key: cord-0321213-g4o6nafa
authors: Rafiq, Muhammad; Rivieccio, Flora; Zimmermann, Ann-Kathrin; Visser, Corissa; Bruch, Alexander; Krüger, Thomas; Rojas, Katherine González; Kniemeyer, Olaf; Blango, Matthew G.; Brakhage, Axel A.
title: PLB-985 neutrophil-like cells as a model to study Aspergillus fumigatus pathogenesis
date: 2021-11-18
journal: bioRxiv
DOI: 10.1101/2021.07.28.454178
sha: 7562dd9b803aed9540046c8188d75bfcb3eafc46
doc_id: 321213
cord_uid: g4o6nafa

Fungal infections remain a major global concern. Emerging fungal pathogens and increasing rates of resistance mean that additional research efforts and resources must be allocated to advancing our understanding of fungal pathogenesis and developing new therapeutic interventions. Neutrophilic granulocytes are a major cell type involved in protection against the important fungal pathogen Aspergillus fumigatus, where they employ numerous defense mechanisms, including production of antimicrobial extracellular vesicles. A major draw-back to work with neutrophils is the lack of a suitable cell line system for the study of fungal pathogenesis. To address this problem, we assessed the feasibility of using differentiated PLB-985 neutrophil-like cells as an in vitro model to study A. fumigatus infection. We find that dimethylformamide-differentiated PLB-985 cells provide a useful recapitulation of many aspects of A. fumigatus interactions with primary human polymorphonuclear leukocytes. We show that differentiated PLB-985 cells phagocytose fungal conidia and acidify conidia-containing phagolysosomes similar to primary neutrophils, release neutrophil extracellular traps, and also produce antifungal extracellular vesicles in response to infection. In addition, we provide an improved method for the isolation of extracellular vesicles produced during infection by employing a size-exclusion chromatography-based approach. Advanced LC-MS/MS proteomics revealed an enrichment of extracellular vesicle marker proteins and a decrease of cytoplasmic proteins in extracellular vesicles isolated using this improved method. Ultimately, we find that differentiated PLB-985 cells can serve as a genetically tractable model to study many aspects of A. fumigatus pathogenesis. IMPORTANCE Polymorphonuclear leukocytes are an important defense against human fungal pathogens, yet our model systems to study this group of cells remains very limited in scope. In this study, we established that differentiated PLB-985 cells can serve as a model to recapitulate several important aspects of human polymorphonuclear leukocyte interactions with the important human fungal pathogen Aspergillus fumigatus. The proposed addition of a cultured neutrophil-like cell line to the experimental toolbox to study fungal pathogenesis will allow for a more mechanistic description of neutrophil antifungal biology. In addition, the easier handling of the cell line compared to primary human neutrophils allowed us to use PLB-985 cells to provide an improved method for isolation of neutrophil-derived extracellular vesicles using size-exclusion chromatography. Together, these results provide significant tools and a baseline knowledge for the future study of neutrophil-derived extracellular vesicles in the laboratory.

Fungal infections remain a major global concern. Emerging fungal pathogens and increasing rates of resistance 24 mean that additional research efforts and resources must be allocated to advancing our understanding of fungal 25 pathogenesis and developing new therapeutic interventions. Neutrophilic granulocytes are a major cell type 26 involved in protection against the important fungal pathogen Aspergillus fumigatus, where they employ numerous 27 defense mechanisms, including production of antimicrobial extracellular vesicles. A major draw-back to work with 28 neutrophils is the lack of a suitable cell line system for the study of fungal pathogenesis. To address this problem, 29

we assessed the feasibility of using differentiated PLB-985 neutrophil-like cells as an in vitro model to study A. 30 fumigatus infection. We find that dimethylformamide-differentiated PLB-985 cells provide a useful recapitulation 31 of many aspects of A. fumigatus interactions with primary human polymorphonuclear leukocytes. We show that 32 differentiated PLB-985 cells phagocytose fungal conidia and acidify conidia-containing phagolysosomes similar to 33 primary neutrophils, release neutrophil extracellular traps, and also produce antifungal extracellular vesicles in 34 response to infection. In addition, we provide an improved method for the isolation of extracellular vesicles 35 produced during infection by employing a size-exclusion chromatography-based approach. Advanced LC-MS/MS 36 proteomics revealed an enrichment of extracellular vesicle marker proteins and a decrease of cytoplasmic 37 proteins in extracellular vesicles isolated using this improved method. Ultimately, we find that differentiated PLB-38 985 cells can serve as a genetically tractable model to study many aspects of A. fumigatus pathogenesis. 39 40 IMPORTANCE 41

Polymorphonuclear leukocytes are an important defense against human fungal pathogens, yet our model systems 42

to study this group of cells remains very limited in scope. In this study, we established that differentiated PLB-985 43 cells can serve as a model to recapitulate several important aspects of human polymorphonuclear leukocyte 44 interactions with the important human fungal pathogen Aspergillus fumigatus. The proposed addition of a 45 cultured neutrophil-like cell line to the experimental toolbox to study fungal pathogenesis will allow for a more 46 mechanistic description of neutrophil antifungal biology. In addition, the easier handling of the cell line compared 47 to primary human neutrophils allowed us to use PLB-985 cells to provide an improved method for isolation of 48 neutrophil-derived extracellular vesicles using size-exclusion chromatography. Together, these results provide 49 significant tools and a baseline knowledge for the future study of neutrophil-derived extracellular vesicles in the 50 laboratory. Fungal infections remain a tremendous source of global morbidity and mortality. More than 1 billion individuals 56 are affected by fungal infections per year, with invasive infections killing numbers comparable to other leading 57 bacterial pathogens (>1.5 million per year; gaffi.org; (1)). Deadly invasive infections are caused by a relatively small 58 number of fungi, with most of these attributed to members of the genera Candida, Pneumocystis, Cryptococcus, 59 and Aspergillus (2). Aspergillus fumigatus is the major cause of aspergillosis and is particularly dangerous to 60 immunocompromised individuals suffering from neutropenia (3). There is also emerging evidence to suggest that 61 invasive aspergillosis may contribute to COVID-19-related deaths (4, 5), but challenges in safely obtaining 62 bronchoalveolar lavage samples from these patients have often made confirmatory diagnosis difficult. Despite the 63 obvious importance of A. fumigatus in the clinic, our understanding of this important pathogen remains lacking in 64 many aspects, in part due to a lack of tractable experimental systems in the laboratory. 65

Mammals are continuously challenged by fungal pathogens. In fact, asexual spores of A. fumigatus, 66 termed conidia, are thought to be inhaled by humans on a scale of hundreds per day (6). For most fungi, the body 67 temperature of mammals is too high to allow for growth, but for organisms like A. fumigatus that thrive in compost 68 piles at high temperatures, the human host provides fertile ground in the absence of a functional immune system 69 (7). However, humans do have multiple additional defenses, including a mucociliary escalator to remove particles 70 from the lungs, a robust epithelium, resident alveolar macrophages that eliminate the majority of the remaining 71 fungal conidia, and infiltrating polymorphonuclear leukocytes (PMNs) that aid in clearance of conidia and 72 destruction of fungal hyphae, among others (3, 8) . Neutrophils play an essential role in antifungal defense, due to 73 their importance in killing fungal hyphae. This is well-illustrated by the high susceptibility of neutropenic patients 74 to A. fumigatus infections in the clinics (reviewed in (3, 9) ). 75

Studies in primary human neutrophils have revealed the capacity of these cells to phagocytose conidia 76 and release granules, neutrophil extracellular traps (NETs), and extracellular vesicles in response to invading 77 pathogens (10-12). Phagocytosis occurs in conjunction with recognition of pathogen-associated molecular 78 patterns by host pathogen recognition receptors. Studies in zebrafish and mice have shown that these internalized 79 conidia can even be passed from neutrophils to macrophages for destruction of the fungus (13), implying complex 80 intracellular trafficking. In neutrophils and macrophages however, internalized wild-type A. fumigatus conidia are 81 capable of stalling phagolysosomal acidification to facilitate outgrowth. 82

In addition to phagocytosis, NETs are an important mechanism of defense against A. fumigatus that are 83 produced in response to fungal recognition in a CD11b-dependent manner (11, 14) . NET production is most 84 abundant against hyphae, but NETs are also sometimes produced in response to resting and swollen conidia (15). 85

In vivo, NETs were shown to be present in mouse lungs during infection but were generally dispensable for fungal 86

Differentiated PLB-985 neutrophil-like cells phagocytose opsonized A. fumigatus conidia. 120

We set out to assess the feasibility of using N,N-dimethylformamide (DMF)-differentiated PLB-985 (dPLB) cells as 121 a model for neutrophil phagocytosis, NET production, and extracellular vesicles release in response to A. 122 fumigatus infection. Upon infection, neutrophils are known to rapidly phagocytose and process A. fumigatus 123 conidia. We therefore challenged dPLB cells with opsonized A. fumigatus resting conidia and assessed various 124 parameters of infection. 125

First, we determined the phagocytic capacity of PLB-985 cells using confocal laser scanning microscopy 126 following incubation of cells with fluorescein isothiocyanate (FITC)-labelled, opsonized conidia (green). We 127 observed that dPLBs were capable of phagocytosis, as evidenced by a phagosomal membrane surrounding the 128 conidia ( Fig. 1A) . A similar result was confirmed for primary human PMNs with this experimental setup (Fig. S1A) . 129

Interestingly, dPLB cells only infrequently phagocytosed unopsonized conidia, whereas primary human 130 neutrophils indiscriminately phagocytosed both unopsonized and opsonized conidia (data not shown). To better 131 quantify phagocytosis, we analyzed 5,000 host cells post-infection by single-cell analysis using imaging flow 132 cytometry. For this we counterstained with calcofluor white (CFW; blue) to elucidate non-phagocytosed conidia 133 ( Fig. 1B and Fig. S1B to C). Quantification of imaging flow cytometry data revealed that approximately 22% of 134 wild-type and 26% of non-pigmented ΔpksP conidia were phagocytosed by dPLBs after 2 hours, while 135 undifferentiated PLB-985 cells exhibited limited phagocytosis of both wild-type and non-pigmented conidia (Fig.  136 1C). As expected, these phagocytosis percentages were lower than those of primary human PMNs, which 137 phagocytosed 62% of wild-type and 54% of ΔpksP conidia after 2 hours and 90% of wild-type and 85% of ΔpksP 138 conidia after 4 hours of infection (Fig. S1D) . We also measured the induction of cell damage following infection 139 using lactate dehydrogenase release assays and found levels below the limit of detection for dPLB cells after 2 and 140 4 hours of incubation (data not shown). To determine if infection also resulted in an activated immune response, 141 we measured proinflammatory cytokine levels by enzyme-linked immunosorbent assay (ELISA). Co-incubation of 142 dPLB cells with wild-type spores resulted in production of significantly increased levels of interleukin (IL)-8, but 143 not IL-1β after 24 hours of co-incubation, comparable to primary neutrophils at the corresponding earlier time 144 points ( Fig. 1D and Fig. S2 ). Together, these results suggested that DMF-differentiated PLB-985 cells could serve 145 as a suitable model for A. fumigatus pathogenesis and warranted further investigation. 146 147 Internalized A. fumigatus conidia are processed inside dPLB phagolysosomes. 148

Following internalization of conidia by professional phagocytes like macrophages and neutrophils, conidia-149 containing phagosomes fuse with lysosomes to acidify the compartment and aid in fungal killing. Numerous 150 additional enzymes are activated upon phagolysosomal acidification to degrade and digest the internalized fungal 151 conidia. The acidification and maturation of phagolysosomes are regulated by protein complexes assembled on 152 the phagolysosomal membrane, including lysosomal-associated membrane protein (LAMP)-1, 2, and 3; several 153 vacuolar ATPases (V-ATPases); Ras-related protein (RAB) 5 and 7; Flotillin 1 and 2; and numerous others (24). DHN-154 melanin on the surface of conidia can interfere with these processes in alveolar macrophages, monocytes, and 155 primary neutrophils to delay fungal processing and phagolysosomal acidification (25). To test whether dPLB cells 156 process conidia similarly to primary human PMNs, we first stained the cells with LAMP-1, a general endocytic 157 marker on the membrane of phagolysosomes. LAMP-1 showed a clear signal around phagolysosomes of infected 158 cells ( Fig. 2A) , and the intensity of the signal did not differ between wild-type and ΔpksP conidia (Fig. 2B) . 159

Furthermore, loading of dPLB cells with Lysotracker, a weak base that becomes fluorescent under acidic 160 conditions, showed that the ΔpksP conidia-containing phagosomes were significantly more acidified than those 161 containing wild-type conidia, revealing that DHN-melanin can also block the acidification process in the dPLB 162 model as well (Fig. 2C to D) . 163

The interference of the acidification pathway in alveolar macrophages by fungal DHN-melanin occurs 164 through the inhibition of V-ATPase assembly (26, 27) . This multiprotein complex plays a major role in lowering the 165 pH from 6 to <4.5 by pumping H + ions across the phagolysosomal membrane. Staining of the V-ATPase V1 subunit 166 in dPLB cells revealed that the percentage of recruitment to conidia-containing phagolysosomes was fairly similar 167 for wild-type and ΔpksP conidia ( Fig. S3A to B) , suggesting that other proton pumps may contribute to acidification 168 of these phagolysosomes in neutrophil-like cells. An additional defense mechanism for intra-phagosomal 169 degradation of pathogens occurs via reactive oxygen species (ROS) production. As expected, we observed dPLB 170 cells to be capable of producing ROS after staining with CellROX Orange (Fig. S3C) , consistent with the literature 171 (28, 29) . 172 173

One defense mechanism employed by neutrophilic granulocytes to fight against pathogens is the formation of 175

NETs. These structures consist of condensed chromatin and various enzymes that are released into the 176 extracellular space and typically correlate with death of the cell (30). NETs contain several anti-fungal proteins, 177 which are responsible for the fungistatic effect against A. fumigatus hyphae (15). To test if dPLBs are also able to 178 form NETs in response to A. fumigatus and serve as a model for this pathway, we co-incubated dPLBs cells with 179 hyphae and stained for nucleic acid with 4′,6-diamidino-2-phenylindole (DAPI; Fig. 3A ). As a positive control, we 180 induced NET formation using phorbol myristate acetate, a known trigger of NET formation in PLB-985 cells (Fig. 181 3B; (21, 31)). We observed the presence of histone H3 embedded in the DNA fibers and an association of NETs 182 with fungal hyphae (Fig. 3B) . Taken together these results showed that A. fumigatus is able to trigger NET 183 formation in dPLB cells and that these NETs are specifically directed against the hyphae. 184 185 dPLB cells produce extracellular vesicles in response to infection. 186

Recently, a new defense mechanism from neutrophilic granulocytes was discovered, the production of 187 antimicrobial extracellular vesicles (32, 33). These small lipid-enclosed nanoparticles are released from primary 188

PMNs after contact with microorganisms and, in the case of A. fumigatus conidia, can inhibit fungal growth after 189 coincubation (10). This effect is likely due in part to their protein cargo, which consists of antimicrobial peptides 190 such as myeloperoxidase, azurocidin, and cathepsin G. To assess if dPLB cells can produce extracellular vesicles 191 spontaneously or in response to A. fumigatus, we incubated cells with or without conidia for 2 and 4 hours. Next, 192 we isolated extracellular vesicles using two different methods; first using a previously described differential 193 centrifugation-based approach (DC; (10)) that enriches for medium-sized extracellular vesicles and a second 194 approach that relies on size-exclusion chromatography (SEC) to purify a more selective population of smaller 195 extracellular vesicles. Using these methods, dPLB cells were observed to actively secrete extracellular vesicles over 196 time in a manner comparable to primary human PMNs ( Fig. 4A to B). Using nanoparticle tracking analysis, we 197 observed the median size of particles to be around 200 nm, similar to extracellular vesicles derived from primary 198 neutrophils, a feature that was comparable between infection-derived and spontaneously released extracellular 199 vesicles ( Fig. 4C to D) . 200 We next compared the protein content of dPLB-derived extracellular vesicles isolated, at the 2 hours 201 timepoint, using each isolation method with and without fungal infection by LC-MS/MS-based proteomics 202 analysis. We were able to identify 1,984 unique proteins across all four samples (Dataset S1). The majority of 203 identified proteins (737 proteins) were found in all four samples (Fig. 5A) . We expected that isolation using size-204 exclusion chromatography would improve the quality of the isolated particles as has been shown previously (34), 205 and this was in fact the case. We observed an increase in extracellular vesicle markers like the tetraspanins CD63 206 and CD81, and tumor susceptibility gene 101 (TSG101), and a decrease in cytoplasmic proteins like calnexin (CANX; 207 Table 1) . 208 Extracellular vesicles produced by primary PMNs in response to A. fumigatus infection are known to be 209 enriched for antimicrobial cargo proteins like azurocidin, cathepsin G, and defensin (10). In line, with the data 210 from primary neutrophils we found that dPLBs also contained many of the same proteins in both infection-derived 211 and spontaneously released extracellular vesicles (Table 1) , as evidenced by an UpSetR plot showing overlapping 212 proteins cohorts ( Fig. S4; (10)), although with some exceptions. For example, dPLBs appeared to lack defensin, 213 neutrophil elastase, and some histone proteins previously observed. Ultimately, improvements in LC-MS/MS 214 technology and the advantage of higher input amounts of extracellular vesicle protein using large amounts of 215 dPLB cells in culture resulted in significantly more proteins detected in the dataset provided here compared to 216 efforts using primary neutrophils (10), proving that dPLBs offer a scalable system for the elucidation of novel 217 mechanisms of neutrophil extracellular vesicle biology. 218 219 dPLB extracellular vesicles produced against A. fumigatus limit fungal growth. 220

The most compelling feature of infection-derived extracellular vesicles of human neutrophils is likely their 221 antifungal capacity, as we previously reported (10). We set out to determine if the extracellular vesicles produced 222 by dPLB cells in response to A. fumigatus opsonized conidia are antifungal to a mitochondrial-GFP reporter strain 223 used previously as a marker of fungal viability (35). First, we incubated the reporter strain with dPLB cells and 224 primary human PMNs and assessed the capacity of the cells to control infection. PMNs were capable of minimizing 225 fungal outgrowth after 22 hours, whereas dPLBs did not completely contain fungal growth (Fig. 6A) . We then 226 assessed the antifungal capacity of the dPLB extracellular vesicles. For this experiment, extracellular vesicles from 227 equal numbers of dPLBs and primary human PMNs were isolated using differential centrifugation and found to be 228 antifungal against the mitochondrial-GFP reporter strain. This was evidenced by fragmentation of fungal 229 mitochondria after administration of extracellular vesicles to germinating conidia, which were allowed to 230 germinate for 6 hours prior to overnight incubation with extracellular vesicles, 3 mM H2O2 as a positive control, 231 or left untreated as a negative control (Fig. 6B) . Although, the mitochondrial reporter clearly indicated 232 fragmentation, the extracellular vesicles from dPLB did appear to be in some cases less effective in limiting the 233 length development of the hyphae than those from primary PMNs. It is important to note that dPLBs produced 234 slightly fewer extracellular vesicles, which could potentially explain the decreased antifungal activity in these 235 assays (Fig. 6B) . Additional representative images of dPLB-derived extracellular vesicles are shown to indicate the 236 spectrum of phenotypes observed for each experimental condition (Fig. S5) . Collectively, these results suggest 237 that dPLB infection-derived extracellular vesicles can limit A. fumigatus hyphal growth, similar in fashion to 238 primary human PMNs (10). 239

Fungal strains cultivation and opsonization. 242

Cultivation of A. fumigatus strains CEA10 (Fungal Genetics Stock Center; A1163), CEA17 ΔakuB ku80 ΔpksP (36), and 243

AfS35/pJW103 (35) was performed on malt agar plates (Sigma-Aldrich) supplemented to a final concentration of 244 2% (wt/vol) agar for 5 days at 37 °C. Conidia were harvested in sterile, ultrafiltrated water, filtered through a 30-245 µm pore filter (MACS, Miltenyi Biotec). Prior to confrontation with PLB-985 cells, conidia were opsonized with 246 normal human serum (Merck Millipore). Briefly, 900 µl of spore suspension was mixed with 100 µl of normal 247 human serum and incubated in a thermomixer at 37 °C for 30 min shaking at 500 rpm. The spore suspension was 248 washed three times by collecting spores via centrifugation at 1800 x g for 1 min at 4 °C and resuspending in fresh 249 PBS 1X. Following washing, spores were enumerated in a Thoma chamber in preparation for infection assays. To assess cell viability after confrontation with A. fumigatus conidia, the release of LDH was determined using the 275 CyQUANT™ LDH cytotoxicity assay kit (Thermo Fisher Scientific) according to the manufacturer´s instruction. Cells 276 treated with A. fumigatus conidia with a multiplicity of infection (MOI) of 5 for 2 hours were tested for their LDH 277 activity, which was compared to the spontaneous LDH activity and the maximum LDH activity. Release of LDH was 278 below the limit of detection for all three independent biological replicates performed. 279 280 Phagocytosis assays. 281

To assess phagocytic ability, we used a combination of imaging flow cytometry and confocal fluorescence 282 microscopy. For both methods the conidia were first stained with fluorescein isothiocyanate (FITC) and then after 283 confrontation of dPLBs or primary neutrophils, counterstained with calcofluor white (CFW; Sigma -Aldrich). The 284 FITC solution was obtained by dissolving FITC powder (Sigma-Aldrich) in 5 mL of 0.1 M sodium carbonate (Na2CO3) 285 followed by filtration through a 0.22-µm pore size filter (Carl Roth). Afterwards 1 ml of this solution was mixed 286 with 500 µl of spore suspension and incubated for 30 min at 37 °C while shaking at 1000 rpm in the dark. The 287 spores were then pelleted and washed three times with PBS 1X with 0.001% (vol/vol) Tween 20 (Carl Roth). During 288 the last washing step Tween was removed to avoid residual detergent in the samples (25, 37). Before counting, 289 the conidia were opsonized following the protocol described above. They were then added to the cells and co-290 incubated for 2 and 4 hours at 37 °C with 5% (vol/vol) CO2. At the end of each time point CFW was added to a final 291 concentration of 1 µg/ml and incubated for 1 min. Afterwards the cell suspension was transferred to 292 microcentrifuge tubes, centrifuged at 600 x g for 2 min at 4 °C and washed with PBS 1X twice. After discarding the 293 supernatant, the pellet was fixed with 150 µL of 3.7% (vol/vol) formaldehyde in PBS at room temperature for 15 294 min and subsequently washed again as described above. For imaging flow cytometry measurements, the cells 295 were resuspended in 150 µL of PBS 1X and analyzed immediately or stored at 4 °C for no more than 24 hours. Four 296 independent experiments were performed and for each replicate 5000 cells were analyzed using the ImageStream 297 X Mark II (Luminex). A 488 nm laser was used to detect FITC staining and a 405 nm laser for the CFW. The laser 298 voltage was adjusted depending on the sample. For wild-type conidia, we used 10 mW of 488 nm laser and 2 mW 299 for the 405 nm laser, and for ΔpksP conidia 1 and 2 mW of the respective lasers. The fluorescence intensities of 300 the samples were compensated and analyzed with the IDEAS software (Luminex). An example of the gating 301 strategy used for analysis can be found in the supplement files ( Fig. S1B to C) . For the fluorescence microscopy 302 analysis, cells were seeded in 8-well µ-slides (Ibidi) and visualized using a Zeiss LSM 780 confocal microscope (Carl 303

Zeiss) from at least three biological replicates as described above. 304 305 Immunofluorescence assays. 306

For immunofluorescence, cells were allowed to adhere in a 24-well plate with poly-L-lysine-coated glass coverslips 307 for 1 hour (Merck). After infection with labelled A. fumigatus spores and incubation for the noted times, cells were 308 fixed using 3.7% (vol/vol) formaldehyde in PBS for 10 min, rinsed three times with PBS 1X, permeabilized for 15 309 min using 0.1% (vol/vol) Triton X-100 in PBS 1X or 0.1% (wt/vol) saponin in PBS 1X, and then blocked for 30 min 310 with 2% (wt/vol) bovine serum albumin (BSA). After permeabilization, washed cells were incubated with primary 311 rabbit anti-Lamp-1 antibody (Abcam 24170, 1:100 dilution), anti-V-ATPase V1 subunit antibody (Abcam 73404, 312 1:100 dilution), or anti-Histone H3 (Cell signaling DIH2, 1:200 dilution) antibody in 1% (wt/vol) BSA in PBS 1X, 313 followed by incubation with secondary goat anti-rabbit IgG antibody DyLight 633 (Thermo Fisher Scientific). The 314 glass cover slips were mounted onto microscopy slides and visualized using a Zeiss LSM 780 confocal microscope 315 (Carl Zeiss). LAMP-1 recruitment was quantified by comparing the positive signal from stained phagolysosomes to 316 non-stained phagolysosomes. For the detection of IL-8 and IL-1β, 2x10 5 dPLB cells or primary neutrophils were seeded in 24-well plates. After 326 addition of conidia at a MOI of 5, plates were incubated at 37 °C with 5% (vol/vol) CO2 for 2, 4, or 6 hours. An 327 additional 24-hour timepoint was included for dPLB cells. As a positive control, cells were treated with 5 µg/mL of 328 lipopolysaccharide (LPS; Sigma-Aldrich L4516). At the appropriate end point, samples were collected, centrifuged 329 at 300 x g for 5 min to remove cell debris, and frozen at -20°C and analyzed within 3 days. Cytokines were 330 measured using human ELISA Max deluxe kits (BioLegend) according to the manufacturer´s instruction. 331 332 Acidification assays. 333 Acidification assays were performed as mentioned previously (27) After infection with A. fumigatus the supernatant of dPLB cells was collected, and extracellular vesicles were 341 isolated using two different methods: a differential centrifugation-based approach or a size exclusion 342 chromatography-based approach. The first method was described previously for the isolation of neutrophil-343 derived extracellular vesicles (10, 32). In both approaches samples were centrifuged at 3000 x g for 15 min at 4 °C 344 and then filtered through 5-µm pore size filters (Carl Roth). In the first method, samples were then centrifuged at 345 19,500 x g for 20 min at 4 °C to collect extracellular vesicles. For the second approach, the clarified filtrate was 346 concentrated using Amicon Ultra-15 centrifugal filters (Merck) with a molecular mass cut-off (MWCO) of 100 kDa 347 for 10 min at 4 °C and 3220 x g and finally loaded on size-exclusion chromatography qEV 70 nm columns (Izon). 348

After discarding the 3 mL void volume, 1.5 mL of extracellular vesicle sample were collected and measured. When 349 necessary, extracellular vesicles were further concentrated using 10 kDa cutoff Amicon Ultra-0.5 ml filters (Merck). After isolation as described above, extracellular vesicles were delipidated as described previously (10) Fragger 3.2. Two missed cleavages were allowed for the tryptic digestion. The precursor mass tolerance was set 394 to 10 ppm and the fragment mass tolerance was set to 0.02 Da. Modifications were defined as dynamic Met 395 oxidation, and protein N-term acetylation with and without methionine-loss as well as static Cys 396 carbamidomethylation. A strict false discovery rate (FDR) < 1% (peptide and protein level) were required for 397 positive protein hits. If only 1 peptide per protein has been identified the hit was accepted if the Mascot score 398 was >30 or the MS Amanda score was >300 or the Sequest score was >4 or the MS Fragger score was >8. The 399

Percolator node of PD2.4 and a reverse decoy database was used for qvalue validation of spectral matches. Only 400 rank 1 proteins and peptides of the top scored proteins were counted. Label-free protein quantification was based 401 on the Minora algorithm of PD2.2 using a signal-to-noise ratio >5. Imputation of missing quan values was applied 402 by setting the abundance to 75% of the lowest abundance identified for each sample. Normalization was based 403 on a replicate median total peptide sum approach, which was calculated based on the sum of all identified peptide 404 abundance values per replicate sample. The sums of each of the three replicates from the four sample groups 405 were used to calculate median values. Normalization factors were calculated by dividing median values of the 406 respective sample group by the abundance sum of each sample. Normalization factors were multiplied with single 407

protein abundance values of each replicate/sample. The p-values are based on a Student`s t-test. Ratio-adjusted 408

p-values were calculated by dividing p-values with the log4ratio of the protein abundance levels. Significant 409 differences in protein abundance were defined when the following three requirements were reached: At least a 410 4-fold change in abundance (up and down), a ratio-adjusted p-value <0.05, and at least identified in 2 of 3 411

replicates of the sample group with the highest abundance. Intersection plots were created using the UpSetR 412 package (39) and only include proteins that were detected in at least two replicates of a given sample. Volcano 413 plots were created using ggplot2 in R using the replicate median total peptide sum normalized (RMN) data for all 414 proteins detected in Dataset S1. 415

To characterize growth inhibition and killing of hyphae by fungal-induced extracellular vesicles an A. fumigatus 417 strain expressing a mitochondrial GFP reporter (AfS35/pJW103; (35)) has been used as described previously (10). Neutrophils are critical players in the immune response to fungal pathogens stemming from their numerous 437 antimicrobial capacities (10, 41). To study neutrophil functions, numerous cell lines that model these abilities in 438

vitro have been considered with limited success. The most promising remains the HL-60 leukemia cell line and 439 PLB-985 sublineage, which have been used as models for aspergillosis and bacterial killing (21) (22) (23) 28) . In the 440 present study, we analyze different aspects of the interaction between DMF-differentiated PLB-985 myeloid cells 441 and the fungus A. fumigatus for the first time, making frequent comparisons with data from primary neutrophils. 442

Primary neutrophils are capable of phagocytosing and processing A. fumigatus conidia, features that were 443 maintained in dPLB cells. As expected for cultured cells, the observed phagocytosis rate of A. fumigatus conidia 444 by dPLB cells was lower than that of primary human PMNs. It is possible that additional differentiation methods 445 will ultimately reveal higher rates of phagocytosis, as PLB-985 cells differentiated with DMF are known to exhibit 446 lower phagocytic activity than cells differentiated with dimethyl sulfoxide (42). In terms of cytokine release, we 447 observed increased release of IL-8 from dPLBs after infection with A. fumigatus opsonized conidia, but not IL-1β, 448 consistent with our observations in human PMNs. These results are also in agreement with those collected from 449 primary PMNs co-incubated with L-ficolin opsonized conidia for 8 hours, where secretion of IL-8 was also much 450 higher than IL-1β (43). 451

We next stained dPLB cells for the endosomal marker LAMP-1 and measured the acidification of the 452 phagolysosomes via Lysotracker to assess intracellular processing of fungal conidia. These experiments confirmed 453 that around 20% of wild-type conidia are completely internalized inside the phagosomes and that lysosomal fusion 454 occurs as a next step of conidial processing. Interestingly, and as already demonstrated for primary monocytes 455 and neutrophils (25), melanized conidia showed less acidification around the phagolysosomal membrane than the 456 mutant lacking DHN-melanin. This effect in monocytes is caused by inhibition of V-ATPase assembly. Surprisingly, 457 staining for the V1 subunit of the V-ATPase did not reveal a significant difference between the wild-type and ΔpksP 458 strains in dPLB cells as has been observed in other cell systems. These results suggest that neutrophils potentially 459 employ additional mechanisms to acidify phagolysosomes in neutrophilic granulocytes after A. fumigatus 460 infection or that the methods used were not sufficiently sensitive to detect slight differences in acidification. 461

Together, these results suggest that dPLBs can serve as an intriguing model to study various aspects of neutrophil 462 phagocytosis and intracellular processing in a more tractable in vitro model system. 463

A conidium that escapes phagocytosis can sometimes germinate into a hypha, a morphotype that is much 464 more difficult to eliminate and requires the antifungal activity of neutrophils. One mechanism that aids in control 465 of A. fumigatus hyphae is the release of fungistatic NETs composed of DNA and antimicrobial proteins. By staining 466 for extracellular DNA and NET constituent histone H3, we could clearly show that dPLB cells also produce NETs in 467 response to A. fumigatus hyphae, comparable to primary human PMNs and in agreement with other studies 468 challenging dPLBs with bacterial pathogens (21, 22) . 469

Production of extracellular vesicles from primary neutrophils co-incubated with Aspergillus conidia or 470 bacteria like Staphylococcus aureus is an important defense strategy against microorganisms (10, 32). We found 471 that dPLB cells were also able to generate comparable populations of extracellular vesicles to PMNs upon contact 472 with opsonized conidia. Accumulation of extracellular vesicles of approximately 200 nm in diameter was observed 473 over time, independently of the isolation method we applied. Proteomic analysis revealed that by using a size-474 exclusion chromatography-based isolation approach, the extracellular vesicles population could be enriched for 475 extracellular vesicle marker proteins like CD63, CD81, and TSG101. We observed that the samples obtained by 476 this method had a higher abundance of proteins and showed more differences between the spontaneously 477 In the proteomic analysis of extracellular vesicles derived from primary neutrophils infected with A. 486 fumigatus, several antimicrobial peptides, such as cathepsin G and azurocidin, were detected (10). Co-incubation 487 of these extracellular vesicles from primary neutrophils with fungal hyphae arrested growth, which was confirmed 488 in this study. Using dPLB cells, we identified extracellular vesicles containing cathepsin G and azurocidin; however, 489 the distribution remained relatively equal between spontaneously released and infection-derived extracellular 490 vesicles. We suspect that this is due to the differentiation procedure of dPLB cells with DMF, which provides a 491 mild inflammatory stimulus. Nevertheless, we observed mitochondrial damage in hyphae treated with infection-492 derived but not spontaneously released extracellular vesicles. We can postulate several reasons for this result. 493

First, it is possible that immunoglobulins are playing a role in targeting extracellular vesicles to fungal hyphae. In 494 this case, the protein cargo may be the same, but targeting would be inefficient in spontaneously released 495 extracellular vesicles. Of note, opsonization of the bacterial pathogen Staphylococcus aureus was not found to 496 influence the antimicrobial capacity of neutrophil-derived extracellular vesicles (32). A second option is that 497 additional extracellular vesicle cargo molecules such as RNA or lipids may play a role in the antifungal activity. It 498 has been shown in many instances that extracellular vesicles can contain small RNAs that exert distinct functions 499 during infection, especially in regard to plant fungal pathogens (46, 47) . We believe that a combination of factors 500 is likely involved in the antifungal effect of extracellular vesicles. 501

Part of the explanation may also come from similarities between neutrophilic granules and extracellular 502 vesicles. Granules and extracellular vesicles are distinct cellular features, but do share some biogenesis pathways 503 and cargo molecules. For example, the azurophilic granules contain myeloperoxidase to aid in pathogen killing in 504 the endocytic pathway via fusion of the granules to pathogen-containing phagosomes (48), but myeloperoxidase 505 is also released in a CD63-negative population of extracellular vesicles known as microvesicles (49). Based on the 506 proteomics analysis of this study, as well as our previous work (10), it seems possible that microvesicle-cargo 507 proteins like myeloperoxidase contribute at least in part to the antifungal activity. However, the presence of 508 myeloperoxidase in spontaneously released extracellular vesicles and the lack of susceptibility of patients lacking 509 myeloperoxidase to fungal infections (50) hint that other factors are also involved. Besides, the shared marker 510 protein CD63 nicely highlights another link between granules and extracellular vesicles, the shared biogenesis 511 from multivesicular bodies of azurophilic granules and the extracellular vesicle subset called exosomes (51). 512

In conclusion our results suggest that DMF-differentiated PLB-985 cells can be used as a model to study 513 aspects of the interaction of the human pathogenic fungus A. fumigatus with neutrophilic granulocytes. Although 514 they will never substitute for all experiments with neutrophils, we do believe that this system will serve as a useful 515 tool for the genetic dissection of A. fumigatus pathogenesis in the future. 516 517 ACKNOWLEDGEMENTS 518

We would like to thank Johannes Wagener for providing the A. fumigatus mitochondrial GFP reporter strain. We 519 also thank Natascha Wilker for excellent technical assistance. The work presented here was generously 520 supported by the Deutsche Forschungsgemeinschaft (DFG)-funded Collaborative Research Center/Transregio 521 

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COVID-19-Associated Pulmonary Aspergillosis

Dectin-543 1 diversifies Aspergillus fumigatus-specific T cell responses by inhibiting T helper type 1 CD4 T cell 544 differentiation

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Essential role for neutrophils but not 550 alveolar macrophages at early time points following Aspergillus fumigatus infection

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NETs 557 formed by human neutrophils inhibit growth of the pathogenic mold Aspergillus fumigatus

Effect of Complement and of the Carbohydrate Components of Sputum on Phagocytosis 560 by

beta-glucan-dependent shuttling of conidia from neutrophils to 563 macrophages occurs during fungal infection establishment

Mac-1 triggers 565 neutrophil DNA extracellular trap formation to Aspergillus fumigatus independently of PAD4 histone 566 citrullination

Production of extracellular traps against Aspergillus fumigatus in vitro and in infected 569 lung tissue is dependent on invading neutrophils and influenced by hydrophobin RodA

Regulate Aspergillus fumigatus and beta-Glucan-Induced Neutrophil Extracellular Trap Formation, but 573 Hyphal Killing Is Dependent Only on CR3

Human polymorphonuclear leukocytes 575 inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion

Human Neutrophils Use Different Mechanisms 579

To Kill Aspergillus fumigatus Conidia and Hyphae: Evidence from Phagocyte Defects

Human neutrophil kinetics: modeling of stable isotope labeling data supports short blood neutrophil 583 half-lives

Studying Neutrophil Function in vitro: Cell Models and Environmental 585 Factors

Afa/Dr diffusely adhering Escherichia coli strain C1845 induces 588 neutrophil extracellular traps that kill bacteria and damage human enterocyte-like cells

Cell-Cycle Proteins Control Production of Neutrophil Extracellular Traps

Loaded Leukocytes as a Novel Treatment Strategy Targeting Invasive Pulmonary Aspergillosis

Proteomic characterization of phagosomal membrane 598 microdomains during phagolysosome biogenesis and evolution

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Brakhage 630 AA. 2015. Virulence determinants of the human pathogenic fungus Aspergillus fumigatus protect against 631 soil amoeba predation

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ELISA detection of IL-8 cytokine released from infected dPLBs and primary human PMNs at different time 678 points. Lipopolysaccharide (LPS) was included for comparison of a bacterial stimulus

±SD, from six biological replicates

FIG 2. Processing of A. fumigatus conidia inside phagolysosomes

A) dPLB cells were stained with LAMP-1 (red) after infection with wild-type or ΔpksP conidia (FITC labeled

Images are representative of three biological replicates. (B) Quantification of LAMP-1 co-localization with 685

Percentage of acidified conidia in dPLB cells after 2-and 4-hours 686 post-infection with wild-type and ΔpksP conidia

Colocalization of conidia (CFW; blue) in acidified compartments labeled with Lysotracker (red)

Confocal scanning laser microscopy of nucleic acid stained with DAPI (blue) released from dPLB cells stained 694 with Cell Mask (red) after challenge with fungal hyphae containing a mitochondrial GFP reporter (A. fumigatus 695 strain AfS35 / pJW103 expressing a mitochondrial GFP reporter; green). Phorbol myristate acetate (PMA) was 696 used as a positive control. Data representative of three biological replicates. (B) Confocal micrographs of NET 697 markers, Histone H3 (red) and nucleic acid stained with DAPI (blue)

Images are representative of three biological replicates

Extracellular vesicles were quantified 707 from (A) dPLB cells or (B) primary human PMNs at 0, 2, and 4 hours post infection and show the average of at 708 least six biological replicates and three biological replicates, respectively. Representative size histograms from five 709 biological replicates are shown for extracellular vesicles derived from (C)

/MS proteomics analysis was performed on extracellular vesicles isolated from dPLB cells using a differential 715 centrifugation-based approach (DC) or a size-exclusion chromatography-based approach (SEC) in the presence or 716 absence of infection with opsonized A. fumigatus conidia. (A) Proteins identified in spontaneously released 717 extracellular vesicles (sEVs) and infection-derived extracellular vesicles (iEVs) from at least two replicates of a 718 given sample were intersected using UpSetR

Volcano plots show the log2 ratio of infection-derived extracellular vesicles (iEVs) versus spontaneously released 720

EVs (sEVs) for (B) DC-based isolation and (C) SEC-based isolation. Input data included values from all replicates 721 using the RMN data included in Dataset S1. Plots were created using ggplot2 in R. Proteomics data is from three 722 analytical replicates of three independent biological replicates. Orange circles represent proteins with greater 723 than 2-fold change

Infection-derived extracellular vesicles from dPLBs are antifungal to A. fumigatus hyphae

AfS35 containing plasmid pJW103 expressing a mitochondrial GFP reporter 729 (green) were opsonized and co-incubated with freshly harvested human PMNs or dPLBs. After overnight 730 incubation (22 hours) samples were stained with CFW for 10 min and images were taken using a Zeiss

B) The A. fumigatus strain AfS35 expressing a mitochondrial GFP 732 reporter (green) was first grown for 6 hours and then stained with calcofluor white (blue) and incubated overnight 733 with spontaneously released extracellular vesicles or infection-derived extracellular vesicles isolated from primary 734 human PMNs or dPLBs. As a control

CFW-stained; false-colored green) phagocytosed by primary 757 human PMNs after 2 hours co-incubation. Membranes were stained with Cell Mask (red) to indicate cell 758 membranes. Images are representative of at least two independent biological experiments. Scale bars are 10 m. 759 (B and C) indicate the gating strategy for determining phagocytosis rates of dPLB cells and PMNs using imaging 760 flow cytometry. (B) Single cell populations were selected based on their area and aspect ratio in the bright field 761 channel. (C) Phagocytosed conidia were gated based on their higher FITC fluorescence intensity and low CFW 762 fluorescence intensity. Cells without conidia had low FITC fluorescence intensity. The data subject to analysis 763 included only cells in focus and were pre-gated during data acquisition based on their high root mean square 764 gradient in the bright field channel. (D) Quantification of phagocytosis by primary human PMNs using the IDEAs 765 software on 5,000 cells per condition with either wild-type or ΔpksP conidia from four independent experiments

ELISA detection of the IL-1β cytokine released from infected dPLBs or primary human PMNs at different time 770 points. Lipopolysaccharide (LPS) was included as a comparison to a bacterial stimulus. Data are presented as mean 771 ±SD, from six biological replicates

Scale bars are 10 µm. 778 (B) Quantification of V-ATPase V1 positive phagolysosomes after 4 hours of infection with wild-type or ΔpksP 779 conidia. Graphs are from three biological replicates. (C) Confocal microscopy images of dPLB cells infected with 780 wild-type conidia (CFW-stained; blue), left uninfected

Intersection of proteomics data with previously reported neutrophil-derived EV proteomes

DC) or a size-exclusion chromatography-based approach (SEC) in the presence or 787 absence of infection with opsonized A. fumigatus conidia and compared to data from (10)

Representative images of dPLB-derived extracellular vesicles effects on A. fumigatus hyphae

Additional 799 representative images are included to show extent of variability that occurs in regard to extracellular vesicle killing 800 experiments shown in Fig. 6. As a control, untreated hyphae and hyphae treated with 3 mM H2O2 to induce cell 801 death are included. An intact mitochondrial network is shown by a filamentous network