of April 4, 2018.This information is current as

neoformansCryptococcusResponse to Ingestion of

Macrophage Mitochondrial and Stress

Gonçalves and Arturo CasadevallRosiris Leon-Rivera, Anamelia Lorenzetti Bocca, Teresa

Wang,Silveira Derengowski, Carlos de Leon-Rodriguez, Bo Carolina Coelho, Ana Camila Oliveira Souza, Lorena da

http://www.jimmunol.org/content/194/5/2345doi: 10.4049/jimmunol.1402350February 2015;

2015; 194:2345-2357; Prepublished online 2J Immunol 

MaterialSupplementary

0.DCSupplementalhttp://www.jimmunol.org/content/suppl/2015/02/02/jimmunol.140235

Referenceshttp://www.jimmunol.org/content/194/5/2345.full#ref-list-1

, 33 of which you can access for free at: cites 64 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Errata

/content/199/5/1944.full.pdfor:

next pageAn erratum has been published regarding this article. Please see

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2015 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

The Journal of Immunology

Macrophage Mitochondrial and Stress Response toIngestion of Cryptococcus neoformans

Carolina Coelho,*,†,‡ Ana Camila Oliveira Souza,x Lorena da Silveira Derengowski,x

Carlos de Leon-Rodriguez,* Bo Wang,*,{ Rosiris Leon-Rivera,‖,#

Anamelia Lorenzetti Bocca,x Teresa Goncalves,†,‡ and Arturo Casadevall*

Human infection with Cryptococcus neoformans, a common fungal pathogen, follows deposition of yeast spores in the lung alveoli.

The subsequent host–pathogen interaction can result in eradication, latency, or extrapulmonary dissemination. Successful control

of C. neoformans infection is dependent on host macrophages, but macrophages display little ability to kill C. neoformans in vitro.

Recently, we reported that ingestion of C. neoformans by mouse macrophages induces early cell cycle progression followed by

mitotic arrest, an event that almost certainly reflects host cell damage. The goal of the present work was to understand macro-

phage pathways affected by C. neoformans toxicity. Infection of macrophages by C. neoformans was associated with alterations in

protein translation rate and activation of several stress pathways, such as hypoxia-inducing factor-1-a, receptor-interacting

protein 1, and apoptosis-inducing factor. Concomitantly we observed mitochondrial depolarization in infected macrophages, an

observation that was replicated in vivo. We also observed differences in the stress pathways activated, depending on macrophage

cell type, consistent with the nonspecific nature of C. neoformans virulence known to infect phylogenetically distant hosts. Our

results indicate that C. neoformans infection impairs multiple host cellular functions and undermines the health of these critical

phagocytic cells, which can potentially interfere with their ability to clear this fungal pathogen. The Journal of Immunology,

2015, 194: 2345–2357.

The interaction of the pathogenic fungus Cryptococcusneoformans with macrophages is thought to be a criticalevent in the course of cryptococcal infection (1–8).

However, host macrophages show little fungicidal activity in vitro(7, 9) and instead allow C. neoformans to reside in a mature acidicphagolysosome, where it replicates. C. neoformans is believed touse macrophages for extrapulmonary dissemination in a Trojanhorse strategy (10). Moreover, the ability for replication within thephagosome is correlated with increased virulence (1, 11, 12),originating the notion that C. neoformans is a facultative intra-cellular pathogen.Survival of C. neoformans in the phagolysosome has been

attributed to various fungal characteristics (13, 14), of which the

most prominent is a large polysaccharide capsule, but manyothers are essential for infection, such as melanin and phos-pholipase B1. Although ingestion of C. neoformans by macro-phages is followed by many hours in which the host cell isviable, several studies have reported damage to host cellularprocesses, including: increased phagosome permeability (1),inhibition of cyclin D1 (15), and DNA instability (16), followedby mitotic arrest (17). Furthermore, intracellular residence of C.neoformans decreases Ag presentation, T cell proliferation, andcytokine production by macrophages (18, 19). Additional evi-dence of host cell damage is apparent when large residualvacuoles are observed in macrophages from which C. neofor-mans has exited by nonlytic exocytosis (20). However, the

*Department of Microbiology and Immunology, Albert Einstein College of Medicineof Yeshiva University, Bronx, NY 10461; †Centre for Neuroscience and Cell Biology,University of Coimbra, 3004-504 Coimbra, Portugal; ‡Faculty of Medicine, Univer-sity of Coimbra, 3004-504 Coimbra, Portugal; xCell Biology Department, BiologyScience Institute, University of Brasilia, Brasilia CEP 70910-900, Brazil; {MD Pro-gram, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461;‖Department of Biology, University of Puerto Rico, Rıo Piedras Campus, San Juan,Puerto Rico 00931; and #Undergraduate Research Program, Albert Einstein Collegeof Medicine of Yeshiva University, Bronx, NY 10461

ORCID: 0000-0002-7523-3031 (C.C.).

Received for publication September 25, 2014. Accepted for publication December26, 2014.

This work was supported by National Institutes of Health Grants 5R01HL059842,5R01AI033774, 5R37AI033142, and 5R01AI052733 (to A.C.) and a FundacaoCiencia e Tecnologia grant. C.C. was a recipient of a Ph.D. grant by FundacaoCiencia e Tecnologia (SFRH/BD/33471/2008) under the Ph.D. Program in Exper-imental Biology and Biomedicine of the Centre for Neuroscience and Cell Biol-ogy, University of Coimbra, Portugal, and Fundacao Ciencia e Tecnologia GrantPEst-C/SAU/LA0001/2013-2014. R.L.-R. was supported through Minority Accessto Research Careers Program Grant 5T34GM007821-31. Genomic data experimentswere performed at the Genome Technology Access Center in the Department ofGenetics at Washington University School of Medicine, partially supported byNational Cancer Institute Cancer Center Support Grant P30 CA91842 to theSiteman Cancer Center and by Institute for Clinical and Translational Science/Clinical Translational Studies Awards Grant UL1RR024992 from the National

Center for Research Resources, a component of the National Institutes of Health,and the National Institutes of Health Roadmap for Medical Research. The Ana-lytical Imaging Facility of Albert Einstein College of Medicine is supportedthrough National Cancer Institute Cancer Center Support Grant P30CA013330.The data in this paper are from a thesis submitted by C.C. in partial fulfillment ofthe requirements for a Ph.D. in the Faculty of Medicine, University of Coimbra,Coimbra, Portugal.

The microarray data presented in this article have been submitted to the Gene Ex-pression Omnibus (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57720) underaccession number GSE57720.

Address correspondence and reprint requests to Dr. Arturo Casadevall, Albert Ein-stein College of Medicine of Yeshiva University, Forchheimer 411, 1300 Morris ParkAvenue, Bronx, NY 10461. E-mail address: arturo.casadevall@einstein.yu.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: AIF, apoptosis-inducing factor; BMDM, bonemarrow–derived macrophage; CCCP-1, carbonyl cyanide m-chlorophenyl hydrazine;HIF-1a, hypoxia-inducing factor–1a; HK, heat killed; Jc-1, 5,59,6,69-tetrachloro-1,19,3,39-tetraethylbenzimidazolylcarbocyanine iodide; LDH, lactate dehydrogenase;Dwm, mitochondrial potential; PDGFBB, platelet-derived growth factor BB; RIP,receptor-interacting protein; ROS, reactive oxygen species; RPM, ribopuromycyla-tion; TMRE, tetramethylrhodamine; TREM1, triggering receptor expressed on my-eloid cells 1.

Copyright� 2015 by TheAmerican Association of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402350

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

mechanisms by which C. neoformans damages cells have notbeen investigated in detail.Intracellular pathogens have evolved strategies to manipulate

host machinery for their survival (21). Interference with signaltransducer activity, manipulation of the lysosomal compartment,and host cell survival versus death are a few examples of com-monly targeted processes. For example, both Francisella andMycobacterium possess virulence factors that decrease caspase-1activation, therefore decreasing production of caspase-1–derivedinflammatory IL-1b (22). Cell death pathways rely on mito-chondrial mediators for, at least, a portion of the pathway, andtherefore many survival versus death decisions are integrated inthe mitochondria. In addition, mitochondria are no longer regardedsolely as the cell’s powerhouse but also are recognized asplaying a role in immune function, producing reactive oxygenspecies (ROS) (23) for activation of the inflammasome (24).Consequently, viral, bacterial, and protozoan pathogens havemyriad factors that manipulate host cell mitochondria (25, 26),but comparable information is not yet available for fungalpathogens.Current views of C. neoformans intracellular pathogenesis

posit a passive resistance of fungi to host attack, but little has beendone to explore active fungal attack on the host. Survival of thehost cell after nonlytic exocytosis and the absence of widespreadhost cell death in C. neoformans–macrophage studies have en-couraged the view that host cells suffer little or no damage fromthis organism. In this work, we have investigated macrophageinjury after C. neoformans infection. Our results indicate thatC. neoformans phagocytosis results in modifications of criticalcellular functions, including impaired mitochondrial function,activation of caspase-1 and cellular stress pathways, and alteredprotein synthesis rate. The accumulation of cellular damage as-sociated with C. neoformans intracellular residence could promoteand potentiate C. neoformans survival in macrophages and con-tribute to cryptococcal virulence.

Materials and MethodsFungal strains

C. neoformans var. grubii strain H99 (serotype A), acapsular mutantcap59, and original wild-type K99 were a kind gift of J. Heitman (Durham,NC). Yeast cells for infection were grown for 2 d in Sabouraud dextrosebroth (Difco, Carlsbad, CA) at 37˚C.

Macrophage and macrophage-like cells

Three types of macrophages were used for most experiments: themacrophage-like murine cell line J774.16 (27), bone marrow–derived mac-rophages (BMDM), and peritoneal macrophages. J774.16 macrophages werekept in DMEM complete media consisting of DMEM (CellGro), 10%NCTC-109 Life Technologies medium (LifeTechnologies), 10% heat-inactivated FBS (Atlanta Biologicals), and 1% nonessential amino acids(CellGro). BMDM were obtained by extracting bone marrow from hind legbones of 6- to 8-wk-old BALB/C female mice (National Cancer Institute)and maturing them in vitro for 6–8 d in DMEMmedia with 20% L-929 cell–conditioned media, 10% FBS, 2 mM L-glutamine (CellGro), 1% nonessentialamino acids (CellGro), 1% HEPES buffer (CellGro), and 2-MΕ (LifeTechnologies). Peritoneal macrophages were extracted by injecting 10 mlice-cold PBS into the peritoneal cavity of the mice, and then were culturedand infected in the same conditions as J774.16 cells. The peritoneal mac-rophage population was defined as adherent CD11b+ cells. In all assays,macrophages were plated to achieve a density of 1 3 105 cells per milliliterat the time of C. neoformans infection. C. neoformans organisms were addedat a multiplicity of infection of 1:2 (unless otherwise noted) along withcapsular mAb 18B7 (28) at 10 mg/ml. For some experiments, C. neoformanswas heat killed (HK) by incubation at 60˚C for 60 min or with oxidativedamage by incubating yeast pellet with 30% (w/w) H2O2 for 30 min. Allanimal experiments were conducted according to ethical guidelines, with theapproval of the Institutional Animal Care and Use Committee of AlbertEinstein College of Medicine.

Killing and caspase activation assays

For fungal killing assays, the cells were detached by vigorous pipetting anddiluted in sterile water onto Saboraud agar plates. CFU were counted after2 d at 30˚C. Specific caspase activity was detected using a fluorochromeinhibitor of caspases (Immunochemistry Technologies) assay. Briefly,carboxyfluorescein-labeled inhibitor peptide was added 1 h before thetermination of phagocytosis and stained with 0.1 mg/ml Hoechst for de-tection of necrotic/late apoptotic cells. Cells were analyzed immediatelyby laser scanning cytometry in the iCys Research Imaging Cytometer(CompuCyte Corporation) (17). Caspase activation was measured by im-aging 20–40 fields in each well with the 340 objective at 0.5 mm reso-lution, allowing a field size of 500 mm 3 192 mm.

ATP, lactate dehydrogenase, and glucose measurements

Total ATP content and lactate dehydrogenase (LDH) release can be used toestimate cell numbers and cellular viability. ATP measurements wereperformed by adding Triton X-100 at 0.25% and 2 mM EDTA (Sigma-Aldrich) to the wells containing infected cells. Next, 25 ml of this ex-tract was incubated with Enliten ATP assay system (Promega), and countsper second were measured in a standard luminometer. We confirmed ineach assay the extraction of ATP solely from mammalian cells and noextraction of C. neoformans–derived ATP, as indicated by the manufac-turer. LDH and glucose were measured by enzymatic reactions (Abcam)according to the manufacturer’s instructions. Briefly, for LDH 10 mlmacrophage supernatants was added to 100 ml enzymatic mix, and ab-sorbance was read after 30 min at 450 nm. For glucose 1:100 dilutions ofcell supernatants were mixed with the assay mix, and the glucose contentwas determined by comparison with glucose standards.

Immunoblot analysis

Cytosolic protein extracts were obtained by resuspending the cell pellet in250 mM sucrose, 50 mM Tris-HCl pH 7.4, and 5 mMMgCl2, and total cellextracts were obtained in RIPA buffer. Both were supplemented withcOmplete Mini Protease Inhibitor Cocktail (Roche Applied Science),followed by 20 strokes with a Dounce homogenizer and centrifugation at3000 rpm for 10 min for cytosolic extracts or 14,000 rpm for 30 min fortotal cell extracts. Western blot was performed in a NuPAGE SDS-PAGE(LifeTechnologies) system for the following primary Abs: rabbit anti–apoptosis-inducing factor (AIF) and mouse anti–receptor-interacting pro-tein (RIP) 1 (1:2000–1:200; from BD Biosciences), cleaved PARP D-214(Cell Signaling), mouse anti–cytochrome c (1:200) (clone 7H8.2C12;Abcam), and MFN1 (H-65) and MFN2 (H-68; Santa Cruz Biotechnology).b-actin Ab coupled to peroxidase (1:5000; Santa Cruz Biotechnology) wasused as a loading control.

Gene expression

Macrophages were plated to achieve a density of 83 106 cells at the time ofinfection on 10-cm2 dishes and infected as before. RNA was extractedusing a RNeasy Mini Kit (QIAGEN) and stored at 280C until analysis.Gene expression analysis was performed at the Genome Technology Ac-cess Center in the Department of Genetics at Washington UniversitySchool of Medicine. Briefly, a total of 10 mg RNAwas converted to cDNAusing the Nugen Pico SL (Nugen) and labeled using Nugen Encore Biotinkit. Target cRNAwas hybridized to the murine genome Affymetrix MouseGene 1.0 ST (Affymetrix). Biological replicates were performed fourtimes, and each experimental set was analyzed separately and then aver-aged for data analysis. All microarray datasets (CEL files) were normal-ized with the ExonRMA algorithm. Data variance was stabilized andtransformed on a logarithmic scale, and quality control was performed.Genes were considered differentially expressed if p , 0.05 and foldchange was .1.5. Genes satisfying these conditions were imputed intoIngenuity Pathway Analysis (Ingenuity Systems; www.ingenuity.com) foranalysis of upstream regulators and Gene Ontology. Gene expressionanalysis was confirmed by RT quantitative PCR for selected genes (list ofprimers is given in Supplemental Table I).

Ribopuromycylation and immunofluorescence

Cells were infected as described above, and 15 min before the end of theexperiment media were supplemented with 91 mM puromycin and 208 mMemetine. One well was left without puromycin and used as negative control(C2). Cells were fixed with 3% paraformaldehyde, protease inhibitorsmixture cOmplete Mini (Roche), 0.015% digitonin, and 10 U/ml RNase-OUT in 50mM Tris-HCl buffer for 15 min at room temperature, blockedin a solution of 0.05% saponin, 1 mg/ml Fc block Ab (BD Biosciences),10 mM glycine, and 5% FBS in PBS for 10 min, followed by the addition

2346 C. NEOFORMANS MEDIATES MACROPHAGE DAMAGE

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

of 1:100 of 2D10-conjugated to Alexa 488 or Alexa 647 fluorophores,a kind gift of J. W. Yewdell (National Institute of Allergy and InfectiousDiseases). Cells were counterstained with Hoechst at 0.1 mg/ml, andfluorescence was quantified in an iCys Compucyte LSC. For mitochondrialmorphology, cells were plated in optical quality dishes (Maktek Corpo-ration); stained for ribopuromycylation (RPM) as before or with Alexa488–conjugated cytochrome c Ab, clone 6H2.B4 (BDPharmingen); andimaged at 0.2-mm steps with the Inverted Olympus IX71 coupled toa Photometrics CoolSnap HQ charge-coupled device camera and analyzedusing Volocity 3D (PerkinElmer).

Mitochondrial potential and ROS measurements

5,59,6,69-Tetrachloro-1,19,3,39-tetraethylbenzimidazolylcarbocyanine io-dide (Jc-1) was added 15 min before the termination of the assay, ac-cording to the manufacturer’s instructions (Immunochemistry Technologies).Jc-1 dye accumulates as a red aggregate in healthy mitochondria and asa green monomer in depolarized mitochondria. ROS were measured withCellROX Deep Red Reagent (LifeTechnologies) for 30 min at a 5-mMconcentration. Tetramethylrhodamine (TMRE) and MitoTracker greenwere added to the cells for 45 min before termination of the experiment at200 and 25 nM, respectively. For in vivo experiments, mice were infectedwith 1 3 107 C. neoformans i.p. or sterile PBS (vehicle) for the indicatedtime, and 30 min before sacrifice each mouse received 30 ml of Jc-1 dyediluted in PBS, which was also injected i.p. Peritoneal lavage cells wereimmunostained with CD45-PercP-Cy5.5 (eBiosciences) and F4/80–Alexa647 (Life Technologies) and Uvitex for exclusion of extracellular yeasts.Fluorescent signal from viable cells, as measured by DAPI exclusion, wasdetected with a Becton Dickinson LSRII instrument (BD Biosciences).

Transmission electron microscopy

Macrophages were infected with C. neoformans for 24 h and fixed with 2%glutaraldehyde, 4% paraformaldehyde in 0.1 M cacodylate at room tem-perature for 2 h, followed by overnight incubation in 4% formaldehyde, 1%glutaraldehyde in PBS. The samples were subjected to postfixation for 90min in 2% osmium, serially dehydrated in ethanol, and embedded in Spurrsepoxy resin. Sections (70–80 nm thick) were cut on a Reichart Ultracut UCTand stained with 0.5% uranyl acetate and 0.5% lead citrate. Samples wereviewed in a JEOL 1200EX transmission electron microscope at 80 kV.

Statistical analysis and plotting

Graphs, statistical analysis, and figures were assembled in Prism version6.00 for Mac OS X (GraphPad Software, San Diego, CA). Fig. 4 wasgenerated using String database (29).

ResultsGene expression changes upon infection of J774.16macrophage-like cells following opsonic ingestion ofC. neoformans

We investigated gene expression changes occurring in the J774.16macrophage-like cell line at 2 and 24 h after ingestion of yeast cells(Fig. 1 and full gene list in Supplemental Table II). Initial geneexpression changes could be observed already at 2 h post infection,with much larger changes becoming apparent at 24 h of infection.Ingestion of live C. neoformans by macrophages differentiallymodulated the expression of 110 genes, whereas infection with HKC. neoformans affected 61 genes. Very few genes were differentiallymodulated by both live and dead yeast cells: Srrt was downregulatedat 24 h, and Ccl2, Ccl7, Gas5, Glipr1, Gprc5a, Hmgb1, Phlda1, Id2,Sh3pxd2a, Mup2, Rhob, and Snhg1 levels were altered after 2 h.When macrophages were infected with live C. neoformans, threegenes remained upregulated throughout the course of infection:Emp1, Serpine1, and Id2 (confirmed by RT-PCR in SupplementalFig. 1). Ingenuity Pathway Analysis of gene expression changesshowed that after 2 h of infection the macrophages upregulatedpathways involved in cell death and survival, cellular movement,proliferation, and cell-to-cell signaling. Predicted upstream regu-lators activated at this time are platelet-derived growth factor BB(PDGFBB), TNFS11 or RANKL (receptor activator of NF k-B li-gand), and triggering receptor expressed on myeloid cells 1 (TREM1).PDGFBB is a mitogenic factor, TNFS11 is an antiapoptotic cellsurvival factor (30), and TREM1 was recently described as a hypoxia-

responsive factor (31) that is involved in the response to As-pergillus fumigatus (32). Analysis of genes affected at 24 hrevealed activation of such cellular functions as amino acidmetabolism, posttranslational modifications, small-moleculebiochemistry, cell growth and proliferation, and cell deathand survival. Predicted upstream regulators activated at 24 hwere IL-4, IL-5, hypoxia-inducible factor–1a (HIF-1a), andCD38. Both IL-4 and IL-5 are Th2-associated cytokines, andboth have been found in Cryptococcus-infected mouse lungs(33). HIF-1a is a major regulator of response to hypoxia thatwas associated with fungal pathogenesis (34). CD38 is an en-zyme responsible for the synthesis and hydrolysis of cyclicADP-ribose from NAD+, and loss of this enzyme impairs im-mune responses to Listeria monocytogenes (35). In summary,gene expression changes in J774.16 cells upon C. neoformansinfection were consistent with those reported in vivo and indicatedsimultaneous activation of immune and stress responses.

C. neoformans infection of murine macrophages alters proteintranslation rate in host cells

To further explore possible mechanisms that could eventually leadto C. neoformans–induced cell stress, we investigated proteinsynthesis, activation of cellular death pathways, and mitochon-drial function in C. neoformans–infected macrophages. Conse-quently, we measured macrophage protein synthesis rate usingimmunofluorescence to detect puromycin binding to nascentmRNA chains (36). Quantification of ribosome-bound puromy-cin revealed a decrease in the rate of translation in J774.16macrophage-like cell line infected with C. neoformans (Fig. 2A).This observation agrees with the gene expression data suggestinga modulation of posttranslational modifications. However, whenwe replicated the experiments in primary macrophages extractedfrom the peritoneal cavity, we observed an increase in rate oftranslation upon C. neoformans infection (Fig. 2B). None of thecells showed alterations in the cellular distribution of the activeribosomes as seen by fluorescence microscopy (Fig. 2C). Thus,although C. neoformans infection can modulate the overall rateof protein synthesis in macrophages, we did not observe a uni-form pattern of increased or decreased protein synthesis in theinfected macrophage subsets.

C. neoformans infection of murine macrophages and J774.16macrophage-like cell line results in major changes inmacrophage metabolism and only partial restriction ofC. neoformans growth in vitro

C. neoformans infection of murine macrophages results in re-striction of fungal growth and some degree of fungal killing forthe first 24 h of infection (Fig. 3A). Live cell microscopy hasshown that during the first 24 h of infection C. neoformans andmacrophage cells coexist in vitro with .90% of macrophagesremaining viable (C. de Leon-Rodriguez, unpublished observa-tions). We found that commonly used methods to measure cellviability by colorimetry (tetrazolium dye reduction and nucleardye incorporation) were unreliable because we could not sep-arate yeast and macrophage contributions to measurements.Consequently, we used total ATP levels as a measure of mac-rophage number based on selective extraction of ATP frommacrophages and not from yeast cells. With this approach weconfirmed that ATP levels in infected cells decreases to 60–80% when compared with uninfected cells in the first 24 h(Fig. 3B), indicating that C. neoformans infection is accom-panied by severe biochemical changes in the surviving cells,a small degree of macrophage cell death, or both of theseprocesses.

The Journal of Immunology 2347

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

FIGURE 1. Gene expression changes upon infection of macrophage-like J774.16 cells following opsonic ingestion of C. neoformans. Gene expression

changes in J774.16 macrophage-like cells owing to their opsonic ingestion of live C. neoformans or HK C. neoformans were analyzed. (A) Venn diagrams

of gene expression changes occurring in uninfected J774.16 cells (Ctrl) compared with changes occurring in Live or HK C. neoformans–infected cells. (B)

STRING database illustration of reported associations between the genes that were found differentially regulated after 24 h infection with live C. neo-

formans. Orange boxes show empirical gene functional clusters. Different line colors represent types of evidence found (see legend) for the association

between proteins (nodes).

2348 C. NEOFORMANS MEDIATES MACROPHAGE DAMAGE

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

C. neoformans infection can activate programmed cell deathpathways in murine macrophages and macrophage-likeJ774.16 cells

To further explore whether C. neoformans–induced intracellularstress could result in accelerated macrophage death, we assessedactivation of programmed cell death effector molecules, such ascaspases, AIF, and RIP1. The J774.16 macrophage-like cell lineshowed a trend to activate caspase-1 and caspase-3 throughoutinfection, but this result did not reach statistical significance(Fig. 4), whereas BMDM activated caspase-1,-3, and -8. Fur-

thermore, we determined that killing of yeast cells by J774.16 orBMDM macrophages in vitro was not affected by pan-caspase

inhibition but was decreased by inhibition of NO formation

(Supplemental Fig. 2). When we assessed protein expression of

apoptosis pathway intermediates, J774.16 macrophage-like cells

increased expression of RIP and AIF early in the course of in-

fection (Fig. 4B, 4C), whereas BMDM showed activation of AIF

and released cytochrome c into the cytosol. Furthermore, there

was an increase of J774.16 cells with Annexin Vexternalization as

well as cells with both Annexin V externalization and propidium

FIGURE 2. C. neoformans (Cn) infection ofmurinemacrophages alters protein translation rate in host cells.Murinemacrophageswere infectedwith opsonized

liveC. neoformansorHKC.neoformans (HKCn) for 24 h, andprotein translationwasmeasured by theRPMmethod asdescribedbyDavid et al. (36).RPMconsists

of puromycin tagging of nascent ribosomes. The amount of bound puromycin is ascertained bymeasuring puromycin-bound 2D10Ab, and it is proportional to the

amount of actively translating ribosomes. Quantification of actively translating ribosomes by RPM for (A) J77.16 macrophage–type cells and (B) peritoneal

macrophages. Experiments were repeated three times for each cell type. Shown are the mean and SEMof all experiments. *p, 0.05, ****p, 0.001 for two-way

ANOVAwith Bonferroni multicomparison correction. (C) Representative images of RPM staining in peritoneal macrophages. Original magnification363; scale

bar, 20 mm. Experiments were repeated three times for a minimum of 30 cells per condition per experiment, and representative images are shown.

The Journal of Immunology 2349

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

iodide permeability (Fig. 4D). In addition, J774.16 cells in-creased LDH levels in supernatants (Fig. 4E), providing a cluethat C. neoformans infection in J774.16 cells caused a type ofnecrotic death. No detectable LDH release was found in BMDM(data not shown), which together with the activation of caspase-3indicates classical features of apoptosis in BMDM. In addition, wemeasured glucose concentration in supernatants and found nodifference of infected macrophages relative to noninfected cells,ruling out glucose depletion due to fungal growth as a mechanismfor macrophage toxicity (Fig. 4F). We then proceeded to testprimary peritoneal macrophages, observing that peritoneal mac-rophages activated caspase-3 and increased their LDH levels in thesupernatant. Results are summarized in Table I. Thus, althoughonly a small subset of macrophages died upon the first 24 h ofC. neoformans infection, we showed that C. neoformans inducedcellular stress pathways and necrotic and apoptotic features ina variety of infected macrophages; however, we also observe thatthe type of stress and death pathway activated was dependent onmacrophage type.

Murine macrophages and J774.16 macrophage-like cellsdepolarize mitochondria postinfection with C. neoformans

Given that mitochondria are critical for many aspects of cellularhomeostasis, we studied mitochondrial potential (Dwm) alterations(37). The Dwm can be measured through incorporation of fluo-rescent dyes for which brightness of TMRE is proportional toDwm. For the dye Jc-1, the color of cells will shift from green to

red as mitochondria hyperpolarize. Therefore, the ratio of red/green will allow for semiquantitative measurement of Dwm (37).As experimental controls, we added carbonyl cyanide m-chlor-ophenyl hydrazone (CCCP-1) to depolarize mitochondria or ro-tenone to hyperpolarize mitochondria. All macrophages depolarizedtheir mitochondria at 24 h of infection (Fig. 5A–C). We note that forBMDM Dwm was decreased to a much lower extent than for theother cell types (Fig. 5B), and because this cell type was also re-sistant to CCCP-1 depolarization, we suggest the existence of a celltype–specific resistance to depolarization. Transmission electronmicroscopy revealed no alterations in mitochondrial ultrastructure(Fig. 5D), but depolarization of mitochondria was accompanied byalterations in the mitochondrial network, with increased fission ininfected cells (Fig. 5E). This finding suggests that mitochondria arenot directly damaged by infection but, rather, have their activitymodulated by prolonged C. neoformans residence. Our attempts tomodulate Dwm in macrophages were unsuccessful because mito-chondrial inhibitory drugs were highly toxic to C. neoformanscells (data not shown). We investigated whether Acanthamoebacastellanii could modulate their mitochondria upon ingestion ofC. neoformans but found no difference in Dwm in these unicel-lular organisms (data not shown).

Mitochondrial alterations do not correlate with oxidative burstbut are mediated by NO

We evaluated whether mitochondrial modulation was related toROS (24) and NO levels (38). We found that neither J774.16 nor

FIGURE 3. Murine macrophages and the macrophage-like J774.16 cell line manifest reduced viability upon C. neoformans (Cn) infection and only

partially restrict C. neoformans growth in vitro. Murine macrophages were exposed to opsonized C. neoformans for the indicated times. Macrophage

viability was assessed by homogenate ATP levels, and fungicidal activity was measured in parallel by quantifying CFU. (A) C. neoformans growth post

infection of murine macrophages. Red line indicates amount of C. neoformans that was used to infect macrophages (initial inoculum). Experiments were

repeated two to three times for each cell type. CFU determinations were performed in triplicate and ATP determinations in quintuplicates. (B) Total ATP

levels of murine macrophages after exposure to live or HK C. neoformans, normalized to ATP levels of uninfected macrophages (% of control). CCCP-1 is

a positive control causing extensive cell death at 24 h. Shown are mean and SEM of all experiments for BMDM and peritoneal macrophages. For J774.16

cells, one representative experiment is shown. *p , 0.05, **p , 0.01 for two-tailed Student t test.

2350 C. NEOFORMANS MEDIATES MACROPHAGE DAMAGE

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

FIGURE 4. Programmed cell death pathways are activated in murine macrophages and the macrophage-like J774.16 cell line infected with C. neo-

formans (Cn). Cell death in murine macrophages infected with C. neoformans was characterized by caspase activation, measured through binding of

a fluorescent caspase-specific peptide and immunoblot quantification of molecules involved in cell death pathways. Cell death was quantified by measuring

externalization of Annexin V and by release of LDH into the extracellular media. (A) Measurement of caspase activation after C. neoformans infection of

J774.16 cells (left), BMDM macrophages (center), and peritoneal macrophages (right). Experiments were repeated three to five times for J774.16 mac-

rophages and BMDM with duplicate wells and twice for peritoneal macrophages. (B) Quantification of protein expression for RIP, AIF, cleaved PARP, and

release of cytochrome c from the cytosol post infection of J774.16 cells and BMDM. Expression levels were normalized for b-actin content. (C) Rep-

resentative immunoblots for J774.16 cells. (D) Quantification of Annexin V+ and propidium iodide (PI)+ cells after C. neoformans infection of J774.16 cells

(left) and peritoneal cells (right). (E) LDH release for J774.16 cells (left) and peritoneal cells (right). (F) Glucose quantification (Figure legend continues)

The Journal of Immunology 2351

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

BMDM produced significant amounts of ROS after phagocytosisof C. neoformans (Fig. 6A, data not shown for BMDM). To dis-card the possibility that the observed lack of ROS production wasdue to a quenching effect of C. neoformans, we exposed macro-phage cells to zymosan, a preparation of Saccharomyces cer-evisiae cell wall, and H2O2-killed C. neoformans, but none ofthese conditions triggered ROS production in J774.16 macrophage-like cells (Fig. 6B). Peritoneal macrophages were capable ofreleasing ROS in response to C. neoformans (data not shown)and when exposed to zymosan or H2O2-killed C. neoformans(Fig. 6C). However, ROS production by mitochondria occurs uponmitochondrial hyperpolarization, and, therefore, in the particularcase of C. neoformans infection in peritoneal macrophages, it islikely that Dwm modulation is disconnected from ROS production.When S-ethyl-iso-thiourea, an inhibitor of NO, was added to H99-infected J774.16 macrophage-like cells and to peritoneal macro-phages (data not shown), depolarization was decreased at 24 h ofinfection (Fig. 6D), suggesting a role for NO in mitochondrialdepolarization.

Intraperitoneal infection of mice with C. neoformans causesa decrease in mitochondrial polarization in peritoneal totalleukocytes and macrophages

We proceeded to investigate if the mitochondrial depolarizationobserved in vitro was also observed in vivo. In a model ofperitoneal C. neoformans infection, we observed that peritoneallavage cells, both total leukocytes (CD45+) and macrophages(F4/80+), were depolarized at 24 h post infection in comparisonwith mock-infected mice (Fig. 7), validating our in vitro findingsin a murine model of infection. Overall, our results show mul-tiple alterations in macrophage functions upon C. neoformansinfection, namely, gene expression changes, alterations of pro-tein translation rates, and alteration in mitochondrial dynamics(summarized in Table I).

DiscussionConsiderable evidence now exists that intracellular residence ofC. neoformans is associated with damage to macrophages, as evi-denced by phagosomal leakiness (1), giant vacuole formation (20),and cell cycle arrest (17). Because progressive accumulation ofcellular damage can result in cell death, we set out to investigatethe mechanisms of macrophage cell damage after C. neoformansphagocytosis, with a specific focus on mitochondrial function,a major integrator of cellular decisions such as survival, oxidativeburst, and immune responses (24). Our initial studies focused ondetermining whether host cell gene expression changes couldpotentially explain the results previously reported. The transcriptlevel of dozens of genes was affected, many of those genes relatedto a stress response, and these changes were consistent withphenotypic observations following C. neoformans infection ofmacrophages. For example, previous work has shown that initialproliferation of macrophages infected with C. neoformans is fol-lowed by cell cycle arrest (17, 39). Gene expression data from thisstudy provide information consistent with and supportive of thecell cycle changes observed. PDGFBB, which belongs to the samefamily as CSFR1 and is a mitogenic factor for endothelial cells(40), could be a mediator of the initial proliferation, whereas ac-tivation of HIF-1a (41) and cyclin G2 (42) at later times mightcontribute to cyclin D1 inhibition (15) and the subsequent cellcycle arrest upon C. neoformans infection (17). The protein syn-thesis machinery is a target for viral (43), bacterial, and parasiticpathogens (44, 45). Translation interference by fungal pathogenswas suggested for Candida albicans and in C. neoformans in-fection (discussed in Ref. 45), but had not been shown for fungalinfections thus far.Next we demonstrated that C. neoformans intracellular resi-

dency affected cell stress, survival, and death responses. We ob-served a low mortality of infected macrophages, and no pathwaypredominated to trigger wholesale cell death, as determined by the

in cell supernatants for J774.16 cells. Experiments were repeated three times for each cell type. Data were normalized to percent of uninfected macrophages

(No Cn). Shown are mean and SEM of all experiments. *p , 0.05, **p , 0.01,***p , 0.005, ****p , 0.001 for two-way ANOVA with Bonferroni

multicomparison correction.

Table I. Summary of findings

J774.16 Macrophage-like Cells BMDM Peritoneal Macrophages

Fungal control ++ ++ +ATP levels ↓ ↓ ↓ ↓ ↓PCD pathwaysLDH release Yes No YesAnnexin V binding Yes a YesActivation ofCaspases 28 21, 23, 28 23RIP Yes No b

AIF Yes Yes b

PARP No No b

Release of cytochrome c No Yes b

Predicted PCD activated Necrosis Apoptosis Apoptosis/NecrosisGene expression Stress pathways activatedMitochondrial characteristicsDepolarization ++ + ++Morphology Fragmented Unchanged FragmentedROS production No No YesMitochondrial-derived ROS No No NoNO dependence Yes a Yes

Protein translation ↓ a ↑

Italics represent conclusions from the data, not results of the experiment.aNot pertinent for conclusions of study.bNot performed owing to technical limitations.↓, downregulated; ↓ ↓, strongly downregulated; PCD, programmed cell death.

2352 C. NEOFORMANS MEDIATES MACROPHAGE DAMAGE

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

mixed pattern of activation of death pathways. It is possible thatsimultaneous interferences in multiple pathways, despite beingsmall in magnitude, could result in significant effects in macro-phage health and ability to clear the pathogen when combined.C. neoformans resides and replicates within host macrophages,but the host cells do not typically display features of necrotic orapoptotic deaths. Of interest, Th1 activation of macrophagesdecreases the amount of lysosomal damage to the host cells, andtherefore macrophage damage can be modulated by the cytokinemilieu [as shown in an accompanying article (46)], providingfurther evidence that equilibrium between host and pathogen ismodulated by the cytokine milieu (33, 47). The absence of a pre-dominant death pathway, combined with the inability of macro-

phages to eliminate C. neoformans, phagosomal leakiness, andcell cycle arrest, may be caused by multiple subtle alterations incellular function, including mitochondrial function. Our results areconsistent with the capacity of C. neoformans to cause latent in-fection in granulomas, with long-term survival of both host andpathogen (3, 8), whereas concomitantly C. neoformans residencyin macrophages causes sufficient dysfunction to explain the sub-optimal stimulation of immune responses reported previously (18,19). Finally, our data are consistent with the scenario that mac-rophage death is likely to occur in a small subset of cells, in whichthe cumulative damage induced by C. neoformans exceeds a cer-tain threshold, and depending on the type of cellular response, thiscell death can become either apoptotic or necrotic.

FIGURE 5. Murine macrophages and macrophage-like J774.16 cells depolarize mitochondria post infection with C. neoformans (Cn). Murine mac-

rophages and macrophage-like J774.16 cells were infected with opsonized C. neoformans, and their Dwm was measured at several time intervals. CCCP-1,

which causes rapid depolarization of mitochondria by uncoupling the proton gradient, was used as a depolarization control. Representative histograms for

(A) J774.16, (B) BMDM, and (C) peritoneal macrophages. Numbers represent percent of cells with polarized mitochondria in the experimental condition

shown. Shaded gray represents noninfected macrophages (No Cn), and each experimental condition is shown by the red line. Dwm was measured by flow

cytometry analysis of the Jc-1 red/green ratio or by TMRE dye accumulation. Ratio of red/green fluorescent signal reflected mitochondrial polarization for

Jc-1. TMRE accumulation is proportional to mitochondrial polarization. Experiments were repeated two to three times for each macrophage cell type, and

a representative experiment is shown. (D) Mitochondrial morphology was studied by electron microscopy. No alteration of mitochondria morphology, such

as nonexistent or swollen cristae, was observed. Scale bar, 500 nm; bottom row is magnification of mitochondria on top row. (E) Peritoneal macrophages

infected with C. neoformans show a fragmented mitochondrial network, shown by cytochrome c immunostaining (green). Images were counterstained with

Hoechst nuclear dye (blue). Original magnification 363; scale bar, 50 mm. Experiments were repeated three times, and representative images are shown.

The Journal of Immunology 2353

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

C. neoformans infection caused a decrease in Dwm in allmacrophage cell types tested, an observation previously reportedfor bacterial pathogens Shigella flexneri (48) and Mycobacteriumtuberculosis (49). Mitochondrial depolarization occurred in everymacrophage type tested and in a mouse model of infection, sug-gesting that this is a conserved response to C. neoformans infec-tion. The necessity for mitochondrial modulation derives bothfrom regulation of fuel and energy requirements and from theemerging role of mitochondria as integrators of cellular decisionsof death, survival, and immune activation (24). For example,mitochondrial depolarization reflects a metabolic switch essentialfor T lymphocyte (50) and dendritic cell activation, allowingimmune cells to survive in hypoxic environments and undergorapid proliferation (38). Similarly, mitochondrial signaling isnecessary for macrophage activation and might even contributedirectly to microbicidal abilities (23). In our model, such a meta-bolic switch in vitro could be triggered by TREM1 (51), HIF-1a,or CD38 (52, 53), as suggested by the gene expression data, be-cause each of them can regulate mitochondrial activity and me-tabolism. It was suggested that a possible role of mitochondria isto produce ROS for activation of the inflammasome (24); however,our data do not support a role for mitochondrial ROS in C. neo-formans infection. Instead, our data suggest a role for NO in mito-

chondrial modulation (38, 54) where prolonged inhibition of NOattenuates mitochondrial depolarization. An added complexity tothe mitochondrial equation is that C. neoformans requires both theglycolytic pathway and the mitochondria for full virulence (55,56), and therefore mitochondrial functions are crucial for bothsides of host–pathogen interaction (57).Macrophage heterogeneity based on different anatomical sour-

ces (58) or culture conditions (59) is well established, a fact thathas been attributed to adaptations to their local microenvironment.Given this heterogeneity, we evaluated three macrophage types fortheir interaction with C. neoformans and observed that eachresponded differently. Our study did not investigate alveolarmacrophages owing to concerns about the relevance of this cell inhost defense. Although alveolar macrophages are presumably thefirst to come into contact with C. neoformans, their role in hostdefense in mice is unclear because their depletion reduces vul-nerability to infection (7). In fact, alveolar macrophages are ina minority in the infected lungs, as they become rapidly out-numbered by bone marrow (monocyte)-derived exudate macro-phages during the inflammatory response to infection. Consequently,exudate macrophages are the major subset of mononuclear effectorcells in the lungs (60), and therefore our studies focused onmacrophage types that resemble exudate macrophages. Finally,

FIGURE 6. Mitochondrial alterations do not correlate with oxidative burst but are mediated by NO. Peritoneal macrophages, but not J774.16 macro-

phage-like cells, produced ROS upon C. neoformans (Cn) infection. However, the pattern of ROS production did not follow the pattern of mitochondrial

depolarization, suggesting non–mitochondrial-derived ROS. As a positive control, we used rotenone (Rot), which blocks the mitochondrial respiratory

chain, resulting in accumulation of electrons, hyperpolarization, and an increase in cellular ROS. (A) J774.16 cells were infected for 24 h with either H99 or

an acapsular strain (cap59). (B) J774.16 cells were exposed to opsonized H2O2 killed C. neoformans or zymosan, resulting in mitochondrial hyperpo-

larization, but no ROS production. (C) Peritoneal cells were exposed to opsonized H2O2 killed C. neoformans or zymosan, resulting in Dwm decrease and

abundant ROS. (D) Addition of 500 mM of S-ethyl-iso-thiourea (SEITU) prevented mitochondrial depolarization in peritoneal macrophages and J774.16

cells (not shown). Total ROS were measured by CellROX Deep Red Reagent, and Dwm was measured by the ratio of Jc-1 red/green fluorescence, using flow

cytometry. Numbers represent percent of cells within each region. Shaded gray represents noninfected macrophages (No Cn), and each experimental

condition is shown by the red line. Each experiment was repeated twice for both J774.16 cells and peritoneal macrophages. Shown are plots of repre-

sentative experiments.

2354 C. NEOFORMANS MEDIATES MACROPHAGE DAMAGE

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

one must consider that virulence is a complex equation (61) andacknowledge that a limitation of this work is that we studied onlyone C. neoformans strain and that there may be strain-specificfactors that could produce strain-dependent outcomes in C. neo-formans–macrophage interactions (12). Given the observed het-erogeneity in responses of the host to the pathogen, we proposetwo scenarios to explain these results. Toxicity might have startedwith disruption of one cellular process that then cascaded to affectmultiple cell processes. For example, mitochondrial alterationscan potentiate ER stress (62) or, alternatively, mitochondrialalterations could be caused by deregulation of cyclin D1 (63). Inany case, our data provide multiple explanations for the previouslyreported cell cycle arrest upon C. neoformans ingestion (17).According to this view, the differences observed in activation ofprogrammed cell death pathways would reflect a heterogeneousresponse to C. neoformans–inflicted stress owing to cell type an-atomical origin, and the differences between J774.16 and the othermacrophages could reflect differences between an immortalizedcell line and primary cells. Alternatively, one might hypothesizethat C. neoformans causes damage to various cellular systems,which in turn results in multiple hits to the host cell. The absenceof a specific damage pathway is intriguing until one considersthat C. neoformans can infect a great variety of hosts, includingmammals, worms, insects, plants, and amoebae, and that itsvirulence for mammals may be accidental (64). In this regard,C. neoformans is very different from pathogenic microbes withnarrow host ranges, such as certain viruses that depend on onehost, which have evolved precise mechanisms to manipulate hostbiology and immune responses. In contrast, our results show thatC. neoformans inflicts damage to the cell by multiple mechanisms,and when this distinction is considered, such a cell pathogenicstrategy can be understood in the sense that it allows survival inmany different cell types. The fact that in contrast to macro-phages C. neoformans–infected amoebae did not manifest mito-chondrial depolarization is consistent with this view, which positsdifferent damage to different hosts. These scenarios are not mu-tually exclusive.In summary, our results establish that macrophages activate

several immune defense mechanisms upon C. neoformans infec-tion but are unable to effectively clear fungal infection in vitro.Residency of fungal cells in macrophages was associated witha progressive deterioration in host cellular functions. Specifically,C. neoformans ingestion activated several stress pathways, af-fected protein translation, and caused mitochondrial depolariza-

tion. Our results provide evidence for subtle cytopathic effects ofC. neoformans on macrophages that with time could progress toeventual cell death, as evidenced by the small percentage ofmacrophage deaths at 24 h. This damage could help the persis-tence of C. neoformans within macrophages, the inability to clearC. neoformans infection, and inefficient immune responses,translating into chronic infections. We established that mito-chondrial modulation is a significant factor in fungal infection ofmacrophages, as shown for bacterial infections (38, 65). Themultiple-hit intracellular survival strategy, illustrated in this arti-cle, provides a paradigm for understanding how pathogens, suchas C. neoformans, are able to infect such evolutionarily distanthosts, ranging from Kingdom Protozoa to Kingdom Animalia.

AcknowledgmentsWe thank Juliana Rizzo for help with amoeba experiments not included

in the final version of the manuscript; Dr. Jonathan W. Yewdell for help

with ribopuromycylation experiments; all personnel at the Analytical Im-

aging Facility—in particular, Vera DesMarais, Hillary Guzik, Ben Clark,

and Geoffrey Perumal—for technical assistance; and the Computational

Genomics Facility, Department of Genetics, Albert Einstein College of

Medicine, Bronx, New York, for help with data analysis.

DisclosuresThe authors have no financial conflicts of interest.

References1. Tucker, S. C., and A. Casadevall. 2002. Replication of Cryptococcus neoformans

in macrophages is accompanied by phagosomal permeabilization and accumu-lation of vesicles containing polysaccharide in the cytoplasm. Proc. Natl. Acad.Sci. USA 99: 3165–3170.

2. Osterholzer, J. J., J. E. Milam, G. H. Chen, G. B. Toews, G. B. Huffnagle, andM. A. Olszewski. 2009. Role of dendritic cells and alveolar macrophages inregulating early host defense against pulmonary infection with Cryptococcusneoformans. Infect. Immun. 77: 3749–3758.

3. Feldmesser, M., Y. Kress, P. Novikoff, and A. Casadevall. 2000. Cryptococcusneoformans is a facultative intracellular pathogen in murine pulmonary infec-tion. Infect. Immun. 68: 4225–4237.

4. Qin, Q. M., J. Luo, X. Lin, J. Pei, L. Li, T. A. Ficht, and P. de Figueiredo. 2011.Functional analysis of host factors that mediate the intracellular lifestyle ofCryptococcus neoformans. PLoS Pathog. 7: e1002078.

5. Gordon, S. 2003. Alternative activation of macrophages. Nat. Rev. Immunol.3: 23–35.

6. Brown, G. D. 2011. Innate antifungal immunity: the key role of phagocytes.Annu. Rev. Immunol. 29: 1–21.

7. Shao, X., A. Mednick, M. Alvarez, N. van Rooijen, A. Casadevall, andD. L. Goldman. 2005. An innate immune system cell is a major determinant ofspecies-related susceptibility differences to fungal pneumonia. J. Immunol. 175:3244–3251.

FIGURE 7. Intraperitoneal infection of mice with C.

neoformans (Cn) causes a decrease in mitochondrial

polarization in peritoneal total leukocytes and macro-

phages. C57/B6 mice were infected with C. neoformans

i.p., and after 24 h peritoneal lavage cells had decreased

Dwm. (A) Flow cytometry gating strategy for evaluat-

ing Dwm in peritoneal lavage CD45+ (total leukocytes)

and F4/80+ (macrophages) cells. Shaded area shows the

fluorescence minus one (F4/80 FMO) tube, used as

negative control. (B) Quantification of Dwm in perito-

neal lavage cells infected with C. neoformans for 24 h.

Jc-1 dye was injected in the peritoneum to measure

Dwm in vivo. Peritoneal lavage was performed, and

cells were immunostained for CD45 and F4/80 in

a flow cytometer. Experiments were performed twice,

and each data point represents 1 mouse, with a total of

6 mice in No Cn and 13 mice in Cn for both experi-

ments. Also shown are mean and SD. **p , 0.01 for

two-way ANOVA with Bonferroni correction.

The Journal of Immunology 2355

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

8. Goldman, D. L., S. C. Lee, A. J. Mednick, L. Montella, and A. Casadevall. 2000.Persistent Cryptococcus neoformans pulmonary infection in the rat is associatedwith intracellular parasitism, decreased inducible nitric oxide synthase expres-sion, and altered antibody responsiveness to cryptococcal polysaccharide. Infect.Immun. 68: 832–838.

9. Vecchiarelli, A., D. Pietrella, M. Dottorini, C. Monari, C. Retini, T. Todisco, andF. Bistoni. 1994. Encapsulation of Cryptococcus neoformans regulates fungicidalactivity and the antigen presentation process in human alveolar macrophages.Clin. Exp. Immunol. 98: 217–223.

10. Kechichian, T. B., J. Shea, and M. Del Poeta. 2007. Depletion of alveolarmacrophages decreases the dissemination of a glucosylceramide-deficient mu-tant of Cryptococcus neoformans in immunodeficient mice. Infect. Immun. 75:4792–4798.

11. Levitz, S. M., S. H. Nong, K. F. Seetoo, T. S. Harrison, R. A. Speizer, andE. R. Simons. 1999. Cryptococcus neoformans resides in an acidic phag-olysosome of human macrophages. Infect. Immun. 67: 885–890.

12. Alanio, A., M. Desnos-Ollivier, and F. Dromer. 2011. Dynamics of Cryptococcusneoformans-macrophage interactions reveal that fungal background influencesoutcome during cryptococcal meningoencephalitis in humans. MBio 2: 2.

13. Coelho, C., A. L. Bocca, and A. Casadevall. 2014. The tools for virulence ofCryptococcus neoformans. Adv. Appl. Microbiol. 87: 1–41.

14. Vecchiarelli, A., E. Pericolini, E. Gabrielli, S. Kenno, S. Perito, E. Cenci, andC. Monari. 2013. Elucidating the immunological function of the Cryptococcusneoformans capsule. Future Microbiol. 8: 1107–1116.

15. Luo, Y., and A. Casadevall. 2010. Intracellular cryptococci suppress Fc-mediatedcyclin D1 elevation. Commun. Integr. Biol. 3: 390–391.

16. Ben-Abdallah, M., A. Sturny-Leclere, P. Ave, A. Louise, F. Moyrand, F. Weih,G. Janbon, and S. Memet. 2012. Fungal-induced cell cycle impairment, chro-mosome instability and apoptosis via differential activation of NF-kB. PLoSPathog. 8: e1002555.

17. Coelho, C., L. Tesfa, J. Zhang, J. Rivera, T. Goncalves, and A. Casadevall. 2012.Analysis of cell cycle and replication of mouse macrophages after in vivo andin vitro Cryptococcus neoformans infection using laser scanning cytometry.Infect. Immun. 80: 1467–1478.

18. Vecchiarelli, A., M. Dottorini, D. Pietrella, C. Monari, C. Retini, T. Todisco, andF. Bistoni. 1994. Role of human alveolar macrophages as antigen-presentingcells in Cryptococcus neoformans infection. Am. J. Respir. Cell Mol. Biol. 11:130–137.

19. Retini, C., A. Vecchiarelli, C. Monari, F. Bistoni, and T. R. Kozel. 1998. En-capsulation of Cryptococcus neoformans with glucuronoxylomannan inhibits theantigen-presenting capacity of monocytes. Infect. Immun. 66: 664–669.

20. Alvarez, M., and A. Casadevall. 2007. Cell-to-cell spread and massive vacuoleformation after Cryptococcus neoformans infection of murine macrophages.BMC Immunol. 8: 16.

21. Thi, E. P., U. Lambertz, and N. E. Reiner. 2012. Sleeping with the enemy: howintracellular pathogens cope with a macrophage lifestyle. PLoS Pathog. 8:e1002551.

22. Bergsbaken, T., S. L. Fink, and B. T. Cookson. 2009. Pyroptosis: host cell deathand inflammation. Nat. Rev. Microbiol. 7: 99–109.

23. Wiese, M., R. G. Gerlach, I. Popp, J. Matuszak, M. Mahapatro, K. Castiglione,D. Chakravortty, C. Willam, M. Hensel, C. Bogdan, and J. Jantsch. 2012.Hypoxia-mediated impairment of the mitochondrial respiratory chain inhibits thebactericidal activity of macrophages. Infect. Immun. 80: 1455–1466.

24. Tschopp, J. 2011. Mitochondria: sovereign of inflammation? Eur. J. Immunol.41: 1196–1202.

25. Rudel, T., O. Kepp, and V. Kozjak-Pavlovic. 2010. Interactions between bacterialpathogens and mitochondrial cell death pathways. Nat. Rev. Microbiol. 8: 693–705.

26. Hwang, I.-Y., J. H. Quan, M.-H. Ahn, H. A. H. Ahmed, G.-H. Cha, D.-W. Shin,and Y.-H. Lee. 2010. Toxoplasma gondii infection inhibits the mitochondrialapoptosis through induction of Bcl-2 and HSP70. Parasitol. Res. 107: 1313–1321.

27. Damiani, G., C. Kiyotaki, W. Soeller, M. Sasada, J. Peisach, and B. R. Bloom.1980. Macrophage variants in oxygen metabolism. J. Exp. Med. 152: 808–822.

28. Casadevall, A., W. Cleare, M. Feldmesser, A. Glatman-Freedman,D. L. Goldman, T. R. Kozel, N. Lendvai, J. Mukherjee, L. A. Pirofski, J. Rivera,et al. 1998. Characterization of a murine monoclonal antibody to Cryptococcusneoformans polysaccharide that is a candidate for human therapeutic studies.Antimicrob. Agents Chemother. 42: 1437–1446.

29. Franceschini, A., D. Szklarczyk, S. Frankild, M. Kuhn, M. Simonovic, A. Roth,J. Lin, P. Minguez, P. Bork, C. von Mering, and L. J. Jensen. 2013. STRINGv9.1: protein-protein interaction networks, with increased coverage and inte-gration. Nucleic Acids Res. 41: D808–D815.

30. Theill, L. E., W. J. Boyle, and J. M. Penninger. 2002. RANK-L and RANK:T cells, bone loss, and mammalian evolution. Annu. Rev. Immunol. 20: 795–823.

31. Bosco, M. C., D. Pierobon, F. Blengio, F. Raggi, C. Vanni, M. Gattorno, A. Eva,F. Novelli, P. Cappello, M. Giovarelli, and L. Varesio. 2011. Hypoxia modulatesthe gene expression profile of immunoregulatory receptors in human maturedendritic cells: identification of TREM-1 as a novel hypoxic marker in vitro andin vivo. Blood 117: 2625–2639.

32. Buckland, K. F., H. Ramaprakash, L. A. Murray, K. J. Carpenter, E. S. Choi,S. L. Kunkel, N. W. Lukacs, Z. Xing, N. Aoki, D. Hartl, and C. M. Hogaboam.2011. Triggering receptor expressed on myeloid cells-1 (TREM-1) modulatesimmune responses to Aspergillus fumigatus during fungal asthma in mice.Immunol. Invest. 40: 692–722.

33. Arora, S., M. A. Olszewski, T. M. Tsang, R. A. McDonald, G. B. Toews, andG. B. Huffnagle. 2011. Effect of cytokine interplay on macrophage polarization

during chronic pulmonary infection with Cryptococcus neoformans. Infect.Immun. 79: 1915–1926.

34. Grahl, N., T. M. Dinamarco, S. D. Willger, G. H. Goldman, and R. A. Cramer.2012. Aspergillus fumigatus mitochondrial electron transport chain mediatesoxidative stress homeostasis, hypoxia responses and fungal pathogenesis. Mol.Microbiol. 84: 383–399.

35. Lischke, T., K. Heesch, V. Schumacher, M. Schneider, F. Haag, F. Koch-Nolte,and H.-W. Mittr€ucker. 2013. CD38 controls the innate immune response againstListeria monocytogenes. Infect. Immun. 81: 4091–4099.

36. David, A., B. P. Dolan, H. D. Hickman, J. J. Knowlton, G. Clavarino, P. Pierre,J. R. Bennink, and J. W. Yewdell. 2012. Nuclear translation visualized byribosome-bound nascent chain puromycylation. J. Cell Biol. 197: 45–57.

37. Perry, S. W., J. P. Norman, J. Barbieri, E. B. Brown, and H. A. Gelbard. 2011.Mitochondrial membrane potential probes and the proton gradient: a practicalusage guide. Biotechniques 50: 98–115.

38. Everts, B., E. Amiel, G. J. W. van der Windt, T. C. Freitas, R. Chott, K. E. Yarasheski,E. L. Pearce, and E. J. Pearce. 2012. Commitment to glycolysis sustains survival ofNO-producing inflammatory dendritic cells. Blood 120: 1422–1431.

39. Luo, Y., S. C. Tucker, and A. Casadevall. 2005. Fc- and complement-receptoractivation stimulates cell cycle progression of macrophage cells from G1 to S. J.Immunol. 174: 7226–7233.

40. Battegay, E. J., J. Rupp, L. Iruela-Arispe, E. H. Sage, and M. Pech. 1994. PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF beta-receptors. J. Cell Biol. 125: 917–928.

41. Wen, W., J. Ding, W. Sun, K. Wu, B. Ning, W. Gong, G. He, S. Huang, X. Ding,P. Yin, et al. 2010. Suppression of cyclin D1 by hypoxia-inducible factor-1 viadirect mechanism inhibits the proliferation and 5-fluorouracil-induced apoptosisof A549 cells. Cancer Res. 70: 2010–2019.

42. Zimmermann, M., A. S. Arachchige-Don, M. S. Donaldson, R. F. Dallapiazza,C. E. Cowan, and M. C. Horne. 2012. Elevated cyclin G2 expression intersectswith DNA damage checkpoint signaling and is required for a potent G2/Mcheckpoint arrest response to doxorubicin. J. Biol. Chem. 287: 22838–22853.

43. Williams, B. R. 1999. PKR; a sentinel kinase for cellular stress. Oncogene 18:6112–6120.

44. Chakrabarti, S., P. Liehl, N. Buchon, and B. Lemaitre. 2012. Infection-inducedhost translational blockage inhibits immune responses and epithelial renewal inthe Drosophila gut. Cell Host Microbe 12: 60–70.

45. Mohr, I., and N. Sonenberg. 2012. Host translation at the nexus of infection andimmunity. Cell Host Microbe 12: 470–483.

46. Davis, M. J., A. J. Eastman, N. Qiu, B. Gregorka, T. R. Kozel, J. J. Osterholzer,J. L. Curtis, J. A. Swanson, and M. A. Olszewski. 2015. Cryptococcus neofor-mans–induced macrophage lysosome damage crucially contributes to fungalvirulence. J. Immunol. 194: 2219–2231.

47. Coelho, C., A. L. Bocca, and A. Casadevall. 2014. The intracellular life ofCryptococcus neoformans. Annu. Rev. Pathol. 9: 219–238.

48. Koterski, J. F., M. Nahvi, M. M. Venkatesan, and B. Haimovich. 2005. VirulentShigella flexneri causes damage to mitochondria and triggers necrosis in infectedhuman monocyte-derived macrophages. Infect. Immun. 73: 504–513.

49. Jamwal, S., M. K. Midha, H. N. Verma, A. Basu, K. V. S. Rao, and V. Manivel.2013. Characterizing virulence-specific perturbations in the mitochondrial functionof macrophages infected with Mycobacterium tuberculosis. Sci Rep 3: 1328.

50. van der Windt, G. J. W., D. O’Sullivan, B. Everts, S. C.-C. Huang, M. D. Buck,J. D. Curtis, C.-H. Chang, A. M. Smith, T. Ai, B. Faubert, et al. 2013. CD8memory T cells have a bioenergetic advantage that underlies their rapid recallability. Proc. Natl. Acad. Sci. USA 110: 14336–14341.

51. Syed, M., D. Panchal, M. Joo, and M. Colonna. 2013. Trem-1 modulates ex-pression of mitofusins and promotes macrophage survival. Am. J. Respir. Crit.Care Med. 187: A5220 (Abstr.).

52. Frases, S., B. Pontes, L. Nimrichter, N. B. Viana, M. L. Rodrigues, andA. Casadevall. 2009. Capsule of Cryptococcus neoformans grows by enlarge-ment of polysaccharide molecules. Proc. Natl. Acad. Sci. USA 106: 1228–1233.

53. Semenza, G. L. 2011. Hypoxia-inducible factor 1: regulator of mitochondrialmetabolism and mediator of ischemic preconditioning. Biochim. Biophys. Acta1813: 1263–1268.

54. Beltran, B., M. Quintero, E. Garcıa-Zaragoza, E. O’Connor, J. V. Esplugues, andS. Moncada. 2002. Inhibition of mitochondrial respiration by endogenous nitricoxide: a critical step in Fas signaling. Proc. Natl. Acad. Sci. USA 99: 8892–8897.

55. Ma, H., and R. C. May. 2010. Mitochondria and the regulation of hypervirulencein the fatal fungal outbreak on Vancouver Island. Virulence 1: 197–201.

56. Price, M. S., M. Betancourt-Quiroz, J. L. Price, D. L. Toffaletti, H. Vora, G. Hu,J. W. Kronstad, and J. R. Perfect. 2011. Cryptococcus neoformans requiresa functional glycolytic pathway for disease but not persistence in the host. MBio2: e00103–e00111, e00103–e00111.

57. Grahl, N., K. M. Shepardson, D. Chung, and R. A. Cramer. 2012. Hypoxia andfungal pathogenesis: to air or not to air? Eukaryot. Cell 11: 560–570.

58. Schaffner, A., H. Douglas, A. I. Braude, and C. E. Davis. 1983. Killing of As-pergillus spores depends on the anatomical source of the macrophage. Infect.Immun. 42: 1109–1115.

59. Vogt, G., and C. Nathan. 2011. In vitro differentiation of human macrophageswith enhanced antimycobacterial activity. J. Clin. Invest. 121: 3889–3901.

60. Osterholzer, J. J., G.-H. Chen, M. A. Olszewski, Y.-M. Zhang, J. L. Curtis,G. B. Huffnagle, and G. B. Toews. 2011. Chemokine receptor 2-mediated ac-cumulation of fungicidal exudate macrophages in mice that clear cryptococcallung infection. Am. J. Pathol. 178: 198–211.

61. Casadevall, A., and L. A. Pirofski. 1999. Host-pathogen interactions: rede-fining the basic concepts of virulence and pathogenicity. Infect. Immun. 67:3703–3713.

2356 C. NEOFORMANS MEDIATES MACROPHAGE DAMAGE

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

62. Stavru, F., A. E. Palmer, C. Wang, R. J. Youle, and P. Cossart. 2013. Atypicalmitochondrial fission upon bacterial infection. Proc. Natl. Acad. Sci. USA 110:16003–16008.

63. Sakamaki, T., M. C. Casimiro, X. Ju, A. A. Quong, S. Katiyar, M. Liu, X. Jiao,A. Li, X. Zhang, Y. Lu, et al. 2006. Cyclin D1 determines mitochondrial functionin vivo. Mol. Cell. Biol. 26: 5449–5469.

64. Casadevall, A., and L. A. Pirofski. 2007. Accidental virulence, cryptic patho-genesis, martians, lost hosts, and the pathogenicity of environmental microbes.Eukaryot. Cell 6: 2169–2174.

65. Stavru, F., F. Bouillaud, A. Sartori, D. Ricquier, and P. Cossart. 2011. Listeriamonocytogenes transiently alters mitochondrial dynamics during infection. Proc.Natl. Acad. Sci. USA 108: 3612–3617.

The Journal of Immunology 2357

by guest on April 4, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

Corrections

Coelho, C., A. C. Oliveira Souza, L. da Silveira Derengowski, C. de Leon-Rodriguez, B. Wang, R. Leon-Rivera, A. Lorenzetti Bocca,T. Goncalves, and A. Casadevall. 2015. Macrophage mitochondrial and stress response to ingestion of Cryptococcus neoformans. J. Immunol.194: 2345–2357.

The author list for reference 51 was incomplete as published. The corrected reference 51 is shown below.

51. Yuan, Z., M. Syed, D. Panchal, M. Joo, M. Colonna, and R. T. Sadikot. 2013. Trem-1 modulates expression of mitofusins and promotes macrophage survival. Am. J. Respir.Crit. Care Med. 187: A5220 (Abstr.).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700948

Copyright � 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00

1944 The Journal of Immunology