Effect of Preservative-Free Artificial Tears on the Antimicrobial Activity of Human ??-Defensin-2 and Cathelicidin LL-37 In Vitro

Abstract

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. KeywordsCarboxymethylcellulose; Cationic antimicrobial peptides; Human β-defensin-2; Human cathelicidin LL-37; Preservative-free artificial tears; Pseudomonas aeruginosa Secretion of small cationic antimicrobial peptides by epithelia is an important component of the innate immune response. 1 These peptides, which include β-defensins and cathelicidins, insert themselves into microbial cell membranes and, by pore formation or electrostatic disruption, cause release of intracellular contents leading to death of the organism. 2 By regulating mammalian cell functions, such as chemotaxis and proliferation, defensins and

The in vitro activity of selected defensins against an isolate of Pseudomonas in the presence of human tears

“…Reversible binding of the peptides to anionic substances present in the tears is a possibility, as has been suggested for loss of b-defensin antimicrobial activity in the presence of carboxymethylcellulose (an anionic molecule)-containing artificial tear solutions. 19 Likely candidates in tear fluid include the anionic lipocalins and mucins. However, as human tear fluid is composed of hundreds of different molecules, many yet to be definitively identified, there may be several other components that can potentially interfere with defensin antimicrobial activity.…”

Dry Eye Disease and Microbial Keratitis: Is There a Connection?

“…191,192,202 However, it has been observed that both hBD-2 and LL-37 lose their ability to kill P. aeruginosa in the presence of carboxymethylcellulose-containing artificial tears in vitro, possibly adding to apparent detrimental effects of artificial tear solutions on ocular surface immunity. 230 Clinically, we often observe that dry eye patients use artificial tear solutions several times per day, which may itself contribute to the flushing out of pathogenic microbes.…”

Toll-Like Receptors and Corneal Innate Immunity

“…In addition to recognizing pathogens and producing proinflammatory cytokines and chemokines, the corneal epithelium is also known to function in the innate immune response through the secretion of antimicrobial peptides [109][110][111][112][113][114][115][116]. Although most β-defensins are typically constitutively expressed, human β-defensin-2 (hBD-2) is remarkable in that it is inducible in a variety of epithelial cell types including those of the cornea [115,116].…”