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Published in final edited form as: Exp Eye Res. 2009 Dec 11;90(3):413–419. doi: 10.1016/j.exer.2009.12.001

Proinflammatory Chemokines during Candida albicans Keratitis

Xiaoyong Yuan 1, Xia Hua 1, Kirk R Wilhelmus 1,*
PMCID: PMC2824024  NIHMSID: NIHMS165097  PMID: 20005222

Abstract

Chemotactic cytokines mediate the recruitment of leukocytes into infected tissues. This study investigated the profile of chemokines during experimental Candida albicans keratitis and determined the effects of chemokine inhibition on leukocyte infiltration and fungal growth during murine keratomycosis. Scarified corneas of BALB/c mice were topically inoculated with C. albicans and monitored daily over one week for fungal keratitis. After a gene microarray for murine chemokines compared infected corneas to controls, real-time reverse transcription polymerase chain reaction (RT-PCR) and immunostaining assessed chemokine expression in infected and mock-inoculated corneas. An anti-chemokine antibody was then administered subconjunctivally and evaluated for effects on clinical severity, corneal inflammation, fungal recovery, and cytokine expression. Of 33 chemokine genes examined by microarray, 6 CC chemokines and 6 CXC chemokines were significantly (P<0.05) upregulated more than two-fold. Chemokine (CC-motif) ligand 3 (CCL3) was upregulated 108-fold (P=0.03) by real-time RT-PCR within one day after fungal inoculation and remained increased 28-fold (P=0.02) at one week, and its in situ expression increased in the epithelium and stroma of infected corneas. Compared to the control antibody-treated group, eyes treated with anti-CCL3 antibody showed reduced clinical severity (P<0.05), less corneal neovascularization (P=0.02), and fewer inflammatory cells infiltrating corneal tissue, but the amount of recoverable fungi was not significantly (P=0.4) affected. Anti-CCL3 treatment significantly (P=0.01) reduced the expression of tumor necrosis factor and interleukin-1β in infected corneas. These results indicate that chemokines, especially the CC chemokine CCL3, play important roles in the acute inflammatory response to C. albicans corneal infection.

Keywords: cornea, fungi, innate immunity, keratitis, mycology, pathogenesis

1. Introduction

Candida albicans, a component of the normal flora, is a fungal opportunist for the eye (Kercher et al., 2001). Trauma and disease predispose to corneal infection (Sun et al., 2007). During C. albicans keratitis yeasts transition into filamentary forms that breach the eye’s defenses and penetrate the stroma (Jackson et al., 2007). Hyphal invasion elicits host responses that bring about corneal inflammation and ulceration (Yuan et al., 2009).

Innate immunity and acute inflammation actively participate in the pathophysiology of fungal keratitis. The cornea detects the presence of invasive C. albicans by toll-like receptors and other pathogen-recognition molecules (Yuan and Wilhelmus, 2010). Corneal epithelial cells, keratocytes, and phagocytes are involved in distinguishing pathogen-associated molecular patterns, and this interaction activates innate responses. Neutrophils, key effector cells for controlling fungal growth, afford a first line of defense during mucosal C. albicans infection.

Leukocytes are drawn into the cornea by the local production of chemotactic cytokines during the initial stages of fungal keratitis (Zhong et al., 2009). Chemokines are released at an early stage of fungal infection and bind to receptors that trigger the synthesis of interleukins and other cytokines that promote fungal clearance (Traynor and Huffnagle, 2001). Members of the CC chemokine subfamily such as CCL2 and CCL3 have potent chemotactic and activating properties for neutrophils and are rapidly induced in bacterial keratitis (Xue et al., 2007).

This study examined the profile of chemokines after the onset of experimental C. albicans keratitis and followed the relative expression of selected CC chemokines during the first week of fungal keratitis in mice. We also studied the effect of an anti-CCL3 antibody on inflammatory severity, fungal growth, and cytokine expression during C. albicans keratitis. Our findings suggest that chemokines such as CCL3 facilitate innate immune responses in the pathogenesis of fungal keratitis.

2. Methods

2.1. Fungi and media

C. albicans strain SC5314, a clinical isolate capable of producing experimental keratomycosis, was cultured on Sabouraud dextrose agar (Difco, Detroit, MI) for 3 days at 25°C. Colonies were harvested after 3 days of inoculation and diluted in sterile phosphate-buffered saline (PBS) to yield 2 × 105 colony-forming units (CFU)/μL based on the optical density (OD) at 600 nm, using an OD600 conversion factor of 3 × 107 CFU/mL.

2.2. Animal model

Animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research under protocols approved by the Baylor College of Medicine Institutional Animal Care and Use Committee. Female BALB/c mice 6 to 8 weeks of age (Harlan Sprague-Dawley, Houston, TX) were anesthetized with an intraperitoneal injection of ketamine, xylazine, and acepromazine. The corneas of right eyes were superficially scarified with a 22-gauge needle. A 5-μL inoculum of either C. albicans containing 1 × 106 CFU or sterilized PBS was topically applied to eyes of infected and control groups, respectively. Mice were monitored daily for 7 days postinoculation (p.i.) using a dissecting microscope to categorize corneal inflammation and angiogenesis. The severity of keratitis was graded by a scoring system that consisted of the relative area of corneal infiltrate, density of corneal opacity, and surface regularity (Wu et al., 2003). The amount of corneal vascularization was assessed by a neovascularization scoring system that assigned grades of 0 to 4 for the number, density, and length of visible corneal blood vessels (Yuan and Wilhelmus, 2009). Corneal photographs with the eye positioned en face or in lateral profile were captured with a Zeiss photo slit-lamp and Nikon digital camera.

2.3. RNA extraction

Mice were sacrificed 1 day p.i. After enucleation corneas were excised and dissected from surrounding conjunctiva and uvea. Pools of 5 corneas were prepared in triplicate from C. albicans-infected and control groups at day 1 p.i. RNA was extracted by a previously reported procedure (Yuan et al., 2009) and was isolated with RNeasy MicroKit columns (Qiagen, Valencia, CA). Samples were treated with DNase (Qiagen, Valencia, CA) and stored at −80°C.

2.4. Gene microarray

Genetic analysis was performed at the Microarray Core Facility, Baylor College of Medicine, as reported (Yuan et al., 2009). After checking RNA samples for quality assurance, Genechip (Affymetrix, Santa Clara, CA) microarray protocols were applied to qualified samples of 3 five-cornea pools from each group for two cycles of amplification. Images and quality control metrics were recorded using Affymetrix GCOS software version 1.4, and signal-intensity data were adjusted and analyzed with BioConductor software. The criterion for significance of differentially regulated genes was established as > 2-fold change with adjusted P < 0.05.

2.5. Quantitative polymerase chain reaction

Total RNA isolated from corneas at 1, 3, and 7 days p.i. was quantified by absorbance at 260 nm. The first-strand cDNA was synthesized from 0.4 μg RNA with Ready-To-Go You-Prime First-Strand Beads (GE Healthcare, Princeton, NJ) and random hexamers (Applied Biosystems, Foster City, CA). Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed using TaqMan Gene Expression Master Mix and Assays (Applied Biosystems). Primers specific for CCL3, CCL2, CXCL1, TNF, IL-1β, IL-6, VEGF-A, MMP8, MMP13, and CRAMP (Applied Biosystems) were used to quantify gene expression levels. The threshold cycle (CT) for each target mRNA was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and averaged. Three five-cornea pools were processed for each group. Two-group comparisons were done using the Student t-test, and three-group comparisons used one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.

2.6. Immunofluorescence

Eyes obtained 1 day p.i. were embedded in OCT compound (Sakura Finetek, Torrance, CA), snap-frozen in liquid nitrogen, and sectioned at 15-μm thickness. Sections were thawed, dehydrated, and fixed in 2% paraformaldehyde then blocked with 10% normal donkey serum (Jackson ImmunoResearch Laboratories, Philadelphia, PA). Immunofluorescent staining was performed as reported (Yuan et al., 2009). Goat serum antibody to the N-terminus of murine CCL3 (R&D Systems, Minneapolis, MN) was diluted 1:100, and applied to blocked sections that were incubated overnight at 4°C. Secondary Alexa-Fluor 488-conjugated donkey anti-goat immunoglobulin (Invitrogen, Carlsbad, CA) was applied to sections that were then incubated in a dark chamber for 1 hour and counterstained with propidium iodine (Invitrogen) in Gel/Mount (Biomeda, Foster City, CA). Sections were observed with a laser-scanning confocal microscope (LSM 510, Zeiss, Thornwood, NY) with 488- and 543-nm excitation and emission filters. Images were acquired with a 40× oil-immersion objective and processed using Zeiss LSM-PC software.

2.7. Anti-CCL3 treatment

Goat anti-mouse CCL3 and control goat immunoglobulin were obtained (R&D Systems). Five mice were allocated to treatment and control groups, respectively. Each mouse was administered 10 μg of antibodies subconjunctivally 30 minutes before superficial corneal scarification. Scarified corneas of the treated group were topically inoculated with C. albicans at a dosage of 1 × 106 CFU/5μL. Control mice received 5-μL of normal goat IgG antibody. Eyes were observed daily with a dissecting microscope to grade the severity of keratitis. Ten additional mice were treated subconjunctivally with either anti-CCL3 antibody or goat IgG 30 minutes before fungal inoculation and then sacrificed one day p.i. for fungal recovery.

2.8. Quantitative fungal recovery

Quantitative fungal cultures were performed on excised corneas by a previously reported method (Yuan and Wilhelmus, 2009). In brief, corneas were homogenized in a frosted-glass grinder with 500 μL PBS, and the homogenated aliquot was diluted 10-fold with PBS. The entire aliquot was then inoculated onto Sabouraud dextrose agar that was incubated for 4 days at 25°C. Visible colonies were counted, and the number of CFUs per cornea was compared between treated and control groups with the Student t-test.

3. Results

3.1. Experimental fungal keratitis

All eyes inoculated with C. albicans developed clinical signs of keratitis. Corneal inflammation began 1 day p.i. (mean keratitis score ± SD, 7.4 ± 0.5), peaked 3 days p.i. (8.0 ± 0.7), and then diminished by 7 days p.i. (6.2 ± 0.8). Capillary budding of limbal vessels began 3 days p.i (mean neovascularization score ± SD, 6.3 ± 0.6) and progressed until 7 days p.i (8.7 ± 0.6). Histopathological evaluation of infected eyes 1 day p.i. revealed partial to total loss of epithelial integrity, the infiltration of neutrophils and macrophages in the stroma, the presence of acute inflammatory cells within the anterior chamber, and the invasion of fungal pseudohyphae and hyphae within the anterior stroma (Wu et al., 2003). No corneal inflammation or neovascularization was found in eyes from mock-inoculated controls or normal mice.

3.2. Chemokine gene expression profile

Gene arrays of C. albicans-infected corneas and mock-inoculated control corneas were compared for chemokine expression. Average ratios of expression levels at 1 day p.i. (Table 2) showed that 12 chemokine genes increased more than 2-fold (P<0.05) and that 2 chemokines decreased more than 2-fold (P<0.05). Compared to mock-infected controls, CCL2, CCL3, CCL4, CCL6, CCL7, and CCL24 were upregulated among CC group chemokines. CXCL1, CXCL3, CXCL4, CXCL12, CXCL14, and CXCL16 were upregulated in the CXC group. CXCL13 and CX3CL1 were slightly downregulated.

Table 2.

Microarray analysis of chemokine gene expression ratios comparing C. albicans keratitis to mock-infected controls.

Systematic name Original name(s) Relative regulation P value
CC group
 CCL1 TCA3, I-390 −1.0 0.74
 CCL2 MCP-1 38.6 0.015
 CCL3 MIP-1α 374.3 0.04
 CCL4 MIP-1β 145.2 0.011
 CCL5 RANTES −2.1 0.36
 CCL6 MPR-1 17.4 0.0011
 CCL7 MCP-3 47.1 0.012
 CCL8 MCP-2 −1.5 0.37
 CCL12 MCP-5 2.1 0.32
 CCL17 TARC −1.0 0.96
 CCL19 MIP-3β, ELC, exodus-3 −1.2 0.80
 CCL20 MIP-3α, LARC, exodus-1 −1.0 0.12
 CCL21 6Ckine, SLC, exodus-2 −2.2 0.84
 CCL22 ABCD-1, MOC 1.1 0.31
 CCL24 MPIF-1 3.8 0.024
 CCL25 TECK −1.0 0.88
 CCL27 CTACK 1.4 0.42
 CCL28 MEC 1.0 0.94
CXC group
 CXCL1 KC 83.6 0.037
 CXCL2 MIP-2α 122.7 0.093
 CXCL3 MIP-2B 291.7 0.044
 CXCL4 PF4 5.4 0.016
 CXCL5 LIX, ENA78 132.4 0.052
 CXCL9 MIG −1.1 0.039
 CXCL10 IP-10, CRG-2 −1.6 0.58
 CXCL11 ITAC −2.6 0.086
 CXCL12 SDF-1 5.5 0.022
 CXCL13 BLC, BCA-1 −2.1 0.0095
 CXCL14 BRAK 68.3 0.0045
 CXCL15 Lungkine −1.0 0.82
 CXCL16 SR-PSOX 2.7 0.039
C group
 XCL1 Lymphotactin-α −1.0 0.62
CXXXC group
 CX3CL1 Fractalkine −2.7 0.0022
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Transcript levels of CCL3 and CCL2 were detected by quantitative real-time RT-PCR on total RNA extracted from groups of five-cornea pools of infected or mock-infected corneas (Fig. 1). Differences of CCL3 transcript were found between C. albicans keratitis and scarified controls at day 1 (108.1-fold, P=0.03), day 3 (41.7-fold, P=0.03), and day 7 (27.6-fold, P=0.02) p.i. In infected corneas, CCL3 transcripts were upregulated highly on day 1 p.i. then declined toward a baseline level but remained significantly increased after 3 days p.i. CCL2 was upregulated at day 1 p.i. (11.0-fold, P=0.01) and remained relatively upregulated in infected corneas at day 3 p.i. (9.3-fold, P=0.03) but not at day 7 p.i. (9.0-fold, P=0.13).

Figure 1.

Figure 1

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Differential gene expression ratios of CCL3 and CCL2 in C. albicans-infected corneas compared to mock-inoculated corneas.

3.3. CC chemokine protein expression pattern

The in situ protein expression pattern determined by immunofluorescent staining at day 1 p.i. was consistent with transcript levels measured by real-time RT-PCR (Fig. 2). CCL3 was present in the epithelium of control corneas. Immunostaining for CCL3 was relatively more intense in the epithelium of infected corneas and was present throughout the stroma. CCL2 was present in mock-inoculated corneas, with moderate epithelial staining and minor stromal staining, and was increased in infected corneas, with enhanced staining of the epithelial and stromal layers. Negative controls in which no primary antibody was used demonstrated no detectable staining by immunofluorescence.

Figure 2.

Figure 2

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Molecular expression patterns in situ in C. albicans keratitis (Infected) or mock-infected controls (Mock). Negative controls included no primary antibody (Control). Sections were processed one day after fungal inoculation for indirect immunofluorescence using a cyan-green-labeled secondary antibody and propidium iodine as a nuclear counterstain.

3.4. Treatment effect of CCL3 neutralizing antibody on C. albicans keratitis

Mice treated with anti-CCL3 antibody showed significantly reduced severity (Fig. 3) at day 1 (mean keratitis score ± SD, 5.4 ± 1.1), day 3 (6.6 ± 0.5), and day 7 (4.0 ± 0.7), indicating reduced corneal inflammation during C. albicans corneal infection compared to the goat IgG control antibody-treated group at day 1 (7.2 ± 0.8, P=0.022), day 3 (7.4 ± 0.5, P=0.049), and day 7 (5.6 ± 0.9, P=0.014). Slit-lamp biomicroscopy also showed a substantially reduced corneal opacity in anti-CCL3 antibody-treated mice in comparison to the control antibody-treated group (Fig. 4). No difference was detected between the goat IgG control antibody-treated group and the untreated infected group during the clinical course (P>0.05). Corneal neovascularization was less in anti-CCL3-treated mice than in goat IgG antibody-treated controls (Fig. 4). An inhibitory effect was apparent by 3 days p.i., and treated animals continued to have a lower corneal neovascularization score on day 1 (4.0±1.0, P=0.64) and day 3 p.i. (5.3 ± 0.6, P=0.047). At 7 days p.i. the average vascularization score (5.33±1.52) in treated mice remained significantly less (P=0.024) than that (8.3 ± 0.6) of controls.

Figure 3.

Figure 3

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Clinical evaluation of C. albicans keratitis in anti-CCL3 treated, goat IgG antibody-treated, and untreated mice. Each point represents the mean severity score with standard deviation.

Figure 4.

Figure 4

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Clinical appearance of C. albicans keratitis among anti-CCL3-treated, goat IgG antibody-treated, and untreated mice at day 1 and day 7 p.i., with corneal profile views showing the extent of corneal neovascularization at 7 days.

Histopathological examination demonstrated less inflammatory cell infiltration and less structural disruption in corneal tissue in the anti-CCL3-treated group compared to the goat IgG-treated group (Fig. 5). Cultures from excised corneas at 1 day p.i. showed no significant difference (P = 0.35) in the mean number (± SD) of viable fungi recovered from control corneas (13,750 ± 9,311 CFU/cornea) compared to those treated with anti-CCL3 antibody (9,250 ± 3,739 CFU/cornea). Treatment with anti-CCL3 antibody significantly reduced RNA expression of CCL3 (−4.30-fold, P=0.01), TNF (−2.81-fold, P=0.01), and IL-1β (−2.23-fold, P=0.01) compared to the goat IgG-treated group at day 1 p.i. (Table 3). The expression of CCL2, CXCL1, IL-6, VEGF-A, MMP-13, MMP-8, and CRAMP did not change significantly.

Figure 5.

Figure 5

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Histological examination showed that infected corneas pretreated with anti-CCL3 antibody (A) had relatively mild stromal infiltration while control eyes (B) had marked infiltration by neutrophils in the stroma and anterior chamber.

Table 3.

Real-time RT-PCR comparison of relative gene expression for corneal inflammatory molecules in C. albicans keratitis, comparing anti-CCL3-treated group to controls.

Gene Treated* Control* Fold-change** P value
CCL3 5.33±0.52 3.23±0.61 −4.30 0.01
CCL2 6.07±0.12 5.83±0.20 −1.18 0.14
CXCL1 4.37±0.32 3.76±0.31 −1.53 0.08
TNF 6.70±0.28 5.21±0.43 −2.81 0.01
IL-1β 3.94±0.41 2.78±0.17 −2.23 0.01
IL6 6.23±0.35 5.76±0.52 −1.38 0.27
VEGF-A 5.50±0.06 5.10±0.06 −1.32 0.001
MMP13 4.69±0.29 4.46±0.17 −1.17 0.31
MMP8 9.90±0.37 9.30±0.31 −1.51 0.10
CRAMP 9.14±0.21 10.21±1.96 2.10 0.40
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*

Mean threshold cycle ± standard deviation for each target mRNA, normalized to GAPDH mRNA.

**

Relative fold-change of the comparison between normalized threshold cycles for each target mRNA in anti-CCL3-treated group compared to goat IgG antibody-treated group.

4. Discussion

4.1. Leukocyte recruitment during corneal infection

Innate immunity is a pivotal protective mechanism of the eye that involves corneal cells and leukocytes. Immunosuppression increases susceptibility to keratomycosis and aggravates fungal proliferation (Wu et al., 2003). Host immune responses are initiated by pathogen-recognition receptors such as toll-like receptors (TLRs) (Herring and Huffnagle, 2002). In C. albicans keratitis, TLRs recognize β-glucan of the C. albicans cell wall and lead to the expression of interleukins and chemotactic cytokines (Yuan and Wilhelmus, 2010). Synthesis of these soluble mediators actuates inflammatory reactions at the site of fungal infection (Traynor and Huffnagle, 2001).

Chemokines are secreted proteins that mediate cell migration, differentiation, and activation (Charo and Ransohoff, 2006). In a systematic nomenclature based on molecular structure (Zlotnik et al., 2006), chemokines are classed into subfamilies according to the arrangement of amino-terminal cysteine motifs. Members of the CC subfamily have two adjacent cysteine residues while those of the CXC subfamily have two cysteines separated by an amino acid. Produced by a variety of cells, CC and CXC chemokines actively participate in the immunopathological reactions contributing to the severity of microbial keratitis (Xue et al., 2003; Xue et al., 2007)

Certain chemokines influence the recruitment of leukocytes during fungal infection by enabling integrin-mediated diapedesis through local blood vessels and tissue infiltration (Romani, 2003). Neutrophils account for most of the leukocytes permeating the cornea during early fungal keratitis (Vemuganti et al., 2002). As in bacterial keratitis (Matsumoto et al., 2005), leukocytes are guided by chemokines that increase immediately after the onset of fungal corneal infection (Zhong et al., 2009). Since chemokines bind the glycosaminoglycan moiety of proteoglycans (Hamel et al., 2009), the extracellular matrix of the corneal stroma likely participates in the chemotactic gradient responsible for the directional movement of neutrophils and other inflammatory cells into the infected cornea (Yuan and Wilhelmus, 2010).

4.2. Chemokines in fungal keratitis

Chemokines mediate the recruitment of neutrophils and macrophages during experimental cryptococcosis, aspergillosis, and candidiasis (Saavedra et al., 1999; Herring and Huffnagle, 2002; Phadke and Mehrad, 2005) CCL3, formerly known as macrophage inflammatory protein-1 (MIP-1α), is a member of the CC chemokine subfamily that is made by the cornea during inflammatory stress (Menten et al., 2002; Yoon et al., 2007). Exposure to C. albicans induces the expression of CC chemokines (Inoue et al., 2007), and our studies demonstrate that CCL3 expression increases within one day after the onset of C. albicans keratitis. Immunofluorescent staining confirmed the presence of increased CCL3 in the corneal epithelium and stroma during early fungal infection. Leukocytes may augment the release of CCL3 (Sallusto and Baggiolini, 2008), but corneas inflamed by a perforating injury have minimally increased CCL3 levels (Spandau et al., 2003). Thus, corneal epithelial cells and fibroblasts appear to be directly involved in the substantial increase of selected chemokines that occurs in response to replicating microorganisms (Menten et al., 2002; Xue et al., 2007).

The profile of CC chemokines upregulated during early C. albicans keratitis is comparable to that of other corneal infections (Kernacki et al., 1998; Huang and Hazlett, 2003; Xue et al., 2007). In addition to CCL3, CCL2, also called monocyte chemoattractant protein-1 (MCP-1), is part of the inflammatory response during corneal infection (Melgarejo et al., 2009; Xue et al., 2007). Receptors for CCL3, such as CCR1 and CCR5, are expressed on tissue cells and leukocytes (Bacon et al., 2002). CC chemokines may also participate in corneal angiogenesis that can complicate fungal and bacterial keratitis. Corneal vessels form soon after the onset of C. albicans keratitis (Yuan and Wilhelmus, 2009), and both CCL3 and CCL2 promote neovascularization associated with corneal inflammation (Ogawa et al., 1999). Our studies with an experimental murine model indicate that CCL3 and other CC chemokines are involved in the pathogenesis of C. albicans keratitis.

CCL3 mediates acute corneal inflammation and chemoattracts leukocytes (Takano et al., 1999; Xue et al., 2007). Neutrophils predominate during early C. albicans keratitis and aid in fungal eradication (Hohl et al., 2006). Macrophages may also be important (Hu et al., 2009; Zhang et al., 2009). In addition to a role in directing innate responses, chemokines may assist in the development of adaptive immunity during fungal infection (Herring and Huffnagle, 2002). An interactive network of immune responses is likely responsible for the clinical manifestations of ocular inflammation caused by invasive fungi.

We hypothesize that the release of CCL3 leads to the recruitment of neutrophils and macrophages that may, in turn, produce additional CCL3 potentiating the inflammatory reaction and controlling fungal growth. The relative upregulation of chemokine expression in the cornea declines as innate immunity eradicates fungi. In the setting of antifungal therapy, CCL3 may result in undesirable inflammation contributing to stromal destruction and corneal opacification. Modifying the activity of CCL3 and other chemokines may offer a way to modify the outcome of fungal keratitis.

4.3. Chemokines as a therapeutic target in fungal keratitis

We studied how an anti-CCL3 antibody affected the progression of corneal inflammation and fungal infection during C. albicans keratitis. Inhibiting CCL3 activity modulated the severity of leukocytic infiltration, but a deleterious effect on fungal proliferation was not found. Our findings suggest that blocking components of the chemokine system may be an adjunctive therapeutic strategy to alter destructive inflammation during anti-infective treatment (Diab et al., 1999; Xue et al., 2007).

Several proinflammatory pathways are set into play by chemokines. Cytokines such as tumor necrosis factor-α and IL-1β increase during candidal infection (Kullberg et al., 1990; Murray et al., 2005; van der Graaf et al., 2005; Vonk et al., 2006). TNF-α, produced by neutrophils and macrophages, regulates leukocyte recruitment and promotes fungicidal activity during disseminated candidiasis (Romani, 2003). IL-1β also promotes neutrophil infiltration to the site of infection. The expression of these cytokines is downregulated after anti-CCL3 antibody is administered, suggesting that one or more CC chemokines help to direct the production of molecular intermediates during fungal keratitis. Our findings indicate that chemokines and cytokines have pivotal roles in intercellular signaling and that selective inhibition can modulate destructive responses during keratomycosis.

Table 1.

Primers Used for Real-time RT-PCR

Gene name Gene symbol GenBank Accession No. Assay ID Amplicon length (bp)
CCL chemokine ligand 3 CCL3 NM_011337.2 Mm99999057_m1 81
CCL chemokine ligand 2 CCL2 NM_011333.3 Mm99999056_m1 96
CXCL chemokine ligand 1 CXCL1 NM_008176.2 Mm00433859_m1 74
Tumor necrosis factor TNF NM_013693.2 Mm99999068_m1 63
Interleukin 1-β IL1β NM_008361.3 Mm00434228_m1 90
Interleukin 6 IL6 NM_031168.1 Mm00446191_m1 124
Vascular endothelial growth factor A VEGFA NM_001025250.3 Mm00437304_m1 77
Matrix metalloproteinase 8 MMP8 NM_008611.4 Mm00772335_m1 127
Matrix metalloproteinase 13 MMP13 NM_008607.1 Mm00439491_m1 65
Cathelicidin CAMP NM_009921.1 Mm00438285_m1 77
Glyceraldehyde-3-phosphate dehydrogenase GAPDH NM_008084.2 Mm99999915_gl 107
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Assay ID is from Applied Biosystems.

Acknowledgments

This work was supported by the National Institutes of Health (EY02520), the Research to Prevent Blindness, Inc., and the Sid W. Richardson Foundation.

Abbreviations

CCL2

chemokine (CC-motif) ligand 2

CCL3

chemokine (CC-motif) ligand 3

IL-1β

interleukin-1β

TNF-α

tumor necrosis factor-α

Footnotes

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