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Infection and Immunity, June 2005, p. 3714-3724, Vol. 73, No. 6
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.6.3714-3724.2005

Expression of Genes Encoding Innate Host Defense Molecules in Normal Human Monocytes in Response to Candida albicans

Hee Sup Kim,¹ Eun Hwa Choi,^1,2 Javed Khan,¹ Emmanuel Roilides,¹ Andrea Francesconi,¹ Miki Kasai,¹ Tin Sein,¹ Robert L. Schaufele,¹ Kenichi Sakurai,^1,3 Chang Gue Son,¹ Braden T. Greer,¹ Stephen Chanock,¹ Caron A. Lyman,¹ and Thomas J. Walsh^1*

National Cancer Institute, National Institutes of Health, Bethesda, Maryland,¹ Seoul National University, College of Medicine, Seoul, Korea,² Nihon University, Tokyo, Japan³

Received 6 October 2004/ Returned for modification 10 November 2004/ Accepted 10 February 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Little is known about the regulation and coordinated expression of genes involved in the innate host response to Candida albicans. We therefore examined the kinetic profile of gene expression of innate host defense molecules in normal human monocytes infected with C. albicans using microarray technology. Freshly isolated peripheral blood monocytes from five healthy donors were incubated with C. albicans for 0 to 18 h in parallel with time-matched uninfected control cells. RNA from monocytes was extracted and amplified for microarray analysis, using a 42,421-gene cDNA chip. Expression of genes encoding proinflammatory cytokines, including tumor necrosis factor alpha, interleukin 1 (IL-1), IL-6, and leukemia inhibitory factor, was markedly enhanced during the first 6 h and coincided with an increase in phagocytosis. Expression of these genes returned to near baseline by 18 h. Genes encoding chemokines, including IL-8; macrophage inflammatory proteins 1, 3, and 4; and monocyte chemoattractant protein 1, also were strongly up-regulated, with peak expression at 4 to 6 h, as were genes encoding chemokine receptors CCR1, CCR5, CCR7, and CXCR5. Expression of genes whose products may protect monocyte viability, such as BCL2-related protein, metallothioneins, CD71, and SOCS3, was up-regulated at 4 to 6 h and remained elevated throughout the 18-h time course. On the other hand, expression of genes encoding T-cell-regulatory molecules (e.g., IL-12, gamma interferon, and transforming growth factor ß) was not significantly affected during the 18-h incubation. Moreover, genes encoding IL-15, the IL-13 receptor (IL-13Ra1), and CD14 were suppressed during the 18-h exposure to C. albicans. Thus, C. albicans is a potent inducer of a dynamic cascade of expression of genes whose products are related to the recruitment, activation, and protection of neutrophils and monocytes.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Candida albicans is a ubiquitous opportunistic pathogen that causes serious disseminated infections in immunocompromised patients. Candida spp. currently rank as the fourth leading cause of nosocomial bloodstream infections (24). Attributable mortality in patients with hematogenously disseminated candidiasis is estimated to be approximately 38 to 49% (17, 54).

An effective host response to C. albicans is critical for a successful outcome. As a result, mortality remains high despite the introduction of new antifungal agents (3). Various studies have examined the expression of individual genes encoding innate host defense molecules in response to Candida spp. However, gene expression analysis of the immune response to Candida spp. has been limited and primarily restricted to expression of specific genes at limited times of infection (29, 34, 40).

Monocytes are one of the initial phagocytic cell populations mediating host defenses against Candida albicans (4). These cells produce an intricate pattern of cytokines and chemokines that enhance chemotaxis, phagocytosis, and microbicidal activity, as well as activating T cells through antigen processing and presentation (5). However, it has become increasingly evident that the complex response to microbial pathogens varies depending upon the specific pathogen encountered, the host response pathway invoked, and the duration of exposure (9, 32). Furthermore, the expression of multiple genes is likely involved in response to these different pathogens over time (26). Little is known, however, about the sequence or kinetics of gene expression in the complex response of normal human monocytes to C. albicans. Moreover, the small amount of data available has been derived from murine cells or human monocytic neoplastic continuous cell lines.

Thus, in the present study, we sought to elucidate the temporal cascade of gene expression by normal human monocytes in response to C. albicans over an 18-h time course. Using microarray technology, we assessed the kinetic profiles of expression of host response genes, including those involved in inflammation, chemotaxis, and immune transcription, as well as those involved in synthesis of prostaglandins, Toll-like receptors (TLR), and T-cell regulation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Culture and preparation of Candida albicans. A well-characterized clinical isolate of C. albicans (CA86-21), originally recovered from a patient with disseminated candidiasis, was used throughout this study. C. albicans was grown overnight at 37°C on Sabouraud glucose agar. Three isolated colonies of the organism were subsequently suspended in 50 ml of Sabouraud glucose broth in 250-ml Erlenmeyer flasks and grown overnight at 37°C in a gyratory water bath to obtain log-phase blastoconidia. The blastoconidia were washed with normal saline two times, resuspended in Hanks balanced salt solution (HBSS) containing calcium and magnesium, and counted on a hemacytometer (Neubauer Improved Bright-line; Supe-Rior, Germany). The blastoconidia were opsonized in 50% pooled human AB-positive sera for 30 min at 37°C, washed twice with HBSS without calcium and magnesium, and resuspended in HBSS to a final concentration of 10⁷ organisms/ml. The organisms were maintained on ice until they were used.

Isolation of peripheral blood monocytes. Peripheral blood monocytes were isolated from healthy human donors by automated leukophoresis and counterflow elutriation (model J-6 M centrifuge; Beckman Instruments, Fullerton, CA) (29). The cell population isolated was 95% monocytes, as determined by morphology and nonspecific esterase staining. The monocytes were washed once, resuspended in HBSS containing calcium and magnesium, and kept on ice until they were used.

Incubation of C. albicans with monocytes. Monocytes (10⁷) were incubated at an effector-to-target ratio of 1:1 with 10⁷ serum-opsonized C. albicans cells in six-well flat-bottom polypropylene plates. Samples were incubated for 2, 4, 6, 8, 12, or 18 h on a rotator at 37°C and 5% CO₂. Monocytes from five separate donors were examined for each time point. For the zero-hour time point, monocytes and C. albicans were kept on ice and mixed immediately prior to being centrifuged at 4°C, followed by RNA extraction (see below). For the phagocytosis assay, 16-mm sterile circular glass cover slides were placed in the six-well plate, and cells and organisms were incubated as described above. At each time point, the glass cover slides were removed and gently washed with prewarmed HBSS to remove extracellular organisms, and the cells were fixed and stained with modified Wright-Giemsa stain. The percent phagocytosis was calculated by determining microscopically the percentage of monocytes containing organisms.

mRNA amplification. At each time point, cells were harvested and total RNA was extracted immediately using Trizol (Life Technologies, Grand Island, NY). RNA was further purified with RNeasy minikits (QIAGEN, Valencia, CA) according to the manufacturer's instructions. Antisense RNA T7-(dT)₂₄ primer (0.5 µg/µl) (5'-GGC-CAG-TGA-ATT-GTA-ATA-CGA-CTC-ACT-ATA-GGG-AGG-CGG-(dT)₂₄-3') was prepared by Genset Oligo (Boulder, CO). Five micrograms of total RNA was incubated with 1 µg of T7-(dT)₂₄ primer in 9 µl diethyl pyrocarbonate-treated water at 70°C for 5 min. For cDNA synthesis 4 µl first-strand buffer, 2 µl 0.1 M dithiothreitol, 2 µl 10 mM deoxynucleoside triphosphate, 2 µl SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA), and 1 µl RNasin (Promega, Madison, WI) were added, and the mixtures were incubated for 1 h at 42°C. The second strand was made by the addition of 91 µl diethyl pyrocarbonate-treated water, 30 µl second-strand buffer, 3 µl 10 mM deoxynucleoside triphosphate, 4 µl E. coli DNA polymerase I, 1 µl E. coli DNA ligase, and 1 µl RNase H (Invitrogen) and incubation at 16°C for 2 h. Two mocroliters T4 DNA polymerase (Invitrogen) was added, and an additional incubation for 5 min was performed under the same conditions. The reaction was stopped by the addition of 10 µl 0.5 M EDTA and 10 µl 1 M NaOH with incubation at 70°C for 10 min and then was neutralized with 25 µl Tris-HCl, pH 7.5. DNA was extracted by centrifugation in Phase Lock Gel (Eppendorf, Hamburg, Germany) with phenol-chloroform-isoamyl alcohol and precipitated in ethanol with 1 µg linear acrylamide (Ambion, Austin, TX). Transcription in vitro was carried out at 37°C for 15 h in a 40-µl reaction volume using the T7 Megascript kit (Ambion) according to the manufacturer's instructions (14). Amplified RNA was purified in an RNeasy minicolumn (QIAGEN).

Microarray hybridization and data analysis. Microarray probes were synthesized with 2.5 µg amplified RNA using random hexamers for priming. cDNA was generated using Cy3- or Cy5-dUTP (Amersham Biosciences, Piscataway, NJ) and SuperScript II reverse transcriptase. Cy3- and Cy5-containing probes were combined and redissolved in 27 µl 1x hybridization solution (240 µl H₂O, 250 µl 20x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 500 µl formamide) and denatured at 97°C with 13 µg human COT-1 DNA (Invitrogen), 5 µg yeast tRNA (Sigma, St. Louis, MO), and 10 µg poly(A) (Amersham Biosciences). Glass slides displaying over 40,000 genes were prepared in the laboratory of Javed Khan. The glass slides were prehybridized at 42°C for 1 h in 5x SSC, 0.1% SDS, and 1% BSA (Sigma). The arrays were hybridized with a probe for 16 h at 42°C. The slides were then washed for 4 min in 1x SSC-0.2% SDS at 42°C, 4 min in 0.1x SSC-0.2% SDS at room temperature, and 4 min in 0.06x SSC at room temperature and were then spun dry at 100 x g. Images were acquired by an Agilent DNA microarray scanner (Agilent, Palo Alto, CA) and analyzed using the Microarray Suite program, as previously described (7).

The data for each array were normalized to the distribution of all genes on that array (per-chip normalization). The data were quality filtered by removing those genes that had poor-quality measurements (quality < 0.75) and further refined by assignment to unique UniGene clusters (build 172). After these processes, there were 12,269 genes remaining from the initial 42,421 genes. For the genes that passed these filters, mean ratio values were calculated as the relative gene expression in monocytes mixed with C. albicans versus monocytes only at the given time point. The data are expressed as the mean change (n-fold) from baseline ± standard error of the mean (SEM) for the five donors tested. Genes with a change in expression of 2.5-fold were defined as up- or down-regulated.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The viability of monocytes was greater than 82% for all donors at all time intervals tested. The percent phagocytosis increased progressively over the 18-h time course from 0% to 93% (Fig. 1).

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FIG. 1. Percent phagocytosis of Candida albicans by normal human monocytes over an 18-h time course. Data represent the means ± SEM for five separate donors at each time point.

As would be expected, 99.9% of genes in normal human monocytes were neither up- nor down-regulated at the zero-hour time point. There were 785 genes with expression ratios of 2.5 measured over 2 to 18 h, and 87 of these genes were related to immune functions according to Gene Ontology (http://www.geneontology.org) and recent references (2, 10, 12, 18, 19, 25, 31, 41, 43, 47, 57, 59). Figure 2 illustrates the gene expression from 0 to 18 h in normal human monocytes in response to C. albicans for genes filtered by a ratio of 2.5. Some up-regulated genes were markedly expressed, especially at the 4-h and 6-h time points, showing expression ratios greater than 30.

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FIG. 2. Gene expression heat map of up-regulated genes in normal human monocytes in response to Candida albicans, demonstrating 785 genes that were up-regulated 2.5-fold during the 18-h time course.

Figure 3 demonstrates the expression of genes belonging to immunologically defined classes of molecules of the innate host response elicited by C. albicans over the 18-h time course.

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FIG. 3. Heat map of gene expression for immunologically defined classes of molecules of innate host response in normal human monocytes in response to Candida albicans during the 18-h time course.

The temporal sequences of expression of genes encoding innate host defense proinflammatory and chemotactic regulatory molecules are depicted in Fig. 4 and 5, respectively. Genes encoding proinflammatory molecules were highly expressed in response to C. albicans (Fig. 4). Genes expressing cytokines, such as tumor necrosis factor alpha (TNF-), interleukin 1 (IL-1) ( and ß), IL-6, the colony-stimulating factors macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, and leukemia inhibitory factor (LIF), were highly induced during the first 6 h. Among these genes, up-regulation of TNF was earliest, showing a marked increase by the 2-h time point and increasing to a 50- ± 10-fold change at 6 h. Levels of TNF expression steadily declined over the subsequent time points, reaching 2.9 ± 0.4 by 18 h. In comparison, expression of the gene encoding LIF peaked at 2 h (12- ± 2.4-fold change) and returned to near-baseline levels by 8 h (1.4- ± 0.07-fold change).

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FIG. 4. Kinetics of expression of genes encoding proinflammatory molecules of normal human monocytes in response to Candida albicans over 18 h. Data represent the mean changes (n-fold) in expression for five separate donors at each time point.

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FIG. 5. Kinetics of expression of genes encoding chemotaxis regulatory molecules in normal human monocytes in response to Candida albicans over 18 h. Data represent the mean changes (n-fold) in expression for five separate donors at each time point.

Additionally, the expression of genes encoding several chemotaxis-regulatory molecules was highly elevated in response to C. albicans, initially reaching peak expression at 4 to 6 h of exposure (Fig. 5). For example, there was a 48- ± 6.9-fold change in MIP1B expression in response to C. albicans at 2 h and 115- ± 28-fold change by 6 h. These levels of expression were not sustained, however, returning to near baseline (2.9- ± 0.94-fold change) by 18 h. The expression of genes encoding CC chemokines, including those for macrophage inflammatory protein 1 (MIP-1), MIP-1ß, MIP-3, and MIP-4, also was highly elevated in response to C. albicans. Additionally, genes encoding the CXC chemokines, GRO-, GRO-, and IL-8, were highly expressed in the first 6 h.

Genes encoding chemokine receptors were also examined (Table 1). In response to C. albicans, expression of genes encoding CCR1, CCR5, CCR7, and CXCR5 were elevated 3- to 5-fold, while the gene encoding CCR2 was highly suppressed by >100-fold.

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TABLE 1. Expression of genes encoding chemokine receptors

Genes related to other host response functional groups were also examined in depth. The expression of genes encoding prostaglandin-related molecules in response to C. albicans was of smaller magnitude than the expression of genes encoding inflammatory and chemotaxis factors (Table 2). The exception to this was the COX2 gene for prostaglandin G/H synthase, whose change in expression increased over the first 6 h (23- ± 4.3-fold change). While there was a steady decline thereafter, the expression of COX2 remained elevated (4.5- ± 0.59-fold change) at 18 h (Table 2). Conversely, expression of genes encoding prostacyclin receptor (PTGIR) and prostaglandin E receptor 4 (PTGER4) did not change in response to C. albicans over the 18-h time course.

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TABLE 2. Expression of genes encoding prostaglandin-related molecules

Among 10 key genes involved in T-cell regulation, including IL12A, IFNG, and TGFB2, only 3 genes increased or decreased in expression in response to C. albicans over the 18-h time course (Table 3). Expression of the genes encoding IL-23 and CD80 was up-regulated at 2 to 4 h but returned to baseline by 18 h. Expression of IL-15 was suppressed maximally at 6 h and also returned to near baseline by 18 h.

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TABLE 3. Expression of genes encoding T-cell-regulatory molecules

Four of the 16 genes encoding immune receptors and toll-like receptors were substantially affected by C. albicans (Table 4). Of note, genes encoding CD14 and IL-13R were down-regulated beginning at 4 to 6 h, with a steady decline in expression thereafter. Genes encoding CD83 and IL-6R were up-regulated between 2 and 4 h and remained elevated throughout the 18-h time course (Table 4).

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TABLE 4. Expression of genes encoding immune receptors

A group of genes encoding molecules that may play a role in protecting monocyte viability were also examined (Table 5). These genes, including those encoding metallothioneins, apoptosis inhibitors, CD71, and suppressor of cytokine signaling 3 (SOCS3), were up-regulated in the first 2 to 4 h of exposure to C. albicans, and while expression decreased from peak levels achieved at 4 to 6 h, they all remained elevated at the end of the 18-h incubation period.

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TABLE 5. Expression of genes encoding molecules that may protect monocyte viability

Genes encoding several heat shock proteins (HSPs) in the monocytes were also found to be elevated, reaching peak levels between 6 and 8 h (Table 6). Genes expressing connexins 26 and 31 were highly up-regulated in the monocytes following the 2-h incubation with C. albicans and remained elevated throughout the 18-h time course (Table 7). In contrast, there was no change in expression of genes encoding connexin 43 over the course of 18 h.

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TABLE 6. Expression of genes encoding heat shock proteins

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TABLE 7. Expression of genes encoding connexins

Among the genes expressing transcription factors, those for NF-B1, NF-B3, and RELB did not meet our filter criteria for changes over the 18-h time course (data not shown).

Top Abstract Introduction Materials and Methods Results Discussion References

 
To our knowledge, this is the first study to investigate the kinetic profile of gene expression for innate host defense molecules of normal human monocytes in response to C. albicans. Previously published studies have evaluated expression of innate host defense molecules in response to opportunistic pathogens, including those of Listeria monocytogenes, Escherichia coli, influenza virus, and C. albicans (8, 23). However, these studies have analyzed expression profiles only at single points in time and have utilized neoplastic monocytic cell lines or dendritic cells. By comparison, our studies were designed to assess the kinetics of expression of genes encoding multiple innate host defense molecules over an 18-h time course following initial interaction between normal human monocytes and C. albicans. Characterization of the kinetics of gene expression over time provides a more accurate understanding of the dynamic innate host responses than a single time point. Moreover, given the potential alteration of complex signal transduction pathways in transformed cell lines, the importance of utilizing normal human monocytes in understanding the pattern of normal human innate host response to Candida infections is paramount.

The increased expression of genes encoding the proinflammatory cytokines TNF-, IL-6, and IL-1ß within the first 6 h of exposure to C. albicans correlated well with the chronological sequence of neutrophil infiltration into infected tissue, which is known to peak within the first 6 h of an inflammatory response (16). TNF- is an essential molecule for the successful control of infection and the development of a Th1-dependent response. Using TNF- knockout mice, Netea et al. have shown that neutrophil recruitment and phagocytosis of Candida are impaired in animals lacking TNF- (37). IL-6, which is a multifunctional cytokine, is also known to play an important role in innate host response against C. albicans by eliciting an effective neutrophil response (51). Again using a murine model, Romani et al. have demonstrated increased susceptibility to Candida infection in IL-6-deficient animals (42). IL-1 is a key proinflammatory cytokine involved in the induction of adhesion molecules on endothelial cells, an important step in the development of a local inflammatory process. Like TNF- and IL-6, IL-1 is an important activator of phagocytes, and it is highly expressed by monocytes infected with C. albicans (27).

The expression of genes encoding several chemokines associated with the recruitment and activation of phagocytes was also evaluated in these studies. Our data showed that yeast cells of C. albicans induced strong expression of genes encoding MIP-1, MIP-1ß, IL-8, and monocyte chemoattractant protein 1, chemokines that are known to be induced in response to yeast phase organisms (50). Considerably less is known about other chemokines in relationship to C. albicans. This study reveals that genes encoding MIP-3, MIP-4, GRO-, and GRO- are also involved in the early host response to this organism. Expression of genes encoding these chemokines may be beneficial for the early recruitment of inflammatory cells against C. albicans while still in its yeast phase. In contrast, expression of the gene encoding the chemokine receptor CCR2 was progressively down-regulated in response to C. albicans. This is consistent with previously published data showing a rapid and drastic reduction in CCR2 mRNA levels in a monocytic cell line in response to lipopolysaccharide (LPS) (46). To our knowledge, this is the first study to demonstrate the kinetics of the coordinated expression of multiple genes encoding chemokines, as well as key proinflammatory cytokines, in response to C. albicans.

By comparison, expression levels of most T-cell-related genes remained unchanged or were down-regulated in our study. For example, IL-12- and gamma interferon (IFN-)-related genes were not elevated over the 18-h time course. Expression of the gene encoding IL-15, a pleotropic proinflammatory cytokine previously shown to up-regulate the antimicrobial activity of human monocytes against C. albicans (52), was down-regulated in the first 6 h of incubation. Recently, IL-15 was shown to be involved in T-cell responses, as well as the generation and maintenance of CD8⁺ memory T cells (45). It may be hypothesized that IL-15 gene expression was suppressed initially to prevent early activation of T cells.

In contrast, there was up-regulation in the expression of the genes encoding CD80 and IL-23. CD80 is induced by microbial products involved in priming naïve T cells but also may inhibit T-cell responses (30). While IL-23 shows similarities to IL-12, its gene transcription and protein secretion do not always parallel those of IL-12. Verreck et al. have shown that stimulation of human type 1 macrophages with LPS activates gene transcription and secretion of IL-23, but not IL-12, suggesting different roles for IL-23 and IL-12 in macrophage-dependent host defense (53). This is consistent with our findings of up-regulation in expression of IL-23 but not IL-12 in monocytes in response to C. albicans. Thus, these microarray findings indicate that genes encoding T-cell-regulatory molecules may not play a significant role within the first 18 h of monocyte exposure to C. albicans. These genomic data are consistent with previous functional studies that indicate that the initial immune response to C. albicans appears to be more dependent on innate immunity than on adaptive immunity (4, 41).

There also was no appreciable change in the expression of genes encoding Toll-like receptors in our studies. Toll-like receptors are important pattern recognition receptors involved in sensing pathogenic microorganisms (49). TLR2 and TLR4 may be involved in the host response to Candida infection (36). Both TLR2 and TLR4 are involved in the recognition of C. albicans, and TLR2-derived signals appear to mediate increased production of IL-10, thus inducing an impaired immune response to invasive candidiasis (35). Our findings suggest that the interaction between TLR2 and C. albicans may occur at a posttranscriptional level, without affecting gene expression within the first 18 h. This is further supported by the observation that expression of genes encoding NF-B and p38, which are involved in signal pathways for TLRs (21), also was not significantly altered during this time course (data not shown). Signal transduction may occur at a posttranslational level without increased expression of the genes encoding these molecules.

These microarray studies reveal changes in expression of a number of surface receptors and signaling molecules on monocytes that heretofore have not been demonstrated to have a role in invasive candidiasis. Expression of the gene encoding a transferrin receptor in monocytes, CD71, increased in response to C. albicans. CD71 has been found to be distributed on a wide range of cells and to be particularly abundant on proliferating lymphoid and erythroid cells (22). The production of reactive oxygen and nitrogen intermediates is modulated by intracellular iron concentrations. Ferritin and the transferrin receptors are coordinately regulated in response to oxidative stress, which has been associated with the expansion of the intracellular free-iron pool and protection against tissue injury (15). Given the importance of iron in the virulence of C. albicans (33), increased expression of CD71 may increase intracellular iron concentrations and decrease extracellular iron, thereby depriving C. albicans of a critical element.

In contrast to CD71, expression of CD31 was decreased approximately 10-fold by 18 h in response to C. albicans. Expression of CD31 on monocytes has been shown to decrease in response to C-reactive protein, which may affect their binding and diapediesis at sites of inflammation (55). Also, there are findings that implicate CD31 as an inhibitor of cellular activation via the protein tyrosine kinase-dependent signal pathway, an activator of integrins, and a suppressor of cell death via pathways that depend on damage to the mitochondria (38). The progressive down-regulation of CD31 over the 18-h time course, as observed in our study, may allow activation of monocytes against C. albicans. Further studies are necessary to define the specific role of CD31 in Candida infections.

Several genes involved in apoptosis also were found to be responsive to C. albicans within the first 18 h of infection. XIAP, which is a potent inhibitor of cell death, was up-regulated in the monocytes, along with other genes inhibiting apoptosis, such as BCL2A1. Additionally, genes encoding caspases were not activated, also suggesting that apoptosis was inhibited. Over the past several years, it has become evident that programmed death of cells of the monocyte/macrophage lineage also may be differentially affected by microorganisms (11, 16). Induction of apoptosis by microorganisms may allow evasion of the innate cellular host response. On the other hand, resistance by monocytes to organism-induced apoptosis may confer an enhanced host response. Heidenreich et al. showed that infection by C. albicans inhibited apoptosis of human monocytes (20). The combined genetic and functional studies collectively indicate that normal human monocytes are resistant to induction of apoptosis by C. albicans.

The gene encoding SOCS3 was elevated by 2 h in response to C. albicans and remained elevated through the remaining time points. SOCS proteins have been characterized as feedback inhibitors of cytokine receptor signaling mediated by the JAK family kinases and thus may protect the host by limiting excessive cytokine production (1, 57). Consistent with our observations of no change in expression of the gene encoding IL-12, SOCS3 has been reported to inhibit IL-12 response (56).

Other genes that encode proteins possibly conferring protection of monocytes against oxidative and nonoxidative injury were found to be up-regulated in response to C. albicans. For example, expression of genes encoding metallothioneins 1H, 1L, 1G, and 2A increased in response to Candida, peaking at 4 to 6 h of exposure. Metallothionein is the primary zinc-binding protein within cells, responsible for zinc transfer to a number of enzymes and transcription factors (58). Treatment of human monocytes with LPS results in a rapid increase in metallothionein mRNA and protein expression (28). Since metallothionein proteins have been shown to scavenge both hydroxyl and superoxide radicals in vitro, they may play a protective role in monocytes by scavenging the oxygen free radicals produced by the respiratory burst during activation in response to C. albicans. Conversely, C. albicans may increase metallothionein levels in order to counteract the oxidative host defense mechanism, as is thought to be the case with L. monocytogenes (8). Further study is necessary to evaluate the role of metallothioneins in C. albicans infection.

Connexins are a family of homologous proteins that provide permeability and regulatory properties to the gap junction channels they form, allowing cells to communicate by intercellular transfer of ions and small compounds (6). Their role in the pathogenesis of candidiasis is unknown. Monocyte/macrophage responses may be mediated by connexin-formed membrane channels that are expressed transiently at inflammatory sites where these cells aggregate. The induction of connexin 43 expression has been shown in monocytes treated with either LPS plus IFN- or TNF- plus IFN- (13). In the studies reported here, expression of genes encoding connexins 26 and 31 was up-regulated in response to C. albicans, while there was no change in expression of the gene encoding connexin 43. The up-regulation of genes encoding connexins 26 and 31 suggests that other members of the family of connexins may be involved in gap junction communication between activated monocytes and endothelial cells, neutrophils, or other monocytes/macrophages in response to Candida. The possible functional role of hemichannels in the immune response of monocytes to C. albicans has not been reported, and further studies are required to understand the roles of these genes in host defense against C. albicans.

The expression of genes encoding several heat shock proteins was increased in monocytes in response to C. albicans in our studies. HSPs are abundant and ubiquitous soluble intracellular proteins that are present in virtually all cells. The interaction between human HSP-peptide complexes and antigen-presenting cells leads to their participation in innate and adaptive immune responses (47). HSPs may participate in innate immunity by inducing the secretion of inflammatory cytokines and chemokines (48). Although Candida HSPs have been studied (39, 44), the role of human HSPs in host defense against candidiasis has not been investigated.

These data demonstrate the utility of the microarray as a powerful method for assessing kinetic changes in expression of innate host response genes in monocytes when exposed to a fungal pathogen. Microarray analysis also identifies genes heretofore not known to be involved in the immunopathogenesis of invasive candidiasis. There are limitations, however, in the interpretation of microarray data. For example, expression of mRNA may not necessarily coincide with or reflect extracellular protein release either quantitatively or qualitatively. A given signal transduction pathway may lead to an increase in protein release through posttranslational modification, such as glycosylation, without an increase in expression of mRNA. Furthermore, increased production of mRNA transcripts may not immediately result in protein synthesis, nor does increased protein synthesis necessarily imply extracellular release or cell membrane surface expression. Nonetheless, these microarray data do offer for the first time an insight into the kinetics of the coordinated expression of genes in response to C. albicans.

Another potential problem in the interpretation of these data are the limited interaction of the organism with a single cell type, in this case, monocytes. In vivo, there is a complex interaction between C. albicans and monocytes, neutrophils, lymphocytes, dendritic cells, and epithelial cells, as well as immunoglobulins and mannose binding lectins. However, as detailed earlier, we found that the data collected in this study correlated well with the limited but important data derived from animal model systems.

These microarray data provide a conceptual framework for the kinetic profiles of groups of genes encoding innate host defense molecules that may permit the model in Fig. 6. During the first 6 h of infection of normal human monocytes by C. albicans, genes encoding proinflammatory cytokines, chemokines, and some chemokine receptors, as well as COX2, IL-23, and heat-shock proteins, are up-regulated, leading to cellular recruitment and activation. In concert with this proinflammatory response, genes encoding antiapoptosis molecules, metallothioneins, and SOCS3 are up-regulated, possibly to protect monocytes from cytokine- and organism-mediated injury. Expression of the gene encoding the transferrin receptor (CD71) is also increased, perhaps to sequester iron within the monocyte and away from C. albicans. On the other hand, the chemokine receptor CCR2 is down-regulated during this early time course of infection.

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FIG. 6. Summary kinetic profile of expression of immunologically important classes of genes in monocyte host defense against C. albicans over an 18-h time course. The expression of genes encoding proinflammatory cytokines, chemokines, some chemokine receptors, COX2, heat shock proteins, antiapoptosis molecules, connexins, metallothioneins, transferrin receptor, and SOCS3 are all up-regulated in association with early monocyte response to C. albicans. By comparison, the gene encoding the chemokine receptor CCR2 is down-regulated within the first 18 h of exposure to C. albicans. Data represent mean changes (n-fold) for genes in each group with 2.5-fold change.

Thus, our findings demonstrate that C. albicans is a potent inducer of genes encoding proinflammatory cytokines and chemokines, as well as numerous other molecules involved in the initial host response to this organism. Defense mechanisms against C. albicans clearly involve regulation of several subsets of genes simultaneously. The kinetic approach used in this microarray study has elucidated a coordinated and temporal basis of host defense molecules elicited against C. albicans infections. Characterization of the unique kinetic profile of gene expression in response to Candida also offers a greater understanding of the early disease process and may lead to more innovative methods of immunotherapy.

 
We thank Gene M. Shearer (NCI, NIH) for his helpful advice and for reviewing the manuscript.

 
* Corresponding author. Mailing address: Immunocompromised Host Section, Pediatric Oncology Branch, NCI, NIH, CRC, Room 1-5740, Bethesda, MD 20892. Phone: (301) 402-0023. Fax: (301) 402-0575. E-mail: walsht{at}mail.nih.gov.

Editor: T. R. Kozel

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, June 2005, p. 3714-3724, Vol. 73, No. 6
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.6.3714-3724.2005

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