Investigação do perfil de macrófagos M1 e M2 e de citocinas em modelos experimentais da doença pulmonar obstrutiva crônica

Abstract

Alveolar macrophages are differentiated from circulating monocytes and play a central role in the pathophysiology of chronic obstructive pulmonary disease (COPD) and are a major target for thepapy. In this study, we investigated the profile of M1 and M2 macrophages and cytokines in experimental models of COPD. First, we evaluated the effect of cigarette smoke (CS) in monocytes from blood and bone marrow and lung macrophages in mice. In addition, we investigated the profile of human macrophages after exposure to a cigarette smoke extract (CSE) and we also analyzed, in COPD patients, the profile of blood monocytes. Male mice (C57BL/6, n = 54) were divided in groups: control and exposed to 12 Marlboro cigarettes (CS 5 days, CS14 days and CS 30 days). Human peripheral blood monocytes were obtained for centrifugation by a Ficoll gradient and differentiated into macrophages after incubation with GM-CSF for 7 days. Monocytes from patients with COPD and controls were also obtained from centrifugation by a Ficoll gradient. The exposure to CS for 5 days induced a pronounced influx of neutrophils and macrophages in the lung associated with increased levels of keratinocyte chemoattractant (KC), tumor necrosis factor-α (TNF-α), nitric oxide (NO), and metalloproteinase from the matrix (MMP) -12. After 14 days of exposure to CS, neutrophil recruitment and cytokine production were reduced. After 30 days of exposure to CS, there was an increase in the recruitment of alveolar macrophages and transforming growth factor- β (TGF-β) production. CS also increased the Ly6Clow monocytes in both bone marrow and blood. Human macrophages derived from monocytes (MDM) were exposed to CSE at different concentrations (2% to 10%) alone or associated with LPS (0.1μg/mL) and IL-4 (10 ng/mL) for 24 hours and cytokine profile was evaluated by ELISA. CSE increased the IL-8/CXCL8 release in a dose-dependent manner, but only CSE 4% was able to increase the MDC/CCL22 release and MMP-12 expression. The challenge with CSE 4% and LPS increased the IL-8/CXCL8 production when compared to LPS only however, IL-6 and Gro -α/CXCL1 release were significantly reduced. We observed lower in macrophages stimulated with LPS. Pretreatment of MDM with JAK 2 inhibitor tyrphostin AG 490 (25 g/mL) for 1 hour decreased the IL-8/CXCL8 production induced by CSE 4%. We confirmed the results obtained in vitro and in vivo models with data obtained from monocytes from COPD patients. We observed a greater number of intermediate monocytes in COPD patients when compared to healthy donors, associated with a lower release of Gro-α/CXCL1 and a greater release of TARC/CCL17 and MDC/CCL22, however, a lower release of IL-8/CXCL8 was observed in COPD patients. Our results showed that, initially, CS induces a pro-inflammatory response marked by M1 cytokine production, however, the chronic exposure to CS favors the M2 cytokine production, inducing a profile e "like" M2 of the macrophages.