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Satya Sree Kolar, Hasna Baidouri, Alison McDermott; Antimicrobial Peptides in the In Vitro Corneal Response to Fungal Pathogens. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5997.
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© ARVO (1962-2015); The Authors (2016-present)
Antimicrobial peptides (AMPs), including defensins and cathelicidins form an integral part of the initial mucosal defense but little is known about their actions against fungal pathogens. In an effort to understand their role during fungal keratitis, we investigated AMP efficacy against Candida albicans (CA) and Fusarium solani (FS) and human corneal epithelial cell (HCEC) AMP expression following fungal challenge in-vitro.
Antifungal activity was tested using broth micro-dilution assays. Fungal cells were incubated with 0-50 µg/ml of cathelicidin LL-37 or defensin hBD-2 for 24h at 30°C then reaction mixtures plated to determine fungal growth. Sytox green uptake was used to determine if AMP antifungal activity occurred via membrane disruption. For the Sytox assay, fungal cells were pre-incubated with 1 µM sytox green, 25 µM LL-37 added and fluorescence intensity recorded over time. To test AMP expression in response to fungal or TLR2 treatments, HCEC (primary cultures and cell lines) were exposed to heat-inactivated CA or FS or 10 µg/ml TLR2 agonist zymosan for 24h. RT-PCR and immunoblotting were performed to determine the expression of hBD-2 and LL-37. Expression of Dectin-1, a fungal pattern recognition receptor, was also investigated by RT-PCR.
Antifungal assays showed that 25 µg/ml LL-37 and 10 µg/ml hBD-2 resulted in >3 log units of killing of CA and FS (n=2). LL-37 promoted uptake of Sytox green indicating an antifungal mechanism involving membrane disruption. Exposure of immortalized HCECs to heat inactivated FS and CA upregulated the expression of LL-37 by 2.78 and 1.86 log2 fold and of hBD-2 by 3.78 and 2.13 log2 fold compared to controls (n=3). Additionally, immunoblotting showed markedly increased LL-37 and hBD-2 protein secretion in supernatants of fungal treated cells compared to control. Primary cultures showed comparable data. RT-PCR showed robust HCEC expression of the functional isoform of dectin-1. Zymosan upregulated HCEC expression of hBD-2 and LL-37 by 1.78 and 2.15 log2 fold compared to untreated cells suggesting a role for TLR2 in AMP modulation.
LL-37 and hBD-2 have potent antifungal activity and were upregulated in HCEC in response to fungal challenge, likely via activation of dectin-1 and TLR2. These in vitro studies suggest that AMPs are an important defence against fungal keratitis in vivo.
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