Papel do receptor ativado por pretease (PAR)2 e de proteases endógenas ativadoras de PAR2 no recrutamento de neutrófilos induzido por 1ps em pulmão de camundongos c57BL/6

Abstract

Innate immunity is the first line of defense against pathogens triggered by the recognition of molecular structures present in the microorganisms like LPS. Proteinase-activated receptors (PARs) are part of a family of metabotropic receptors activated by serine proteases through proteolytic cleavage in their Nterminal portion named PAR 1-4. Among these, PAR2 is expressed in leukocytes and resident lung cells and its activation has been related to the development of inflammation and cell recruitment, however, the involvement of PAR2 activation by its endogenous agonists in inflammatory response induced by LPS is not well understood. The aim of this work was to evaluate the role of PAR2 and endogenous proteases in neutrophils recruitment to the lung of C57BL/6 mice in response to intranasal (i.n.) instillation of LPS. The experimental protocols were approved by the animal ethics committee (CEUA/UFMG, 150/2017). The results show that intranasal administration of LPS or mast cell (MC) tryptase induced an increase in the number of neutrophils recovered in bronchoalveolar lavage (BAL) 4h later, and pretreatment with intraperitoneal injection of PAR2 antagonist (ENMD-1068) or with intranasal instillation of protease inhibitor (aprotinin) 1h prior to stimulus with LPS reduced the presence of these cells in BAL. In contrast, pre-treatment with ENMD-1068 increased the number of neutrophils in peripheral blood of animals. PAR2 antagonist also decreased the production of CXCL1 chemokine measured in supernatants of BAL obtained 4h after LPS instillation. Moreover, histopathological analysis in mouse lungs stained with hematoxylin and eosin showed that ENMD-1068 treatment reduced pulmonary inflammation induced by LPS. In addition, PAR2 expression was increased in lungs of mice 4h after LPS stimulation when compared to PBS-instilled mice. Mast cell depletion by pretreatment with compound 48/80 reduced the number of neutrophils recovered in BAL of animals treated by intranasal instillation with LPS. The amplitude of calcium signals and the number of responsive cells to LPS were reduced in RAW 264.7 cells preincubated with ENMD-1068 during 40 min. before stimulus with LPS or treated with LPS in the presence of protease inhibitors cocktail. In conclusion, our results suggest a role for PAR2 and MC tryptase on neutrophil recruitment into the lung of mice induced by LPS.