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CpG motifs in bacterial DNA delay apoptosis of neutrophil granulocytes

Research Center, Maisonneuve-Rosemont Hospital and Department of Medicine, University of Montréal, Montréal, QC, Canada

²Correspondence: Research Center, Maisonneuve-Rosemont Hospital, 5415 boulevard de l’Assomption, Montréal, Québec, Canada H1T 2M4. E-mail: janos.g.filep{at}umontreal.ca

SPECIFIC AIMS

Neutrophil granulocytes die rapidly and their survival is contingent upon rescue from programmed cell death by signals from the environment. We investigated the impact of bacterial DNA, containing unmethylated CpG motifs (CpG-DNA), on human and rat neutrophil apoptosis and studied the underlying molecular mechanisms.

PRINCIPAL FINDINGS

1. Human neutrophils express toll-like receptor-9 (TLR-9)
Western blot analysis of lysates from high-purity (>99.9%) human neutrophils revealed the presence of TLR-9 in neutrophils, which was further confirmed by flow cytometry using permeabilized neutrophils and an R-PE-tagged antibody.

2. CpG-DNA prolongs neutrophil survival by delaying apoptosis
Isolated neutrophils developed prominent features of apoptosis, including loss of membrane asymmetry (assessed by annexin V binding), chromatin condensation, and internucleosomal cleavage of DNA, resulting in hypodiploid nuclei, within 24 h of culture. CpG-DNA concentration-dependently (EC₅₀ value:170-320 ng/mL) suppressed the development of apoptotic morphology and increased neutrophil viability (Fig. 1 ). The apparent maximum inhibition that can be achieved with CpG-DNA was similar to that seen with lipopolysaccharides (LPS). Calf thymus DNA or methylated Escherichia coli DNA (Fig. 1) was without detectable effect.

View larger version (50K): [in this window] [in a new window]   Figure 1. CpG-DNA delays apoptosis of human neutrophils. a) Morphologic features of neutrophils maintained in suspension culture. Cytospin preparations of neutrophils were stained immediately after isolation of neutrophils (0 h) or after culture for 24 h in the absence (untreated) or presence of CpG-DNA, calf thymus DNA (both at 1.6 µg/mL) or LPS (1 µg/mL). b, c) Kinetic analysis and concentration dependence of the effects of CpG-DNA on neutrophil viability and development of apoptotic morphology. Aliquots of neutrophils were stained with propidium iodide (to assess viability) or with annexin V, or processed for nuclear DNA content analysis. d) Neutrophils were cultured for 24 h at 37°C in the absence (untreated) or presence of CpG, calf thymus or methylated bacterial DNA (met-CpG-DNA) (all at 1.6 µg/mL) or LPS (1 µg/mL), then stained with propidium iodide or annexin V. Results are the mean ± SE for 5–8 experiments using neutrophils from different blood donors. *P < 0.05; **P < 0.01; ***P < 0.001 (compared with untreated). e) CpG-DNA attenuates chromatin cleavage in neutrophils maintained in culture for 24 h. Lane 1, DNA kilobase marker standards, and values for selected standards are shown on the left margin; lane 2, unstimulated (control); lane 3, CpG-DNA, 400 ng/mL; lane 4, CpG-DNA, 800 ng/mL; lane 5, CpG-DNA, 1.6 µg/mL; lane 6, LPS, 1 µg/mL). The experiments were repeated 3 times.

3. Endosomal acidification inhibitors block the anti-apoptotic action of CpG-DNA
Inhibition of endosomal acidification of CpG-DNA with bafilomycin A, chloroquine, and monensin, which have different mechanisms of action, almost completely reversed the apoptosis suppressing action of CpG-DNA, indicating a pH-dependent step in CpG-DNA signaling. None of these inhibitors showed detectable effects on LPS suppression of neutrophil apoptosis.

4. CpG-DNA induces phosphorylation of BAD via MAPK kinase/ERK and PI 3-kinase/Akt signaling pathways
CpG-DNA induced transient, concentration-dependent phosphorylation of ERK 1/2 and Akt, reaching a peak within 15 min. Neither the MAPK/ERK kinase inhibitor PD98059 nor the PI 3-kinase inhibitor wortmannin alone affected neutrophil apoptosis, whereas the p38 MAPK inhibitor SB203580 significantly decreased the percentage of apoptotic cells. Both PD98059 and wortmannin effectively, though never completely, blocked the apoptosis-delaying action of CpG-DNA, and their inhibitory effects were not additive. CpG-DNA plus SB203580 resulted in suppression of neutrophil apoptosis similar to those observed with either CpG-DNA or SB203580 alone. CpG-DNA induced phosphorylation of BAD at Ser112 and Ser136. PD98059 inhibited CpG-DNA-induced phosphorylation at Ser112, but not at Ser136; wortmannin blocked phosphorylation at Ser136 without affecting phosphorylation at Ser112.

5. CpG-DNA inhibits disruption of mitochondrial transmembrane potential (_m), cytochrome c release and activation of caspase-3
Culture of neutrophils for 4 and 22 h was associated with disruption of _m and release of cytochrome c, which was partially inhibited by CpG-DNA (Fig. 2 a–d) but not by calf thymus DNA. The inhibitory action of CpG-DNA was concentration dependent and the apparent maximum inhibition that could be achieved with CpG-DNA was comparable to that of 1 µg/mL LPS (Fig. 2a) . Caspase-3 activity was barely detectable in freshly isolated neutrophils. In vitro culture of neutrophils for 24 h resulted in marked increases in caspase-3 activity, which was reduced by CpG-DNA (EC₅₀ value: 380 ng/mL) (Fig. 2e ). Both PD98059 and wortmannin attenuated the caspase-3 inhibitory action of CpG-DNA, though complete reversal was not achieved, not even in the presence of both PD98059 and wortmannin (Fig. 2f ).

View larger version (30K): [in this window] [in a new window]   Figure 2. CpG-DNA attenuates disruption of m, cytochrome c release, and activation of caspase-3. Neutrophils were incubated with or without CpG-DNA, then aliquots of 105 cells were incubated for 15 min with JC-1 (10 µg/mL) and analyzed by cytoflourometry. a) Representative plots. Percentages on representative plots reflect the reduction in m; 0 h refers to staining immediately after isolation of neutrophils. The effect of LPS (1 µg/mL) is shown for comparison. b) Time dependency of the effect of CpG-DNA (1.6 µg/mL) on m. c) Concentration-dependent effects of CpG-DNA on m assessed after 8 h incubation. The results are mean ± SE of 6 experiments. d) Mitochondrial and cytosolic cytochrome c levels in freshly isolated (0 h) neutrophils and in neutrophils cultured for 24 h in the absence or presence of CpG-DNA (1.6 µg/mL) or LPS (1 µg/mL). Results are mean ± SE of 4–7 experiments. *P < 0.05; **P < 0.01. e) Concentration-dependent inhibition of caspase-3 activity. Caspase-3 activity was determined using Ac-DEVD-AMC as a substrate and expressed as fluorescence units (FU). No fluorescence was detected in the presence of Ac-DEVD-aldehyde, an inhibitor of caspase-3 activity. f) Reversal of the CpG-DNA effect on caspase-3 activity by PD98059 (50 µM) and wortmannin (100 nM). Values are the mean ± SE of 5 independent experiments. *P < 0.05; **P < 0.01 (vs. untreated); #P < 0.05 (vs. CpG DNA-treated)

6. CpG-DNA inhibits neutrophil apoptosis in rats
To investigate whether CpG-DNA could suppress neutrophil apoptosis in vivo, we injected CpG-DNA into conscious rats. Since neutrophils are removed from the circulation upon expression of phosphatidylserine on their surface (an early sign of apoptosis), we collected blood 60 min postinjection of CpG-DNA, isolated neutrophils, and studied apoptosis ex vivo. Injection of CpG-DNA (200 µg/kg) evoked transient 15 to 29 mmHg decreases in mean arterial blood pressure and a modest neutrophilia, whereas calf thymus DNA was without detectable effects. Neutrophils isolated from CpG-DNA-treated rats showed a higher percentage of viability and a lower percentage of annexin V positivity than neutrophils prepared from rats treated with calf thymus DNA or vehicle. Ex vivo culture of isolated neutrophils from vehicle or calf thymus DNA-treated rats resulted in similar time-dependent reductions in cell viability and development of apoptotic morphology. By contrast, significantly fewer neutrophils showed signs of apoptosis in CpG-DNA-treated group even after 20 h culture than in the vehicle- or calf thymus DNA-treated group.

CONCLUSIONS AND SIGNIFICANCE

Progression to apoptosis appears to be the normal default state for circulating neutrophils, whereas prolonged neutrophils survival is required for excessive leukocyte trafficking into inflamed tissues. Neutrophil survival is contingent upon rescue from programmed cell death by signals from the environment. Here we report that a novel signal for delaying neutrophil apoptosis is bacterial DNA, and unmethylated CpG motifs in particular.

Consistent with the commitment of neutrophils to apoptosis, CpG-DNA delayed, rather than blocked, apoptosis, resulting in prolonged neutrophil survival as demonstrated both in vitro and ex vivo after injection of CpG-DNA into rats. Our results indicate that differences in methylation patterns would enable neutrophils to recognize bacterial DNA, because methylation of cytosines in CpG dinucleotides in bacterial DNA completely abolished its apoptosis suppressing activity and calf thymus DNA did not reproduce the effects of bacterial DNA.

Pharmacological inhibition of endosomal acidification rendered the apoptotic machinery unresponsive to CpG-DNA, but not to LPS. Endosomal acidification of CpG-DNA is thought to be necessary to initiate intracellular signaling through binding to TLR-9. Human neutrophils have been reported to express TLR-9 mRNA and protein. Our analysis of high-purity (>99.9%) neutrophils confirms the presence of TLR-9. CpG-DNA induces concurrent phosphorylation of ERK and Akt (the target of PI 3-kinase). We found that pharmacological inhibition of either MAPK/ERK kinase or PI 3-kinase markedly attenuated, though never fully reversed (even in combination), the apoptosis-delaying action of CpG-DNA, indicating that ERK and Akt work in concert to delay neutrophil apoptosis. Spontaneous neutrophil apoptosis is associated with p38 MAPK-mediated activation of caspase-3. Conversely, inhibition of p38 MAPK partially rescued neutrophils from apoptosis. However, our data indicate that neither ERK nor Akt interferes with p38 MAPK signaling. Indeed, these pathways converge on BAD, a member of the Bcl-2 family. We observed CpG-DNA-induced phosphorylation of BAD at Ser112 and Ser136 through activation of ERK and Akt, respectively. Phosphorylated BAD then dissociates from Bcl-2, thereby enhancing the anti-apoptotic effects of the Bcl-2 family proteins, including prevention of loss in _m. We found that CpG-DNA can partially prevent collapse of _m and cytochrome c release in neutrophils undergoing spontaneous apoptosis. Moreover, the degree of CpG-DNA reduction of neutrophil apoptosis and inhibition of mitochondrial membrane perturbation was similar. Preservation of _m and mitochondrial cytochrome c by CpG-DNA is consistent with decreased caspase-3 activation, as detected in this study. Thus, a major mechanism by which CpG-DNA inhibits the neutrophil intrinsic pro-apoptotic machinery is ERK and PI 3-kinase-dependent prevention of mitochondrial dysfunction and repression of caspase-3 activity (Fig. 3 ).

View larger version (28K): [in this window] [in a new window]   Figure 3. Proposed mechanism for the apoptosis delaying action of bacterial DNA in human neutrophils. m, mitochondrial transmembrane potential. Broken arrows indicate yet undefined pathways.

The present findings could have a profound effect on the way we think about neutrophil activation and survival. If bacterial DNA is proinflammatory as indicated by the present and previous studies, then simply killing bacteria may not be sufficient to counter inflammation. Indeed, DNA from dead bacteria would be still present, as was shown in the sputum of cystic fibrosis patients, and could contribute to persistent inflammation. Our results provide evidence that nonmethylated CpG motifs in bacterial DNA promote neutrophil survival by suppressing the intrinsic apoptotic machinery both in vitro and ex vivo and underscore the importance of bacterial constituents other than LPS in the regulation of neutrophil functions.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2048fje;

¹ L.J. and T.K. contributed equally to this work.

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