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Transcriptome Profile of the Vascular Endothelial Cell Response to Candida albicans

  1. Katherine S. Barker1,3,a,
  2. Hyunsook Park5,a,
  3. Quynh T. Phan5,
  4. Lijing Xu4,
  5. Ramin Homayouni4,
  6. P. David Rogers1,2,3 and
  7. Scott G. Filler5,6
  1. 1Department of Clinical Pharmacy, College of Pharmacy, Tennessee
  2. 2Department of Pharmaceutical Sciences, College of Pharmacy, and Department of Pediatrics, College of Medicine, University of Tennessee Health Science Center, Tennessee
  3. 3Children's Foundation Research Center, Le Bonheur Children's Medical Center, Tennessee
  4. 4Division of Bioinformatics, College of Arts and Sciences, University of Memphis, Tennessee
  5. 5St. John's Cardiovascular Research Center, Division of Infectious Diseases, Department of Medicine, Los Angeles Biomedical Research Institute at Harbor-University of California, Los Angeles (UCLA), Medical Center, Torrance
  6. 6The David Geffen School of Medicine at UCLA, Los Angeles, California
  1. Reprints or correspondence: Katherine S. Barker, PhD, Room 304 WPT Le Bonheur Children's Medical Center, 50 North Dunlap Street, Memphis, TN 38103 (ksbarker{at}utmem.edu).
 
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Abstract

Background. During hematogenously disseminated candidiasis, bloodborne Candida albicans interacts with vascular endothelial cells (ECs), which have the capacity to influence the local inflammatory response to this organism.

Methods. To elucidate the EC response to C. albicans, we determined the transcriptional profile of ECs infected with wild-type C. albicans strain SC5314 and a hypovirulent cph1Δ/cph1Δ efg1Δ/efg1Δ mutant, CAN34. These EC responses were also compared to our previously published data on the response of the macrophage-like THP-1 cell line to C. albicans.

Results. Infection with strain SC5314 induced upregulation of EC genes involved in chemotaxis, stress response, angiogenesis, and inhibition of apoptosis. Infection with CAN34 induced weaker expression of fewer genes. The angiogenic and anti-apoptotic response of ECs to C. albicans did not occur in THP-1 cells. However, there was upregulation of CCL3 and CCL4 expression in both cell types. Because CCR5 is the receptor for CCL3 and CCL4, we tested the susceptibility of CCR5−/− mice to disseminated candidiasis. The survival and renal fungal burden of the CCR5−/− mice were similar to that of wild-type control mice.

Conclusions. ECs respond significantly differently to infection with C. albicans, compared with THP-1 cells. CCR5 is dispensable for the host defense against disseminated candidiasis in immunocompetent mice.

Hematogenously disseminated candidiasis (HDC) is initiated when Candida albicans enters the bloodstream. The fungi are carried throughout the body, causing microabscesses in virtually all organs. The mortality of patients with HDC approaches 50%, even if they receive antifungal therapy [1]. During the initiation of HDC, bloodborne C. albicans must adhere to and invade the endothelial cell (EC) lining of the vasculature to invade the deep tissues. ECs are therefore among the first host cells with which C. albicans comes into contact as it disseminates. In response to C. albicans, ECs secrete proinflammatory cytokines and express leukocyte adhesion molecules [2, 3]. Thus, ECs have the potential to significantly influence the local host response to vascular invasion by C. albicans.

C. albicans can interconvert between blastospores and filamentous hyphae. Mutants of C. albicans that grow predominantly as blastospores have a reduced capacity to invade ECs, and they stimulate a weaker proinflammatory response, compared with wild-type organisms [4]. Indeed, the capacity of C. albicans to form hyphae is a key virulence factor of this organism. C. albicans mutants with defective hyphae formation have significantly attenuated virulence in the mouse model of HDC [5–7].

Previous studies on the interactions of C. albicans with ECs have focused on a limited number of EC responses. It is known that the adherence of C. albicans hyphae to ECs stimulates them to endocytose the organisms [8, 9]. Endocytosis is induced when the C. albicans invasin, Als3p, binds to N-cadherin and other proteins on the EC surface [10, 11]. In vitro, ECs that have endocytosed C. albicans secrete prostacyclin, tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), IL-1β, IL-6, IL-8, and monocyte chemoattractant protein 1 [2, 3, 12, 13]. These ECs also have increased surface expression of leukocyte adhesion molecules, such as E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) [2, 3].

To more fully elucidate the EC response to C. albicans, we analyzed the transcriptional profile of ECs infected with a wild-type C. albicans or the hyphae-deficient mutant strain of C. albicans. We compared the responses of these ECs to those of ECs stimulated with TNF-α and to our previous data on the macrophage-like THP-1 cell line response to wild-type C. albicans [14].

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Methods

Cells and growth conditions. The C. albicans strains used in this study were SC5314 (wild type), CAN34 (cph1Δ/cph1Δ efg1Δ/efg1Δ) and CAN35 (cph1Δ/cph1Δ efg1Δ/efg1Δ::EFG1. CAN34 and CAN35 were kindly provided by Dr. Gerald Fink [7]). All strains were grown in yeast extract peptone dextrose medium (1% yeast extract, 2% peptone, and 2% glucose) at 30°C overnight. They were harvested, washed, and counted as described elsewhere [13, 15]. Immediately before being added to the ECs, the organisms were suspended in M-199 medium (Gibco) containing 10% pooled human serum (Gemini Bio-products).

ECs were harvested from umbilical cord veins and cultured as described elsewhere [8, 16]. The night before the experiments, the medium above the ECs was changed to M-199 medium with 10% pooled human serum.

Assessment of C. albicans morphology and EC damage. The morphology of the different strains of C. albicans after 3 h and 8 h of incubation with ECs was determined as described elsewhere [4]. The extent of EC damage caused by the different C. albicans strains at both time points was determined using our standard 51Cr release assay [8, 17].

RNA extraction, microarray hybridization, and data analysis. Confluent ECs in 6-well tissue culture plates were infected with 5 × 105 blastospores of C. albicans. Two wells of ECs were infected with each strain at each time point. Parallel wells of ECs were exposed to medium alone. In one experiment, ECs were also exposed to 1 ng/mL recombinant TNF-α (Sigma-Aldrich). After 3 h and 8 h of incubation, the medium was aspirated, and ECs were rinsed once with Hanks' balanced salt solution (Irvine Scientific). Next, EC RNA was isolated using RNAwiz (Ambion), in accordance with the manufacturer's protocol, and RNA from duplicate wells was combined. The isolated EC RNA contained no detectable C. albicans RNA (data not shown). Each set of cocultures was performed 3 times using ECs from different umbilical cords to generate 3 biological replicates for the microarray studies.

For microarray hybridization, 10 µg of total EC RNA was used to produce biotin-labeled cRNA, which was hybridized with Affymetrix U133A and U133 Plus 2.0 human gene arrays, in accordance with the manufacturer's instructions. Gene expression values were normalized by using GeneSpring software (Agilent Technologies), and each experimental condition was compared to its corresponding control to calculate n-fold changes in gene regulation. A differentially expressed gene was defined as being upregulated or downregulated at least 2-fold and was called “present” in accordance with the Affymetrix criteria in at least 2 experiments. The expression data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO, available at http://www.ncbi.nlm.nih.gov/geo/) and is accessible through GEO Series accession number GSE8166. 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 (http://david.abcc.ncifcrf.gov/home.jsp), using a significance level of P ⩽.01.

cDNA synthesis and real-time reverse-transciptase polymerase chain reaction (RT-PCR). Real-time RT-PCR was used to verify the expression levels of selected EC genes in response to 8 h of infection with the 3 C. albicans strains. cDNA was synthesized from EC RNA treated with DNase I (Ambion) using RETROscript kit (Ambion), in accordance with the manufacturer's protocol. Real-time PCR was performed using TaqMan Gene Expression Assay Probes for the selected genes and TaqMan Universal PCR Master Mix (Applied Biosystems), in accordance with the manufacturer's protocol. Fold changes were calculated using the 2−ΔΔCT method [18], in which expression in each condition was compared to expression in the medium-only sample by using GAPDH1 as an endogenous control.

Measurement of EC chemokine production. ECs were grown in 24-well tissue culture plates and exposed for 8 h to 105 blastospores of C. albicans, medium alone, or 1 ng/mL TNF-α. Each stimulus was coincubated with 2.5 µg/mL neutralizing anti-TNF-α murine monoclonal antibody or an isotype matched control (R&D Systems) [2]. The concentration of IL-8, MIP-1α, and MIP-1β in the supernatants was determined by ELISA (Biosource). Each experiment was performed in triplicate on 3 separate occasions. Differences in chemokine production by ECs exposed to various stimuli were compared by analysis of variance.

Assessing C. albicans virulence in CCR5−/− mice. To determine the role of CCR5 in the host defense against HDC, 5-week-old to 6-week-old B6.129P2-Ccr5tm1Kuz/J (CCR5−/−) mice and wild-type C57BL/6J mice (The Jackson Laboratory) were used. Sixteen mice from each strain were inoculated intravenously with 5 × 105 or 105 SC5314 blastospores. Ten mice were monitored for survival 2 times per day for 30 days. All experiments were approved by the Institutional Animal Care and Use Committee. Differences in survival between different strains of mice were analyzed using the log rank test.

Determination of renal fungal burden. Six mice from each strain were sacrificed, after which their kidneys were harvested and samples were quantitatively cultured. The renal fungal burden was determined after 2 and 4 days of infection in mice inoculated with 5 × 105 and 105 organisms, respectively.

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Results

Interactions of C. albicans strains with ECs. First, the interactions of C. albicans and ECs were characterized at the same time points and under the same conditions as were used in the transcriptional profiling experiments. The ECs were infected with blastospores of a wild-type strain of C. albicans (SC5314) and a cph1Δ/cph1Δ efg1Δ/efg1Δ mutant strain (CAN34). CAN34 does not form hyphae under most conditions; it adheres to and invades ECs poorly, and it has severely attenuated virulence in the mouse model of HDC [4, 5, 7]. As reported elsewhere [4, 5], SC5314 formed hyphae and caused significant damage to ECs (figure 1). In contrast, CAN34 grew as chains of blastospores and did not cause detectable EC damage, even after 8 h of infection. These defects in hyphae formation and EC damage were reversed when CAN34 was complemented with a wild-type copy of EFG1.

Figure 1
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Figure 1

Morphology of Candida albicans strains and the extent of endothelial cell damage they induce.

Gene expression in ECs and THP-1 cells. Gene expression profiles of ECs after 3 h and 8 h of infection with SC5314 and CAN34 were compiled. To provide a context for the C. albicans infection data, we also determined the EC response to incubation for 3 h and 8 h with the prototypical stimulus, TNF-α. We also compared these EC data to previous microarray data of the macrophage-like THP-1 cell line response to SC5314 infection for 3 h [14]. These THP-1 studies used the same experimental design and microarray data analysis as were employed in the current experiments.

Hierarchical clustering demonstrated that the EC response to SC5314 at 3 h clustered together with the EC response to CAN34 at 3 h (figure 2A). Similarly, the EC responses to both strains at 8 h clustered together. The EC responses to TNF-α exposure for 3 h and 8 h also clustered together, and this TNF-α cluster was most similar to the EC response to both strains at 8 h. The EC response profiles to C. albicans or TNF-α at both time points were quite different from the response of THP-1 cells to C. albicans at 3 h, indicating a fundamental difference in THP-1 and EC responses to C. albicans.

Figure 2
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Figure 2

A, Cluster analysis of genes detected by Affymetrix microarrays of endothelial cells (ECs) exposed to 2 different strains of Candida albicans or tumor necrosis factor α (TNF-α), and the macrophage-like THP-1 cell line exposed to SC5314. ECs were exposed for 3 h and 8 h to TNF-α, SC5314, and CAN34. THP-1 cells were exposed for 3 h to SC5314. Shown is a hierarchical clustering analysis (average linkage method) of 4377 genes detected as “present” in at least 2 samples and either upregulated or downregulated at least 2-fold. B, Venn diagram showing the relationship among EC genes that were differentially expressed in response to SC5314, CAN34, and TNF-α. C, Venn diagram showing the relationship among EC and THP-1 cell genes that were differentially expressed in response to infection with SC5314. Genes were considered to be differentially expressed if they were upregulated or downregulated at least 2-fold, compared to expression in cells exposed to medium alone and were “present” according to Affymetrix criteria in at least 2 samples for cells infected with C. albicans and in at least 1 sample for ECs exposed to TNF-α.

Genes that were differentially expressed in response to each stimulus at each time point were analyzed (figure 3). More EC genes were differentially expressed in response to either SC5314 or CAN34 after 8 h, compared with the response at 3 h (figure 2B). At each time point, there was little overlap between the SC5314-responsive genes and the CAN34-responsive genes. TNF-α exposure altered the expression of many more genes at both time points than did either C. albicans strain, and the EC response to TNF-α was greatest at 3 h. Approximately half of the genes that were differentially expressed in response to C. albicans were also differentially expressed in response to TNF-α. The majority of these genes were upregulated.

Figure 3Figure 3
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Figure 3

Differential gene expression in endothelial and THP-1 cells, in response to stimulus with tumor necrosis factor α (TNF-α) and Candida albicans.

The EC genes that were differentially expressed in response to SC5314 at 3 h and/or 8 h were compared to the THP-1 cell genes that were differentially expressed at 3 h. Only 8 genes were upregulated in both cell types, and no genes were downregulated (figure 2C).

Gene Ontology analysis of the biological significance of differentially expressed genes. To explore the biological significance of the genes that were differentially expressed in response to C. albicans, we determined the Gene Ontology (GO) terms of these genes [19]. This analysis was performed using a significance level of P < .01, and only pathways that were enriched in ECs infected with C. albicans and relevant to documented host-pathogen interactions were examined (table 1).

The genes that were upregulated in response to SC5314 at 8 h were enriched in GO terms, including inflammation, cell proliferation, and response to stress or wounding (table 1). Very few GO terms were overrepresented in the EC response to SC5314 at 3 h. Also, there was no significant enrichment in any specific GO term among the genes that were upregulated in response to CAN34 or downregulated in response to either strain of C. albicans.

Many of the EC genes involved in immune response and inflammatory response at 8 h specified proteins involved in chemotaxis (table 2). CAN34 induced the expression of most of these genes, although at a lower level compared to ECs infected with SC5314. All chemotaxis-associated genes that were upregulated in response to C. albicans were also upregulated by TNF-α, which elicited a generally earlier and stronger response (table 2). Many chemotaxis-related genes were upregulated in response to TNF-α but were not induced by C. albicans (table 1).

There was only a partial overlap among the chemotaxis-related genes that were upregulated in ECs and THP-1 cells in response to SC5314. CCL3, CCL4, and CXCL2 were upregulated in both cell types in response to C. albicans. CXCL1, CXCL3, IL1A, and VEGF were upregulated only in ECs, and CXCL10 was upregulated only in THP-1 cells. ECs, but not THP-1 cells, also responded to SC5314 by upregulating genes encoding the leukocyte adhesion molecules, ICAM-1 and E-selectin (figure 3).

C. albicans induces significant EC damage (figure 1B) [4, 5, 8, 13], thus, it was interesting that the genes related to the response to stress or wounding were overrepresented in response to SC5314 (table 1). The majority of these genes encode proinflammatory mediators, such as IL-1α, calgranulin C, E-selectin, and prostaglandin-endoperoxide synthase 2 (table 3). MAP4K5, which activates the stress-activated protein kinase pathway [20], and SGK, which enhances cell survival in response to stress, were also upregulated [21]. SC5314 also induced EGR1 (figure 3), which encodes a transcription factor that governs the EC response to mechanical injury [22]. CAN34, which did not cause detectable EC damage (figure 4), had minimal effect on the expression of stress response genes (tables 2–4).

Figure 4
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Figure 4

Survival rates of wild-type and CCR5−/− mice with hematogenously disseminated candidiasis. Wild-type C57BL/6J mice and CCR5−/− B6.129P2-Ccr5tm1Kuz/J mice were infected with 5 × 105 (A) and 105 (B) blastospores of SC5314 via the tail vein and monitored for survival for 30 days. The survival of CCR5−/− mice was not significantly different from that of the wild-type mice.

Notably, TNF-α induced different stress-related and wounding-related genes than were induced by C. albicans (table 1). Of the 11 stress response genes that were upregulated in response to C. albicans, 7 were not upregulated in response to TNF-α. Also, only 1 of these stress response genes was upregulated in THP-1 cells. These results suggest that SC5314 induces a unique stress response in ECs.

Figure 5
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Figure 5

Stimulation of chemokine secretion by endothelial cells (ECs) in response to Candida albicans. ECs were exposed to medium alone, the strains of C. albicansindicated, or tumor necrosis factor α (TNF-α) in the presence of either an anti-TNF-α neutralizing monoclonal antibody or its isotype control. After 8 h, the conditioned medium above the cells was collected and the concentrations of interleukin 8 (IL-8), MIP-1α, and MIP-1β were determined by ELISA. Results are the mean ± SD of 3 independent experiments, each performed in triplicate. *P ⩽ .03 compared to control cells exposed to medium alone, †P < .05 compared to CAN34 exposed to the same antibody, ‡P < .005 compared to the same stimulus in the presence of the isotype control.

Table 1
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Table 1

Gene Ontology (GO) analysis of endothelial cell genes upregulated in response to wild-type Candida albicans.

Three angiogenesis pathway genes were upregulated in response to SC5314: VEGF, IL8, and ANGPTL4 (tables 2–4). IL-8 stimulates angiogenesis by binding to CXCR2 on ECs [23]. CXCL1/GROα and CXCL3/GROγ, both upregulated in response to SC5314 (table 2), also bind to this receptor and induce angiogenesis [24]. Sixteen genes in the apoptosis pathway were upregulated in response to C. albicans (table 4), 12 of which encode products with anti-apoptotic functions. Collectively, these results suggest that ECs respond to SC5314 infection by proliferating.

Table 2
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Table 2

Genes involved in chemotaxis that were upregulated in endothelial and/or THP-1 cells infected with C. albicans.

Table 3
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Table 3

Genes involved in stress response that were upregulated in endothelial and/or THP-1 cells infected with C. albicans

Table 4
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Table 4

Genes involved in apoptosis that were upregulated in endothelial and/or THP-1 cells infected with C. albicans.

We used real-time PCR to verify that selected EC genes were upregulated in response to 8 h of infection with C. albicans. We found that ANGPTL4, BCL2A1, CXCL3, F3, SGK, and VEGF were upregulated by 2.9-fold to 52.9-fold in ECs infected with SC5314 (table 5). CAN34 induced much lower expression of these genes (1.2-fold to 3.3-fold). Importantly, infection with the EFG1-complemented strain, CAN35, restored expression of these genes to at least wild-type levels. Therefore, the reduced EC stimulation induced by CAN34 resulted from the absence of EFG1.

Table 5
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Table 5

Real-time polymerase chain reaction-verified endothelial cell gene expression in response to 8 h of infection with different strains of C. albicans.

EC secretion of MIP-1α and MIP-1β is induced by a different mechanism than secretion of IL-8. The finding that CCL3 and CCL4 (encoding MIP-1α and MIP-1β, respectively) were upregulated in both ECs and THP-1 cells in response to C. albicans suggested that MIP-1α and MIP-1β may play a key role in the host defense against invasive candidal infections. Therefore, we investigated whether MIP-1α and MIP-1β are also required for the host defense against C. albicans. First, we verified that C. albicans induced ECs to secrete MIP-1α and MIP-1β into the medium. IL-8 secretion was measured as a control, because we have previously shown that C. albicans induces EC secretion of IL-8 [2, 3]. SC5314 stimulated ECs to secrete IL-8, MIP-1α, and MIP-1β (figure 5). Interestingly, CAN34 stimulated significantly less secretion of IL-8, but significantly greater levels of MIP-1α and MIP-1β secretion than did SC5314. These results indicate that C. albicans stimulates IL-8 secretion by a different mechanism than the one governing the production of MIP-1α and MIP-1β.

C. albicans stimulates ECs to synthesize TNF-α, which then induces IL-8 synthesis [2]. We found that TNF-α also stimulated ECs to accumulate CCL3 and CCL4 mRNA and secrete MIP-1α and MIP-1β into the medium (table 2; figure 5). Therefore, we used a neutralizing anti-TNF-α monoclonal antibody to investigate the role of EC-derived TNF-α in MIP-1α and MIP-1β production. The anti-TNF-α antibody had no effect on the synthesis of either chemokine in response to SC5314 but significantly reduced the synthesis of both chemokines in response to CAN34 (figure 5). Collectively, these results suggest that the production of MIP-1α and MIP-1β by ECs in response to SC5314 is independent of TNF-α. However, MIP-1α and MIP-1β production in response to CAN34 is likely mediated by both a TNF-α-dependent mechanism and a TNF-α-independent mechanism.

CCR5 was not essential for host defense against HDC. To investigate further the role of MIP-1α and MIP-1β in the host defense against HDC, we used CCR5−/− mice, which lack the common receptor for these chemokines. The CCR5−/− mice had survival similar to that of wild-type control mice after intravenous infection with either a high or a low inoculum of C. albicans (figure 4). Also, the renal fungal burdens of the CCR5−/− mice and the control mice were found to be similar after 2 days of infection in the high inoculum experiment and after 4 days of infection in the low inoculum experiment (table 6). Therefore, CCR5 is not essential for the host defense against HDC in this model.

Table 6
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Table 6

Renal fungal burdens of mice with hematogenously disseminated candidiasis.

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Discussion

The transcriptional profiling data indicate that infection with C. albicans induces a unique EC response, consisting of upregulation of genes involved in inflammatory response, stress response, angiogenesis, and inhibiting apoptosis. This profile is significantly different from the THP-1 response to C. albicans. In addition, we found that C. albicans induces a more limited EC response than does TNF-α. This result was particularly interesting because C. albicans stimulates ECs to synthesize TNF-α [2]. Furthermore, even though some of the responses induced by both SC5314 and TNF-α were similar, they were induced by different mechanisms. For example, both stimulated ECs to secrete MIP-1α and MIP-1β. However, the anti-TNF-α antibody did not inhibit the secretion of these chemokines in response to C. albicans. One possible explanation for these results is that C. albicans may activate additional signal transduction pathways in ECs that mimic some TNF-α responses while inhibiting others.

Some of the inflammatory response genes that were upregulated in response to C. albicans in the current study have been previously shown by Northern blot analysis to be upregulated in response to this organism [3]. However, in our previous study, we found that infection with a spontaneous hyphae-deficient strain of C. albicans did not induce the upregulation of any of the inflammatory response genes mentioned above [3]. In the current study, the hyphae-deficient CAN34 strain also induced little or no upregulation of these genes. However, the transcriptional profiling experiments revealed that CCL3 and CCL4 must be regulated differently from the other inflammatory response genes because they were strongly induced by CAN34. In fact, we found that MIP-1α and MIP-1β were secreted in higher amounts by ECs infected with CAN34, compared with ones infected with SC5314. These results suggest that MIP-1α and MIP-1β secretion is governed differently than the secretion of the other chemokines. Consistent with this model, we found that SC5314 induced secretion of MIP-1α and MIP-1β by a mechanism that was independent of EC-derived TNF-α. In contrast, secretion of IL-8 in response to SC5314 required the presence of TNF-α.

The basis for the different EC response induced by CAN34 compared to the wild-type strain is likely multifactorial. In addition to being hyphae-deficient, CAN34 has very low expression of many hyphae-specific genes, such as ALS3 and HWP1, that encode cell surface proteins. Moreover, it has aberrant expression of genes involved in cell wall synthesis even when grown under non-hyphae inducing conditions [25]. As a result, both the cell surface carbohydrates and proteins of CAN34 are different from those of SC5314. These differences likely account for CAN34's defective EC adherence and invasion, as well as its limited damage to ECs [4] and the unique EC transcriptional profile induced. Previously, we have found that many EC responses to wild-type C. albicans, such as expression of IL6, IL8, PTGS2, SELE, and ICAM1 genes are only induced when ECs endocytose the organism [3]. As the wild-type strain adheres to and is endocytosed by ECs, it likely engages multiple endothelial cell receptors, such as N-cadherin [10,11]. However, because CAN34 neither adheres to nor is endocytosed by ECs [4], it unlikely to interact with these receptors. Therefore, is possible that EC stimulation by CAN34 is mediated via a soluble factor that is released from the mutant cells.

The EC transcriptional profiling data also indicated that SC5314 induced a stress response that was distinct from that induced by TNF-α and from that induced in THP-1 cells infected with this organism. The stress response induced by SC5314 consisted of the upregulation of proinflammatory genes encoding chemokines, leukocyte adhesion molecules, and cyclooxygenase, which likely help to recruit activated leukocytes to foci of infection.

Our data also suggested that ECs respond to C. albicans by expressing angiogenesis and anti-apoptotic genes. These responses were likely stimulated by C. albicans-induced damage to the ECs, as they were not induced by CAN34. The expression of genes whose products induce angiogenesis and inhibit apoptosis likely results in EC proliferation, as it has been reported that mice infected with C. albicans have EC proliferation in the regions adjacent to microabscesses in the kidney and brain [26].

ECs and THP-1 cells respond differently to C. albicans. The angiogenesis-related and apoptosis-related genes were overrepresented in the transcriptional profile of ECs infected with C. albicans, but not in that of THP-1 cells infected with C. albicans. We also found that although the responses of both cell types were enriched in many of the same GO terms, there was little overlap among the genes that were contained within these GO terms. For example, although SC5314 induced the expression of numerous genes involved in chemotaxis in both ECs and THP-1 cells, only 3 of these genes were expressed in both types of cells.

Two of the 3 chemotaxis-related genes that were upregulated in both cell types were CCL3 and CCL4. Transcriptional profiling of peripheral blood mononuclear cells indicates that wild-type C. albicans induces the upregulation of not only CCL3 and CCL4, but also CCR5 [27]. Therefore, we investigated whether the CCR5 signaling pathway is necessary for normal host defense against HDC. We found that CCR5−/− mice infected with C. albicans had similar mortality and fungal burden to those of wild-type mice. Huffnagle et al [28] found that CCR5−/− mice are more susceptible to cryptococcosis. In mice infected with C. neoformans, CCR5 is required for normal leukocyte recruitment into the brain, but not the lungs, suggesting that CCR5 has an organ-specific role in leukocyte recruitment. There are 2 nonexclusive explanations for our findings that CCR5 is not essential for the host defense against HDC. First, CCR5 may not be required for leukocyte recruitment into the kidney, which is the main target organ in mice with HDC [29, 30]. Second, CCR5 mediates recruitment of mononuclear cells, but not neutrophils [28]. Thus, in the CCR5−/− mice, any impairment in mononuclear cell recruitment was compensated by the presence of neutrophils, which are known to be the key host effector cell for defense against HDC [15, 31].

The microarray data suggest other interesting hypotheses to be investigated, such the role of angiogenesis in the pathogenesis of disseminated candidiasis, and the contribution of other chemokine pathways to the host defense against this disease. We are currently exploring these hypotheses.

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Acknowledgments

We thank Norma Solis for technical assistance and the nurses at the Harbor-UCLA Medical Center Pediatric Clinical Research Center for umbilical cord collection. Confocal microscopy was performed at the Henry L. Guenther Cell Molecular Core facility at the Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center.

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Footnotes

  • ↵a K. S. B. and H. P. contributed equally to this work.

  • Potential conflicts of interest: S. G. F. has received research support from Amgen, Merck, and Pfizer, and has equity in NovaDigm Therapeutics. All other authors report no relevant conflicts.

    Presented in part: 8th Meeting on Candida and Candidiasis, Denver, Colorado, 13–17 March 2006 (abstract S4:3).

  • Financial support: National Institutes of Health (grants R01AI054928 to S.G.F., R01AI019990 to S.G.F., R01AI058145 to P.D.R., and MO1RR00425 to S.G.F.); Toyota USA (donation of the Olympus phase-contrast microscope used for these studies).

  • Received October 25, 2007.
  • Accepted February 5, 2008.
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  1. J Infect Dis. (2008) 198 (2): 193-202. doi: 10.1086/589516
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