Phenotypic, Morphological, and Functional Heterogeneity of Splenic ...

Phenotypic, Morphological, and Functional Heterogeneity of Splenic Immature 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-1ⴙ CD11bⴙ cell phenotype with functional immunosuppression in diverse pathological conditions, including bacterial and parasitic infections and cancer. Gr-1ⴙ CD11bⴙ cells were the largest population of cells present in the spleens of mice infected with sublethal doses of the Francisella tularensis live vaccine strain (LVS). In contrast, the number of T cells present in the spleens of these mice did not increase during early infection. There was a significant delay in the kinetics of accumulation of Gr-1ⴙ CD11bⴙ cells in the spleens of B-cell-deficient mice, indicating that B cells play a role in recruitment and maintenance of this population in the spleens of mice infected with F. tularensis. The splenic Gr-1ⴙ CD11bⴙ cells in tularemia were a heterogeneous population that could be further subdivided into monocytic (mononuclear) and granulocytic (polymorphonuclear) cells using the Ly6C and Ly6G markers and differentiated into antigen-presenting cells following ex vivo culture. Monocytic, CD11bⴙ Ly6Chi Ly6Gⴚ cells but not granulocytic, CD11bⴙ Ly6Cint Ly6Gⴙ cells purified from the spleens of mice infected with F. tularensis suppressed polyclonal T-cell proliferation via a nitric oxide-dependent pathway. Although the monocytic, CD11bⴙ Ly6Chi Ly6Gⴚ cells were able to suppress the proliferation of T cells, the large presence of Gr-1ⴙ CD11bⴙ cells in mice that survived F. tularensis infection also suggests a potential role for these cells in the protective host response to tularemia.

F

rancisella tularensis is a small, aerobic, nonmotile, Gram-negative, pleomorphic coccobacillus. It is a facultative intracellular organism that replicates in macrophages and hepatocytes (4, 5, 7, 14, 16, 27–29, 46, 47). Four subspecies have been identified. The most virulent subspecies in humans is F. tularensis subsp. tularensis (also known as type A), and it is the predominant cause of tularemia in North America. F. tularensis subsp. holarctica (type B) predominates in Eurasia and causes less severe human disease than does type A. F. tularensis subsp. novicida and F. tularensis subsp. mediasiatica are not important pathogens for humans. The F. tularensis live vaccine strain (LVS) is an attenuated type B strain and is infectious and virulent in mice but not in humans. This murine infection model has served as a very useful surrogate for the human illness (27). The clinical severity of tularemia, its protean manifestations, and its lethality, particularly in type A infections, are the main reasons for the inclusion of F. tularensis in the category A group of agents of bioterrorism (http://www.bt.cdc .gov/agent/agentlist.asp). The basis for the virulence and clinical severity of infection with F. tularensis is not completely understood. The bacteremia and hepatitis of tularemia are undoubtedly contributors to the clinical severity, but there is also evidence suggesting that early dysfunction of the immune system could play a role. The immune response to this bacterium is being scrutinized closely, but gaps remain in understanding the mechanisms that depress the adaptive response (23). Immune suppression during infection with F. tularensis could delay the development of adaptive immunity and contribute to high morbidity and mortality. The composition of the cellular immune response in the livers of infected mice has provided a potential clue to immune suppression. The histopathology of hepatic tularemia is characterized by the formation of granuloma-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

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livers using specific cell surface markers showing several types of cells that express the myeloid cell marker CD11b (also known as Mac-1) (55). The largest subpopulation of cells infiltrating the infected livers expressed both Gr-1 and CD11b. Recent studies have linked the accumulation of cells with the Gr-1⫹ CD11b⫹ phenotype to functional immunosuppression in bacterial and parasitic infections, acute and chronic inflammation, and cancer. Most attention has been focused on the role of Gr-1⫹ CD11b⫹ myeloid cells in cancer since they accumulate in large numbers in tumors in practically all tested experimental models, as well as in patients with different types of cancer, and cause a global and profound immune suppression (2, 8–11, 42–45, 57, 64). Gr-1⫹ CD11b⫹ cells are a heterogeneous population that have been referred to as myeloid-derived suppressor cells (31). We refer to this cell phenotype here as immature myeloid cells (IMC) to avoid a functional connotation. Although there are some differences among the results and the experiments that have been done in the context of IMC and infection, 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, Boise State University, Boise, Idaho, USA, and Nihal A. Okan, Department of Microbiology 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. doi:10.1128/IAI.00365-12

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pression associated with the Gr-1⫹ CD11b⫹ phenotype. Early observations of precursor myeloid cells being involved in immunosuppression were made in a Salmonella infection model in 1991. In this study, the appearance of macrophage precursors was shown to play an important regulatory role in the immune response to these bacteria (3). Burns infected with Pseudomonas aeruginosa have large populations of Gr-1⫹ CD11b⫹ cells that inhibit the production of antimicrobial peptides by keratinocytes (40). A recent study showed that Gr-1⫹ CD11b⫹ cells cause immunosuppression in experimental sepsis after passive transfer (19). Gr-1⫹ CD11b⫹ cells have also been implicated in immune suppression in protozoal (34) and fungal (50) infections. The heterogeneous Gr-1⫹ CD11b⫹ cells can be subdivided into functionally and morphologically distinct subpopulations. Murine monocytes are composed of two distinct subpopulations characterized by expression or absence of Gr-1 (32, 33). Gr-1 is expressed on neutrophils, inflammatory monocytes, and some populations of dendritic cells (69), and monoclonal antibodies to Gr-1 recognize both Ly6C and Ly6G isoforms (26). More recently, monoclonal antibodies have been used to separate Gr-1⫹ subpopulations into neutrophil-like (CD11b⫹ Ly6Cint Ly6G⫹) and inflammatory monocytic (CD11b⫹ Ly6Chi Ly6G⫺) cells (18, 74). In fact, Gr-1⫹ CD11b⫹ cells form two separate myeloid lineages with apparently opposing functions that develop as a result of infection with mycobacteria (20). In infections with Trypanosoma gondii, Listeria monocytogenes, or Aspergillus fumigatus, the monocytic component is required for an effective host response (17, 21, 35, 37, 54, 58, 59, 61, 62). Thus, the heterogeneous IMC play a complex role in host-pathogen interactions. Both monocytes/macrophages and neutrophils are important in innate immunity to F. tularensis. F. tularensis replicates within macrophages and are thus shielded from the humoral immune response (27). Mice that are depleted of neutrophils with antisera to Gr-1 succumb to sublethal doses of F. tularensis LVS (41, 67). However, these results are difficult to interpret as multiple cell types express the Gr-1 surface marker. Due to their potentially immunosuppressive nature and their accumulation in large numbers in the livers of mice infected with F. tularensis, we considered that Gr-1⫹ CD11b⫹ cells could play an important role in the host response. In the present study, we demonstrate that Gr-1⫹ CD11b⫹ cells can be elicited in the spleens of mice infected with sublethal doses of F. tularensis, purified by cell sorting, differentiated into antigen-presenting cells, and subdivided into granulocytic (CD11b⫹ Ly6Cint Ly6C⫹) and monocytic (CD11b⫹ Ly6Chi Ly6C⫺) subpopulations. The monocytic subpopulation inhibited T-cell proliferation via a nitric oxide-dependent mechanism, suggesting that elicitation of these cells by F. tularensis may contribute to disease progression by suppressing host immune function. However, the large presence of Gr-1⫹ CD11b⫹ cells in surviving mice also suggests that these cells may contribute to host protection. Thus, the Gr-1⫹ CD11b⫹ cells could in fact have dual roles in infection, providing a balance of immunosuppressive and protective functions where the tipping of this balance may be an important factor influencing the outcome of infection. MATERIALS AND METHODS Bacteria. F. tularensis LVS (29684; American Type Culture Collection Manassas, VA) was cultured in Mueller-Hinton (MH) broth (BD Biosciences, Sparks, MD) supplemented with 2% IsoVitaleX Enrichment (BD

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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. Bacteria were cultured as previously described (55). To enumerate the bacteria, 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 phosphate-buffered saline (PBS), plated on Chocolate II agar (BD Biosciences), and incubated at 37°C and 5% CO2 for 48 h before the CFU were counted. Mice. Female C3H/HeN, C57BL/6 and BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA) and used at 6 to 10 weeks 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 B cells because they lack the expression of membrane-bound IgM (39). All mice were housed in microisolator cages with free access to food and water. Mice received intradermal injections of 105 to 106 CFU of F. tularensis LVS. At various times postinoculation, mice were euthanized, and their blood and organs were used for determination of bacterial burdens. Mice were weighed immediately after euthanization to calculate a ratio of spleen to body weight. The Institutional Animal Care and Use Committee at Stony Brook University approved all animal procedures. Flow cytometry. Excised spleens were teased apart to a single cell suspension and collected in Dulbecco modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA). Bone marrow cells were collected from mice by removing the femur and tibiae of both legs and cutting the epiphysis to expose the marrow. The cavities of the bones were flushed with DMEM to collect the bone marrow cells. Both spleen and bone marrow cells were treated with NH4Cl buffer (144 mM NH4Cl and 17 mM Tris [pH 7.4] in water) to lyse red blood cells before resuspension in fluorescence-activated cell sorter (FACS) buffer (0.2% bovine serum albumin [Sigma] and 0.09% NaN3 [Sigma] in PBS). Cells (106) were then incubated with antiFc␥R antibody (clone 2.4G2; BD Pharmingen, San Diego, CA) before appropriate amounts of conjugated antibodies or isotype-matched control antibodies were added, followed by incubation in the dark for 30 min at 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 a FACSCalibur flow cytometer with CellQuest Pro Software (BD Biosciences, San Jose, CA). Additional analysis was performed using WinList software (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 sections and frozen tissue sections were stained with the antibodies listed below, as previously described (55). F. tularensis in paraffin sections was detected with polyclonal rabbit antisera, followed by alkaline phosphatase-conjugated anti-rabbit IgG and Vulcan Fast Red chromogen (Biocarta, San Diego, CA). For immunofluorescence assays, secondary fluorescein isothiocyanate (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 freezing compound (Richard-Allan Scientific, Kalamazoo, MI), frozen in isopentane that had been cooled with liquid nitrogen, cut at 5 ␮m in the cryostat at ⫺25°C, air dried, and fixed in acetone for 30 s. After application of the appropriate primary and secondary antibodies, slides were washed and mounted in Opti-Mount (Richard-Allan Scientific). Slides were examined by phase-contrast and epifluorescence microscopy using a Nikon Eclipse E600 microscope, and images were captured using a Spot camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Slides for confocal microscopy were analyzed using a Leica DM IRE2 confocal microscope. Images of the red, green, and blue emission signals were captured sepa-


Heterogeneity of Immature Myeloid Cells in Tularemia

rately with the Leica LCS software package. Images were then processed with Adobe Photoshop software. Antibodies for flow cytometry and confocal microscopy. The following antibodies were used for flow cytometry and confocal microscopy: fluorescein isothiocyanate (FITC) anti-mouse CD11c (clone HL3), FITC anti-mouse CD49b/Pan NK cells (clone DX5), FITC anti-mouse CD21 (clone 7G6), R-phycoerythrin (PE) anti-mouse CD3 (clone 17A2), PE anti-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 RB68C5), PE anti-mouse CD138 (Syndecan-1; clone 281-1), peridinin chlorophyll-a protein (PerCP) anti-mouse CD4 (clone RM4-5), PerCP-Cy5.5 anti-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 APC anti-mouse CD8 (clone 53-6.7) from BD Pharmingen; APC anti-mouse CD23 (clone 2G8) from Southern Biotech (Birmingham, AL); Alexa Fluor 647 anti-mouse CD11b (Mac-1; clone M1/70) from Biolegend (San Diego, CA); and Alexa Fluor 488 anti-mouse F4/80 (clone CI:A3-1) from Serotec (Raleigh, NC). Isotype-matched antibodies, as well as secondary antibodies (all from BD Pharmingen), were used as a control for nonspecific binding in all experiments. The following antibodies were used for experiments with T cells: PerCP anti-mouse CD11b (clone M1/70), PE anti-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 from both uninfected and infected spleens to purify cells expressing Gr-1⫹ and CD11b⫹. First, CD11b⫹ cells were collected in magnetic cell sorting buffer (2 mM EDTA and 0.5% bovine serum albumin in PBS at pH 7.2) from a suspension of spleen cells by using magnetic beads specific for CD11b according to the protocol of the manufacturer (Miltenyi Biotec, Auburn, CA). Magnetically purified CD11b⫹ cells were stained with APC antimouse Ly-6G and Ly-6C (anti-Gr-1; clone RB6-8C5) and sorted by the FACSAria cell sorting system (BD Biosciences). Purity of sorted cell populations was verified by flow cytometry and was consistently ⬎98%. Staining of cells with an annexin V and propidium iodide apoptosis detection kit (BD Pharmingen) was used to evaluate cell viability. Single cell suspensions of sorted cell populations were either fixed in methanol for Giemsa stain or stained with DAPI (4=,6=-diamidino-2-phenylindole; Invitrogen) and mounted with Vectashield (Vector Laboratories, Burlingame, CA) for microscopy. Ex vivo culture of Gr-1ⴙ CD11bⴙ cells. Purified Gr1⫹ CD11b⫹ cells were cultured ex vivo in RPMI 1640 medium (Invitrogen) supplemented with 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 culture plates (BD Biosciences) at a density of 3 ⫻ 105 cells/well. For all ex vivo experiments, aliquots of the sorted cells, as well as medium, were plated and cultured on Chocolate II agar for the detection of contaminating residual bacteria. Cells were stimulated with 10 ng of recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems, Minneapolis, MN)/ml for 7 days after sorting, detached with porcine 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 infected with 105 CFU of F. tularensis LVS. After 10 days, the splenocytes were harvested and stained with conjugated monoclonal antibodies against CD11b, Ly6G, and Ly6C (Biolegend). CD11b⫹ Ly6Cint Ly6G⫹ granulocytic IMC and CD11b⫹ Ly6Chi Ly6G⫺ monocytic IMC were identified by flow cytometry. For experiments on T-cell proliferation, the splenocytes were enriched for CD11b⫹ cells using magnetic microbeads and separation columns (Miltenyi Biotec). The resulting populations enriched for CD11b⫹ cells were stained with antibodies against CD11b, Ly6G, and Ly6C (Biolegend) and sorted by flow cytometry as described above. T cells were enriched from spleens of uninfected, syngeneic mice using antiCD90.2 magnetic microbeads and separation columns (Miltenyi Biotec)


and labeled with the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) at 5 ␮M, as described previously (48, 49). The CFSE-labeled T cells were then seeded into tissue culture plates coated with 3 ␮g of anti-CD3 (Biolegend)/ml and cultured with 5 ␮g of antiCD28 (Biolegend)/ml. Where indicated, granulocytic or monocytic IMC were added at a ratio of 10 IMC per T cell. In some samples, the nitric oxide (NO) synthase inhibitor 1400W (Sigma) was added to the cultures at a final concentration of 200 ␮M. Cultures were incubated for 96 h at 37°C and 5% CO2. After 96 h, the cells were stained with antibodies against CD90.2 and analyzed by flow cytometry, as described above. Dilution of CFSE was used as a measure of cell proliferation. Statistics. Statistical analysis was performed using unpaired one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison post test. P values were calculated by comparing data from uninfected mice to data from infected mice, and the significance was determined using InStat software (GraphPad, San Diego, CA). For experiments involving T cells, two-way ANOVA with the Bonferroni post test (to compare the two IMC populations) and one-way ANOVA with the Tukey post test (to compare the levels of T-cell proliferation) were used (InStat software; GraphPad).

RESULTS

Gr-1ⴙ CD11bⴙ cells constitute the majority of the cellular infiltrate of the spleens in mice infected with F. tularensis. The spleen enlarged during acute tularemia due to increased cellularity and underwent changes in architecture and histopathology, including lymphoid follicle disintegration and involution with associated expansion of the red pulp (Fig. 1A and B). Vulcan Fast Red staining was used to detect the presence of bacteria in the spleen (Fig. 1C and D). The spleen/mouse weight ratio increased almost 5-fold in the first 10 days after intradermal inoculation of a sublethal dose of F. tularensis (Fig. 1E and F). The composition of spleen cells for the first 20 days of the infection showed that the greatest increase was in CD11b⫹ cells (Table 1). Some of these cells expressed 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 subpopulation of CD11b⫹ cells expressed both F4/80 and MHC-II, all of which are markers of monocytes/macrophages. Three- and two-fold increases in dendritic cells (CD11c⫹ CD11b⫹) and NK cells (DX5⫹ NK1.1⫹), respectively, were also noted in the spleens of mice infected with F. tularensis (Table 1). By day 10 postinfection, T lymphocytes reached numbers above those in uninfected spleens (Table 1). The T-lymphocytic compartment (CD3⫹) increased in both the CD8⫹ and CD4⫹ subsets along the same ratio present in the normal spleen (Table 1). Gr-1⫹ CD11b⫹ cells were the most prominent population in the spleens of infected mice by day 10. Their accumulation in the spleen was apparent from the first day after inoculation and progressed rapidly to a peak in numbers by day 10 postinoculation (Table 1). After 20 days, the numbers of these cells were reduced from the levels at 10 days after inoculation. In addition, the population of monocyte/macrophages (F4/80⫹ MHC-II⫹ Gr-1⫺ CD11b⫹) showed large increases (from 0.7 ⫻ 106 cells at day 1 to 8.8 ⫻ 106 cells at day 5) in the infected spleens. Although the presence of this antigen-presenting cell phenotype is likely important for host defense, these cells were not major contributors to the increase in cellularity at 10 days postinoculation (Table 1). The increase in the splenic Gr-1⫹ CD11b⫹ cell population over time in F. tularensis-infected C3H/HeN mice is documented in Fig. 2A. Although similar increases in splenic Gr-1⫹ CD11b⫹ cell populations 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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FIG 1 Hematoxylin-and-eosin-stained sections of spleens of mice infected with sublethal doses of F. tularensis. (A) Normal spleen. (B) By 7 days postinoculation, an influx of mononuclear cells in the red and white pulp disrupts normal splenic architecture. (C) Detection with Vulcan Fast Red of vast growth of F. tularensis in the red pulp at 5 days after infection. (D) High-power view as in panel C. Bars, 200 ␮m (A to C) and 50 ␮m (D). (E) Means and standard deviations of spleen/mouse body weight ratios. The increase is due to both an increase in spleen weight and a loss of mouse total body weight during infection. (F) Comparison of an age- and sex-matched spleen from a mouse that was not infected (NI) with a spleen from mouse infected 10 days earlier shows splenomegaly of the latter. The data shown are from a single experiment using three mice per experimental group. Asterisks indicate statistically significant differences (*, P ⬍ 0.05; **, P ⬍ 0.01).

were not as large as in C3H/HeN mice. To determine bacterial loads, organ burden assays were performed during the progression 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 subsequent investigations using C3H/HeN mice, except for experiments that required genetically modified mice in the C57BL/6 background (see below). Nonetheless, there are noted differences in the responses of different strains of mice to infection with F. tularensis (30), and the role of Gr-1⫹ CD11b⫹ cells in this differential response may be of importance. We used several markers to document the location of F. tularensis relative to the Gr-1⫹ CD11b⫹ cells in the spleen. Because the histological architecture of the spleen changed dramatically during infection, the bacteria appeared to be distributed throughout the organ and not specifically associated with either the red or the white pulp (Fig. 1). Some bacteria colocalized with Gr-1⫹ CD11b⫹ cells in the spleen (Fig. 2E, arrows), suggesting these cells are infected, and the high numbers of both bacteria and cells of this phenotype were evident in spleens of mice infected 5 days earlier. Similar observations were made for liver granuloma-like lesions containing large numbers of Gr-1⫹ CD11b⫹ cells with superimposed F. tularensis (55). Significant increases in the percentages of Gr-1⫹ CD11b⫹ cells in the bone marrow of mice infected with F. tularensis also occurred (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 infection. Shift in splenic B-cell populations during acute infection with F. tularensis. The number of B cells in the spleens of mice with tularemia showed an initial decrease until day 10, when all B-cell populations rebounded, along with the appearance of plasmablasts (IgM⫹ CD138⫹). Follicular (FO) B cells (IgM⫹ CD21⫹/⫺ CD23⫹) were found to decrease significantly in numbers until day 10 (Fig. 3A). Marginal zone (MZ) B cells (IgM⫹ CD21⫹ CD23⫺) followed a similar trend (Fig. 3B). This loss in numbers of FO B cells and MZ B cells could have resulted from their differentiation into plasmablasts, which increased in number significantly by day 10 (Fig. 3C). Alternatively, the loss might have been due to their egress into the red pulp. The simultaneous decreases of the FO and MZ B cells, together with the large accumulation of Gr-1⫹ CD11b⫹ cells, led us to the premise that these opposing shifts might be associated. To investigate this premise,

TABLE 1 Flow cytometric analysis of surface marker expression by splenocytes from mice that were either not infected or infected with F. tularensis at various days postinoculation Mean no. of splenocytes (106) ⫾ SDa Cell surface marker(s) ⫹

⫹

⫺

Gr-1 CD11b MHC-II Gr-1⫹ CD11b⫹ F4/80⫺ F4/80⫹ MHC II⫹ Gr-1⫺ CD11b⫹ CD3⫹ CD3⫹ CD4⫹ CD3⫹ CD8⫹ CD11c⫹ CD11b⫹ DX5⫹ NK1.1⫹ a

NI

Day 1

Day 5

Day 10

Day 20

5.7 ⫾ 0.3 5.4 ⫾ 0.5 0.4 ⫾ 0.0 27.8 ⫾ 2.5 18.2 ⫾ 1.9 7.5 ⫾ 0.8 0.8 ⫾ 0.3 0.6 ⫾ 0.2

10.5 ⫾ 2.5 9.9 ⫾ 2.6 0.7 ⫾ 0.1 17.1 ⫾ 3.5 11.5 ⫾ 2.6 4.9 ⫾ 0.9 0.6 ⫾ 0.2 0.3 ⫾ 0.1

16.1 ⫾ 3.3 18.2 ⫾ 7.2 8.8 ⫾ 4.4** 35.6 ⫾ 3.1 20.8 ⫾ 1.0 9.0 ⫾ 0.2 1.7 ⫾ 1.1 0.9 ⫾ 0.6

70.7 ⫾ 9.3** 78.4 ⫾ 10.8** 7.6 ⫾ 1.0** 40.2 ⫾ 9.0* 26.1 ⫾ 7.1* 14.8 ⫾ 4.2** 2.4 ⫾ 0.5* 1.3 ⫾ 0.2

14.0 ⫾ 2.0 13.3 ⫾ 1.6 1.2 ⫾ 0.3 33.8 ⫾ 10.2 20.1 ⫾ 5.7 11.9 ⫾ 3.1 1.2 ⫾ 0.3 0.5 ⫾ 0.2

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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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 various days 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). Splenocytes were collected, stained fluorescently for the cell surface markers Gr-1 and CD11b, and analyzed by flow cytometry. (D) Bacterial loads per gram of spleen tissue harvested from C3H/HeN mice (s), C57BL/6 mice () and BALB/c mice (䊐) 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 Fluor 647, 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 and standard 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 femurs and 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 per experimental group. Asterisks indicate statistically significant differences (*, P ⬍ 0.05; **, P ⬍ 0.01).

B6.129S2-Igh-6tmlCgn/J mice (B cell deficient) and the corresponding C57BL/6 wild-type (WT) mice were infected with different doses of the LVS in order to establish a 50% lethal dose (LD50). The LD50s of the WT and B6.129S2-Igh-6tmlCgn/J mice were not different, which is consistent with earlier studies (22, 24). In agreement with these findings, WT and B-cell-deficient mice harbored similar levels of F. tularensis in spleen and blood throughout the experimental course of infection (Fig. 3D and data not shown). Splenocytes from WT and B-cell-deficient mice infected with F. tularensis were harvested and analyzed by flow cytometry for expression of surface Gr-1 and CD11b. There was a difference in the kinetics of splenic accumulation of Gr-1⫹ CD11b⫹ cells between F. tularensis-infected WT and B-cell-deficient mice, with a significant delay in the accumulation of these cells by day 5 in the animals that lacked B cells (Fig. 3E). This delay could be due to (i) reduced splenic infiltration, (ii) reduced proliferation outside of the bone marrow, or (iii) the lower numbers of Gr1⫹ cells present in B-cell-deficient mice prior to infection. In contrast, Gr-1⫹ CD11b⫹ cell numbers remained significantly elevated in B-celldeficient mice by day 20, at a time when these cells had returned to


normal levels in the WT mice. Collectively, these results suggest a role for B cells in the control of the rate of Gr-1⫹ CD11b⫹ cell accumulation in spleens of mice infected with F. tularensis. Gr-1ⴙ CD11bⴙ cells from both infected and uninfected mouse spleens can differentiate into cells with macrophage- and dendritic cell-like phenotypes following ex vivo culture. Using FACS, we were able to purify a morphologically heterogeneous population of Gr-1⫹ CD11b⫹ cells from both infected and normal mouse spleens. Giemsa staining of the purified cells showed both a mononuclear cell type and a neutrophil-like cell type with a ring nucleus but with scant granulation (Fig. 4A). Mature polymorphonuclear cells were not seen in these purified preparations. The heterogeneous nature of the Gr-1⫹ CD11b⫹ cells has been documented before in infection (19, 20, 51) and in cancer (2, 9, 74). Flow cytometric analysis using forward scatter and side scatter showed 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 and CD11b markers (Fig. 4D to F). For baseline comparisons, aliquots of the Gr-1⫹ CD11b⫹ cells purified from infected mice at day 10 postinoculation were rean-

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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 that were 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 cell subpopulation 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 tissue harvested from B-cell-deficient mice (circles) and C57BL/6 wild-type (WT) mice (squares) infected with F. tularensis. (E) Kinetics of accumulation and maintenance 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 single experiment using three mice per experimental group. Asterisks indicate statistically significant differences (*, P ⬍ 0.05; **, P ⬍ 0.01).

alyzed immediately by flow cytometry for expression of markers associated with monocyte/macrophages (F4/80⫹ CD11b⫹) and dendritic cells (CD11c⫹), as well as for Gr-1. As expected, these assays reflected the purity levels obtained after sorting with Gr-1 and CD11b markers (Fig. 4D to F). The numbers of freshly purified Gr-1⫹ CD11b⫹ cells expressing F4/80 or CD11c were less than 1% (Fig. 5B and C, D0 infected). Aliquots of the Gr-1⫹ CD11b⫹ cells from uninfected and infected mice were cultured in RPMI 1640 medium supplemented with 10 ng of GM-CSF/ml for 7 days to characterize the ability of these cells to differentiate ex vivo. As shown in Fig. 5A, the cells increased in size and granularity over the 7 days, comparably in cells isolated from either infected or uninfected spleens. Flow cytometric analysis revealed that Gr-1⫹ CD11b⫹ cells from infected mice differentiated into F4/80⫹ CD11b⫹ cells (Fig. 5B, D7 infected) and into CD11c⫹ dendritic cells (Fig. 5C, D7 infected) and almost totally lost Gr-1 expression (Fig. 5D, D7 infected). This

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same differentiation pattern was seen for Gr-1⫹ CD11b⫹ cells from uninfected mice, where monocyte/macrophage and dendritic cell markers were acquired with a concomitant loss of Gr-1 (Fig. 5B and D, D7 uninfected). Expression of CD11b was maintained at high levels (95 to 99%) for the 7 days of ex vivo culture with GM-CSF (Fig. 5B). Therefore, splenic Gr-1⫹ CD11b⫹ cells from both infected and uninfected mice could differentiate into monocyte/macrophages and dendritic cells in the presence of the appropriate growth factor. Although GM-CSF is essential for the ex vivo culture of sorted Gr-1⫹ CD11b⫹ cells, we cannot exclude the possibility that GM-CSF induced the differentiation of the heterogeneous Gr-1⫹ CD11b⫹ cells, resulting in survival and enrichment of newly differentiated monocyte/macrophages and dendritic cells. Monocytic IMC but not granulocytic IMC purified from spleens of mice infected with F. tularensis can inhibit T-cell proliferation. CD11b⫹ cells enriched from spleens of infected or un-



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 earlier

and incubated with anti-CD11b microbeads to collect CD11b⫹ cells by magnetic separation. CD11b⫹ cells were then stained with a fluorescent antibody to Gr-1 and 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 detected by flow cytometry. (D to F) Three separate experiments demonstrating high levels of purity of sorted Gr-1⫹ CD11b⫹ IMC, as detected by flow cytometry. Images are representative of at least three independent experiments, each using cells pooled from three or four mice.

infected mice were stained with anti-mouse Ly6G and anti-mouse Ly6C antibodies. The granulocytic IMC component (CD11b⫹ Ly6Cint Ly6G⫹) represented 15% ⫾ 1.2% (mean ⫾ the standard deviation) of the total number of cells from infected spleens, whereas uninfected spleens had 5% ⫾ 1%. In contrast, the monocytic component (CD11b⫹ Ly6Chi Ly6G⫺) represented 4% ⫾ 0.2% of cells from infected spleens and 1% ⫾ 1% of cells from uninfected organs (Fig. 6A). Given that there is evidence to suggest that IMC can mediate the suppression of T-cell responses in cancer, we used an in vitro assay (70) to determine whether IMC purified from spleens of mice infected with F. tularensis could suppress the proliferation of T cells. T cells enriched from spleens of uninfected mice were labeled with CFSE and seeded in tissue culture plates coated with anti-CD3 and supplemented with anti-CD28 (both part of the T-cell immunological synapse and required for the proliferation of T cells in vitro). CFSE is a fluorescent dye that is distributed evenly among daughter cells with each round of cell division, providing a measure of cell proliferation. Monocytic IMC or granulocytic IMC purified from spleens of mice infected with F. tularensis were then added to the T cells. After 96 h, the cells were harvested, stained, and analyzed by flow cytometry. As shown in Fig. 6, T cells cultured with monocytic IMC but granulocytic IMC exhibited significantly reduced levels of proliferation in response to anti-CD3/CD28 (Fig. 6B and C). The suppressive activity of IMC is often associated with production of nitric oxide (NO). To determine whether F. tularensis-induced monocytic IMC inhibit proliferation of T cells via NO, a selective inhibitor of inducible NO synthase, 1400W, was added to the cultures. The addition of


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

The splenomegaly of experimental infection with F. tularensis was characterized 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 contributor to splenomegaly. These cells were not at all prominent in uninfected mice. Most Gr-1⫹ CD11b⫹ cells in the tularemia-infected spleen were negative for the expression of MHC-II and F4/80, emphasizing their immature myeloid nature (Table 1). Simultaneous analysis of the splenocytes for four myeloid markers (F4/80, MHC-II, Gr-1, and CD11b) provided further evidence that the majority of these cells had not developed into the monocyte/macrophage lineage. Moreover, typical granular polymorphonuclear cells were generally absent from the sorted Gr-1⫹ CD11b⫹ population isolated 10 days after infection, indicating that there were very few mature neutrophils. The splenic responses to experimental tularemia centered on the expansion of the Gr-1⫹ CD11b⫹ population and, as such, parallel the expansion of these cells in the hepatic granulomas characteristic of this infection (55). Although cells of the myeloid lineage are derived from the bone marrow, extramedullary proliferation of these cells (i.e., proliferation of these cells outside of the bone marrow) in the spleen in response to tumor-derived growth factors, proinflammatory proteins, and

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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 of

uninfected 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, these cells increase in size and granularity comparably in samples derived from infected and uninfected spleens. (B and C) Gr-1⫹ CD11b⫹ cells differentiate into cells expressing 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 infected or uninfected mice. Images are representative of at least three independent experiments, each using cells pooled from three or four mice.

sepsis has been documented (19, 66). A parallel expansion of Gr-1⫹ CD11b⫹ cells in the bone marrow was also noted in our mice, although in the case of the bone marrow, a portion of this expanded 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 the proliferation of Gr-1⫹ CD11b⫹ cells in both the spleen and the liver (55). The large numbers of Gr-1⫹ CD11b⫹ cells in the spleens of tularemia-infected mice suggest the possibility of splenic pro-

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liferation, a notion bolstered by the presence of mitotic cells in both spleens and livers (data not shown). Immature myeloid cells with suppressor functions have been observed previously in the spleens and tumors of mice (8, 10), in models of chronic inflammation (25), and in polymicrobial experimental sepsis where large splenic accumulations were noted (19). Specifically, these cells have been shown to inhibit T-cellmediated immune responses (45, 56, 64). We considered the possibility that this could be the case in murine tularemia. In mice



FIG 6 Purified monocytic IMC (mIMC) but not granulocytic IMC (gIMC) suppress antigen-independent polyclonal T-cell proliferation via a NO-dependent mechanism. (A) Percentages of gIMC (CD11b⫹ Ly6Cint Ly6G⫹ cells) and mIMC (CD11b⫹ Ly6Chi Ly6G⫺ cells) present in the spleens of mice left uninfected (n ⫽ 6) or infected with F. tularensis for 10 days (n ⫽ 9). The results are pooled from two independent experiments. (B) Proliferation of CFSE-labeled T cells stimulated by anti-CD3/CD28 in the presence of either purified gIMC or purified mIMC, as determined by flow cytometry. The leftmost panel shows control T cells that were not stimulated with anti-CD3/CD28. In the fourth panel from the left, the inducible NO synthase inhibitor 1400W was added to the cultures. Values indicate the percentages of cells that have undergone at least one round of proliferation. (C) Compilation of results generated as in panel B from three independent experiments, each using cells pooled from three or four mice. Asterisks indicate statistically significant differences (*, P ⬍ 0.05; ***, P ⬍ 0.001). n.s., not significant.

infected with F. tularensis, there was only a modest expansion of T cells 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 same period of time, the numbers of lymphoid cells (expressing CD3, CD4, CD8, CD21, CD23, CD138, and/or IgM) tended to decrease within the first day, and, by day 10 postinfection, increase to levels less than 2-fold different from the normal controls (Table 1 and Fig. 3). In this context, while there were absolute increases in the numbers of Gr-1⫹ CD11b⫹ cells in the spleens of mice of different strains, the percentages of this cell population were not as high in BALB/c and C57BL/6 mice compared to C3H/HeN mice (Fig. 2A to C). Lethality of F. tularensis infection depends on the mouse strain, infecting dose, and route of inoculation. Earlier studies showed that the intraperitoneal and intravenous LD50 for


C57BL/6 and BALB/c mice are higher than those for C3H/HeN mice (30). The levels of splenic bacteria among these three strains of mice were comparable (Fig. 2D), so there was not a linear relationship with the levels of CFU and IMC. Likewise, there are strain differences 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 pathogens. As such, they are a first line of defense against blood-borne pathogens based on their anatomical localization in the marginal sinus of the spleen. FO B cells constitute the majority of recirculating B cells in the body. They produce the canonical antibody response to protein antigens and require T-cell help to generate high-affinity, isotype-switched, somatically mutated antibodies and B-cell memory. We demonstrated that after infection of mice with F. tularensis, the numbers of MZ and FO B cells tended to decrease initially (Fig. 3A and B), possibly because these cells differentiated into plasmablasts during the first 10 days of infection. These changes are in line with what is known about the behavior of splenic B cells in other blood-borne infections, where there is a loss of splenic MZ and FO B cells by egress and cellular differentiation before robust expression of IgM⫹ plasmablasts (1). Our data further suggest a role for B cells in the control of the rate of Gr-1⫹ CD11b⫹ cell accumulation in spleens of mice infected with F. tularensis. The delay in the accumulation of the Gr-1⫹ CD11b⫹ cells in the spleens of B-cell-deficient mice (Fig. 3E) could be due to reductions in infiltration or extramedullary proliferation or to the lower numbers of Gr1⫹ cells present in these mice prior to infection. By day 20 postinfection, cells expressing Gr-1 and CD11b decreased to uninfected levels in WT mice (Table 1, Fig. 2A and C, Fig. 3E). Along with decreased bacterial burden (Fig. 2D, 3D), these results indicate a general trend toward regaining a normal splenic architecture. In contrast, B-cell-deficient mice maintained Gr-1⫹ CD11b⫹ cells at significantly higher levels (Fig. 3E). Thus, our data clearly suggest that, during infection with F. tularensis, there is an interdependence between the splenic B cells and the kinetics of Gr-1⫹ CD11b⫹ cell accumulation and maintenance. Other studies have documented a connection between the accumulation of Gr-1⫹ CD11b⫹ cells and B lymphocytes using alum injection (38) and infection with T. cruzi (52) or Leishmania (68). Ex vivo differentiation of Gr-1⫹ CD11b⫹ cells with GM-CSF resulted in the development of antigen-presenting cells, since markers of mature macrophages and dendritic cells were highly expressed after 7 days, with a concurrent decrease in Gr-1 expression (Fig. 5). Ex vivo differentiation of tularemia-induced Gr-1⫹ CD11b⫹ cells into antigen-presenting cells agrees with other results 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. If similar differentiation of Gr-1⫹ CD11b⫹ cells into antigen-presenting cells occurs in vivo, then this would be advantageous to the host in that the newly differentiated monocyte/macrophage and dendritic cell types could contribute to protection. In recent years, it has become clear that F. tularensis targets multiple host pathways to induce acute immunosuppression and subvert the mammalian immune system. For example, F. tularensis infection inhibits production of IFN-␥ by inducing expression of a negative regulator of IFN-␥, SOCS3, in both murine and human monocytes (53). Also, F. tularensis suppresses the adaptive

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immune response by inducing the release of prostaglandin E2, which induces anti-inflammatory cytokine production and blocks T-cell proliferation (72). Interestingly, the release of prostaglandin E2 in cancer has been shown to induce the accumulation of IMC (65). Given the modest expansion of T cells (Table 1), we considered the possibility that IMC might contribute to T-cell immunosuppression. To this end, the functional heterogeneity of granulocytic (CD11b⫹ Ly6Cint Ly6G⫹) and monocytic (CD11b⫹ Ly6Chi Ly6G⫺) IMC was evaluated. The more numerous granulocytic IMC did not have an inhibitory effect on T-cell proliferation, whereas the less-abundant monocytic IMC induced inhibition of T-cell proliferation via a NO-dependent mechanism (Fig. 6). This inhibitory effect could be associated with an early unresponsiveness of T cells that would allow for initial immune suppression, causing disease progression. It will be of interest to determine whether the monocytic IMC subpopulation is larger in the first few days of infection, making the balance between the monocytic and granulocytic IMC a determinant of the disease outcome. Despite the inhibition of T-cell proliferation by monocytic IMC (Fig. 6), our results raise the possibility that Gr-1⫹ CD11b⫹ cells aid in controlling the infection by providing an initial barrier to bacterial dissemination (60, 63). Several lines of evidence suggest that IMC could contribute to protection against tularemia. The greatest accumulation of these cells in spleen and liver occurred by day 10 postinfection (Table 1 and Fig. 2 and 3), at a time of frank recovery of the mice and when bacteria in the spleen are either absent or not numerous. The interdependence between splenic 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 of Gr-1⫹ CD11b⫹ cells into antigen-presenting cells occurs in the same manner as our ex vivo experiments suggest. Taken together, we suggest that Gr-1⫹ CD11b⫹ cells have a complex role in F. tularensis infection, one that may be related to the actions of specific subsets of cells within this heterogeneous population or the different and changing functional states of these cells (74). Furthermore, we suggest that Gr-1⫹ CD11b⫹ cells could in fact have dual roles in infection, providing a balance of immunosuppressive and protective functions where the tipping of this balance may be an important factor influencing the outcome of infection.

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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-Lipkin). We thank Susan Malkiel and Marc Golightly for their assistance with flow cytometric analysis and Gloria Monsalve for her assistance with animal experimentation.

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