Role of myosin IXB in the regulation of inflammasome activation

• Grant number: 22/12443-3
• Support Opportunities: Regular Research Grants
• Start date: February 01, 2023
• End date: January 31, 2026
• Field of knowledge: Biological Sciences - Immunology - Cellular Immunology
• Principal Investigator: Bruna Cunha Gondim de Alencar
• Grantee: Bruna Cunha Gondim de Alencar
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated researchers: Alessandra Pontillo ; Denise Morais da Fonseca

Abstract

Myosins are actin-dependent motor proteins. Cells of the immune system express a range of different myosins that have a role in processes like endo- and exocytosis, migration and cell division. The participation of myosins in the regulation and signaling by innate immunity cytoplasmic receptors, however, is unknown. The unconventional myosin IXB is highly expressed in leukocytes and, in addition to being a motor capable of moving on actin filaments, it also incorporates a RhoGAP domain that inhibits the activation of small GTPases of the Rho family, especially RhoA, which modulate actin polymerization. Polymorphisms in the MYO9B gene have been associated to higher susceptibility to inflammatory diseases, especially inflammatory bowel disease (IBD).Inflammatory diseases are also related to the innate immunity sensors known as inflammasomes. Inflammasomes are multi-protein complexes formed in response to the activation of pattern recognition receptors that lead to the production and release of the inflammatory cytokines IL-1b and IL-18, in addition to inducing the inflammatory cell death known as pyroptosis. Genetic variants of both NLRP3 and MEFV genes (that code for sensors NLRP3 and pyrin, respectively) have been correlated to the development of several infectious and non-infectious inflammatory diseases, including IBD. Pyrin senses the inhibition of RhoA activity, while NLRP3 senses other pathogenic changes in the cell induced by the presence of microorganisms or cell damage, and its activation seems to be regulated by both microtubules and the actin network.Therefore, as Myo9b participates in the regulation of the actin cytoskeleton, as the actin cytoskeleton modulates the activation of NLRP3 and pyrin inflammasomes, and as there are evidences for the involvement of the three proteins in the same inflammatory diseases, this project aims to investigate the role of Myo9b in the modulation of NLRP3 and pyrin inflammasomes' activation. For that, Myo9b will be silenced or knocked-out in Thp1 monocytic cells or in cells derived from human blood monocytes. Silenced cells will be stimulated with NLRP3 and/or pyrin inflammasome activators, and we will analyze the production of IL-1b, the formation of ASC specks, in addition to cell death. Myo9b-silenced cells will also be exposed to the bacteria Yersinia pseudotuberculosis, which contain toxins that activate the pyrin inflammasome. We will then investigate the response of the monocytic cells to the bacteria using the above-mentioned parameters.Hence, this project intends to better understand inflammasome regulation by the cytoskeleton, an interaction that has been little explored so far, especially concerning myosins. There is no published study regarding myosins, particularly unconventional myosins, in the regulation of inflammasomes or the intrinsic immune response, therefore opening the possibility of elucidating an unexplored regulation level. Finally, such information might allow the conception of new treatments for inflammatory diseases involving inflammasomes. (AU)