Characterization of the purinergic receptor P2X7 and the NLRP3 inflammasome in the inflammatory response induced by antimicrobial peptides and infections

Zusammenfassung

This Thesis describes how the antimicrobial peptide melittin induces the canonical activation of NLRP3 inflammasome by a membrane permeabilization and a decrease of the intracellular K+ concentration. After melittin treatment, ASC protein was necessary to activate caspase-1 and induces IL-1Beta release. However, cell death induced directly by membrane permeabilization induced by melittin inhibited the caspae-1 activation amplification, IL-18 release and pyroptosis. Thus, the NLRP3 inflammasome activation induced by melittin resulted in an attenuated response of the inflammasome without caspase-1 dependent cell death induction. In addition, a cohort of intra-abdominal origin septic patients (n=35) was studied, where we found that a NLRP3 inflammasome activation mediated by ATP was defected in a group of patients between the first 24h od sepsis development. These patients presented an immunocompromised immune response and they accumulated the major part of death associated to sepsis. In immunocompromised septic patients, P2X7 receptor was associated with mitochondrial dysfunction in monocytes, but not to the NLRP3 inflammasome activation. P2X7 receptor activation in monocytes without microbial stimulation resulted in an inhibition of the NLRP3 inflammasome mediated by a mitochondrial damage. Several markers for sepsis severity and circulating cytokines were determined without this high-risk of mortality group of patients' stratification. Our data revealed a molecular mechanism initiated by P2X7 receptor, which contributes to the immunosuppression in monocytes from septic patients that could be considered as a new early biomarker to predict the mortality in sepsis. Not least important, we found that P2X7-activated macrophages and HEK293 cells reduced its ability to adhere to substrate in a short time and a dose-dependent manner. These data indicated that cell adhesion to a substrate by non-activated macrophages is higher than cell adhesion of non-treated HEK-cells, into the culture plate and different extracellular matrix (ECMs). This phenomenon was carried out the cellular release of the Beta1 integrin, induced by the P2X7 receptor activation, resulting in a loss of cell adhesion to the tissue culture plate and type I collagen, but not to fibronectin and laminin. Although cell adhesion rapidly increased after macrophages were treated with Escherichia coli LPS , after ATP stimulation, cell adhesion radically decreased independently of the adherent substrate where macrophages were plated. Under these conditions, cell death induced by pyroptosis after P2X7 receptor activation, induced non-selectively the loss of cell adhesion. These results revealed that the P2X7 receptor is actively associated to the macrophages adhesion process.