H-ficolin binds Aspergillus fumigatus leading to activation of the lectin complement pathway and modulation of lung epithelial immune responses

Abstract

Aspergillus fumigatus is an opportunistic fungal pathogen that typically infects the lungs of immunocompromised patients leading to a high mortality. H-Ficolin, an innate immune opsonin, is produced by type II alveolar epithelial cells and could participate in lung defences against infections. Here, we used the human type II alveolar epithelial cell line, A549, to determine the involvement of H-ficolin in fungal defence. Additionally, we investigated the presence of H-ficolin in bronchoalveolar lavage fluid from transplant patients during pneumonia. H-Ficolin exhibited demonstrable binding to A. fumigatus conidia via l-fucose, d-mannose and N-acetylglucosamine residues in a calcium- and pH-dependent manner. Moreover, recognition led to lectin complement pathway activation and enhanced fungal association with A549 cells. Following recognition, H-ficolin opsonization manifested an increase in interleukin-8 production from A549 cells, which involved activation of the intracellular signalling pathways mitogen-activated protein kinase MAPK kinase 1/2, p38 MAPK and c-Jun N-terminal kinase. Finally, H-ficolin concentrations were significantly higher in bronchoalveolar lavage fluid of patients with lung infections compared with control subjects (n = 16; P = 0·00726). Receiver operating characteristics curve analysis further highlighted the potential of H-ficolin as a diagnostic marker for lung infection (area under the curve = 0·77; P < 0·0001). Hence, H-ficolin participates in A. fumigatus defence through the activation of the lectin complement pathway, enhanced fungus-host interactions and modulated immune responses.

Keywords: Aspergillus; complement; ficolin; innate immunity.

Figure 1

Binding characteristics of H-ficolin to Aspergillus fumigatus conidia. H-Ficolin (18·4 μg/ml) was incubated with 5 × 10⁵ conidia in the presence or absence of calcium, in a range of pHs from pH 3·7 to 10·7, or following pre-incubation with a variety of carbohydrates. (a) Representative histogram of H-ficolin binding to A. fumigatus conidia in the presence of calcium. (b) Representative histogram of H-ficolin binding to A. fumigatus conidia in the absence of calcium. (c) Binding of H-ficolin to A. fumigatus conidia in the presence and absence of calcium indicating the median fluorescence intensity (MFI). (d) Binding of H-ficolin to A. fumigatus conidia in a range of pHs from pH 3·7 to pH 10·7. (e) Carbohydrate binding specificity of H-ficolin to A. fumigatus conidia. H-Ficolin was pre-incubated with a range of carbohydrates (glucose, galactose, l-fucose, d-mannose and N-acetylglucosamine) at various concentrations (0–100 mm) before conidial binding at pH 5·7. Results are representative of the average of all the data points gained from three independent experiments. Error bars represent the SD and significance was determined via two-tailed Students t-test. For carbohyrdrate inhibition, significance was determined via one-way analysis of variance and post hoc analysis was conducted using the Tukey test. *P < 0·05.

Figure 2

H-Ficolin opsonization enhances association of Aspergillus fumigatus conidia with A549 epithelial cells. FITC-labelled A. fumigatus conidia (5 × 10⁵) were opsonized with 18·4 μg/ml H-ficolin before incubation with A549 cells (conidia : A549 ratio of 5 : 1) in pH 5·7 and pH 7·4 conditions for 16 hr. (a) P1 gate on the A549 population. (b) Representative histogram depicting the fluorescence (FL1-A) of the A549 population following challenge with non-opsonized or H-ficolin opsonized A. fumigatus conidia. (c) The relative number of associated FITC-labelled conidia (based upon the median fluorescence intensity (MFI) either non-opsonized (− H-ficolin), BSA-opsonized (+ BSA) or H-ficolin opsonized (+ H-ficolin). Results are representative of the average of all the data points gained from three independent experiments. Error bars represent the SD and significance was determined via two-tailed Students t-test. *P < 0·05.

Figure 3

H-Ficolin activates the lectin complement pathway on Aspergillus fumigatus conidia. The restoration of lectin complement pathway C3 deposition was observed on A. fumigatus following the reconstitution of ficolin-depleted human serum with recombinant H-ficolin. Serum dilutions of serum were incubated in Maxisorb microtitre 96-well coated plates and deposited C3 was detected by using rabbit anti-human C3c complement antibodies and goat anti-rabbit IgG-alkaline phosphatase conjugate. N-acetyl BSA was used as a positive control for C3 deposition (*P < 0·05). HS, human serum; HF, H-ficolin; AF, A. fumigatus; NBSA, N-acetyl BSA; rHF, recombinant H-ficolin. All data points are represented as mean absorbance measured at 405 nm in the presence of 1·25% serum and are representative of the data obtained from three independent experiments. Significance was determined via one-way analysis of variance and post hoc analysis was determined using the Tukey test.

Figure 4

Interleukin-8 (IL-8) secretion from A549 epithelial cells is enhanced following challenge with H-ficolin opsonized Aspergillus fumigatus conidia and is reliant upon mitogen-activated protein kinase (MAPK) kinase 1/2 (MEK 1/2), p38 MAPK and c-Jun N-terminal kinase (JNK) activation. (a) Supernatants from A549 cells were collected 24 hr after challenge with H-ficolin (18·4 μg/ml) alone, conidia (1 × 10⁶) alone or H-ficolin (18·4 μg/ml) -opsonized A. fumigatus (1 × 10⁶ conidia), before cytometric bead array. (b) To investigate the cell signalling pathways involved, A549 cells were pre-incubated with 10 μm of the MEK 1/2 inhibitor (U0126), the p38 MAPK inhibitor (SB202190) or the JNK inhibitor (SP600125) before A. fumigatus challenge. Media from A549 cells without stimulus and lipopolysaccharide (LPS) (100 ng/ml; grey bar) were used as negative and positive controls, respectively. DMSO was used as a vehicle control for MAPK inhibitors (white bar). All data are represented as the average concentration of IL-8 produced (pg/ml) and are representative of the averages of all the data points gained from three independent experiments. Error bars represent the SD. Significance was determined via two-tailed Students t-test. *P < 0·05.

Figure 5

Bronchoalveolar lavage (BAL) H-ficolin concentrations from infected lung transplant patients are higher than those found in non-infected patients. The BAL fluid was collected following bronchoscopies on lung transplant patients before the measurement of H-ficolin concentrations by ELISA. (a) The concentration of H-ficolin (ng/ml) in BAL samples. BAL samples were considered positive (n = 16) or negative (n = 16) for infection dependent upon patients classification according to EORTC/MSG criteria, Aspergillus antigen detection, radiology and bacterial, viral or fungal cultures. (b) H-Ficolin concentration in the BAL fluid of patients testing culture positive for Penicillium (Pen; n = 1), Pseudomonas aeuriginosa (PsA; n = 3), A. fumigatus (AsF; n = 4), Staphylococcus aureus (StA; n = 1), rhinovirus (Rhi; n = 1) or culture negative (Nil; n = 6). (c) Receiver operating characteristics curve analysis for H-ficolin detection in infected transplant patients compared with non-infected transplant patients. Results are representative of the data points gained from three independent experiments (16 positive and 16 negative patients). Bars represent the median and significance was determined via two-tailed Students t-test. *P < 0·05.