Proinflammatory chemokines during Candida albicans keratitis

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-1beta 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.

Figure 1

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

Figure 2

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.

Figure 3

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

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.

Figure 5

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.