Involvement of CD14 and toll-like receptors in activation of human monocytes by Aspergillus fumigatus hyphae

Invasive fungal infections represent an increasing problem associated with high mortality. The present study was undertaken to identify leukocyte subs ets that are activated by hyphal fragments in a whole-human-blood model, as well as to examine the involvement of CD14 and Toll-like receptors (TLRs) in activation of monocytes by hyphae. Incubation of whole human blood with hyphal fragments from Aspergillus fumigatus and Scedosporium prolificans fo r 6 h caused induction of mRNAs for tumor necrosis factor alpha (TNF-alpha) , interleukin-1 beta (IL-1 beta), and IL-6 in T cells, B cells, and monocyt es, but not in granulocytes, as analyzed by reverse transcription-PCR with mRNA isolated from very pure populations of these leukocyte subsets. In pri mary adherent human monocytes, induction of TNF-alpha by hyphal fragments w as dependent on plasma. Heat treatment of plasma at 56 degreesC for 30 min strongly reduced the ability of plasma to prime for activation. Pretreatmen t of human monocytes with different concentrations (1, 3, and 10 mug/ml) of monoclonal antibody (MAb) HTA125 (anti-TLR4) or MAb 18D11 (anti-CD14) for 30 min inhibited the release of TNF-alpha. induced by hyphal fragments in a dose-dependent manner. Maximal inhibitions of 35 and 70% were obtained wit h 10 mug of HTA125 and 18D11 per mi, respectively. In contrast, pretreatmen t with MAb TL2.1 (anti-TLR2) did not affect signaling induced by hyphae. Pr etreatment with the lipid A antagonist B975 blocked lipopolysaccharide sign aling but did not inhibit TNF-alpha production induced by hyphal fragments. Our results suggest that T cells, B cells, and monocytes are involved in t he innate immune response to invasive fungal pathogens and that serum compo nents are relevant for activation of monocytes by hyphae. CD14 and TLR4 may be involved in signaling of Aspergillus hyphae in monocytes, but further s tudies to elucidate this issue are warranted.