The role of SOCS and STATs proteins in the differentiation of TCD4+ naive cells in an experimental model of Asthma and Chronic Obstructive Pulmonary Disease

• Grant number: 21/02477-5
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: May 01, 2021
• End date: October 31, 2022
• Field of knowledge: Biological Sciences - Physiology - Physiology of Organs and Systems
• Principal Investigator: Fernanda Degobbi Tenorio Quirino dos Santos Lopes
• Grantee: Juliana Dias Lourenço
• Host Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 18/02537-5 - Characterization of asthma-COPD overlap syndrome (ACOS): experimental and clinical studies, AP.TEM

Abstract

Although asthma and chronic obstructive pulmonary disease are described as distinct clinical entities, more recently, discussions about when COPD and asthma are expressions of the same disease that affects the respiratory system have intensified (1). The use of animal models helps to better understand the pathophysiological mechanisms involved in the development of different diseases, including asthma and COPD. To date, few studies have described the use of animals to simulate the overlap of asthma and COPD, as well as the role of adaptive immunity in this process. Generally, asthmatic patients have a Th2 response profile, with increased TCD4 + cells and increased eosinophils around the airways, while COPD patients have a Th1 immune response profile, increased TCD8 + cells and increased neutrophils (3; 4 ). Our group has already demonstrated in studies of an experimental model of COPD and in tissues of COPD patients, the role of the TH17 response in this disease progession with decreased Tregulatory cells. Aims: Our aim in this study is to evaluate the role of adaptive immunity in in an asthma and COPD overlap model, and more specifically the role of Th17 and Th2 response in this process, as well as the involvement of the intracellular STAT "Signal Transducer and Activator of Transcription") and SOCS ("Suppressor Of Cytokine Signaling") proteins in the differentiation of TCD4+ naive cells into their subtypes. Work Plan and Methodology: Male BALB/c mice (average weight 26g), from the Bioterio Central from Medical School of University of Sao Paulo (FMUSP), will be used. The animals will be submitted to the Asthma and Emphysema Overlap Induction protocol; experimental groups: A) SF group: Inhalations with sterile saline (SF) (n = 16); B) OVA group: Inhalations with ovoalbumin solution (OVA) (n = 16); C) ELA group: intratracheal instillations of elastase (n = 16); D) ACOS group: Inhalations with OVA solution and intratracheal instillations of elastase (n = 16). Collection and processing of the pulmonary tissue and blood samples: The animals in each group will be anesthetized with Thiopental (50 mg/kg, intraperitoneally) to draw blood through the abdominal vena cava. Then, still anesthetized, the animals will be euthanized through the exsanguination of this same route to remove the lungs. The removed lung tissue will be frozen in liquid nitrogen and then kept in a -70ºC temperature and will only be defrosted by the time to perform the analyzes. The blood samples will be centrifuged and the supernatant will be recovered and storage in a -70ºC temperature (Toledo et al., 2013). Immunoblotting of the pulmonary tissue: As described in project 1, but using the primary antibodies for SOCS 1, 3 and STATS 3, 5, 6 and beta-actin as endogenous control. Immunohistochemistry of the pulmonary tissue: Method as described in project 1, but using the following primary antibodies: Stat3 (F-2), p-Stat3 (B-7), Stat 5, SOCS-1 (H93), SOCS-3 ( H-103), IL-17, FOXP3, Stat6, pStat6, IL-4 and IL-13. After the specific staining, morphometric analyzes will be performed to quantify the density of positive cells by tissue area. ELISA: As described in project 1: Specific kits for mice from R&D Systems (Boston, USA), for detection of IL-6 (M6000B), IL_10 (M1000B), IL-17 (M1700), TGF-beta (MGD150), IL-4 (MAB404R) and IL-13 (M1300 CB) will be used. STATISTICAL ANALYSIS: Statistical analyzes will be performed using the Sigma Stat 11.0 software, in which the T-student test will be applied. A value of p <0.05 will be considered statistically significant. (AU)