Characterization of the mechanism by which the intestinal microbiota including Proteus mirabilis activates the NLRP3 inflammasome

• Grant number: 14/20384-0
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: March 01, 2015
• End date: February 29, 2016
• Field of knowledge: Biological Sciences - Immunology - Applied Immunology
• Principal Investigator: Vera Lucia Garcia Calich
• Grantee: Claudia Feriotti
• Supervisor: Gabriel Nuñez
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: University of Michigan, United States
• Associated to the scholarship: 13/02396-9 - The role of the NLRP3 inflammasome in pulmonary paracoccidioidomycosis, BP.PD

Abstract

Characterization of the mechanism by which the intestinal microbiota including Proteus mirabilis activate the NLRP3 inflammasome. P. mirabilis is a member of the Enterobacteriaceae family, and it is a robust inducer of IL-1beta release via NLRP3 and this resident bacterium induces NLRP3 activation by the production of the hemolysin hmpA. Based on studies using conventional, germ-free (GF) mice and antibiotic-treated mice, the intestinal microbiota triggers IL-1beta and this is mediated by the NLRP3 inflammasome. In this aim, we propose a series of studies to further understand the mechanism by which the microbiota and P. mirabilis, a resident bacterium that triggers robust IL-1beta secretion elicit IL-1beta via NLRP3. We will begin by stimulating bone marrow-derived macrophages deficient in both MyD88/TRIF and RIP2, the adaptors required for all TLR and NOD1/2 signaling, respectively, with fecal contents or P. mirabililis and the expression of pro-IL-1beta mature IL-1beta and NLRP3 will be determined by qPCR and immunoblotting analyses. To determine whether hemolysin is required for caspase-1 activation, macrophages will be stimulated with wild-type and hpmA-deficient P. mirabilis and cell extracts will be immunoblotted with anti caspase-1 (p20 subunit) antibody. To examine whether hpmA mediates K+ efflux, macrophages will be stimulated with wild-type and hpmA-deficient P. mirabilis and the intracellular levels of K+ will be measured. To determine whether NLRP3 and ASC oligomers are induced in response to the microbiota or P. mirabilis, we will use size-exclusion chromatography to fractionate extracts from primed macrophages stimulated with fecal contents or P. mirabilis,and fractions will be run on SDS-PAGE and immunoblotted with anti-NLRP3, anti-ASC and anti-caspase-1 antibodies.To determine whether the microbiota and P. mirabilis triggers speck formation and ASC oligomerization, we will incubate macrophages with intestinal contents or P. mirabilis and formation of ASC speckles and ASC oligomers will be detected by fluorescence microscopy.We will treat macrophages with the cell-permeable pancaspase inhibitor zVAD-FMK to assess whether NLRP3 and ASC requires caspase-1 activation. Experiments in vivo will be performed, to test whether TLR/NOD signaling is required for the priming of the inflammasome and if hemolysin is required for IL-1beta secretion in the intestine by using a triply-deficient MyD88-/-TRIF-/-RIP2-/- mice and GF mice that will be colonized with hpmA-deficient P. mirabilis strains and treated with dextran sodium sulphate (DSS) in the drinking water, and assess expression of NLRP3 and IL-1beta mRNA by qPCR and IL-1beta by ELISA. We expect that TLR and/or Nod1/Nod2 signaling will mediate priming of the NLRP3 inflammasome. The methodology, materials and mechanistic aspects are in place, so we do not have problems with the experiments proposed in this aim. (AU)