Topical flagellin-mediated innate defense against Candida albicans keratitis

Abstract

Purpose: This study was conducted to investigate whether flagellin, the sole ligand of Toll-like receptor-5 (TLR5), induces an innate defense that is sufficient to protect injured corneas from Candida albicans.

Methods: Scarified corneas of adult B6, TLR5(-/-), Camp(-/-) (cathelicidin-related antimicrobial peptide), or PMN-depleted mice were pretreated with Pseudomonas aeruginosa flagellin or a mutant and then were inoculated with C. albicans. The corneas were compared for disease progression, cytokine and Camp expression, and PMN infiltration before and after C. albicans infection. Disease progress was recorded by digital photography and clinical scoring, cytokine levels were determined by ELISA, the levels of Camp gene product were assessed by Western blot, and PMN infiltration was measured by MPO determination and immunohistochemistry.

Results: Topical application of flagellin induced profound protection against Candida keratitis in a TLR5-dependent manner. The improved disease outcome including reduced tissue inflammation and rapid functional recovery can be attributed to a marked decrease in fungal burden at the early stage of C. albicans infection in flagellin-exposed B6 mouse corneas. Although both PMN infiltration and Camp upregulation contributed to corneal innate defense against fungal infection, Camp ablation totally, and PMN depletion partially, abrogated flagellin-induced fungal clearance in B6 mouse corneas.

Conclusions: Flagellin induces a strong innate defense and promotes robust resistance to C. albicans infection in the cornea. Topical flagellin or its mimetic may become a new prophylactic agent for preventing contact lens or trauma/injury-associated microbial keratitis.

Figure 1.

Topical application of flagellin was effective in inducing protection against C. albicans keratitis. The center of B6 mouse corneas were gently scratched with 26-gauge needles (1-mm long, three lines) followed by topical application of flagellin (500 ng) or PBS to the injury sites. The corneas were scratched again 24 hours after pretreatment and inoculated with 1.0 × 10⁵ CFU of C. albicans. The infected corneas were photographed at 1, 3, and 5 dpi and representative micrographs from each group are shown (A). Disease severity is represented by clinical scores, where the horizontal line represents the mean clinical score. A nonparametric Mann-Whitney U test was performed to compare each flagellin pretreatment to the PBS group (**P < 0.01, n = 5) (B). Results are representative of six independent experiments.

Figure 2.

Flagellin pretreatment increased fungal clearance in the B6 mouse cornea. B6 mouse corneas were pretreated with topically applied flagellin or PBS for 24 hours, as described in Figure 1. At the indicated time after C. albicans inoculation, the eyes were enucleated and subjected to fungal culture by colony counting. The results are presented as the number of CFU per cornea. A nonparametric Mann-Whitney U test was performed to compare each flagellin pretreatment to the PBS group (*P < 0.05, **P < 0.01, n = 5). Results are representative of three independent experiments.

Figure 3.

Flagellin pretreatment dampened cytokine production and PMN infiltration in C. albicans-infected corneas of B6 mice. B6 mouse corneas were pretreated with topically applied flagellin or PBS for 24 hours, as described in Figure 1, followed by C. albicans inoculation. The eyes were enucleated at 6 hours or 1 or 3 dpi for IL-1β (A), MIP-2 (B) ELISA, and MPO determination (units/cornea) (C), respectively. The results are representative of three independent experiments, and the indicated P values were generated using the Student's t-test. *P < 0.01; **P < 0.01, n = 5.

Figure 4.

TLR5 ablation obliterated flagellin-induced protection in B6 mice. TLR5^−/− mice were pretreated with topical flagellin (500 ng) or PBS. After 24 pretreatment, the cornea was scarified and inoculated with 1.0 × 10⁵ CFU of C. albicans. The infected corneas were photographed (A), and disease severity was scored from 0 to 12, with the results represented as individual and mean clinical scores (B). At 3 dpi, the corneas were excised and subjected to fungal counting (C) with the results presented as CFU (×1000) C. albicans per cornea and to MPO determination (units/cornea) (D). The results are representative of two independent experiments.

Figure 5.

Flagellin mutated in the TLR5-binding region failed to induce protection against C. albicans infection in B6 mice. B6 mice were pretreated topically with either PAK flagellin or L94A mutant flagellin (500 ng) for 24 hours, and the cornea was inoculated with 1.0 × 10⁵ CFU of C. albicans. The infected corneas were photographed (A) and the disease severity was scored from 0 to 12 with the results represented as individual and mean clinical scores (B). At 3 dpi, the corneas were excised and subjected to fungal counting, and the results are presented as CFU C. albicans (×1000/cornea) (C). The results are representative of two independent experiments.

Figure 6.

Flagellin pretreatment induced PMN infiltration and CRAMP production in the injured B6 mouse corneas. B6 mouse corneas were needle wounded and topically challenged with flagellin (flagellin or F) or PBS (PBS or P). At the indicated time points, the corneas were either OCT snap frozen, followed by sectioning and immunostaining with the antibody NIMP-R14, to detect PMNs (A), or homogenized, followed by Western blot analysis of CRAMP (B). For immunohistochemistry (A), the photographs were taken at the center of the cornea with DAPI as the counterstain for nuclei. For Western blot (B), two corneas were pooled, and two samples were used for each condition with immunoreactivity of β-actin used as the internal control for protein loading.

Figure 7.

Flagellin pretreatment did not protect PMN-depleted mice against C. albicans infection. B6 mice were made neutropenic by intraperitoneal injection of the mAb RB6-8C5, and the controls were injected with the IgG isotype. The control and neutropenic B6 mouse corneas were pretreated with topical flagellin for 24 hours and infected with 1.0 × 10⁵ CFU of C. albicans. (A) Micrographs of infected corneas were performed at 1 and 2 dpi. (B) Fungal load was determined at 2 dpi and the indicated P values were generated with Student's t-test, **P < 0.01, n = 5. The results are representative of two separate experiments.

Figure 8.

Camp^−/− mouse corneas were more susceptible than the wild type to C. albicans. Camp^−/− or WT (B6) mice were scarified and infected with either 1.0 × 10⁴ (A) or 1.0 × 10⁵ (B) CFU of C. albicans. The disease severity was scored from 0 to 12 at 1, 3, and 5 dpi and represented as mean clinical scores. A nonparametric Mann-Whitney U test was performed to compare each flagellin pretreatment to the PBS group (*P < 0.05, **P < 0.01, n = 5). The results are representative of three independent experiments.

Figure 9.

Topical flagellin was ineffective in protecting the cornea from C. albicans in Camp^−/− mouse cornea. Camp^−/− mice were pretreated with a topical application of flagellin (500 ng) or PBS. After 24 pretreatment, the cornea was inoculated with 1.0 × 10⁵ CFU of C. albicans. The infected corneas were photographed at 1 and 3 dpi (A), and disease severity was scored from 0 to 12 and represented as the mean clinical score (B). The results are representative of three independent experiments. At 3 dpi, the corneas were excised and subjected to fungal counting (C), with the results presented as CFU (×1000) C. albicans per cornea, and to MPO determination (units/cornea) (D). No statistically significant differences were observed in the analyses in (B), (C), and (D) .