-. Biolegend, AMs and IMs were sorted by flow cytometry based on their differential F4/80/CD11c/MHCII expression, as previously described 54 . PMNs were sorted as MHCII?/CD11b+/Ly6G+ cells. Isolated cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 50 ?M ?-mercaptoethanol, 50 ?g/ml streptomycin, and 50 IU/ml penicillin, Clone BM8, Bio-Legend, #123109, 1:100 dilution), anti-CD11c-PerCP-Cy5.5 (Clone N418, BioLegend, #117328, 1:100 dilution), anti-CD11b-PE-Cy7 (Clone M1/70, BioLegend, #101215, 1:200 dilution), anti-Ly6G-PE (Clone 1A8, BioLegend, #127607, 1:200 dilution), and anti-Ly6C-FITC (Clone HK1.4, BioLegend, #128005, 1:200 dilution)

, anti-CD11c-PerCP-Cy5.5 (Clone N418, BioLegend, #117328, 1:200), anti-I-A/I-E-PE-Cy7 (M5/ 114.15.2, BioLegend, #107630, 1:200), and anti-Singlec-F Alexa Fluor ® 647 (E50-2440, BD Pharmingen, #562680, 1:200). Cells were acquired in a FACS Aria IIu (BD Biosciences) and data were analyzed with the FACSDIVA software (BD Biosciences), 0200.

, Endogenous peroxidase was blocked with 3% H 2 O 2 for 10 min. The slides were then incubated in blocking solution (serum-free protein block, Dako X0909) for 20 min to block nonspecific binding. The primary antibodies were added to the slides and incubated overnight in a humidified chamber at 40°C. Detection was accomplished using an Envision_Horseradish Peroxidase Kit (Dako K0679). Immunostaining was revealed using 3,3?-diaminobenzidine. The slides were lightly counterstained with hematoxylin, progressively dehydrated through graded alcohols and xylene, and finally covered with a coverslip after mounting in DPX mounting medium. Slides were examined under an Olympus light microscope, Histopathological and immunohistochemistry studies. Lungs were fixed in 10% formalin, paraffin embedded, cut in 4-?m sections, and stained with hematoxylin and eosin. For immunohistochemistry studies, the anti-CD11c Abs (HL3; BD Biosciences, 1:200 dilution) and anti-CD68 (FA-11, 137001, BioLegend, vol.1

, × 10 6 cells per condition) obtained following 6 days of differentiation of BM cells of 12-week-old female C57BL/6 mice were seeded in 12-well plates and left overnight at 37°C in DMEM media. Next, BMDMs were washed 2× with culture media, infected at an MOI of 1:2 (macrophage:fungal conidia) with R. delemar (strain 99-880), and 1 h later washed five times to remove extracellular conidia, RNA isolation from Rhizopus-infected BMDMs. BMDMs

, × 1 s (set 40). Afterwards, isolation of RNAs was performed according to the manufacturer's instructions

, One hundred and fifty nucleotide sequences were determined from both ends of each complementary DNA fragment using the HiSeq platform (Illumina) as per the manufacturer's protocol. Sequencing reads were annotated and aligned to the UCSC mouse reference genome (mm10, GRCm38.75) as well as the R. delemar (strain 99-880) genome using TopHat2 55 . The alignment files from TopHat2 were used to generate read counts for each gene and a statistical analysis of differential gene expression was performed using the DE-seq package from Bioconductor 56 . A gene was considered differentially expressed if the P value for differential expression was <0.05 and the absolute log, All RNA-seq libraries (strand-specific, paired-end) were prepared with the TruSeq RNA Sample Prep Kit (Illumina)

, ?he patient provided written informed consent in accordance with the Declaration of Helsinki Statistical analysis. The data were expressed as mean ± SEM. Statistical significance of differences was, 2014.

G. D. Mann-whitney-;-brown, test and one-way analysis of variance with the indicated post hoc test for multiple comparisons (P < 0.05 was considered statistically significant), Sci. Transl. Med, vol.4, pp.165-113, 2012.

D. P. Kontoyiannis, Prospective surveillance for invasive fungal infections in hematopoietic stem cell transplant recipients, Clin. Infect. Dis, vol.50, pp.1091-1100, 2001.

P. G. Pappas, Invasive fungal infections among organ transplant recipients: results of the Transplant-Associated Infection Surveillance Network (TRANSNET), Clin. Infect. Dis, vol.50, pp.1101-1111, 2010.

D. P. Kontoyiannis, Zygomycosis in a tertiary-care cancer center in the era of Aspergillus-active antifungal therapy: a case-control observational study of 27 recent cases, J. Infect. Dis, vol.191, pp.1350-1360, 2005.

S. M. Trifilio, Breakthrough zygomycosis after voriconazole administration among patients with hematologic malignancies who receive hematopoietic stem-cell transplants or intensive chemotherapy, Bone Marrow Transplant, vol.39, pp.425-429, 2007.

M. M. Roden, Epidemiology and outcome of zygomycosis: a review of 929 reported cases, Clin. Infect. Dis, vol.41, pp.634-653, 2005.

B. Spellberg, J. Edwards, and A. Ibrahim, Novel perspectives on mucormycosis: pathophysiology, presentation, and management, Clin. Microbiol. Rev, vol.18, pp.556-569, 2005.

A. S. Ibrahim, B. Spellberg, T. J. Walsh, and D. P. Kontoyiannis, Pathogenesis of mucormycosis, Clin. Infect. Dis, vol.54, pp.16-22, 2012.

A. S. Ibrahim, The high affinity iron permease is a key virulence factor required for Rhizopus oryzae pathogenesis, Mol. Microbiol, vol.77, pp.587-604, 2010.

M. Liu, Fob1 and Fob2 proteins are virulence determinants of Rhizopus oryzae via facilitating iron uptake from ferrioxamine, PLoS Pathog, vol.11, p.1004842, 2015.

J. R. Boelaert, Mucormycosis during deferoxamine therapy is a siderophore-mediated infection. In vitro and in vivo animal studies, J. Clin. Investig, vol.91, pp.1979-1986, 1993.

J. R. Boelaert, J. Van-cutsem, M. De-locht, Y. J. Schneider, and R. R. Crichton, Deferoxamine augments growth and pathogenicity of Rhizopus, while hydroxypyridinone chelators have no effect, Kidney Int, vol.45, pp.667-671, 1994.

T. Gebremariam, Bicarbonate correction of ketoacidosis alters host-pathogen interactions and alleviates mucormycosis, J. Clin. Invest, vol.126, pp.2280-2294, 2016.

T. Gebremariam, CotH3 mediates fungal invasion of host cells during mucormycosis, J. Clin. Invest, vol.124, pp.237-250, 2014.

M. Liu, The endothelial cell receptor GRP78 is required for mucormycosis pathogenesis in diabetic mice, J. Clin. Invest, vol.120, pp.1914-1924, 2010.

M. S. Lionakis, I. D. Iliev, and T. M. Hohl, Immunity against fungi. JCI Insight, vol.2, p.93156, 2017.

N. Uwamahoro, The pathogen Candida albicans hijacks pyroptosis for escape from macrophages, vol.5, pp.3-00014, 2014.

T. R. O&apos;meara, Global analysis of fungal morphology exposes mechanisms of host cell escape, Nat. Commun, vol.6, p.6741, 2015.

A. Shah, Calcineurin orchestrates lateral transfer of Aspergillus fumigatus during macrophage cell death, Am. J. Respir. Crit. Care Med, vol.194, pp.1127-1139, 2016.

C. H. Li, Sporangiospore size dimorphism is linked to virulence of Mucor circinelloides, PLoS Pathog, vol.7, p.1002086, 2011.

I. Kyrmizi, Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling, J. Immunol, vol.191, pp.1287-1299, 2013.

T. Akoumianaki, Aspergillus cell wall melanin blocks LC3-associated phagocytosis to promote pathogenicity, Cell. Host Microbe, vol.19, pp.79-90, 2016.

R. Levin, S. Grinstein, and J. Canton, The life cycle of phagosomes: formation, maturation, and resolution, Immunol. Rev, vol.273, pp.156-179, 2016.

J. P. Latge, The cell wall: a carbohydrate armour for the fungal cell, Mol. Microbiol, vol.66, pp.279-290, 2007.

J. D. Nosanchuk, R. E. Stark, and A. Casadevall, Fungal melanin: what do we know about structure?, Front. Microbiol, vol.6, p.1463, 2015.

M. M. Mircescu, L. Lipuma, N. Van-rooijen, E. G. Pamer, and T. M. Hohl, Essential role for neutrophils but not alveolar macrophages at early time points following Aspergillus fumigatus infection, J. Infect. Dis, vol.200, pp.647-656, 2009.

N. Van-rooijen and A. Sanders, Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications, J. Immunol. Methods, vol.174, pp.83-93, 1994.

H. Drakesmith, The hemochromatosis protein HFE inhibits iron export from macrophages, Proc. Natl Acad. Sci. USA, vol.99, pp.15602-15607, 2002.

S. Recalcati, Differential regulation of iron homeostasis during human macrophage polarized activation, Eur. J. Immunol, vol.40, pp.824-835, 2010.

G. Corna, Polarization dictates iron handling by inflammatory and alternatively activated macrophages, Haematologica, vol.95, pp.1814-1822, 2010.

L. P. Partida-martinez and C. Hertweck, Pathogenic fungus harbours endosymbiotic bacteria for toxin production, Nature, vol.437, pp.884-888, 2005.

G. Chamilos, Drosophila melanogaster as a model host to dissect the immunopathogenesis of zygomycosis, Proc. Natl Acad. Sci. USA, vol.105, pp.9367-9372, 2008.

K. Voelz, R. L. Gratacap, and R. T. Wheeler, A zebrafish larval model reveals early tissue-specific innate immune responses to Mucor circinelloides, Dis. Model Mech, vol.8, pp.1375-1388, 2015.

N. Kumar, Gain-of-function signal transducer and activator of transcription 1 (STAT1) mutation-related primary immunodeficiency is associated with disseminated mucormycosis, J. Allergy Clin. Immunol, vol.134, pp.236-239, 2014.

A. Fahimzad, Z. Chavoshzadeh, H. Abdollahpour, C. Klein, and N. Rezaei, Necrosis of nasal cartilage due to mucormycosis in a patient with severe congenital neutropenia due to HAX1 deficiency, J. Investig. Allergol. Clin. Immunol, vol.18, pp.469-472, 2008.

D. C. Vinh, Mucormycosis in chronic granulomatous disease: association with iatrogenic immunosuppression, J. Allergy Clin. Immunol, vol.123, pp.1411-1413, 2009.

S. M. Levitz, M. E. Selsted, T. Ganz, R. I. Lehrer, and R. D. Diamond, In vitro killing of spores and hyphae of Aspergillus fumigatus and Rhizopus oryzae by rabbit neutrophil cationic peptides and bronchoalveolar macrophages, J. Infect. Dis, vol.154, pp.483-489, 1986.

P. G. Jorens, Human and rat macrophages mediate fungistatic activity against Rhizopus species differently: in vitro and ex vivo studies, Infect. Immun, vol.63, pp.4489-4494, 1995.

A. R. Waldorf, S. M. Levitz, and R. D. Diamond, In vivo bronchoalveolar macrophage defense against Rhizopus oryzae and Aspergillus fumigatus, J. Infect. Dis, vol.150, pp.752-760, 1984.

A. R. Waldorf, N. Ruderman, and R. D. Diamond, Specific susceptibility to mucormycosis in murine diabetes and bronchoalveolar macrophage defense against Rhizopus, J. Clin. Invest, vol.74, pp.150-160, 1984.

J. Guarner and M. E. Brandt, Histopathologic diagnosis of fungal infections in the 21st century, Clin. Microbiol. Rev, vol.24, pp.247-280, 2011.

S. M. Lehar, Novel antibody-antibiotic conjugate eliminates intracellular S. aureus, Nature, vol.527, pp.323-328, 2015.

S. Davoudi, P. Anderlini, G. N. Fuller, and D. P. Kontoyiannis, A long-term survivor of disseminated Aspergillus and Mucorales infection: an instructive case, Mycopathologia, vol.178, pp.465-470, 2014.

K. Volling, A. Thywissen, A. A. Brakhage, and H. P. Saluz, Phagocytosis of melanized Aspergillus conidia by macrophages exerts cytoprotective effects by sustained PI3K/Akt signalling, Cell Microbiol, vol.13, pp.1130-1148, 2011.

W. M. Artis, J. A. Fountain, H. K. Delcher, and H. E. Jones, A mechanism of susceptibility to mucormycosis in diabetic ketoacidosis: transferrin and iron availability, Diabetes, vol.31, pp.1109-1114, 1982.

A. S. Ibrahim, The iron chelator deferasirox protects mice from mucormycosis through iron starvation, J. Clin. Invest, vol.117, pp.2649-2657, 2007.

M. P. Soares and G. Weiss, The iron age of host-microbe interactions, EMBO Rep, vol.16, pp.1482-1500, 2015.

G. Weiss and U. E. Schaible, Macrophage defense mechanisms against intracellular bacteria, Immunol. Rev, vol.264, pp.182-203, 2015.

C. D. Skory and A. S. Ibrahim, Native and modified lactate dehydrogenase expression in a fumaric acid producing isolate Rhizopus oryzae 99-880, Curr. Genet, vol.52, pp.23-33, 2007.

F. Fava, D. D. Gioia, and L. Marchetti, Characterization of a pigment produced by Pseudomonas fluorescens during 3-chlorobenzoate co-metabolism, Chemosphere, vol.27, pp.825-835, 1993.

S. Ito and K. Wakamatsu, Chemical degradation of melanins: application to identification of dopamine-melanin, Pigment Cell Res, vol.11, pp.120-126, 1998.

K. L. Wozniak and S. M. Levitz, Cryptococcus neoformans enters the endolysosomal pathway of dendritic cells and is killed by lysosomal components, Infect. Immun, vol.76, pp.4764-4771, 2008.

J. E. Strasser, Regulation of the macrophage vacuolar ATPase and phagosome-lysosome fusion by Histoplasma capsulatum, J. Immunol, vol.162, pp.6148-6154, 1999.

D. Bedoret, Lung interstitial macrophages alter dendritic cell functions to prevent airway allergy in mice, J. Clin. Invest, vol.119, pp.3723-3738, 2009.

D. Kim, TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions, Genome Biol, vol.14, p.36, 2013.

S. Anders and W. Huber, Differential expression analysis for sequence count data, Genome Biol, vol.11, p.106, 2010.