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Originally published online as doi:10.1189/jlb.1104637 on March 9, 2005

Published online before print March 9, 2005
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(Journal of Leukocyte Biology. 2005;77:893-897.)
© 2005 by Society for Leukocyte Biology

The live vaccine strain of Francisella tularensis replicates in human and murine macrophages but induces only the human cells to secrete proinflammatory cytokines

Courtney E. Bolger*, Colin A. Forestal*, Jaime K. Italo*, Jorge L. Benach*,{dagger} and Martha B. Furie*,{ddagger},1

* Center for Infectious Diseases and Departments of
{dagger} Molecular Genetics and Microbiology and
{ddagger} Pathology, School of Medicine, Stony Brook University, New York

1Correspondence: Center for Infectious Diseases, Room 240 CMM, Stony Brook University, Stony Brook, NY 11794-5120. E-mail: Martha.Furie{at}stonybrook.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 REFERENCES
 
Francisella tularensis is the highly infectious agent of tularemia, a disease that can prove fatal in humans. An attenuated live vaccine strain (LVS) of this bacterium is avirulent in man but produces lethal illness in mice. As a step toward understanding the species specificity of the LVS, we compared its interactions with murine and human leukocytes. The bacterium replicated within murine bone marrow-derived macrophages (muBMDM), human monocyte-derived macrophages (huMDM), and freshly isolated human monocytes. However, the murine and human phagocytes differed in their ability to secrete proinflammatory cytokines in response to the LVS. The huMDM released large amounts of CXC chemokine ligand 8 (CXCL8) and CC chemokine ligand 2 when incubated with live or killed LVS organisms, and live bacteria also elicited production of interleukin-1ß (IL-1ß). Furthermore, human monocytes secreted CXCL8, IL-1ß, and tumor necrosis factor {alpha} in response to various bacterial preparations. In contrast, muBMDM produced little to no proinflammatory cytokines or chemokines when treated with any preparations of the LVS. Clearly, human and murine macrophages support growth of this bacterium. However, the greater proinflammatory response of human leukocytes to F. tularensis LVS may contribute to the avirulence of this strain in the human host.

Key Words: bacterial infection • chemokines • inflammatory cytokines • monocytes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
REFERENCES
 
Francisella tularensis, the bacterial agent of tularemia, is highly infectious and can cause acute, severe disease in humans. Consequently, there is concern that this organism could be exploited as a weapon of biological terrorism [1 ]. Most infections in humans are caused by two main subspecies, tularensis and holarctica [2 ]. An attenuated live vaccine strain (LVS) derived from the F. tularensis subspecies holarctica is avirulent in humans but causes lethal disease in mice that is similar to human tularemia [3 ]. In the infected host, F. tularensis is thought to replicate primarily within macrophages, thereby evading assault from the immune system [3 ]. In vitro, the LVS grows in murine macrophages [3 , 4 ], human peripheral blood monocytes [3 ], and human monocyte/macrophage-like cell lines [4 , 5 ]. However, the capacity of the LVS to replicate in primary human macrophages has not been reported.

To determine whether its attenuation in humans stems from a reduced ability to multiply in the macrophages of this host, we compared the growth of the LVS in murine bone marrow-derived macrophages (muBMDM) and human monocyte-derived macrophages (huMDM). The muBMDM were isolated from femurs of female C57BL/6 mice [6 ], and huMDM were prepared by culturing peripheral blood monocytes [7 ] for 5–7 days in the presence of 10 ng/ml human macrophage colony-stimulating factor (Sigma Chemical Co., St. Louis, MO). F. tularensis LVS (American Type Culture Collection 29684, Manassas, VA) was kindly provided by Karen L. Elkins (Center for Biologics, Evaluation, and Research, Food and Drug Administration, Rockville, MD). The LVS was grown in broth [8 ], resuspended in the appropriate macrophage culture medium, and added at various multiplicities of infection (MOI) to muBMDM (7.5x104 cells/cm2) or huMDM (5x104 cells/cm2) cultured on coverslips. The MOI were initially estimated by measuring the optical density at 600 nm of the broth cultures and quantitated precisely by plating on agar [8 ]. Plates containing the cocultures were centrifuged for 3.5 min at 200 g and incubated for 2 h to permit uptake of the bacteria by the macrophages. Some of the cultures were fixed immediately, permeabilized, and stained with rabbit antiserum to F. tularensis and a fluorescein-conjugated secondary antibody (both from BD Biosciences, Lincoln Park, NJ). Other cultures were washed, treated with 5 µg/ml gentamicin for 1 h to eliminate extracellular bacteria [8 ], incubated in antibiotic-free media for 15 h more, and then immunostained. There was little spread of the LVS from leukocyte to leukocyte during the assay, as similar percentages of macrophages harbored bacteria at 2 h and 16 h.

As expected, the LVS replicated extensively in the muBMDM (Fig. 1A 1B 1C 1D ). However, it also grew very well in the huMDM (Fig. 1E 1F 1G 1H) . In fact, the huMDM ingested the LVS much more avidly than did the murine cells: a MOI approximately tenfold greater was needed to infect muBMDM to the same extent as the human macrophages (Fig. 1 and Table 1 ). Although Clemens et al. [5 ] noted a requirement for opsonization for efficient uptake of F. tularensis by human phagocytes, our experiments were performed using media supplemented with heat-inactivated fetal bovine serum, which should not result in opsonization. Our microscopic observations were confirmed by colony-forming unit (CFU) assays, in which macrophages cultured in 24-well plates were lysed with 0.1% deoxycholate immediately after infection for 2 h and treatment with gentamicin for 1 h or after incubation for 15 h more in antibiotic-free medium. Numbers of viable bacteria were then determined by plating diluted lysates on agar. When the LVS was added to huMDM for 2 h at a MOI of 5, three independent CFU assays showed an increase in bacterial numbers of 11.1 ± 6.1-fold during the 15-h interval after removal of extracellular organisms. Replication in muBMDM (MOI {approx}50) was more variable, ranging from 18- to 183-fold over the 15-h incubation. The numbers of viable bacteria that we obtained after replication of the LVS in huMDM were less than we expected based on microscopic evaluation (Fig. 1G and 1H) . A possible explanation is that LVS organisms might replicate extensively in huMDM but are eventually killed intracellularly. Alternatively, infected huMDM were noticeably more rounded than the muBMDM (Fig. 1) and may have detached in greater numbers during washing prior to lysis.



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  		Figure 1. F. tularensis LVS replicates within murine and human macrophages. Cultured muBMDM or huMDM were incubated for 2 h with the LVS at MOI of 50 and 5, respectively. Some samples were then washed and fixed immediately, whereas others were washed and incubated for an additional 16 h. Bacteria were detected by indirect immunofluorescence, and samples were visualized by epifluorescence (A, C, E, G) and phase microscopy. Some panels depict epifluorescent microscopic images superimposed on the phase microscopic images of the same field (B, D, F, H, I, J). Arrows indicate examples of internalized bacteria (B, F). Two hours after bacteria were added to the macrophages, small numbers of F. tularensis LVS were seen associated with muBMDM (A, B) and huMDM (E, F). Sixteen hours later, muBMDM (C, D) and huMDM (G, H) contained markedly increased numbers of bacteria. Magnified views confirm extensive intracellular replication of the LVS after 16 h in huMDM (I) and muBMDM (J).

 

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  		Table 1. Death of Human and Murine Macrophages after Infection with F. tularensis LVS

 
In agreement with a previous report [3 ], we found that the LVS also grew within freshly isolated human monocytes. However, as determined by immunofluorescent staining, it was apparent that only a limited subset of these leukocytes (~15%) was able to take up bacteria, even at a MOI as high as 100. In three CFU assays, numbers of F. tularensis LVS increased 7.8 ± 5.5-fold over 15 h, following infection of the monocytes at a MOI of ~40. Freshly isolated human neutrophils took up the LVS avidly, but as observed by others [9 ], the bacteria did not survive to any appreciable degree within these cells (data not shown).

Intracellular replication of F. tularensis LVS leads to apoptosis of J774 cells, a murine macrophage-like line [10 ]. We compared the death rates of infected muBMDM and huMDM by measuring release of lactate dehydrogenase (Cytotox 96® nonradioactive cytotoxicity assay, Promega, Madison, WI). In three initial experiments, the LVS was added to the macrophages at a MOI of ~25 and allowed to replicate intracellularly and extracellularly for 24 h. Under these conditions, a substantially greater proportion of the huMDM died compared with the muBMDM (69±11% vs. 33±12%). However, this difference likely reflects the greater ability of the huMDM to ingest the LVS. As shown by the experiment summarized in Table 1 , populations of murine and human macrophages that were infected intracellularly to similar extents also displayed comparable levels of cellular death.

Our results indicate that the avirulence of the LVS in humans cannot be attributed to an inability to replicate within or kill human macrophages. We next considered whether the specificity of the LVS might be correlated with its capacity to induce macrophages to secrete proinflammatory cytokines. The lipopolysaccharide (LPS) of the LVS has an atypical lipid A and core structure [11 ] and does not induce production of tumor necrosis factor {alpha} (TNF-{alpha}), interferon-{gamma}, interleukin (IL)-12, IL-10, or nitric oxide by murine macrophages [12 , 13 ]. The purified LPS also causes minimal production of TNF-{alpha} and no production of IL-1{alpha} by human monocytes [14 ]. Like its LPS, living F. tularensis LVS induces little to no secretion of proinflammatory mediators by murine macrophages [15 , 16 ], but its activity toward human macrophages has not been reported.

We therefore assessed the capacity of huMDM to secrete IL-1ß, TNF-{alpha}, and the chemokines CXC chemokine ligand 8 (CXCL8; also known as IL-8) and CC chemokine ligand 2 (CCL2; also called monocyte chemoattractant protein 1) in response to the LVS. IL-1ß and TNF-{alpha} are pleiotropic host proinflammatory cytokines, whereas CXCL8 and CCL2 are potent chemoattractants for neutrophils and monocytes, respectively. All are produced by macrophages in response to a variety of stimuli and could reasonably be expected to play roles in the innate immune response to F. tularensis. The huMDM were treated with the LVS using three different protocols: LVS was added at a MOI of ~25 and allowed to replicate extracellularly and intracellularly for 24 h (denoted "Live Ft" in Figs. 2 and 3 ); LVS at a MOI of ~25 was first killed with 50 µg/ml gentamicin for 1 h and then added to the huMDM for 24 h ("Killed Ft"); or LVS was added at a MOI of ~25 for 2 h, cultures were washed, and incubation was continued for 22 h in the presence of 5 µg/ml gentamicin ("Intracellular Ft"). Amounts of the cytokines released into cell-free, conditioned media were then measured using ELISA.



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  		Figure 2. Human, but not murine, macrophages secrete large amounts of proinflammatory cytokines in response to F. tularensis LVS. In separate experiments, huMDM or muBMDM were incubated with the LVS at an initial MOI of ~25 for 24 h at 37°C in the absence (Live Ft) or presence (Killed Ft) of 50 µg/ml gentamicin. In some samples, cultures were washed 2 h after infection was initiated and incubated for 22 h more in medium containing 5 µg/ml gentamicin (Intracellular Ft). Other samples were treated with Escherichia coli LPS (Sigma Chemical Co.) at 10 ng/ml for muBMDM or 25 ng/ml for huMDM, medium only (Unstim), or a sham preparation of bacteria. This sham consisted of uninoculated LVS culture broth that was processed simultaneously with and in the same manner as the inoculated broth. Cell-free, conditioned media were collected, and amounts of IL-1ß (A), TNF-{alpha} (B), CXCL8 or murine macrophage inflammatory protein 2 (muMIP-2; C), or CCL2 (D) were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (Antigenix America, Franklin Square, NY, and R&D Systems, Minneapolis, MN). Bars represent the means ± SD of three replicate samples. Data were subjected to an unpaired ANOVA and Tukey-Kramer multiple-comparison test using GraphPad Instat Version 3.01 (GraphPad Software, San Diego, CA). *, P < 0.001; **, P < 0.05, compared with unstimulated and sham controls. These experiments were repeated twice for the huMDM and three times for the muBMDM. Donor-to-donor variability in total amounts of cytokines produced by the huMDM was observed, in accordance with observations of others [17 ], but the patterns of up-regulation were consistent in all experiments. For the muBMDM, levels of TNF-{alpha} and muMIP-2 were significantly increased in one and three of the four experiments, respectively, but only in response to live bacteria that were allowed to replicate extracellularly.

 


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  		Figure 3. Human monocytes secrete proinflammatory cytokines in response to F. tularensis LVS. Freshly isolated human monocytes were incubated for 24 h at 37°C with medium alone (Unstim), 10 ng/ml E. coli LPS, or various preparations of the LVS, as described in the legend to Figure 2 . Amounts of IL-1ß (A), TNF-{alpha} (B), and CXCL8 (C) in the cell-free, conditioned media were measured by ELISA. Bars represent the means ± SD of three replicate samples. *, P < 0.001; **, P < 0.05, compared with unstimulated and sham controls. This experiment was repeated twice, save for the intracellular infection, which was performed once more. Although the total amounts of cytokines produced varied from donor to donor, similar patterns of up-regulation were seen in all experiments. The sole exception was secretion of IL-1ß in response to killed LVS, which was significantly increased in only one of the three experiments.

 
As seen in Figure 2 , living LVS induced secretion of substantial amounts of IL-1ß, CXCL8, and CCL2 by the huMDM within the 24-h period of incubation. When extracellular growth of the LVS was unrestricted, increased secretion of CXCL8 was seen by 8 h after addition of the bacteria. Levels of CXCL8 continued to rise between 8 h and 16 h postinfection but remained constant between 16 h and 24 h (data not shown). These results indicate that decay of the chemokine as a result of death of the huMDM was not a complicating factor in our assays. Amounts of IL-1ß, CXCL8, and CCL2 produced by the huMDM after 24 h were similar, whether growth of the bacterium was restricted to the intracellular compartment, or replication in the extracellular medium also was allowed (Fig. 2) . This observation suggests that intracellular organisms may be the predominant stimulus for production of cytokines. Killed bacteria were even more effective than the living organisms in promoting secretion of CXCL8 and CCL2, but they did not induce IL-1ß (Fig. 2) . Previously, we have shown that killed LVS organisms induce a wider array of proinflammatory changes in human umbilical vein endothelial cells than do the living bacteria [8 ]. It is thus possible that the dead bacteria release proinflammatory factors that are distinct from those produced by their viable counterparts. Potentially, then, the inflammatory response in the human host is enhanced as infection with LVS progresses, and bacteria begin to die. Notably, the potency of preparations of LVS equaled or even exceeded that of 25 ng/ml E. coli LPS with respect to stimulating secretion of IL-1ß, CXCL8, and CCL2 by the huMDM (Fig. 2) .

In separate experiments, the ability of muBMDM to be stimulated by the LVS was evaluated, again by measuring release of proinflammatory cytokines. Cytokines measured for the murine macrophages were IL-1ß, TNF-{alpha}, CCL2, and muMIP-2, which was chosen as a representative CXC chemokine for this species, as mice have no known homologue of CXCL8. In agreement with the literature [12 , 13 , 15 , 16 ], the muBMDM produced little to none of these cytokines in response to any preparation of LVS (Fig. 2) . Of four independent experiments, muMIP-2 was slightly but significantly elevated in three and TNF-{alpha} in one. These increases were seen only when the muBMDM were treated with LVS that was allowed to replicate in the extracellular medium, and the total amounts secreted were quite small (Fig. 2) . These results imply that mice infected with the LVS may produce limited quantities of proinflammatory cytokines compared with vaccinated humans. Nonetheless, TNF-{alpha} appears to participate in the innate immune response of mice to the LVS, as blockade of this cytokine soon after infection results in an inability of the animals to survive otherwise sublethal intradermal inocula [18 , 19 ].

Although the huMDM and muBMDM were cultured in different media [6 , 7 ], the LVS replicated extracellularly to a similar extent in both. In a typical experiment, the number of CFU increased 61-fold in the huMDM culture medium, compared with 69-fold for the muBMDM medium. Thus, the murine macrophages failed to respond even when confronted with large numbers of the LVS. Additionally, there was little-to-no release of TNF-{alpha} and muMIP-2 when the LVS was added to muBMDM at a MOI of 400 and replication was subsequently restricted to the intracellular compartment (data not shown). We cannot rule out the possibility that the difference in responsiveness of the murine and human macrophages reflects a difference in their developmental stage rather than a distinction between the two species. However, it is noteworthy that the muBMDM produced more TNF-{alpha} and CXC chemokine in response to 10 ng/ml E. coli LPS than the huMDM did in response to 25 ng/ml of the LPS. The murine macrophages, then, were not intrinsically incapable of responding to proinflammatory stimuli.

As an extension of our studies with human macrophages, we examined the ability of freshly isolated human monocytes to release cytokines in response to the LVS. Although none of the LVS preparations stimulated huMDM to produce TNF-{alpha} (Fig. 2) , killed bacteria consistently augmented secretion of this cytokine, as well as CXCL8, by the monocytes (Fig. 3) . Enhanced release of CXCL8 and IL-1ß also was seen when the monocytes were incubated with LVS that was allowed to replicate both intracellularly and extracellularly (Fig. 3) . In contrast, freshly isolated human neutrophils produced little-to-no IL-1ß or TNF-{alpha} when challenged with the bacteria. Although the neutrophils did secrete significantly increased levels of CXCL8 in response to living or killed LVS, the amounts were small when compared with an equal number of mononuclear phagocytes (data not shown).

In summary, we have found that although human and murine macrophages took up F. tularensis LVS, the human cells did so more readily than their murine counterparts. Once internalized by the macrophages, however, the bacteria were able to replicate well regardless of whether the leukocytes were of murine or human origin. This observation is consistent with reports that F. tularensis LVS escapes from phagosomes and enters the cytoplasm of murine peritoneal macrophages [4 ] and huMDM [5 ], and it rules out the possibility that attenuation of the LVS in man is a result of its failure to thrive in differentiated human macrophages.

Our results raise an alternative possibility, namely that a more robust inflammatory response on the part of the human host may foster the more rapid elimination of the LVS. We have shown that the LVS directly up-regulates expression of adhesion molecules and secretion of chemokines by human endothelial cells [8 ]. Production of IL-1ß and TNF-{alpha} by infected monocytes and macrophages might further stimulate endothelium in an indirect manner, thus enhancing recruitment of leukocytes to infected tissues. Secretion of large amounts of CXCL8, a potent attractant for neutrophils, by resident macrophages at sites of infection might be particularly important for clearance of the bacterium, as we and others [9 ] have shown that human neutrophils readily ingest and destroy the LVS. However, further experiments are needed to clarify the degree to which the virulence of F. tularensis and its ability to provoke inflammation are linked. In this context, extension of our studies to encompass strains of F. tularensis that are fully virulent in mouse and man may prove particularly illuminating.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grants AI48492 and AI055621. C. E. B. and C. A. F. contributed equally to this work. We thank Celine Pujol for assistance with culture of the murine macrophages, Dan Furie for help in preparing figures, and Jim Bliska and David Thanassi for critical review of the manuscript.

Received November 4, 2004; revised February 1, 2005; accepted February 10, 2005.


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M. A. Gavrilin, I. J. Bouakl, N. L. Knatz, M. D. Duncan, M. W. Hall, J. S. Gunn, and M. D. Wewers
Internalization and phagosome escape required for Francisella to induce human monocyte IL-1{beta} processing and release
PNAS, January 3, 2006; 103(1): 141 - 146.
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