In Vitro Activity of Human β-Defensin 2 against Pseudomonas aeruginosa in the Presence of Tear Fluid▿

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

MyD88 contribution to ocular surface homeostasis

<div><p>The cornea must maintain homeostasis, enabling rapid response to injury and microbial insult, to protect the eye from insult and infection. Toll-like receptors (TLRs) are critical to this innate immune response through the recognition and response to pathogens. Myeloid differentiation primary response (MyD88) is a key signaling molecule necessary for Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R)-mediated immune defense and has been shown to be necessary for corneal defense during infection. Here, we examined the intrinsic role of TLR signaling in ocular surface tissues by determining baseline levels of inflammatory mediators, the response to mechanical stimuli, and corneal infection in MyD88-deficient mice (MyD88^-/-). In addition, cytokine, chemokine, and matrix metalloproteinase (MMP) expression was determined in ocular surface cells exposed to a panel of TLR agonists. Compared to wild-type (WT) animals, MyD88^-/- mice expressed lower MMP-9 levels in the cornea and conjunctiva. Corneal IL-1α, TNFα, and conjunctival IL-1α, IL-2, IL-6, and IL-9 levels were also significantly reduced. Additionally, CXCL1 and RANTES expression was lower in both MyD88^-/- tissues compared to WT and IL-1R^-/- mice. Interestingly, MyD88^-/- mice had lower corneal sensitivities (1.01±0.31 gm/mm²) than both WT (0.59±0.16 gm/mm²) and IL-1R^-/- (0.52±0.08 gm/mm²). Following <i>Pseudomonas aeruginosa</i> challenge, MyD88^-/- mice had better clinical scores (0.5±0.0) compared to IL-1R^-/- (1.5±0.6) and WT (2.3±0.3) animals, but had significantly more corneal bacterial isolates. However, no signs of infection were detected in inoculated uninjured corneas from either MyD88 or IL-1R-deficient mice. This work furthers our understanding of the importance of TLR signaling in corneal defense and immune homeostasis, showing that a lack of MyD88 may compromise the baseline innate response to insult.</p></div

MyD88^-/- mice have decreased levels of cytokines in the conjunctiva.

<p>IL-1α (A), IL-6 (B), IL-2 (C), and IL-9 (D) expression were determined in untreated conjunctival homogenates from wild-type (WT), IL-1R^-/-, and MyD88^-/- mice by Luminex multiplex assay. Data represent mean ± SEM of 3 independent experiments with each sample pooled from 5 mice. Analysis was performed by ANOVA with Bonferroni’s test for multiple comparisons, with comparison to WT samples. p<*0.05, **0.01.</p

MyD88^-/- mice infected with PA were protected from inflammatory corneal infiltrates; however, more bacteria were isolated from MyD88^-/- corneas compared to both WT and IL-1R^-/-.

<p>Corneas of WT (C57), IL-1R^-/-, and MyD88^-/- mice were scratched and infected with 1.0 × 10⁶ CFU GFP-PA01. (A-B) After 24 hours, eyes were imaged by slit lamp microscopy and graded based on severity of infection. Images are representative of 3 animals per group. Analysis was performed by ANOVA with Bonferroni’s test for multiple comparisons. p<*0.05, **0.01 (B) For bacterial counts, corneas were harvested 24 hours post-infection, homogenates plated, and incubated for 16 hours at 37°C. The number of colonies was counted and data represent 3 corneas per group.</p

Lack of TLR signaling (MyD88^-/-) results in decreased levels of cytokines in the cornea.

<p>IL-1α (A, C) and TNFα (B, D) gene expression was determined in untreated corneal lysates by RT-PCR (top) and protein expression quantitated in corneal homogenates by Luminex multiplex assay (bottom), in wild-type (WT), IL-1R^-/-, and MyD88^-/- mice. Graphs represent mean ± SEM of 3 independent experiments with each sample pooled from 5 mice. Analysis was performed by ANOVA with Bonferroni’s test for multiple comparisons, with comparison to WT samples. p<*0.05, **0.01, nd = not determined.</p

<i>In vivo</i> wound closure is delayed with vitamin D treatment.

<p>Mice were wounded and treated with vehicle (0.02% ethanol/PBS) or 1,25D₃ (10^-7M) every 6 hours or left untreated (control) for 24 hours. (A) Corneal wound areas were monitored by fluorescein staining every 6 hours. (B) Wound area remaining open was determined as a percentage of original wound area. Data represent mean ± SEM and were analyzed with two-way repeated measures ANOVA and Bonferroni’s correction for multiple comparisons. * = p<0.05 (comparison between vehicle and vitamin D treatments; n = 9 mice/group).</p

MyD88-deficient mice have lower levels of MMP expression in the cornea and conjunctiva.

<p>(A) MMP-9 expression was determined in untreated corneal lysates by RT-PCR. Graph represent mean ±SEM of 3 independent experiments with each sample pooled from 4 mice. (B) MMP-9 protein expression was quantitated in conjunctival homogenates by Luminex multiplex assay. Samples represent 10 pooled corneas per genotype. Graph represent mean ±SEM of 3 independent experiments (n = 3). Analysis was performed by ANOVA with Bonferroni’s test for multiple comparisons. p < *0.05, **0.01.</p

TLR activation increases levels of MMPs in human corneal and conjunctival cells.

<p>Primary HCEC, hTCEpi, and primary HConjEpi were left untreated (control), or were treated with TLR agonists [TLR2/1 (PAM), TLR2 (HKLM), TLR4 (LPS), TLR5 (FLAG), TLR6/2 (FLS-1), TLR7 (IMQ), TLR8 (ssRNA), or TLR9 (ODN)] for 24 hours. Pro-MMP-9 (A), MMP-1 (B), and MMP-10 (C) expression was determined in cell supernatants by Luminex bead assay. Graphs represent mean ±SEM of 3 independent experiments (n = 3). Analysis was performed by ANOVA with Bonferroni’s test for multiple comparisons. p < *0.05, +0.01, #0.0001.</p

MyD88^-/- mice have decreased corneal sensitivity.

<p>Corneal sensitivity was measured in untreated mice using a Cochet-Bonnet esthesiometer, with greater pressure (gm/mm²) indicating lower sensitivity. Graph represents mean ± SEM of 9 mice per genotype. Analysis was performed by ANOVA with Bonferroni’s test for multiple comparisons. p<***0.001.</p