Abstract
Surfactant protein D (SP-D), a C-type lectin and pattern-recognition soluble factor, plays an important role in immune surveillance to detect and eliminate human pulmonary pathogens. SP-D has been shown to protect against infections with the most ubiquitous airborne fungal pathogen, Aspergillus fumigatus, but the fungal surface component(s) interacting with SP-D is unknown. Here, we show that SP-D binds to melanin pigment on the surface of A. fumigatus dormant spores (conidia). SP-D also exhibited an affinity to two cell-wall polysaccharides of A. fumigatus, galactomannan (GM) and galactosaminogalactan (GAG). The immunolabeling pattern of SP-D was punctate on the conidial surface and was uniform on germinating conidia, in accordance with the localization of melanin, GM, and GAG. We also found that the collagen-like domain of SP-D is involved in its interaction with melanin, whereas its carbohydrate-recognition domain recognized GM and GAG. Unlike un-opsonized conidia, SP-D-opsonized conidia were phagocytosed more efficiently and stimulated the secretion of proinflammatory cytokines by human monocyte-derived macrophages. Furthermore, SP-D-/- mice challenged intranasally with wildtype conidia or melanin ghosts (i.e. hollow melanin spheres) displayed significantly reduced proinflammatory cytokines in the lung compared with wildtype mice. In summary, SP-D binds to melanin present on the dormant A. fumigatus conidial surface, facilitates conidial phagocytosis, and stimulates the host immune response.
Highlights
Surfactant protein D (SP-D) has been shown to bind various fungal pathogens through its carbohydrate recognition domain (CRD), as fungi are endowed with a cell wall rich in different carbohydrate moieties [6, 32]
We show for the first time that surfactant proteins (SP)-D binds to A. fumigatus dormant conidial surface melanin pigment and two cell-wall polysaccharides, GM, which is masked by the surface melanin-rodlet layers in dormant conidia [28, 29], and GAG, which is only synthesized during germination [27]
The binding of GM or GAG to SP-D is mediated by its CRD in a calcium-dependent manner, whereas melanin binding is through the collagen-like domain of SP-D that is independent of calcium
Summary
Previous study employing flow cytometry showed that SP-D binds to A. fumigatus conidia [14, 21]. We confirmed rhSP-D binding to A. fumigatus conidial surface by flow cytometry and immunolabeling and binding to the melanin by ELISA (Fig. S3). We cultured MDMs with SP-D alone, in the absence of conidia, that did not produce any proinflammatory immune response, suggesting that the observed immune response is due only to the conidial or melanin ghost-opsonizing capacity of SP-D. conidia or extracted melanin ghost induced significantly higher transcripts of proinflammatory (tnf-␣, il-6, il-8, il-1, and il-12a) and anti-inflammatory (il-10) cytokines (Fig. 5A). Wildtype (WT) or SP-D knockout (SP-DϪ/Ϫ) mice were intranasally challenged with A. fumigatus conidia or melanin ghost, and SP-DϪ/Ϫ mice showed significantly reduced cytokine transcripts in the lung homogenates during the early infection (6 h) compared with the WT mice (Fig. 6, A and B). Our results show that SP-D acts as an immunomodulator, overcoming the immunological inertness of conidial melanin
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