Nanoparticle decoration impacts airborne fungal pathobiology

Nanoparticle decoration impacts airborne fungal pathobiology

Dana Westmeiera,1, Djamschid Solouk-Saranb, Cecilia Valletc, Svenja Siemera, Dominic Doctera, Hermann G?tzd, Linda M?nnb, Anja Hasenbergb, Angelina Hahlbrocka, Kathrin Erlerb, Christoph Reinhardte, Oliver Schillingf, Sven Beckera, Matthias Gunzerb, Mike Hasenbergb, Shirley K. Knauerc, and Roland H. Staubera,1

aDepartment of Nanobiomedicine/ENT, University of Mainz Medical Center, 55101 Mainz, Germany; bInstitute for Experimental Immunology and Imaging, University Hospital, University of Duisburg-Essen, 45141 Essen, Germany; cInstitute for Molecular Biology, Centre for Medical Biotechnology, University Duisburg-Essen, 45117 Essen, Germany; dCore Facility Biomaterials, University of Mainz Medical Center, 55101 Mainz, Germany; eCenter for Thrombosis and Hemostasis, University of Mainz Medical Center, 55101 Mainz, Germany; and fInstitute of Molecular Medicine and Cell Research, University of Freiburg,

79104 Freiburg, Germany

Edited by Catherine J. Murphy, University of Illinois at Urbana?Champaign, Urbana, IL, and approved May 17, 2018 (received for review March 19, 2018)

Airborne fungal pathogens, predominantly Aspergillus fumigatus, can

cause severe respiratory tract diseases. Here we show that in environ-

ments, fungal spores can already be decorated with nanoparticles.

Using representative controlled nanoparticle models, we demonstrate

that various nanoparticles, but not microparticles, rapidly and stably

associate with spores, without specific functionalization. Nanoparticle-

spore complex formation was enhanced by small nanoparticle size rather than by material, charge, or "stealth" modifications and was concentration-dependently reduced by the formation of environmen-

tal or physiological biomolecule coronas. Assembly of nanoparticle-

spore surface hybrid structures affected their pathobiology, including

reduced sensitivity against defensins, uptake into phagocytes, lung cell

toxicity, and TLR/cytokine-mediated inflammatory responses. Follow-

ing infection of mice, nanoparticle-spore complexes were detectable in

the lung and less efficiently eliminated by the pulmonary immune defense, thereby enhancing A. fumigatus infections in immunocom-

promised animals. Collectively, self-assembly of nanoparticle-fungal

complexes affects their (patho)biological identity, which may impact

human health and ecology.

| | nanomedicine fungal infection nanoparticles

Bioaerosols are composed of biotic and abiotic particulates, including microbes such as fungal spores and (nano) particles, and are known to influence human health and the Anthropocene (1?4). Besides naturally occurring nanoparticles (NPs), humans and ecosystems are increasingly exposed to unintentional and engineered anthropogenic NPs (2, 5?7).

In addition to their importance for ecosystems, fungi are associated with various diseases (3, 8?10). The clinically most relevant airborne fungal pathogen, A. fumigatus, can be found in decaying vegetation and (almost) every building, and is dispersed through the air as asexual spores, known as conidia (3, 8?10). Not only in the clinic, but also in other workplaces, such as construction sites, fungal contaminations are considered a potential health hazard (3, 8, 9). A. fumigatus can cause multiple diseases, ranging from allergies to invasive pulmonary aspergillosis (IPA), a life-threatening infection in immunocompromised patients (8, 11). Accordingly, organ transplant recipients or patients receiving anticancer chemotherapy represent a predisposed population at high risk for IPA, due to their immunosuppressive treatment (8, 12). Moreover, respiratory problems, including allergic reactions, aspergilloma, asthma, and hypersensitivity pneumonitis, have been associated with exposure to A. fumigatus and other fungal species (12, 13). In healthy individuals, inhaled conidia are cleared mainly by alveolar macrophages and neutrophils (3, 8? 10), cells also known to play a major role in the body's response to NPs (2, 4, 6); however, effective strategies to improve the outcomes for patients with mycoses present an enormous medical challenge. Besides antimycotic drugs, natural antifungal peptides, such as human defensins, have been considered as future therapeutics (14).

Inhalation is also considered the most relevant exposure route for NPs (4, 15). In addition to adverse effects of fine particulates, the potential toxicologic relevance of NPs is under intense

investigation, particularly for individuals with preexisting respiratory diseases (2, 6, 7, 16).

Surprisingly little is known on the direct crosstalk of NPs with spores and its pathobiological consequences even though simultaneous exposure of humans and ecosystems to both occurs. Studies to date have examined sequential exposure scenarios, in which lung disease models were first treated with NPs and then subsequently challenged with bacteria or other pathogens, resulting in increased cytokine responses (17, 18).

Consequently, we here performed a comprehensive tiered analysis of the self-assembly of nanomaterial-fungal hybrid structures and its pathobiological relevance from in situ to in vitro and in vivo.

Results NPs Adsorb Rapidly and Stably to Fungal Conidia. Electron microscopy studies demonstrated that conidia harvested from a construction site with fungal contamination in the clinic or outdoors were already partially covered with nanosized particles (Fig. 1A). Energy-dispersive X-ray spectroscopy (EDX) analysis detected silica and other elements on the surface of these specimens, as was expected from the collection from realistic although complex environments (SI Appendix, Fig. S1A and Table S1). As the number of variables during sampling and the complexity of the environments do not allow analysis of the underlying mechanisms and pathobiological relevance of NPconidia complexes, we next used tiered complementary analytical approaches under standardized conditions.

The physicochemical characteristics of NPs define their biological identity, and also potentially their interaction with conidia (4, 15, 19). As environmental NPs are mainly complex

Significance

In this work, we demonstrate that nanoparticles rapidly assemble on spores under physiologically and ecologically relevant conditions. We provide in vitro and in vivo evidence that nanoparticle coating of the clinically most relevant airborne fungal pathogen, Aspergillus fumigatus, can affect the pathobiological identity and fate of both fungal spores and nanoparticles. Our findings suggest that nanoparticle coating of bioaerosols may be relevant for ecology and human health.

Author contributions: D.W., M.G., M.H., and R.H.S. designed research; D.W., D.S.-S., C.V., S.S., D.D., H.G., L.M., A. Hasenberg, A. Hahlbrock, K.E., C.R., O.S., and S.B. performed research; D.W. analyzed data; and D.W., S.K.K., and R.H.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

1To whom correspondence may be addressed. Email: danawestmeier@uni-mainz.de or roland.stauber@unimedizin-mainz.de.

This article contains supporting information online at lookup/suppl/doi:10. 1073/pnas.1804542115/-/DCSupplemental.

Published online June 20, 2018.

MEDICAL SCIENCES

cgi/doi/10.1073/pnas.1804542115

PNAS | July 3, 2018 | vol. 115 | no. 27 | 7087?7092

Fig. 1. NP physicochemical properties affect their assembly on spores. (A) SEM images of NP-covered conidia harvested from a construction site. Arrows indicate NPs. (Scale bar: 2 m.) (B) A. fumigatusGFP conidia incubated with fluorescent silica (SiOR) or polymer NPs (OSiRN or OSiRPEG). Negatively charged SiOR or PEGylated OSiRPEG efficiently adsorbed to conidia in situ, whereas positively charged OSiRN bound less efficiently. (Scale bar: 2 m.) (C) TEM indicates better fitting of small ( 30 nm; Left) compared with larger ( 140 nm; Right) SiO NPs into groove structures on the conidia surface. (Scale bars: 50 nm.) (D) SEM showing assembly of SiO ( 30/140 nm) and ZnO. (Scale bars: 200 nm.) (E) Kinetic analysis of complex formation demonstrating rapid NP binding (within ................
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