The role of the NALP3 inflammasome in pulmonary Paracoccidioidomycosis

Abstract

The recognition of P. brasiliensis components by pathogen recognition receptors (PRRs) present on macrophages and other cells of innate immunity plays an important role in the innate and adaptive immune response against this fungal pathogen. Toll like receptors (TLRs), C-type Lectin Receptors (CLRs) and the NOD-like receptors (NLR, Nucleotide binding Oligomerization Domain Receptors), may function alone or in synergy to recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). The NLRs include some families of receptors that promote cellular activation following interaction with pathogen PAMPs or cell DAMPs. Some NLRs components (NALP3 or IPAF) can oligomerize forming multimolecular compounds called inflamassomes, which induce the activation of caspase-1 and the maturation of pró-IL-1beta and pró-IL-18 to their active forms. It is well known that macrophages require two signals for the efficient production of IL-1beta and IL-18; one that induces the production of pro-inflammatory cytokines and another that induces the activation of caspase-1. Our project aims to study the role of the NALP3 in the recognition of P. brasiliensis by murine macrophages, and its function in the innate and adaptive phases of immunity against this fungal pathogen. Using in vitro approaches, the production of IL-1beta and IL-18 as well as the activation of caspase-1 will be evaluated after the interaction of macrophages with P. brasiliensis yeasts. The fungicidal ability and nitric oxide production will be also utilized as tools to evaluate the macrophage function. Muramyl dipeptide, ATP and LPS will be employed as control or first signal for macrophage activation. The assays employing caspase-1, Syk quinase and P2X7R inhibitors will help to clarify some cell signaling pathways. More direct approaches using cells from knockout mice for caspase-1, NALP3, ASC, Dectin-1, MyD88 and P2X7R will clarify the contribution of several PRRs to macrophage activation and IL-1 production. Furthermore, caspase-1-/- and NALP3-/- mice infected by the i.t. route will be studied at some postinfection periods regarding several features of innate and adaptive immunity. Altogether, this proposal intends to obtain a comprehensive view on the role of NALP3 in pulmonary paracoccidiodomycosis. (AU)