Involvement of toll-like receptor 2 in experimental invasive pulmonary aspergillosis

Abstract

Aspergillus fumigatus, an opportunistic fungal pathogen, causes severe and usually fatal invasive pulmonary aspergillosis in immunocompromised hosts. Interestingly, Drosophila cells lacking the Toll protein are prone to A. fumigatus infection. In the current study, we looked for the involvement of Toll-like receptor 2 (TLR2) in the recognition of A. fumigatus by analyzing the in vivo and ex vivo responses of immunocompromised TLR2(-/-) and TLR2(+/+) mice to this fungus. Upon intratracheal administration of conidia, survival and tumor necrosis factor alpha (TNF-alpha), interleukin-12, and macrophage inhibitory protein-2 alpha concentrations in the airspaces of TLR2(-/-) mice were significantly lower than those of TLR2(+/+) animals. In vitro analysis of TNF-alpha production by conidia-challenged alveolar macrophages from TLR2(-/-) revealed a significant deficiency in comparison with macrophages from TLR2(+/+) mice. Infected TLR2(-/-) mice also have a higher respiratory distress and a higher pathogen burden than TLR2(+/+) mice. These data demonstrate that TLR2 plays a significant role in the defense of the host against A. fumigatus infection.

FIG. 1.

Lethality induced by A. fumigatus conidia in TLR2^−/− and TLR2^+/+ mice. Age-matched TLR2^−/− (n = 22) and TLR2^+/+ (n = 24) male mice received 3 × 10⁶ conidia through the intratracheal route. Mortality was assessed daily for 10 days. Wilcoxon test for comparisons of Kaplan-Meier survival curves indicated a significant decrease in the survival of TLR2^−/− mice compared to that of TLR2^+/+ mice (P = 0.0005).

FIG. 2.

TNF-α, IL-12, and MIP-2α recovered from the BALF of A. fumigatus-infected TLR2^−/− and TLR2^+/+ mice. Age-matched TLR2^−/− and TLR2^+/+ male mice received 3 × 10⁶ conidia (A. fumigatus +) or an equivalent volume of the conidium vehicle (A. fumigatus −) through the intratracheal route. After 24 h, BALF were collected and TNF-α, IL-12, and MIP-2α were assayed in the cell-free supernatants. Results are expressed as the means ± SEM obtained from three distinct animals. *, P < 0.05.

FIG. 3.

TNF-α production by A. fumigatus-activated alveolar macrophages collected from TLR2^−/− and TLR2^+/+ mice. Alveolar macrophages collected from TLR2^−/− (n = 3) and TLR2^+/+ (n = 3) male mice were incubated or not (CONTROL) with an equivalent number of swollen conidia (A. fumigatus) for 6 h. Culture supernatants were then assayed for TNF-α concentrations. Results are expressed as means ± SEM of three distinct experiments, each one performed with a pool of cells collected from three mice, allowing us to perform each point in triplicate. *, P < 0.05.

FIG. 4.

Lung histology from A. fumigatus-infected TLR2^−/− and TLR2^+/+ mice. Lung sections from mice 48 h after intratracheal inoculation of 3 × 10⁶ conidia. (A) TLR2^+/+ mouse with hematoxylin-eosin stain at a magnification of ×40; (B) TLR2^−/− mouse with hematoxylin-eosin stain at a magnification of ×40; (C) TLR2^+/+ mouse with hematoxylin-eosin stain at a magnification of ×200; (D) TLR2^−/− mouse with hematoxylin-eosin stain at a magnification of ×200; (E) TLR2^+/+ mouse with methenamine silver stain at a magnification of ×40; (F) TLR2^−/− mouse with methenamine silver stain at a magnification of ×40; (G) TLR2^+/+ mouse with methenamine silver stain at a magnification of ×200; (H) TLR2^−/− mouse with methenamine silver stain at a magnification of ×200.

FIG. 5.

Chitin and galactomannan content of lungs and/or serum from A. fumigatus-infected TLR2^−/− and TLR2^+/+ mice. Age-matched TLR2^−/− (n = 4) and TLR2^+/+ (n = 4) male mice received 3 × 10⁶ conidia through the intratracheal route. After 24 and 48 h, lungs and blood were collected and chitin-derived glucosamine and/or galactomannan contents were assayed. Background glucosamine values obtained from the lungs of noninfected animals have been subtracted. Results are expressed as the means ± SEM (n = 4). Significant differences (*, P < 0.05) were observed between TLR2^−/− and TLR2^+/+ mice for the three studied parameters and time points, except for galatomannan measured at 24 h in serum (NS, P > 0.05).