Rachel L. Redfern

Toll-like receptors in ocular surface disease.

The ability of the ocular surface to mount an immune response is in part attributed to a family of proteins called toll-like receptors (TLRs). The latter are evolutionary conserved receptors that recognize and respond to various microbes and endogenous ligands. In addition to their recognition function, TLR activation triggers a complex signal transduction cascade that induces the production of inflammatory cytokines and co-stimulatory molecules, thus initiating innate and adaptive immunity. Toll-like receptor expression at the ocular surface is modulated during infection (e.g. Herpes simplex, bacterial keratitis and fungal keratitis) as well as during various inflammatory conditions (allergic conjunctivitis and dry-eye syndrome). Here recent findings regarding TLR expression and their involvement in various ocular surface diseases are discussed.

Human ß-defensin 2 is up-regulated during re-epithelialization of the cornea

Purpose. Human ß-defensins 1 and 2 (hBD-1, -2) are antimicrobial peptides found in several epithelia including corneal epithelium. Breach of the epithelium leaves the cornea vulnerable to infection so we sought to determine if there is an increase in defensin expression after injury. Methods. The epithelium from human cadaver corneas was collected by scraping (original samples). The corneas were then placed into organ culture to permit regeneration of the epithelium. Samples of re-grown epithelium were collected when healing was partially and 100% complete as determined by fluorescein staining. Total RNA was extracted from original and re-grown samples and used in RT-PCR reactions using primers specific for hBD-1 and hBD-2 and the constitutively expressed gene glyceraldehyde-3-phosphate dehydrogenase. Immunoblotting was performed to detect defensin peptide in original and re-grown samples. Results. hBD-1 mRNA was detected in all original epithelial tissue samples (n = 10) examined suggesting that it is constitutively expressed. hBD-2 mRNA was detectable in only two of the ten samples. Of six corneas placed in to organ culture, hBD-1 mRNA expression was unchanged in the re-grown epithelial samples compared to the original epithelium samples, however the expression of hBD-2 mRNA was markedly increased. hBD-1 and hBD-2 peptides showed the same pattern of expression as their respective transcripts. Conclusions. These data show that hBD-2 mRNA and peptide is up-regulated in the corneal epithelium during re-epithelialization which is in keeping with the role of this defensin as an antimicrobial peptide.

Toll-like receptor activation modulates antimicrobial peptide expression by ocular surface cells.

The ability of the ocular surface to respond to pathogens is in part attributed to toll-like receptors (TLRs) that recognize conserved motifs on various microbes. This study examines TLR expression on various ocular surface cells, if TLR agonists can modulate the expression of antimicrobial peptides (AMPs), human beta defensins (hBD) and cathelicidin (hCAP-18/LL-37) which maybe functionally active against Pseudomonas aeruginosa (PA) and if TLR agonists or AMPs can modulate TLR mRNA expression. TLR1-10 mRNA expression was examined in corneal epithelial, corneal stromal cells and conjunctival epithelial cells by RT-PCR. To confirm protein expression flow cytometry or immunostaining was performed for selected TLRs on some cell cultures. Ocular surface cells were cultured with a range of TLR agonists and then hBD-1, 2, 3, or hCAP-18 mRNA and protein expression was determined by RT-PCR and immunoblotting. In some experiments, cells were cultured with a cocktail of agonists for TLR3, 5 and 6/2 and the antimicrobial activity of the culture media was tested against PA. TLR mRNA expression was also examined in primary human corneal epithelial cells (HCEC) treated with either 3 μg/ml of hBD-2, 5 μg/ml of LL-37 or TLR4, 5 and 9 agonists. Overall, the ocular surface cells expressed mRNA for most of the TLRs but some differences were found. TLR2 was not detected in corneal fibroblasts, TLR4 was not detected in primary cultured or freshly isolated HCEC, TLR5 was not detected in conjunctival epithelial cells (IOBA-NHC) and corneal fibroblasts, TLR7 was not detected in freshly isolated HCEC and TLR10 was not detected in HCEC and IOBA-NHC. TLR8 mRNA was not expressed by any of the samples tested. Immunostaining of cadaver corneas revealed TLR5 and 9 expression throughout the cornea while TLR3 was significantly expressed only in the epithelium. Flow cytometry and immunostaining revealed cultured fibroblasts expressed TLR9 but had no significant TLR3 expression. hBD-2 expression was upregulated by TLR1/2, 3, 4, 5 and 6/2 agonists depending on the cell type, whereas only the TLR3 agonist upregulated the expression of hCAP-18 in primary HCEC. The combination of TLR3, 5 and 6/2 agonists in primary HCEC, upregulated hBD-2 and hCAP-18 mRNA and peptide expression and secretion into the culture media, which significantly killed PA. This antimicrobial activity was primarily attributed to LL-37. TLR agonists did not modulate TLR expression itself, however, LL-37 or hBD-2 downregulated TLR5, 7 and/or 9 mRNA depending on the cell type. TLRs are expressed on the ocular surface and TLR agonists trigger the production of LL-37 and hBD-2, with LL-37 being particularly important for protecting the ocular surface against PA infection.

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.

The TFOS International Workshop on Contact Lens Discomfort: report of the definition and classification subcommittee.

The Ocular Surface Institute, University of Houston College of Optometry, Houston, Texas Louisiana State University Health Sciences Center, Louisiana State University School of Medicine, Louisiana State University Eye Center, New Orleans, Louisiana HealthPartners Medical Group, Minneapolis, Minnesota Centre for Contact Lens Research, School of Optometry and Vision Science, University of Waterloo, Waterloo, Ontario, Canada Corneal Consultants of Colorado, PC, University of Colorado Medical School, Denver, Colorado La Jolla BioConsulting, San Diego, California Brien Holden Vision Institute, Sydney, New South Wales, Australia Vision Co-operative Research Centre, Sydney, New South Wales, Australia School of Optometry and Vision Science, University of New South Wales, Sydney, New South Wales, Australia

Toll-Like Receptor Expression and Activation in Mice with Experimental Dry Eye

PURPOSE To investigate the expression and/or function of toll-like receptors (TLRs) and antimicrobial peptides (AMPs) in dry eye inflammation. METHODS Experimental dry eye (EDE) was induced in C57BL/6 mice and TLR mRNA and protein expression were determined at the ocular surface and lacrimal gland. TLR agonist cocktail was applied to the ocular surface in untreated (UT), corneal scratched, and EDE mice. The corneal expression of cathelin-related antimicrobial peptide (CRAMP; human LL-37 orthologue), and mouse beta defensin (mBD)-3 and -4 (human BD-2 orthologue) was compared. LL-37, hBD-2, TLR4, 5, and TLR9 mRNA expression was examined in patients with dysfunctional tear syndrome (DTS) via conjunctival impression cytology. Murine central corneal thickness (CCT) and inflammatory cell recruitment into the stroma was determined by in vivo imaging. RESULTS EDE upregulated TLR2-4 and 9 mRNA expression in the palpebral conjunctiva and with the exception of TLR4, a similar expression, occurred in the corneal epithelium. TLR2 and 5 were upregulated in lacrimal gland and overall, there was a corresponding change in TLR protein. EDE decreased CRAMP mRNA and protein. hBD-2 and TLR9 expression were modulated in DTS subjects. Topical TLR agonist increased inflammatory cells recruitment and CCT in mice with a cornea scratch. In EDE, TLR agonist treatment downregulated corneal mBD-4 protein caused corneal epithelial loss, and stromal ulceration resulting in decreased CCT. CONCLUSIONS DTS modulates the expression of TLR and CRAMP and topical application of TLR agonists in EDE mice resulted in corneal epithelial loss and thinning. These results suggest that TLRs are involved in DTS inflammation.

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

Dry eye is a common ocular surface disease of multifactorial etiology characterized by elevated tear osmolality and inflammation leading to a disrupted ocular surface. The latter is a risk factor for ocular surface infection, yet overt infection is not commonly seen clinically in the typical dry eye patient. This suggests that important innate mechanisms operate to protect the dry eye from invading pathogens. This article reviews the current literature on epidemiology of ocular surface infection in dry eye patients and laboratory-based studies on innate immune mechanisms operating at the ocular surface and their alterations in human dry eye and animal models. The review highlights current understanding of innate immunity in dry eye and identifies gaps in our knowledge to help direct future studies to further unravel the complexities of dry eye disease and its sequelae.

Functions of Peptidoglycan Recognition Proteins (Pglyrps) at the Ocular Surface: Bacterial Keratitis in Gene-Targeted Mice Deficient in Pglyrp-2, -3 and -4

Purpose Functions of antimicrobial peptidoglycan recognition proteins (Pglyrp1-4) at the ocular surface are poorly understood. Earlier, we reported an antibacterial role for Pglyrp-1 in Pseudomonas aeruginosa keratitis. Here we investigated functions of three other related genes Pglyrp-2, -3 and -4 in a mouse model of P. aeruginosa keratitis. Methods Wild type (WT) and each of the Pglyrp-null genotypes were challenged with P. aeruginosa keratitis. The eyes were scored in a blinded manner 24 and 48h post infection. Viable bacterial counts and inflammatory factors (IL-12, TNF-α, IFN-γ, CCL2, IL-6 and IL-10) were measured in whole eye homogenates using cytometric bead arrays. Expressions of Pglyrp-1-4, mouse beta defensins (mBD)-2,-3, cathelicidin-related antimicrobial peptide (CRAMP) were determined by qRTPCR in total RNA extracts of uninfected and infected eyes of WT and each of the Pglyrp-null mouse types. Results The Pglyrp-2 -/- mice showed reduced disease and lower induction of pro-inflammatory TNF-α (p = 0.02) than WT or the other Pglyrp null mice. Viable bacterial yield was significantly lower in the Pglyrp-2-/- (p = 0.0007) and the Pglyrp-4-/- (p = 0.098) mice. With regards to expression of these antimicrobial genes, Pglyrp-2 expression was induced after infection in WT mice. Pglyrp-3 expression was low before and after infection in WT mice, while Pglyrp-4 expression was slightly elevated after infection in WT, Pglyrp-2 and -3 null mice. Pglyrp-1 expression was slightly elevated after infection in all genotypes without statistical significance. Transcripts for antimicrobial peptides mBD2, mBD3 and CRAMP were elevated in infected Pglyrp-2 -/- males without statistical significance. Conclusions Efficient resolution of keratitis in the Pglyrp-2 -/- mice may be due to a reduced pro-inflammatory microenvironment and synergistic antibacterial activities of defensins, CRAMP and Pglyrp-1. Therefore, in ocular infections the pro-inflammatory functions of Pglyrp-2 must be regulated to benefit the host.

MyD88 contribution to ocular surface homeostasis

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 Pseudomonas aeruginosa 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.

Neutrophils cause obstruction of eyelid sebaceous glands in inflammatory eye disease in mice

Neutrophils obstruct the meibomian gland in a mouse model of inflammatory eye disease and correlate with disease severity in patients with meibomian gland dysfunction. Inflammatory obstruction in the eye Obstruction of eyelid glands called meibomian glands (MGs) is a risk factor for developing chronic inflammation of the eyelids. The function of these glands is to secrete oils onto the surface of the eye. The etiology of MG obstruction is not completely understood. Now, Reyes et al. have discovered that polymorphonuclear neutrophils (PMNs) promoted MG obstruction in a mouse model of inflammatory eye disease. Furthermore, PMNs were increased in tears of patients with MG obstruction, and PMN number correlated with the severity of the obstruction. The data suggest that PMNs might contribute to the etiology of MG obstruction in inflammatory eyelid disease. Meibomian glands (MGs) are sebaceous glands of the eyelid margin that secrete lipids needed to avert tear evaporation and to help maintain ocular surface homeostasis. Obstruction of MGs or other forms of MG dysfunction can promote chronic diseases of the ocular surface. Although chronic eyelid inflammation, such as allergic eye disease, is an associated risk factor for obstructive MG dysfunction, it is not clear whether inflammatory processes contribute to the pathophysiology of MG obstruction. We show that polymorphonuclear neutrophils (PMNs) promoted MG obstruction in a chronic inflammatory model of allergic eye disease in mice. Analysis of leukocytes in tears of patients with MG dysfunction showed an increase in PMN numbers compared to healthy subjects. Moreover, PMN numbers in tears positively correlated with clinical severity of MG dysfunction. Our findings point to a role for PMNs in the pathogenesis and progression of MG dysfunction.