Assessing the role of Caspase-8 in the inflammasome

Abstract

The innate immune system is composed of several sensors and effectors widely expressed in macrophages, dendritic cells and epithelial cells. These molecules can rapidly induce inflammatory responses by triggering the production of cytokines, chemokines and release of danger signals present in the cytoplasm. In this context, host cell death has been reemerged as an important effector mechanism that operates during infection with intracellular pathogens. Our group has demonstrated that NLRP3, ASC and caspase-1 are involved in host protection against Leishmania amazonensis, by mechanisms that involve IL-1b and IL-1R signaling. Preliminary data obtained by our group suggested that caspase-8 may play an important role during inflammasome activation in response to L. amazonensis infection. Therefore, we hypothesize that caspase-8 can be important to protect the host by two main mechanisms: induction of inflammatory gene expression and through inflammasome activation and cell death. Thus, we aim to gain expertise in caspase-8 biology in a laboratory containing the most up-to-date available techniques to study this enzyme and its related pathways. In this sense, we will employ in vitro systems to characterize the biology of caspase-8 during inflammasome activation. This includes the maintenance, genotyping and breeding of several gene-target mice for further obtaining of macrophages from these lineages. Initially, we aim to establish tissue culture from CRISPR-Cas9 gene-targeted mice lacking caspase-8 in caspase-1 and gasdermin-D deficient backgrounds. We will also apply live cell imaging to evaluate kinetics and dynamics of caspase-8 responses during inflammasome activation in vitro. During the BEPE program, the Ph.D student will gain expertise and learn novel technologies, including live cell imaging and gene editing to obtain tools for the evaluation of effector mechanisms by which caspase-8 controls Leishmania infection. Ultimately, this development of this BEPE project will promote student training and further bring novel tools to our laboratory in Brazil.