Aims: To explore the innate response of human corneal epithelial cells (HCECs) exposed to fungus by producing antimicrobial peptides LL-37 and β-defensins. Methods: Primary HCECs were treated with heat-killed Candida albicans (HKCA) at different doses (103-106 cells/ml) for 2-48 h. The cells were subjected to total RNA extraction, reverse transcription and quantitative real-time PCR for mRNA expression. Cells treated for 48 h were used for immunofluorescent staining and ELISA. Results: Human LL-37 and β-defensins (hBDs) 1-4 were detected in normal HCECs. The mRNA expression of LL-37, hBD2, and hBD3 was dose-dependently induced by HKCA with their peak levels at 4 h. HKCA (106 cells/ml) stimulated the mRNA of LL-37, hBD2, and hBD3 4.33 ± 1.81, 3.75 ± 1.31, and 4.91 ± 1.09 fold, respectively, in HCECs. The stimulated production of LL-37, hBD2, and hBD3 by HKCA was confirmed at protein levels by immunofluorescent staining and ELISA. The protein production of LL-37, hBD2, and hBD3 significantly increased to 109.1 ± 18.2 pg/ml, 4.33 ± 1.67 ng/ml, and 296.9 ± 81.8 pg/ml, respectively, in culture medium of HCECs exposed to HKCA (106 cells/ml) compared to untreated HCECs. Conclusions: HCECs produce antimicrobial peptides, LL-37, hBD2 and hBD3, in response to stimulation of HKCA, which suggests a novel innate immune mechanism of the ocular surface in defense against fungal invasion.

Keywords:
Ocular surface, Epithelium, Antimicrobial peptides, LL-37, Defensin, Fungus, Innate immunity
1.
Meyer T, Stockfleth E, Christophers E: Immune response profiles in human skin. Br J Dermatol 2007;157(suppl 2):1-7.
2.
Muller CA, Autenrieth IB, Peschel A: Innate defenses of the intestinal epithelial barrier. Cell Mol Life Sci 2005;62:1297-1307.
3.
Alexopoulou L, Kontoyiannis D: Contribution of microbial-associated molecules in innate mucosal responses. Cell Mol Life Sci 2005;62:1349-1358.
4.
Ueta M, Kinoshita S: Ocular surface inflammation mediated by innate immunity. Eye Contact Lens 2010;36:269-281.
5.
Cole AM, Ganz T: Human antimicrobial peptides: analysis and application. Biotechniques 2000;29:822-831.
6.
McDermott AM: The role of antimicrobial peptides at the ocular surface. Ophthalmic Res 2009;41:60-75.
7.
Lai Y, Gallo RL: AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense. Trends Immunol 2009;30:131-141.
8.
Huttner KM, Bevins CL: Antimicrobial peptides as mediators of epithelial host defense. Pediatr Res 1999;45:785-794.
9.
Tjabringa GS, Vos JB, Olthuis D, Ninaber DK, Rabe KF, Schalkwijk J, Hiemstra PS, Zeeuwen PL: Host defense effector molecules in mucosal secretions. FEMS Immunol Med Microbiol 2005;45:151-158.
10.
Haynes RJ, Tighe PJ, Dua HS: Antimicrobial defensin peptides of the human ocular surface. Br J Ophthalmol 1999;83:737-741.
11.
McDermott AM: Defensins and other antimicrobial peptides at the ocular surface. Ocul Surf 2004;2:229-247.
12.
Kumar A, Yin J, Zhang J, Yu FS: Modulation of corneal epithelial innate immune response to pseudomonas infection by flagellin pretreatment. Invest Ophthalmol Vis Sci 2007;48:4664-4670.
13.
Narayanan S, Manning J, Proske R, McDermott AM: Effect of hyperosmolality on beta-defensin gene expression by human corneal epithelial cells. Cornea 2006;25:1063-1068.
14.
Sack RA, Nunes I, Beaton A, Morris C: Host-defense mechanism of the ocular surfaces. Biosci Rep 2001;21:463-480.
15.
McIntosh RS, Cade JE, Al-Abed M, Shanmuganathan V, Gupta R, Bhan A, Tighe PJ, Dua HS: The spectrum of antimicrobial peptide expression at the ocular surface. Invest Ophthalmol Vis Sci 2005;46:1379-1385.
16.
Abedin A, Mohammed I, Hopkinson A, Dua HS: A novel antimicrobial peptide on the ocular surface shows decreased expression in inflammation and infection. Invest Ophthalmol Vis Sci 2008;49:28-33.
17.
Mohammed I, Yeung A, Abedin A, Hopkinson A, Dua HS: Signalling pathways involved in ribonuclease-7 expression. Cell Mol Life Sci 2011;68:1941-1952.
18.
Srinivasan M: Fungal keratitis. Curr Opin Ophthalmol 2004;15:321-327.
19.
Florcruz NV, Peczon I Jr: Medical interventions for fungal keratitis. Cochrane Database Syst Rev 2008;23:CD004241.
20.
Thomas PA: Fungal infections of the cornea. Eye (Lond) 2003;17:852-862.
21.
Thomas PA, Geraldine P: Infectious keratitis. Curr Opin Infect Dis 2007;20:129-141.
22.
Chern KC, Meisler DM, Wilhelmus KR, Jones DB, Stern GA, Lowder CY: Corneal anesthetic abuse and Candida keratitis. Ophthalmology 1996;103:37-40.
23.
Jackson BE, Wilhelmus KR, Mitchell BM: Genetically regulated filamentation contributes to Candida albicans virulence during corneal infection. Microb Pathog 2007;42:88-93.
24.
Yuan X, Wilhelmus KR, Matoba AY, Alexandrakis G, Miller D, Huang AJ: Pathogenesis and outcome of Paecilomyces keratitis. Am J Ophthalmol 2009;147:691-696.
25.
Yuan X, Wilhelmus KR: Toll-like receptors involved in the pathogenesis of experimental Candida albicans keratitis. Invest Ophthalmol Vis Sci 2010;51:2094-2100.
26.
Masinick SA, Montgomery CP, Montgomery PC, Hazlett LD: Secretory IgA inhibits Pseudomonas aeruginosa binding to cornea and protects against keratitis. Invest Ophthalmol Vis Sci 1997;38:910-918.
27.
Ni M, Evans DJ, Hawgood S, Anders EM, Sack RA, Fleiszig SM: Surfactant protein D is present in human tear fluid and the cornea and inhibits epithelial cell invasion by Pseudomonas aeruginosa. Infect Immun 2005;73:2147-2156.
28.
Fleiszig SM, Zaidi TS, Ramphal R, Pier GB: Modulation of Pseudomonas aeruginosa adherence to the corneal surface by mucus. Infect Immun 1994;62:1799-1804.
29.
Kim HS, Jun SX, de Paiva CS, Chen Z, Pflugfelder SC, Li DQ: Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures. Exp Eye Res 2004;79:41-49.
30.
de Paiva CS, Pflugfelder SC, Li DQ: Cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells. Stem Cells 2006;24:368-375.
31.
Luo L, Li D-Q, Doshi A, Farley W, Corrales RM, Pflugfelder SC: Experimental dry eye stimulates production of inflammatory cytokines and MMP-9 and activates MAPK signaling pathways on the ocular surface. Invest Ophthalmol Vis Sci 2004;45:4293-4301.
32.
Yoon KC, de Paiva CS, Qi H, Chen Z, Farley WJ, Li DQ, Pflugfelder SC: Expression of Th-1 chemokines and chemokine receptors on the ocular surface of C57BL/6 mice: effects of desiccating stress. Invest Ophthalmol Vis Sci 2007;48:2561-2569.
33.
Chen Z, de Paiva CS, Luo L, Kretzer FL, Pflugfelder SC, Li D-Q: Characterization of putative stem cell phenotype in human limbal epithelia. Stem Cells 2004;22:355-366.
34.
Li D-Q, Tseng SC: Three patterns of cytokine expression potentially involved in epithelial-fibroblast interactions of human ocular surface. J Cell Physiol 1995;163:61-79.
35.
Macdonald TT: The mucosal immune system. Parasite Immunol 2003;25:235-246.
36.
Gao N, Kumar A, Jyot J, Yu FS: Flagellin-induced corneal antimicrobial peptide production and wound repair involve a novel NF-kappaB-independent and EGFR-dependent pathway. PLoS One 2010;5:e9351.
37.
McDermott AM, Redfern RL, Zhang B, Pei Y, Huang L, Proske RJ: Defensin expression by the cornea: multiple signalling pathways mediate IL-1beta stimulation of hBD-2 expression by human corneal epithelial cells. Invest Ophthalmol Vis Sci 2003;44:1859-1865.
38.
McNamara NA, Van R, Tuchin OS, Fleiszig SM: Ocular surface epithelia express mRNA for human beta defensin-2. Exp Eye Res 1999;69:483-490.
39.
Sack RA, Nunes I, Beaton A, Morris C: Host-defense mechanism of the ocular surfaces. Biosci Rep 2001;21:463-480.
40.
Haynes RJ, Tighe PJ, Dua HS: Innate defence of the eye by antimicrobial defensin peptides. Lancet 1998;352:451-452.
41.
Lehmann OJ, Hussain IR, Watt PJ: Investigation of beta defensin gene expression in the ocular anterior segment by semiquantitative RT-PCR. Br J Ophthalmol 2000;84:523-526.
42.
Mendez-Samperio P: The human cathelicidin hCAP18/LL-37: a multifunctional peptide involved in mycobacterial infections. Peptides 2010;31:1791-1798.
43.
Bowdish DM, Davidson DJ, Lau YE, Lee K, Scott MG, Hancock RE: Impact of LL-37 on anti-infective immunity. J Leukoc Biol 2005;77:451-459.
44.
Gordon YJ, Huang LC, Romanowski EG, Yates KA, Proske RJ, McDermott AM: Human cathelicidin (LL-37), a multifunctional peptide, is expressed by ocular surface epithelia and has potent antibacterial and antiviral activity. Curr Eye Res 2005;30:385-394.
45.
Huang LC, Reins RY, Gallo RL, McDermott AM: Cathelicidin-deficient (Cnlp-/-) mice show increased susceptibility to Pseudomonas aeruginosa keratitis. Invest Ophthalmol Vis Sci 2007;48:4498-4508.
46.
Nijnik A, Hancock RE: The roles of cathelicidin LL-37 in immune defences and novel clinical applications. Curr Opin Hematol 2009;16:41-47.
47.
Lopez-Garcia B, Lee PH, Yamasaki K, Gallo RL: Anti-fungal activity of cathelicidins and their potential role in Candida albicans skin infection. J Invest Dermatol 2005;125:108-115.
48.
Yuan X, Hua X, Wilhelmus KR: The corneal expression of antimicrobial peptides during experimental fungal keratitis. Curr Eye Res 2010;35:872-879.
49.
Wu M, McClellan SA, Barrett RP, Zhang Y, Hazlett LD: Beta-defensins 2 and 3 together promote resistance to Pseudomonas aeruginosa keratitis. J Immunol 2009;183:8054-8060.
50.
Wu M, McClellan SA, Barrett RP, Hazlett LD: Beta-defensin-2 promotes resistance against infection with P. aeruginosa. J Immunol 2009;182:1609-1616.
51.
Kumar A, Hazlett LD, Yu FS: Flagellin suppresses the inflammatory response and enhances bacterial clearance in a murine model of Pseudomonas aeruginosa keratitis. Infect Immun 2008;76:89-96.
52.
McDermott AM, Redfern RL, Zhang B: Human beta-defensin 2 is up-regulated during re-epithelialization of the cornea. Curr Eye Res 2001;22:64-67.
53.
Leal SM Jr, Roy S, Vareechon C, Carrion S, Clark H, Lopez-Berges MS, diPietro A, Schrettl M, Beckmann N, Redl B, Haas H, Pearlman E: Targeting iron acquisition blocks infection with the fungal pathogens Aspergillus fumigatus and Fusarium oxysporum. PLoS Pathog 2013;9:e1003436.
54.
Li Z, Jhanji V, Tao X, Yu H, Chen W, Mu G: Riboflavin/ultraviolet light-mediated crosslinking for fungal keratitis. Br J Ophthalmol 2013;97:669-671.
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2014
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