Laminin Triggers Neutrophil Extracellular Traps (NETs) and Modulates NET Release Induced by Leishmania amazonensis

Abstract

Neutrophils are recruited from the blood and transmigrate through the endothelium to reach tissues, where they are prone to respond through different mechanisms, including the release of neutrophil extracellular traps (NETs). These responses occur in close contact with proteins from the basement membrane and extracellular matrix, where laminins are abundant. Thus, we investigated the interactions between neutrophils and different laminin (LM) isoforms and analyzed the induction of NETs. We showed that neutrophils stimulated with LM isoforms 111, 211, 332, 411, 421, and 511 released NETs. The same occurred when neutrophils interacted with polymerized LMs 111, 411, and 511. LM-induced NETs were partially inhibited by pretreatment of neutrophils with an anti-α6 integrin antibody. Furthermore, NETs triggered by laminins were dependent on elastase and peptidylarginine deiminase (PAD)-4, enzymes that participate in chromatin decondensation. We also found that LMs 411 and LM 511 potentiated the NET release promoted by promastigotes of the protozoan parasite Leishmania, and that NETs stimulated by LMs alone display leishmanicidal activity. The ability of LM to induce NET release may have potential implications for the course of inflammation or infection.

Keywords: Leishmania amazonensis; extracellular matrix; laminin; neutrophil extracellular traps (NET).

Figure 1

Laminin isoforms 111, 411, and 511 induce NET release. Neutrophils were incubated with the LM isoforms 111 (A–D), 411 (F–I), and 511 (K–N) at 1 µg/mL for 90 min. The cells were then fixed and stained for detection of DNA (with DAPI) (A,F,K), elastase (B,G,L), and LM isoforms 111, 411, and 511 (C,H,M). Enlarged images show merged staining for LMs 111 (D), 411 (I), and 511 (N). Bars: 20 µm. NETs in neutrophil culture supernatants stimulated with LMs 111 (E), 411 (J), and 511 (O) for 90 min were quantified using PicoGreen. The data were normalized according to spontaneous release of DNA (Nil) and are represented as the mean ± SEM of 4 experiments (E,J,O). * p < 0.05; ** p < 0.004. *** p < 0.001.

Figure 2

Neutrophils express the α6 integrin chain (CD49f/VLA-6), and NETs are released as a result of recognition of LM isoforms by this integrin. Neutrophils were incubated with anti-α6 chain antibody (GOH3) or the isotype control (2 µg/mL) for 20 min at 4 °C, and CD49f expression was evaluated. The data are shown as the mean ± SEM of the percentage of CD49f+ neutrophils (A) in samples from six different donors. Neutrophils were either untreated (Nil) or treated with anti-α6 integrin antibody (GoH3) for 20 min and then incubated with LMs 111, 411, and 511 for 60 min. NETs in culture supernatants were quantified using PicoGreen (B). The data were normalized according to spontaneous release of DNA (Nil) from unstimulated neutrophils and are presented as the mean ± SEM of 7 (LM-111), 10 (LM-411) and 8 (LM-511) donors. *** p < 0.001; * p < 0.05.

Figure 3

Involvement of Elastase and PAD4 in NET induction by the LM isoforms 111, 411, and 511. Neutrophils were either untreated (Nil) or treated for 30 min with elastase inhibitor (E.I.–A) or PAD-4 inhibitor, chloroamidine (Cl-A–B) and then incubated with LMs 111, 411, and 511 in suspension for 60 min. NETs in culture supernatants were quantified using PicoGreen. The data were normalized according to spontaneous release of DNA from unstimulated neutrophils (Nil) and are presented as the mean ± SEM of 9, 3, and 3 donors for LMs 111, 411, and 511, respectively (A); 8, 4, and 4 donors for LMs 111, 411, and 511, respectively (B). The data from PMA, which were used as a control, are from 4 donors (B) * p < 0.05.

Figure 4

Laminin in solution or adsorbed onto plates induces NET release. Neutrophils were incubated with the indicated LM isoforms in suspension (A) or on coated plates (B) for 90 min, and NETs were quantified in culture supernatants using PicoGreen. The data were normalized according to spontaneous (Nil) release of DNA and are presented as the mean ± SEM of 7 (A) and 3 donors (B) for each isoform. * p < 0.05; ** p < 0.004.

Figure 5

Polymerized laminins (polyLM) 111, 411, and 511 trigger NET release. Neutrophils were incubated on polyLM 111, 411, and 511 (50 μg/mL)-coated plates for 90 min and then fixed and stained for histone (A,F,K); polyLM 111, 411, and 511 (B,G,L); and DNA (C,H,M). Enlarged images show merged staining for polyLM 111 (D), 411 (I), and 511 (N). Bars: 20 µm. NETs in the culture supernatants of neutrophils stimulated with polyLM 111 (E), 411 (J), and 511 (O) for 90 min were quantified using PicoGreen. The data were normalized according to spontaneous release of DNA (Nil) and are represented as the mean ± SEM of 4 experiments (E,J,O). * p < 0.05.

Figure 6

LMs 111, 411, and 511 modulate NETs induced by Leishmania amazonensis (La). Neutrophils were incubated with or without LMs 111, 411, and 511 in solution for 30 min and then further incubated in the presence or absence of La (10³) for 60 min. NETs in culture supernatants were quantified using PicoGreen. The data were normalized according to spontaneous release of DNA (Nil) and are represented as the mean ± SEM of 6 (A), 8 (B), and 10 donors (C). * p < 0.05.

Figure 7

Leishmania killing induced by NET-rich supernatants. Promastigotes (1 × 10⁶) were incubated or not for 2 h with NET-rich supernatants obtained after 4 h of stimulation with L. amazonensis or LM 511 at 37 °C and 5% CO₂. Then, parasites were stained with ethidium homodimer-1 (EthD-1) for 30 min at 37 °C and 5% CO₂ and analyzed via flow cytometry. Leishmania fixed with 4% formaldehyde (Fla, dead parasites) served as the positive control (100% of death). The results are shown as the mean  ±  SEM of 2 donors. * p < 0.05.