Depletion of Extracellular Chemokines by Aspergillus Melanin

Abstract

Aspergillus fumigatus is an environmental fungus that can cause life-threatening pulmonary disease. Infections initiate when conidia are inhaled and land deep inside the small airways and alveoli of the lungs, where they interact with epithelial cells. These cells provide a physical barrier and secrete chemokines to attract innate immune cells to the site of infection. Melanin, a key constituent of the conidial cell wall, is required for the establishment of invasive infection due to its ability to inhibit the function of innate immune cells recruited to clear the infection. Here, we provide evidence for an additional mechanism by which A. fumigatus can alter host innate immune responses. In vitro infection of a normal human small airway epithelial cell line (HSAEC1-KT) caused a decrease in extracellular protein levels of CXCL10 and CCL20, two proinflammatory chemokines that are required for the host defense against aspergillosis, despite a dramatic increase in the levels of each mRNA. A. fumigatus depleted recombinant human CXCL10 and CCL20 from medium in the absence of host cells, suggesting that the block in accumulation is downstream of protein translation and secretion. Melanin is both necessary and sufficient for this chemokine-depleting activity because a dihydroxynaphthalene (DHN)-melanin-deficient strain of A. fumigatus is defective in depleting chemokines and purified melanin ghosts retain potent depletion activity. We propose that A. fumigatus, through the action of melanin, depletes important chemokines, thereby dampening the innate immune response to promote infection. IMPORTANCE Aspergillus fumigatus is the major airborne fungal pathogen that affects humans. In order to cause an invasive infection, inhaled spores must avoid killing by innate immune cells that are recruited to the site of infection. Understanding how A. fumigatus achieves immune evasion is important for the development of novel therapeutics. We provide evidence that melanin, a pigment contained in the spore cell wall, can remove certain chemokines from the extracellular space to suppress the host inflammatory response that is responsible for clearing fungal infection.

Keywords: Aspergillus fumigatus; CCL20; CXCL10; airway epithelial cells; chemokines; melanin.

FIG 1

A. fumigatus can deplete CXCL10 and CCL20 from culture supernatants of airway epithelial cells. (A to D) HSAEC1-KT cells were infected with A. fumigatus conidia (strain Af293) for 24 h, after which the levels of CXLC10 and CCL20 protein levels were determined in the culture supernatants by ELISA (A and C) and the mRNA levels for each gene were determined by quantitative reverse transcription-PCR (qRT-PCR) (B and D). (E to H) HSAEC1-KT cells were treated with PMA in the presence or absence of A. fumigatus conidia (Af293) for 6 h, after which the levels of CXLC10 and CCL20 protein levels were determined in the culture supernatants by enzyme-linked immunosorbent assay (ELISA) (E and G) and the mRNA levels for each gene were determined by qRT-PCR (F and H). (I and J) Recombinant human CXCL10 (I) or CCL20 (J) was added to tissue culture medium and incubated in the presence or absence of A. fumigatus conidia (Af293). Protein levels were measured by ELISA following 6 h of incubation. (K and L) The experiment from panels E and G was repeated with either live or heat-killed conidia of isolate Af293. Values represent the mean ± standard error of the mean (SEM) from at least 2 experiments, each performed in triplicate. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant; HK, heat killed; Af, A. fumigatus; (−), untreated control. Detailed methods are described in Text S1 in the supplemental material.

FIG 2

A. fumigatus melanin is necessary and sufficient for depletion of CXCL10 and CCL20. (A to D) HSAEC1-KT cells were treated with PMA in the presence or absence of the designated A. fumigatus conidia (from the B5233 isolate background) for 6 h, after which the levels of CXLC10 and CCL20 protein levels were determined in the culture supernatants by ELISA (A and C) and the mRNA levels for each gene were determined by qRT-PCR (B and D). (E and F) The experiment from panels A and C was repeated with the addition of purified melanin ghosts from A. fumigatus (Af293) with a multiplicity of 3 melanin ghosts per host cell. (G) Schematic of our proposed model by which A. fumigatus dampens innate immunity by depleting CXCL10, CCL20, and potentially other cytokines from the extracellular space. Values represent the mean ± SEM from at least 2 experiments, each performed in triplicate. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant; (−), untreated control; MG, melanin ghosts. Detailed methods are described in Text S1 in the supplemental material.