Non-canonical NLRP3 inflammasome activation and IL-1β signaling are necessary to L. amazonensis control mediated by P2X7 receptor and leukotriene B4

Abstract

Leishmaniasis is a neglected tropical disease affecting millions of individuals worldwide. P2X7 receptor has been linked to the elimination of Leishmania amazonensis. Biological responses evoked by P2X7 receptor activation have been well-documented, including apoptosis, phagocytosis, cytokine release, such as IL-1β. It was demonstrated that NLRP3 inflammasome activation and IL-1β signaling participated in resistance against L. amazonensis. Furthermore, our group has shown that L. amazonensis elimination through P2X7 receptor activation depended on leukotriene B4 (LTB4) production and release. Therefore, we investigated whether L. amazonensis elimination by P2X7 receptor and LTB4 involved NLRP3 inflammasome activation and IL-1β signaling. We showed that macrophages from NLRP3-/-, ASC-/-, Casp-1/11-/-, gp91phox-/- , and IL-1R-/- mice treated with ATP or LTB4 did not decrease parasitic load as was observed in WT mice. When ASC-/- macrophages were treated with exogenous IL-1β, parasite killing was noted, however, we did not see parasitic load reduction in IL-1R-/- macrophages. Similarly, macrophages from P2X7 receptor-deficient mice treated with IL-1β also showed decreased parasitic load. In addition, when we infected Casp-11-/- macrophages, neither ATP nor LTB4 were able to reduce parasitic load, and Casp-11-/- mice were more susceptible to L. amazonensis infection than were WT mice. Furthermore, P2X7-/- L. amazonensis-infected mice locally treated with exogenous LTB4 showed resistance to infection, characterized by lower parasite load and smaller lesions compared to untreated P2X7-/- mice. A similar observation was noted when infected P2X7-/- mice were treated with IL-1β, i.e., lower parasite load and smaller lesions compared to P2X7-/- mice. These data suggested that L. amazonensis elimination mediated by P2X7 receptor and LTB4 was dependent on non-canonical NLRP3 inflammasome activation, ROS production, and IL-1β signaling.

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. Proposed Mechanism.

ATP activates the P2X7 receptor, which in turn leads to the production and release of LTB₄. LTB_4, 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.