Evaluation of inflammasome requirement in neutrophilic inflammation induced by pore-forming toxins, Natterins

• Grant number: 19/10500-7
• Support type: Scholarships abroad - Research
• Effective date (Start): September 01, 2019
• Effective date (End): September 30, 2019
• Field of knowledge: Biological Sciences - Immunology
• Principal Investigator: Carla Lima da Silva
• Grantee: Carla Lima da Silva
• Host: Bernhard Ryffel
• Home Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Local de pesquisa : Institut de Transgénose Orléans-Villejuif, France
• Associated research grant: 13/07467-1 - CeTICS - Center of Toxins, Immune-Response and Cell Signaling, AP.CEPID

Abstract

Neutrophils, the most abundant immune cells in the circulation, are key players in acute inflammation. Excessive neutrophil recruitment is observed in traumatic and ischemic injuries, autoimmunity, and sterile liver injury and excessive neutrophil activation results host tissue injury. Therefore, understanding mechanisms of neutrophil recruitment is of major physiological and pathophysiological importance. Neutrophil trafficking is orchestrated by adhesion molecules, such as b2 integrins, chemokines, and cytokines as IL-1b. The processing of the 31-kDa inactive cytosolic pro-IL-1² precursor into the bioactive 17-kDa IL-1², mostly depends on autocatalytic cleavage of the cysteine protease caspase-1. Such cleavage is triggered by several structures termed as inflammasome, which consist of a group of related multiprotein cellular complexes, among which the best characterized include receptors of the NLR family of proteins, namely NLRP1, NLRP3 and NLRC4. While canonical inflammasome activation results in caspase-1 cleavage and activation, the activation of a non-canonical inflammasome results in activation of pro-caspase-11 that promotes cell death as well as IL-1a and IL-1b secretion and neutrophil recruitment during infection response. The family Natterins was first discovered in the venom gland of Brazilian venomous fish Thalassophryne nattereri with five orthologues 1-4 and -P. Natterin proteins contain at the N-terminal region two DM9 domains first discovered in Drosophila, followed by an aerolysin domain at the C-terminal. The aerolysin family belongs to the large class of pore-forming proteins/toxins (b-PFTs) and is considered to be the principal virulence factors contributing to severe inflammatory diseases in humans and animals. If it is described for the other aerolysins derived from bacteria (nigericin, maitotoxin and aerolysin of P. aeruginosa) an action in the activation of the inflammatory complex NLRP3, is expected that for the Natterins this action also occurs. However, preliminary results from experiments with wild-type mice treated with anti-IL-1² neutralizing antibodies and with MCC950 inhibitor and inhibitors for caspase-1 and -11 have shown that neutrophilia induced by Natterins is caspase/IL-1² dependent, but in contrast to other aerolysins, does not depend on the activation of NLRP3. In this project, our objective is to verify the role of the activation of canonical or non-canonical platform members of the inflammasome in the recruitment of neutrophils to the site of injury induced by pore-forming toxins Natterins as well as the direct influence of these members in the activation of neutrophils modulating their capacity to produce mediators that amplify the inflammatory response.