DNA and RNA are then detected by, respectively, TLR7 and TLR9 and trigger MyD88- and IRF1-dependent responses. Interestingly, our data indicating that actin polymerization and phagocytosis are not required for dectin-1-dependent cytokine induction (e.g. for S. cerevisiae-induced TNF-α secretion) are in agreement with a recent report showing that dectin-1 can be activated by β-glucan immobilized on a nonphagocytosable surface (such as a culture plate), occurs prior to initiation of phagocytic cup formation and BEZ235 ic50 is not dependent on actin dynamics [55]. Altogether our data, pointing to the importance in anti-fungal defenses of the latter pathway, may be useful to better
understand the strategies used by C. albicans to evade the innate immune system and to devise alternative Alvelestat nmr therapeutic strategies. Knock-out mice were originally obtained from T. Taniguchi (IRF1−/−, IRF3−/−, and IRF7−/−), and S. Akira (TLR2−/−, TL3−/−, TLR4−/−, TLR7−/−, TLR9−/−, MyD88−/−, Mal−/−, TRAM−/−
and TRIF−/−) as previously described [29]. Dectin 1−/−, 3d and TLR7/9 double Ko (TLR7−/−/TLR9−/−) mice were provided by, respectively, G. Brown [11], B. Beutler [35] and S. Bauer. C57BL/6 WT mice, used as controls, were purchased from Charles River Laboratories (Calco, Italy). The mice were housed and bred under pathogen-free conditions in the animal facilities of the Elie Metchnikoff Department, University of Messina. All studies were performed in agreement with the European Union guidelines of animal care and were approved by the Ethics Committee of the Metchnikoff Department of the University of Messina (CESA) and by the relevant national authority (Istituto Superiore di Sanità). C. albicans (ATCC 90028) was purchased from the American Type Culture Collection. S. cerevisiae strain A11 was isolated in the clinical mycology
laboratory of the Elie Metchnikoff Department, University Rho of Messina [22]. For in vivo and in vitro experiments, these two strains were grown in a chemically defined medium as previously described [22]. CFU numbers used in each experiment were determined after plating on Sabouraud dextrose agar (Difco Laboratories). Heat killed C. albicans strains were prepared as previously described [22], followed by washing with PBS and resuspension to the original volume. Depleted zymosan (i.e. hot alkali-treated zymosan, which is devoid of TLR-dependent stimulating properties) and control stimuli (poly I:C, Escherichia coli ultrapure LPS, CpG B, CL264) were purchased from InvivoGen. Curdlan was purchased from Wako Pure Chemicals and detoxified using cold NaOH treatment [22]. Fungal cell extracts were obtained by vortexing of C. albicans (grown in the mid-log phase) in the presence of glass beads 425–600 μm in diameter (Sigma).
No related posts.