Interações imunoendócrinas: efeito do hormônio ouabaína em neutrófilos

Abstract

Ouabain is a carditonic steroid initially described as a secondary metabolite from plants. However, this molecule was later described as an endogenous substance present in mammalian plasma. In addition to its classic role as a Na+ /K+ -ATPase inhibitor, which is associated with its cardiovascular effects, ouabain can activate different signaling pathways mediated by this pump. Numerous evidences indicate that ouabain regulates several immune functions. Our group demonstrated that this hormone can negatively modulate many aspects of the inflammatory response, such as vascular permeability, cytokine production, and neutrophil migration in different models. However, little is known about the effects of ouabain on neutrophils, as well as the possible signaling pathways activated by the interaction of this hormone with Na+ /K+ -ATPase present in these cells. In this context, the aim of this study was to evaluate the effect of ouabain on neutrophils, functionally and molecularly, in order to understand the role of this hormone in inflammatory processes. Therefore, this study was performed with mouse bone marrow and human peripheral blood neutrophils. Initially, a cytotoxicity assay was performed, and it was observed that ouabain (1, 10, and 100 nM) did not alter mouse neutrophils viability at the evaluated times (2, 4, and 24 h). However, neutrophils baseline viability at 4h (78.7%) and 24 h (35.2%) were lower than at 2h (88.1%). Thus, the following protocols were performed using the shortest treatment time. Subsequently, it was analyzed whether ouabain would modulate the migratory function of neutrophils extracted from mice in vitro (transwell assay). As previously demonstrated in vivo studies, ouabain also reduces neutrophil migration in vitro (inhibition: 1 nM = 80.12%; 10 nM = 76.24%; 100 nM = 56.36%). In assessing whether ouabain is modulating molecules related to cell migration, it was seen that this hormone, at 1 nM concentration, reduces CD18 adhesion molecule (β2-integrin) expression (30%) in mouse neutrophils, however, without interfering with CXCR2 chemokine receptor expression. Ouabain also did not modulate CXCL-1 levels in culture of macrophages, which is a chemokine-producing tissue cell. Considering that the interaction between ouabain and Na+ /K+ -ATPase may modulate different signaling pathways, it was then evaluated whether these pathways would be regulated in neutrophils. Treatment with ouabain was found to induce an increase of 55.18% (1 nM), 98.56% (10 nM) and 59.75% (100 nM) in Src protein kinase phosphorylation. However, this treatment did not modulate the phosphorylation levels of ERK and p38, and NF-κB transcription factor. It was further studied whether ouabain could modulate another neutrophil function, the neutrophil extracellular traps (NETs) release. Ouabain treatment (1, 10, and 100 nM) did not modulate PMA-induced NETs levels in human neutrophils. However, only 100nM ouabain treatment induced NETs production. Thus, in addition to reducing the migration of neutrophils possibly via CD18 expression reduction, the activation of Src kinase and the induction of NETs observed in this work may represent one of the mechanisms of action of the anti-inflammatory effect of ouabain.