Abstract
BackgroundToll-like receptor (TLR) activation is hypothesized to contribute to inflammatory eye disease including uveitis, yet the distribution pattern of TLRs in human uveal tissues remains poorly described. The purpose of this study was to investigate the expression profile of TLRs in human iris pigment epithelial cells (IPE) at the gene and protein level and examine the effect of pathogen-associated molecular patterns (PAMPs), such as Pam3CSK4.3HCl, Poly(I:C), lipopolysaccharides (LPS from E. coli serotype O111:B4), Flagellin, MALP-2 (macrophage activating lipopeptide-2), Poly(U) and CpGODN2395 on the production of inflammatory mediators including interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) from human IPE and retinal pigment epithelial cells (RPE).MethodsRT-PCR and Western blotting was employed to investigate the expression of TLRs 1–10 in primary IPE and RPE. Secretion of IL-8 or MCP-1 following treatment with PAMPs was measured by ELISA. The role of TLR2, TLR3 and TLR4 in mediating an inflammatory response was investigated using pharmacological TLR inhibitors.ResultsIPE and RPE expressed transcripts for TLR1-6 and 8–10; and proteins for TLR1-6 and 9. IPE secreted IL-8 or MCP-1 in response to Pam3CSK4.3HCl, Poly(I:C), LPS and MALP-2, whereas RPE produced IL-8 only after Poly(I:C), LPS or MALP-2 treatment. TLR inhibitors (OxPAPC, CI-095 and chloroquine) blocked IL-8 secretion in Poly(I:C), LPS or MALP-2-treated IPE and RPE.ConclusionsOcular pigment epithelial cells respond to PAMPs through activation of TLRs, particularly TLR2, TLR3 and TLR4. Expression of TLRs in human IPE cells provides a basis for responses to many ocular pathogens and their activation may be involved in the pathogenesis of ocular inflammation.
Highlights
Toll-like receptor (TLR) activation is hypothesized to contribute to inflammatory eye disease including uveitis, yet the distribution pattern of Toll-like receptors (TLRs) in human uveal tissues remains poorly described
This study demonstrated that human iris pigment epithelial cells (IPE) and retinal pigment epithelial cells (RPE) secrete IL-8 and monocyte chemotactic protein 1 (MCP-1) in response to pathogen-associated molecular patterns (PAMPs), which was partially mediated through TLR activation
Human IPE and RPE express TLRs Real time-PCR analysis revealed that human IPE and RPE expressed mRNA for TLR2, −3, −4 and −6 (Figure 1A) which were expressed in positive controls ARPE-19 and THP-1 cell lines (Figure 1B and C)
Summary
Toll-like receptor (TLR) activation is hypothesized to contribute to inflammatory eye disease including uveitis, yet the distribution pattern of TLRs in human uveal tissues remains poorly described. The purpose of this study was to investigate the expression profile of TLRs in human iris pigment epithelial cells (IPE) at the gene and protein level and examine the effect of pathogen-associated molecular patterns (PAMPs), such as Pam3CSK4.3HCl, Poly(I:C), lipopolysaccharides (LPS from E. coli serotype O111:B4), Flagellin, MALP-2 (macrophage activating lipopeptide-2), Poly(U) and CpGODN2395 on the production of inflammatory mediators including interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) from human IPE and retinal pigment epithelial cells (RPE). Activation of TLRs by PAMPs due to an initiating mucosal infection and the subsequent immune response has been hypothesised to play a key role in the pathogenesis of anterior uveitis [2]. Studying TLRs on iris pigment epithelial cells (IPE) and their response to PAMPs may provide an insight into pathogenesis of ocular inflammation, anterior uveitis
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