Novel anti-bacterial activities of β-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation

Abstract

Human β-defensins (hBD) are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus), forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET) formation by target polymorphonuclear leukocytes (PMNs), which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Platelets sequester S. aureus.

Cultured S. aureus were incubated in the presence or absence of platelets (240 minutes). At the end of this period, an equal volume of fixative was added to the suspension and the samples were prepared for confocal (A) or transmission electron (B) microscopy. (A) The left panel shows S. aureus (magenta - stained with topro-3) only. The middle panel displays the pattern of S. aureus (magenta) growth in the presence of platelets, which are not visible in this panel but shown in an overlay that is displayed in the far right panel (bacteria, magenta; platelets, green – stained with phalloidin). Scale bars = 5 µm. (B) Transmission electron microscopy of S. aureus in the presence (right panel) or absence (left panel) of platelets. Scale bars = 10 µm. (C) Differential interference contrast (left panel) and transmission electron (right panel) microscopy of clusters of S. aureus (arrows) surrounded by platelets. Scale bars = 5 µm. The panels in figure 1 are representative of three to four independent experiments. Sa, S. aureus; P, platelets.

Figure 2. Platelets impede the growth of S. aureus.

Cultured S. aureus were incubated in the presence or absence of platelets for 240 minutes and bacterial growth was then measured as described in Materials and Methods. (A) The blood agar plates are representative examples of four independent studies. (B) The bars in the lower panel represent the mean±SEM of the fold increase in S. aureus growth (240 minutes) in the presence or absence of platelets over baseline (horizontal line). The single asterisk indicates a significant increase (p<0.05) in growth over baseline. The double asterisks indicates a significant (p<0.05) reduction in S. aureus growth in the presence of platelets when compared to S. aureus growth by itself.

Figure 3. Platelets express and release β-defensin 1 in response to α-toxin.

(A) The curves show real-time PCR results for integrin αIIb and each of the defensin family members in platelets using the same template RNA. The inset shows semi-quantitative PCR for hBD-1, -2 and -3 in platelets and HeLa cells. This figure is representative of three independent experiments. (B) Immunocytochemical analysis of hDB-1 (green) and WGA in unstimulated platelets. Inset (top row, hBD-1): area selected for enlargement and merge. This figure is representative of five independent experiments. Scale bars = 5 µm. (C) hBD-1 protein in lysates and supernatants from platelets that were stimulated with α-toxin (50 ng/ml), thrombin (0.1 U/ml), TRAP (20 µM), or PAF (100 nM) for 30 minutes. The bars represent the mean±SEM of three independent experiments.

Figure 4. Platelet-derived β-defensin 1 inhibits the growth of S. aureus.

(A) S. aureus growth was determined in the presence of recombinant hBD-1 (400 ng/ml) or its vehicle. The bars represent the mean±SEM of the fold increase in S. aureus growth (240 minutes) over baseline (horizontal line). The single asterisk indicates a significant increase (p<0.05) in growth over baseline. The double asterisks indicates a significant (p<0.05) reduction in S. aureus growth in the presence of recombinant hBD-1 when compared to S. aureus incubated with its vehicle. (B) S. aureus growth in the presence of immunoprecipitates that were captured from platelet lysates incubated with anti-hBD-1 or its control IgG. The bars represent the mean±SEM of the fold increase in S. aureus growth (240 minutes) over baseline (horizontal line). The single asterisk indicates a significant increase (p<0.05) in the growth of S. aureus that was treated with IgG immunoprecipitates over baseline. The double asterisks indicates a significant (p<0.05) reduction in S. aureus growth in the presence of anti-hBD-1 immunoprecipitates when compared to IgG immunoprecipitates. (C) The growth of S. aureus in the absence or presence of platelets that were co-incubated with a neutralizing antibody against hBD-1 or its IgG control. The bars represent the mean±SEM of the fold increase in S. aureus growth (240 minutes) over baseline (horizontal line). The single asterisk indicates a significant increase (p<0.05) in the growth of S. aureus alone or S. aureus co-incubated with platelets and anti-hBD-1 over baseline. The double asterisks indicates a significant (p<0.05) reduction in S. aureus growth in the presence of platelets incubated with control IgG when compared to S. aureus alone or S. aureus co-incubated with platelets and anti-hBD-1.

Figure 5. β-defensin 1 induces NET formation by PMNs.

(A) NET formation in the presence of control immunoglobulin (left panel) or hBD-1 (right panel) immunoprecipitates that were captured from human platelets. The NETs (arrows) were detected by live cell imaging as previously described. (B) NET formation in untreated PMNs (control) or PMNs incubated with 100 ng/ml of recombinant hBD-1 alone or in the presence of anti-hBD-1 or its control IgG. Figure 5A and 5B are representative of three independent experiments.

Figure 6. β-defensin 1, but not other β-defensin family members, induce PMNs to form NETs.

PMNs were left untreated (control) or incubated with 100 ng/ml of recombinant hBD-1, hBD-2, or hBD-3. After 60 minutes, NETs (arrows) were detected by live cell imaging as previously described. Increasing concentrations of hBD-2 or hBD-3 failed to induce NET formation (data not shown). Images are representative of three independent experiments.

Figure 7. β-defensin 1 mediated NET formation is dependent on reactive oxygen species.

(A) PMNs were incubated with diphenylene iodonium (DPI) and then left untreated (control) or stimulated with 100 ngl/ml of LPS or hBD-1 for 1 hour. Images are representative of three independent experiments. (B) PMNs were left alone or treated with 100 ng/ml of LPS or recombinant hBD-1 for 1 hour and then stained with trypan blue to determine cell viability. (C) PMNs were left alone or incubated with 100 ng/ml of LPS or recombinant hBD-1. After 1 hour, neutrophil elastase activity was measured. The bar graph represents the percent increase in neutrophil elastase activity over untreated neutrophils. The bars in panel B and C represent the mean±SEM of three independent experiments.