Ficolins Promote Fungal Clearance in vivo and Modulate the Inflammatory Cytokine Response in Host Defense against Aspergillus fumigatus

Abstract

Aspergillus fumigatus is an opportunistic fungal pathogen that causes severe invasive infections in immunocompromised patients. Innate immunity plays a major role in protection against A. fumigatus. The ficolins are a family of soluble pattern recognition receptors that are capable of activating the lectin pathway of complement. Previous in vitro studies reported that ficolins bind to A. fumigatus, but their part in host defense against fungal infections in vivo is unknown. In this study, we used ficolin-deficient mice to investigate the role of ficolins during lung infection with A. fumigatus. Ficolin knockout mice showed significantly higher fungal loads in the lungs 24 h postinfection compared to wild-type mice. The delayed clearance of A. fumigatus in ficolin knockout mice could not be attributed to a compromised recruitment of inflammatory cells. However, it was revealed that ficolin knockout mice exhibited a decreased production of proinflammatory cytokines in the lungs compared to wild-type mice following A. fumigatus infection. The impaired clearance and cytokine production in ficolin knockout mice was independent of complement, as shown by equivalent levels of A. fumigatus-mediated complement activation in ficolin knockout mice and wild-type mice. In conclusion, this study demonstrates that ficolins are important in initial innate host defense against A. fumigatus infections in vivo.

Fig. 1

Increased pulmonary fungal loads in ficolin-deficient mice 24 h after A. fumigatus infection. WT and ficolin KO mice were intranasally inoculated with 2 × 10⁷A. fumigatus conidia. a Mice were sacrificed at the indicated time points for quantification of the fungal load (CFU/lung) in whole-lung homogenates. b Weight changes in mice during A. fumigatus infection. Results (mean ± SD) were obtained from two independent experiments (n = 5-7/group in each experiment). * p < 0.05 for ficolin KO to WT comparisons at each time point (unpaired two-tailed Student's t test).

Fig. 2

Lung histology of mice following A. fumigatus infection. WT and ficolin KO mice were intranasally inoculated with PBS as controls (a) or 2 × 10⁷A. fumigatus conidia (b) and sacrificed 24 h p.i. Lung tissues were harvested, fixed, and HE stained for histological examination. Representative lung sections from WT or ficolin KO mice are shown. Images are representative of 2 mice in PBS mock-infected groups and 6 mice in the A. fumigatus-infected groups obtained from one experiment.

Fig. 3

Lung MPO concentration following A. fumigatus infection. WT and ficolin KO mice were intranasally inoculated with 2 × 10⁷A. fumigatus conidia and sacrificed 24 h p.i. Control mice received PBS. Lung tissues were harvested and the MPO concentration in supernatants of lung homogenates was measured by ELISA. Results represent means ± SEM from one experiment with n = 2 (PBS group) or n = 6 (A. fumigatus group).

Fig. 4

Impaired production of proinflammatory cytokines in the lungs of ficolin-deficient mice following A. fumigatus infection. WT and ficolin KO mice were intranasally inoculated with 2 × 10⁷ A. fumigatus conidia and sacrificed 24 h p.i. Control mice received PBS. a Lungs were removed and the expression of proinflammatory cytokines in lung homogenates was measured by qPCR; data represent means ± SEM as the fold increase over the control group (WT mock infected with PBS). b In separate experiments BALF was collected and cytokine concentrations in BALFs were measured by ELISA. The figures illustrate results from one experiment, representative of two independent experiments showing similar results, with n = 2 (PBS group) or n = 6 (A. fumigatus group) in each experiment. * p < 0.05 for ficolin KO to WT comparisons (unpaired two-tailed Student's t test).

Fig. 5

Complement deposition in the lungs of mice following A. fumigatus infection. WT and ficolin KO mice were intranasally inoculated with PBS as controls (a) or 2 × 10⁷A. fumigatus conidia (b) and sacrificed 24 h p.i. Lungs were harvested, fixed, and immunohistochemically stained with an anti-C3d antibody (upper panels) or a control antibody (lower panels). Sections were counterstained with hematoxylin. Representative lung sections from WT or ficolin KO mice are shown. Images are representative of 2 mice in the PBS mock-infected group and 6 mice in the A. fumigatus-infected group obtained from one experiment.

Fig. 6

Complement activation in lungs of mice following A. fumigatus infection. WT or ficolin KO mice were intranasally inoculated with 2 × 10⁷A. fumigatus conidia and sacrificed 24 h p.i. Control mice received PBS. The BALF was collected and C3a concentration in BALFs was measured by ELISA. Results were obtained from two experiments (n = 4-8 mice in the A. fumigatus groups per experiment).