In vitro activity of human beta-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 beta-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.

FIG. 1.

Bactericidal effect of hBD-2 against Pseudomonas aeruginosa. A total of 10⁷ CFU/ml P. aeruginosa (ATCC 27853) was incubated with hBD-2 (0.05, 0.1, 1, 10, 25, 50, 75, 100, and 200 μg/ml) in the absence and presence of 150 mM NaCl for 2 h. The average data from three experiments are shown, and a concentration-dependent antimicrobial effect of hBD-2 is shown. The EC₅₀ values were 2.5 ± 1.0 μg/ml and 32.5 ± 1.2 μg/ml for ATCC 27853 in the absence and presence of NaCl, respectively.

FIG. 2.

Antimicrobial activity of hBD-2 in the presence of human tears or NaCl solutions. Ciloxan, ciprofloxacin HCl ophthalmic solution. (A) A total of 10⁷ CFU/ml P. aeruginosa (ATCC 27853) was incubated with 0.01% acetic acid, hBD-2 (100 μg/ml), or hBD-2 (100 μg/ml) and tears (17.5%, 35%, or 70% [vol/vol]) at 37°C for 2 h. Mean data, expressed as the percentage of bacteria killed by hBD-2 in tears obtained from five subjects, are shown. (B) A total of 10⁷ CFU/ml P. aeruginosa (ATCC 27853) was incubated with 0.01% acetic acid, hBD-2 (100 μg/ml), or hBD-2 (100 μg/ml) and NaCl solutions at 37°C for 2 h. Average data from three experiments, expressed as the percentage of bacteria killed by hBD-2 in the presence of NaCl, are shown.

FIG. 3.

hBD-2 is not degraded in tears. Western blotting for hBD-2 was performed by using synthetic peptide (hBD-2) and reaction mixtures containing hBD-2 and tears (hBD-2 + Tears). Representative data from one of three subjects are shown.

FIG. 4.

Effect of heat-inactivated or filtered human tears on antimicrobial activity of hBD-2. A total of 10⁷ CFU/ml P. aeruginosa (ATCC 27853) was incubated with 0.3% ciprofloxacin HCl ophthalmic solution (Ciloxan), hBD-2 (100 μg/ml), hBD-2 with 70% vol/vol tears (hBD-2 + Tears), hBD-2 with 70% (vol/vol) heat-inactivated tears (hBD-2 + HI Tears), or hBD-2 with 70% (vol/vol) filtered tears (hBD-2 + Filtered Tears) at 37°C for 2 h. Mean data from five experiments, expressed as the percentage of bacteria killed by hBD-2, are shown.

FIG. 5.

Antimicrobial activity of hBD-2 in the presence of cationic and anionic tear fractions. A total of 10⁷ CFU/ml P. aeruginosa (ATCC 27853) was incubated with 0.3% ciprofloxacin HCl ophthalmic solution (Ciloxan), hBD-2 (100 μg/ml), hBD-2 with 70% (vol/vol) tears (hBD-2 + Tears), hBD-2 with 70% (vol/vol) cationic tear fraction [hBD-2 + Tears (Cationic)], or hBD-2 with 70% (vol/vol) anionic tear fraction [hBD-2 + Tears (Anionic)] at 37°C for 2 h. Mean data obtained from three experiments, expressed as the percentage of bacteria killed by hBD-2, are shown.

FIG. 6.

(A) IP of mucin MUC5AC. Tears (100 μl) were immunoprecipitated with anti-MUC5AC antiserum or goat preimmune serum and electrophoresed on a 4 to 15% gradient gel for MUC5AC immunodetection. Lane 1, nontreated tears; lane 2, anti-MUC5AC antiserum immunoprecipitate; lane 3 residual tears after anti-MUC5AC IP; lane 4, preimmune goat serum immunoprecipitated pellet; lane 5, residual tears after preimmune goat serum IP. Representative data from one of three experiments are shown. (B) Antimicrobial activity of hBD-2 in tears in the presence and absence of MUC5AC. A total of 10⁷ CFU/ml P. aeruginosa (ATCC 27853) was incubated with hBD-2 (100 μg/ml), hBD-2 with 70% (vol/vol) tears (hBD-2 + Tears), hBD-2 with 70% (vol/vol) anti-MUC5AC immunoprecipitated tears [hBD-2 + IP Tears (no MUC5AC)], or hBD-2 with 70% (vol/vol) goat preimmune serum immunoprecipitated tears [hBD-2 + IP tears (with MUC5AC)] at 37°C for 2 h. Mean data from five experiments, expressed as the percentage of bacteria killed by hBD-2, are shown. (C) Direct interaction of hBD-2 and MUC5AC in human tears. Tears (50 μl) preincubated with hBD-2 (100 μg/ml) were immunoprecipitated with anti-hBD-2 polycolonal antibody or rabbit serum and electrophoresed on a 4 to 15% gradient gel for MUC5AC immunodetection. Lane 1, nontreated tears; lane 2, anti-hBD-2 antibody immunoprecipitated pellet; lane 3, rabbit serum immunoprecipitated pellet.