Calcineurin Orchestrates Lateral Transfer of Aspergillus Fumigatus During Macrophage Cell

AJRCCM Articles in Press. Published on 10-May-2016 as 10.1164/rccm.201601-0070OC Page 1 of 63

Calcineurin orchestrates lateral transfer of Aspergillus fumigatus during macrophage cell

death

Anand Shah 1*, Shichina Kannambath 1*, Susanne Herbst 1, Andrew Rogers 2, Simona Soresi 3,

Martin Carby 3, Anna Reed 3, Serge Mostowy 4, Matthew C. Fisher 5, Sunil Shaunak 6, Darius P.

Armstrong-James 1+

1 National Heart and Lung Institute, Imperial College London, London SW7 2AZ UK

2 Histopathology Department, Royal Brompton and Harefield Hospitals, London SW3 6NP

3 Lung Transplant Unit, Royal Brompton and Harefield Hospitals, Middlesex, UB9 6JH UK

4 MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London

SW7 2AZ UK

5 School of Public Health, Imperial College London, London W2 1PG UK

6 Department of Infectious Diseases and Immunity, Imperial College London, London W12

0NN UK

* These authors contributed equally

+ Corresponding Author

Tel: +44207 594 3187

Email: [email protected]

Fax: unavailable

Address: Fungal Pathogens Laboratory, National Heart and Lung Institute, Sydney Street

Wing, Royal Brompton Hospital, London UK SW3 6NP

Impact: Pulmonary aspergillosis is a life-threatening complication in transplantation and chronic respiratory disease. Here we report necroptosis as an early response to germination of Aspergillus fumigatu s in human macrophages, and show that this results in lateral transfer of Aspergillus fumigatus between macrophages as a mechanism to limit hyphal escape. We define calcineurin as a key phosphatase that regulates necroptotic transfer of

Aspergillus fumigatus , yielding important insights into the pathogenesis of pulmonary aspergillosis in the immunocompromised host.

Author contributions: D.A-J, A.S., S.K., S.S. and S.M. designed the experiments. D.A-J, A.S,

S.K., S.H., and A.Ro. conducted the experiments. S.S, A.Re. and M.C. consented the patients and undertook the bronchoscopies. A.S. and S.K. wrote the first draft of the manuscript. All authors contributed to the data analysis and the writing of the manuscript .

Funding: This work was supported by an MRC Clinician Scientist Fellowship to DAJ

[G0902260/1] and an MRC Clinical Research Fellowship [MR/K002708/1] to AS. SM is supported by a Wellcome Trust Research Career Development Fellowship [WT097411MA] and by the Lister Institute for Preventative Medicine.

Running Head: Macrophage necroptosis in pulmonary aspergillosis

Descriptor number: 10.6 Host Defenses to Microbial Pathogens

Manuscript word count: 3442/3500

At a Glance Commentary

Scientific Knowledge on the Subject: Macrophages are the front-line defense against

pulmonary infection with the mould pathogen Aspergillus fumigatus . Phagocytosis and

generation of reactive oxygen species are known to be crucial for control of infection.

However, the cellular outcome from progressive macrophage infection with Aspergillus

fumigatus has not been systematically studied.

What This Study Adds to the Field: Here we present a systematic analysis of human

macrophage infection with Aspergillus fumigatus . We show that successful fungal

germination in the phagosome leads to necrotic cell death and can result in cell-cell transfer

of germinating Aspergillus fumigatus between macrophages. Ultimately this process assists

control of fungal germination, and is orchestrated through the calcium-responsive serine-

threonine phosphatase calcineurin. Our systematic analysis of the macrophage response to

Aspergillus fumigatus defines necroptosis as a key early event in the pathogenesis of

pulmonary aspergillosis.

This article has an online data supplement, which is accessible from this issue's table of

content online at www.atsjournals.org

iii

Abstract

Rationale: Pulmonary aspergillosis is a lethal mould infection in the immunocompromised host. Understanding initial control of infection, and how this is altered in the immunocompromised host, is a key goal for understanding the pathogenesis of pulmonary aspergillosis.

Objectives: To characterise the outcome of human macrophage infection with Aspergillus fumigatus , and how this is altered in transplant recipients on calcineurin inhibitor immunosuppressants.

Methods: We defined the outcome of human macrophage infection with Aspergillus fumigatus , and the impact of calcineurin inhibitors, through a combination of single cell fluorescence imaging, transcriptomics, proteomics, and in vivo studies.

Measurements and Main results: Macrophage phagocytosis of Aspergillus fumigatus enabled control of 90% of fungal germination. However fungal germination in the late phagosome led to macrophage necrosis. During programmed necroptosis, we observed frequent cell-cell transfer of Aspergillus fumigatus between macrophages which assists subsequent control of germination in recipient macrophages. Lateral transfer occurred through actin-dependent exocytosis of the late endosome in a vasodilator-stimulated phosphoprotein (VASP) envelope. Its relevance to the control of fungal germination was also shown by direct visualisation in our zebrafish aspergillosis model in vivo . The calcineurin inhibitor FK506/tacrolimus reduced cell death and lateral transfer in vitro by 50%. This resulted in uncontrolled fungal germination in macrophages and hyphal escape.

Conclusions: These observations identify programmed necrosis-dependent lateral transfer

of Aspergillus fumigatus between macrophages as an important host strategy for controlling

fungal germination. This process is critically dependent on calcineurin. Our studies provide

fundamental insights into the pathogenesis of pulmonary aspergillosis in the

immunocompromised host .

Word count: 248

Key Words: Pulmonary Fungal Diseases; Macrophage; Necrosis; Aspergillus; Calcineurin

Introduction

Aspergillus fumigatus (Af ) is a mould with small airborne conidia that are a ubiquitous component of the aerial mycobiota (1). Normally inhaled conidia are cleared by alveolar macrophages to prevent fungal germination, tissue invasion and destructive lung disease (2,

3). Our previous work using murine and zebrafish models of invasive aspergillosis has shown the importance of macrophages in control of Af early post infection, with neutrophil influx a later event (4, 5). Previous studies have highlighted the importance of inflammatory monocytes in murine invasive aspergillosis (6).

A spectrum of immunocompromised states predispose individuals to high-mortality invasive or chronic forms of pulmonary aspergillosis (PA) (7). Organ transplant recipients are at high risk of PA, with lung transplant recipients being particularly susceptible with a mortality of

>40% (8, 9). There are an estimated 3 million individuals with chronic PA, and 4.5 million with allergic bronchopulmonary aspergillosis globally (10, 11). Af has also been implicated in the pathogenesis of asthma, chronic obstructive airways disease and bronchiectasis (11-13).

Treatment options remain limited, and high mortality rates persist (14). Better understanding of susceptibility to PA in the non-neutropenic host is a key clinical research priority.

Calcineurin-NFAT signalling is the target of the calcineurin inhibitor immunosuppressants

(CNIs) used in organ transplantation (15). We have shown that CNIs (FK506/tacrolimus) increase susceptibility to murine PA through inhibition of the macrophage TLR9-calcineurin-

NFAT pathway (4, 5). This pathway is crucial for activation of inflammatory responses to Af , and recruitment of fungicidal neutrophils to the site of infection. Calcineurin-NFAT signalling has also been shown to be critical for the innate immune response to Candida albicans and

Escherichia coli , through endocytic mechanisms convergent on phospholipase C-ɣ-

dependent calcium flux (16, 17). These observations underscore the importance of

calcineurin-NFAT signalling for myeloid immunity, and reveal a novel relationship between

innate sensing and calcineurin-NFAT signalling in the lung that requires further clinical

definition.

Here we show that host programmed necrosis is an important outcome of Af germination in

the human macrophage. Cell death occurred in response to phagosomal germination, with

necroptosis-associated lateral transfer of Af between macrophages. Cell death-dependent

transfer was calcineurin-dependent, and enabled control of germination in recipient

macrophages. Crucially, inhibition of either calcineurin-dependent cell death, or lateral

transfer, enabled hyphal escape from the macrophage. These studies identify programmed

necrosis as a key cellular response in PA, and show that it is likely to be critically impaired in

organ transplant recipients on calcineurin inhibitors. Our studies reveal cell-cell transfer as a

novel and important cell death associated defence mechanism that enables control of

fungal germination in the myeloid compartment.

Methods

Ethics Statement

Studies were approved by the Biomedical Research Unit (NRES reference 10/H0504/9),

Royal Brompton and Harefield NHS Trust (AS1). All experiments conformed to the WMA declaration of Helsinki. Written informed consent was obtained from all participants.

Zebrafish care and maintenance

Experiments were approved by the United Kingdom Home Office in accordance with the project license PPL 70/7446. Full details are given in the Supplemental Experimental

Procedures.

Fungal strains and culture

A. fumigatus CEA10 was used for Western blot, Luminex, fungal burden, Imagestream, phosphoproteomic and RNA sequencing experiments. ATCC46645-eGFP, a gift from Frank

Ebel, was used for microscopy experiments. See Supplemental Experimental Procedures.

Isolation of human macrophages hAMs were isolated from bronchoalveolar lavage by adherence. Purified hAMs were rested for 3 days. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) from healthy volunteers by Ficoll-Paque gradient centrifugation and negative magnetic bead isolation (Pan-monocyte isolation kit, Miltenyi Biotech, CA, USA). hMDMs were differentiated using 5ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10% human serum for 7 days. See Supplemental Experimental Procedures.

RNA-seq analysis

Illumina RNA-seq libraries were prepared from hMDMs ( n = 6, healthy donors) pre-treated

with 10 ng/ml FK506 for 1h or vehicle and stimulated with live Af swollen conidia (SC)

(MOI=1) for 1h and 6h. Illumina paired-end reads were mapped to the human reference

genome (hg19) using TopHat2 (18) (GEO database accession number GSE74924) and

differential expression analyses performed using Cufflinks v2.2.1 (19). Gene analyses were

carried out using DAVID v6.7 (20). Human PPI network modules were obtained using

MCODE (21) analysis in Cytoscape. See Supplemental Experimental Procedures.

Phosphoproteomic analysis

A Phospho Explorer Antibody Microarray was conducted by Full Moon BioSystems Inc

(California, USA). hMDMs were differentiated at 1 x 10 7 cells in 75mm 2 tissue culture flasks.

At day 7, cells were stimulated with live Af swollen conidia (SC) (MOI=1) following pre-

treatment for 1h with FK506 (10ng/ml, Calbiochem) or vehicle (DMSO diluted in RPMI). At

1h post-infection, cells were washed (x5) with 10mls ice-cold phosphate-buffered saline

(PBS) containing protease inhibitors (Cell Signalling), and collected by centrifugation (250g

for 10min at 4°C). Cells were frozen at -80°C and transferred to Full Moon Biosystems on dry

ice. The array consisted of 1,318 phospho-specific antibodies. Proteins were labelled with

biotin and adhered to pre-blocked microarray slides. After washing, detection of total and

phosphorylated proteins was conducted using Cy3-conjugated streptavidin. Expression of

phosphorylated proteins was normalized to the corresponding total protein abundance.

Fold change was calculated as the phospho- to non-phospho-protein ratio of FK506-treated

cells/the phospho- to non-phospho-protein ratio of untreated cells.

Statistical analysis

Results are presented as mean ± SEM and were analyzed using GraphPad Prism software

(version 6.0; GraphPad). Significance was determined using a Student’s t test for unpaired observations; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. If 3 or more groups were compared a one-way ANOVA with Bonferroni correction was used.

Full details of Experimental Procedures are available in the Supplemental Information.

Results

Aspergillus fumigatus activates calcineurin -NFAT-dependent inflammatory responses in human macrophages

Af phagocytosis activates NFAT nuclear translocation in murine macrophages (4). We therefore characterised the role of NFAT in human monocyte-derived macrophages

(hMDMs). hMDMs were infected with live Af swollen conidia (SC) or resting conidia (RC) and NFAT translocation determined by Western blot and confocal microscopy. SC phagocytosis led to sustained NFAT translocation after 30min (Figures 1A, B and E1A). RC induced a weak transient NFAT activation response (Figure E1B, E1C). NFAT translocation in response to SC was confirmed in alveolar macrophages (hAMs) from lung transplant recipients (Figure E1D). Maximum translocation of NFATc2 to the nucleus was seen in response to Af at 60 minutes p.i. NFAT translocation was blocked by the calcineurin inhibitor

FK506 (Figures 1B, 1C, E1E), but NFκB Rel65 translocation was unaffected (Figure 1B).

NFAT transcriptional activity was defined by measuring expression of the NFAT-specific

target Regulator of Calcineurin 1 (RCAN1 ). SC infection increased RCAN1 expression, which

was abrogated by FK506 (Figures 1D, E1F). To determine the role of the calcineurin-NFAT

pathway in inflammatory responses, we quantified chemokine and cytokine production in

hMDMs during SC infection. TNF-α, GM-CSF, MCP-1, MCP-1α and MCP-1β expression were

attenuated by calcineurin inhibition (P=0.02; P=0.02; P=0.03; P=0.0003 and P=0.009

respectively) (Figures 1E, E1G). Flow cytometry characterisation of hMDMs and lung

transplant hAMs revealed similar inflammatory activation status with CD11c +MHCII +CD206 +

surface expression. There were significant differences in surface CD11b (P≤0.001) and CD86

(P≤0.001) expression (Figure E1H). Blockade of the fungal C-type lectin receptor Dectin-1

(P=0.001) or inhibition of downstream Syk signalling (P=0.001) impaired TNF-α responses,

but there was no effect on NFAT translocation (Figures E1I, E1J). These results indicate that

the calcineurin-NFAT signalling pathway is critical for early macrophage inflammatory

responses to Af , independent of Dectin-1 and Syk, in humans.

Human macrophage phagocytosis, reactive oxygen species production and killing of A.

fumigatus are calcineurin-dependent

Next we characterised the role of calcineurin-NFAT in killing of Af. hMDMs were infected

with SC following treatment with FK506 or vehicle, and fungal growth analysed at 1 and 6h

p.i.. Both hMDMs and hAMs pre-treated with FK506 exhibited impaired control of fungal

growth (P<0.001 and P<0.0001 respectively) (Figures 2A, E2A). Time-lapse video microscopy

demonstrated increased Af hyphal transition in FK506-treated macrophages (P=0.02) (Figure

2B). To identify the calcineurin-dependent mechanism for fungal growth inhibition, we

assessed efficiency of Af phagocytosis. Calcineurin inhibition delayed phagocytosis of SC in

hMDMs (P<0.01) and hAMs (P=0.04) early post-infection but phagocytosis of RC was not affected (Figures 2C, E2B, E2C). We measured reactive oxygen species (ROS) production and

Cathepsin B (a lysosomal cysteine protease) activation, which are critical for Af killing (22,

23). ROS production was partially calcineurin-dependent (P<0.0001) with no effect on mitochondrial ROS (Figures 2D, E2D-E) or activated Cathepsin B production (Figures 2E-F).

Calcineurin-dependent effects on phagosomal maturation were analysed by phagosomal acidification and recruitment of the late phagosomal maturation marker, LAMP-1 to Af - containing phagosomes. Calcineurin inhibition increased LAMP-1 recruitment at early time points after infection (P<0.05) (Figure E2F, E2G). Phagosomal acidification was not calcineurin-dependent (Figures E2H, E2I). These results indicate that inhibition of the calcineurin-NFAT pathway impairs the ability of human macrophages to control fungal hyphal transition, delays fungal phagocytosis, and reduces ROS production.

Macrophages traffic Af -containing endosomes to neighbouring cells through calcineurin- dependent lateral transfer

During live time-lapse confocal imaging, we observed lateral transfer of germinating Af between hMDMs (Figure 3A, Movie 1). This was confirmed in alveolar macrophages from lung transplant recipients (Movie 2). Lateral transfer events occurred in 3.0 ± 0.4% of cells infected with SC, peaking at 4 to 6h p.i. (Figure 3B). Calcineurin inhibition markedly reduced lateral transfer (53.9% +/- 10.3) (P= 0.002) (Figure 3C).

We assessed the role of fungal germination in lateral transfer of Af by time-lapse microscopy of metabolically inactive RC or dead fixed SC. Lateral transfer was dependent on both live and swollen Af (P<0.001) (Figure 3D). We observed co-localisation of actin and

dynamin to conidia undergoing lateral transfer (Figures 3E,3F) suggesting an actin and

dynamin-dependent process. This was confirmed by treating hMDMs with actin

polymerisation or dynamin inhibitors (Cytochalasin D or Dynasore respectively) following

phagocytosis (P<0.001) (Figure 3D). Fungal burden was increased after inhibition of lateral

transfer post-phagocytosis using Dynasore, suggesting transfer enables recipient

macrophage control of hyphal germination (P<0.01) (Figure E3A). Transmission electron

microscopy showed that transfer of Af -containing cargo between hMDMs occurred in

membrane-bound compartments (Figure E3B). The Af -containing cargo transferred between

hMDMs was further characterized by staining for endo-lysosomal markers (Rab 5, Rab7 and

LAMP-1). This showed that Af was transferred between hMDMs in Rab7-positive and Rab5

and LAMP-1-negative compartments, suggesting late endosomal trafficking (Figure 3F and

E3C).

To determine whether this phenomenon occurred in vivo , we exploited our zebrafish

invasive aspergillosis model (4). Using an mpeg:mCherry transgenic zebrafish line and eGFP-

expressing Af, we examined the macrophage-Af interaction in vivo using time-lapse high-

resolution confocal microscopy. We observed similar macrophage Af lateral transfer in

zebrafish larvae, consistent with an evolutionarily conserved macrophage response to Af

infection that occurs in vivo (Figure 3G and Movie 3). Taken together, these observations

indicate that successful fungal germination in the late endosome triggers exocytic transfer

of Af between macrophages.

Macrophages undergoing calcineurin-dependent programmed necrosis transfer Af conidia

to neighbouring cells to control fungal germination

hMDMs transferring Af had increasingly translucent cytoplasm, organelle swelling and increased cell volume, suggesting programmed cell death during transfer. This was confirmed by confocal microscopy using propidium iodide (PI) (Figure 4A, Movie 4). Around

90% of macrophages successfully control fungal germination and do not die (Figure 4B). PI staining revealed that in dying macrophages (13±2%), lateral transfer occurred in 37 ± 5%

(Figure 4D). This represents ~3-4% of total macrophages as previously described infected with Af SC. Transfer of Af conidia from dying hMDMs enhanced control of germination, compared to dying hMDMs where conidia were not transferred (P= 0.003) (Figure 4B).

Germination of Af in live hMDMs was well controlled when compared to cells undergoing programmed necrosis (Figure E3D) , further supporting a model where programmed cell death and lateral transfer occur in response to successful fungal germination in macrophages, to enable containment of infection in recipient macrophages (Figure 4B).

Consistent with programmed cell death, we observed massive vacuole formation adjacent to the Af -containing compartment (Figure E3E, Movie 5) (24). Transmission electron microscopy also showed conidia residing within large vacuoles (Figure E3F). Vacuole formation was inhibited by pre-treatment with the vacuolar H+ ATPase inhibitor Bafilomycin

(P=0.003), with a trend towards decreased transfer (P=0.06) (Figure E3G).

To define the relationship between cell death and transfer, induction of necroptosis with the pan-caspase inhibitor Z-VAD-FMK (an inhibitor of caspase-dependent apoptosis which increases necroptosis) was performed (25, 26). This increased lateral transfer (P=0.003)

(Figure 4C). Addition of Necrostatin-1, an inhibitor of RIP1 kinase-dependent necroptosis, to

Z-VAD-FMK, inhibited both cell death (P=0.078) and lateral transfer (P=0.028) induced by Z-

VAD-FMK alone (Figure 4C). However, induction of necroptosis with Z-VAD-FMK led to

increased fungal burden due to recipient macrophage death (P<0.01) (Figure E3H).

Necrostatin-1 alone or necrosulphonamide could not inhibit macrophage programmed cell

death or lateral transfer (Figure 4C). As pyroptosis has recently been shown following

macrophage infection with C. albicans , we blocked pyroptosis with an irreversible cell

permeable caspase-1 inhibitor, (Z-YVAD-FMK), which did not affect cell death or lateral

transfer (Figure 4C) (27). These observations are consistent with transfer of Af between

macrophages during a programmed cell death process with hallmarks of necroptosis.

As calcineurin regulates cell death, we assessed its role in macrophage necrosis following

infection (28). Calcineurin inhibition reduced macrophage programmed necrosis by 57% +/-

13.7 (P=0.01) (Figure 4D) and was associated with accelerated fungal germination at late

time-points in macrophages that failed to undergo necrosis-dependent transfer (P<0.001)

(Figures E3I, E3J). This is consistent with a calcineurin-dependent checkpoint for

programmed necrosis during Af germination.

Together, these results indicate that hMDMs infected with live Af SC undergo calcineurin-

dependent cell death with hallmarks of programmed necroptosis and transfer Af -containing

endosomes to neighbouring cells to facilitate control of fungal germination.

Calcineurin regulates cell death and the MAP kinase pathway during Af infection

To further define the macrophage response to Af , we undertook next generation transcript

profiling of hMDMs during phagocytosis. hMDMs pre-treated with FK506 were infected

with SC for 1h or 6h, and differentially expressed genes compared to baseline and untreated

infection controls were identified (Figure E4A). The major gene sets regulated by calcineurin

at 6h p.i. were associated with cell death, inflammatory responses and transcriptional

regulation (Figure E4B). 51 genes associated with the cell death pathway were calcineurin- dependent at 6h p.i (Table E1).

Over-representation analysis using ConsensusPathDB (CPDB) identified calcineurin- dependent control of the programmed cell death, MAPK signalling and cytokine-cytokine interaction pathways at 6h p.i. (Figure E4C). To assess the interaction between the pathways regulated by calcineurin, a protein-protein interactions (PPI) network was created. This identified significant interactions and clustering of transcriptional regulation pathways, immune response pathways, apoptosis and the AP-1 transcription factor network (Figure

4E). Studies have suggested a critical role for the MAPK-AP-1 pathway, and dynamin-related protein 1(Drp1)-mediated mitochondrial fission in necroptosis (25, 29). As mitochondrial translocation of Drp1 and MAPK-AP-1 pathway regulation are thought to be calcineurin- dependent, this was explored in Af -infected hMDMs (30-32). Significant modulation of

MAPK-AP-1 pathway activation and Ser637 P-Drp1 phosphorylation was observed (Figure

E4D). Thus, RNA- and protein-level analyses further support a role for calcineurin in the macrophage programmed cell death response to Af .

Af containing endosomes transferred from macrophages to macrophage are enveloped within actin-VASP rich cargo

Calcineurin phosphatase activity has multiple NFAT-independent effects on cytoskeletal reorganisation and regulation of MAPK pathways (31, 33, 34). To identify calcineurin phosphatase targets, we performed a comparative phosphoproteomic array on FK506 vs. vehicle pre-treated hMDMs infected with SC (Figure 5A). There were differences in phosphorylation of proteins within the MAPK pathway including c-Jun, and the cell cycle

regulatory protein CDC25A. This is consistent with our transcriptomic findings of cross-

control between calcineurin, and MAPK pathways and cell death pathways.

The analysis revealed a 26-fold increase in Ser238 phosphorylated vasodilator-stimulated

phosphoprotein (VASP) levels in FK506-treated hMDMs (Figure 5A). VASP is an actin

polymerase that promotes assembly of actin networks, and is important for directional

motility and phagocytosis (35-37). Its function is tightly regulated with Ser238

dephosphorylation required for actin filament formation. We therefore postulated that

VASP is a direct calcineurin target. As calcineurin is the only Ca 2+ -activated serine-threonine

phosphatase, we measured Ser238 P-VASP following the addition of the calcium ionophore

ionomycin (to selectively activate calcineurin) to hMDMs with phosphorylated VASP

(induced using a cell permeable cGMP analogue) in the presence or absence of FK506

(Figure 5B). This confirmed that VASP is a major dephosphorylation target of calcineurin.

As VASP is an important actin cytoskeletal regulator, and calcineurin is required for optimal

phagocytosis and transfer of Af, we hypothesized that VASP is involved in actin-dependent

phagocytosis and lateral transfer of Af. Using high-resolution confocal microscopy, we

observed co-localisation of VASP to Af at phagocytic cups, which disappeared following

internalisation (Figures 5C, 5D and Movie 6). Localisation of VASP to Af was not calcineurin-

dependent. During Af lateral transfer at late time-points, high intensity co-localisation of

VASP and actin staining was observed to Af -containing endosomes (Figure 5E). High-

resolution 3D reconstruction revealed that Af containing endosomes trafficked between

macrophages are enveloped within actin-VASP rich cargo (Figure 5C-G and Movie 7).

To determine the role of VASP in macrophage Af phagocytosis, siRNA knockdown of VASP

was performed in human macrophages, followed by analysis of uptake of calcofluor white-

stained live Af SC compared to control. This showed that phagocytosis of live Af SC at early time-points is VASP-dependent (P<0.01) (Figure 5H), consistent with the defect seen for calcineurin inhibition (Figure 2B). Taken together, these results indicate that calcineurin has a key role in macrophage actin cytoskeleton re-organisation during fungal infection, by direct de-phosphorylation and activation of the actin polymerase VASP. This enables optimal actin-dependent Af phagocytosis and lateral transfer of Af containing endosomes during programmed necrosis, which are critical to achieve control of germinating conidia.

Discussion

Pulmonary aspergillosis has emerged as a serious infectious complication of a range of immunocompromised states and chronic respiratory diseases. Overt infection is typically characterised by hyper-inflammatory tissue destruction, with more complex relationships between airway colonization and progression of chronic respiratory diseases (38). Here we report human macrophage programmed necrosis as an important response to germination of Af. Whilst necrosis has been recognized as a form of cell death since the mid-nineteenth century, it has recently become clear that programmed necrosis may occur through receptor-interacting protein kinase 3 (RIPK3) necroptotic cell death (39). Necroptosis occurs as a first line defense to intracellular pathogens, and is central to the pathogenesis of a number of chronic inflammatory conditions including emphysematous change (40, 41).

Remarkably, we observed frequent lateral transfer of Af during necroptosis which ultimately enabled control of hyphal escape from the macrophage. Transfer occurred through a late endosomal compartment, suggesting that this process may have similarities to exocytosis

(42). Whilst lateral transfer has previously been observed for Cryptococcus neoformans at

low frequency, the molecular basis for transfer and its relationship to cell death or control

of infection have not been previously defined (43). The pathogen escape and transfer

processes described to date are pathogen-mediated, and enhance either tissue

dissemination, or evasion from immune attack (47). However, our observations are

consistent with host-dependent lateral transfer of Af , to limit hyphal escape during

macrophage death. The observation of cell-cell transfer of Af supports a model whereby

inhaled conidia may persist in the myeloid compartment as spores for prolonged periods of

time. This has potentially important implications for individuals who subsequently undergo

immunosuppression.

Notably, we observed that both fungal-driven programmed necrosis and lateral transfer

were calcineurin-dependent, indicating that necroptotic control of infection is likely to be

impaired in organ transplantation. Calcineurin has been postulated to have a role in the

regulation of programmed cell death through interaction with Bcl-2 family proteins (25, 26).

Consistent with this, we found significant calcineurin-dependent expression of Myeloid Cell

Leukaemia 1 (MCL1), a Bcl-2 protein that inhibits apoptosis, and Bcl2 Modifying Factor

(BMF), a member of the pro-death BH3-only subgroup of Bcl-2 family proteins required for

TNFα-induced necroptosis (27). Transcriptomic and phosphoproteomic analysis defined

calcineurin as a key phosphatase mediating cross-control of cell death, the MAPK-AP-1

innate pathway, and VASP-dependent cytoskeletal remodelling during macrophage

infection. Calcineurin inhibition impaired these cellular processes, leading to enhanced

fungal germination in the late phagosome, impaired lateral transfer, and ultimately hyphal

escape. Interestingly, virulent Burkholderia spp. have recently been shown to mimic VASP

actin polymerases to enable cell fusion and spread (44). Whether fungi are able to directly manipulate host actin polymerases remains to be determined.

In summary, we have identified macrophage programmed necrosis as an important response to progressive germination of Aspergillus fumigatus in the human macrophage, and report a previously undescribed phenomenon of cell death-dependent lateral transfer as a co-operative macrophage behaviour that assists in limiting hyphal escape. We show that calcineurin is a key orchestrator of this process, further defining the importance of this pathway in innate immunity. Our findings yield novel insights into host innate immunity to

Af in the lung, extend current understanding of the pathogenesis of PA in organ transplantation, and have broader implications for transplant immunity and chronic lung disease.

Acknowledgements

We thank the patients of the Royal Brompton and Harefield Hospitals UK who participated in this study. This project was supported by the NIHR Respiratory Disease Biomedical

Research Unit and the Imperial College Academic Health Science Centre. We thank Avinash

Shenoy and Jason Mercer for helpful discussions and the Imperial Facility for Imaging and

Light Microscopy and Amelia Shoemark for technical support.

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Figure Legends

Figure 1. A. fumigatus activates calcineurin -NFAT-dependent inflammatory responses in

human macrophages

(A) hMDMs were stimulated with live Af SC (MOI=1). hMDM nuclear extracts were collected

at 30, 60 and 120min p.i. and probed by Western blot for NFATc2. Maximum translocation

of NFATc2 to the nucleus was observed at 60 minutes p.i. Western shown representative of

data from n= 4.

(B-C) hMDMs were pre-treated with FK506 (10ng/ml) and stimulated with live Af SC

(MOI=1). Nuclear extracts were probed by western blot for NFACTc2 and whole cell extracts

for RCAN1. NFAT translocation was assessed by confocal microscopy. NFAT shown in red,

nuclei in blue and Af conidia are seen in green. Representative images are shown of live

conidia stimulating NFAT nuclear translocation. Western shown representative of data from

n= 4.

(D) hMDMs were pre-treated with FK506 (10ng/ml) and stimulated with live Af SC (MOI=1).

FK506 completely inhibited RCAN1 production. Western shown representative of data from

n= 4.

(E) hMDMs were pre-treated with FK506 (10ng/ml) and stimulated with live Af SC (MOI=1).

Culture supernatant cytokines were measured at 6hr p.i. by Luminex. N =7

Figure 2. Human macrophage phagocytosis, reactive oxygen species production and

control of A. fumigatus growth are calcineurin dependent

(A) hMDMs pre-treated with FK506 (10ng/ml) or vehicle were stimulated with live Af SC

(MOI=1), and total RNA isolated at 1 and 6 hour p.i. Fungal growth was assayed by RT-PCR of fungal RNA. N = 5

(B) hMDMs pre-treated with FK506 (10ng/ml) or vehicle were infected with live eGFP- expressing Af SC (MOI=1), and hyphal transition assessed using time-lapse video microscopy over a 10h period. N = 4

(C) hMDMs pre-treated with FK506 (10ng/ml) or cytochalasin D (5nM) or vehicle were stimulated with live Af SC (MOI=1) prestained with Calcofluor white. Phagocytosis was quantified by Imagestream. p.i. N=4

(D) hMDMs pre-treated with FK506 (10ng/ml) or vehicle were stimulated with live Af SC

(MOI=1) and ROS production assayed by luminescence. N=4

(E) hMDMs pre-treated with FK506 (10ng/ml) or vehicle were stimulated with live Af SC

(MOI=1) and Cathepsin B activation assayed by live confocal time-lapse microscopy.

Phagosomal activated Cathepsin B activation was quantified by ImageJ . N=4

(F) Representative confocal microscopy image showing Cathepsin B activation in Af conidia containing phagosomes in MDMs. Activated Cathepsin B is shown in red, nuclei in blue and

Af in green. Data are represented as mean ± SEM.

Figure 3. Macrophages infected with swollen A. fumigatus undergo calcineurin-dependent lateral transfer.

(A) Representative time-lapse widefield microscopy images of Af lateral transfer in MDMs

infected with live Af SC (MOI=1, shown in green). Transferring macrophage labelled ‘a’ and

receiving macrophage labelled ‘b’. Images taken at 10 minute intervals.

(B) hMDMs were stimulated with live eGFP-Af SC (MOI=1) and time-lapse widefield imaging

performed at 10min intervals for 12h p.i.. Timing of lateral transfer events per hour in

macrophages infected with live Af SC is shown (N=3).

SC (MOI=1) and lateral transfer events quantified by time-lapse widefield imaging at 10

minute intervals for 12 hour p.i. (N=5). Total lateral transfer events over the 12h period

were quantified for each biological replicate.

(D) hMDMs were stimulated with live or fixed Af SC or RC (MOI=1) and lateral transfer

events quantified by time-lapse widefield microscopy. Cytochalasin D (5nM) and Dynasore

(100μM) were added at 90mins p.i. with unbound conidia washed away 45mins p.i.. N=4

(E) Representative confocal microscopy image of dynamin-dependent lateral transfer in

monocyte-derived macrophages stimulated with live Af SC. Dynamin shown in red, nuclei in

blue and Af in green.

(F) MDMs were stimulated with live Af SC (MOI=1) with non-phagocytosed conidia washed

away at 45mins p.i.. Lateral transfer events were captured by fixing cells every 10mins from

120-180mins p.i.. Characterisation of Af lateral transfer was performed by staining for Rab 5,

Rab 7 and LAMP-1 (shown in red) alongside actin (shown in cyan). Nuclei are shown in blue

and live swollen Af in green. Representative images show positive Rab7 localisation to Af

containing endosomes during lateral transfer with little Rab 5 localisation and no LAMP-1 localisation.

(G) MPEG:mCherry Zebrafish larvae were infected with ~50 eGFP-expressing conidia of Af and time-lapse confocal microscopy performed. Representative images show Af lateral transfer between macrophages. The transferring macrophage is labelled ‘a’ and the receiving macrophage labelled ‘b’. Images shown are at 7 minute intervals. Data are represented as mean ± SEM.

Figure 4. Macrophages undergoing programmed necrosis laterally transfer germinating A. fumigatus conidia in endosomes through an actin-dependent process.

(A) Representative time-lapse confocal microscopy images of lateral transfer of live Af SC

(shown in green) between MDMs. Propidium iodide staining (red) showing cell death of macrophage transferring Af conidia (labelled ‘a’) to neighbouring macrophage (labelled ‘b’).

Images taken at 10 minute intervals.

(B) Time-lapse confocal microscopy was used to quantify the fate of transferred vs non- transferred conidia from dying human MDMs and to conidia within live macrophages.

Germination was assessed over a 12-hour period. Control of fungal germination was significantly increased in dying hMDMs that transferred conidia to neighbouring cells compared to those that did not. N=4

(Necrostatin-1 10μM), caspase-1 inhibtor (Z-YVAD-FMK 50µM), FK506 10ng/ml or vehicle were stimulated with live Af SC (MOI=1) and lateral transfer events and cell death quantified by propidium iodide fluorescence based time-lapse confocal microscopy. Treatment with Z-

VAD-FMK induced necroptotic cell death coupled to increased Af lateral transfer, which was

inhibited by the addition of a RIPK-1 inhibitor (Necrostatin-1). N=3

(D) hMDMs pre-treated FK506 (10ng/ml) or vehicle were stimulated with live Af SC (MOI=1)

and cell death assayed by propidium iodide fluorescence quantified by time-lapse confocal

microscopy over a 6h period. The graph also shows the proportion of lateral transfer events

in dying cells (~30% of dying cells). N=4

(E) The network modules of the significantly expressed genes in FK506 pre-treated

macrophages stimulated with live Af SC (MOI=1) for 6hrs. Significantly expressed genes

were mapped on to PPI network and network modules identified using the MCODE plugin

application of Cytoscape. Over represented pathway involving these network modules were

identified from the KEGG and Wikipathways databases. N=6. Data are represented as mean

± SEM.

Figure 5. Lateral transfer of Aspergillus fumigatus occurs through VASP tunnel-like

structures.

(A) A phosphoprotein array analysis of hMDMs pre-treated with FK506 and stimulated with

live Af SC (MOI=1) for 1hr p.i was performed. Proteins with significant fold change

differences in the phosphoprotein to non-phosphoprotein ratio between FK506 and control

macrophages stimulated with live Af SC are shown.

(B) hMDMs were pre-treated with a cell-permeable cGMP analogue (8-pCPT-cGMP 25mM)

to induce Ser238 VASP phosphorylation and FK506 (10ng/ml) or vehicle. They were then

stimulated with a calcium ionophore (Ionomycin 2μg/ml) to activate calcineurin.

Macrophage whole cell extracts were probed by western blot for Ser238 VASP and total

VASP.

(C) Representative confocal microscopy images of hMDM phagocytosis and lateral transfer of live Af SC (MOI=1). VASP shown in red, nuclei in blue and Af in green. 1: Uninfected hMDMs; 2: Initial phagocytosis of Af by MDMs; 3: Late phagocytosis of Af by hMDMs; 4:

Lateral transfer of Af between hMDMs.

(D) 3D reconstruction of confocal microscopy images of phagocytosis and lateral transfer of live Af SC (MOI=1) between hMDMs. Image of Af phagocytosis at 30min p.i. with VASP shown in red, Af in green and nuclei in blue.

(E) 3D reconstruction of confocal microscopy image of Af lateral transfer with VASP shown in yellow, actin in magenta, Af in green and nuclei in blue.

(F-G) Representative z-slice confocal microscopy image (F) and 3D reconstruction (G) of Af lateral transfer. VASP shown in red, Af in green.

(H) THP-1 macrophages treated with either control or VASP siRNA (50µM) were infected with live Af SC and stained with calcofluor-white (Sigma) (MOI=1). Phagocytosis was quantified by flow cytometry. N=3. Data are represented as mean ± SEM.

Figure 1. A. fumigatus activates calcineurin-NFAT-dependent inflammatory responses in human macrophages

Figure 2. Human macrophage phagocytosis, reactive oxygen species production and control of A. fumigatus growth are calcineurin dependent

PPP P PPPP

PPPP P

b b b b b

B C P = 0.003 P = 0.028 P = 0.026 P = 0.078 D 25 Total dying cells Live cells Dying cells undergoing lateral transfer Transferred conidia from dying cells 20 Non-transferred conidia within dying cells P = 0.01

P = 0.003 15

5 PI positive cells %

Lateral transfer/ 300 cells 0 Swollen AF conidia - PI positive cells % % Control % of germination + + + + + + + + + + + + Z-VAD-FMK - + + -- - - - + + -- - FK506 - + Necrostatin-1 - - + + -- - - - + + -- A. fumigatus Necrosulphonamide ---- + ------+ - Z-YVAD-FMK - --- - + ------+

Caspase cascade in apoptosis E NOD pathway

IL2RG

SPI1

CEBPB SOCS7 MEF2A NCOA1 USO1 KAT2B RXRB EHMT1 CTBP2 FOS RUNX3 IRF1 ACTN4 ATR CDK7 PIN1 BAG6 E2F1 ADRB2 STAT1 EGR1 TFE3 SYK HSF1 PHB2 AIFM1

GOSR1 DDB2 MDM2

STX5 NACA Immune system ACLY Immune response mRNA processing GET4 Apoptosis Transcription IL-1 Signalling pathway MCPH1 PTMS

ASAP1 PCGF2 COG8

SOS1 TAF6 COG6

MBD3 TBP TAF1 COG1 COG5 TAF10 ABL1 CHD4 CRK KDM1A KAT2A TAF4 PHC1 TOP1 COG2 BCAR1 MTA2 RING1 TAF5 PARK7 IL23A COG7 TRRAP CBX4 MTA1 BATF3 SF3B3 PHC2 SNW1 ATF4 RIOK2 CBX8

DDX3X SETD5

BMI1 CINP ATF3

EBPL PHC3 COG3 CAND1 MAML2 LCP2 SRSF2SF3B2 RRAS DPYD NADK RXRA RET INSR NBPF1 BSCL2 STRN3 GCNT3 FRS2 SF3B4 TIMP3 PLCG2 DHX15 PDE4B SSBP3 RFC1 ORC6 MKNK1 FPR1 BRMS1

TNIP3 SRPK2 NCF4 PEF1 AVL9 EPHB2 SRF MMP10 ELOF1 FYB BCL6ING1 WDFY4 SHC1 FBXL3 EHD4 SAP30 RUNX1 ABL2 SRC TRAF1 HDAC7 PARL NR2C2 SGK3 TAP2 DCTN5 MTA3 ARAF UBP1 BLNK MET CD33 NCOR2 ORC4 HERC4 TFPT BTBD6 SIKE1 COG4 TRAF3 PML SIN3A DNMT1 LST1 GMIP CBL ABI1 TDG NMT1 ZGPAT TFEB TEC ARL2 FPR2 G2E3 DCTN6 CCNC CFLAR TRAF2 HDAC2 GAKGALM USE1 BCR ATG2B TPST1 VPRBP IL7 WDR89 CYTIP ALG3 HDAC1 CASC4 TESK1 AZI2 BTK BIRC2 DDX17 MGA ZFAT ADCY7 IQCK TEN1 FIBP PHF1 DUS1L PLCG1 GRB2 RBBP4 FCAR LARP7 DPP4 TEX14 PDE2A RRN3 MST4DDA1 SRRM1 MBP TAF7 LYN PAQR3 NEMF STK25 ORAI2 WIPI1 TGIF2 NOP58 SCRN3 PIAS1FCN1 MLH1 TAF1D CDCA4 RAB12 AIM1 ORC5 ORC2 BIN2 IRF5 NFYC PRG2 ZG16B TBRG4 NPHP3 CEP78 PRKDC XPO6 ZZZ3 BIRC3 PAIP1 PIR CCL8 HDAC3 ATF1 GAB2 RNPS1 TPRN PTAFR SMAD7 RBP1 BUB3 U2AF1 BRWD3 IL15 STRN4 ATF2 KCP KIFC3 MLEC API5 CCNY MIER1 ETV7 EMB BAG1 WDR5 NACC2 CCL24 SBNO2 DDX5 ABCB8 APOM ZNF57 FAM3A DHX9CUL4A U2AF2 KCTD6 RBM22 SUMO1 AP3M2 BBX SAR1A RBM19 PHF10 CTDP1 ZNF23 NSF KDM2A SLMAP DDX1 STRN NID1 WDR24 CD151 FKBP4CCL17 ENY2 PEX19 MTCP1 UHMK1 SNX4 GMPPB SMAD1 AP1M2 TSLP ELMO2 S1PR4 ARL4D DDX27 ERCC8 HERC6 THEM4 HPS5 PI4KA ICAM4 PRUNE TLR5 FBXL5 TXNL1 GFRA2 ECE2 PGM1 GBP2 RCN1 WWOX NLRC5 TLR6 ZNF92 ETHE1 NAB2 UCHL5 CBX6 RWDD3AREG PER2 RAP2A GALC MTMR2 GNL3 TRAK2 HIPK1 SUZ12 ODF2L CMC1 SRSF5 IMP3 ASCC2 TTLL5 RBM23 CD22 DHX33 IL12B RFX7 PVRL2 PXDC1 CRY1 APOE SRRT FNIP1PCF11 CBR3 GAS7 IDH3A ACRBP TESC J AG1 TMX1 CDK3RGS16 PCSK6 LARP1 CD226 ZNF35 RRAGC CUL3 SUMO2 CASS4 ASB1 CSPP1 HEXDCCAMP HIPK3 PCNPTADA3 NUDC HIC1ATRX CDK1CCNB2 MLKL ISOC2 LAT CUL1 CNNM2 MORC2 GPR3EVI5L KIF1B RIPK1 MBD2 KLHL2 CKAP4 AHI1 GNPAT RBM14 MOCS3 LY9 RRP12 IFT88 GRASP NLRC4 PDK2 XIRP1 TTC33 MALT1 HSBP1 MZT1 YIPF1 TSR2 TAF5L PRRC1 MDM1 CSF3R ADO ECSIT SRA1 SMYD2 DTD2 CLMN FER KCNN4 TXLNA SF3B4 MAEA HIPK2 CKAP5 SRSF7 GNAQ CCND3 SKIL LRRC3 PRDM4 SCRN2 ID1 NOM1 STIL TTC4NSUN2 CCL1 J UN AP2A1 LTBP3UBE3C ASXL1 DDAH2 STK10 SMC1AID2 CDK6CCNA2 COASY SRSF1 PVRL4 NOXA1 SOCS5 GCH1 MPP7 RAP2C BCCIP ARL15 SKP1 TAOK1 PDGFA TGFB2 EXOC5 TOX4 NUP93MYCAP1S1 BCL9L SF3A1 UTY ID3 FOSB PRDX2 LAMA2 BAD GMPPA PUF60 SSH3 CARD9 CHD6 RELB DVL1 ZNF17 PDE4APLECANXA2 PINK1 LIME1 TIE1 PELI3 DPP3 ATN1 FXR2 GSTK1NSDHL CEBPA CCNK FNBP1 CPEB2 GSTCD ADRM1 FNTB RAI1 ADCK4 MMP7 AP1M1C1R GRWD1 ZFHX3 OFD1 BLMH GPS2 EPC2 AP1G1 MYL9 MAFB EHD3 NELFB LAIR1 TTYH3 FLOT2 ITCH NCBP2 AP2B1VDR SFPQSRRM2ILF3 ZBTB9 STAT3 STMN1 CASP3 SIAH1 EZH2CDK2ETS1 FKBP3 ZNF18 PTX3 SSR1 CDK16 DPEP2 LIN52 SKA2 LYRM4 ACSL5 CHD1 NAA10 FGFR1 MRGBP DDX18 TYRO3 CTIF SCRN1 TIAF1 MSTO1 FGD2 DOK3 PPM1G ELK1 MAF UTP6 AFF1 DNMBP RBM12 TLR1 PGM2 NSRP1 MFSD5 PVRL1 GPSM1 QSER1 MBD4 MELK LRP11 SYTL1 SH2B2 HEG1 RNF11 HGF PARG EXT1 NAV1 NSD1 EEA1 GPN1 LSM12 IFIT5 NOC2L VPS72 BOP1 DSC2 XAF1 OSTM1 SLTM MEMO1 ZFP82TFDP1 LIN7A FARP2 RERE ITPK1 PPM1BPTPRE LTV1 SLMO2 CCNG1 DTD1 ILF2 AAK1 DSN1 NCK2 MYO1F CDK5 AIMP1 ACOT2 NOD1PHF7 SCYL2 MEP1A SCOC CD72 PDGFB PNISR CPSF2SP1 KDM5C SF3A3 CADM1 PKP4 PRKX PEBP4ISCU CKS2 CEP68 CHAC2 SIAH2 STAG3 DGKA ATG4C MBTD1 RTN1 TTC3 RIC8B MEGF6 PHF19 J ADE2 SPSB2 MT1A ALS2 FES BICD1ETV5 NQO2 BECN1 AGO2 RAP1A HMOX2 RNF6 KNTC1 BRPF3 PKN2 TAF6L NRP2CRK NGLY1 ADCY9 IL1R2 ALG2 MACF1 SEC62TEF PCGF1 NUMBL SMYD3 FLNB APEX1 E2F3 SRSF3 DYSF ERCC2 PGAM2 TRIM8 ILVBL NEK11SLBPSYT11 CD244 BCL2 SF3A2 GRB10SENP1 SATB1 RFX2 ASTE1 SDF2 EBP OPHN1 NOL3RBM45 ZFP1 CXXC5 CRADDEGLN3IP6K2 CPNE1HUWE1 KIFC2 WDR92CSAD ING5 MEX3D BORA IFT46 P2RX7 BIN1 HMGCL TRIP4 RIN2 GAB3 PDGFC ATR IGF1R GNL1 FUBP3 ERN1 DBP TPST2 SON FZD5 RORASOCS2 BATF TFB1M ITSN2ENSA AXIN1 BACE1 BMP6 WDR3 ETFB CRTC2 BICD2 OPA1 MCMBP GAS6 OASL MCPH1IL2RG ASB2 RCAN1 IRF9 CYLD J ADE1 ARRB1 OAZ3 VAV3 RCOR3 TTC5 ERI2 ADCY3 CCL23EMD CELF1 MS4A7RFX3 SRPK1 MPP5 PRAF2 ALDOC IQCE HNMT PYGO2 TNIKE2F4TNKS2 APOL2 NQO1 APH1A SKAP2 AKT2 NR1D2 S100B CCM2 PA2G4 CCR7 UBE2K DDX42 GNL3L KAT2B CDK7 WEE1 NACC2 DCTN4 NAA38 TMX4 SHB J MY EXTL3 CTBP2J AK2 TULP3 FHL1 HDAC9CREB1 HERC5 RUFY2 ZMIZ1 STAU1 SAFB2 IREB2 TAF11 EFNB1 PIAS2 NEU1 RBBP6 CCL3 BTAF1 FZD4 PIAS3 STAT1 WDHD1 HAUS3 PLTP SPC25 ZHX3CEP97 DGKD RUNX3 HSF1 LMNB1 EHMT2 CENPV MZF1 C1QC CXCR5 ATG13 CSRP2 BBS10 DHRS7 MMP14 MGMT AQP3 SDC1 KCNQ1 NIN FZD1RGS3 EHMT1 TFE3 MIB1 PELO TRAK1 CREM ATG5 RAE1 EDC4 MERTKPLD2 NCOA1 GSE1 CENPJ UBR7 HMGB2 NBR1 PRC1 GSTM4 NMI BAI1 AIFM1 HDAC4 ZNF45 SAMD1RSU1 PECR GPC4 QKI LENG8 WIPI2MCM8 CDK9 RUSC1 ACD PUS7 TLR7 HBEGF TOB1 TPD52 CDK14 EMC6 DNPEP LUC7L WIBG EPHB6 ADRB2 J DP2 TAF1 TRA2A RGS14 PSME4 GGA2 ERI1 NEK3 RARGCEBPBDDB2 EGR1 MTA3 TAF5 SOX13 RAB37 ATRN NACC1 SYS1 PILRB ACTN4MEF2APIN1 E2F1 SMAD6CCNE1 PCNT ATAD2 PSMG2 UBA3 RBM15 PHB2USO1 FOS SPI1 CASP6 TAF8TAF1A CAB39 SNX6 SESN2 GNA13 ATP5J WDR34 MITF ADNP DLG4 XPA NFYB CLOCK KDM6B CIZ1 GNAI1 NOB1 FGD3 TRAF5 MDM2LEPRGET4 PI4KB ACOX2 WDR53 SMG5 DKC1 RING1 AES SNTA1 RILP MARK4 SOCS7 NCOA2 CENPC NAIF1 PQBP1 IMP4 PRKRADHX8 CEP55BAG6ACLYSYK IRF1 NPAT SEPP1 ELK3 TAP1 DEXI BCL10 KLF13 RXRB TOR3A TLR2ACSL4 BOLA1 RIN1 WDR13 ZNF8 EXT2 RAB23 GOSR1DAAM1 LRRK1 TNNI2 CLN5 DNM1L SRPRGSTM2 SSBP2 CNN2AHR USP9X SAMD9 STX5 SRSF8 MECR DHX36TACC1DDX23 SYTL3 CD79A CEBPZ GATA2 IWS1BCL7A CASKMEX3C TFPI2 DGKQ KIF22 FXYD6 RRAS2 BUD31 ATG7 CNPY4 DCAF8 LIN7B ADAM9 SNX1 ICK DGKG XRCC1 MTAP RFC4 TAF4B RCC2 TTBK2 HUS1 MKL2 UTP15 ACTN2 TADA1 SFXN3 RALY HPS4 AKAP9NACA KAT2A ZMAT3TAF4 SHPRH MYOZ1 GNA15 TPRKBSYVN1CRTAP WWC2 TDRD3 TUSC2 PDE7A NDOR1 IFT57 MCM4 LRWD1 MMGT1 SRP54 RAB36 ITPR1 PLD1 RAI14 LAGE3 NEK1 MAVS SMAD5 RAD18 SMTN FOSL2 SSNA1 ZNF16 MCM2 NEDD1 PCSK5HACL1 FRS3 CHD7 HIC2 TRAF6 KIFC1 TWF1 FNBP4 NUAK2 YIF1A NEK8 RASA1 SVIL FKBPL ATM KRI1 CENPQ PLCL2 YIPF5 EIF2D GIPC1 RIC8A TAF13 LIMK2CUL9 TAB1 CBX5 SNX30 AP1S2 RUFY1 PRR5L NAGPA WWP2 RAB4B IFRD1 DLL1 SCLY PTMS KCTD3 ZNF91 IDH3B RHOGTGFB1 CASP1 FAM3C TMEM5 EXOC1 NINL MFN1 RAD9A PWP2 FOXK1 HMGN5CLIP1 CNOT8 GLRX3 PAK4 VAV2 TAF1C WBP1 HACE1 CDC6 ZFP28 HTRA1BMF SNTB2 CUL7 PSIP1 PCGF6 PDCD1 PRCC CD63RND1 SVIP AP4M1 PTRF GNA11DDX58 RNF8 MAFA STX7FGD5 IDH3G PAOX SUN2 RIT1 UBFD1 ACACA LRP1 LTBP4 PAN2 CBX8 GCFC2 MED20 PEX5 TRPT1 GPNMB PSMF1 GSTM3 GIT2 NGDN RBM34 MCM3 MYO9BASAP1 URM1 HMGN2 CORO7 MSRA WWTR1 AURKA HLTFMEGF8 ADAT3 BTG3 RRBP1 UCHL1 SUGT1 PHF2 WASH1 TERF1 ATE1RAD1 HMGN4 NOSIP SETD8 XPO4 RFC5NLK RFC3 DECR2 THOC5 NUDT5POLH P2RX4 NR4A1 SCRIB ABI3 PKD1 GRK5 ASF1B CNTLNCENPU WNK1ELF4 TES NOLC1 TNPO3 CCNH STX11 DTWD2 TRPV1 CKAP2 FKBP9 ABI2 TPM1 LACE1 FUT4 SOCS3 STX10 TTC17 PDK1 GSK3B KAT5 FOXO3 ATRIP SMG1 ADCK1 PARP2 FANCA UTP20 ADCK3UVSSA YPEL2 APOL6 SPSB1 CD4 CLCN6 LSM10 STX16EXOC4 BZW2 SRPRB CPNE2 GCDH PELP1 DTX4 RBM3 PDE12 DUS4L CRIPT STX17 ITSN1 AURKBHERC2 DZIP3 EID2 GOSR2 LMF1 PTPRC PSD4 RAB5B SYNE1 TBK1 RBBP7 ESF1 PIBF1 NDE1 ARL3 MPRIP ULK1 ASB3 ENOX2 HCLS1 ESYT2 RGS2 APOO RETN CRIP1 DTL HSF2TAF3 TAF9BPCGF2 PTPRM IRAK1HES6 FRAT2FLII CIB1 TLK1 HBB CHID1 PPT2 RPAP2 WSB1 SNX9PSMD9 HMGN1 BBS12 RAB9A PYGB RFTN1 ABCF3 BMI1PHKG2 OCEL1MIER3LYL1 TLR8 J AK1 TEX10 CUTA ZEB2 CNOT7 TRAP1 HLX GLE1 MIS12 CEP89 IL2RA PHF12 TRPS1 MUC20 CENPK RBM41 CD47DTX2 DGKZ ANXA1 MBD1 ISCA1 RBL1CHD4 LRP3 ACAP1 MCTS1 MSH2 MEN1 HBG2 CENPL RGS12 REEP3 RAD52 PMF1PSMG1 LRBA TRIM3 ECT2 ELP4 PEX1 PMM1 LIMK1 RARA KCMF1 SMG7 MYBPH UBR1 WRN RHOQ TTC28 RRP36 BTBD7 ACTB BRD3 MLF1MYCBP CBR1 DDX3Y CDS1 CERS2 NAF1 DDX20 NOL12 ZNF71 SSRP1G3BP2 CEP85 HRH1 SFXN1 GNB1L SH2B3 VPS41 G3BP1 MEF2D PHC1 NFS1CLN8 RASA3 S1PR3 HSF4 CWC15 TPM2 MTERF HAUS7 RBBP9 C1S J UND TRIM9 OLA1 PLOD2TACC3DHX58 TLN1 FCER2 AP4S1 STX1A CD80 SRBD1 ERMAP MBNL3 STIM2 TACO1 EIF3M ACAP2 TRAF7 TAB3 LGR4 BAG2ISG15 HECW2SPIN1 CCNE2 LNX2 NIP7 PPARG FIS1 CDC20 MCRS1 PPIH BRD7 TCTA TEX30 SPHK2 ARAP3 TRAM1 EDEM1 NFAT5 MBD5 ACVR1 BMP2K NIPBL USF1ACADM CHFR MON1A FCHO1 RRP15 PURA FURIN UBAC2 LCORL BRD2 CCAR1PATZ1 ABCC9 NUDT7 PEX7 UAP1RALB DERL2 TAF9 CLPP KLF8 SKP2 XPO1 PCNAUBAP1 CDC26 NOL9 RLF KIF27 ZFP30 TTI1 KLC4 AAMP CD36 FMNL3 RHOB PPTC7 OGFR NARG2 SRPX BTG2 TMCO1 NEIL2 CAND1 ILKAP CENPM IPO11 TOP3B CDCP1 BACH1 EGLN1 RFFL NMB MBD3 REEP6 THOC3 HMGB3 MSH3 UBTD2 ZNF7SPHK1CPVL FADS2 EIF3G TNIP2 SNX2 AP3B1 ARL1 MTMR6 NOP16SEC63 LRMP CNNM3 CDK20 CEP76 KIF3C SRSF9 PDCL3 THOC6 POP1 RPAP1 KLC2 HOMEZ WDR37 MFFSMG9ST5 PNMA1 TAF12 PDRG1 EME2 TANC2 LDHD BAG4HABP4 PMS1 CLYBL J KAMP ZCRB1 OPTN PAN3 KCTD2 CMIP SCNM1 THAP3 BRCA2 ZFR SACS TNNT1IFIT1 GDAP1 PARP3 EIF3A FAN1 E4F1 PHC3 MTX1 KAT6B MEF2C TTF1 BRF2 FYCO1 MDN1 COMT ELK4 ESYT1 EI24 MYO5A UBE3A IGBP1 UBAP2OXR1 DENR NTHL1 HAUS4 EPAS1 SLU7 SAT2 APBB3 SMC2 TCF19 CDAN1 TTC7B TAF10 VPS18 VPS11 EIF3J PEX14 PNPO UMPS DCPS AVEN LMTK2 BDP1 NCS1 ENC1 RNF5 NMD3 DHX57 TMCC3 MEI1 SNTB1 EIF3I SRSF2 FOXO1 MFSD1ECI1 HELZ PTCD3 DDX28 J MJ D4 HDHD3 DDX3X PSMD2 PCBP2 FBXL6 PISD KANK1 VWF UCK1 GGT7 ZP3 AP3S2 FOXP1 NPRL2 PXMP2 OSTF1 TMED1 EIF3D GBP1 PTBP2ZFY SCMH1 LDLR NEK6 CXCL5 PAIP2 RAD21 RRAGB CBY1 S1PR2 SOX4FLOT1 SSR4 RAB1B BEX4 ZMYM6 CLCN2 NEK7MGRN1 AP1S3 TCHP AEBP2 PHF6 SHOC2 UBL7 CBS RUSC2 EPM2A TCEA2 FBXL4 AP3S1 GLRX5 PIGS TCFL5 MUTYH AMACR PDE8ATOP1 FADS1 RAC3TPP2 MDM4TAOK3 SMYD4 WDR41 ADA RGS10 VASP KLHL5 TGIF1 NUPL1 UST NVL TTF2 DCP1B TULP4 NCOA7 NOMO3 STAP2TRNT1 GAN MYO9A STS GLRX2 ABCB6 ACE ARMC5 PUM1 ABTB2 TRUB1 SPCS2 CLK1 RPP25 MKS1 ROR1 HSPB1 ZBTB1 SIPA1 ALDH2 TLN2 HHLA3 WDR60 DDX11TEP1 SPSB3 WBP2RHOC EVL STAT4NMT2 PTBP1 UBR4 FKTN PPOX CLCN5 ASB13 NFXL1 SET VDAC2 PHC2 SF3B5 AHDC1 MINA NOL10 TRPV2 AIDA LIN7C BAG3SUN1 MTF1 SAE1 FTO URB2 ACOT1 RPRD2PEX3 GFOD1 ARAP1 PODXLPRDX5 DHX15 ZBTB5 NIT2 MTO1 UACA MMP2 C1D MYNN SFXN2 WDR19 CCZ1B CALU CIRBP PSMD4 SGOL2 RBM38 STAG2RPP21 NAA40 UCK2 ASB7 RTEL1 CTU2 CDR2 CRY2 TOR1A SSU72 EAF1 CANT1 RBMS2 FASTK MPZL1 MCAT CSF3CENPT SNRK ANXA4 CASP4 FRG1 PLK3 LFNG HOOK2 KIF23 RBM8A CHP1 MIIP CDK18 TMED4 KCNA3 NAGK RYBP ERH NKTR CCL7 RHOU DCAF6 FANCG MTM1 TM2D3 MYO1B A1BG PUSL1 SNX33 POGK PSME1EIF3F RHOH MTCH2 DPM2 HAUS8 CFDP1 TRPV4 NCF1C KIF3B CETN3 ETFA CLN6 TEAD3 DDI2 HPS1 NFIL3 ARMC9XRCC3 RMI1 MRM1 AKAP1 SGSM2 SND1 SF3B1 UBE2F ACSF3 MMAB CD59 FKBP8 SPG21 CNOT2 RTCA NPHP4 STK39 TYW1 QSOX2 UFC1 MIER2 PURBGDE1 FAF2 PEX6 DHRS4 VDAC3 UBE2Z CCNG2CD68 MPV17ASCC1 RDH11SLK PCCB PUM2 PKN1 PDP1 MLH3 ZNF84 SETD4PVR DGCR6 TDRD6 FANCE EDRF1 FKRP KRIT1 UNKL SIK2 CHKA ZNF28 PDZD8 MLF2 UXS1 VAPA HAT1 KLHL8 HERC1 COPG1 LSR MUM1 NCLN PARP4 CTU1 GMCL1 LMAN2 SOCS6 ERCC1 UBAC1 ELMO1 DHX40 TTC37 COQ2 LENG9 SCFD2 PAQR4 AUP1IDE VPS29 TECR BRAT1 CCL13 DPP9 BROX ICAM5 BPHL ZBTB6 HEY1 UXT EPS8 CPT1A OTUD5 GMPR2 LY96 PIGA FRMD6 MEX3BRBM42 SIRPD OGG1 VTI1BDGKH NCDNCCNI IL6 GLYR1 CUX1 MTMR3 SNX12 FLCN HELQ GANAB NOD2 CUTC UBE2T CASZ1 SFR1 DFFB AATF AIFM2 RBAK VAMP3DEAF1 MOB1ATRAF4 ING4 TPBG RNF31 LIN54 EP400CTR9 SHPK ATG10 FNTA RRAGA EID1 FOXP4 STK19 ASGR1 BIRC7 UBXN6 ELF2 ADAM8 NLRX1 RBFA POMT2 CLIP4 TIRAP TGFA NSUN5 ZNF41 RAD17 EPN1IRAK4 WSB2 BBIP1BBS5 OSTC TTYH2 PRDX4PCM1IRAK2TLR4 NOMO2 TTC8ARL6MYO7ANENF CCL22TRIB1 AGFG1 ZMYM1 SMAP2 TRIOSPG20 ALG1 ZNF12 GSTM1 NF1 CASP2MYOF TBL1X PEX2 N4BP2 PTPRA RAB2A CHD2 LPAR1 FEM1C APBB2 SH2B1 PEPD LATS2 EXOC8 CCNT1 IRAK3 LEO1 FHITRFX1 NRIP1 BTBD9FOCAD SFI1 ABCD4 LAMC1 DMAP1FYNSLIRPRIPK2E2F6CREB3 IL1R1 SSR2 OTUD1 MAPK3 SNX8GSK3A TEFM GZF1 PDK3 ING2 LATS1SAV1 XIAPSNX17UIMC1MYD88PELI2 PAF1 MTF2 NUBP1 FBXW9 PAPD7 ARL8B WWC3 NDRG2 ZER1 OTUD3 FZD7 KDM4ABLMCTBP1AP2A2PELI1STAM2CBLBYAF2FKBP5UBE2ALBRHCFC2TREX1 EXD2 PTCD1 CRLS1 NBNNCK1EP300MYO1ESYNJ 2 RIN3 BBS1 CENPH MON1B FOPNL RPP40 NDST1 MSRB2 NHEJ 1 GORAB FHL3COPE MSH6CASP8GNSDAB2CDC27PSMD3EXOC3ARPC2ISOC1BCORDOCK1 RTN3 NFU1 TFEC INVS MT1M PUS3 VMAC ERCC6CASP9STK3WDR82MDC1XRCC6WASLNCOR1MAXRGL2RBL2BRCC3 SMC6 CEP44 DSE KPTN RYK AGK BRD4LRCH3XRCC5HGSPMS2TLE1WIZPSMD1ASH2LEXOC7CSKRELSTK4TBCBLIN37JDAG1 UNBPTTG1BCL3 FGR NCOA3 SMCR8 GFPT2 PDIA5 HDHD1 RRP7A PARP1HIF1ACSF1RNAT10PAK2DOK1EPORDOCK3NR2C1BRD8BIDCASP7ZC3H3EPC1 RTF1ESPL1 COX14 FOLR2 FADDDDB1SOS2MCM7RPP38PDS5AEEDPDIA4FBXW8PHF8HCKSNX3BBS2MAFKBBS4 RASA2 WDFY2 GIN1DDTLEVI5 PPWD1 RAB31 DOCK7MCM6CBX7C1QBPGAB1NKRFSAP18RBBP5SUMO3UBE2BPAAF1LZTS2TFG WDR61 MAP4 ZFP91 NPTN TIA1 OTUB1UBE2CLMNADDX21OXSR1GTF2ITWF2RCC1PSMD6CD2APBBS7CRKLPPARAMEPCERFWD2SRSF6NFKB2CDC73 ARPC5CDC16 UNC50 DOCK2 FUT11 UBTD1 ABCG2 ARMC7 LZTR1 STK16CPSF7NONONEDD8CUL2AGO1PCBP1ASAP2IKZF1ILKPSMD8PLOD1FOSL1PPARDPPIATRADD STT3B NFIC CETN2 NSA2 AIP GCSH NSUN4 SURF4 CIAO1RALARBM10HDGFCXXC1RTCBPHF5ADDIT3GLMNDPY30PDS5BCDC23 FSTL3KDM4B CCL17 ZNF32 PPM1K BRPF1 BAMBIDLG1 RPTORLIMD1 RCOR1 STAG1NOP2AKAP2CLIC4FCHO2RBM25ARPC3 ZADH2 OPA3 BNIP3 ZNF14 ZNRF1 NOP56TRA2BDOCK6 SIN3B EMC10 SRR SCFD1 ACAD8 PER1 ORC3TANKAPBB1PROSC LMNB2BRWD1 TAF6 DDX51 MMP1 CCR1 SNAI3 USP9Y SOCS1 RBM4GRSF1WDTC1WBP4PLRG1CUL4BDOCK8TOE1ASNS QPCT AP1B1 AKT1 DAXX MFAP1 DCTD KLF7 RC3H1 TCEA1ABCF1NXF1RBM39UBXN7RRP1BCBX4LSM1 DET1 IL1A PLCB2 ITFG2 PICK1 ULK2IRF3 KLF5HDAC8LRCH1BRELSM5 UBN2 CLK3 NGRN BCAT2 ARSK SMC5RBM48 CRLF3RRS1 WDR26 KRCC1 MMAA TRAM2 BBC3 WDR6 ABL1 PSME2 LSM3 LSM6 RAB32 ACOT4 MPC2 MUCL1 LRFN4 GOPC NOL8 BAZ2A DAPP1 PNPT1 IKZF5 NUP62 IP6K1 KLF4 RASD1 DEGS1STK11 SNIP1 SRSF4 RGAG4 TMA16 DVL2 ZMYM5MED31 CREB5PEX13 SDC4 TIAM1 WBP11 RNF24 MXRA7 BMP1 TGDS NISCH TBPL1 ZBTB4 ARSBTINF2 DCTN1 PPIG UTP23 IQCB1 EAF2 ARID2 ALPK3 CD14 MITD1 PHAX MDFIC PPCS SNX27 CBR4 TMED2 WDR36 LARP4 ALG8 OSBP2CIDEB J AZF1 EXOC6 TRRAP DDX31 VPS25 UTP18 MANEA LTN1 TSC2 SMAD4 TFAM PILRA CLCF1 BSDC1 RRAGD KAT6A PIGY APBA3DTNAPLCB3CAAP1 RSF1 NRP1CNPY3 DVL3 MTA2 IRF2 AMY2B PLEK2 KDM8 STK35 ACBD6 HDAC6 BMPR2 BRK1 CEP19 MIB2 IL16 CD276 GHITM SUMF1 GALE NUPR1 DDX47 CCR7 WDR25 RPGR FPGT CCNB1 CDK13 CSTF2 ZWINT REPS1 PPID PIM3 MYO1G MFSD8 SP110 SNX15 DOCK4 ATP7A PDE1B SYNJ 1 RAD50 TGS1 IDI2 SYPL1 SSFA2 LEMD2 GPD1LEVC NUP43 MTOR COIL FBXW2 DFNA5 DAPK1 NUP98 XPOT ENDOV PIM2 RIOK1 MYO10 CD82 CCR5 DDX6 TIPIN HTRA2 MTA1 KLF2 ACAP3 THTPA KTI12 THAP8 ADTRP PSME3 UBE2H MICU1 EGLN1 CD37MGST3 NR4A3 DDX55 SDC2 RAB10 ACSL3 COX18 TTC13 MFSD9 MOV10 HDAC5TERF2 ACSL1 IAH1 MBNL2 DIP2A MIA3 ZC3H4 HBS1L ETS2 TMCO6 DHRS1 OSGEP PGAM5 NAA50FEN1 TMED7 MECP2 TLE3 CCL22 TXLNG PIGT ERP44 TTC1 TSR3 PTBP3 J TB PLP2 ZC3H6 IMPA2 PACS2 SMU1HINFP ABCE1 SGTA STK24 NPM3 ASXL2 CCL15 MYSM1 CUL5 MED7 SIL1 LRP8 PHF14 LLPH S100P STAC3 XRN1 ZMYM2 SLMO1 PTH2R OSCAR MAT2B MYPOP CBX1 PPME1 ACYP2OAS3 AP4B1 TOP3A FOXO4 RAB21 CHD1L SATB1 RBM33 MT1X TBCA KLF11 FLI1 SOAT1 TEX2 ASCC3 SENP2 WDR11 PMP22 EDF1 IPO8 WDR73NOC3L CENPO CSF2 IPPK SH2B2 ETS2 GGT1 MKLN1 IL36B VAMP5 LPP AKTIP WDR18 THAP7 WDR59RBM18 EGR2 CNOT1 ATF6B IFT80 STX6 WDFY3 YIPF4 GMDS ST14 SGTB TIGD6 ACSF2 SMC4 NCBP1 FSD1L POC1A KDM3A DTNB COQ3 GSTT1RNF38 NUP37 ZXDC UVRAG UBE2O BRD1 PIGP CDIPT DAD1 EXOG RAB14KLC1 RDH13 PBX2 DDX52 ISY1 KCNC3 PPDPF HELBGNAI2 CCNL2 SENP3 NRF1 TXNIP PARK7 SPAG5 KDM5A PHF3 DOLK IPO9 RFC2 DGCR8 NBPF3GLOD4 SRP19 SURF1 HHEX KDM5B DFFA STON2 MAGT1 RCOR3 COQ6 TAF1B PEX26 ASPH PMM2 PRPF3 ABCD1 CCL1 CCR5 MKL1 HSDL2 IFFO1GMEB2 USPL1 IPO4 ACTN1 CHDH SNX14 KCTD9 ANKH ICAM3 WDR70 YLPM1 NOA1 NOL6 PGAM1 PSTK CXCR4 AFF1 SOGA1 ITPR2 BCAR1 LCORTBL3 IFI27 SOS1 NUP85 ZNF2 TIPRL ARNT ULK3 OLR1 VMP1 ROCK1 MARK3 SNX12 PGS1 WDR4 SP2 CLK4 NFKB1 IBTK ADCK5 CLN3 CHMP3 SCML1 UHRF2 BTBD2 ZMYM4 DCAF4TET3 CXCL3 RAB5C NR1H3 MRS2 USF2 NHP2 EGLN2 PSPC1 CCL2 GMEB1 TTC27 MIPEPSELK LYRM1 TRIM5 HAUS6 URGCP RAC1 PANX1 BET1 COPG2 TMED5 MCM9 PNRC1 TNIP1 B9D1 EFHD2 CCND2 PIAS4BAG5TFCP2 KDM2B HBDPNRC2 ADPGK DESI1 REEP5IRF8 LPIN1 SETD8 RRN3 MON2 PAX8 NAA25 RBM26 GSTP1NUCB2 UBE2S SHB PACS1 SURF2 MGST2 TIMP2 KLF10 ZW10 PALB2 LAS1L CSTF1 SNURF GPSM3 RBM5 SPAST UNG NUPL2 EZH1 TMED3 COA5 AP2M1 PUS7L SRP68 NR1H2 MKL1 PPM1D VRK1 DPH1 NUCB1 MAFF TMOD3 CHTOP SOCS5 ASB8 P4HA2 RHEB ATF5 DRG1 PEAK1 AP1G2 HOOK3 FRYL TELO2 WNT2B LIMS1 DCXR VOPP1 MUL1 XBP1 PREB GPS2 RIPK3 FMR1 HERC3 DHX37 BMS1 DPCD LSM7 THOC7 CHD3 WDR91 VAPB SGPL1 FANCL BFAR PWP1 NIPA2 TAF1A RNF41 TDRD7 ZNF3 PKD2 AMPD3DCP2 VAC14 MATK ZYX CD97 CCL5 TYK2 ATN1 NFYC PPARG CEP63 OCRL ST7 HEBP1 COX15 OS9 CD86 FEZ2 MFN2 SOCS1 NR1H3 TSC1BIRC6 CCDC6 RPP30 STAU2 PDIA6 NCOA4 LSM4 APCTRIP6 HAUS1KLF3 AP4E1 RSBN1 STIP1 GRHPR LMAN1 FEM1B RPA1 OVOL1 COPZ2 EDC3 POLK RGL1 LDHBMYH9 EIF3H PARVG NCOA3 FBXW7 KDM6ACLK2 UBE3B GCC2BIN3 CLPB RINT1 RP9 ARL5B IL18 WDR43 NELFE FBRS PTPRJ MED13 ASB6 IFI6 MED15 TNPO2 ICAM2 RBM27 RFWD3 SERP1 RREB1 PHIP ARF4 SETD7 SUGP1 CDC37 DDX41 WWP1 RAD9A RCL1 CDKN3 CENPN MTG1 RNF13 ARRB2 DGKH HDAC9 ELL2 ALG13 TRUB2 NDRG1 MCFD2 GPAA1 STAT6 TAF1C ELL EXOC2 REV1 YPEL3 HEXA VEGFA HINFP RBM4B TUFT1 VSIG4 RAB13 CEP57 TAF2 PPM1F TTC9C CDK10 IVD LAMP2BTG1 BST2 CSDE1 HBP1 SYF2 TFPT CARD6 CR1 PCCA SSR3 MAK16 PDIA3 IGF1R J AK1 PIAS1 NFYB DMTF1 GOLM1 CYHR1 EIF3K G6PD ECM1 DLG4 RIF1 WDR44 TTI2 UBR3 MOAP1EHD1 EIF3L SOCS4 CSRP1 MOB2 RENBP CPSF2EGLN2 TOP2B BPNT1 SENP6 CHD9 VEGFB ARPC4 SKI RARA MRGBP IGSF8 DIDO1 ZBED4 VAMP1 EMP3 RAD50 ARMC8 FSCN1 THOP1 SLIT3 NAB1ELP6 NPRL3 EEPD1 EIF3E ETV3 FOXO3 PRDM1 SMG8POMT1 ELF1 J AK2 KDM6A MED27 TTC14 KLHL9 ACADS RAB6A VPS28 GPBP1 PIGF PQLC2 CD14 ACTN2 RNF8 PIAS2 MED23 RIOK2 MED30 DBR1 GMFB MKRN1 AFF4 VPS52 RPAP3 CHD8 C2CD3 IST1 MED13 MTSS1 PHF11 YRDC HIP1 VMA21 CXCL2 GNA12 MBNL1PRDX6 DCTN3 LGMN SH2B1 BCL10 HOOK2 VPS41 MED29 MED18 RELL1 PIGC GON4L CDYL2 CCNL1 TRAF5 NFRKB SRA1 MED18 FBXW7 MED24 MED16 ARMC1 BUD13 MAZ STAT3 MED24 TM2D2 PTGDS PDCD7 LASP1NEK4 CD79A NELFA RER1 MNDA PCID2 GIT1 NFKB1 MED27 TBCD CRBN MED14 NEK9 SLFN5 GRK6 HCFC1 MED11 MED28 MED12 CDK19 TBX21 CBWD2 NARFL SCAP RAB35 RWDD1 MED1 MED17 CLCN7 EIF2A KTN1 RSRC2 RFC2 MED8 LAMB2 SHQ1 PEX16 FBXW2 MED6MED15MRC2MED10 THAP5 VPS72 VAMP4 HPS6 THYN1 NIT1 STX4 CDV3 CAPZB RAB1A PALLD LAT ITCH HDAC4 CDK8 MED25 MOB3B PIGO CNPY2DEDD2 STT3A INO80 ING3 XPO5 CPNE8 AP2S1 RPN2IPO7 FEM1B LMNB1 MED17 MED26MED23 BRAP ARMC6 VASH1 AIF1 THOC5 MED21 MED16 PINX1 MED22 CARD8 NT5C LPPR2 TCF25 MZT2B NAA38DVL2 MED9 RAPH1 RPIA SAAL1 TPMT PA2G4 PRDX1 VPS54 SYNC ABCD3 NDC1 ZMIZ2 MMP9BAZ2B SP1 MED19 MTMR4 MINK1 B9D2 STAT6 SYNJ 1 SMC2TRIM8 SNAI1 RPP14 PDCD4 MED4 PIGQ SMSNUP54 NAV2 PGP POMP SRP72 MXI1 UBA5 KIF1C CCL18 HMGB1 ATP5L GGCT NCOA2 ZNF83 SENP5 HFE IFT20 LRP10 PEBP1 VDAC1 SIAH1 LETM1 NOC4L DDX49 REEP4 SNF8 SUMF2 COPG1 HDAC3 TAF1D UEVLD TET2 PIGX BANP BTG1 MED6 PEX10KCNE1 SASH1 RNF34 NANP SNX19 WDR1BAX FBXW5 NOL3 RIPK1 KLF9 DCTN2 CNOT6 LYST NDST2 DPH2MTMR9 NDEL1 RPN1 CNBP DGKZ GBP2 NR4A1 PTER DOCK5 TMF1 PON2 RBX1 MED9 IFIT2 LPAR2 SDC3 RIOK3 COX17 MYL6 CHTOP ATG2A MTFR1 STOM MEN1 CCNJ HDHD2 BRAF CISD1 ZNRF2 EPT1 NUMA1 KRR1 CISH PIGV GPX1 DDX24 ELL2 ANXA7 KAT8 BBC3 PANK4 XRCC4 APEX2 ERF PER3 COTL1 RAB5A PDCD5 PLCD1 MEF2D CASP6 LEP ADPRH WDR55 ATMIN ANXA6RAF1 OCRL UBE2N MED26 RAB2B AKT3 PMVK KCNE3 SARNPTAB2 SSRP1 SRCAP TKT RRAGD APTX MFNG PARP9 BTBD3 COX5A HAX1 PRDX3 CBX3 MIDN MED1 CCNT2 CENPW HMGN3 RIPK3 WAC UCKL1 C1QB ALG12 SSBP4 SET TCF20 SPG7 IFIH1KDM1B DTX3L BRD9 MAFG SKP2 RNF10 EAF1 TPGS1 PIGG P4HA1 PTPRJUGDH DCAF7 TAB1 FUS TAOK2RBMX2 ZZEF1 XYLT1 CCL4 DBNL PCNA DDX10 FABP4 TRIB3 CICDIEXF ITM2B HCST OXSM RGCC FKBP2 GGA3 SREK1 KDM3B RAB38 DYRK2 STAT2 PRDM2 IPO13 MAML1 CEPT1 FUBP1 PHB CDK4 PNKD WRN PCGF6UHRF2 ASB8 SRCAPCPSF4 COG8 ACER3 VAV2 IFT27 NCF1IRS2 GCC1 APOC2 PCIF1 RNF4BAZ1A VHL RAD1 DTL RC3H2 PDE3B CLCC1 CEBPDCEBPG KLHL6 FANCA CCNE2 MED19 NXT1 TYK2 ETV6 TIMP1 LRP12 TMED9DPM3 RNF10 BCAT1 EDEM2 LRRK1 CUL5 BCAR3 MED22 CEP70 TNPO1 ARF6 FUS ECE1 PRR13 PDIA6 DOHH PRR5 DEDD ESCO1 IL1B RAB8A MAF1 ZNFX1 HDAC5 MNT LDB1 ARV1 NCOA6 TBCE ZUFSP LMO4 RESTSUGP2 J MJ D6 BIRC3 BAHD1BPTF FOXK2 ICMT ACOX3ACOT8 CCL2 ARF5 COG3 VPS35 ANXA7 NPC1 AFF4 TPR GATA2 VRK2 SGSM3 NUB1 MAP7 GBF1 SETD3 TOM1 KEAP1 OAT THOC6 MCRS1 CSF1 FYB RAB18ECH1 RBX1 SNW1PDCD6 STK24 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FKBP8 TREX1 TESK2 TMEM9 DHX29IRF7 NUP50 CFLARSHC1TRAF1BIRC2 BATF3 IMMT DGKA NCOA6HN1L GSTO1 HDAC2DDX5PSD3STRN MATR3 RPA2 AKAP8 C1QACXCR4 NCF2 RBBP4ERCC8PUF60NELFB AP3D1 VPS4A UBXN8 KAT7 SBNO1 SUMO2GRWD1SUZ12CUL4ARNPS1 NRBF2UBE2N MSH2 BAD TERF1 RPF2 DOK2 TAZ WWP1 MYO1D MX1 ING3 NARF ILF3TECSRSF7SF3A1SF3A2 WIPF1 TPP1PLIN3 AHSA1 RRAGC TLK2 IFIT3 SUMO1SAT1SIN3AAGO2SF3A3SFPQCUL1U2AF2ABHD6STRN3 SBDS UCHL3 LSM2 SNX18 PLCD1 MKKS ADNP2MSL1 CUL3SONWWOX SRRM2 RBMS1 KCTD5 TBPL1 LAMB3 PPIC GIT1 PTENCTCF HCFC1 CNIH4 BUB3LARP1SRSF5 SCFD1 SSU72 GGA3 KDM4C TSNAX RBM6 VPRBPDDA1 U2AF1SRRM1BOP1 PDE6D ZHX2FHOD1 ELP2 DHX30 PLEK SRGN SRSF3 COPA STAU1 UBB TOX4 LRCH4 MAST2 FXR1TBP STRN4STK25 ATG3 GGA1 MED10 UCHL5 CNDP2 DEK XRN2 OTUD4 MVP SMC4 ATF2 SEC13 COPB2EXOC5 GGH KLF16 SREK1 BNIP2 SNX5 ULK1 IGBP1 RFC4 SRP72 PDPR ERCC3 FOXJ 3 THOC7 SPOP MARK2 GABPA KDM1A DCK PRR14 RTN4 LSM4 MED30 RGS20MBD6 SMAD2 WDR33 MADD PYGL PLOD3 SURF6 CPNE3 MCM5 CTDP1 PARP6 BTBD1 CDS2 TPR ZBTB2LOXL3 CNNM4 ZPR1 PGK1 SRXN1 SIK1 CARM1 NFRKB ADRM1STX3 STX18 APMAP SAE1 WDR7 WDR47 ABCA1 PAK1 PNKPOTUB2 DNAL4 AIMP2 ATF7 SETD7 MTMR1 EDN1 MSMO1 KLF6 DDIT4 UFM1GNPTG CMC2 AP3S1 MATR3 ELP4 RAB24 SYNRG REXO1 CD53 MED11 WIPF2 SP3 EPS15 LRIF1 KDM5DRAB7A HINT1 WDR33 PRDX6 AKTIP GEM CPSF6 PJDUS3L A1 RAC2 RTN4R ABR MED28 TRABD CASC3 SIK3 YKT6 NIPBL LPIN2 VHL SF3B3 AMPD2 CLIC1 DAPK3 SMC3 CD9 PJ A2 CHMP5 HES4 IMPA1 PTPRO RSRC1 ZGPAT NUDT9 SRPK2 NCOA5ELL PSMG3 PDCD2CWC27 BCL7B SRSF9 BAX PDPK1 CD44 ANXA5 FMNL2 GGA2 CPSF3 PADI2 NFYA SKI CPSF1 DCP1A CDC5LZHX1 XPC ERCC5 SELT MSH3 LUZP1 AZIN1MANF NUMB FIG4 CDK19 TSN ATG12 LYAR ASF1A CRTC3 SRP68 LARP7 PREX1 DUT SYMPKUBR5 PPIL4 RAP1B CPEB4 ZMAT2 EXOC4 COPA YBEY WDR48 POGZ NOL7 FGD6 THAP1 RGS1MUS81 NUDT3 PHRF1 DERL1 THOC3 AGO3 LPXN PATL1 VPS16 EYA3 GYS1 RNF14 OAZ1 TOR2A OXA1L RRAGB LPP CCNA1 ATAD1 PDXK ARF3 THOC1 MYO1C FAF1 PEA15 AMZ2 VAMP2 FASRFX5 IPO5 XPO7 CNOT4 NAT9CD81 ALDOA FABP5 HMOX1 MLX SYMPK TSSC4 CSTF3 OAZ2 MGST1 ARC ACY1 NASP PREP DYRK4THOC1 ADAP1 RBM7CISD2 RBM8A HOOK3 PARNRHOA TPRA1 CKLF TPK1 N4BP1MOGSCDK17 HMGA1 MYL6 SPNS1 RALB TDG MMS19 FOXN3 UBB CHIC2 LSM7 USPL1 STRBP UBL4A CNOT3 BAZ1BSART3LENG1 KIF2ASTIM1 NAA20 ATG4B PSMD5 GPN3 AKIP1 SNN CREG1 FLII MED21 TBL2 UBE4B CENPB CDK12 NDRG3 LAMP1 TPM4 APLP2 TMX2 CDK8 VTI1A UBA6 DDX50 ELP3 CDC42 DCP2 MED14 MLXIP DDX56CCDC9 HAUS2 ZNF76 MGAT1 AP3M1 TIFA SNRPAMSL2 STK38EMC1 ICT1 ECI2PCGF3 DPM1 RRAGA MED8 RND3 RCN2 TOP2B NKAPLIMA1 MCL1ARL5A PRDX1 COPG2 PRPS2 DHCR7 THOC2HIRA DDX54 VPS36 TOB2 PIM1 GRN SHPRH DDX46 ACOX1 SSH2 WDFY1 TMUB1 NFX1 AGO4 SCO2 MIF CIR1 TOR1B CEP95 ZNF24 PTMA AP3M1 CCNT2 BET1L MOB1B INO80 AAAS AGGF1 ICAM1 UBL5 MORC3 MTCH1 HM13 RBM28 UBE4A NTPCRNR4A2 CHERP FLNA RSPH3 ATP5H ANXA5 RTN2 VPS53 SSH1 WDR75 SGK1BTF3 VPS45 VPS16 PPM1A BCL7C CBWD1 MOB3A UBR2 UBE2WLSM2 POP7NAA15 GFPT1 CALR RAN EIF1B ERI3 AP3S2 SFXN4 PTOV1 THADA ERAL1POC1B CAP1 PGK1 DMXL2 ACAD9 TFB2M CD209 UNK ASUNRAB34 BLVRB LSG1 CLPX NUBP2VPS39 ABCF2 LDHA WDR20 RTN4 SASH3 PRPS1 SESN1 GAR1 VGLL4 CHKB CPT2 STAG2 MTX2 DCAF5 DRG2 WDR83 TGM2 ARF1KYNU IDS DSCR3 WDR81GALK1 SETX ARL16SRXN1 ATP5I NUP88 PPIL1 BZW1 PPIB TPM3 MYLIP PIN4 SRP14 DHX34 UBN1 HEBP2 UBXN4 EAPP STX2 WDR12 DHX16MKNK2 HEXB PPIL2 EIF1 TSSC1 NOMO1 NAA16 DPP8 WDR74 CECR5 ZMAT5 RUFY1 PTGR1 SARDH EXOC2 PPIL3 NAMPT CAPG OST4 ASH1L VRK3 ABCB7SCO1 PHF23 HMHA1 PPT1 NOP14 VAMP8 DIP2B ABHD5 HYPK TSR1 MTRR RRP1 TOR4A PPIE SMAP1 USMG5 CWC25 RWDD4 MCU FEM1A CYB5B DIRC2 CCS CWC22LXN KLHL7 GBA2 PHKG1 MKRN2 PLD3 COX7C HN1 UFL1 COX5B KCTD7 YIPF3 NOL11CNST TRIM4 TMX3 PPIF LYRM2 ASAH1 LSP1 PIGH ZBED5 ERMP1 PLIN2 PANK2 PIGU VAT1 TTC32 INHBA NOP10 ARSA CHMP7 WRB TTC7A FMN1 MTFP1 FRMD8

SERF2 COX7B ZFX FTSJUPP1 1 DNA damage response SMUG1 VPS8 Transcriptional regulation DHDDS AHSA2

GPX4 NPC2 YIPF2

IPMK YIPF6 Lysosome vesicle biogenesis AP-1 transcription factor network Cellular response to stress

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Movie Legends

Movie 1. Human monocyte-derived macrophages infected with swollen A. fumigatus undergo lateral transfer.

Representative time-lapse widefield microscopy images of Af lateral transfer in human

MDMs infected with live Af SC (MOI=1, shown in green). Lateral transfer highlighted with black arrow. Images taken at 10 minute intervals.

Movie 2. Human alveolar macrophages infected with swollen A. fumigatus undergo lateral transfer.

Representative time-lapse widefield microscopy images of Af lateral transfer in human AMs infected with live Af SC (MOI=1, shown in green). Lateral transfer highlighted with black arrow. Images taken at 10 minute intervals.

Movie 3. In vivo confirmation of macrophage lateral transfer in zebrafish macrophages infected with swollen A. fumigatus .

MPEG:mCherry Zebrafish larvae were infected with ~50 eGFP-expressing conidia of Af and time-lapse confocal microscopy performed. Representative three-dimensional reconstructed images show Af lateral transfer between macrophages. The transfer process is highlighted with a yellow arrow. Images shown are at 7 minute intervals.

Movie 4. Macrophages infected with germinating A. fumigatus conidia undergo lateral transfer during programmed cell death.

Representative time-lapse confocal microscopy images of lateral transfer of live Af SC

(shown in green) between MDMs. Propidium iodide staining (red) showing cell death of

macrophage transferring Af conidia to neighbouring macrophage. Transfer process

highlighted by black arrow. Images taken at 10 minute intervals.

Movie 5. Massive vacuole formation occurs during A. fumigatus germination in human

monocyte-derived macrophages

Representative time-lapse widefield microscopy images of vacuole formation in human

MDMs infected with live Af SC (MOI=1, shown in green). Images taken at 10 minute

intervals.

Movie 6. VASP co-localises to the human monocyte-derived macrophage A. fumigatus

phagocytic cup.

3D reconstruction of confocal microscopy image of phagocytosis of live Af SC (MOI=1) by

hMDMs. Image of Af phagocytosis at 30min p.i. with VASP shown in red, Af in green and

nuclei in blue.

Movie 7. Lateral transfer of germinating A. fumigatus conidia between human monocyte-

derived macrophages occurs within VASP-enveloped endosomes.

3D reconstruction of confocal microscopy image of hMDM Af lateral transfer with VASP

shown in green and Af in magenta.

Calcineurin orchestrates lateral transfer of Aspergillus fumigatus during macrophage cell death

Anand Shah, Shichina Kannambath, Susanne Herbst, Andrew Roger, Simona Soresi, Martin

Carby, Anna Reed, Serge Mostowy, Matthew C. Fisher, Sunil Shaunak, Darius Armstrong-

James

Online Data Supplement

Supplementary Experimental Procedures

Zebrafish care and maintenance

All zebrafish experiments were approved by the United Kingdom Home Office and

performed in accordance with the project license PPL 70/7446. Wild-type adult breeders

were purchased from the Zebrafish International Resource Center (Eugene, OR). The

Tg(UAS-E1b:Eco.NfsB.m Cherry)c24 (referred to as mpeg:mCherry) zebrafish lines have been

described elsewhere (1). Embryos were raised in Petri dishes containing 0.5 × E2 medium

supplemented with 0.3 lg/ml of methylene blue. For imaging studies, from 24 h post-

fertilization (hpf), 0.003% 1-phenyl-2-thiourea (Sigma-Aldrich) was added to the medium to

prevent melanin synthesis. Embryos were reared at 30°C, and larvae were anaesthetized

with 200 lg/ml tricaine (Sigma-Aldrich) during the injection and imaging procedures

Fungal strains and culture

Aspergillus fumigatus AFCEA10 was used for the Western blot, Luminex, fungal burden,

Imagestream, phosphoproteomic and RNA sequencing experiments. An eGFP-expressing

strain (ATCC46645-eGFP, a gift from Frank Ebel) was used for all microscopy experiments.

All strains were cultured on Sabouraud dextrose agar (Oxoid). Conidia were harvested in

0.1% Tween/H 2O and filtered through MIRACLOTH (Calbiochem, UK). Conidial suspensions

were washed twice in phosphate-buffered saline (PBS) and resuspended in RPMI (Sigma) at

concentrations shown. To generate swollen conidia (SC), resting conidia (RC) were

suspended in RPMI at 1 × 10 6 conidia/ml and swollen at 37°C for 4 h. Fixed SC were

generated by fixing in 2% formalin for 30 min at 4°C, followed by quenching in 0.1M

ammonium chloride for 10 minutes and further washes in PBS x4.

Isolation of human alveolar macrophages

A total of 240 ml saline bronchoalveolar lavage (BAL) was performed by flexible bronchoscopy and return transferred on ice for processing. BAL fluid was passed through a

100μm cell strainer (BD Bioscience, UK) and centrifuged at 400g for 10 min to pellet cells, which were subsequently washed in cold PBS and resuspended in pre-warmed RPMI 1640.

Macrophages were plated at the desired concentration and adhered for 1hour. These purified AMs were then further rested for 3 days to ensure any residual calcineurin inhibitor effect was removed.

Isolation of monocyte-derived macrophages

Peripheral blood mononuclear cells (PBMCs) were isolated from 60 ml of healthy volunteer blood or from leukocyte cones purchased from the National Blood Service, Colindale, UK by mixing 3:1 in warm RPMI and layering over Ficoll-Paque plus (GE healthcare) and centrifuging at 450g for 45 minutes. PBMCs were washed at 200g in cold PBS to remove platelets and monocytes subsequently purified by negative magnetic bead selection using a pan-monocyte isolation kit (Miltenyi Biotech, Auburn, CA). To obtain monocyte-derived macrophages (MDMs), freshly isolated monocytes were cultured with RPMI 1640 medium supplemented with 10% human serum (Sigma, UK) and 5 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF) (Peprotech, UK) for 7 days at 37°C.

Western blotting

For whole-cell lysates, cells were lysed in 150 mM NaCl, 50 mM Tris (pH 8), 1% Triton X-100 containing proteinase/phosphatase inhibitor cocktail (Cell Signalling, US). Cytoplasmic and

nuclear extracts were prepared using the NE-PER extraction reagents (Thermo Scientific).

Extracts were separated by 4–15% Bis-Tris gel (BioRad), and proteins transferred to PVDF

membranes activated with methanol. Membranes were probed with anti-NFATc2 (sc-7296 ,

Santacruz), anti-NF-κB p65 (C22B4), anti-HDAC1 (10E2), anti-Histone H3 (D1H2), anti-β-actin

(8H10D10), anti-DSCR1 (D6694, Sigma, UK), anti-VASP (9A2), anti-phospho-VASP (Ser239)

(3114), anti-SAPK/JNK (9252), anti-phospho-SAPK/JNK (Thr183/Tyr185) (81E11), anti-p38

MAPK (D13E1), anti-phospho-p38 MAPK (Thr180/Tyr182) (D3F9), anti-DRP1 (D6C7) and anti-

phospho-DRP1 (Ser637) (GTX5091, Genetex, US) antibodies. All antibodies, where not

specified, were from Cell Signalling, US.

Luminex and TNF-α ELISA assay

MDMs were plated at 1x10 5 per well in a 96-well plate and pre-treated with FK506

(10ng/ml, Calbiochem), human Dectin-1 blocking antibody (3µg/ml, R&D Systems),

piceatannol (20µM, Cayman Chemical) or vehicle for 1h followed by stimulation with live Af

SC (MOI=1) for 6h. Luminex analysis of culture supernatants was performed using the

Milliplex human cytokine/chemokine magnetic bead panel kit from Merck Millipore.

Supernatants were assayed for TNF-α, GM-CSF, MCP-1, MIP-1α and MIP-1β. TNF-α

production was additionally assayed using a human TNF-α ELISA kit (R&D Systems)

according to manufacturer’s instructions.

Real-time PCR

AMs and human MDMs were stimulated with live Af SC (MOI=1) with FK506 or vehicle pre-

treatment for 1h. A total of 5x10 5 macrophages were lysed in Tri Reagent 1 and 6h after

stimulation and whole RNA extracted using TRIzol and purified further using RNAeasy kit

(QIAGEN). RNA was reverse-transcribed using the QuantiTect kit according to the manufacturer’s instruction (Qiagen). Fungal burden was analysed by measuring A. fumigatus

18S rRNA (GenBank accession number AB008401) by real time PCR as described (2). For

RCAN1.4 and TNF-α mRNA expression, RNA was extracted as above from human MDMs stimulated with live Af SC (MOI=1) pre-treated with or without FK506. RCAN1.4 expression was measured using the primer set, forward primer 5’-AGAAAGCAAGATGCATTTTAGAAAC-3’ and reverse primer 5’-CGCTGAAGATATCACTGTTTGC-3’, and TNF-α was measured using forward primer 5’-CGAGTGACAAGCCTGTAGCC-3’ and reverse primer 5’-

TTGAAGAGGACCTGGGAGTAG-3’. mRNA expression was normalised to β-actin.

Macrophage flow cytometry phenotyping

AMs were harvested at D3 after isolation and MDMs at D7 after differentiation and stained with Zombie-aqua cell viability stain (Biolegend, UK) as per manufacturer’s guidelines. Prior to fixation, cells were blocked on ice for 20mins with human FcR block (1 in 100 dilution in

PBS/0.1%BSA, BD Biosciences) and subsequently stained with antibodies against CD11b,

CD11c, CD206, CD86, HLA-DR, and Dectin-1 for 30mins at RT in the dark (see table below).

All antibodies were from Biolegend, UK. Cells were washed with PBS/0.1%BSA x1, fixed as above and then acquired on a Fortessa flow cytometer. Results were analysed using Flow Jo

(Oregon, US).

Antibody description Manufacturer Clone anti-CD11b-PerCP/Cy5.5 Biolegend ICRF44 anti-CD11c-PE/Cy7 Biolegend 3.9 anti-CD206-AF647 Biolegend 15-2

anti-CD86-Brilliant violet Biolegend IT2.2

605

anti-HLA-DR-APC/Cy7 Biolegend L234

anti-Dectin-1-PE Biolegend 15E.2

Imagestream analysis of phagocytosis

5 x 10 5 MDMs pre-treated with FK506 (10ng/ml) and/or cytochalasin D (5µM, Sigma) for 1h

were stimulated with live Af SC (MOI=1) pre-stained with Calcofluor White (Sigma).

Infections were synchronised by incubating cells at 4°C for 30min pre and post-infection

before starting the experiment by incubation at 37°C. At specified time-points, macrophages

were washed twice with warm PBS to remove non-internalised Af conidia. Cells were

harvested by incubation with ice-cold PBS and repeat pipetting and centrifugation at 400 x g

for 10 minutes. Cells were stained with nuclear stain DRAQ5 (Cell signalling) and fixed with

2% paraformaldehyde. Fixed cells were run on ImageStreamX (Amnis) and data analysed

using IDEAS software. Single cells with in-focus nuclei were chosen for analysis and

percentage of macrophages with internalised conidial quantified according to Calcofluor

White fluorescence within the cell.

Reactive oxygen species (ROS) production analysis

MDMs were plated at 1x10 5/well in 96-well TC-treated black clear bottom plates (Fisher

Scientific). Cells were pre-treated with 10 ng/ml FK506 and 5µM cytochalasin D for 1h and

stimulated with live Af SC (MOI=1). Infections were synchronised by centrifugation at

1000rpm for 5 min. 10μM dihydrorhodamine 123 (Life technologies) was added to each well

and the reactive oxygen species production measured over 24h using a fluorescent plate

reader (Tecan). For analysis of mitochondrial ROS production, MDMs were plated at

5x10 5/well in 24-well plates and infected with live Af SC (MOI=1) following pre-loading with

Mitotracker Orange CM-H2TMRos (Invitrogen) according to the manufacturer’s instructions and pre-treatment with FK506 (10ng/ml), or vehicle, for 1h. Cells were harvested at the required time-points using ice-cold PBS/5mM EDTA and fluorescence quantified by flow cytometry (Fortessa). Results were analysed using Flow Jo (Oregon, US).

Confocal and time-lapse video microscopy for in vitro studies

For confocal microscopy, cells seeded on coverslips were fixed in 2% PFA for 15 minutes followed by quenching in 50mM NH 4Cl for 10 min. Cells were blocked and permeabilised in

PBS containing 10% goat serum (Sigma, UK) and 0.1% Saponin (Sigma, UK) for 2h at room temperature and incubated overnight at 4°C with a primary antibody (anti-NFATc1, clone

7A6, BD Biosciences; anti-Rab 5, clone C8B1, Cell signalling; anti-Rab 7, clone D95F2, Cell

Signalling; anti-LAMP-1, clone H4A3, Biolegend; anti-VASP, clone 9A2, Cell Signalling) in blocking buffer. After washing with PBS, cells were incubated with an anti-rabbit AF555 (Life

Technologies), anti-rabbit Cy5 or anti-mouse Cy5 antibody with or without Phalloidin AF555 or AF643 (Life Technologies) for 45min at room temperature in the dark and mounted with

Vectashield mounting medium containing DAPI (Vector laboratories). Imaging of activated

Cathepsin B and phagosomal acidification was performed on live cells using a Magic Red

Cathepsin B staining kit (Immunochemistry Technologies) and with MDMs pre-stained using

1μM Lysotracker Red DND-99 respectively. Colocalisation of acidification, activated

Cathepsin B and phagosomal markers was assessed by quantifying the average pixel intensity around single conidia using ImageJ. Live time-lapse imaging was performed by plating human MDMs at 1 x 10 5 cells/well in 8-well μ-slides (Ibidi) and stimulating with live

Af SC e-GFP expressing Af, resting Af or fixed Af (MOI=1). Cells were pretreated for 1h with

FK506 (10ng/ml, Calbiochem), Bafilomycin (100nM, Sigma), Z-VAD-FMK (50μM, Promega),

Necrostatin-1 (10μM, Calbiochem), Necrosulphonamide (5μM, Calbiochem) or vehicle.

Following stimulation with Af, propidium iodide was added to the media (1.25μg/ml,

Invitrogen) and lateral transfer events visualised by confocal time-lapse imaging using a

Leica SP5 inverted confocal microscope. To avoid effects on Af uptake, media was replaced

to contain cytochalasin D (5μM, Sigma) and Dynasore (100μM) 1h p.i. and un-internalised

conidia removed by washing with warm RPMI x3. Cells were maintained at 37°C with 5%

CO 2 during imaging and images analysed using ImageJ with 3D reconstructions performed

using Velocity.

Electron microscopy

Human MDMs in 24-well plates were infected with live swollen CEA10 Af conidia (MOI=1).

Un-internalised conidia were removed by washing with warm RPMI x3 at 1h p.i., and

cultures were subsequently fixed in 2.5% glutaraldehyde in 0.05M cacodylate buffer at

15min intervals from 105min p.i. to 180min p.i.. Samples were post-fixed in 1.0% osmium

tetroxide, dehydrated through a methanol series (70-100%) and embedded in epoxy resin.

Regions of interest were selected from 1.0 μm thick toluidine blue-stained resin survey

sections. Ultrathin sections (70–80 nm) were contrast-stained with uranyl acetate and lead

citrate then examined by transmission electron microscope (JEOL 1400+, Jeol Ltd, UK).

Digital images were captured using an AMT 16X camera (Deben, UK).

Zebrafish infection experiments and imaging

At 2 days post-fertilization (dpf), zebrafish larvae were transferred into 0.5 × E2. For zebrafish infection studies, 1x10 8 resting e-GFP Af conidia were stained overnight at 4°C with Alexa-flour 488 5-SDP ester (Life Technologies) at 400µg/ml in 250μl of 0.1M NaHCO3 pH 8.3 buffer, washed twice with 0.1%Tween/H 2O followed by PBS and resuspended in PBS containing 1% polyvinylpyrrolidone (Sigma, UK). At Day 3, up to 10μl of prestained AF resting conidia from a stock concentration of 5 x 10 7/ml was injected into the hindbrain resulting in an inoculum of 10 to 50 conidia per embryo. Infected larvae were embedded in

1% agarose in 35mm live-imaging μ-dish (Ibidi) and covered with 0.5 x E2 media.

Macrophage recruitment and AF lateral transfer was imaged with live time-lapse imaging in a humidified incubation chamber at 30°C using a Leica SP5 resonant inverted confocal microscope. Images were analysed using ImageJ and Velocity.

RNA extraction, RNA library preparation and sequencing

5x10 5 MDMs ( n = 6, healthy donors) were plated in 24 well plates and half of the plates pre- treated with 10 ng/ml FK506 for 1h. Cells were stimulated with live Af SC (MOI=1) for 1h and

6h and total RNA extracted using TRIzol reagent (Life technologies). RNA was further purified using an RNAeasy kit (QIAGEN) with an additional purification step by on-column

DNase treatment using the RNase-free DNase Kit (QIAGEN) to ensure elimination of genomic DNA. RNA quality was analysed using a 2200 Tape station (Agilent Technologies).

RNA with a RIN greater than 9.0 was used for library preparation. One microgram of total

RNA was used to generate RNA-seq libraries using the mRNA seq kit v2 (Illumina, Essex, UK), according to the manufacturer’s instructions. Briefly, RNA was purified and fragmented using poly-T oligo-attached magnetic beads using two rounds of purification, followed by the first and second cDNA strand synthesis. Next, cDNA 3′ ends were adenylated and

adapters were ligated, followed by 10 cycles of library amplification. Finally, the libraries

were selected by size using AMPure XP beads (Beckman Coulter) and purified. Constructed

libraries were assessed with a 2200 Tape station and quantified using a KAPA Illumina SYBR

Universal Lib QPCR kit (Anachem Ltd, Bedfordshire, UK) and broad range Qubit analysis

using the QuantiT dsDNA BR assay (Life technologies). Libraries were then sequenced to

generate 150-bp paired-end reads on an Illumina HiSeq 2500 (Genome facility, MRC Clinical

Science Centre).

Mapping of sequenced reads and differential gene expression analysis

Quality filtered Illumina paired-end reads were mapped to human reference genome (hg19)

using the read alignment software TopHat2 with default parameters (3). Genes that have

zero counts across all samples were removed from the dataset. Sequencing and mapping

were controlled for quality using standard tools provided in the FastQC software

(http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc ). Differential expression analyses

were done with Cufflinks v2.2.1 (4). Genes differentially expressed (DE genes) with FK506

treatment and Af stimulation (at 1h and 6h time points) were identified with 2 fold gene

expression changes (both up and down) with less than 5% false discovery rate (FDR). 492

genes were identified with significant differential expression at 6 hours of FK506 treatment

DE genes were used to construct regulatory networks in Cytoscape and STRING v9.05 (5, 6).

In Cytoscape the DE genes were mapped onto the BioGrid and IntAct protein-protein

network database. Nodes were coloured according to the maximal log 2 ratio across the time

points and placed into functional groups. Gene ontology enrichment, functional

classification of genes and KEGG pathway enrichment analysis were carried out using

DAVID v6.7 (7). All GO categories over-represented with adjusted P-value of <0.05 were

obtained for analysis. The Cytoscape Enrichment Map plugin was used to visualize GO biological processes.

PPI network construction and network module analysis

A human PPI network was constructed from the HIPPIE database and the PPI network for the genes expressed in FK506 pre-treated samples were visualised with Cytoscape (8). The network modules were obtained based on the Molecular Complex Detection (MCODE) analysis using the Cytoscape app. Default parameters (Degree Cutoff: 2, Node Score Cutoff:

0.2, K-Core: 2) were used as the cutoff criteria for network module screening (9). GO term enrichment of network modules with MCODE score >10 above were further analysed using

BinGO plugin in cytoscape (10).

Phosphoproteomic analysis

The Phospho Explorer Antibody Microarray was conducted by Full Moon BioSystems Inc

(California, USA). Human MDMs were differentiated at 1 x 10 7 cells in 75mm 2 tissue culture flasks. At day 7, cells were stimulated with live Af SC (MOI=1) following pre-treatment for 1h with FK506 (10ng/ml, Calbiochem) or vehicle. At 1h post stimulation, cells were washed (x5) with 10mls ice-cold PBS with protease inhibitor (Cell signalling), and collected by gentle scraping and centrifugation (250g for 10min at 4°C). Cells were frozen at -80°C and transferred to Full Moon Biosystems on dry ice. The array consists of 1,318 phospho-specific antibodies. In brief, proteins were labelled with biotin and placed on pre-blocked microarray slides. After washing, detection of total and phosphorylated proteins was conducted using

Cy3-conjugated streptavidin. Expression of phosphorylated proteins was normalized to

corresponding total protein expression. Fold change was calculated as follows:

phosphorylation of FK506-treated cells/phosphorylation of untreated cells.

siRNA transfection of THP-1 cells and flow cytometry analysis of phagocytosis

3x10 5 THP-1 cells were plated in 24-well plate wells and differentiated with 100ng/ml PMA

(phorbol myristate acetate) overnight. The next day, cells were transfected with 50µM

Signal Silence human VASP siRNA (Cell signalling) and 6µl HiPerfect siRNA transfection

reagent/well according to manufacturers’ instructions. Cells were left for 72h before

washing away remaining complexes with warm RPMI and performing experiments. Cells

were infected with swollen CEA10 conidia pre-stained with Calcofluor White (Sigma) as

above. Infections were synchronised at 4°C. At 30min and 1h p.i. uninternalised conidia

were washed away with warm RPMI and cells were harvested in ice cold PBS/5mM EDTA,

fixed in 2% paraformaldehyde (PFA) and uptake analysed by flow cytometry (Fortessa).

Results were analysed using Flow Jo (Oregon, US).

Statistical analysis

Data were presented as mean ± SEM and were analyzed using GraphPad Prism software

(version 6.0; GraphPad). Significance was determined using a Student’s t test for unpaired

observations; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. If 3 or more groups were compared One-

way ANOVA with Bonferroni correction was used. P < 0.05 was considered statistically

significant.

Supplemental Table E1. List of genes involved in human apoptosis pathway that are regulated by calcineurin in hMDMs stimulated with Af at 6 hours p.i.

log2 fold

Gene Gene description change p value q value

ADRB2 adrenergic, beta-2-, receptor, surface 2.03666 5.00E-05 0.0025039

ANGPTL4 angiopoietin-like 4 3.14403 5.00E-05 0.0025039

ARF6 ADP-ribosylation factor 6 1.03967 5.00E-05 0.0025039

BAG5 BCL2-associated athanogene 5 1.09531 0.00015 0.00622643

BMF Bcl2 modifying factor -1.63738 5.00E-05 0.0025039

BTG2 BTG family, member 2 1.64023 5.00E-05 0.0025039

CARD6 caspase recruitment domain family, member 6 1.5241 5.00E-05 0.0025039

CEBPB CCAAT/enhancer binding protein (C/EBP), beta 1.30165 5.00E-05 0.0025039

colony stimulating factor 2 (granulocyte-

CSF2 macrophage) 1.3387 5.00E-05 0.0025039

EGLN3 egl nine homolog 3 (C. elegans) 1.77097 5.00E-05 0.0025039

FASTKD5 FAST kinase domains 5 1.66276 5.00E-05 0.0025039

FEM1B fem-1 homolog b (C. elegans) 1.23424 5.00E-05 0.0025039

FOXO1 forkhead box O1 -1.13244 0.0006 0.0195528

GREM1 G protein-coupled receptor 183 1.57284 5.00E-05 0.0025039

heat shock 70kDa protein 1A; heat shock 70kDa

HSPA1B protein 1B -1.63925 5.00E-05 0.0025039

IER3 immediate early response 3 1.94819 5.00E-05 0.0025039

IL1A interleukin 1, alpha 1.19629 5.00E-05 0.0025039

IL7 interleukin 7 1.69211 0.00015 0.00622643

MCL1 myeloid cell leukemia sequence 1 (BCL2-related) 1.20625 0.0001 0.00451116

monocyte to macrophage differentiation-

MMD associated 1.32196 5.00E-05 0.0025039

MOAP1 modulator of apoptosis 1 1.48072 5.00E-05 0.0025039

NLRP3 NLR family, pyrin domain containing 3 1.30533 5.00E-05 0.0025039

NUAK2 NUAK family, SNF1-like kinase, 2 -1.53623 0.00055 0.0180311

OSM oncostatin M 1.7967 5.00E-05 0.0025039

pleckstrin homology-like domain, family A,

PHLDA1 member 1 1.54668 5.00E-05 0.0025039

pleckstrin homology-like domain, family A,

PHLDA2 member 2 1.07479 0.0003 0.0112934

PHLPP1 -1.01121 0.00015 0.00622643 PH domain and leucine rich repeat protein

phosphatase 1

phosphoinositide-3-kinase, catalytic, gamma

PIK3CG polypeptide 1.15058 0.00015 0.00622643

phorbol-12-myristate-13-acetate-induced protein

PMAIP1 1 1.3955 5.00E-05 0.0025039

RNF144B ring finger protein 144B -1.06347 5.00E-05 0.0025039

RRAGA Ras-related GTP binding A 1.08069 5.00E-05 0.0025039

serpin peptidase inhibitor, clade B (ovalbumin),

SERPINB2 member 2 1.95908 5.00E-05 0.0025039

solute carrier family 33 (acetyl-CoA transporter),

SLC33A1 member 1 1.16431 5.00E-05 0.0025039

tumor necrosis factor (TNF superfamily, member

TNF 2) 1.284 0.0002 0.00811673

TRAF4 TNF receptor-associated factor 4 -1.2598 5.00E-05 0.0025039

References:

1. Mostowy S, Boucontet L, Mazon Moya MJ, Sirianni A, Boudinot P, Hollinshead M, Cossart P, Herbomel P, Levraud JP, Colucci-Guyon E. The zebrafish as a new model for the in vivo study of shigella flexneri interaction with phagocytes and bacterial autophagy. PLoS pathogens 2013;9:e1003588. 2. Werner JL, Metz AE, Horn D, Schoeb TR, Hewitt MM, Schwiebert LM, Faro-Trindade I, Brown GD, Steele C. Requisite role for the dectin-1 beta-glucan receptor in pulmonary defense against aspergillus fumigatus. Journal of immunology 2009;182:4938-4946. 3. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. Tophat2: Accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome biology 2013;14:R36. 4. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L. Differential gene and transcript expression analysis of rna-seq experiments with tophat and cufflinks. Nature protocols 2012;7:562-578. 5. Cline MS, Smoot M, Cerami E, Kuchinsky A, Landys N, Workman C, Christmas R, Avila- Campilo I, Creech M, Gross B, Hanspers K, Isserlin R, Kelley R, Killcoyne S, Lotia S, Maere S, Morris J, Ono K, Pavlovic V, Pico AR, Vailaya A, Wang PL, Adler A, Conklin BR, Hood L, Kuiper M, Sander C, Schmulevich I, Schwikowski B, Warner GJ, Ideker T, Bader GD. Integration of biological networks and gene expression data using cytoscape. Nature protocols 2007;2:2366-2382. 6. Jensen LJ, Kuhn M, Stark M, Chaffron S, Creevey C, Muller J, Doerks T, Julien P, Roth A, Simonovic M, Bork P, von Mering C. String 8-a global view on proteins and their functional interactions in 630 organisms. Nucleic acids research 2009;37:D412-D416. 7. Dennis G, Jr., Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA. David: Database for annotation, visualization, and integrated discovery. Genome biology 2003;4:P3. 8. Schaefer MH, Fontaine JF, Vinayagam A, Porras P, Wanker EE, Andrade-Navarro MA. Hippie: Integrating protein interaction networks with experiment based quality scores. PloS one 2012;7. 9. Bader GD, Hogue CW. An automated method for finding molecular complexes in large protein interaction networks. BMC bioinformatics 2003;4:2. 10. Maere S, Heymans K, Kuiper M. Bingo: A cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 2005;21:3448-3449.

Figure E1. FK506 inhibits A. fumigatus mediated activation of the calcineurin/NFAT pathway in human lung transplant alveolar macrophages and healthy monocyte-derived macrophages.

(A) MDMs were stimulated with live Af SC (MOI=1) and macrophage nuclear extracts probed by western blot for NFATc1. NFATc1 was normalised to HDAC1 and nuclear translocation quantified. N =3

(B) AMs isolated from human lung transplant recipients after bronchoscopy were stimulated with live Af RC (MOI=1) and macrophage nuclear extracts probed by western blot for NFATc2. N =5

(C) MDMs were stimulated with live Af RC (MOI=1) and total RNA extracted at various time points p.i. mRNA expression of the NFAT-specific regulatory target RCAN1.4 was normalised with β-actin and quantified. N =5

(D) AMs isolated from human lung transplant recipients after bronchoscopy were stimulated with live Af SC (MOI=1) and macrophage nuclear extracts probed by western blot for NFATc2. N =5

(E) MDMs pre-treated with FK506 (10ng/ml) or vehicle were stimulated with live Af SC

(MOI=1) macrophage nuclear extracts probed by western blot for NFATc1. NFATc1 nuclear translocation was normalised with HDAC1 and quantified. N =4

(F) MDMs pre-treated with FK506 (10ng/ml), NF-κB inhibitor SC514 (10 μM) or vehicle were stimulated with live Af SC (MOI=1) and total RNA was extracted at various time point p.i. mRNA expression of the NFAT-specific regulatory target RCAN1.4 was normalised with

β-actin and quantified. N =5

(G) TNF-α mRNA expression quantified from total RNA isolated from MDMs infected

with live Af SC (MOI=1) and pre-treated with FK506 (10ng/ml) or vehicle. N =4

(H) Healthy hMDMs (N=4) and AMs (N=5) isolated from lung transplant recipients were

characterised by flow cytometry. Cells were gated on live-dead stain -, forward and size

scatter and CD45 +. Both healthy MDMs and lung transplant AMs are CD11c +, MHC + and

CD206 +. MDMs are CD11b + whereas AMs are CD11b -.

(I-J) TNF-α production measured by ELISA and NFATc1 nuclear translocation was

analysed by confocal microscopy shown in MDMs pre-treated with Dectin-1 blocking

monoclonal antibody (3µg/ml) or the Syk inhibitor piceatannol (20µM) and infected with

live Af SC (MOI=1). N=4. Data are represented as mean ± SEM

Figure E2. Calcineurin inhibition leads to impaired phagocytosis and ROS production in

human macrophages infected with Aspergillus fumigatus but does not affect phagosomal

maturation.

(A) AMs isolated from human lung transplant recipients after bronchoscopy were pre-

treated with FK506 (10ng/ml) or vehicle and stimulated with live Af SC (MOI=1). Total RNA

was isolated at 1 and 6 hour p.i. Fungal growth was assayed by RT-PCR of fungal RNA. N =5

(B) MDMs pre-treated with FK506 (10ng/ml) or cytochalasin D (5nM) or vehicle were

stimulated with live Af RC (MOI=1) and pre-stained with Calcofluor white. N =3

treated with FK506 (10ng/ml), Cytochalasin D (5nM) or vehicle and stimulated with live Af

SC (MOI=1). Af SC (pre-stained with Calcoflour White) phagocytosis by AMs (pre-stained with Draq5, Abcam) were quantified using Imagestream. N =6

(D) MDMs pre-treated with FK506 (10ng/ml) or vehicle were stimulated with live Af RC

(MOI=1) and ROS production assayed by luminescence. N=4

(E) MDMs stained with Mitotracker Orange CM-H2TMRos (Invitrogen) were pre-treated with FK506 (10ng/ml) or vehicle and stimulated with live Af SC (MOI=1). Cells were analysed at 0, 2 and 4h p.i. by flow cytometry and fluorescence quantified by Flow Jo (Oregon, US).

N=3

(F) MDMs pre-treated with FK506 (10ng/ml) or vehicle were stimulated with live Af SC

(MOI=1) and LAMP-1 expression analysed by confocal microscopy at 30minute intervals.

Phagosomal LAMP-1 intensity was quantified by ImageJ . N=3

(G) Representative confocal microscopy image showing LAMP-1 recruitment to Af conidia containing phagosomes in MDMs. LAMP-1 is shown in red, nuclei in blue and Af in green.

(H) Representative confocal microscopy image showing acidification in Af conidia containing phagosomes in MDMs. Lysotracker Red can be seen in red, and Af in green.

(I) MDMs stained with Lysotracker Red (Invitrogen) and pre-treated with FK506

(10ng/ml) or vehicle were stimulated with live Af SC (MOI=1) and fluorescence assayed by live confocal microscopy with phagosomal intensity quantified by ImageJ . N=3

Data are represented as mean ± SEM

Figure E3. Characterisation of calcineurin-dependent lateral transfer and programmed

necrosis in human macrophages infected with Aspergillus fumigatus .

(A) MDMs were stimulated with eGFP live Af SC (MOI=1) and treated with Dynasore

(100µM) or vehicle following phagocytosis. Fungal burden was quantified by analysis of

eGFP fluorescence over time during time-lapse confocal microscopy.

(B) Representative electron microscopy image showing two conidia (indicated by red

arrows) contained within specialised compartments being transferred between

macrophages.

confocal microscopy in MDMs infected with live Af SC undergoing lateral transfer and 30 p.i.

was quantified by ImageJ. N=3

(D) Time-lapse confocal microscopy was performed of human MDMs infected with live

SC (MOI=1). Propidium iodide was used to analyse cell necrosis. The conidial area (μm2) as a

marker of fungal germination was quantified within cells undergoing programmed necrosis

and surrounding live cells. Conidia that undergo lateral transfer are highlighted in red. N=14

(E-F) Time-lapse widefield microscopy images (E) and representative transmission

electron microscopy images (F) showing vacuole development in Af containing

compartments in macrophages infected with live Af SC. Electron microscopy image was

taken at 180min p.i.. Conidia are indicated by red arrows. Widefield microscopy images

taken from 45min p.i. and subsequently at 45min intervals. (See Movie 4)

(G) Quantification of vacuolar development and lateral transfer in MDMs pre-treated

with Bafilomycin (100nM) and infected with eGFP live Af SC (MOI=1). N=3

(H) MDMs pre-treated with a pan-caspase inhibitor (Z-VAD-FMK 50μM), RIPK-1 inhibitor

(Necrostatin-1 10μM), both or vehicle were stimulated with eGFP live Af SC (MOI=1) and fungal burden was quantified by analysis of eGFP fluorescence over time. N=3

(I-J) MDMs pre-treated with FK506 (10ng/ml) or vehicle were stimulated with eGFP- expressing live Af SC (MOI=1) and time-lapse confocal microscopy performed. Fungal burden was quantified by eGFP fluorescence quantification over time (I) and average conidial size 6h p.i. quantified to assess fungal germination (J). N=4

Figure E4. Transcriptome analysis of FK506 pre-treated monocyte-derived macrophages in response to live A. fumigatus infection.

(A) Venn diagram indicating the number of significantly regulated genes after FK506

(10ng/ml) pre-treatment of macrophages stimulated with live Af SC (MOI=1). Results shown for 1 and 6 hours together with the overlap between each set of genes.

(B) Selected GO enrichment terms for the differentially expressed genes in MDMs pre- treated after FK506 (10ng/ml) and stimulated with live Af SC (MOI=1) for 6 hours.

Differentially expressed genes were analysed using the functional annotation tool in DAVID.

(C) Visualisation of selected over represented pathways of differentially expressed genes analysed using ConsensusPathDB (CPDB) interaction map. The node size reflects the total number of components in a pathway; the node colour reflects the p-value of the pathway representation analysis with a darker colour corresponding to a lower p-value.

(D) Monocyte-derived macrophages pre-treated with FK506 (10ng/ml) or vehicle were

stimulated with live Af SC (MOI=1) and whole cell lysates probed by western blot for DRP-1

phosphorylation and MAPK pathway activation.

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