IFN-β Increases Listeriolysin O-Induced Membrane Permeabilization and Death of Macrophages

Listeriolysin O

Plasmids encoding six-histidine tagged rLLO, either full-length (rLLO529) or N-terminally truncated (rLLO493; a gift of M. Mitsuyama, Kyoto University, Kyoto, Japan) (32) within the pQE31 vector, were transformed into XL-10 Gold Escherichia coli (Stratagene). rLLO expression in cultures for purification was induced by addition of 0.3 mM isopropyl β-d-thiogalactoside, and the culture was grown for a further 3 h at 28°C. The pellet from a 600-ml culture was resuspended in 10 ml of lysis buffer (0.5 M sodium chloride, 50 mM sodium dihydrogen phosphate (pH 7.0)), lysozyme (end concentration 1 mg/ml) was added, and incubated on ice for 30 min. This was followed by sonication for 6 × 10 s and centrifugation at 12,000 × g for 30 min at 4°C. The supernatant was passed through a 27 G needle, and 250 mM imidazole elution buffer was added such that the end imidazole concentration was 20 mM. A total of 500 μl of prewashed His-bind beads (Qiagen) were added and the mixture rotated for 1 h at 4°C. Beads were captured by pouring through a column, washed with 5 × 10 ml of lysis buffer (pH 7.0) containing 20 mM imadazole, then trapped protein was eluted with 5 ml of lysis buffer (pH 7.0) containing 250 mM imadazole. The eluate was dialyzed twice into storage buffer (0.5 M sodium chloride, 10 mM sodium phosphate (pH 7.0), 0.5 mM EDTA (33)). Endotoxin was removed by multiple passes through Detoxi-Gel endotoxin removal columns (Pierce), and end concentration was measured as <20 pg/ml for the 90 μg/ml stock solution (Rapid Endotest Service; Cambrex). Protein was stored frozen in aliquots at −80°C. Protein concentration was measured by Bradford assay (Bio-Rad), using BSA standards.

Cells, reagents, and infection

Bone marrow-derived macrophages were obtained by culture of bone marrow from 7- to 11-wk-old mice of C57BL/6 genetic background in L cell-derived CSF-1 as described (34). MyD88-deficient (35) and TLR4-deficient mice (36) were as described and provided by S. Akira (Osaka University, Osaka, Japan). Stat1-deficient (37) mice were as described and provided by D. Levy (New York University School of Medicine, New York). Caspase 1-deficient mice (38) were a gift of A. Zychlinsky (Max Planck Institute for Infection Biology, Berlin, Germany). Recombinant murine IFN-β (Calbiochem) was added to a final concentration of 500 U/ml. SB203580 (Calbiochem) was added 30 min before LLO addition to a final concentration of 10 μM. LLO treatment of cells was done in DMEM medium with 3% FBS. Dextran 500 (m.w. 500,000; GE Healthcare) was dissolved in this medium where indicated before use. Infection of macrophages with Listeria monocytogenes (strain LO28 (39)) was as described in (31), using wild-type or LLO-deficient (Δhly) bacteria. Infection of macrophages with Salmonella typhimurium strain SR11 was as described in Ref. 40 .

Assays of cell death

Crystal violet staining and measurement of lactate dehydrogenase (LDH) release was as described in (31).

Western blot analysis

Western blot analysis was done as described in Ref. 41 . Rabbit phosphospecific Ab against p38 MAPK and rabbit antiserum against p38 MAPK were obtained from New England Biolabs and used at dilution 1/1000, analyzing separate blots for the same samples to prevent overlapping signals. Ab recognizing Thr²⁰² and Tyr²⁰⁴ phosphorylated ERKs (phospho-ERK) was obtained from Cell Signaling Technologies, Ab recognizing ERKs (pan-ERK) was obtained from BD Transduction Laboratories, both used 1/1000. Ab recognizing IκBα was obtained from Santa Cruz Biotechnology and used 1/1000. Ab recognizing Tyr⁷⁰¹ phosphorylated Stat1 was obtained from New England Biolabs and used 1/1000, while Ab recognizing the C terminus of Stat1α was as described in Ref. 41 . Ab recognizing precursor caspase 1 and the p10 subunit from cleavage was from Santa Cruz Biotechnology and used 1/100. All primary Abs except caspase 1 for short exposure were probed with fluorescence-labeled secondary Ab (Molecular Probes). This was detected by the infrared imaging system Odyssey (LI-COR) and quantified using associated software. Caspase 1 for short exposure was probed with HRP-labeled secondary Ab and visualized using ECL.

Patch clamp measurements

Macrophages were plated onto glass coverslips and treated where indicated with IFN-β overnight. For measurement, macrophages on coverslips were superfused with an extracellular solution containing (in millimoles) 150 NaCl, 5.4 KCl, 0.5 MgCl₂, 1.8 CaCl₂, 10 HEPES, 5 glucose (pH 7.4) adjusted with NaOH. The recording pipette contained (in millimoles) 140 potassium aspartate, 2 MgCl₂, 2 CaCl₂, 10 HEPES, adjusted to pH 7.4 with KOH. Currents were recorded in the perforated patch clamp configuration using amphothericin B (600 μg/ml) in the pipette solution. All measurements were performed at 20–22°C. Electrodes pulled of borosilicate capillaries had a tip resistance of 5–6 M Ω. After patch formation, an equilibrium period of 5 min followed, which was succeeded by a control period of 5 min at a holding potential of −30 mV during which no electrical activity could be observed. Then, LLO was added to the bathing solution at concentrations of 2.5 or 5.0 μg/ml. First channel openings were detected ∼1 min after addition of LLO. Electrophysiological measurements were conducted with an Axopatch-1D patch clamp amplifier (Axon Instruments) at a cutoff frequency (−3 dB) of 2 kHz. Currents were filtered at 5 kHz and sampled at 10 kHz. Data acquisition was executed directly to a PC with pCLAMP 6 software (Axon Instruments). Current analysis was performed with ASCD software (G. Droogmans, Katholieke Universiteit Leuven, Leuven, Belgium).

Reverse transcription and real-time PCR

Total RNA extraction, cDNA synthesis, and real-time PCR was conducted as described in Ref. 42 , using the GAPDH housekeeping gene as an endogenous control with primers as described. Primers for HMGCoA reductase and fatty acid synthase were as described in Ref. 43 . Primers for caspase 1 were as described in Ref. 44 , primers for caspase 11 were as described in Ref. 45 , primers for apoptosis-associated speck-like protein containing a CARD were as described in Ref. 46 .

FACS analysis

For FACS analysis, macrophages were harvested by incubation for 3–5 min in citric saline (0.135 M potassium chloride, 0.015 M sodium citrate), centrifugated together with the medium from the plate to collect cells in suspension, spun for 7 min at 300 × g at 4°C, and resuspended in PBS containing 0.2% BSA before analysis by FACSCalibur (BD Biosystems). Annexin V/7-aminoactinomycin D (7AAD) staining was conducted according to the manufacturer’s instructions using annexin V-allophycocyanin and 7-AAD (BD Biosciences).

Statistics

Statistical significance was assessed where indicated using the Student t test, two-tailed, assuming equal variance, and was calculated using Microsoft Excel software. Significance is indicated in figures using the convention: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

IFN-β enhances the lytic activity of LLO

Addition of LLO to primary macrophages triggered cell death in a concentration-dependent manner (Fig. 1⇓, A and B). The time course of LDH release indicated that onset of death was rapid, within the first few hours after toxin addition. The long-term survival of cells was assayed by staining adherent cells with crystal violet 24 h after toxin addition. Cell death was visible in this assay at LLO concentrations at 0.25 μg/ml and above. IFN-β treatment alone did not cause cell death, but a larger number of pretreated macrophages died after LLO addition. IFN-β triggers Stat1-dependent as well as Stat1-independent pathways (47). IFN-β pretreatment of Stat1-deficient macrophages was without consequences for the lytic activity of LLO (Fig. 1⇓C), showing that the sensitization effect is mediated by Stat1.

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FIGURE 1.

IFN-β pretreatment increases macrophage death upon exposure to LLO in a Stat1-dependent manner. A, With or without pretreatment with IFN-β, bone marrow macrophages (BMMs) were exposed to LLO at the concentrations shown. At the time points indicated, LDH released into the medium was measured, relative to the total LDH release from lysis of untreated cells. The degree of statistical significance is indicated, comparing IFN-β pretreated and not-pretreated cells for the same concentration of toxin. B, BMMs were pretreated or not with IFN-β. Surviving adherent cells were stained 24 h after LLO addition with crystal violet. C, Wild-type (Wt) or Stat1-deficient BMMs, pretreated or not with IFN-β, were exposed to LLO at the concentrations shown. LDH release was measured after 3 h of LLO treatment. D, With or without pretreatment with IFN-β, BMMs were exposed to 1.0 μg/ml LLO. One hour after addition, cells were harvested, stained with allophycocyanin-conjugated annexin V and 7AAD, and analyzed by FACS. The mean and SD (n = 3) of the percentage of total cells in each quadrant is indicated; the dot plot shows one representative result. Statistical significance is indicated, comparing the percentage of total cells for each quadrant between IFN-β-pretreated and not-pretreated samples.

Addition of the pan-caspase inhibitor z-VAD-fmk before LLO exposure led to only a small reduction in death, and oligonucleosomal DNA fragmentation (typical of caspase-mediated cell death) was detected only at the highest LLO concentration tested (1.0 μg/ml; data not shown). Cell death, therefore, may occur by both caspase-dependent and -independent mechanisms. The exposure of the membrane lipid phosphatidylserine (PS) on the outer leaflet of the cell membrane is associated with apoptosis, and can be monitored by assaying binding of its ligand annexin V. Combining this assay with monitoring membrane permeability with the nuclear dye 7-AAD, we saw a rapid increase (within the first hour) in the population of annexin V-positive cells (Fig. 1⇑D). Upon IFN-β pretreatment, more of this annexin V-positive population was also simultaneously 7-AAD positive. This pattern did not change at later time points. It suggests that IFN-β does not increase an apoptotic signal actuated by LLO.

Signal transduction by LLO differs from that stimulated by viable L. monocytogenes

Given the range of signaling pathways reported to be activated by LLO in various cell types, we examined which of these were triggered by LLO treatment of primary macrophages. We observed robust and rapid activation of p38MAPK at concentrations below those causing notable death (Figs. 2⇓A and 3⇓B). A truncated form of LLO, missing domain 4 and hence lacking cytolytic activity (32) did not produce this response. As this was purified in parallel and stored in the same buffer, this also indicates contaminating endotoxin is not responsible for the signaling seen. p38MAPK activation occurred after LLO addition in MyD88-deficient and TLR4-deficient cells. In contrast, early activation of p38MAPK by Listeria or LPS required MyD88 or TLR4, respectively (Fig. 2⇓, A and B). p38MAPK activation by Listeria was not dependent on LLO secretion, as activation was also seen upon infection with LLO-deficient bacteria. This is consistent with the demonstrated TLR2 dependence of early p38 activation upon Listeria infection (48), with TLR2 recognizing Listeria cell wall components (49). Partial MyD88 independence of p38MAPK activation was noted only at later stages of Listeria infection (Fig. 2⇓B). The MyD88-dependent activation of p38MAPK by Listeria, or TLR4-dependent activation by LPS, was maximal at 30 min, while a maximal signal was present already at 10 min after LLO treatment. TLR4 independence of LLO-induced signaling questions the reported role of TLR4 as an LLO receptor (21).

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FIGURE 2.

LLO treatment of BMMs induces p38 MAPK activation due to osmotic stress on the membrane, independent of MyD88 and TLR4. Early signaling induced by Listeria is dependent on MyD88 but not on TLR4. A, Wild-type (Wt), MyD88-deficient, or TLR4-deficient BMMs were treated as indicated with 0.2 μg/ml full-length or truncated LLO, or LPS (10 ng/ml or 10 pg/ml), or infected with either wt or LLO-deficient L. monocytogenes at a multiplicity of infection (MOI) of 10. Cells were harvested at 10 or 30 min time points and protein extracts analyzed by Western blot for phosphorylated p38 MAPK. B, Wt, MyD88-deficient, or TLR4-deficient BMMs were infected with Listeria at MOI 10. Cells were harvested at the time points indicated. Extracts were analyzed by Western blot for the phosphorylation of p38MAPK. C, BMMs were treated with 0.2 μg/ml LLO for 20 min, in medium containing 0, 5, or 10% dextran, and analyzed for p38MAPK activation as before. D, BMMs were treated with LLO at the concentrations shown. Where indicated, 10 μM SB203580 (inhibitor of p38MAPK) was added 30 min before LLO addition. LDH release was measured after 3 h of LLO treatment. E, BMMs were treated with 0.5 or 1.0 μg/ml LLO, or LPS (10 ng/ml or 10 pg/ml), or infected with L. monocytogenes at MOI 10. Cells were harvested after 3 h, and extracts were analyzed by Western blot for phosphorylation of Tyr⁷⁰¹ of Stat1, an indication of Stat1 activation, using Stat1α as a loading control. F, BMMs were treated with 1.0 μg/ml LLO, with or without IFN-β pretreatment, or LPS (10 ng/ml or 10 pg/ml), or infected with L. monocytogenes at MOI 10. Cells were harvested at the time points indicated. Extracts were analyzed by Western blot for the presence of IκBα, and phosphorylation of ERK1 (upper band) and ERK2 (lower band). G, BMMs were infected as for B, and analyzed by Western blot for the presence of IκBα, and phosphorylation of ERK1 (upper band) and ERK2 (lower band).

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FIGURE 3.

IFN-β pretreatment increases damage to the cell membrane induced by LLO. A, With or without IFN-β pretreatment, bone marrow macrophages (BMMs) were exposed to LLO at the concentrations indicated in medium containing 5 μg/ml propidium iodide. After 1 h, cells were harvested and analyzed for propidium iodide uptake by FACS. The division between dim (D) and bright (B) populations is shown in the upper histogram plots. In the plots below, not-pretreated cells are shown in gray. Cells pretreated with IFN-β are shown by the black line. B, Current flow due to pore formation in single cells upon LLO treatment was measured using the perforated patch clamp configuration. Illustrative original recordings at a holding potential of −30 mV are shown during control (upper trace), in presence of 0.5 μg/ml LLO (middle trace), and with 0.5 μg/ml LLO in IFN-β-pretreated macrophages (lower trace). Downward deflections represent channel openings, and C indicates the closed state of the pore. Single channel and multiple openings are seen in the recording of the IFN-β-pretreated cells. The horizontal and vertical calibration bars indicate 1 s and 25 pA, respectively. For further details, see text section describing the perforated patch clamp experiment. C, With or without IFN-β pretreatment, cells were harvested after 20 min LLO exposure and analyzed by Western blot for phosphorylation of p38. This was quantified by standardizing against p38 amount measured by Western blot. The extent of phosphorylation was expressed as a percentage setting the value for 0.1 μg/ml as 100%, and the appropriate untreated sample as 0%: this is indicated below the blots.

p38MAPK activation can be stimulated by treatment with detergents (17). Similarly, exposure of epithelial cells to pore-forming toxins results in osmotic stress-induced activation of p38MAPK (50). In line with these results, activation of the enzyme by LLO did not occur when cells were treated in medium containing dextran (Fig. 2⇑C), which offers protection against osmotic stress and hemolysis caused by toxin pore formation (51).

The p38MAPK pathway was shown to support cell survival when innate immune cells are exposed to bacterial products including LPS or anthrax lethal toxin (52, 53). We used the specific inhibitor SB203580 to test whether p38MAPK regulates the viability of cells treated with LLO. In agreement with a previous report for another cholesterol-dependent cytolysin, streptolysin O (54), p38MAPK inhibition did not influence the lytic action of LLO (Fig. 2⇑D).

Contrasting p38MAPK, other examined signaling pathways were not or only marginally stimulated by LLO. Tyrosine-phosphorylated Stat1, a readout for IFN production and signaling, was undetectable even at the highest concentration of LLO tested (Fig. 2⇑E). Weak activation of ERK2, and some slight degradation of IκBα, could be detected only when a high concentration of the toxin was used (Fig. 2⇑F and data not shown). These signals were far weaker than those induced by Listeria infection or LPS treatment, which both lead to IFN production, robust IκB degradation, and activation of both ERKs 1 and 2. As in the case of p38MAPK, IκB degradation or ERK activation by viable bacteria showed partial dependence on MyD88, particularly in the early phase of infection (Fig. 2⇑G).

IFN-β increases transient pore formation and the loss of plasma membrane integrity

Our previous results show that IFN-β increases the activity of cellular pathways contributing to Listeria-induced macrophage death (63). Results shown in Fig. 1⇑ suggest that IFN-I may additionally increase the damage caused to the infected cell by LLO. To further address this possibility, the amount of pore formation upon toxin treatment was assessed by adding toxin to cells with the membrane-impermeant nucleic acid stain propidium iodide present in the medium. Cells were harvested after 1 h and analyzed by FACS to measure propidium iodide uptake. A very bright population, labeled region B, increased in frequency with LLO concentration and with IFN-β pretreatment (Fig. 3⇑A). This corresponded to cells which had lost significant membrane integrity to allow propidium iodide to enter freely, and were counted as dead. The dim population, labeled region D, corresponded to living cells. This population displayed a small increase in mean fluorescence when toxin was added, compared with untreated cells, though remained distinct from the dead cell population. This is most likely due to temporary pore formation in the cell membranes allowing limited propidium iodide entry, but with later repair of these pores to prevent unlimited entry. Such limited entry has been previously described in the context of perforin-mediated damage (55). As expected, the mean fluorescence of this population increased with LLO concentration, as more pore formation could occur. Notably, IFN-β pretreatment resulted in a higher mean fluorescence than for nonpretreated cells at the same LLO concentration, suggesting it increases membrane damage suffered by cells upon exposure to LLO. Such a phenomenon would explain the increased death observed.

To further confirm these results, we measured ion flux from pore formation in macrophages exposed to LLO, with or without IFN-β pretreatment, using the perforated patch clamp technique to measure currents generated across the plasma membrane of one cell (Fig. 3⇑B). After a control period without electrical activity, LLO was added to the external solution bathing the cells and, after ∼1 min, single channel currents could be observed. The pore formation progressed rapidly and multiple openings occurred. At least three elementary pore current amplitudes of 16.5 ± 1.5, 32.0 ± 2.0, and 64.1 ± 6.2 pA were measured at a holding potential of −30 mV, being multiples of the smallest pore current amplitude. Pore openings were rare and occurred in bursts. At a concentration of 0.5 μg/ml, LLO caused channel openings only of small amplitude (n = 3). The current was markedly increased in IFN-β-pretreated macrophages (n = 3). Multiple openings were observed, which were not seen in not-pretreated cells under the same experimental conditions. At 1.0 μg/ml LLO (n = 2), obviously more pores were incorporated into the cell membrane as multiple openings were frequently seen, and again membrane current was larger in IFN-β-pretreated cells (n = 3) (data not shown). The frequency of pore openings slowly decreased after ∼10 min.

Quantification of p38MAPK activation by low concentrations of LLO showed greater activation in IFN-β-pretreated cells compared with nonpretreated for the same concentration (Fig. 3⇑C). As this is dependent on membrane permeabilization (Fig. 2⇑C), increased activation suggests greater membrane damage.

Caspase 1 activation by LLO is enhanced in IFN-β-stimulated macrophages, but makes no contribution to the lytic activity of LLO

The action of pore-forming agents, such as nigericin and aerotoxin, can act as a trigger for inflammasome activation through decreasing intracellular potassium levels (56, 57, 58), and it is suggested that LLO may be important in activation of the inflammasome during Listeria infection (59). Low levels of cleavage of caspase 1 were apparent upon LLO addition to IFN-β-pretreated cells, but not detectable in those not pretreated (Fig. 4⇓A). In this experiment, we also observed that protein expression of full-length caspase 1 was greater upon IFN-β pretreatment. Up-regulation of caspase 1 gene expression in a Stat1-dependent manner was shown by real-time PCR (Fig. 4⇓B). Likewise, IFN-β increased the expression of another potential inflammasome component, caspase 11. By contrast, expression of apoptosis-associated speck-like protein containing a CARD, a key inflammasome adapter protein, remained unchanged (Fig. 4⇓C). Caspase 1 has been linked to cell death caused by infection with Salmonella or Shigella. However, we saw no difference in LLO-induced cell death, or in the up-regulation of death by IFN, in caspase 1-deficient macrophages when compared with wild type (Fig. 4⇓, D and E).

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FIGURE 4.

Caspase 1 is cleaved upon LLO addition to bone marrow macrophages (BMMs) pretreated with IFN-β. Expression of caspases 1 and 11 is up-regulated by IFN-β. LLO-induced death is not dependent on caspase 1. A, BMMs, pretreated or not with IFN-β, were exposed to 1.0 μg/ml LLO and harvested at the time points shown. As a control, BMMs were infected at a multiplicity of infection (MOI) of 25 with S. typhimurium. Protein extracts were analyzed by Western blot, probing with an Ab that recognizes both full-length caspase 1and the p10 product of cleavage. A far stronger signal was seen for full-length caspase 1 than for the cleaved product: a short exposure after probing with an HRP-labeled secondary Ab is shown to illustrate levels of this protein. B and C, Wild-type (Wt) or Stat1-deficient BMMs were treated with IFN-β for 16 h at the concentrations shown. Total RNA was extracted, reverse transcribed, and analyzed by real-time PCR or RT PCR for expression of the genes indicated. Inducibility was calculated by comparison with the appropriate untreated sample. D and E, Wt or caspase 1-deficient BMMs, pretreated or not with IFN-β, were exposed to LLO at the concentrations shown. After 24 h, surviving adherent cells were stained with crystal violet (D). Alternatively, LDH release into the medium was measured after 3 h of incubation (E). The background LDH release from untreated cells is subtracted from the values shown. The degree of statistical significance is indicated, comparing IFN-β-pretreated and not-pretreated cells of each genotype.

Up-regulation of lipogenesis pathways (via caspase 1 activation) is reported to promote cell survival in response to aerolysin (58), presumably by promoting membrane repair. Interestingly, we observed a considerable down-regulation by IFN-β of HMG CoA reductase and fatty acid synthase (Fig. 5⇓A), two genes involved in lipogenesis, whose up-regulation was linked to cell survival after aerolysin treatment. The basal levels of expression in Stat1-deficient cells were reduced, but the decrease seen was Stat1 dependent (Fig. 5⇓B). Unlike for aerolysin, we did not observe up-regulation of these genes upon LLO treatment, with or without IFN-β pretreatment (data not shown). This corresponds with the comparatively low level of caspase 1 activation observed in our system, and may be attributed to a difference in toxin, cell type, or species.

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FIGURE 5.

IFN-β down-regulates genes linked to lipogenesis. A, Bone marrow macrophages (BMMs) were treated with IFN-β for 16h at the concentrations shown. Expression of the genes indicated was analyzed by real-time PCR as described before, compared with the levels in untreated cells. B, Wild-type (Wt) or Stat1-deficient BMMs were treated with 500 U/ml IFN-β for 8 h and gene expression analyzed as before.