Leishmania amazonensis induces AIM2 inflammasome activation in macrophages via endoplasmic reticulum stress and mitochondrial DNA

Author(s)

Leonardo Lima dos Santos

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-05-04

Examining board members

Dario Simões Zamboni; Valéria de Matos Borges; Karina Ramalho Bortoluci; Niels Olsen Saraiva Câmara

• Advisor: Dario Simões Zamboni

Abstract

The endoplasmic reticulum is an organelle involved in processes of lipid synthesis, translation and protein processing. In homeostasis, it promotes the proper folding of proteins to allow them to perform their physiological functions. However, lack of nutrients, hypoxia, protein mutations, high levels of reactive oxygen species (ROS), intra-reticular calcium level imbalance and infections, can lead to the accumulation of misfolded proteins in the ER lumen and consequent induction of endoplasmic reticulum stress (ERS) effector pathways. Among several consequences of ERS activation are the production of proinflammatory cytokines, induction of mitochondrial damage and inflammasome activation. The relationship between ERS induction and NLRP3 inflammasome activation has already been described in the literature. However, despite being reported that mitochondrial DNA release occurs during ERS activation, there is no data showing the connection between ERS, mitochondrial DNA (mtDNA) release in the cytosol and AIM2 activation. Thus, we hypothesized that inflammasome activation by ERS induction would be mediated through the recognition of mitochondrial DNA by the AIM2 inflammasome. To test this hypothesis, we used pharmacological models of ERS activation - Thapsigargin (TG) and Tunicamycin (TM) - as well as a model of ERS and inflammasome activation that is the parasite L. amazonensis. An initial analysis demonstrated that TG and TM-treated BMDMs lead to IL-1 secretion, caspase-1 cleavage and ASC puncta formation in an AIM2- dependent manner. Furthermore, we demonstrated that AIM2 inflammasome activation also occurs during Leishmania spp. infection, in BMDMs, BMDCs, it is microbiota-independent, MOI, time-dependent, and species-specific. Finally, we observed that Leishmania amazonensis-induced IL-1 secretion is reduced in the context of ERS inhibition, as well as in AIM2 deficiency and that in Aim2/ BMDMs ERS inhibition does not promote changes in IL-1 levels, suggesting that these mechanisms operate in the same pathway. These data support the first part of our hypothesis, in which ERS leads to AIM2 inflammasome activation. Then, using a biologically relevant model of L. amazonensis in vitro infection in BMDMS, we supported the hypothesis that ERS could lead to AIM2 activation and this pathway would be important for in vitro infection control. To test the second part of the hypothesis, in which ERS induces the activation of AIM2 inflammasome would be mediated by mtDNA, we used a model of mtDNA depletion in BMDMs. This model is functional, once the cells maintain cell viability and mitochondrial membrane potential intact during the experiments. Initially, we observed that L. amazonensis, as well as TG and TM, promote changes in mitochondrial membrane potential in an ERSdependent and AIM2-independent manner. Suggesting that the DNA sensor would be downstream of ERE and mitochondrial damage. Comparing cells depleted or not for mitochondrial DNA, we observed that the secretion of IL-1B induced by TG, TM or L. amazonensis, and the control of the infection of this parasite are reduced in the WT background but not in the Aim2/, suggesting that these molecules also operate in the same pathway. Taken together, the data from this work support the hypothesis that ERS induction leads to an activation of AIM2 mediated by mitochondrial damage and mtDNA and that this pathway is biologically relevant for the intracellular control of a pathogen. (AU)

• FAPESP's process: 17/20687-1 - Evaluation of the AIM2 role in the innate immune response against Leishmania amazonensis
• Grantee: Leonardo Lima dos Santos
• Support Opportunities: Scholarships in Brazil - Doctorate