3'-nucleotidase/nuclease activity allows Leishmania parasites to escape killing by neutrophil extracellular traps

Abstract

Leishmaniasis is a widespread neglected tropical disease caused by parasites of the Leishmania genus. These parasites express the enzyme 3'-nucleotidase/nuclease (3'NT/NU), which has been described to be involved in parasite nutrition and infection. Bacteria that express nucleases escape the toxic effects of neutrophil extracellular traps (NETs). Hence, we investigated the role of 3'NT/NU in Leishmania survival of NET-mediated killing. Promastigotes of Leishmania infantum were cultured in high-phosphate (HP) or low-phosphate (LP) medium to modulate nuclease activity. We compared the survival of the two different groups of Leishmania during interaction with human neutrophils, assessing the role of neutrophil extracellular traps. As previously reported, we detected higher nuclease activity in parasites cultured in LP medium. Both LP and HP promastigotes were capable of inducing the release of neutrophil extracellular traps from human neutrophils in a dose- and time-dependent manner. LP parasites had 2.4 times more survival than HP promastigotes. NET disruption was prevented by the treatment of the parasites with ammonium tetrathiomolybdate (TTM), a 3'NT/NU inhibitor. Inhibition of 3'NT/NU by 3'-AMP, 5'-GMP, or TTM decreased promastigote survival upon interaction with neutrophils. Our results show that Leishmania infantum induces NET release and that promastigotes can escape NET-mediated killing by 3'-nucleotidase/nuclease activity, thus ascribing a new function to this enzyme.

FIG 1

Promastigotes of Leishmania infantum induce release of neutrophil extracellular traps. (A and B) Neutrophils were incubated with HP promastigotes (A) or LP promastigotes (B) of L. infantum at different cell ratios (1NΦ:0.1, 1 neutrophil-to-0.1 parasite ratio) and time points as indicated. Supernatants were recovered, and NETs were quantified as described in Materials and Methods. Controls (Ctrl) were incubated for 120 min. Results of at least 6 independent experiments are shown as means ± standard errors of the means (SEM). *, P < 0.01 for control versus experimental results; **, P < 0.05. (C) Interdonor variations in NET release. Neutrophils from different healthy blood donors were incubated with HP or LP promastigotes of L. infantum at a ratio of 1 neutrophil to 0.1 parasite. After 120 min at 35°C, supernatants were recovered and NETs quantified. The control (Ctrl) condition was the spontaneous release of NETs. Results of 18 independent experiments are shown as means ± SEM. *, P < 0.01 in relation to control. (D) Neutrophils were incubated with HP or LP promastigotes of L. infantum at a 1 neutrophil-to-0.1 parasite ratio. After 60 min, slides were fixed and stained with DAPI, and images were taken in a Zeiss Axioplan. The control condition was neutrophils incubated without parasites. Bar, 50 μm.

FIG 2

Susceptibility of parasites grown in HP and LP media to neutrophil killing. Neutrophils were incubated in the absence (A, C) or presence (B) of DNase (20U/ml) for 20 min, followed by the addition of stationary-phase HP and LP promastigotes (A, B) or metacyclic promastigotes (C) (1 neutrophil-to-0.1 parasite ratio) for 2 h at 35°C. Fetal calf serum was added to the cultures to a final concentration of 10%, and live parasites were counted after 2 days at 26°C. (D) Neutrophils were incubated with HP or LP metacyclic and procyclic promastigotes at a 1 neutrophil-to-0.1 parasite ratio. After 120 min of incubation at 35°C, supernatants were recovered and NETs quantified. The control raw numbers for the experiment whose results are shown in panel B were 528.5 × 10⁴ ± 139.5 HP promastigotes/ml and 1,288.0 × 10⁴ ± 255.4 LP promastigotes/ml. Results are shown as means ± SEM; n = 20 (A), n = 16 (B), n = 6 (C), n = 4 (D). *, P < 0.01, and **, P < 0.05, for the statistical difference between experimental and control results.

FIG 3

Leishmania 3′-nucleotidase/nuclease activity digests NETs from human neutrophils and allows the parasites to evade NETosis. (A) HP and LP parasites (2 × 10⁶) were pretreated or not with ammonium tetrathiomolybdate (TTM, 100 μM) and incubated with NET-enriched supernatants (1,000 ng of DNA). After 180 min, tubes were centrifuged at 4,000 rpm for 10 min, and supernatants were resolved by electrophoresis in a 1% agarose gel with GelRed staining. (B) Intact HP or LP promastigotes (1 × 10⁶) were pretreated with different concentrations of TTM and then incubated for 1 h at 30°C in NaCl (116 mM), KCl (5.4 mM), glucose (5.5 mM), and HEPES-Tris (50 mM) buffer with 3′-AMP as the substrate. Inorganic phosphate was quantified in culture supernatants as described in Materials and Methods. In the absence of TTM, considered 100% of 3′NU/NT activity, HP and LP parasites had enzymatic activities of 139.2 ± 11.8 and 220.7 ± 20.6 nmol P_i/h/10⁶ cells, respectively. Results of 10 experiments are shown as percentages of control ± SEM. (C) Promastigotes (5 × 10⁶ in 500 μl) were pretreated with different doses of TTM for 20 min, and then 2 × 10⁵ (20 μl) parasites were added to 2 × 10⁶ neutrophils and cocultured for 2 h at 35°C. FCS was added to the cultures to a final concentration of 10%, and live parasites were counted after 2 days at 26°C. (D, E) The inhibitors 3′-AMP and 5′-GMP were added to the coculture (ratio of 1 neutrophil to 0.1 parasite) together with HP and LP promastigotes. After 2 h at 35°C, FCS was added to the cultures to a final concentration of 10%, and live parasites were counted after 2 days at 26°C. Results of at least 5 independent experiments are shown as means ± SEM. *, P < 0.01; **, P < 0.05. (F) TTM and 5′-GMP were similarly tested in L. amazonensis and L. donovani promastigotes. Results from 5 independent experiments are shown as means ± SEM. *, P < 0.01.

FIG 4

3′-Nucleotidase/nuclease inhibitors do not induce or interfere with NET formation and are not toxic to neutrophils or parasites. (A to C) Neutrophils were incubated with inhibitors for 120 min at 35°C, supernatants were recovered, and NETs were quantified. (B) HP or LP promastigotes pretreated or not with ammonium tetrathiomolybdate (TTM) were incubated with neutrophils for 120 min at 35°C, supernatants were recovered, and NETs were quantified. Results of 6 independent experiments are shown as means ± SEM. *, P < 0.01 in relation to control. (D) Neutrophils were treated with 3′NT/NU inhibitors at the indicated concentrations for 2 h at 35°, and then supernatants were recovered and the activity of lactate dehydrogenase enzyme was measured. Control neutrophils without treatment were considered 100% viable. Results are expressed as percentages of viable neutrophils and shown as means ± SEM of 4 independent experiments. (E) Growth of L. infantum cultivated as described in Materials and Methods for 5 days in the absence (closed circles) or presence of 1,000 μM 3′-AMP (open squares), 500 μM 5′-GMP (closed triangles), or 6.7 μM TTM (open diamonds). The cell proliferation was determined daily by counting cell numbers in a hemocytometer. Results are shown as means ± SEM of three experiments. (F) Parasites were treated with TTM (100 μM) for 20 min, stained with propidium iodide, and analyzed on a FACSCalibur flow cytometer. Results are expressed as percentages of viable parasites which were negative for PI staining. Results are shown as means ± SEM of 2 independent experiments performed in triplicates.