Role of Defensins in Corneal Epithelial Barrier Function against Pseudomonas aeruginosa Traversal

Abstract

Studies have shown that epithelium-expressed antimicrobial peptides (AMPs), e.g., ␤-defensins, play a role in clearing bacteria from mouse corneas already infected with Pseudomonas aeruginosa. Less is known about the role of AMPs in allowing the cornea to resist infection when healthy. We previously reported that contact lens exposure, a major cause of P. aeruginosa keratitis, can inhibit the upregulation of human ␤-defensin 2 (hBD-2) by corneal epithelial cells in response to P. aeruginosa antigens in vitro. Here, we studied the role of AMPs in maintaining the corneal epithelial barrier to P. aeruginosa penetration using both in vitro (human) and in vivo (mouse) experiments. Results showed that preexposing human corneal epithelial multilayers to bacterial antigens in a culture supernatant (known to upregulate AMP expression) reduced epithelial susceptibility to P. aeruginosa traversal up to 6-fold (P < 0.001). Accordingly, small interfering RNA (siRNA) knockdown of any one of four AMPs expressed by human epithelia promoted P. aeruginosa traversal by more than 3-fold (P < 0.001). The combination knockdown of AMPs further enhanced susceptibility to bacterial traversal by ϳ8-fold (P < 0.001). In vivo experiments showed that the loss of murine ␤-defensin 3 (mBD-3), a murine ortholog of hBD-2, enhanced corneal susceptibility to P. aeruginosa. The uninjured ocular surface of mBD-3 ؊/؊ mice showed a reduced capacity to clear P. aeruginosa, and their corneal epithelia were more susceptible to bacterial colonization, even when inoculated ex vivo to exclude tear fluid effects. Together, these in vitro and in vivo data show functional roles for AMPs in normal corneal epithelial cell barrier function against P. aeruginosa.

The Importance of the Pseudomonas aeruginosa Type III Secretion System in Epithelium Traversal Depends upon Conditions of Host Susceptibility

“…When blotting was followed by 1 h of EGTA treatment (100 mM in phosphate-buffered saline [PBS]), the bacteria could traverse the epithelium all the way to the underlying basal lamina (46). Using gene knockout mice, we have found that MyD88 is important for protecting the corneal epithelium against both adhesion and subsequent traversal (47) and that mBD3, the mouse equivalent of human hBD2 and a MyD88-dependent factor (51,52), is involved in this protective effect (49). We further showed that these novel methods for studying traversal of in situ-grown corneal epithelium could be adapted for ex vivo use, which allows exclusion of tear fluid and infiltrating phagocytic responses when desirable.…”

“…The susceptibility of MyD88 Ϫ/Ϫ corneas to ExsA-independent traversal led us to investigate if MyD88 deficiency impaired antimicrobial activity of the corneal epithelium. Our choice to focus on antimicrobial activity, rather than other potential defense factors, was based on our earlier discoveries that corneally expressed antimicrobials (including mBD3 and keratin-derived antimicrobial peptides [KDAMPs]) contribute to maintaining the corneal epithelial barrier against P. aeruginosa (49,59). Thus, the corneal epithelium of normal healthy wild-type and MyD88…”

“…Our published data obtained using these models suggest that multiple levels of defense protect the healthy cornea. They show that uninjured normal mouse ocular surfaces rapidly cleared P. aeruginosa, even when enormous inocula (Ͼ10 11 /ml) were applied (48,49), contrasting scratch injury, after which the corneas succumb to destructive keratitis with 6 to 8 orders of magnitude fewer bacteria (28,30,31,50). We showed that blotting the corneal surface with tissue paper (Kimwipe), which disrupts tight junctions while otherwise leaving corneas morphologically similar, made corneas susceptible to bacterial adhesion without subsequent epithelial traversal.…”

Cytokeratins mediate epithelial innate defense through their antimicrobial properties

“…In animal models, even large inocula of potentially pathogenic bacteria are rapidly cleared from the ocular surface when it is healthy (10)(11)(12). Various antimicrobial peptides are expressed at ocular, and other, surfaces of humans and animals.…”

Defensins and Other Antimicrobial Peptides and a Role of Neutrophil Protein-Synthesing Function Disorders for Pathogenesis of Respiratory Diseases

“…α-HD обладают широким спектром антибактери-альной активности в отношении как грамположи-тельных, так и грамотрицательных микроорганиз-мов, а также вирусов [5,12] Кроме названных микробных агентов, β-HD-3 действует бактерицидно в отношении Streptococcus pyogenes, S. aureus, включая мультирезистентные штаммы S. aureus, и даже vancomycin-резистентный Enterococcus faecium [13][14][15]. Активность β-HD-4 в отношении P. aeruginosa более выражена, чем у дру-гих молекул HD [8].…”