demonstrating high levels of purity of sorted Gr-1� CD11b� IMC, as detected by flow cytometry. Imagesare representative of at least three independent experiments, each using cells pooled from three or four mice.

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sepsis has been documented (19, 66). A parallel expansion ofGr-1� CD11b� cells in the bone marrow was also noted in ourmice, although in the case of the bone marrow, a portion of thisexpanded cell population could have been neutrophils (Fig. 2F).Nevertheless, it is possible that medullary proliferation of Gr-1�

CD11b� cells was occurring more or less simultaneously with theproliferation of Gr-1� CD11b� cells in both the spleen and theliver (55). The large numbers of Gr-1� CD11b� cells in the spleensof tularemia-infected mice suggest the possibility of splenic pro-

liferation, a notion bolstered by the presence of mitotic cells inboth spleens and livers (data not shown).

Immature myeloid cells with suppressor functions have beenobserved previously in the spleens and tumors of mice (8, 10), inmodels of chronic inflammation (25), and in polymicrobial ex-perimental sepsis where large splenic accumulations were noted(19). Specifically, these cells have been shown to inhibit T-cell-mediated immune responses (45, 56, 64). We considered the pos-sibility that this could be the case in murine tularemia. In mice

FIG 5 Ex vivo differentiation of purified Gr-1� CD11b� cells stimulated with GM-CSF. For comparison, Gr-1� CD11b� IMC were purified from spleens ofuninfected or infected mice. (A) Cellular differentiation as detected by forward scatter and side scatter of Gr-1� CD11b� cells. After 7 days of ex vivo culture, thesecells increase in size and granularity comparably in samples derived from infected and uninfected spleens. (B and C) Gr-1� CD11b� cells differentiate into cellsexpressing F4/80 and CD11b (B) and CD11c (C) after 7 days of ex vivo culture. (D) Expression of Gr-1 decreases after 7 days of ex vivo culture in cells from infectedor uninfected mice. Images are representative of at least three independent experiments, each using cells pooled from three or four mice.

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infected with F. tularensis, there was only a modest expansion of Tcells in the spleen (Table 1) and no expansion at all in the liver(55). Ten days after infection, the absolute numbers of Gr-1�

CD11b� cells in the spleen had increased markedly. In the sameperiod of time, the numbers of lymphoid cells (expressing CD3,CD4, CD8, CD21, CD23, CD138, and/or IgM) tended to decreasewithin the first day, and, by day 10 postinfection, increase to levelsless than 2-fold different from the normal controls (Table 1 andFig. 3). In this context, while there were absolute increases in thenumbers of Gr-1� CD11b� cells in the spleens of mice of differentstrains, the percentages of this cell population were not as high inBALB/c and C57BL/6 mice compared to C3H/HeN mice (Fig. 2Ato C). Lethality of F. tularensis infection depends on the mousestrain, infecting dose, and route of inoculation. Earlier studiesshowed that the intraperitoneal and intravenous LD50 for

C57BL/6 and BALB/c mice are higher than those for C3H/HeNmice (30). The levels of splenic bacteria among these three strainsof mice were comparable (Fig. 2D), so there was not a linear rela-tionship with the levels of CFU and IMC. Likewise, there are straindifferences in responses to vaccines that parallel the LD50 studies(12).

MZ B cells provide an initial and prompt antibody response,mainly to T-cell-independent antigens of different types of patho-gens. As such, they are a first line of defense against blood-bornepathogens based on their anatomical localization in the marginalsinus of the spleen. FO B cells constitute the majority of recircu-lating B cells in the body. They produce the canonical antibodyresponse to protein antigens and require T-cell help to generatehigh-affinity, isotype-switched, somatically mutated antibodiesand B-cell memory. We demonstrated that after infection of micewith F. tularensis, the numbers of MZ and FO B cells tended todecrease initially (Fig. 3A and B), possibly because these cells dif-ferentiated into plasmablasts during the first 10 days of infection.These changes are in line with what is known about the behavior ofsplenic B cells in other blood-borne infections, where there is aloss of splenic MZ and FO B cells by egress and cellular differen-tiation before robust expression of IgM� plasmablasts (1). Ourdata further suggest a role for B cells in the control of the rate ofGr-1� CD11b� cell accumulation in spleens of mice infected withF. tularensis. The delay in the accumulation of the Gr-1� CD11b�

cells in the spleens of B-cell-deficient mice (Fig. 3E) could be dueto reductions in infiltration or extramedullary proliferation or tothe lower numbers of Gr1� cells present in these mice prior toinfection. By day 20 postinfection, cells expressing Gr-1 andCD11b decreased to uninfected levels in WT mice (Table 1, Fig. 2Aand C, Fig. 3E). Along with decreased bacterial burden (Fig. 2D,3D), these results indicate a general trend toward regaining a nor-mal splenic architecture. In contrast, B-cell-deficient mice main-tained Gr-1� CD11b� cells at significantly higher levels (Fig. 3E).Thus, our data clearly suggest that, during infection with F. tula-rensis, there is an interdependence between the splenic B cells andthe kinetics of Gr-1� CD11b� cell accumulation and mainte-nance. Other studies have documented a connection between theaccumulation of Gr-1� CD11b� cells and B lymphocytes usingalum injection (38) and infection with T. cruzi (52) or Leishmania(68).

Ex vivo differentiation of Gr-1� CD11b� cells with GM-CSFresulted in the development of antigen-presenting cells, sincemarkers of mature macrophages and dendritic cells were highlyexpressed after 7 days, with a concurrent decrease in Gr-1 expres-sion (Fig. 5). Ex vivo differentiation of tularemia-induced Gr-1�

CD11b� cells into antigen-presenting cells agrees with other re-sults in tumor-bearing mice (73) and polymicrobial sepsis (19).This ex vivo preference appears to be due to the action of GM-CSF,and the cells do not survive in culture without the growth factor. Ifsimilar differentiation of Gr-1� CD11b� cells into antigen-pre-senting cells occurs in vivo, then this would be advantageous to thehost in that the newly differentiated monocyte/macrophage anddendritic cell types could contribute to protection.

In recent years, it has become clear that F. tularensis targetsmultiple host pathways to induce acute immunosuppression andsubvert the mammalian immune system. For example, F. tularen-sis infection inhibits production of IFN-� by inducing expressionof a negative regulator of IFN-�, SOCS3, in both murine andhuman monocytes (53). Also, F. tularensis suppresses the adaptive

FIG 6 Purified monocytic IMC (mIMC) but not granulocytic IMC (gIMC)suppress antigen-independent polyclonal T-cell proliferation via a NO-depen-dent mechanism. (A) Percentages of gIMC (CD11b� Ly6Cint Ly6G� cells) andmIMC (CD11b� Ly6Chi Ly6G� cells) present in the spleens of mice left unin-fected (n 6) or infected with F. tularensis for 10 days (n 9). The results arepooled from two independent experiments. (B) Proliferation of CFSE-labeledT cells stimulated by anti-CD3/CD28 in the presence of either purified gIMCor purified mIMC, as determined by flow cytometry. The leftmost panel showscontrol T cells that were not stimulated with anti-CD3/CD28. In the fourthpanel from the left, the inducible NO synthase inhibitor 1400W was added tothe cultures. Values indicate the percentages of cells that have undergone atleast one round of proliferation. (C) Compilation of results generated as inpanel B from three independent experiments, each using cells pooled fromthree or four mice. Asterisks indicate statistically significant differences (*, P �0.05; ***, P � 0.001). n.s., not significant.

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immune response by inducing the release of prostaglandin E2,which induces anti-inflammatory cytokine production and blocksT-cell proliferation (72). Interestingly, the release of prostaglan-din E2 in cancer has been shown to induce the accumulation ofIMC (65).

Given the modest expansion of T cells (Table 1), we consideredthe possibility that IMC might contribute to T-cell immunosup-pression. To this end, the functional heterogeneity of granulocytic(CD11b� Ly6Cint Ly6G�) and monocytic (CD11b� Ly6Chi

Ly6G�) IMC was evaluated. The more numerous granulocyticIMC did not have an inhibitory effect on T-cell proliferation,whereas the less-abundant monocytic IMC induced inhibition ofT-cell proliferation via a NO-dependent mechanism (Fig. 6). Thisinhibitory effect could be associated with an early unresponsive-ness of T cells that would allow for initial immune suppression,causing disease progression. It will be of interest to determinewhether the monocytic IMC subpopulation is larger in the firstfew days of infection, making the balance between the monocyticand granulocytic IMC a determinant of the disease outcome.

Despite the inhibition of T-cell proliferation by monocyticIMC (Fig. 6), our results raise the possibility that Gr-1� CD11b�

cells aid in controlling the infection by providing an initial barrierto bacterial dissemination (60, 63). Several lines of evidence sug-gest that IMC could contribute to protection against tularemia.The greatest accumulation of these cells in spleen and liver oc-curred by day 10 postinfection (Table 1 and Fig. 2 and 3), at a timeof frank recovery of the mice and when bacteria in the spleen areeither absent or not numerous. The interdependence betweensplenic B cells and the accumulation and persistence of Gr-1�

CD11b� cells further support a role for these IMC in host defense.However, we cannot be certain that in vivo differentiation ofGr-1� CD11b� cells into antigen-presenting cells occurs in thesame manner as our ex vivo experiments suggest.

Taken together, we suggest that Gr-1� CD11b� cells have acomplex role in F. tularensis infection, one that may be related tothe actions of specific subsets of cells within this heterogeneouspopulation or the different and changing functional states of thesecells (74). Furthermore, we suggest that Gr-1� CD11b� cellscould in fact have dual roles in infection, providing a balance ofimmunosuppressive and protective functions where the tipping ofthis balance may be an important factor influencing the outcomeof infection.

ACKNOWLEDGMENTS

This study was supported by grants from the National Institutes of Health(PO1 AI055621 [J.L.B.], T32 AI007539 [J.W.T.], and R21 AI092165[A.W.M.V.D.V.]) and Northeast Biodefense Center (U54 AI057158-Lip-kin).

We thank Susan Malkiel and Marc Golightly for their assistance withflow cytometric analysis and Gloria Monsalve for her assistance with an-imal experimentation.

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