Abstract
Toll-like receptors (TLRs) play a crucial role for detecting conserved pathogen-associated molecular patterns comprising fungal structures. Activation of polymorphonuclear granulocytes (PMNs) as well as mobilization of antigen presenting cells is important for successful clearance of fungal infections. Here, the role of TLR2 and TLR4 for activation of PMNs and immature dendritic cells (iDCs) through Aspergillus fumigatus was analyzed.
Generation of iDCs was achieved by cultivating monocytes in the presence of GM-CSF and IL-4. PMNs were obtained from fresh blood. In blocking studies, cells were preincubated with anti-TLR2 and / or anti-TLR4 antibody before stimulation with Aspergillus fumigatus germinating conidia. For RNA interference (RNAi) experiments, iDCs were transfected with short-interfering RNA (siRNA) via electroporation and gene expression was controlled by quantitative real-time PCR.
Oxidative burst of PMNs was induced after contact with Aspergillus or TLR2 ligand zymosan. Release of oxygen intermediates could be significantly reduced if PMNs were preincubated with anti-TLR2 antibody.
In contrast to monocytes, cytokine secretion (IL-12 and TNF-α) of iDCs was not altered if anti-TLR antibodies were used. To study the role of TLR2 and TLR4 more detailed, an RNAi system for iDCs was established to downregulate gene expression. Determination of siRNA transfection rate revealed an efficiency of about 85% and allowed significant downregulation of TLR2 and TLR4 (> 90%). In accordance to blocking studies, expression of IL-10, IL-12 and TNF-a was not reduced after TLR2 or TLR4 siRNA transfection and stimulation with Aspergillus.
We conclude that TLR2 plays an important role in the induction of the oxidative burst of PMNs whereas cytokine release of iDCs seems to be independent of TLR2 / TLR4 signalling. The established siRNA system allows the detection of receptors responsible for activation of iDCs.
Disclosures: German Research Foundation.
Author notes
Corresponding author
