Fig 1.
L. amazonensis control via P2X7 receptor was dependent on NLRP3 inflammasome and IL-1R signaling.
Peritoneal macrophages from C57BL/6 (A-C), NLRP3-/- (D-F), ASC-/- (G-I), Casp-1/11-/- (J-L), and IL-1R-/- (M-O) mice were infected with L. amazonensis promastigotes at the ratio of 10:1 (Leishmania:macrophage). After 4 hours, the free parasites were washed and after 24 hours, infected cells were treated (B, E, H, K, and N) or not (A, D, G, J, and M) with 500 μM of ATP. Twenty-four hours later, cells were stained with May-Grunwald-Giemsa and the infection index was determined by direct counting under light microscopy. Normalized values represent means ± SEM of 3–4 independent experiments performed in triplicate. Arrows correspond to vacuoles with L. amazonensis and asterisks represent empty vacuoles. (*P < 0.05) compared to the control group (without treatment).
Fig 2.
L. amazonensis control via LTB4 was dependent on the NLRP3 inflammasome and IL-1R signaling.
Peritoneal macrophages from C57BL/6 (A-C), NLRP3-/- (D-F), ASC-/- (G-I), Casp-1/11-/- (J-L), and IL-1R-/- (M-O) mice were infected with L. amazonensis promastigotes at a ratio of 10:1 (Leishmania:macrophage). After 4 hours, the free parasites were washed and after 24 hours, infected cells were treated (B, E, H, K, and N) or not (A, D, G, J, and M) with 100 nM of LTB4. Twenty-four hours later, cells were stained with May-Grunwald-Giemsa and the infection index was determined by direct counting under light microscopy. Normalized values represent means ± SEM of 3–4 independent experiments performed in triplicate. Arrows correspond to vacuoles with L. amazonensis and asterisks represent empty vacuoles. (*P < 0.05) compared to the control group (without treatment).
Fig 3.
L. amazonensis control via IL-1β was dependent on IL-1R signaling.
Peritoneal macrophages from C57Bl/6 (A-C), ASC-/-(D-F), and IL-1R-/- (G-I) were infected with L. amazonensis promastigotes at the ratio of 10:1 (Leishmania:macrophage). After 4 hours, the free parasites were washed and after 24 hours, infected cells were treated with 100 pg/ml of IL-1β. Twenty-four hours later, cells were stained and the infection index was obtained. Normalized values represent means ± SEM of 3–4 independent experiments performed in triplicate. Arrows correspond to vacuoles with L. amazonensis and asterisks represent empty vacuoles. (*P < 0.05) compared to the control group (without treatment).
Fig 4.
L. amazonensis control via ATP/P2X7 receptor was Casp-11 dependent.
Peritoneal macrophages from C57Bl/6 (panels A-C, G, and H) and Casp-11-/- (D-F) mice were infected with promastigotes of L. amazonensis at a ratio of 10:1 (Leishmania:macrophage). After 4 hours, the free parasites were washed and after 24 hours, infected cells were treated with ATP (500 μM; B and E). Infected macrophages were also treated with Z-YVAD-FMK and Z-LEVD-FMK, Casp-1 and Casp-11 inhibitors respectively, at the concentration of 2 μM 30 minutes before ATP treatment. Twenty-four hours later, the infection index was determined. Standard values represent means ± SEM of 3 independent experiments performed in triplicate. Arrows correspond to vacuoles with L. amazonensis and asterisks represent empty vacuoles. (*P < 0.05) compared to the control group (without treatment).
Fig 5.
L. amazonensis control via LTB4 was Casp-11 dependent.
Peritoneal macrophages from C57Bl/6 (panels A-C, G, and H) and Casp-11-/- (D-F) mice were infected with promastigotes of L. amazonensis at a ratio of 10:1 (Leishmania:macrophage). After 4 hours, the free parasites were washed and after 24 hours infected cells were pretreated with 2 μM of Z-YVAD-FMK (G) or Z-LEVD-FMK (H) for 30 min. Subsequently, cells were treated with LTB4 (100 nM). Twenty-four hours later, the infection index was determined. Standard values represent means ± SEM of 3 independent experiments performed in triplicate. Arrows correspond to vacuoles with L. amazonensis and asterisks represent empty vacuoles. (*P < 0.05) compared to the control group (without treatment).
Fig 6.
Casp-11-/- mice were more susceptible infection and LTB4 and IL-1β restored resistance of P2X7 receptor-/- mice to L. amazonensis.
C57Bl/6 (A, B, C, D, E, F), P2X7-/- (C, D, E, F), Casp-1/11-/- (A, B), and Casp-11-/- (A, B) mice were infected with 106 promastigotes of L. amazonensis for 28 days and were treated with or without 5 ng of LTB4 (C, D) or 300 pg of IL-1β (E, F) twice weekly, for 3 weeks. Lesions was determined by thickening (A, C, E) and number of parasites by LDA as described (B, D, F). A, B correspond to mean ± SEM of a group of 9 animals; C, D correspond to mean ± SEM n = 8 mice in two independent experiments; E, F correspond to mean ± SEM of n = 5–4 animals (*P < 0.05).
Fig 7.
ATP activates the P2X7 receptor, which in turn leads to the production and release of LTB4. LTB4, through a mechanism as yet unknown, activates Casp-11. Casp-11 activates the NLRP3 inflammasome, leading to maturation of Casp-1. Casp-1 cleaves pro-IL-1β into mature IL-1β as it is released into extracellular medium and activates IL-1 receptors. By a mechanism as yet unknown, activation of IL-1R leads to the control of L. amazonensis in macrophages.