Transcriptome profile of the murine macrophage cell response to Candida parapsilosis

Fungal Genetics and Biology 65 (2014) 48–56

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Transcriptome profile of the murine macrophage cell response to Candida parapsilosis Tibor Németh a,1, Adél Tóth a,1, Zsuzsanna Hamari a, András Falus b, Katalin Éder b, Csaba Vágvölgyi a, Allan J. Guimaraes c, Joshua D. Nosanchuk c, Attila Gácser a,⇑ a b c

Department of Microbiology, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary Department of Genetics, Cell- and Immunobiology, Semmelweis University, Nagyvárad tér 4, 1089 Budapest, Hungary Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Ave, New York, NY 10461, USA

a r t i c l e

i n f o

Article history: Received 22 October 2013 Accepted 29 January 2014 Available online 13 February 2014 Keywords: Candida parapsilosis Infection Host response Macrophages

a b s t r a c t Candida parapsilosis is a human fungal pathogen with increasing global significance. Understanding how macrophages respond to C. parapsilosis at the molecular level will facilitate the development of novel therapeutic paradigms. The complex response of murine macrophages to infection with C. parapsilosis was investigated at the level of gene expression using an Agilent mouse microarray. We identified 155 and 511 differentially regulated genes at 3 and 8 h post-infection, respectively. Most of the upregulated genes encoded molecules involved in immune response and inflammation, transcription, signaling, apoptosis, cell cycle, electron transport and cell adhesion. Typical of the classically activated macrophages, there was significant upregulation of genes coordinating the production of inflammatory cytokines such as TNF, IL-1 and IL-15. Further, we used both primary murine macrophages and macrophages differentiated from human peripheral mononuclear cells to confirm the upregulation of the TNF-receptor family member TNFRSF9 that is associated with Th1 T-helper cell responses. Additionally, the microarray data indicate significant differences between the response to C. parapsilosis infection and that of C. albicans. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Candida parapsilosis has recently emerged as a major human pathogen (Pfaller et al., 2010; Trofa et al., 2008; van Asbeck et al., 2009). Despite the increasing clinical importance, little is known about the host response to C. parapsilosis infection. However, defining the interactions between a host cell and C. parapsilosis is crucial to understand the molecular details of pathogenesis. In contrast to the massive amount of work that has been described on host defense against Candida albicans, there is a paucity of information about the immune response against C. parapsilosis. C. parapsilosisis typically a commensal of human skin, and its pathogenicity is limited by intact integument. However, the species is known for its capacity to form biofilms on catheters and other implanted devices, for nosocomial spread by hand carriage, and for persistence in the hospital environment (Trofa et al., 2008). In fact, it is now the second or third most commonly isolated Candida species from blood cultures worldwide (Cisterna et al., 2010; Horasan et al.,

⇑ Corresponding author. Fax: +36 62544823. 1

E-mail address: [email protected] (A. Gácser). T.N. and A.T. contributed equally to this work.

http://dx.doi.org/10.1016/j.fgb.2014.01.006 1087-1845/Ó 2014 Elsevier Inc. All rights reserved.

2010; Peman et al., 2005; Tortorano et al., 2011). C. parapsilosis is of special concern in critically ill neonates (reviewed by Pammi et al. (2013)), causing more than one-quarter of all invasive fungal infections in low-birth-weight infants in the United Kingdom and up to one-third of neonatal Candida bloodstream infections in North America (Benjamin et al., 2003; Clerihew et al., 2007; Neu et al., 2009; Smith et al., 2005). Moreover, it even outranks C. albicans infections in some European hospitals (Montagna et al., 2010). Macrophages are critical cells of the innate immune system, contributing to immediate and robust defense against microbial infections. These cells produce an intricate pattern of cytokines and chemokines that enhance chemotaxis, phagocytosis and microbicidal activity; as well as activate T cells through antigen processing and presentation (Bourgeois et al., 2010; Seider et al., 2010). The pivotal role of macrophages in immune response during candidiasis explains the large number of studies dealing with this interaction, primarily focusing on C. albicans. These analyses use different approaches to address the complex host response to C. albicans, including human and mouse macrophage-like cell lines (Barker et al., 2005; Heidenreich et al., 1996; Kim et al., 2005; Marcil et al., 2002), as well as primary human monocytes (Cummings and Relman, 2000; Kim et al., 2005; Manger and Relman, 2000). In sharp contrast, little is known about the

T. Németh et al. / Fungal Genetics and Biology 65 (2014) 48–56

regulation and coordinated expression of genes in macrophages involved in the host response to C. parapsilosis. In fact, the significant differences in pathobiology and virulence of C. parapsilosis in different patient groups indicate that effective immune responses exists and suggest that they can be different from responses occurring in interactions with C. albicans (Trofa et al., 2008; van Asbeck et al., 2009). In this study, we analyzed the transcriptional responses of murine J774.2 macrophage-like cells to C. parapsilosis infection. It is well known that J774.2 cells are able to efficiently phagocytose and kill both C. albicans and C. parapsilosis (Bertini et al., 2013; Dementhon et al., 2012; Lewis et al., 2012; Tibor Németh et al., 2013). As expected, many genes known to be responsive to C. albicans were up-regulated upon C. parapsilosis infection. Some of these genes were chosen for quantitative real-time PCR (RT-qPCR) analysis to validate the results obtained by microarray. In addition, further analysis using primary murine macrophages and macrophages derived from human peripheral blood mononuclear cells (PBMCs) was performed on the tumor necrosis factor receptor superfamily member 9 (TNFRSF9) gene to confirm that it was highly upregulated, as it had not previously been associated with host responses to Candida infections.

2. Material and methods 2.1. Fungal strains and culture conditions Candida parapsilosis GA1 (Gacser et al., 2007), Candida glabrata CBS 138, Candida albicans ATCC 90028, Candida guilliermondii CBS 566, Candida krusei CBS 573, Candida tropicalis CBS 94, Candida metapsilosis SZMC (Szeged Microbiological Collection) 1548, and Candida orthopsilosis SZMC 1545 were maintained at 80 °C in 35% glycerol. The cells were grown in YPD (1% yeast extract, 2% bactopeptone, 2% glucose). For infection, Candida cells were grown overnight at 30 or 37 °C. Yeast cells in log-phase were washed three times in sterile phosphate-buffered saline (PBS) and counted using a hemacytometer. 2.2. J774.2 cell cultivation The murine macrophage cell line J774.2 (BALB/c) was maintained in DMEM medium (Lonza) supplemented with 10% heat-inactivated fetal bovine serum (Lonza) and 1% 100x Penicillin–Streptomycin solution (Sigma). Macrophages were incubated under the following conditions: 37 °C, 5 v/v% CO2 and 100% relative humidity. J774.2 cells were plated at 5  106 cells per well in six-well plates for RNA isolation, or 5  105 cells on tissue culture coverslips placed into a 24-well plate for scanning electron microscopy (SEM). The J774.2 cells were co-incubated at an effector-to-target ratio of 1:5 with C. parapsilosis cells.

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2.4. Human PBMC isolation and macrophage differentiation Peripherial Blood Mononuclear Cells (PBMCs) were isolated from buffy coat blood sample deriving from a healthy donors by using Ficoll-Paque Plus (GE Healthcare) density gradient centrifugation. Experiments were performed according to the institutional regulation of the independent ethics committee of the University of Szeged. Cells were suspended in RPMI medium (Lonza) containing 10% heat-inactivated human serum (Lonza) and 1% Penicillin– Strepromycin solution (Sigma). Monocytes were allowed to adhere to the surface of the plate for two hours then carefully washed with pre-warmed PBS. They were incubated for five days in the same medium under the conditions mentioned above and allowed to differentiate into macrophages. Differentiated cells were co-incubated at an effector-to-target ratio of 1:5 with C. parapsilosis cells. 2.5. Phagocytosis assay (quantitative imaging) For the analysis of phagocytosis by quantitative imaging, yeast cells were labeled with the fluorescent dye Alexa Fluor 647 carboxylic acid, succinimidyl ester (Invitrogen) as described (Tibor Németh et al., 2013). J774.2 macrophages were co-cultured with the labeled Candida cells in 12-well plastic cell culture plates at an effector:target ratio of 1:5 for 15, 30, 60 or 120 min to allow phagocytosis. Macrophages were washed extensively with PBS after the incubation period in order to eliminate non-phagocytosed Candida cells. Afterwards, macrophages were gently suspended to a single cell suspension by pipetting, harvested by centrifugation, suspended in 50 lL FACS buffer (0.5% FBS in PBS) and measured on a FlowSight instrument (Amnis). Data were analyzed using IDEAS Software (Amnis). 2.6. Scanning electron microscopy J774.2 and C. parapsilosis co-incubation was performed on coverslips (Sarstedt) in a 24 well-plate at 37 °C and 5% CO2 for 1 h. Cells were washed twice with PBS, fixed and dried as described by Van de Velde et al. (2010) with the following modifications. Samples were fixed with 2.5% glutaraldehyde in Sorenson-buffer (pH = 7.5) instead of cacodylate-buffer overnight at 4 °C. The samples were serially dehydrated in 50% ethanol (2  15 min on ice), 70% ethanol (2  15 min on ice), 80% ethanol (2  15 min on ice), 90% ethanol (2  15 min on ice), 95% ethanol (2  15 min on ice), and then absolute ethanol (2  15 min on ice). Samples were then held in tert-butyl alcohol:absolute ethanol 1:2, 1:1, 2:1 one after the other for 1 h each at room temperature. Then 100% tert-butyl alcohol was applied for 1 h at room temperature. Finally, the coverslips were frozen in 100% tert-butyl alcohol at 4 °C, freeze dryed overnight, and fixed on aluminum stubs with double adhesive carbon tapes. Samples were coated with gold in a Quorum Technologies SC 7620 ‘Mini’ sputter coater, and observed by using a Hitachi S-4700 scanning electron microscope.

2.3. Isolation of murine peritoneal macrophages 2.7. Acridine orange/crystal violet staining Peritoneal macrophages were harvested from euthanized BALB/c mice by peritoneal lavage by using 10 ml of sterile ice cold PBS. The cells were collected (120 g, 10 min), suspended in ACK lysis buffer and incubated for 10 min on ice to eliminate red blood cells. The suspension was centrifuged again (120 g, 10 min) and the pellet was suspended in 37 °C DMEM medium (Lonza) supplemented with 10% heat-inactivated FBS and 1% Penicillin–Streptomycin solution. Cells were counted by using a hemocytometer and plated out into 12-well plates. They were allowed to adhere for one hour, then washed with pre-warmed PBS and cultured in DMEM medium. Cells were incubated as described above and co-incubated at an effector-to-target ratio of 1:5 with C. parapsilosis cells.

The staining was performed based on the protocol of Miliotis (Miliotis, 1991) with slight modifications. J774.2 and C. parapsilosis co-incubation was performed on coverslips (Sarstedt) in a 24 wellplate. Samples were stained for 30 s in 0.01% acridine orange solution recovered in PBS, and then washed three times in PBS for 30 s. To quench the extracellular fluorescence 0.15 M crystal violet dissolved in PBS was applied for 30 s, then the dye was removed and the samples were washed three times in PBS. Coverslips were mounted on slides in PBS by using colorless nail polish. The staining protocol was performed at room temperature in dark. Samples were observed by using Olympus DP-72 fluorescent microscope.

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2.8. Lysosome–phagosome co-localization assay The co-localization of lysosomes and phagocytosed fungi was examined using fluorescently labeled yeast cells and Lysotracker Red fluorescent dye (Life Technologies). Yeast cells were incubated overnight, washed two times with PBS, suspended in 2 ml FITC-buffer (100 mMNaCl, 50 mM NaHCO3) supplemented with FITC (Fluorescein-isothiocyanate) in a final concentration of 25 lg/ml and then incubated in the dark overnight at 30 °C in an orbital shaker (200 rpm). Before use, the cells were washed four times with PBS to completely remove all the unbounded dye. To examine lysosome maturation and co-localization, macrophages were plated on coverslips placed in a 24-well plate. After incubation, the cells were washed once with pre-warmed PBS, then a pre-warmed 50 nM Lysotracker Red working solution was added. The cells were incubated for another 2 h under the same culture conditions. The Lysotracker Red solution was then removed, the cells were washed two times with pre-warmed PBS, and the coverslips were mounted onto a clean slide using PBS as a mounting medium. The borders of the coverslips were sealed with nail polish and the samples were examined using an Olympus confocal LSM. 2.9. Flow cytometry analysis C. parapsilosis yeast cells were grown in YPD and washed three times in PBS. J774.2 macrophage cells were placed in a 6 wells plate in DMEM medium at a 2  105 macrophages/well and incubated overnight at 37 °C/5% CO2. The next day, a ratio of 5 C. parapsilosis cells were added to one macrophage considering the doubling time of the macrophages in culture (4  106 C. parapsilosis yeast). Uninfected macrophages, and macrophages obtained after 0, 12, 24 36 and 48 h post-infection were washed, fixed with a 4% paraformaldehyde solution and incubated with a 1:500 dilution in PBS of a PE anti-mouse CD137 (Biolegend Inc., CA, USA) for 1 h at room temperature. Cells were washed and fluorescence intensity analyzed in a FACS can Flow Cytometer. 2.10. Microarray analysis J774.2 cells were co-incubated with C. parapsilosis cells at a ratio of 1:5 for 3 or 8 h. Following incubation, cells were harvested and total RNA was extracted using the RNeasy Mini Kits (QIAGEN) according to the manufacturer’s instructions. Experiments were performed in triplicate and samples were pooled before microarray analysis. One lg quality-checked total RNA was reverse transcribed by the QickAmp Labeling Kit (Agilent Technologies, Palo Alto, CA) and then transcribed to Cy3-labeled cRNA according to the manufacturer. The labeled cRNA was purified (RNeasy kit, Qiagen, Valencia, CA), and the dye content (>9.0 pmol dye/g cRNA) and concentration of cRNA were measured by NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). For each sample, 1.65 g of Cy3-labeled cRNA was hybridized to Whole Mouse Genome (4x44K) Oligo (Agilent Technologies, Palo Alto, CA) microarrays at 65 °C for 17 h, then the slides were washed and treated with Stabilizing and Drying Solution (Agilent Technologies, Palo Alto, CA) and scanned with a Agilent Microarray Scanner. All steps were carried out according to the manufacturer (Agilent Technologies, Palo Alto, CA). Array analysis was performed in triplicate. Data were normalized by the Feature Extraction software version 10.5.1.1 with default parameter settings for Agilent one-color microarrays and then transferred to GeneSpringGX program (Agilent Technologies, Palo Alto, CA) for further statistical evaluation. Normalization and data transformation steps recommended by Agilent Technologies for one-color data were applied. Gene expressions were normalized to the corresponding control

at each time point. Genes with a P2-fold expression change were defined as up- or down-regulated. Differentially expressed genes for each condition were analyzed to identify significantly overrepresented gene ontology pathways using Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatic Resources 2007 (Huang da et al., 2009). 2.11. cDNA synthesis and RT-qPCR RT-qPCR was used to verify the selected macrophage genes in response to 3, 6 8, 12, 24 h of infection with C. parapsilosis. Similarly, RT-qPCR was used to assess TNFRSF9 expression in response to different Candida species at 6 and 12 h. cDNA was synthesized from macrophage RNA treated with DNaseI Fermentas RevertAid™ First Strand cDNA Synthesis Kit according to the manufacturer’s instructions. Reverse transcriptase reactions contained approximately 1–1 lg of RNA samples. RT-qPCR was performed using Fermentas MaximaÒ SYBR Green/Fluorescein qPCR Master Mix (2x) for the selected genes according to the manufacturer’s protocol. Fold changes were calculated using the 2-DDCT method, in which expression in each infection condition was compared to expression in the non-infection condition sample by using b-actin (mouse) and beta-2 microglobulin (human) as a housekeeping endogenous control. All the primers were obtained from Sigma–Aldrich, and their sequences were as follows: Mouse Actb For Actb Rev CD-83 For CD-83 Rev IL1b For IL1b Rev IL15 For IL15 Rev PTGS-2 For PTGS-2 Rev TNFa For TNFa Rev Tnfrsf-9 For Tnfrsf-9 Rev

50 -ACAGCTTCTTTGCAGCTCCTTCG-30 50 -ATCGTCATCCATGGCGAACTGGTG-30 50 -TGGCAACTCTACTGGGCTGTTAC-30 50 -ATGACAGGCATTCGCTCAGCTC-30 50 -CCTGTGTAATGAAAGACGGCACAC-30 50 -ATTGCTTGGGATCCACACTCTCC-30 50 -ATAACCAGCCTACAGGAGGCCAAG-30 50 -AGATGAGCTGGCTATGGCGATG-30 50 -AGCCAGGCAGCAAATCCTTG-30 50 -ACTGGTCAAATCCTGTGCTCATAC-30 50 -AAGATGCTGGGACAGTGACCTG-30 50 -AGGCTCCAGTGAATTCGGAAAGC-30 50 -CGTGTGTGTGTGTGTGTGTGTG-30 50 -ACCAACCCTTTCTCTTCTGACCTC-30

Human B2mg For B2mg Rev Tnfrsf-9 For Tnfrsf-9 Rev

50 -CCGTGTGAACCATGTGACTTTGTC-30 50 -GCTGCTTACATGTCTCGATCCC-30 50 -TCTGTCGACCCTGGACAAACTG-30 50 -CTCCTTCGTCCCATTCACAAGC-30

2.12. Statistical analysis The significance of differences between datasets was determined by unpaired t-test using GraphPad Prism 6 software. Differences were considered statistically significant at p < 0.05. 3. Results 3.1. Phagocytosis of C. parapsilosis cells by J774.2 murine macrophages To investigate the interactions between C. parapsilosis cells and J774.2 macrophages, we first examined the course of phagocytosis using quantitative imaging and fluorescence as well as scanning electron microscopic analysis (Figs. 1 and 2). Using quantitative imaging flow cytometry, we found that phagocytic events could be detected within 30 min of incubation, and the

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Fig. 1. Quantitative imaging of the kinetics of the phagocytosis of C. parapsilosis cells by J774.2 macrophages. J774.2 macrophages were co-incubated with labeled C. parapsilosis cells at an effector/target ratio of 1:5 for the indicated time periods and phagocytosis was assessed by quantitative imaging flow cytometry. R2: phagocytosing macrophage population discriminated by the presence of red fluorescence due to ingestion of Alexa Fluor 647-labeled yeast cells.

rate of yeast cell engulfment most significantly increased between 1 and 2 h of co-incubation (Fig. 1). There was little increase in phagocytosis after 3 h. We also examined the co-localization of phagosomes–lysosomes in macrophages by confocal microscopy, and found that the yeast cell containing phagosomes are not associated with lysosomes at 3 h and that they are co-localized by 8 h (Fig. 2). Moreover, imaging of co-cultures stained with acridin orange dye, which allows the differentiation of live and dead cells, revealed that the majority of intracellular Candida cells were alive at 3 h post-infection, but killed by 8 h (Fig. 2). Based on these results, we decided to perform the microarray analysis at two different time points: 3 h post-infection, when phagocytosis had already taken place but the phagosome maturation is in an early stage; and 8 h post-infection, when the molecular machinery involved in intracellular killing is fully activated in the macrophages. 3.2. Microarray analysis of the transcriptome of C. parapsilosisstimulated J774.2 macrophages The analysis of the transcriptomes of C. parapsilosis-stimulated macrophages as well as non-stimulated cells revealed 155 and 511 differentially expressed genes at the 3 and 8 h co-culture interval, respectively (Supplementary Tables 1 and 2). At 3 h, 117 genes were up-regulated and 38 genes were down-regulated relative to macrophages in the absence of C. parapsilosis. In contrast, 273 genes were highly expressed after 8 h of infection and 238 showed lower expression in comparison to control macrophages. There were only 75 genes which were up-regulated and 23 genes which were down-regulated at both time points. To assess the biological significance of the differentially expressed genes, we determined their Gene Ontology (GO) terms (Huang da et al., 2009). Differentially regulated genes in response to C. parapsilosis at both the 3

and 8 h intervals were enriched in GO terms especially pertaining to responses to stress, inflammation, and chemokine and cytokine activity (Table 1.), with double the number of these GO terms at 8 h compared to 3 h. Notably, genes encoding molecules involved in immune responses – including toll-like receptors (TLRs), cytokines and cytokine receptors – were highly expressed in response to C. parapsilosis (Tables 2–4). 3.3. Validation of microarray results using RT-qPCR To validate the results of microarray analysis, RT-qPCR was used to examine 5 host genes with significant roles in immune responses that were upregulated by C. parapsilosis infection (Fig. 3). For each of the 5 genes, the results of RT-qPCR correlated with those of the microarray analysis. Although IL1B failed to reach the 2-fold difference in microarray analysis, its expression was also examined by RT-qPCR as this cytokine is known to have an important role in host defense during Candida infections. The inflammatory cytokine genes tumor necrosis factor alpha (TNFA), interleukin 1 beta (IL1B) and interleukin 15 (IL15) were up-regulated by 3 h in C. parapsilosis-stimulated macrophages (mean fold change ± S.D., 5.26 ± 1.642, 3.467 ± 1.890 and 7.947 ± 2.501, respectively; TNFA, p < 0.05, IL1B, p = 0.0866, IL15, p < 0.01). After 8 h of stimulation, these genes remained over-expressed, although to a lower extent (3.280 ± 0.4093, 2.243 ± 0.1793 and 2.990 ± 0.2946, respectively, compared to the non-infected control; p < 0.001). The regulation of the CD83 gene showed a similar pattern, being highly expressed after 3 h (mean fold change ± S.D., 45.24 ± 13.76, p < 0.01) and 8 h (4.350 ± 0.8316, p < 0.01) of stimulation. Notably, the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) gene was higher after 8 h (14.26 ± 3.431, p < 0.01) than after 3 h (6.180 ± 0.8697, p < 0.001). Of special interest was our finding that the gene of the co-stimulatory molecule tumor necrosis factor receptor

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Fig. 2. J774.2 murine macrophages phagocytose and kill Candida parapsilosis yeast cells. (A–C) Scanning electron microscopic images taken after one hour of co-incubation demonstrating macrophages in the process of ingesting C. parapsilosis. White arrow (Image C) indicates a Candida cell partially ingested by the macrophage. (D and E) Acridine orange/crystal violet (AO/CV) staining differentiates between live and dead yeast cells by dyeing them green or red, respectively. Green arrows show live yeast cells inside the phagocytes at 3 h of co-incubation (Image D). In contrast, yeast cells are stained red at 8 h post-infection (Image E – red arrows). (F and G) Formation of lysosomes discriminated by LysoTracker Red staining after uptake of FITC-labeled C. parapsilosis cells. After 3 h of co-incubation, ingested yeast cells are not associated with the lysosomes (F, black arrows). At 8 h post-infection, a strong co-localization can be observed between the lysosomes and ingested Candida cells (G, yellow arrows). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1 Gene Ontology (GO) analysis of J774.2 macrophage cell genes differentially expressed in response to C. parapsilosis after 3 h and 8 h. GO ID

TERM

p

No

3h GO:0002376 GO:0016265 GO:0050896 GO:0065007 GO:0009987 GO:0032502

Immune system process Death Response to stimulus Biological regulation Cellular process Developmental process

<0.001 0.001 0.01 0.04 0.04 0.04

17 12 28 61 76 27

8h GO:0006950 GO:0009611 GO:0006954 GO:0042379 GO:0008009 GO:0007049 GO:0009605 GO:0005125 GO:0001664 GO:0006952 GO:0048583 GO:0002376 GO:0050776 GO:0006955

Response to stress Response to wounding Inflammatory response Chemokine receptor binding Chemokine activity Cell cycle Response to external stimulus Cytokine activity G-protein-coupled receptor binding Defense response Regulation of response to stimulus Immune system process Regulation of immune response Immune response

<0.001 <0.001 <0.001 0.001 0.001 0.001 0.003 0.006 0.01 0.02 0.04 0.04 0.06 0.06

19 14 14 9 9 24 14 16 9 18 2 19 2 19

superfamily member 9 (TNFRSF9) was up-regulated both at 3 and 8 h post-infection (mean fold change ± S.D. at 3 h, 6.697 ± 4.593, p = 0.0982; 8 h, 7.777 ± 0.7100, p < 0.0001).

3.4. Expression of TNFRSF9 in J774.2 mouse macrophages induced by different Candida species To examine whether the up-regulation of TNFRSF9 in host cells is a response induced specifically by C. parapsilosis, we investigated the expression of the gene upon stimulation by several other pathogenic Candida species (Fig. 4). Interestingly, by 6 h after co-infection only C. parapsilosis and C. metapsilosis increased TNFRSF9 expression in J774.2 cells. However, by 12 h the amount of TNFRSF9 mRNA was significantly increased with all the Candida spp. used (C. albicans, C. glabrata, C. guilliermondii, C. krusei, C. metapsilosis, C. orthopsilosis, C. parapsilosis and C. tropicalis). However, C. parapsilosis induced the highest increase in TNFRSF9 expression (mean ± S.D., 108.9 ± 27.17-fold change).

3.5. Analysis of TNFRSF9 expression on the surface of host cells by flow cytometry Since the expression of TNFRSF9 was up-regulated at the transcriptional level, we examined the expression of the protein on the surface of macrophages. Flow cytometric analysis showed that the TNFRSF9 protein was highly expressed after 12 h co-culture of C. parapsilosis with the macrophages compared to non-stimulated cells (Fig. 5A). Interestingly, the protein concentration on the surface of infected macrophages was even higher at 24 and 36 h (Fig. 5B). These results suggest that this co-stimulatory molecule may play an important role in immune response upon C. parapsilosis infection.

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T. Németh et al. / Fungal Genetics and Biology 65 (2014) 48–56 Table 2 Genes involved in immune responses that were differentially expressed upon C. parapsilosis infection. Gene

Gene product

Fold change

TNFRSF9 IL1RN CD83 CCLR2 TNFAIP3 IL7R CXCL2 CCL7 PTGS2 CD86 TRAF1 IL12b

Tumor necrosis factor receptor superfamily, member 9 Interleukin 1 receptor antagonist (Il1rn), transcript variant 1 CD83 antigen (Cd83) Chemokine (C-C motif) receptor-like 2 Tumor necrosis factor, alpha-induced protein 3 Interleukin 7 receptor Chemokine (C-X-C motif) ligand 2 Chemokine (C-C motif) ligand 7 Prostaglandin-endoperoxide synthase 2 CD86 antigen (Cd86) Tnf receptor-associated factor 1 Interleukin 12b (Il12b)

Table 3 Cytokine–cytokine receptor interaction. GeneBank accession NM_013652 NM_011333 NM_007778 NM_008352 NM_019568 NM_011610 NM_011337 NM_013693 NM_013653 NM_008359 NM_009140 NM_011331 NM_008357 NM_011612 NM_013654 NM_008372 NM_144547 NM_033622

Gene name

Chemokine (c-c motif) ligand 4 Chemokine (c-c motif) ligand 2 Colony stimulating factor 1 (macrophage) Interleukin 12b Chemokine (c-x-c motif) ligand 14 Tumor necrosis factor receptor superfamily, member 1b Chemokine (c-c motif) ligand 3 Tumor necrosis factor Chemokine (c-c motif) ligand 5 Interleukin 17 receptor Chemokine (c-x-c motif) ligand 2 Chemokine (c-c motif) ligand 12 Interleukin 15 Tumor necrosis factor receptor superfamily, member 9 Chemokine (c-c motif) ligand 7 interleukin 7 receptor Anti-mullerian hormone type 2 receptor Tumor necrosis factor (ligand) superfamily, member 13b

3h

8h

8.98 9.00 8.27 5.74 5.16 4.64 nd 3.12 nd 2.95 2.79 nd

39.34 nd 2.81 3.78 3.84 5.40 4.72 14.5 7.11 nd 2.14 13.62

Table 4 Toll-like receptor signaling pathway. Fold change 3h

8h

nd nd 2.36 nd nd nd

3.35 8.33 2.12 13.62 2.17 2.10

nd nd nd 2.75 nd nd nd 8.98

2.24 2.36 2.48 nd 4.72 6.93 2.48 39.34

3.12 4.64 nd nd

14.50 5.40 3.07 2.87

3.6. TNFRSF9 gene expression in mouse and human primary macrophages In order to examine the expression the TNFRSF9 in primary cells, we isolated mouse peritoneal macrophages from wild type BALB/c mice, and also differentiated macrophages from freshly isolated human PBMCs. TNFRSF9 mRNA increased significantly upon co-culture of mouse peritoneal macrophages with C. parapsilosis, reaching its maximal level by 24 h [mean ± S.D., 16.05 ± 5.745 fold expression at 24 h (p < 0.01) vs. 1.427 ± 0.055 fold change at 6 h (p < 0.001) and 2.710 ± 0.260 fold expression at 12 h post-infection (p < 0.001) compared to non-stimulated cells, Fig. 6A]. TNFRSF9 had similar expression kinetics in human PBMC-derived macrophages with maximal expression at 12 h in C. parapsilosis-stimulated cells [mean ± S.D., 1.92 ± 0.584 fold change at 6 h (p < 0.05) vs 4.70 ± 1.559-fold expression at 12 h (p < 0.05) vs. 3.483 ± 1.547 fold change at 24 h (p = 0.0556), Fig. 6B].

4. Discussion In this study, the complex response of J774.2 murine macrophages to infection with C. parapsilosis was investigated at the level of gene expression using an Agilent mouse microarray. The

GeneBank accession

Gene name

NM_029094

Phosphatidylinositol 3-kinase, catalytic, beta polypeptide Chemokine (c-c motif) ligand 4 Chemokine (c-c motif) ligand 3 Chemokine (c-c motif) ligand 5 Tumor necrosis factor Toll-like receptor adaptor molecule 2 Toll-like receptor 8 cd86 antigen Mitogen activated protein kinase kinase 6 Interleukin 12b

NM_013652 NM_011337 NM_013653 NM_013693 NM_173394 NM_133212 NM_019388 NM_011943 NM_008352

Fold change 3h

8h

nd

2.32

nd nd nd nd nd nd 2.95 nd nd

3.35 2.24 2.48 2.36 2.81 2.11 nd 2.91 13.62

analysis was performed at two distinct time points (3 and 8 h post-infection), that were chosen to represent different stages in the course of phagocytosis. We show that while the rate of Candida cell ingestion is highest in the first 2–3 h of co-incubation, and that full maturation of phago-lysosomes is achieved by 8 h. Using microarray analysis, we identified more than 500 differentially regulated genes in C. parapsilosis-stimulated macrophages compared to non-stimulated macrophages. Many of the up-regulated genes encode molecules that are involved in immune responses, such as transcription, signaling, apoptosis, cell cycle, electron transport and cell adhesion. To confirm the results of the microarray analysis, 6 genes (TNFA, IL1B, IL15, PTGS2, CD83 and TNFRSF9) were chosen and their expression was examined by RT-qPCR. As expected, the expression of genes encoding cytokines such as IL-1b, TNF-a and IL-12 was up-regulated in C. parapsilosis-stimulated J774.2 macrophage cells. TNF-a and IL-1b are key inflammatory cytokines that play an essential role in host defense during Candida infections (Netea et al., 2006). IL12A and IL12B genes encode the p35 and p40 subunits of the heterodimeric cytokine IL-12, respectively, and they are up-regulated in human monocytes in response to yeast forms of C. albicans (Barker et al., 2005). The expression of IL-15 was up-regulated and this cytokine increases human monocyte superoxide production resulting in enhanced killing of C. albicans (Vazquez et al., 1998). IL-15 is also important for the stimulation of natural killer (NK) cells and T-cells, which are involved in the immune response against a broad range of pathogens (Yoshikai and Nishimura, 2000). The cell surface determinant CD83, a marker of mature dendritic cells (DC), was strongly up-regulated in the early stage of C. parapsilosis infection and the activation level decreased over time. Human mononuclear cells infected with C. albicans have increased expression of CD83 both at mRNA and protein level (Barker et al., 2005).

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Fig. 3. Validation of RNA microarray data by using RT-qPCR. J774.2 cells were co-incubated with C. parapsilosis for 3 or 8 h at a ratio of 1:5 and the expression of IL1-b, IL-15, TNFa, CD83, PTGS-2 and TNFRSF9 was determined by RT-qPCR. Data are normalized to uninfected control samples and indicate mean fold change ± SEM of three independent replicates. Cp, C. parapsilosis, *p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001.

Fig. 4. Candida parapsilosis triggers the most robust TNFRSF9 response in J774.2 macrophages. J774.2 cells were stimulated with different Candida spp. for 6 or 12 h at a ratio of 1:5 and the expression of TNFRSF9 was determined by RT-qPCR. Data indicate mean fold change ± SEM relative to uninfected controls. Experiments were performed in triplicate. *p < 0.05, **p < 0.01.

We also detected important differences between gene expression changes induced by C. parapsilosis and those induced by C. albicans. C. albicans induces the expression of a large number of chemokine and chemokine receptor genes, such as CXCL1, CXCL3, CCL2, CCL4, CCR1, CCR5 and CCR7 in human monocytes (Kim et al., 2005). In contrast, we found that in response to C. parapsilosis, only CXCL2, CCL3, CCL4 and CCL5 genes were upregulated, and there

were also several downregulated chemokine genes such as CCL2, CXCL14 and CCL7. In the future, we will further explore the biological significance of these differences in chemokine activation induced by different Candida spp. Interestingly, we found that the most up-regulated gene in J774.2 macrophages upon C. parapsilosis infection was TNFRSF9, the gene of tumor necrosis factor receptor superfamily, member

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Fig. 5. Interaction with Candida parapsilosis promotes the overexpression of TNFRSF9 protein by J774.2 cells. J774.2 cells were co-incubated with C. parapsilosis for 12, 24 or 36 h and the expression of TNFRSF9 (CD137) on macrophages was analyzed by flow cytometry following staining with PE-conjugated anti-mouse CD137 Ab. (A) histogram of expression of TNFRSF9 on J774.2 cells, (B) mean fluorescence intensity levels normalized to the uninfected control.

Fig. 6. Primary macrophages upregulate the expression of TNFRSF9 in response to C. parapsilosis. Mouse peritoneal macrophages (A) and human PBMC-derived macrophages (B) were co-incubated with C. parapsilosis for 3, 6, 12 or 24 h at a ratio of 1:5 and the expression of TNFRSF9 was determined by RT-qPCR. Data are normalized to uninfected control samples and indicate mean fold change ± SEM of three independent replicates. Cp, C. parapsilosis, *p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001.

9. This receptor, also called 4-1BB, ILA or CD137, is an important co-stimulatory molecule originally described as an activator of Tcells (Vinay and Kwon, 2011). Agonistic anti-4-1BB antibodies possess strong anti-tumor activity due to their capability to activate cytotoxic CD8+ T lymphocytes (Vinay and Kwon, 2012). However, TNFRSF9 is not only expressed on T-cells, but also on activated dendritic cells, monocytes, neutrophil granulocytes, B cells and NK cells, indicating its involvement in the activation of innate immune cells as well (Vinay and Kwon, 2011). TNFRSF9 has been implicated in host defense against different viruses including HIV (Kassu et al., 2009), influenza virus (Kwon et al., 2002) and lymphocytic choriomeningitis virus (Kwon et al., 2002), as well as intracellular bacteria, such as Listeria monocytogenes (Lee et al., 2005) and Francisella tularensis (Zhou et al., 2012). However, TNFRSF9 has never been associated with fungal infections; therefore, we decided to further examine the expression profile of this molecule in macrophages in response to stimulation by Candida spp. We found that all Candida spp. (C. albicans, C. glabrata,C. guilliermondii, C. krusei, C. metapsilosis, C. orthopsilosis, C. parapsilosis and C. tropicalis) induced the expression of TNFRSF9 in J774.2 macrophages. However, C. parapsilosis induced the expression earlier and to a significantly greater extent than the other Candida spp. Using flow cytometry, we found that the TNFRSF9 protein was highly expressed on the surface of J774.2 macrophages upon

C. parapsilosis infection. To further establish the relevance of TNFRSF9 up-regulation in the macrophage response to C. parapsilosis, we also examined the expression of the gene in primary mouse and human macrophages. Again, we found that the amount of TNFRSF9 mRNA was significantly increased in C. parapsilosis-stimulated cells. Although the exact role of the molecule is yet to be determined, our data indicate that it may play an important part in host defense during Candida infections. Understanding how macrophages respond to C. parapsilosis at the molecular level may facilitate the development of new therapeutic paradigms. To our knowledge, this is the first report analyzing the dynamic expression of host genes upon C. parapsilosis infection. Acknowledgments JDN is supported in part by an Irma T. Hirschl/Monique WeillCaulier Trust Research Award. AG is supported by OTKA NN100374, NF84006 and by EMBO Installation Grant 1813. AG supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. This research was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP 4.2.4. A/2-11-1-2012-0001 ‘National Excellence Program’.

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Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fgb.2014.01.006.

References Barker, K.S. et al., 2005. Coculture of THP-1 human mononuclear cells with Candida albicans results in pronounced changes in host gene expression. J. Infect. Dis. 192, 901–912. Benjamin Jr., D.K. et al., 2003. Candida bloodstream infection in neonates. Semin. Perinatol. 27, 375–383. Bertini, A. et al., 2013. Comparison of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis adhesive properties and pathogenicity. Int. J. Med. Microbiol. 303, 98–103. Bourgeois, C. et al., 2010. Fungal attacks on mammalian hosts: pathogen elimination requires sensing and tasting. Curr. Opin. Microbiol. 13, 401–408. Cisterna, R. et al., 2010. Nationwide sentinel surveillance of bloodstream Candida infections in 40 tertiary care hospitals in Spain. J. Clin. Microbiol. 48, 4200– 4206. Clerihew, L. et al., 2007. Candida parapsilosis infection in very low birthweight infants. Arch. Dis. Child. Fetal Neonatal Ed. 92, F127-9. Cummings, C.A., Relman, D.A., 2000. Using DNA microarrays to study host–microbe interactions. Emerg. Infect. Dis. 6, 513–525. Dementhon, K. et al., 2012. Development of an in vitro model for the multiparametric quantification of the cellular interactions between Candida yeasts and phagocytes. PLoS ONE 7, e32621. Gacser, A. et al., 2007. Targeted gene deletion in Candida parapsilosis demonstrates the role of secreted lipase in virulence. J. Clin. Invest. 117, 3049–3058. Heidenreich, S. et al., 1996. Infection by Candida albicans inhibits apoptosis of human monocytes and monocytic U937 cells. J. Leukoc. Biol. 60, 737–743. Horasan, E.S. et al., 2010. Increase in Candida parapsilosis fungemia in critical care units: a 6-years study. Mycopathologia 170, 263–268. Huang da, W. et al., 2009. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57. Kassu, A. et al., 2009. Decreased 4-1BB expression on HIV-specific CD4+ T cells is associated with sustained viral replication and reduced IL-2 production. Clin. Immunol. 132, 234–245. Kim, H.S. et al., 2005. Expression of genes encoding innate host defense molecules in normal human monocytes in response to Candida albicans. Infect. Immun. 73, 3714–3724. Kwon, B.S. et al., 2002. Immune responses in 4-1BB (CD137)-deficient mice. J. Immunol. 168, 5483–5490. Lee, S.C. et al., 2005. 4-1BB (CD137) is required for rapid clearance of Listeria monocytogenes infection. Infect. Immun. 73, 5144–5151. Lewis, L.E. et al., 2012. Stage specific assessment of Candida albicans phagocytosis by macrophages identifies cell wall composition and morphogenesis as key determinants. PLoS Pathog. 8, e1002578.

Manger, I.D., Relman, D.A., 2000. How the host ‘sees’ pathogens: global gene expression responses to infection. Curr. Opin. Immunol. 12, 215–218. Marcil, A. et al., 2002. Candida albicans killing by RAW 264.7 mouse macrophage cells: effects of Candida genotype, infection ratios, and gamma interferon treatment. Infect. Immun. 70, 6319–6329. Miliotis, M.D., 1991. Acridine orange stain for determining intracellular enteropathogens in HeLa cells. J. Clin. Microbiol. 29, 830–831. Montagna, M.T. et al., 2010. Invasive fungal infections in neonatal intensive care units of Southern Italy: a multicentre regional active surveillance (AURORA project). J. Prev. Med. Hyg. 51, 125–130. Netea, M.G. et al., 2006. Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors. J. Clin. Invest. 116, 1642–1650. Neu, N. et al., 2009. Epidemiology of candidemia at a Children’s hospital, 2002 to 2006. Pediatr. Infect. Dis. J. 28, 806–809. Pammi, M. et al., 2013. Candida parapsilosis is a significant neonatal pathogen: a systematic review and meta-analysis. Pediatr. Infect. Dis. J.. Peman, J. et al., 2005. Epidemiology and antifungal susceptibility of Candida species isolated from blood: results of a 2-year multicentre study in Spain. Eur. J. Clin. Microbiol. Infect. Dis. 24, 23–30. Pfaller, M.A. et al., 2010. Candida bloodstream infections: comparison of species distribution and antifungal resistance in community onset and nosocomial isolates in the SENTRY antimicrobial surveillance program (2008–2009). Antimicrob. Agents Chemother.. Seider, K. et al., 2010. Interaction of pathogenic yeasts with phagocytes: survival, persistence and escape. Curr. Opin. Microbiol. 13, 392–400. Smith, P.B. et al., 2005. Neonatal candidiasis. Infect. Dis. Clin. North Am. 19, 603– 615. Németh, Tibor et al., 2013. Characterization of virulence properties in the Candida parapsilosis Sensu Lato species. PLoS ONE 8. Tortorano, A.M. et al., 2011. Invasive fungal infections in the intensive care unit: a multicentre, prospective, observational study in Italy (2006–2008). Mycoses. Trofa, D. et al., 2008. Candida parapsilosis, an emerging fungal pathogen. Clin. Microbiol. Rev. 21, 606–625. van Asbeck, E.C. et al., 2009. Candida parapsilosis: a review of its epidemiology, pathogenesis, clinical aspects, typing and antimicrobial susceptibility. Crit. Rev. Microbiol. 35, 283–309. Van de Velde, W. et al., 2010. Plant peptides govern terminal differentiation of bacteria in symbiosis. Science 327, 1122–1126. Vazquez, N. et al., 1998. Interleukin-15 augments superoxide production and microbicidal activity of human monocytes against Candida albicans. Infect. Immun. 66, 145–150. Vinay, D.S., Kwon, B.S., 2011. 4-1BB signaling beyond T cells. Cell. Mol. Immunol. 8, 281–284. Vinay, D.S., Kwon, B.S., 2012. Immunotherapy of cancer with 4-1BB. Mol. Cancer Ther. 11, 1062–1070. Yoshikai, Y., Nishimura, H., 2000. The role of interleukin 15 in mounting an immune response against microbial infections. Microbes Infect. 2, 381–389. Zhou, H. et al., 2012. Genome-wide RNAi screen in IFN-gamma-treated human macrophages identifies genes mediating resistance to the intracellular pathogen Francisella tularensis. PLoS ONE 7, e31752.