Skip Navigation

Resident CD11c+ Lung Cells Are Impaired by Anthrax Toxins after Spore Infection

  1. Aurélie Cleret1,
  2. Anne Quesnel-Hellmann1,
  3. Jacques Mathieu2,
  4. Dominique Vidal1 and
  5. Jean-Nicolas Tournier1
  1. 1Unité d’Immunobiologie, Département de Biologie des Agents Transmissibles, and
  2. 2Unité de Radiobiologie et Inflammation, Département de Radiobiologie et de Radiopathologie, Centre de Recherche du Service de Santé des Armées, La Tronche, France
  1. Reprints or correspondence: Dr. Jean-Nicolas Tournier, Unité d’Immunobiologie, Département de Biologie des Agents Transmissibles, CRSSA, 24 ave. des maquis du Grésivaudan, 38702 La Tronche, France (jntournier{at}crssa.net)
 
Next Section

Abstract

Bacillus anthracis secretes 2 toxins: lethal toxin (LT) and edema toxin (ET). We investigated their role in the physiopathologic mechanisms of inhalational anthrax by evaluating murine lung dendritic cell (LDC) functions after infection with B. anthracis strains secreting LT, ET, or both or with a nontoxinogenic strain. Three lung cell populations gated on CD11c/CD11b expression were obtained after lung digestion: (1) CD11chigh/CD11blow (alveolar macrophages), (2) CD11cintermediate (int)/CD11bint (LDCs), and (3) CD11clow/CD11bhigh (interstitial macrophages or monocytes). After infection with LT-secreting strains, a decrease in costimulatory molecule expression on LDCs was observed. All CD11c+ cells infected with a nontoxinogenic strain secreted tumor necrosis factor (TNF)–α, interleukin (IL)–10, and IL-6. LT-secreting strains inhibited overall cytokine secretion, whereas the ET-secreting strain inhibited only TNF-α secretion and increased IL-6 secretion. Similar results were obtained after preincubation with purified toxins. Our results suggest that anthrax toxins secreted during infection impair LDC function and suppress the innate immune response

Bacillus anthracis is a gram-positive, spore-forming bacterium and is the causal agent of zoonotic anthrax disease. It has recently become a major concern, after its use as a bioweapon in terrorist attacks in 2001. B. anthracis has 2 major virulence factors: (1) the capsule encoded by the plasmid pXO2 and (2) the 2 toxins produced by the association of 3 factors encoded by the plasmid pXO1. These 3 factors are protective antigen (PA), lethal factor (LF), and edema factor (EF). PA associates with LF to form lethal toxin (LT) and associates with EF to form edema toxin (ET). PA binds to at least 2 cellular receptors and, after being cleaved by a furin-like protease, self-assembles to form a heptameric prepore that facilitates the entry of LF/EF moieties into the cytosol, where they exert their toxic activities [1]. LF is a zinc-dependent metalloprotease that cleaves most of the mitogen-activated protein kinase kinases [2], whereas EF is a calcium- and calmodulin-dependent adenylate cyclase that increases intracellular cAMP concentration [3, 4]

The pathogenesis of inhalational anthrax disease, especially the crucial step of crossing the alveolocapillar space, is not well understood [5, 6]. Inhaled spores are phagocytosed in the alveolar spaces by antigen-presenting cells (APCs). Phagocytes then carry the spores from the alveolus to the mediastinal lymph nodes [7, 8]. The spores germinate within their intracellular niche and release vegetative bacteria extracellularly, which causes septicemia associated with a release of the toxins and, ultimately, causes host death. For decades, research focused on alveolar macrophages (AMs), which were thought to be the “Trojan horse” for the pathogen [9]. However, AMs are not thought to play a role in antigen traffic to lymph nodes [10]. Also, macrophage-depleted mice are still susceptible to aerosol infection, demonstrating the crucial role of another, as-yet-unidentified population of cells [11]. Resident lung dendritic cells (LDCs) are strong candidates for this population, since they have been clearly identified among the various lung phagocytes

A crucial function of LDCs is to capture antigens at alveolar sites and deliver them to mediastinal lymph nodes to mount an efficient adaptive immune response [12]. Previous studies have shown that LT impairs murine bone marrow–derived, spleen-derived, and human monocyte–derived DC cytokine secretion [13 –16]. Although these studies have shown the host response mechanism to the pathogen, they have given no clue as to the early interactions after inhalation of the spores, because pulmonary-tract APCs have particular phenotypic and functional properties. For example, the CD11c antigen (integrin αxβ2) is expressed by both LDCs and AMs, whereas it is expressed only by DCs in other organs [17 –20]. Therefore, several studies have shown that the purification of lung parenchymal cells results in several APC populations, depending on the expression of CD11c and CD11b [17, 19] or CD11c and MHC class II molecules [18]. Also, pulmonary CD11c+ cells usually can secrete high levels of anti-inflammatory cytokines, such as interleukin (IL)–10 [21], and of proinflammatory cytokines, such as IL-6 [22] and tumor necrosis factor (TNF)–α. These special functional abilities of CD11c+ cells could be related to the need to protect an infected lung from inflammatory injuries

Given the particular characteristics of LDCs, we hypothesized that anthrax toxins could have significantly different effects on LDCs than on bone marrow–derived DCs (BMDCs). By focusing on LDCs, we were able to obtain more-accurate data on the early effects that toxins have on the primary interactions between B. anthracis spores and resident lung cells. This should provide more physiological data on the early effects of each anthrax toxin after inhalation

Here, we show that LDCs are more efficient than AMs at phagocytosing anthrax spores. Moreover, phagocytosis of LT-secreting spores inhibited LDC maturation and impaired overall cytokine secretion by CD11c+ cells. In contrast, phagocytosis of ET-secreting spores did not affect LDC maturation but inhibited the secretion of TNF-α and up-regulated the secretion of IL-6. Our results strongly suggest that anthrax toxins have specific effects on LDCs

Previous SectionNext Section

Materials and Methods

Mice Male 6–12-week-old BALB/c (H-2d) mice (Janvier) were housed in clean, standard conditions at our animal-care facility. The local ethics committee approved our animal experiments

B. anthracis strains We used the following B. anthracis strains: the parental Sterne strain 7702 (pXO1+); single-mutant derivatives RP10 Δlef and RP9 Δcya which secrete PA-EF and PA-LF, respectively [23]; and double-mutant RP42 Δlef/Δcya which secretes PA only [24] (all from M. Mock, Institut Pasteur, Paris, France)

Isolation of CD11c + cells from lung and generation of BMDCs BALB/c mice were killed, and bronchoalveolar lavage (BAL) was performed 4 times, by intratracheal instillation of 1 mL of PBS (Gibco; Invitrogen) supplemented with 2 mmol/L EDTA (Sigma-Aldrich). The thoracic cavity was opened, and lungs were perfused with 5 mL of PBS–2 mmol/L EDTA via the right ventricular cavity of the heart. The lungs were aseptically removed, cut into small pieces, and digested twice, in RPMI 1640 medium (Sigma-Aldrich) containing collagenase type I (1 mg/mL; Worthington Biochemical) and DNAse I (50 U; Sigma-Aldrich), by incubation for 30 min in a humidified incubator at 37°C in a 5% CO2 atmosphere. Enzyme activity was stopped by washing the digested tissues in PBS–10 mmol/L EDTA for 10 min on ice. The digested lungs were further disrupted by gently forcing the tissue through 2 nylon screens (pore size, 70 μm and 40 μm; BD Biosciences). After red-blood-cell lysis (in NH4Cl solution), cells were separated using CD11c microbeads (Miltenyi Biotec). We collected the positive fraction, and cell suspensions were counted and viability assessed by trypan-blue exclusion. All reagents used for isolation and culture of cells were certified as being endotoxin free. BMDCs were generated as described elsewhere [15, 25]

In vitro infection and toxin-exposure conditions CD11c+ cells were seeded at 1.5×106 cells/mL in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS; Invitrogen) and 2 mmol/L l-glutamine (Sigma-Aldrich) for 1 h in a humidified incubator at 37°C in a 5% CO2 atmosphere. Spores of B. anthracis were heat activated (70°C for 10 min), and cells were infected with the spores for 1 h at an MOI of 20 [15, 26, 27]. The cells were washed twice in RPMI supplemented with 2.5 μg/mL gentamycin (Invitrogen) to kill any remaining extracellular bacteria [26] and were resuspended in RPMI–5% FBS–gentamycin. After 17 h of culture, followed by centrifugation, supernatants were sampled, and cells were harvested. Noninfected CD11c+ lung cells were used as negative controls

For intoxication with purified toxins, the CD11c+ cells were preincubated with PA (1 μg/mL) and/or LF and/or EF at either 1 or 10 ng/mL (all 3 components from List Biological Laboratories) for 2 h. RP42 (LT−/ET−) strain spores were added and incubated for 1 h at an MOI of 20. Cells were then washed twice. Fresh culture medium containing gentamycin (2.5 μg/mL) was added, and the culture supernatants and cells were sampled 17 h later. BMDCs were seeded at 1.5×106 cells/mL and infected as described above

Phenotypic analysis Cells were incubated with FcR blocking antibody (anti–mouse CD16/CD32 antibody, clone 2.4G2; BD Biosciences) to reduce nonspecific binding. We used the following monoclonal antibodies (MAbs; all from BD Biosciences): anti–I-Ad–fluorescein isothiocyanate (FITC) (clone AMS-32.1), anti–CD86-FITC (B7-2, clone GL1), anti–I-A/I-E–phycoerythrin (PE) (clone M5/114.15.2), anti–CD40-PE (clone 3/23), anti–CD80-PE (B7-1, clone 16-10A1), anti–CD11b-FITC or -PE (clone M1/70), and anti–CD11c-biotin (clone HL3). Cells were incubated for 30 min at 4°C with different combinations of MAbs. APC-conjugated streptavidin was used after incubation with biotinylated MAb. Cells were fixed with 4% paraformaldehyde before analysis

Phagocytosis analysis Sterne spores were heat activated (70°C for 10 min) before incubation for 30 min on ice with FITC-labeled anti-BclA MAb (4B7G12; obtained from P. Sylvestre, Institut Pasteur, Paris, France). Cells were preincubated with anti-CD16/32 antibodies to avoid receptor Fc–mediated phagocytosis and were infected for 30 min at 37°C with the Sterne spores stained by anti-BclA antibodies at an MOI of 20. The cells were washed twice and stained for CD11c and CD11b markers as described above. The cells were fixed before analysis

Cytokine and chemokine measurement in supernatants and TNF-α intracellular staining TNF-α was measured in 18-h cell-culture supernatants by use of ELISA kits (R&D Systems). IL-6, IL-10, IL-12p70, IFN-γ, and monocyte chemoattractant protein (MCP)–1 were measured using a cytometric-bead-array multiplex detection system (BD Biosciences)

For intracellular staining, CD11c+ cells were infected as described above. After 1 h in medium supplemented with gentamycin (2.5 μg/mL), brefeldin A (5 μg/mL) was added as described elsewhere [25]. Cells were labeled with CD11c and CD11b surface markers as described above and with an intracellular TNF-α–PE MAb (BD Biosciences) for 1 h at 4°C. The cells were fixed before analysis

Flow cytometry and statistical analysis Cell acquisition was performed on a FC500 (Beckman Coulter), and the data were analyzed by CXP software (version 2.0; Beckman Coulter). The cell distributions were compared using the Kolmogorov-Smirnov test

Statistical analysis was performed using SigmaStat software (version 3.0; Systat). Statistical differences between the groups were determined by a factorial analysis of variance (1-way analysis of variance; Holm-Sidak method)

Previous SectionNext Section

Results

Characterization of LDCs in BALB/c mice We first characterized the major subsets of APCs present in murine lungs. We performed flow cytometry, using antibodies against the CD11b and CD11c antigens that are known to be differentially expressed by AMs and LDCs [17, 28]. After digestion, total lung cells showed 3 different CD11c/CD11b expression patterns, as has been demonstrated in previous studies (figure 1A ) [17 –19]. The AMs, which represented 14.6% of the total cells, characteristically expressed CD11chigh/CD11blow markers. LDCs (3.5%) represented the double-positive population and expressed intermediate (int) levels of the 2 markers. A third population of cells expressed only the CD11b marker (10.3%). These cells were either interstitial macrophages (IMs) [28] or monocytes, as has been proposed elsewhere [18, 29]. The BAL samples contained a large CD11chigh/CD11blow population (64.4%) (figure 1A ). Furthermore, these cells were highly autofluorescent (data not shown), which was consistent with previous data showing that BAL fluid contains mainly AMs [30] and confirmed the AM phenotype observed in the total lung cells

Figure 1
View larger version:
Figure 1

Characterization of lung dendritic cells (LDCs) in BALB/c mice. A Total lung cells and cells from bronchoalveolar lavage fluid (BAL) were stained for CD11b and CD11c markers. Values at the top right of each panel are the percentages of cells in each quadrant. B CD11b/CD11c expression pattern of purified CD11c+ cells; gate R1 represents the alveolar macrophages (AMs), and gate R2 represents the LDCs. Values at the top right of each panel are the percentages of cells in each quadrant. C Autofluorescence (analyzed in the canal FL1) and MHC class II expression of AMs (R1) and LDCs (R2) after positive selection with CD11c microbeads. D Expression of MHC class II molecules and costimulatory molecules CD86 and CD80 by freshly isolated CD11c+ lung cells (dashed line) and after overnight culture (unshaded portion of histogram); shading indicates isotype control staining. Results shown are representative of 3 independent experiments. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin

We next looked at purified AMs and LDCs from lungs. These are thought to play a crucial role in inhalational anthrax. We isolated these 2 APC subsets by using magnetic microbeads to separate cells expressing CD11c. After purification, we obtained enriched populations of CD11c+ total lung cells (figure 1B ): AMs (CD11chigh/CD11blow) represented 67.5% and LDCs (CD11cint/CD11bint) represented ∼10% of the purified cells. We could clearly identify these populations on the basis of cell autofluorescence and the expression of MHC class II molecules [18, 19]. The AMs (gate R1) were highly autofluorescent and did not express MHC class II molecules (figure 1C panel R1), whereas the LDCs (gate R2) were less autofluorescent and expressed an intermediate level of MHC class II molecules (figure 1C panel R2). Freshly isolated LDCs showed an immature phenotype with no expression of the costimulatory molecules CD40, CD80, and CD86 (figure 1D ). However, after overnight culture, the expression of CD80, CD86, and MHC class II molecules was up-regulated in these LDCs (figure 1D ) [31]

Infection of CD11c + cells by spores of B. anthracis.   We studied the early events of inhalational anthrax by infecting CD11c+ lung cells, at an MOI of 20, with spores of the Sterne strain (LT+/ET+) or one of its derivative mutants, RP9 (LT+/ET−), RP10 (LT−/ET+), and RP42 (LT−/ET−). We assessed cell viability by 7-aminoactinomycin D (7-AAD) staining after 18 h of culture. We found that the different strains did not affect either cell viability or the expression of CD40 and CD80 markers (data not shown)

We next compared the efficiency of each major lung APC population in the phagocytosis of anthrax spores (figure 2A ). We observed that more LDCs than AMs phagocytosed anthrax spores (84%±3.5% vs. 61%±9%, respectively). Moreover, LDCs phagocytose more anthrax spores than do AMs, as assessed by the mean fluorescence intensity (MFI) (P<.000001, Kolmogorov-Smirnov test). Spore phagocytosis was also less efficient for IMs and AMs from BAL fluid (figure 2B ). These results suggest that LDCs were the most efficient lung APCs in the phagocytosis of anthrax spores

Figure 2
View larger version:
Figure 2

Infection of CD11c+ cells by Bacillus anthracis spores. A Phagocytosis of Sterne spores stained for exosporium BclA protein by CD11c+ cells. The different cell populations were gated on the expression of CD11b/CD11c markers (upper panels): alveolar macrophages (AMs) (R1) lung dendritic cells (LDCs) (R2) and interstitial macrophages (IMs) (R3); fluorescence in canal FL1 was also analyzed (R1–R3). Values in the quadrants are the percentages of cells, and values in parentheses are the mean fluorescence intensities (MFIs) of the positive cells. The percentages of positive cells in R1 and R2 are significantly different (P<.000001, Kolmogorov-Smirnov test). B Phagocytosis of fluorescein isothiocyanate (FITC)–labeled Sterne spores by AMs. The major cell population of bronchoalveolar lavage (BAL) fluid was gated on the expression of CD11b/CD11c markers (left) and fluorescence in the canal FL1 was analyzed (right). Values in the quadrants are the percentages of cells. Results shown are representative of 2 independent experiments. APC, allophycocyanin; PE, phycoerythrin

Effects of toxins on LDC maturation after spore infection or intoxication We then specifically focused on LDCs, by gating the analysis on CD11c+ cells after magnetic selection. After 18 h of culture, we observed that a subset of noninfected CD11cint/CD11bint LDCs (∼2%–5% of the population) spontaneously expressed high levels of MHC class II molecules and CD86 costimulatory molecules (figure 3A and 3B ). This LDC subset was observed after infection by both the atoxinogenic strain RP42 (LT−/ET−) and the ET-secreting strain RP10 (LT−/ET+) (4.63% and 4.28%, respectively) (figure 3A ). In contrast, the percentage of this LDC subset was decreased after infection by both LT-secreting strains, RP9 (LT+/ET−) and Sterne (LT+/ET+) (to 0.61% and 0.66%, respectively)

Figure 3
View larger version:
Figure 3

Effect of toxins on CD11c+ cell maturation after spore infection (A) or intoxication (B) and on bone marrow–derived dendritic cell (BMDC) maturation after spore infection (C). A Phenotype analysis of the mature subset of lung dendritic cells (LDCs) after infection with RP42 (lethal toxin [LT]−/edema toxin [ET]−), RP10 (LT−/ET+), RP9 (LT+/ET−), and Sterne (LT+/ET+) spores for 18 h. Data were gated on CD11c+ cells. B Phenotype analysis of the mature subset of LDCs after preincubation with protective antigen (PA), edema factor (EF), and/or lethal factor (LF) at 1 or 10 ng/mL for 2 h and infection with RP42 (LT−/ET−) spores for 18 h. Data were gated on CD11c+ cells. C Phenotype analysis of the mature subset of BMDCs after infection with RP42 (LT−/ET−), RP10 (LT−/ET+), RP9 (LT+/ET−), and Sterne (LT+/ET+) spores for 18 h. Values in the quadrants are the percentages of double-positive cells in the gate. Results shown are representative of 3 independent experiments. FITC, fluorescein isothiocyanate; PE, phycoerythrin

Therefore, we preincubated the CD11c+ lung cells with purified toxins, to confirm the direct effect of LT on LDCs during infection. Preincubation with either PA or ET only (1 or 10 ng/mL) did not change the highly mature double-positive MHC class II+/CD86+ population subset (figure 3B ). In contrast, the percentage of this subset decreased in a dose-dependent manner after treatment with LT (to 1.00% at 1 ng/mL of LT and to 0.29% at 10 ng/mL of LT). Furthermore, after treatment with ET and LT together at 1 ng/mL, the highly mature cell subset was not as affected as after treatment with LT alone (1.44% for the ET-LT group vs. 1.00% for the LT group). However, when the ET-LT concentrations reached 10 ng/mL, we observed the same decrease as for LT at 10 ng/mL alone (to 0.21% and 0.29%, respectively)

We next assessed the effect of LT on BMDCs. The atoxinogenic strain induced a mature double-positive MHC class IIhigh/CD86high population. However, none of the LT- and/or ET-secreting strains affected this cell subset (figure 3C ). The same results were obtained after intoxication (data not shown). These results suggested that LT specifically impairs LDC maturation

Effects of toxins on CD11c + lung cell cytokine secretion after spore infection or intoxication We assessed in parallel the secretion of cytokines (TNF-α, IL-6, IL-10, IL-12p70, and IFN-γ) and of the chemokine MCP-1, 18 h after infection, in the CD11c+ lung cell supernatants. After infection with the atoxinogenic strain, RP42 (LT−/ET−), the CD11c+ lung cells secreted proinflammatory cytokines (TNF-α and IL-6) and the anti-inflammatory cytokine IL-10 (figure 4A ), but they did not secrete significant levels of IL-12p70, IFN-γ, or MCP-1 (data not shown)

Figure 4
View larger version:
Figure 4

Effect of toxins on CD11c+ lung cell cytokine secretion after spore infection (A) or intoxication (B) and on bone marrow–derived dendritic cell (BMDC) cytokine secretion after spore infection (C). A Edema toxin (ET) and lethal toxin (LT) secreted during Bacillus anthracis infection differentially impair cytokine secretion by CD11c+ lung cells. CD11c+ cells were infected with spores of RP42 (LT−/ET−), Sterne (LT+/ET+), RP9 (LT+/ET−), or RP10 (LT−/ET+) strains. Tumor necrosis factor (TNF)–α concentration was measured in culture supernatants after 18 h by ELISA (R&D Systems). Interleukin (IL)–10 and IL-6 secretion was assessed using a multiplex detection system. B Differential impairment of CD11c+ lung cell cytokine secretion by purified ET and LT. CD11c+ cells were preincubated with protective antigen (PA), edema factor (EF), and/or lethal factor (LF) at 1 or 10 ng/mL for 2 h and infected with spores of the RP42 (LT−/ET−) strains. Cytokine concentrations were measured in culture supernatants after 18 h, as described above. C Impairment of BMDC cytokine secretion by ET and LT secreted during B. anthracis infection. Cells were infected with spores of RP42 (LT−/ET−), Sterne (LT+/ET+), RP9 (LT+/ET−), or RP10 (LT−/ET+) strains. TNF-α concentration was measured in culture supernatants after 18 h by ELISA. IL-10 and IL-12p70 were assessed using a multiplex detection system. Data are mean±SD cytokine concentrations representative of 3 independent experiments. *P<.0001, vs. RP42 group

After infection with a strain either secreting both toxins (Sterne [LT+/ET+]) or secreting LT only (RP9 [LT+/ET−]), overall cytokine secretion was inhibited (figure 4A ). After infection with the ET-secreting strain, RP10 (LT−/ET+), we observed that TNF-α secretion was intermediately inhibited and that IL-6 secretion was up-regulated, compared with the atoxinogenic strain. We did not observe any modification of IL-10 secretion after infection with the RP10 strain

CD11c+ lung cell supernatants were analyzed using the same multiplex detection system 18 h after preincubation with purified toxins (figure 4B ). We observed that control CD11c+ lung cells infected with RP42 (LT−/ET−) spores secreted high levels of TNF-α, IL-6, and IL-10. Preincubation with PA alone did not modify cytokine secretion, compared with that in the control group. However, preincubation with LT inhibited cytokine secretion by these cells. In contrast, incubation of CD11c+ lung cells with ET did not affect IL-10 or IL-6 secretion, whereas we observed an intermediate inhibition of TNF-α secretion. The combination of LT and ET at 10 ng/mL inhibited all cytokines. These results were highly consistent with our data showing the effect of infection with spores secreting or not secreting the toxins

Simultaneously, we analyzed the effect of LT-secreting strains on BMDCs (figure 4C ). As was observed for LDCs, BMDCs secreted high levels of TNF-α and IL-10 after infection with the atoxinogenic strain. In contrast to LDCs, they also secreted IL-12p70, a major Th1-inducing cytokine. The LT-secreting strains (Sterne and RP9) inhibited overall cytokine secretion, whereas the ET-secreting strain (RP10) inhibited TNF-α secretion, did not modify IL-12p70 secretion, and enhanced IL-10 secretion, in contrast to the effect seen in LDCs

Cytokine detection in supernatants reflects secretion by a heterogeneous cell population. To discriminate each cell population, we investigated the regulation of TNF-α secretion at the single-cell level by intracellular staining. We focused on AMs and LDCs by gating the population for CD11b and CD11c expression, as described above (figure 5A ). Our results showed that both AMs and LDCs secreted TNF-α (figure 5B ). Infection with the Sterne (LT+/ET+) strain inhibited TNF-α secretion by both AMs and LDCs, compared with the RP42 strain. We observed a decrease in TNF-α+ cell percentage (P<.000001, Kolmogorov-Smirnov test), as well as in TNF-α MFIs (P<.000001, Kolmogorov-Smirnov test), for each population. These results showed that the toxinogenic Sterne strain inhibited TNF-α secretion at the single-cell level

Figure 5
View larger version:
Figure 5

Intracellular tumor necrosis factor (TNF)–α secretion by CD11c+ cells after spore infection. A CD11b/CD11c expression pattern of CD11c+ cells: alveolar macrophages (AMs) (gate R1) and lung dendritic cells (LDCs) (gate R2). B Impairment of TNF-α secretion by AMs and LDCs, as a result of edema toxin (ET) and lethal toxin (LT) secreted during Bacillus anthracis infection. CD11c+ cells were infected with RP42 (LT−/ET−) or Sterne (LT+/ET+) spores. TNF-α secretion was analyzed at the single-cell level by intracellular staining. Values in the quadrants are the percentages of cells; values in parentheses are mean fluorescence intensities of positive cells. Results are representative of 3 independent experiments. APC, allophycocyanin; PE, phycoerythrin

Previous SectionNext Section

Discussion

In our study, we investigated—for the first time, to our knowledge—the effects of toxins after incubation of relevant LDCs with B. anthracis spores. Early events in B. anthracis infection are thought to be crucial for the outcome of the infection, because the immune responses were completely abolished in patients who died during the 2001 outbreak of inhalational anthrax [32]. We show that LT and ET impair early LDC functions and show that toxins may play a crucial role in switching off the immune response in inhalational anthrax infection

This is the first study showing how anthrax toxins act after infection of LDCs. Most previous studies have used AMs, because these cells, which are the major cell population in BAL samples, are easier to isolate [27] and because only very recently has a clear distinction between the different subpopulations of CD11c+ cells in murine lungs been described [17 –20]. Here, we show that multiparametric analysis of the cellular populations present in the whole lung is essential for differentiating the LDCs and the AMs. These 2 populations have important phenotypic and functional differences. We have discriminated between LDCs and AMs on the basis of their expression of CD11b/CD11c antigens and MHC class II molecules, as well as their autofluorescence. Using this strategy, we are able to study the differential expression of maturation markers, such as CD86 and MHC class II molecules, by LDCs, in addition to spore phagocytosis

Our results show that LDCs are significantly more efficient than AMs for spore phagocytosis. This has several consequences with regard to host-pathogen interactions. Because LDCs are able to migrate to the mediastinal lymph nodes, their increased capacity to phagocytose spores makes it easier for the pathogen to cross the alveolocapillar wall. A previous study has shown that secretion of anthrax toxin by the germinated spores enables B. anthracis to kill from within [33]. Therefore, the greater ability of LDCs to phagocytose spores may provide a strong advantage for the pathogen to spread by the pulmonary route

LDCs are also highly sensitive to LT. We observed that LT inhibits, in a dose-dependent manner, maturation characterized by the coexpression of CD86 and MHC class II molecules. This maturation inhibition may lead to an aberrant ability to activate and coordinate the adaptive immune response. The presentation of antigens in this way could lead to an anergy of the specific T cell clone or to the development of T regulator cells

Finally, ET and LT have very different effects on CD11c+ lung cells. LT inhibited the secretion of IL-6, IL-10, and TNF-α. Although ET inhibited the secretion of TNF-α, it did not affect IL-10 secretion, and IL-6 secretion was enhanced. A previous study has shown that human monocytes stimulated by lipopolysaccharide and incubated with ET have enhanced IL-6 secretion [34]. The high levels of IL-6 and IL-10 secretion observed with ET clearly favor the development of a Th2 immune response. We have previously shown that ET has a strong adjuvant effect on the humoral and PA response that could be linked to a Th2 shift [35]. Indeed, it has been specifically shown that IL-6 secreted by APCs polarizes T cells toward a Th2 response [22], whereas IL-10 secretion mediates tolerance [21]. ET may further trigger the development of T regulator cells, as has previously been shown for cholera toxin, another bacterial toxin that increases intracellular cAMP concentration [36]

We did not observe phenotypic maturation inhibition in BMDCs. Furthermore, we observed very different cytokine secretion patterns for lung CD11c+ cells and BMDCs. These results stress the need to evaluate the effects of toxins in relevant cells

Finally, the results presented here describe the effects of B. anthracis toxins on LDCs that may be relevant for understanding inhalational anthrax physiopathologic mechanisms. These results require further investigation using an in vivo model of inhalational anthrax to determine which cells are involved in spore migration

Previous SectionNext Section

Acknowledgments

The authors thank F. Desor for her excellent technical assistance and D. Bois and D. Coulon for their excellent animal care. We also thank M. Mock from the Institut Pasteur, Paris, for providing the Bacillus anthracis strains

Previous SectionNext Section

Footnotes

  • ↵Presented in part: 12th International Congress of Mucosal Immunology, Boston, MA, 25–30 June 2005 (abstract 53928)

    Potential conflicts of interest: none reported

    Financial support: Délégation Générale pour l’Armement (grant CO 010808); Service de Santé des Armées (135OP3B LFR EMA)

  • Received November 18, 2005.
  • Accepted February 4, 2006.
Previous Section
 

References

  1. 1.↵
    1. Abrami L,
    2. Reig N,
    3. van der Goot FG
    . Anthrax toxin: the long and winding road that leads to the kill. Trends Microbiol 2005;13:72-8.
    CrossRefMedlineWeb of Science
  2. 2.↵
    1. Duesbery NS,
    2. Webb CP,
    3. Leppla SH,
    4. et al
    . Proteolytic inactivation of MAP‐kinase‐kinase by anthrax lethal factor. Science 1998;280:734-7.
    Abstract/FREE Full Text
  3. 3.↵
    1. Leppla SH
    . Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. Proc Natl Acad Sci USA 1982;79:3162-6.
    Abstract/FREE Full Text
  4. 4.↵
    1. Little SF,
    2. Ivins BE
    . Molecular pathogenesis of Bacillus anthracis infection. Microbes Infect 1999;1:131-9.
    CrossRefMedlineWeb of Science
  5. 5.↵
    1. Mock M,
    2. Fouet A
    . Anthrax. Annu Rev Microbiol 2001;55:647-71.
    CrossRefMedlineWeb of Science
  6. 6.↵
    1. Guarner J,
    2. Jernigan JA,
    3. Shieh WJ,
    4. et al
    . Pathology and pathogenesis of bioterrorism‐related inhalational anthrax. Am J Pathol 2003;163:701-9.
    MedlineWeb of Science
  7. 7.↵
    1. Ross JM
    . The pathogenesis of anthrax following the administration of spores by respiratory route. J Path Bact 1957;73:485-93.
    CrossRef
  8. 8.↵
    1. Vasconcelos D,
    2. Barnewall R,
    3. Babin M,
    4. et al
    . Pathology of inhalation anthrax in cynomolgus monkeys (Macaca fascicularis) . Lab Invest 2003;83:1201-9.
    CrossRefMedlineWeb of Science
  9. 9.↵
    1. Guidi‐Rontani C
    . The alveolar macrophage: the Trojan horse of Bacillus anthracis . Trends Microbiol 2002;10:405-9.
    CrossRefMedlineWeb of Science
  10. 10.↵
    1. Vermaelen K,
    2. Pauwels R
    . Pulmonary dendritic cells. Am J Respir Crit Care Med 2005;172:530-51.
    Abstract/FREE Full Text
  11. 11.↵
    1. Cote CK,
    2. Rea KM,
    3. Norris SL,
    4. van Rooijen N,
    5. Welkos SL
    . The use of a model of in vivo macrophage depletion to study the role of macrophages during infection with Bacillus anthracis spores. Microb Pathog 2004;37:169-75.
    CrossRefMedlineWeb of Science
  12. 12.↵
    1. de Heer HJ,
    2. Hammad H,
    3. Kool M,
    4. Lambrecht BN
    . Dendritic cell subsets and immune regulation in the lung. Semin Immunol 2005;17:295-303.
    CrossRefMedlineWeb of Science
  13. 13.↵
    1. Agrawal A,
    2. Lingappa J,
    3. Leppla SH,
    4. et al
    . Impairment of dendritic cells and adaptive immunity by anthrax lethal toxin. Nature 2003;424:329-34.
    CrossRefMedline
  14. 14.↵
    1. Brittingham KC,
    2. Ruthel G,
    3. Panchal RG,
    4. et al
    . Dendritic cells endocytose Bacillus anthracis spores: implications for anthrax pathogenesis. J Immunol 2005;174:5545-52.
    Abstract/FREE Full Text
  15. 15.↵
    1. Tournier JN,
    2. Quesnel‐Hellmann A,
    3. Mathieu J,
    4. et al
    . Anthrax edema toxin cooperates with lethal toxin to impair cytokine secretion during infection of dendritic cells. J Immunol 2005;174:4934-41.
    Abstract/FREE Full Text
  16. 16.↵
    1. Alileche A,
    2. Serfass ER,
    3. Muehlbauer SM,
    4. Porcelli SA,
    5. Brojatsch J
    . Anthrax lethal toxin‐mediated killing of human and murine dendritic cells impairs the adaptive immune response. PLoS Pathog 2005;1:e19.
    CrossRefMedline
  17. 17.↵
    1. Gonzalez‐Juarrero M,
    2. Orme IM
    . Characterization of murine lung dendritic cells infected with Mycobacterium tuberculosis . Infect Immun 2001;69:1127-33.
    Abstract/FREE Full Text
  18. 18.↵
    1. von Garnier C,
    2. Filgueira L,
    3. Wikstrom M,
    4. et al
    . Anatomical location determines the distribution and function of dendritic cells and other APCs in the respiratory tract. J Immunol 2005;175:1609-18.
    Abstract/FREE Full Text
  19. 19.↵
    1. Vermaelen K,
    2. Pauwels R
    . Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: methodology and new insights. Cytometry A 2004;61:170-7.
    CrossRefMedline
  20. 20.↵
    1. Osterholzer JJ,
    2. Ames T,
    3. Polak T,
    4. et al
    . CCR2 and CCR6, but not endothelial selectins, mediate the accumulation of immature dendritic cells within the lungs of mice in response to particulate antigen. J Immunol 2005;175:874-83.
    Abstract/FREE Full Text
  21. 21.↵
    1. Akbari O,
    2. DeKruyff RH,
    3. Umetsu DT
    . Pulmonary dendritic cells producing IL‐10 mediate tolerance induced by respiratory exposure to antigen. Nat Immunol 2001;2:725-31.
    CrossRefMedlineWeb of Science
  22. 22.↵
    1. Dodge IL,
    2. Carr MW,
    3. Cernadas M,
    4. Brenner MB
    . IL‐6 production by pulmonary dendritic cells impedes Th1 immune responses. J Immunol 2003;170:4457-64.
    Abstract/FREE Full Text
  23. 23.↵
    1. Pezard C,
    2. Berche P,
    3. Mock M
    . Contribution of individual toxin components to virulence of Bacillus anthracis . Infect Immun 1991;59:3472-7.
    Abstract/FREE Full Text
  24. 24.↵
    1. Pezard C,
    2. Duflot E,
    3. Mock M
    . Construction of Bacillus anthracis mutant strains producing a single toxin component. J Gen Microbiol 1993;139:2459-63.
    Abstract/FREE Full Text
  25. 25.↵
    1. Tournier JN,
    2. Hellmann AQ,
    3. Lesca G,
    4. Jouan A,
    5. Drouet E,
    6. Mathieu J
    . Fever‐like thermal conditions regulate the activation of maturing dendritic cells. J Leukoc Biol 2003;73:493-501.
    Abstract/FREE Full Text
  26. 26.↵
    1. Guidi‐Rontani C,
    2. Levy M,
    3. Ohayon H,
    4. Mock M
    . Fate of germinated Bacillus anthracis spores in primary murine macrophages. Mol Microbiol 2001;42:931-8.
    CrossRefMedlineWeb of Science
  27. 27.↵
    1. Guidi‐Rontani C,
    2. Weber‐Levy M,
    3. Labruyere E,
    4. Mock M
    . Germination of Bacillus anthracis spores within alveolar macrophages. Mol Microbiol 1999;31:9-17.
    CrossRefMedlineWeb of Science
  28. 28.↵
    1. Lagranderie M,
    2. Nahori MA,
    3. Balazuc AM,
    4. et al
    . Dendritic cells recruited to the lung shortly after intranasal delivery of Mycobacterium bovis BCG drive the primary immune response towards a type 1 cytokine production. Immunology 2003;108:352-64.
    CrossRefMedlineWeb of Science
  29. 29.↵
    1. Gonzalez‐Juarrero M,
    2. Shim TS,
    3. Kipnis A,
    4. Junqueira‐Kipnis AP,
    5. Orme IM
    . Dynamics of macrophage cell populations during murine pulmonary tuberculosis. J Immunol 2003;171:3128-35.
    Abstract/FREE Full Text
  30. 30.↵
    1. van Rijt LS,
    2. Kuipers H,
    3. Vos N,
    4. Hijdra D,
    5. Hoogsteden HC,
    6. Lambrecht BN
    . A rapid flow cytometric method for determining the cellular composition of bronchoalveolar lavage fluid cells in mouse models of asthma. J Immunol Methods 2004;288:111-21.
    CrossRefMedlineWeb of Science
  31. 31.↵
    1. Swanson KA,
    2. Zheng Y,
    3. Heidler KM,
    4. Zhang ZD,
    5. Webb TJ,
    6. Wilkes DS
    . Flt3‐ligand, IL‐4, GM‐CSF, and adherence‐mediated isolation of murine lung dendritic cells: assessment of isolation technique on phenotype and function. J Immunol 2004;173:4875-81.
    Abstract/FREE Full Text
  32. 32.↵
    1. Quinn CP,
    2. Dull PM,
    3. Semenova V,
    4. et al
    . Immune responses to Bacillus anthracis protective antigen in patients with bioterrorism‐related cutaneous or inhalation anthrax. J Infect Dis 2004;190:1228-36.
    Abstract/FREE Full Text
  33. 33.↵
    1. Banks DJ,
    2. Barnajian M,
    3. Maldonado‐Arocho FJ,
    4. Sanchez AM,
    5. Bradley KA
    . Anthrax toxin receptor 2 mediates Bacillus anthracis killing of macrophages following spore challenge. Cell Microbiol 2005;7:1173-85.
    CrossRefMedlineWeb of Science
  34. 34.↵
    1. Hoover DL,
    2. Friedlander AM,
    3. Rogers LC,
    4. Yoon IK,
    5. Warren RL,
    6. Cross AS
    . Anthrax edema toxin differentially regulates lipopolysaccharide‐induced monocyte production of tumor necrosis factor alpha and interleukin‐6 by increasing intracellular cyclic AMP. Infect Immun 1994;62:4432-9.
    Abstract/FREE Full Text
  35. 35.↵
    1. Quesnel‐Hellmann A,
    2. Cleret A,
    3. Vidal DR,
    4. Tournier JN
    . Evidence for adjuvanticity of anthrax edema toxin. Vaccine 2006;24:699-702.
    CrossRefMedlineWeb of Science
  36. 36.↵
    1. Lavelle EC,
    2. McNeela E,
    3. Armstrong ME,
    4. Leavy O,
    5. Higgins SC,
    6. Mills KHG
    . Cholera toxin promotes the induction of regulatory T cells specific for bystander antigens by modulating dendritic cell activation. J Immunol 2003;171:2384-92.
    Abstract/FREE Full Text
| Table of Contents

This Article

  1. J Infect Dis. (2006) 194 (1): 86-94. doi: 10.1086/504686
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most