Modulation of Toll-like receptor 2 (TLR2) and TLR4 responses by Aspergillus fumigatus

Abstract

Toll-like receptor (TLR)-based signaling pathways in the host may be modulated by pathogens during the course of infection. We describe a novel immunomodulatory mechanism in which Aspergillus fumigatus conidia induce attenuation of TLR2- and TLR4-mediated interleukin (IL)-6 and IL-1beta proinflammatory responses in human mononuclear cells with suppression of IL-1beta mRNA transcription. Background TLR2 and TLR4 mRNA transcription was not influenced. A. fumigatus conidia induced TLR2 internalization and uptake into the phagosome with a resultant decrease in surface receptor expression. A. fumigatus hyphae, on the other hand, selectively downregulated the TLR4-mediated response. These novel immunosuppressive effects may facilitate the invasiveness of A. fumigatus.

FIG. 1.

Effects of preincubation of PBMC (a to h) or MDM (i and j) with heat-killed Aspergillus conidia (a to d, i, and j), hyphae (e and f), or live conidia (g and h) on the host TLR2 (Pam₃Cys)- and TLR4 (LPS)-mediated proinflammatory response, compared to results obtained with the corresponding unprimed controls (RPMI 1640). (a and b) Dose-dependent IL-6 response to TLR2 agonist and TLR4 agonist. Live Aspergillus conidia (g and h) resulted in IL-6 attenuation similar to that obtained with heat-killed components, but the TLR4-induced TNF-α response was attenuated with germination and hyphal development (as anticipated with heat-killed hyphae [f]). The data are the cumulative results of at least three sets of experiments and are means and standard errors of the means. *, P < 0.05 for a comparison with corresponding unprimed PBMC control. Asp, Aspergillus; HK, heat killed.

FIG. 2.

IL-1β and TNF-α mRNA expression in PBMC preincubated with A. fumigatus components for 24 h, followed by RPMI 1640, TLR2 ligand, or TLR4 ligand stimulation for 4 h. IL-1β mRNA expression was suppressed by Aspergillus conidia in the absence (RPMI 1640) and in the presence (Pam3Cys and LPS) of secondary stimulation. The data are the cumulative results of three sets of experiments and expressed as the means ± standard errors of the means. *, P < 0.05 relative to the ratio of the fold increase in the mRNA levels of unprimed cells. Asp, Aspergillus.

FIG. 3.

IL-6 production after TLR2 or TLR4 stimulation by PBMC preincubated with A. fumigatus conidia or hyphae in the presence or absence (Control) of cytochalasin B (CytoB) compared to the results for corresponding unprimed PBMC (RPMI 1640). Inhibiting phagocytosis reversed TLR2-induced IL-6 attenuation (a) but not TLR4-induced IL-6 attenuation (b). The data are the cumulative results of three sets of experiments and are means and standard errors of the means. *, P < 0.05 compared to the corresponding unprimed PBMC following secondary stimulation.

FIG. 4.

IL-6 production after TLR2 or TLR4 stimulation by PBMC preincubated with A. fumigatus conidia or hyphae in the presence or absence (Control) of the specified inhibitor compared to those for the corresponding unprimed PBMC (RPMI 1640). The inhibitors used were mannan (which blocks MR), laminarin (which blocks dectin-1), and Bartonella LPS (TLR4 antagonist). TLR4-mediated attenuation of IL-6 by A. fumigatus hyphae was reversed using mannan (b). Modulatory effects were not reversed by blocking the dectin-1 receptor or TLR4 (c to f). The data are the cumulative results of three sets of experiments and expressed as the means ± standard errors of the means. *, P < 0.05 compared to the respective unprimed PBMC. Bart, Bartonella.

FIG. 5.

Relative TLR2 and TLR4 mRNA expression in PBMC following incubation with Aspergillus conidia and hyphae. The data are the cumulative results of three sets of experiments and expressed as the means ± standard errors of the means. The values are not statistically significant. Asp, Aspergillus.

FIG. 6.

(a) Direct microscopic view of a monocyte with three phagocytosed Aspergillus conidia and an adherent conidium in the external milieu (arrow). (b) Blankophor staining of the same field, except that the externalized conidium (arrow) was fluorescent. (c and d) Light and fluorescent views of a monocyte labeled with Alexa 488-TLR2 MAb obtained by confocal microscopy, showing localization of TLR2 on cell surface. (e and f) Following phagocytosis of Aspergillus conidia (asterisk), concurrent internalization of TLR2 with the phagosome was observed.