Participação da enzima fosfatidilinositol-3-quinase gama (PI3Kγ) em parâmetros clínicos e neuroinflamatórios da encefalomielite autoimune experimental

Abstract

Introduction: Multiple sclerosis (MS) is an autoimmune inflammatory disease that affects the central nervous system (CNS). The Encephalomyelitis Autoimmune Experimental (EAE) is an animal model for the study of MS. The enzyme phosphatidylinositol-3-kinase-γ (PI3Kγ) plays an important role in the motility and survival of leukocytes in various inflammation models. Objective: To evaluate the role of the enzyme PI3Kγ in the development of the immune response of EAE and also, if the inhibition of this pathway would be neuroprotective in the disease. Methods: Wild-type (WT) or PI3Kγ (KO) deficient C57BL/6 female mices were immunized with the Oligodendrocyte Myelin Glycoprotein Peptide (MOG35-55) in Freund's complete adjuvant, supplemented with Mycobacterium tuberculosis H37RA and still, pertussis toxin. First, the EAE was evaluated until the 21st day, and in the 14th and 21st dpi groups of animals were euthanized. The brains, spinal cords and inguinal lymph nodes were removed and submitted to histopathological analysis by H & E staining to cellular infiltration and Luxol Fast-Blue staining to CNS demyelination process. Through ELISA, the IFN-γ, IL-17, IL-6, TGF-β and IL-10 cytokines, CCL5 and CCL20 chemokines levels were measured. Microglial and astrocyte proliferation by labeling for Iba+ and GFAP+, respectively, by immunofluorescence and labeling of apoptotic cells in the spinal cords, by TUNEL, were evaluated. EAE were treated with 1mg / kg FTY720 or its analogue, by gavage, from 10th to 20th dpi. At 21º dpi, the animals were euthanized for histopathological analyzes, cytokines levels by ELISA, in the brains, spinal cords and lymph nodes, and the evaluation of protein phosphorylation by Western Blotting. Results: WT-EAE animals had the peak of EAE around the 19th dpi, whereas those of KO-EAE present a delay in the development of EAE with the peak of EAE reduced on 20º dpi, in relation to the WTEAE group. Reduction of infiltrates and foci of demyelination was associated with a lower production of CCL5, CCL20, IFN-γ, IL-17 in the CNS of KO-EAE mice at 14th and 21st dpi, in relation to the WT-EAE group. The KO-EAE group had lower microglial and astrocytic proliferation at 14ºdpi in relation to the WT-EAE group. In the inguinal lymph nodes, both proinflammatory cytokine production and chemokines were lower in the KO-EAE group than in the WT-EAE group at the two evaluated points. A larger number of apoptotic cells was observed in the CNS of WT-EAE animals at both points. Treatment with FTY720 or its analogue was able to reduce the worsening of clinical signs of EAE, infiltration of inflammatory cells, demyelination, proinflationary cytokines and also, phosphorylation of Akt, which became a less activation of the pathway PI3K/Akt/mTOR. Conclusion: The results indicate that PI3K is importante to survival, proliferation and migration of the leukocytes in EAE. In addition, compounds FTY720 and its lipophilic analog have been able to reduce the neuroinflammation caused by this disease in the CNS.