Ling C. Huang

Human Cathelicidin (LL-37), a Multifunctional Peptide, is Expressed by Ocular Surface Epithelia and has Potent Antibacterial and Antiviral Activity

Purpose: This study determined whether LL-37 (cathelicidin) is expressed by conjunctival and corneal epithelia as part of ocular host defense. The antimicrobial activity of LL-37 was also assessed in vitro against Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), herpes simplex virus type 1 (HSV-1), and adenovirus (Ad). Methods: Expression of LL-37/hCAP 18 mRNA and LL-37 protein was determined by reverse transcription–polymerase chain reaction (RT-PCR) and immunoblotting, respectively, in scraped human corneal epithelium and primary cultured human corneal and conjunctival epithelial cells. The EC50 values for three strains of PA and one each of SA and SE were determined for LL-37. LL-37 antiviral inhibition of HSV-1 and adenovirus was assessed by direct inactivation assays. Toxicity of LL-37 to A549 cells was evaluated by a MTT assay. Results: LL-37/hCAP18 mRNA and LL-37 peptide were expressed by human corneal and conjunctival epithelial cells. Antibacterial activity for LL-37 was demonstrated (EC50 values for the three PA strains were 2.8 ± 1.3, 1.9 ± 0.3, and 3.6 ± 2.1; for SA: 1.6 ± 1.5; for SE: 1.3 ± 1.9 μ g/ml). LL-37 produced a significant reduction (p < 0.001 ANOVA) in HSV-1 and Ad19 viral titers with distinctly different time-kill curves (p < 0.001). LL-37 (up to 111 μ M) produced no toxicity in A549 cells. Conclusions: Corneal and conjunctival epithelia express LL-37 as part of mucosal innate immunity to protect against bacterial and viral ocular infections.

Ocular Surface Expression and In Vitro Activity of Antimicrobial Peptides

Purpose: Human ocular surface epithelia express four antimicrobial peptides (APs): β -defensin (hBD) 1-3 and LL-37. Here the expression of additional APs (hBD 4-6, HE2β 1; histatin-1, -3; liver expressed antimicrobial peptide-1, -2; macrophage inflammatory protein (MIP)-3α, and thymosin (T)β -4) was sought and activity against common ocular pathogens studied. Methods: AP expression was determined in human corneal and conjunctival epithelial cells (HCEC, HCjEC) by RT-PCR and in corneal sections by immunostaining. Antimicrobial assays were performed to assess peptide (hBD 1-3, LL-37, MIP-3α, and Tβ 4) activity against Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), and Staphylococcus epidermidis (SE) in the presence of NaCl or tears. Results: HCEC and HCjEC expressed MIP-3α and Tβ 4. hBD 1-3, MIP-3α, and Tβ 4 showed activity against PA. hBD-3 had potent activity against SA and SE, whereas hBD-2, MIP-3α and Tβ 4 had moderate activity and hBD-1 had none. NaCl markedly attenuated, and tears almost completely inhibited the activity of hBD 1-2 and Tβ 4, but not that of hBD-3. Conclusions: The ocular surface epithelia additionally express MIP-3α and Tβ 4 which have moderate antimicrobial activity. The current data support a role for hBD-3 as an antimicrobial peptide in vivo, but call in to question the effectiveness of some other APs. However, further study is required to conclusively elucidate the physiological role of each AP.

In vitro activity of human β-defensin 2 against Pseudomonas aeruginosa in the presence of tear fluid

ABSTRACT Pseudomonas aeruginosa causes vision-threatening keratitis and is difficult to treat due to emerging resistance. Human β-defensin 2 (hBD-2) is an antimicrobial peptide expressed by ocular surface epithelia with broad-spectrum activity against various pathogens, including P. aeruginosa. The activity of hBD-2 against P. aeruginosa in the presence of human tears or NaCl was studied. In some experiments, tears were heat-inactivated, filtered, and separated into cationic/anionic fractions or mucin MUC5AC was removed by immunoprecipitation before use. Immunoprecipitation was performed to study the interaction between hBD-2 and MUC5AC. hBD-2 activity was reduced by 40 to 90% in the presence of 17.5 to 70% (vol/vol) tears. NaCl reduced hBD-2 activity, but at most it could account for only 36% of the inhibitory effect of tears. Heat inactivation and filtration attenuated the ability of tears to inhibit hBD-2 activity by 65 and 68%, respectively. Anionic tear fractions significantly reduced (86%) the activity of hBD-2, whereas only a 22% reduction was observed with the cationic fractions. In the absence of MUC5AC, the activity of hBD-2 was restored by 64%. Immunoprecipitation studies suggested that the loss of hBD-2 activity in tears is due to a direct binding interaction with MUC5AC. Our data showed that the antimicrobial activity of hBD-2 is sensitive to the presence of human tears and that this is partly due to the salt content and also the presence of MUC5AC. These data cast doubt on the effectiveness of hBD-2 as an antimicrobial peptide, and additional studies are required to conclusively elucidate its role in innate immunity at the ocular surface in vivo.

Effect of preservative-free artificial tears on the antimicrobial activity of human beta-defensin-2 and cathelicidin LL-37 in vitro

Purpose. Human β-defensin-2 (hBD-2) and cathelicidin LL-37 are salt-sensitive cationic antimicrobial peptides expressed by ocular surface epithelia. The goal of this study was to investigate the effect of preservative-free artificial tears on hBD-2 and LL-37 antimicrobial activity against Pseudomonas aeruginosa. Methods. P. aeruginosa was incubated with hBD-2 or LL-37 in the absence or presence (70% vol/vol) of different preservative-free artificial tears-Visine Tears (300 mOsm/kg), Tears Naturale Free (261 mOsm/kg), TheraTears (185 mOsm/kg), and Refresh Plus (325 mOsm/kg)-for 2 hours at 37°C. In some experiments, P. aeruginosa was incubated with hBD-2 or LL-37 and Visine Tears or Tears Naturale Free with or without carboxymethylcellulose (0.5% vol/vol final concentration). Plates were inoculated with samples of each reaction mixture and then incubated for 24 hours at 37°C. Results. Visine Tears and Tears Naturale Free had little or no effect on the antimicrobial activity of 100 μg/mL hBD-2 or LL-37. In the presence of Refresh Plus and TheraTears, the activity of 100 μg/mL hBD-2 or LL-37 was reduced by 90% to 100%. Carboxymethylcellulose, at a concentration comparable to that present in Refresh Plus, reduced the effectiveness of hBD-2 or LL-37 by 40% to 90% in the presence of Tears Naturale Free and Visine Tears. Conclusion. Human β-defensin-2 and cathelicidin LL-37 inhibit the growth of P. aeruginosa in vitro, but this activity is markedly reduced in the presence of Refresh Plus and TheraTears. These results suggest that carboxymethylcellulose-containing artificial tears may reduce the activity of the endogenously produced antimicrobial peptides.