Distinct Functions between Toll-Like Receptors 3 and 9 in Retinal Pigment Epithelial Cells

Abstract

Retinal pigment epithelial cells (RPE cells) are key players in the first-line defense against invading organisms such as viruses and bacteria. The interaction between RPE cells and viral or bacterial components is very important for clearance of these organisms. Toll-like receptors are a family of recognition receptors involved in innate immunity. Each TLR acts as a primary sensor of conserved microbial components and drives the induction of specific biological responses. TLR 3 is involved in the recognition of viral components, such as double-stranded RNA (dsRNA) and poly(I:C), while TLR 9 recognizes viral or bacterial DNA without methylation at CpG motifs. In the present study, we investigated the expression and function of TLR 3 and 9 in RPE cells. PCR analysis revealed expression of genes for TLR 3 and 9 in RPE cells. Expression of TLR 3 and 9 protein was detected in RPE cells by flow cytometry. TLR 3 and 9 showed strong intracellular expression. To detect angiogenetic factors produced by RPE cells, culture supernatant was examined with the Human Angiogenesis Antibody Array, which can simultaneously detect 20 different angiogenetic factors including cytokines, chemokines, soluble cytokine receptors, and growth factors. RPE cells showed high production of interleukin-8 (IL-8) and monocyte chemotactic protein-I (MCP-I). Furthermore, stimulation of RPE cells with the dsRNA analogue poly(I:C) enhanced the secretion of IL-8 and MCP-I, as well as enhancing the expression of junctional adhesion molecule-I (Jam-I) and intracellular adhesion molecule-I (ICAM-I), and promoted the adhesion of monocyte to these cells. In contrast, stimulation with the CpG-DNA motif only enhanced the secretion of IL-8. However, CpG-DNA motif enhanced phagocytosis in RPE cells. These results may indicate that TLR 3 and 9 play a distinct role in the inflammatory response that clears viruses from the retina.

Mitochondrial DNA has a pro-inflammatory role in AMD

“…In other studies, extracellular mtDNA was mildly immunostimulatory in certain cell types, but only at very high concentrations (>10µg/mL) 13,20 . In ARPE-19 cells, extracellular CpG DNA has been shown to induce IL-8 secretion only at the dose of 84µg/mL, almost 2 orders of magnitude higher than the concentration we used, and the authors attributed the effect to CpG DNA that had been internalized by phagocytosis 69 .…”

“…This could be explained by a lack of surface receptors for mtDNA on ARPE-19 cells. Practically all the DNA sensors described to date are intracellular 36 , including Toll-like receptor 9 (TLR9), a transmembrane receptor of unmethylated CpG motifs that has been confirmed to be predominantly intracellular rather than on the cell membrane 67,68 , including in ARPE-19 69 . Our findings are also in concordance with previous evidence that extracellular mtDNA has no immunostimulatory effect unless used with a molecule that aids its uptake into the cell 20,70 .…”

Toll-Like Receptor 3 Activation Initiates Photoreceptor Cell Death In Vivo and In Vitro

“…Consistent with findings in RPE cells, increased expression of proinflammatory cytokines IL-6 and CCL2 have been detected. 18 Meanwhile, signaling factor myeloid differentiation primary response gene 88 (MyD88) in the TLR-link signaling pathway-and cytokines and chemokines such as CXCL10, CCL5, IL-18, CCL4, IFN-a, and IFN-b-found upregulated in poly I:C-treated photoreceptor cells. 26 In line with previous studies, MyD88 plays an important role in retinal degeneration by regulating expression of CCL2, CCL4, CCL7, CXCL10 and CCL5.…”

“…16,17 Previous studies have demonstrated that poly I:C treatment urges secretion of IFNs, IL-6, IL-8, and MCP-1 in RPE cells, which plays a key role in retinal immune defense. 18,19 Provided that photoreceptors are most adjacent to RPE in the retina, the aim of this study was to determine whether immune defense occurs in photoreceptors other than RPE. To the best of our knowledge, it has not been thoroughly investigated so far.…”

Pathogenic Roles of Sterile Inflammation in Etiology of Age-Related Macular Degeneration

“…RPE cells make up the first line of defense against pathogens by expressing almost all TLR iso-forms except TLR-8 (Kumar et al, 2004). Activation of TLR3 and TLR9 leads to the production of inflammatory mediators including cytokines and adhesion molecules in cultured RPE cells (Ebihara et al, 2007). TLR3 detects mRNA (Kariko et al, 2004) and dsRNA (Dogusan et al, 2008) released from necrotic cells while TLR9 senses endogenous DNA (Zhang et al, 2010), triggering sterile inflammatory response and subsequent toxicity.…”