Research Article: PKC/ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection

Date Published: June 26, 2015

Publisher: Public Library of Science

Author(s): Marina Tiemi Shio, Jan Gregor Christian, Jee Yong Jung, Kwang-Poo Chang, Martin Olivier, Barbara A Burleigh.

Abstract: Parasites of the Leishmania genus infect and survive within macrophages by inhibiting several microbicidal molecules, such as nitric oxide and pro-inflammatory cytokines. In this context, various species of Leishmania have been reported to inhibit or reduce the production of IL-1β both in vitro and in vivo. However, the mechanism whereby Leishmania parasites are able to affect IL-1β production and secretion by macrophages is still not fully understood. Dependent on the stimulus at hand, the maturation of IL-1β is facilitated by different inflammasome complexes. The NLRP3 inflammasome has been shown to be of pivotal importance in the detection of danger molecules such as inorganic crystals like asbestos, silica and malarial hemozoin, (HZ) as well as infectious agents. In the present work, we investigated whether Leishmania parasites modulate NLRP3 inflammasome activation. Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1β production upon stimulation. In this context, the expression and activity of the metalloprotease GP63 – a critical virulence factor expressed by all infectious Leishmania species – is a prerequisite for a Leishmania-mediated reduction of IL-1β secretion. Accordingly, L. mexicana, purified GP63 and GP63-containing exosomes, caused the inhibition of macrophage IL-1β production. Leishmania-dependent suppression of IL-1β secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation. The observed loss of ROS production was due to an impaired PKC-mediated protein phosphorylation. Furthermore, ROS-independent inflammasome activation was inhibited, possibly due to an observed GP63-dependent cleavage of inflammasome and inflammasome-related proteins. Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface metalloprotease GP63, can significantly inhibit NLRP3 inflammasome function and IL-1β production.

Partial Text: Leishmania parasites, which are the causative agent of leishmaniasis, are able to both survive and proliferate within macrophages. The protozoan parasites evolved strategies to avoid phagocyte activation during infection by seizing control of key signaling pathways [1,2]. Studies previously implicated the metalloprotease GP63—a major virulence factor of Leishmania parasites—in a variety of parasite survival mechanisms. In this context, GP63 has been suggested to affect amongst others Leishmania binding to macrophages, phagocytosis of parasites, evasion of complement-mediated lysis and protozoan migration through the extracellular matrix [1,3]. Furthermore, GP63 has been identified as a key Leishmania virulence factor that modulates cellular signalling through the subversion of host protein tyrosine phosphatase (PTP) function [4,5,6]. In this context GP63-mediated PTP-cleavage, results in the activation of the respective phosphatases. This mechanism was identified for the SH2 domains-containing protein tyrosine phosphatase (SHP-1) and protein-tyrosine phosphatase 1B (PTP-1B) [6]. Besides phosphatases, GP63 has been shown to cleave other targets within the cells including kinases like TAB 1 and transcription factors, including AP-1 and NF-κB [7,8]. The importance of the host PTP-modulation and the subsequent inhibition of signaling pathways is emphasized by the observation that key pro-inflammatory mediators such as nitric oxide (NO), IL-6 and TNFα were subsequently downregulated by Leishmania [4,5,9].

Leishmania parasites have evolved many mechanisms to hijack macrophage microbicidal functions in order to survive and proliferate within the phagocytes. In the present work we addressed how Leishmania parasites can attenuate IL-1β production through the leishmanial virulence factor GP63 during infection. In the past, the activity of inflammasomes and the associated production of especially IL-1β has been correlated with the host protection against parasitic infections, for instance in the case of T. cruzi or T. gondii [47,48,49]. In the case of Leishmania parasites the importance and the role of inflammasomes and IL-1β is very controversially discussed, mainly due to the use of different Leishmania species, different leishmanial developmental stages and different infection models. Previous work indicated the possibility of a species-dependent dysregulation of inflammasomes and inflammasome-related pathways and implicated different leishmanial virulence factors. Reports showed that L. donovani and L. tropica do not induce IL-1β production, and negatively modulate the capacity of IFN-primed human or LPS-primed murine peritoneal macrophages to produce IL-1β upon activation [10,50,51]. The focus of several studies was a parasite-mediated dysregulation of IL-1β on a transcriptional level after infection of human [52,53] or murine phagocytes [54]. In this context, Hatzigeorgiou and collaborators [55] implicated a LPG-dependent interference with IL-1β mRNA that translated into both decreased stability and production of IL-1β mRNA and consequently reduced transcription of the IL-1β gene [52,55]. However, data of Cillari et al. [54] and Gurung et al. [56] indicated that L. major infections might increase inflammasome activity and cytokine production during long term infections. Some reports indicate that the role the inflammasome is dependent on the model for infection with a possible delay in the resolution of cutaneous lesions in the absence of IL-1β [33,34]. Thus indications exist for a possible inflammasome-mediated host protective mechanism in some murine models of infection. In contradictory reports working with the new world Leishmania species L. amazoniensis, parasites have been described to be able to both suppress and induce IL-1β secretion by infected cells. On the one hand a study by Ji et al. provided data that the infection of C57Bl/6 mice with L. amazoniensis led to a delay in the secretion of chemokines and cytokines including IL-1β in vivo [11]. On the other hand a recent report of Lima-Junior et al. presented data that inflammasomes and IL-1β are involved in the control of L. amazoniensis infections of C57Bl/6 mice as shown by in vitro and in vivo studies using mice and BMDMs of deficient in IL-1β production (including caspase-1 and NLRP3 KO mice) [13].



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