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. 2016 Nov 4:7:484.
doi: 10.3389/fimmu.2016.00484. eCollection 2016.

Neutrophils Discriminate between Lipopolysaccharides of Different Bacterial Sources and Selectively Release Neutrophil Extracellular Traps

Elmar Pieterse  1 , Nils Rother  1 , Cansu Yanginlar  1 , Luuk B Hilbrands  1 , Johan van der Vlag  1
Affiliations

Neutrophils Discriminate between Lipopolysaccharides of Different Bacterial Sources and Selectively Release Neutrophil Extracellular Traps

Elmar Pieterse et al. Front Immunol. .

Abstract

The release of neutrophil extracellular traps (NETs), either during "suicidal" or "vital" NETosis, represents an important strategy of neutrophils to combat Gram-negative bacteria. Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, is a reported stimulus for NET formation. Although it is widely acknowledged that the structural diversity in LPS structures can elicit heterogeneous immune responses, species- and serotype-specific differences in the capacity of LPS to trigger NET formation have not yet been investigated. In the present study, we compared the NET-inducing potential of LPS derived from Escherichia coli (serotypes O55:B5, O127:B8, O128:B12, O111:B4, and O26:B6), Salmonella enterica (serotype enteritidis), and Pseudomonas aeruginosa (serotype 10), under platelet-free and platelet-rich conditions in vitro, and in whole blood ex vivo. Here, we demonstrate that under serum- and platelet-free conditions, mimicking tissue circumstances, neutrophils discriminate between LPS of different bacterial sources and selectively release NETs only in response to LPS derived from E. coli O128:B12 and P. aeruginosa 10, which both induced "suicidal" NETosis in an autophagy- and reactive oxygen species (ROS)-dependent, but TLR4-independent manner. Intriguingly, in whole blood cultures ex vivo, or in vitro in the presence of platelets, all LPS serotypes induced "vital" NET formation. This platelet-dependent release of NETs occurred rapidly without neutrophil cell death and was independent from ROS formation and autophagy but required platelet TLR4 and CD62P-dependent platelet-neutrophil interactions VSports手机版. Taken together, our data reveal a complex interplay between neutrophils and LPS, which can induce both "suicidal" and "vital" NETosis, depending on the bacterial origin of LPS and the presence or absence of platelets. Our findings suggest that LPS sensing by neutrophils may be a critical determinant for restricting NET release to certain Gram-negative bacteria only, which in turn may be crucial for minimizing unnecessary NET-associated immunopathology. .

Keywords: NETosis; cell death; lipopolysaccharides; neutrophil extracellular traps; platelets V体育安卓版. .

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Figures

Figure 1
Figure 1
Neutrophils selectively release NETs in response to LPS structures. (A) LPS-O128 and LPS-PA stimulate neutrophils to release NETs after 180 min of incubation in platelet-free cell cultures (left panel), as measured by fluorometry. The release of DNA by LPS-O128 and LPS-PA coincides with extracellular activity of neutrophil elastase (right panel). (B) NET release in response to LPS-O128 and LPS-PA was confirmed by immunofluorescence microscopy, in which DNA was stained with 100 nM Sytox Orange (yellow). Both “spread” NETs (red arrows) and “diffused” NETs (white arrows) were observed. NET release in response to 100 nM PMA served as positive control. LPS was used at a concentration of 8 pg LPS per neutrophil. Scale bar: 30 μm. *p < 0.05 and ***p < 0.001, when compared to control. Data represent mean values ± SEM of at least three experiments.
Figure 2
Figure 2
LPS-O128 and LPS-PA induce NETosis when present above a threshold value. (A) LPS-O128 and LPS-PA induce NET release only at high LPS concentrations of 8 pg LPS per neutrophil (PMN). (B) Priming of neutrophils for 1 h with recombinant TNF-α (10 ng/ml), IL-6 (10 ng/ml), IFN-α (100 ng/ml), or a mixture of all, does not promote NET release nor prime neutrophils for LPS-induced NETosis by LPS-O111 (bottom left), LPS-O128 (top middle), and LPS-PA (bottom middle) at a concentration of 6 pg LPS per neutrophil (PMN). In these graphs, NETosis induced by LPS-O128 and LPS-PA at 8 pg LPS per neutrophil is shown in dark as positive controls. Cytokines do not enhance NETosis by LPS-O128 or LPS-PA at LPS concentrations of 8 pg LPS per neutrophil (top and bottom right). Quantifications of NET release were performed by fluorometry, as outlined. *p < 0.05, **p < 0.01, and ***p < 0.001, when compared to control. Data represent mean values ± SEM of at least three experiments.
Figure 3
Figure 3
LPS-PA and LPS-O128 induce ROS- and autophagy-dependent “suicidal” NETosis. (A) DNA release in response to 100 nM PMA, LPS-O128, or LPS-PA was monitored by fluorometry using 100 nM Sytox Orange during an incubation period of 5 h. (B) NETosis induced by LPS-O128 and LPS-PA can be inhibited by 5 μM wortmannin (inhibitor of autophagy) or 40 μM diphenyleneiodonium (DPI; inhibitor of ROS), but not by anti-TLR2 and anti-TLR4 neutralizing antibodies (5 μg/ml). (C) The inhibitory effects of wortmannin and DPI on NET release by LPS-PA were confirmed by immunofluorescence microscopy, in which DNA was stained with 100 nM Sytox Orange (yellow). Immunofluorescence imaging also confirmed that anti-TLR2 and anti-TLR4 neutralizing antibodies did not prevent NETosis induced by LPS-PA. LPS was used at a concentration of 8 pg LPS per neutrophil. Scale bar: 30 μm. **p < 0.01 and ***p < 0.001, when compared to control, $p < 0.05 and #p < 0.01, when compared to LPS-PA alone. Data represent mean values ± SEM of at least three experiments.
Figure 4
Figure 4
All LPS structures induce “vital” NETosis in the presence of platelets. (A) Typical extracellular DNA filaments (white arrows) were observed in whole blood cultures ex vivo after 180 min of incubation with the different LPS serotypes. (B) Platelets (panel 2) or LPS-O111 (panel 3) alone does not induce NETosis after 180 min of incubation with neutrophils, whereas the combination of both (panel 4) stimulates NET formation without neutrophil (lytic) cell death. Massive neutrophil cell death is observed in response to LPS-PA alone (panel 5), based on the failure to exclude the vital dye Sytox Orange (yellow), which can be largely prevented by the addition of platelets (panel 6). LPS was used at a concentration of 8 pg LPS per neutrophil. Notably, representative light microscopy images are shown to visualize neutrophil morphology after stimulation and do not correspond in terms of “field of view” to the adjacent representative immunofluorescence images. Scale bars: white = 20 μm and red = 40 μm.
Figure 5
Figure 5
“Vital” NETs lack proteolytic active myeloperoxidase and elastase. (A) “Vital” NETs induced by platelets exposed to LPS-PA, as well as “suicidal” NETs induced by LPS-PA alone, stain positive for both myeloperoxidase (MPO) and neutrophil elastase (NE). Note the highly refined architecture of thinly interwoven DNA filaments of “vital” NETs when compared to “suicidal” NETs. Also note (right panels, inserts) the granular and intact neutrophil phenotype (i.e., lobulated nuclei) for “vital” NETs (white arrows) when compared to the altered neutrophil phenotype (i.e., decondensed chromatin) for “suicidal” NETs (blue arrows). (B) “Vital” NETs induced by platelets exposed to LPS-PA lack proteolytic active myeloperoxidase (MPO) and neutrophil elastase (NE) when compared to “suicidal” NETs induced by LPS-PA alone. For these assays, NETs were isolated through digestion with micrococcal nuclease and normalized on the basis of DNA content in NET-containing supernatants. Scale bars: white = 40 μm and red = 20 μm. ***p < 0.001, when compared to control. Data represent mean values ± SEM of at least three experiments.
Figure 6
Figure 6
Platelet-mediated “vital” NETosis requires platelet TLR4 and CD62P. (A,B) “Vital” NETosis induced by LPS-O111 and platelets is insensitive to inhibition by 5 μM wortmannin (inhibitor of autophagy) or 40 μM diphenyleneiodonium (DPI; inhibitor of ROS) but can be prevented by pretreatment of platelets with anti-TLR4 neutralizing antibodies (5 μg/ml). Representative images are merged pictures of extracellular DNA (yellow, as stained with 100 nM Sytox Orange) and neutrophils (brightfield channel). The images show abundant NETs despite exclusion of Sytox Orange by neutrophils, indicating cell death-independent NET release (i.e., “vital” NETosis). Quantification of NET release (B) was performed by fluorometry, as outlined. (C) PKH26-labeled platelets (red) stimulated with LPS-PA form aggregates with neutrophils (NE, neutrophil elastase) after 30 min of incubation. This aggregate formation is inhibited by anti-CD62P-neutralizing antibodies. (D) Flow cytometry analysis confirms the formation of platelet–neutrophil aggregates, since cells within the predefined neutrophil gate increase in size (FSc, forward scatter) and stain positive for PKH26-labeled platelets after LPS-PA stimulation. (E) Anti-CD62P-neutralizing antibodies decrease platelet-mediated “vital” NETosis induced by LPS-PA, as measured by fluorometry. LPS was used at a concentration of 8 pg LPS per neutrophil. Scale bars: red = 100 μm; white = 30 μm; yellow = 10 μm. *p < 0.05 and ***p < 0.001, when compared to control, where not indicated. Data represent mean values ± SEM of at least three experiments.
Figure 7
Figure 7
Differential regulation of LPS-induced NET release under platelet-free and platelet-rich circumstances. Under serum- and platelet-free conditions, mimicking tissue circumstances, LPS-PA and LPS-O128 trigger ROS- and autophagy-dependent “suicidal” NET release in extravasated neutrophils, whereas other LPS structures (LPS-O26, LPS-O55, LPS-SE, LPS-O127, and LPS-O111) do not. In the presence of platelets, mimicking blood circumstances, neutrophils do no longer discriminate between LPS structures and release “vital” NETs in response to all LPS structures.
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