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. 2015 May 6;11(5):e1004853.
doi: 10.1371/journal.ppat.1004853. eCollection 2015 May.

Brucella Abortus Induces the Premature Death of Human Neutrophils Through the Action of Its Lipopolysaccharide

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Free PMC article

Brucella Abortus Induces the Premature Death of Human Neutrophils Through the Action of Its Lipopolysaccharide

Elías Barquero-Calvo et al. PLoS Pathog. .
Free PMC article

Abstract

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. B. abortus is partially resistant to the killing action of PMNs.
(A) PMNs were isolated from blood and incubated with B. abortus or S. enterica (MOI 5) and CFUs determined at different time points. (B) Heparinized blood was incubated with B. abortus-GFP or fluorescent latex beads for two hours (MOI 10 or 100). Blood smears were then fixed and mounted with ProLong Gold Antifade Reagent with DAPI. At least 100 PMNs were counted per sample and the number of intracellular bacterial or latex particles determined in each PMN and the proportion expressed as % of phagocytized particles. (C) Human PMNs infected with different MOI of B. abortus-GFP and stained as in “B”. Microscope images are at 400 × magnification. Representative PMNs with DAPI-stained nuclei and intracellular green fluorescent B. abortus were photographed under the microscope using the appropriate color filter channel. Images were cut from microscope field, contrasted and saturated using Hue tool to obtain suitable color separation. Images were then merged using Adobe Photoshop 8 software. Experiments were repeated at least three times. Values of p<0.01 (**) are indicated.
Fig 2
Fig 2. B. abortus infection induces PMN cell death in a dose dependent manner.
(A) Heparinized blood was incubated with B. abortus-GFP (MOI 10) for two hours and PMNs population analyzed for cell death using AquaDead and Annexin V markers. GFP fluorescence intensity was used to differentiate among three categories: (a) low, (b) intermediate and (c) high infection. (B) Percentages of PMNs positive for any marker in relation to the number of internalized bacteria are shown. Experiments were repeated at least three times.
Fig 3
Fig 3. Live and heat-killed B. abortus induce PMN cell death.
Heparinized blood was incubated with live or heat-killed (HK) B. abortus for two hours (10 and 100 bacteria/PMN). PMN population was analyzed by flow cytometry for cell death using AquaDead and Annexin V markers, as described in Fig 2. Percentages of PMNs positive for any marker were determined. Experiments were repeated at least three times. No significant differences were detected between live and HK B. abortus.
Fig 4
Fig 4. Br-LPS is released inside PMNs.
Heparinized blood was incubated with B. abortus-RFP for one hour (MOI 2). Blood smears were fixed, stained with anti-Brucella LPS FITC (green) and mounted with ProLong Gold Antifade Reagent with DAPI. (a) B. abortus-RFP, (b) IgG-FITC anti-Brucella LPS staining, (c) PMN DAPI staining and (d) merged images. Shed Brucella LPS (white arrow) is pointed. Representative PMNs with DAPI-stained nuclei and intracellular B. abortus were photographed under the microscope using the appropriate color filter channel. Images were cut from microscope field, contrasted and saturated using Hue tool to obtain suitable color separation. Images were then merged using Adobe Photoshop 8 program. Microscope images are at 1000 × magnification.
Fig 5
Fig 5. Br-LPS released inside cells is mostly found within vacuolar compartments of PMNs.
Purified human PMNs 5 ×106 were infected with B. abortus 2308 at MOI 20. After one hour incubation, infected cells were fixed and processed for immunogold staining and electron microscopy. Detection of Br-LPS was performed using mouse IgG anti Br-LPS in combination with protein-A/protein-G colloidal gold 15 nm. (A) PMN (n, nucleus of cell) with intracellular B. abortus (white asterisk) and immunogold detection of Br-LPS. (B) and (C) correspond to amplified sections pointed with arrows from “A” panel; B. abortus (white asterisk) and immunogold detection of Br-LPS inside vacuoles (pointed by black arrow heads). (D) B. abortus (white asterisk) within a phagosome (ph) and vacuoles containing immunogold labeled Br-LPS (black arrow heads). (E) PMN membrane ruffle showing immunogold detection of Br-LPS associated to the membrane (white arrow heads) and B. abortus (white asterisk) debris inside a phagosome (ph) and immunogold detection of Br-LPS inside vacuoles (black arrow heads). No colloidal gold particles were observed when IgG purified from normal mouse serum was used for controlling the specificity of the reaction. Bar represents 500 nm.
Fig 6
Fig 6. Br-LPS induces cell death of PMN in a dose dependent manner.
(A) Heparinized blood was incubated with 100μg/mL of (a) Ec-LPS (corresponding to 7.5 pmol/mL) or (b) Br-LPS (corresponding to 3 pmol/mL) for two hours and the PMN population was analyzed by AquaDead and Annexin V markers as in Fig 2. (c) Percentages of PMNs positive for any marker treated and with various concentrations of LPS are shown. (B) Human blood was incubated with 3 pmol/mL of Br-LPS for two hours. (a) Lymphocyte and (b) PMN populations were analyzed by AquaDead and Annexin V markers. (c) Percentages of lymphocytes and PMNs positive for any marker treated and with various concentrations of Br-LPS for are shown. Experiments were repeated at least three times. Values of p<0.01 (**) are indicated.
Fig 7
Fig 7. Quantities of Br-LPS interacting with PMNs.
The quantities of Br-LPS associated to PMNs were determined by Western blotting using a monoclonal antibody against Br-LPS conjugated with peroxidase enzyme. All wells were loaded with 15 μL of the respective preparation. The amounts of purified Br-LPS in the left panel were used to estimate the quantities based on a standard curve ranging from 0.06 ng to 12 ng (only wells from 1.3–6 ng are shown). The right panel corresponds to the assay: purified PMNs were incubated with Br-LPS and the associated amounts determined by Western blot (Br-LPS+PMNs). In order to have a saturating positive control, the assay was also performed in the presence of human antibodies against Br-LPS (Br-LPS+PMNs+Ac). Controls included the assay performed with Br-LPS in the absence (C1) or presence of human antibodies (C2) but in the absence of PMNs. PMNs alone did not show any signal. Notice that the Br-LPS molecules associated to PMNs corresponded to the lower molecular weight fraction (~30–40 MW). The amounts of Br-LPS were estimated to be in the range of 5–25 ng/106 PMNs, corresponding to less than 0.25% of the original Br-LPS added. The amounts of associated Br-LPS in the presence of antibodies were between 10–50 ng/106 PMNs. These estimated quantities were from four different experiments. The read-out of the corresponding bands was performed by densitometry.
Fig 8
Fig 8. Brucella lipid A induces PMNs cell death in a dose dependent manner.
(A) Heparinized blood was incubated for two hours with LPSs of Y. enterocolitica O:9 (3 pmol/mL), of E. coli (7.5 pmol/mL), of B. abortus 2308 (3 pmol/mL), of B. abortus ∆WadC (3 pmol/mL) and of O. anthropi (2 pmol/mL), all corresponding to 100μg/mL of LPS. The LPSs differed in at least one of the moieties (O-chain, core and lipid A) with B. abortus 2308 LPS: (a) LPSs possessing lipid As that differ from B. abortus 2308 LPS, (b) LPSs possessing lipid As structures similar to B. abortus 2308 LPS. (B) Heparinized blood treated with different concentrations of B. abortus 2308 lipid A for two hours. In all assays, PMN population was gated and analyzed by Annexin V marker and the geometric means of histograms displayed as relative units. Experiments were repeated at least three times.
Fig 9
Fig 9. Neutralization of CD14 protects against Br-LPS-induced PMN cell death.
(A) Heparinized blood was incubated for two hours with 0.4 pmol/mL of Ec-LPS or 3 pmol/mL of Br-LPS. Prior to LPS stimulation, some samples were previously treated with anti-TLR4 (1 μg/mL) or anti-CD14 (5μg/mL) antibodies and TNF-α secretion quantified in plasma by ELISA. Values of p<0.01 (**) are indicated in relation to their respective LPS control. (B) Heparinized blood was incubated with Br-LPS (3 pmol/mL) alone or previously neutralized with different quantities of anti-CD14. PMN population was gated and analyzed by Annexin V marker. Geometric means of histograms displayed as relative units. Experiments were repeated at least three times. (C) Purified PMNs were incubated for two hours with Br-LPS (1.5 pmol/mL). Prior to LPS stimulation, some samples were previously treated with anti-CD14 (5μg/mL) antibodies and PMN population gated and analyzed by AquaDead marker. Anti-CD14 alone does not have any significant effect in PMN cell death. Value of p<0.01 (**) is indicated in relation to the Br-LPS control.
Fig 10
Fig 10. Br-LPS-induced PMN cell death correlates with low ROS formation.
Purified human PMNs were seeded on serum-uncoated plates and treated with various concentrations of Br-LPS or Ec-LPS for 6.5 h. (A) ROS kinetics production was monitored for 90 minutes by luminol-amplified chemiluminescence and the maximum obtained value for each LPS concentration plotted (black line). (B) Cell death of purified PMNs was monitored by evaluation of Sytox green fluorescence (shown as percentage of cell death relative to PMA-induced cell death) (red dotted line). Figure represents the outcome of a single experiment. Similar results were obtained in repeated experiments. Correlation R 2 was obtained by using the Excel tool facility.
Fig 11
Fig 11. Inhibitory action of various compounds on the Br-LPS-induced PMN cell death.
Prior to Br-LPS stimulation, samples were treated with wortmanin (50 nM), genistein (100 μM), IM-54 (10 μM), tyrphostin (250 μM), thapsigargin (50 nM), NS3694 (10 μM), PD098059 (50 μM), Z-YVAD-FMK (10 μM), Z-LEHD-FMK (10 μM), necrostatin-5 (10 μM), Z-LEVD-FMK (10 μM), BAPTA/AM (10 μM), Z-IETD-FMK (10 μM), Z-WEHD-FMK (10 μM), YVAD-CHO (50 μM), Z-VAD-FMK (10 μg/mL), AZD7762 (30 μM), catalase (2800 U/mL), tiron (2 mg/mL), acetovanillone (100 μg/mL) or PBS. After treatment with the inhibitory compounds, samples were incubated with Br-LPS (1.5 pmol/mL) for 2 hours. Samples were further processed and analyzed by cytometry for cell death with Annexin V as described above. Geometric means of histograms displayed as relative units. In the upright corner, the read out procedure of the inhibitory action of tiron, catalase and acetovanillone is presented. Values were estimated as relative units of the geometric means of histograms. Each experiment was repeated at least three times.
Fig 12
Fig 12. Br-LPS induces activation of caspase 8 and 9 in PMNs.
(A) Heparinized blood was incubated with 0.3 pmol/mL of Br-LPS or PBS for 30 minutes and stained with anti-active caspase 8 or anti-active caspase 9. PMNs population was analyzed by each caspase marker (B) Heparinized blood samples were treated with Z-VAD-FMK or PBS for 1 hour and then incubated with Br-LPS (1.5 pmol/mL) for 2 hours. PMNs population was analyzed by Annexin V. Geometric means of histograms are displayed as relative units. Experiments were repeated at least three times.
Fig 13
Fig 13. Cytokine differences between blood and purified PMNs infected with B. abortus or stimulated with Br-LPS.
The level of the indicated cytokines was determined by ELISA in the plasma of heparinized blood or in the culture supernatants of purified PMNs after treatment with S. enterica, B. abortus or Br-LPS at various concentrations for two hours. Experiments were repeated at least three times.

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References

    1. Pillay J, den Braber I, Vrisekoop N, Kwast LM, de Boer RJ, Borghans JAM, et al. In vivo labeling with 2H2O reveals a human neutrophil lifespan of 5.4 days. Blood. 2010;116: 625–7. 10.1182/blood-2010-01-259028 - DOI - PubMed
    1. Payne CM, Glasser L, Tischler ME, Wyckoff D, Cromey D, Fiederlein R, et al. Programmed cell death of the normal human neutrophil: an in vitro model of senescence. Microsc Res Tech. 1994;28: 327–44. - PubMed
    1. Stark MA, Huo Y, Burcin TL, Morris MA, Olson TS, Ley K. Phagocytosis of apoptotic neutrophils regulates granulopoiesis via IL-23 and IL-17. Immunity. 2005;22: 285–94. - PubMed
    1. Savill JS, Wyllie AH, Henson JE, Walport MJ, Henson PM, Haslett C. Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J Clin Invest. 1989;83: 865–75. - PMC - PubMed
    1. Nauseef WM. How human neutrophils kill and degrade microbes: an integrated view. Immunol Rev. 2007;219: 88–102. - PubMed

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Grant support

This work was partially supported by grants Fondo Especial de la Educación Superior (FEES-CONARE) grant numbers 0500-13, 0504-13, 0505-13, 0248-13, Costa Rica; the Fondation de la Recherche Médicale; The Fondation Méditerranée Infection; the Centre National de la Recherche Scientifique; the Institut National de la Santé et de la Recherche Médicale and the Aix-Marseille Université. EB-C received a fellowship from SEP-UCR and CONICIT-Costa Rica. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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