Toll-like receptors involved in the pathogenesis of experimental Candida albicans keratitis

Abstract

Purpose. To investigate the expression and function of toll-like receptors (TLRs) during experimental keratomycosis. Methods. Scarified corneas of BALB/c mice were topically inoculated with Candida albicans and compared with control corneas by a murine gene microarray and immunostaining. Real-time reverse transcription polymerase chain reaction (RT-PCR) determined relative TLR gene expression in murine and human donor corneas. The scarified corneas of TLR2(-/-) mice, TLR4(-/-) mice, and C57BL/6J control mice were also inoculated with C. albicans, to determine relative severity, fungal load, and cytokine transcript levels. Results. TLR1, -2, -4, -6, and -13 were significantly upregulated (5- to 10-fold; P < 0.01) by microarray, and TLR1, -2, -4, and -13 were significantly increased (4- to 11-fold; P < 0.05) by real-time RT-PCR in BALB/c murine corneas. Similarly, TLR2, -6, and -13 were significantly upregulated (5- to 16-fold; P < or = 0.001) by real-time RT-PCR in C57BL/6J murine corneas the day after inoculation, and TLR2 and -13 remained significantly (P < 0.05) increased after 1 week. TLR2 transcript was also upregulated twofold (P = 0.04) in C. albicans-inoculated explanted human corneas. Although murine keratitis severity scores were similar, significantly more fungi were recovered from TLR2(-/-) mouse corneas (P = 0.04) than from TLR4(-/-) mouse corneas (P = 0.9). Tumor necrosis factor-alpha, interleukin 23, chemokine C-C ligands 3 and 4, and dectin-1 were significantly (P < 0.05) downregulated in C. albicans-infected corneas of TLR2(-/-) mice. Conclusions. TLR2 signals proinflammatory cytokines that control fungal growth during C. albicans keratitis. TLR13 may have an additional role in the innate immune response of murine corneal candidiasis.

Figure 1.

Clinical evaluation of C. albicans keratitis in C57BL/6J, TLR2^−/−, and TLR4^−/− mice. Each point represents the mean severity score with SD.

Figure 2.

Protein expression patterns in situ in corneas with C. albicans keratitis (Infected) or mock-inoculated control corneas (Mock), comparing negative control corneas lacking primary antibody (Control) and those processed with anti-TLR2 or -TLR4 antibodies. Immunofluorescence showed TLR2 in the healed corneal epithelium of mock-infected eyes and of infected eyes 1 day after the onset of experimental C. albicans keratitis. TLR4 was present in the epithelium and stroma. Original magnification, ×400. Scale bar, 20 μm.

Figure 3.

Differential gene expression ratios of TLRs detected by real-time RT-PCR throughout the first week in BALB/c mouse corneas with posttraumatic C. albicans keratitis compared to those in scarified, mock-infected control corneas. On day 3 pi, TLR13 was increased 5.1-fold (P = 0.01). By day 7 pi, TLR2 was upregulated 2.8-fold (P = 0.01), whereas TLR13 had increased 18.1-fold (P = 0.01) compared with levels in scarified control corneas.