DIFFERENTIAL ACTIVATION OF INNATE IMMUNE CELLS BY Candida spp. BIOFILMS

JULIO E ARCE MIRANDA, MARÍA A DA SILVA, JOSÉ BARONETTI, GIULINA MOSCONI , PAULA ICELY, CLAUDIA E SOTOMAYOR, MARÍA G PARAJE.

Rosario, Santa Fe

Congreso; IX Congreso Argentino de Microbiología General; 2013

Sociedad Argentina de Microbiología General

Candida spp. is the major fungal pathogen of humans causing a variety of afflictions. One of the defense mechanisms of this fungus is the capacity to form biofilms. On the other hand, macrophages (Mø) are very important in the control of infections. These cells are capable of phagocytizing and killing microorganisms and secrete an array of cytotoxic products such as hydrogen peroxide and nitric oxide (NO). Therefore, the interaction between biofilms and these immune cells is determinant to the course of the infection. The aim of this study was to investigate the interaction between biofilm and Mø. To this purpose, two models of biofilm-Mø interaction performed and four pathogenic Candida spp. strains were used, and different activation pathways such as NO release and the arginase one, the production of reactive oxygen species (ROS), the levels of antioxidant defenses and the cytokine liberation were evaluated. A murine macrophage-like cell line (RAW 264.7) was attached on the 96 wells plates and then incubated with Candida albicans and Candida no albicans strains at ratio of 1:3 (Mø-Ca) at 37°C in a humid atmosphere for 24 h (Model 1). On the other hand, mature biofilms was used and MØ were added to interact for 24 h (Model 2). As positive and negative controls, cells were incubated with 1µg/ml lipopolysaccharides (LPS) from Escherichia coli or RPMI 1640 medium, respectively. The supernatant of the different co-cultures was separated for extracellular nitrosative stress, which was evaluated as nitrite by a microplate assay method using Griess reagent. For assess the arginase activity, cells were used and one unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1 µmol of urea per min. The supernatant was separated by measuring the superoxide anion (O2?-) production by the reduction of the nitro-blue tetrazolium (NBT) reaction; total superoxide (SOD) activity was assayed photochemically based on the inhibition of NBT reduction; and the total system antioxidant capability was determined through FRAP assay (Ferrous Reduction Antioxidant Potency). Cells were used for quantify catalase (CAT) activity with H2O2, potassium dichromate (K2Cr2O7) in solution in glacial acetic acid. The levels of IL-6, TNF-α, and TGF-β in culture supernatants were determined by ELISA. Our findings show that biofilms are potent stimuli for catabolism of arginine via arginase, but trigger dissimilar profiles for NO synthesis via inducible NO synthase (iNOS) in both models. We also observed in these co-cultures, the enhanced production of TGF-β (Model 2). On the other hand, the production of ROS only was observed in the Model 1. The levels of antioxidant defenses were not observed. These results contribute to a better understanding of the interaction between biofilms and cells of the immune system, which may help to clarify the relevance of biofilms as virulence factors in the immune-pathogenesis of Candida infections.