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Immunomodulatory effect of the probiotic Lactobacillus rhamnosus on macrophages stimulated by LPS in the production of TNF-±, IL-1 ², IL-4, IL-6, IL-10, IL-12 and nitric

Grant number: 12/24904-3
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): March 01, 2013
Effective date (End): February 28, 2014
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Principal Investigator:Luciane Dias de Oliveira
Grantee:Bruna Costa de França Marques
Home Institution: Instituto de Ciência e Tecnologia (ICT). Universidade Estadual Paulista (UNESP). Campus de São José dos Campos. São José dos Campos , SP, Brazil

Abstract

Increasing evidence justify the use of probiotic microorganims in functional foods, dairy products or other dietary supplements for the maintenance and promotion of human health, with a renewed interest in the use of probiotics, which are capable of modulating the immune response cells under microorganisms cell wall antigenic stimulation. It is known that lipopolysaccharide (LPS) is a potent cellular activator and that probiotics help the nonspecific phagocytic activity of macrophages as well as the activation and production of proinflammatory and anti-inflammatory properties, it is of great importance to assess their role in the LPS-activated macrophages, once the immunomodulatory effects of probiotics are not yet clearly defined and the understanding of its mechanism of action in the immune response will enable better indication of its therapeutic use. The purpose of this study is evaluating the immunomodulatory effect of the probiotic Lactobacillus rhamnosus or their products on macrophages (RAW 264.7) activated by LPS with the production of proinflammatory and anti-inflammatory (TNF-±, IL-1 ², IL-4, IL-6, IL-10, IL-12) and nitric oxide. It will be used the standard strain of L. rhamnosus ATCC 7469, seeded Agar Man-Rogosa-Shape (MRS) and grown at 37° C / 5% CO2 for 24 h. It will be used three different preparations of probiotics: 1) Alive probiotic (PV) at a concentration of 5 x 107 cells/mL in standardized in spectrophotometer, 2) Dead probiotic neutral (PM) obtained from previous preparation by autoclaving at 121° C/15 min, 3) Supernatant (SP) of the probiotic suspension, obtained by centrifugation of the PM suspension at 5000 rpm/10 min. It will be used mouse macrophages (RAW 264.7) grown in complete DMEM medium (supplemented with 10% fetal bovine serum). In order to perform the tests, these cells will be distributed in polystyrene microplates at a concentration of 1 x 106 viable cells/well/ml complete DMEM medium. After 24 h incubation (37° C/5% CO2) for cell adhesion, plates will be washed with sterile and nonpyrogenic PBS and cells will be stimulated with Escherichia coli LPS at a concentration of 1 mg/mL and the cells will receive, at the same time, different preparations of probiotics. The plates will be incubated for 8 h (37° C/5% CO2). Culture supernatants will be collected for the detection and quantification of cytokines (TNF-±, IL-1², IL-4, IL-6, IL-10 and IL-12) by immunoenzymatic method (ELISA) and nitric oxide by Griess method. The results will be statistically analyzed (ANOVA and Tukey test, 5%).