Estudo de proteínas induzidas por glicocorticoides: papel de GILZ (Glucocorticoid-Induced Leucine Zipper) e Anexina A1 na resolução da inflamação aguda

Abstract

The inflammatory response is characterized by the initial release of pro-inflammatory mediators and migration of leukocytes to the site of injury in response to sterile stimulus or caused by pathogens. However, persistent or uncontrolled inflammation may be cause of chronic inflammatory, systemic or autoimmune diseases with eventual loss of function of the affected organ, since proteases present in the granules of neutrophils can perpetuate inflammation by acting as phlogistic agent. Thus, this process must be finely controlled by the action of anti-inflammatory and/or pro-resolving mediators and endogenous anti-proteases that assist in resolution of the inflammatory process and the return of tissue homeostasis. In recent years the study of agonists of the resolution, aiming to find molecules that act in the production and termination phase of inflammation, has gained much importance to the findings of lipid and protein pro-resolving molecules. Among these mediators, are highlighted GILZ (glucocorticoid-induced leucine zipper) and annexin A1 (AnxA1), proteins induced by glucocorticoids (GCs) that mediate various functions of this class of drugs. The present study investigated the participation of GILZ and AnxA1, and the effect of natural and synthetic antiproteases in the resolution of the acute inflammatory response. For this purpose, it was used the murine model of LPS-induced pleurisy in BALB/c mice. Subsequently, the cells in the pleural fluid were collected at different post injection intervals and analyzed. It was observed that GILZ and AnxA1 expression was increased during the resolution phase of pleurisy, especially in macrophages with resolutive phenotypes. The prophylactic or therapeutic treatment of mice injected with LPS with the peptide TAT-GILZ (allows the in vivo delivery of GILZ), improved resolution indices, decreased cytokine levels, and promoted apoptosis of neutrophils. TAT-GILZ also decreased the activation/accumulation of the survival proteins ERK1/2, NF-B and Mcl-1. In deficient GILZ (GILZ-/-) mice the resolution of inflammation was associated with an early increase of AnxA1 without changing the influx of neutrophils induced by LPS. Dexamethasone (Dex) solved inflammation inducing GILZ expression, which was dependent of AnxA1. The resolution induced by Dex has not changed in GILZ-/- mice due to a compensatory increase AnxA1 expression. These results shows that despite GILZ deficiency are offset of AnxA1 increase, the therapeutic administration of GILZ effectively induces a pro-apoptotic program by promoting resolution of neutrophilic inflammation induced by LPS. As regards the study of the balance of protease/anti-protease during kinetic resolution, intact AnxA1 was detected in cells challenged with PBS, and its cleavage was maximal between 8-24 hours after LPS, when the recruitment of neutrophils and the expression and elastase activity was maximal. The AnxA1 expression as well as endogenous antiproteases expression (SLPI - secretory leukocyte protease inhibitor and Elafin) was increased during the resolution phase of inflammation. Treatment of mice with SLPI and Elafin promoted the resolution of inflammation associated with increased apoptotic neutrophils and intact AnxA1 in the pleural cavity. Similarly, the synthetic inhibitor of elastase Sivelestat, promoted the resolution of inflammation associated with prevention of AnxA1 cleavage and increased neutrophil apoptosis in a caspases dependent manner. Importantly, Sivelestat incresased the number of macrophages with resolutive phenotype and efferocytosis of apoptotic neutrophils. The AnxA1 neutralization using a neutralizing antibody or blocking its receptor abolished the effect induced by Sivelestat. These results show that inhibition of elastase by anti-proteases promote the resolution of inflammation associated with increased levels of intact AnxA1 and apoptosis. Taken together, these data show that proteins induced by GCs or therapeutic strategies which increases their levels may constitute an interesting strategy to the control of inflammatory diseases.