Epithelial cytokine induction is independent of TLR2, TLR4, dectin-1 and MR.

<p>Human epithelial cells (1×10⁶) were pre-incubated with (<b>A</b>) 10 µg/ml anti-TLR2, anti-TLR4, anti-MR antibodies, laminarin (100 µg/ml) or <i>S. cerevisiae</i> mannan (40 µg/ml) 2 h before epithelial cells were stimulated with <i>C. albicans</i> walls (1×10⁸) for 24 h. (<b>B</b>) Oral epithelial cells (3×10⁵) isolated from wild-type and MyD88−/− mice were incubated for 24 h with isolated walls (3×10⁷). (<b>C</b>) Human epithelial cells (1×10⁶) were incubated with 5 µM cytochalasin D for 30 min prior stimulation with <i>C. albicans</i> walls (1×10⁸) for 24 h (<b>D</b> and <b>E</b>) Cell wall mannoproteins were deproteinized by incubating <i>C. albicans</i> walls (1×10⁸) with proteinase K or deglycosylated by PNGaseF digestion (cleaves <i>N-</i>glycosylation) or NaOH treatment (alkaline β-elimination reduces <i>O</i>-glycosylation). Human epithelial cells (1×10⁶) were incubated for 24 h with isolated walls (as positive control) or proteinase K-, PNGaseF- and NaOH-treated walls. (<b>F</b> and <b>G</b>) Epithelial cells (1×10⁶) were incubated for 24 h with cell walls (1×10⁸) isolated from <i>C. albicans</i> wild type (SC5314), <i>N-</i>glycosylation (<i>och1Δ</i>), <i>O</i>-glycosylation (<i>mnt1Δ/mnt2Δ</i>), <i>N−/O</i>-glycosylation (<i>pmr1Δ</i>) mutant strains or non- pathogenic <i>S. cerevisiae</i>. Human GM-CSF and mouse MIP-2 were quantified by ELISA. TLR4 mRNA up regulation in epithelial cells was determined by quantitative RT-PCR. Data are given as relative mRNA expression compared to mRNA expression of PBS-treated control cells (control = 1.0). (<b>A–G</b>), <i>n</i> = 3 (± <i>SEM</i>), <i>* p</i><<i>0.05, **p<0.01</i>, 2-tailed paired Student’s <i>t</i> test.