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

Abstract

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.

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

“…Candidate ExsA-independent factors driving epithelial traversal include various virulence factors shown to be involved in passaging through cultured cells, such as proteases, rhamnolipids, exotoxin A, and the MexAB/OprM antimicrobial efflux pump (27,38,39,41,61). Whatever the case, ExsA-independent factors driving traversal could differ in the 6-h healing corneas and MyD88-compromised corneas considering that (i) the barrier function with regard to fluorescein is lost in the former (45) but not in the latter (47) situation and (ii) that MyD88-deficient epithelia are defective in expression of many defense factors (51,52,62,63), including those identified in this study.…”

“…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.…”

Pattern recognition receptors in microbial keratitis

“…[8][9][10][11][12] In addition, bathing tear fluid contains mucins, secretory immunoglobulin A (sIgA), and surfactant protein D, all antimicrobial factors that can bind microbes and potentially alter their interactions with corneal epithelial cells. [13][14][15] Epithelial cells also express several antimicrobial peptides, including human β-defensin 2 (hBD-2), cathelicidin LL-37, 11,12,16,17 and cytokeratin 6A. 18 In addition, superficial epithelial cells can internalise bacteria and then desquamate, thereby reducing the infective load.…”

Mouse Bone Marrow Sca-1 + CD44 + Mesenchymal Stem Cells Kill Avirulent Mycobacteria but Not Mycobacterium tuberculosis through Modulation of Cathelicidin Expression via the p38 Mitogen-Activated Protein Kinase-Dependent Pathway

“…tuberculosis and M. bovis BCG infections modulate TLR and cytokine expression in BM-MSCs. Previous studies have shown that bacterial challenge increases the expression levels of TLRs, which subsequently increase the production of AMPs to reduce the risk of microbial infections (23). It has been shown that cathelicidin expression is regulated mainly via TLR2 and TLR4 signaling cascades (24,25).…”