Evaluation of the immunomodulatory and antifungal effects of caffeic acid (CA), caffeic acid phenethyl ester (CAPE) and artepillin C (ARC) on C. albicans and development of candidiasis in in vivo model hosts

Abstract

Currently, treatment options for fungal infections, such as oral candidosis, are limited due to the low availability of antifungal drugs and the emergence of drug resistant strains. Thus, the development of new antifungal compounds and new therapeutic approaches are of great need for the control of these infections. Some constituents of propolis such as caffeic acid (CA), caffeic acid phenethyl ester (CAPE) and artepillin C (ARC) are considered to be promising antimicrobial compounds because they have antifungal and antibacterial activity, as well as immunomodulatory and anti-inflammatory action by inhibition of NF-ºB transcription factor and enzymes involved in the inflammatory process. Therefore, the present work aims to determine if CA, CAPE and ARC are able to stimulate the immune system against C. albicans and inhibit the development of candidosis in experimental models in vivo. Initially, 40 clinical strains of C. albicans, isolated from the oral cavity of patients with Human Immunodeficiency Virus (HIV), will be submitted to the Minimum Inhibitory Concentration (MIC) test for fluconazole, amphotericin, CA, CAPE and ARC. The antifungal effect of these compounds will be verified in two strains resistant to fluconazole through the in vitro biofilm formation and filamentation tests. Also in vitro, the immunomodulatory action of the compounds will be investigated using Candida-activated RAW264.7 cells for quantification of cytokines by fluorescently labeled beads. The prophylactic and therapeutic effects of CA, CAPE and ARC will be investigated in the experimental model in Galleria mellonella through the analysis of the survival curve, hemocyte counts, cellular characterization of hemocytes by flow cytometry, C. albicans counts in the fat body and the expression of antimicrobial peptides (galiomycin, galleriomycin, cepropin D), phagocytosis (C8-Contig 1910) and the regulation of cytokines and the NF-kB cascade (C7 Contig 15362), present in the immune response of this invertebrate. Therefore, the compound with the best antifungal and immunomodulatory effect in the previous tests will be selected for the purpose of expanding and proving such effects in the experimental model of induced oral candidiasis in mice. This compound will be applied to candidiasis lesions induced on the back of the animals tongue. The development of oral candidiasis will be monitored by means of recovery of C. albicans from the oral cavity of the animals, macroscopic evaluation, histological analysis and gene expression of ²-defensins 1 and 3. The results will be submitted to an exploratory analysis for the selection of most appropriate statistical test for each experiment in this study (P d 0.05).