Toll-like receptors at the ocular surface

Abstract

The Toll-like receptor (TLR) family of pathogen recognition molecules has an important role in recognizing microbial pathogens and microbial breakdown products. Activation of TLRs in the corneal epithelium induces CXC chemokine production and recruitment of neutrophils to the corneal stroma. Although essential for pathogen killing, neutrophils can cause extensive tissue damage, leading to visual impairment and blindness. In this review, we examine the role of TLRs in microbial keratitis and in noninfectious corneal inflammation, most commonly associated with contact lens wear. we present recent findings on TLR signaling pathways in the cornea, including MyD88- and TRIF-dependent responses and discuss the role of resident macrophages and dendritic cells. Finally, we examine the potential for targeting the TLR pathway as a potential therapeutic intervention for microbial keratitis and contact lens-associated corneal inflammation.

Figure 1

A model for TLR-induced corneal inflammation. Physical disruption of the corneal epithelium and exposure to microbial products, including lipoproteins (Pam3Cys), dsRNA (Poly[I:C]), LPS or flagellin, initiates MyD88-dependent or TRIF-dependent signaling. The MyD88 pathway activates MAP kinase (p42/p44, JNK, and p38) and NF-κB, resulting in expression of CXC chemokines. In contrast, the TRIF pathway activates IRF-3, MAP kinases JNK and p38, and NF-κB, resulting in production of CCL5/RANTES and type I interferons. Chemokine production stimulates macrophage and neutrophil infiltration into the cornea and subsequent loss of corneal clarity. (Adapted from Johnson A, Pearlman E and Johnson AC, Li X, Pearlman E.)

Figure 2

A. TLR2-, TLR4, and TLR9-induced corneal inflammation is MyD88-dependent. Corneas of control and MyD88^–/– mice were abraded and treated with PBS, Pam₃Cys, LPS, or CpG ODN. After 24 h, cellular infiltration to the corneal stroma and stromal thickness and haze were determined by in vivo confocal microscopy (Confoscan™). Representative images of the central corneal stroma show a cellular infiltrate in wild type mice (upper panels) but not in MyD88^–/– mice (lower panels). (Reprinted from Johnson AC, Heinzel FP, Diaconu E, et al with permission from Invest Ophthalmol Vis Sci.) B and C: Exacerbated TLR3/TRIF responses in MyD88^–/– mice. C57BL/6 or MyD88^–/– corneas were abraded and treated with Poly(I:C) for 72 h, then examined by confocal microscopy or after immunohistochemical analysis for macrophages and neutrophils. Representative Confoscan™ images of the central corneal stroma demonstrate the severity of Poly(I:C)-induced inflammation in MyD88^–/– mice (B), and increased neutrophils and macrophages (C). Note that TLR3/TRIF-induced responses were also enhanced in human corneal epithelial cells treated with siRNA to knockdown MyD88, but not in MyD88^–/– bone marrow-derived macrophages (see Section v). D. TLR3/TRIF-induced corneal inflammation. Poly(I:C) was added topically to the abraded corneal epithelium of C57BL/6, TLR3^–/–, and TRIF^–/– mice. After 72 h, corneas were processed for immunohistochemistry, and macrophages were detected using Ab to F4/80. (B-D reprinted from Johnson AC, Li X, Pearlman E. In accordance with policy of the Rockefeller University Press, the authors retain copyright to material published in J Biol Chem.)

Figure 3

A. Role of MAP kinases in TLR2-induced corneal inflammation. C57BL/6 corneas were gently scarified by three parallel scratches and treated with either H₂O (trauma control) or with the TLR2 ligand Pam₃Cys. At indicated times, corneas were dissected and processed for SDS-PAGE and Western blot analysis. Each sample represents a pool of two corneas and is representative of two repeat experiments. B. SP600125 inhibits TLR2-induced corneal inflammation. C57BL/6 mice were treated topically with either SP600125 or with vehicle alone 1 h prior to, at the same time as, and 6 h after stimulation with Pam₃Cys. After 24 h, neutrophils in the corneal stroma were detected by immunohistochemistry (A), and corneal thickness and haze were measured by in vivo confocal microscopy (B, C). C. TLR2-induced corneal inflammation in JNK1^–/– mice. Corneas of JNK-1^–/– and C57BL/6 mice were scarified as described above and stimulated by topical application of Pam₃Cys. (Reprinted from Adhikary G, Sun Y, Pearlman E with permission from J Leukoc Biol.)

Figure 4

Membrane nanotubes in the corneal stroma. C57BL/6 mice were irradiated and given a bone marrow transplant from eGFP mice as described, and were either untreated or stimulated by topical application of LPS. Corneal whole mounts were immunostained and examined by confocal microscopy. A. Donor-derived (GFP+/green) MHC class II+ (red) and double positive (yellow) cell connecting via a fine membrane nanotube (arrows) to a resident MHC class II+ GFP negative cell (red). B. GFP+ donor-derived cells expressing MHC class II+ cells appear to connect by a membrane nanotube (inset shows higher magnification). C and D. Long, nonbridged membrane nanotubes on MHC class II+ cells in the naive (C) and inflamed (D) mouse corneal stroma. Connected neighboring cells were not identifiable. Scale bars = 20 μm; inset scale bar = 10 μm. E. Frequency of membrane nanotubes. Pooled data from all regions of the cornea reveal a higher density of nanotubes in inflamed corneas (both saline- and LPS-treated eyes) compared with naive corneas. (Reprinted from Chinnery HR, Pearlman E, McMenamin PG with permission from J Immunol).

Figure 5

Effect of topical C6 ceramide in nanoparticle formulation (Lip-C6) on S. aureus- and LPS-induced corneal inflammation. The central corneal stroma of C57BL/6 mice was abraded as described, and 2nMoles (811ng) topical Lip-C6 was given 1 h before and 6 h after exposure to heat-killed S. aureus. After 24 h, neutrophils were detected by immunohistochemistry, and corneal thickness and haze were calculated as described in references 38, 39, and 69. Data points represent individual corneas from groups of 4-8 mice. (Reprinted from Sun Y, Fox T, Adhikary G, et al with permission from J Leukoc Biol.)