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Gene expression regulation in retinal pigment epithelial cells induced by viral RNA and viral/bacterial DNA.

Brosig A, Kuhrt H, Wiedemann P, Kohen L, Bringmann A, Hollborn M - Mol. Vis. (2015)

Bottom Line: We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells.The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina.The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit viral retinal inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.

ABSTRACT

Purpose: The pathogenesis of age-related macular degeneration (AMD) is associated with systemic and local inflammation. Various studies suggested that viral or bacterial infection may aggravate retinal inflammation in the aged retina. We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells.

Methods: Cultured human RPE cells were stimulated with poly(I:C; 500 µg/ml) or CpG-ODN (500 nM). Alterations in gene expression and protein secretion were determined with real-time RT-PCR and ELISA, respectively. Phosphorylation of signal transduction molecules was revealed by western blotting.

Results: Poly(I:C) induced gene expression of the pattern recognition receptor TLR3, transcription factors (HIF-1α, p65/NF-κB), the angiogenic factor bFGF, inflammatory factors (IL-1β, IL-6, TNFα, MCP-1, MIP-2), and complement factors (C5, C9, CFB). Poly(I:C) also induced phosphorylation of ERK1/2 and p38 MAPK proteins, and the secretion of bFGF and TNFα from the cells. CpG-ODN induced moderate gene expression of transcription factors (p65/NF-κB, NFAT5) and complement factors (C5, C9), while it had no effect on the expression of various TLR, angiogenic factor, and inflammatory factor genes. The activities of various signal transduction pathways and transcription factors were differentially involved in mediating the poly(I:C)-induced transcriptional activation of distinct genes.

Conclusions: The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina. The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit viral retinal inflammation.

No MeSH data available.


Related in: MedlinePlus

Effects of viral RNA and viral/bacterial DNA on the expression of complement factor genes. The mRNA levels were determined with real-time RT–PCR analysis after stimulation of the cells for 2, 6, and 24 h (as indicated by the panels of the bars), and are expressed as fold of unstimulated controls. A: Expression of complement factor genes in cells cultured for 2 (1), 6 (2), and 24 h (3), as determined by RT–PCR. Negative controls (0) were done by adding double-distilled water instead of cDNA as a template. B: Near-confluent cultures were stimulated with poly(I:C; 500 µg/ml). C: Dose-dependent effect of poly(I:C) on the expression of the CFB gene. Confluent cultures were stimulated with poly(I:C; 100 and 500 µg/ml, respectively). D: Near-confluent cultures were stimulated with CpG-ODN (500 nM). Each bar represents data obtained in three to five independent experiments using cells from different donors. Each bar represents data obtained in 3 to 5 independent RPE cell lines, each from a different human eye donor; experiments with each cell line were carried out in triplicate: *p<0.05. ●, p<0.05.
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f6: Effects of viral RNA and viral/bacterial DNA on the expression of complement factor genes. The mRNA levels were determined with real-time RT–PCR analysis after stimulation of the cells for 2, 6, and 24 h (as indicated by the panels of the bars), and are expressed as fold of unstimulated controls. A: Expression of complement factor genes in cells cultured for 2 (1), 6 (2), and 24 h (3), as determined by RT–PCR. Negative controls (0) were done by adding double-distilled water instead of cDNA as a template. B: Near-confluent cultures were stimulated with poly(I:C; 500 µg/ml). C: Dose-dependent effect of poly(I:C) on the expression of the CFB gene. Confluent cultures were stimulated with poly(I:C; 100 and 500 µg/ml, respectively). D: Near-confluent cultures were stimulated with CpG-ODN (500 nM). Each bar represents data obtained in three to five independent experiments using cells from different donors. Each bar represents data obtained in 3 to 5 independent RPE cell lines, each from a different human eye donor; experiments with each cell line were carried out in triplicate: *p<0.05. ●, p<0.05.

Mentions: Age-related retinal inflammation is associated with local activation of the alternative complement cascade [2,3] and subretinal deposition of activated complement proteins such as C3a and C5a [32]. We compared the effects of viral RNA and viral/bacterial DNA on the gene expression of various complement proteins in RPE cells (Figure 6A). As shown in Figure 6B, the synthetic viral RNA analog poly(I:C) induced significant (p<0.05) time-dependent increases in the expression of C5, C9, CFB, and CFH genes, and had no effect on the expression of the C3 gene. The effect of poly(I:C) was more pronounced in respect to the CFB gene than to the other complement factor genes investigated (Figure 6B,C). Stimulation of RPE cells with the viral/bacterial DNA analog CpG-ODN induced increased expression of C5 and C9 genes, while it had no effect on the expression of C3, CFB, and CFH genes (Figure 6D). The data suggest that both viral RNA and viral/bacterial DNA induce the expression of distinct complement factor genes in RPE cells.


Gene expression regulation in retinal pigment epithelial cells induced by viral RNA and viral/bacterial DNA.

Brosig A, Kuhrt H, Wiedemann P, Kohen L, Bringmann A, Hollborn M - Mol. Vis. (2015)

Effects of viral RNA and viral/bacterial DNA on the expression of complement factor genes. The mRNA levels were determined with real-time RT–PCR analysis after stimulation of the cells for 2, 6, and 24 h (as indicated by the panels of the bars), and are expressed as fold of unstimulated controls. A: Expression of complement factor genes in cells cultured for 2 (1), 6 (2), and 24 h (3), as determined by RT–PCR. Negative controls (0) were done by adding double-distilled water instead of cDNA as a template. B: Near-confluent cultures were stimulated with poly(I:C; 500 µg/ml). C: Dose-dependent effect of poly(I:C) on the expression of the CFB gene. Confluent cultures were stimulated with poly(I:C; 100 and 500 µg/ml, respectively). D: Near-confluent cultures were stimulated with CpG-ODN (500 nM). Each bar represents data obtained in three to five independent experiments using cells from different donors. Each bar represents data obtained in 3 to 5 independent RPE cell lines, each from a different human eye donor; experiments with each cell line were carried out in triplicate: *p<0.05. ●, p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554413&req=5

f6: Effects of viral RNA and viral/bacterial DNA on the expression of complement factor genes. The mRNA levels were determined with real-time RT–PCR analysis after stimulation of the cells for 2, 6, and 24 h (as indicated by the panels of the bars), and are expressed as fold of unstimulated controls. A: Expression of complement factor genes in cells cultured for 2 (1), 6 (2), and 24 h (3), as determined by RT–PCR. Negative controls (0) were done by adding double-distilled water instead of cDNA as a template. B: Near-confluent cultures were stimulated with poly(I:C; 500 µg/ml). C: Dose-dependent effect of poly(I:C) on the expression of the CFB gene. Confluent cultures were stimulated with poly(I:C; 100 and 500 µg/ml, respectively). D: Near-confluent cultures were stimulated with CpG-ODN (500 nM). Each bar represents data obtained in three to five independent experiments using cells from different donors. Each bar represents data obtained in 3 to 5 independent RPE cell lines, each from a different human eye donor; experiments with each cell line were carried out in triplicate: *p<0.05. ●, p<0.05.
Mentions: Age-related retinal inflammation is associated with local activation of the alternative complement cascade [2,3] and subretinal deposition of activated complement proteins such as C3a and C5a [32]. We compared the effects of viral RNA and viral/bacterial DNA on the gene expression of various complement proteins in RPE cells (Figure 6A). As shown in Figure 6B, the synthetic viral RNA analog poly(I:C) induced significant (p<0.05) time-dependent increases in the expression of C5, C9, CFB, and CFH genes, and had no effect on the expression of the C3 gene. The effect of poly(I:C) was more pronounced in respect to the CFB gene than to the other complement factor genes investigated (Figure 6B,C). Stimulation of RPE cells with the viral/bacterial DNA analog CpG-ODN induced increased expression of C5 and C9 genes, while it had no effect on the expression of C3, CFB, and CFH genes (Figure 6D). The data suggest that both viral RNA and viral/bacterial DNA induce the expression of distinct complement factor genes in RPE cells.

Bottom Line: We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells.The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina.The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit viral retinal inflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Eye Hospital, University of Leipzig, Leipzig, Germany.

ABSTRACT

Purpose: The pathogenesis of age-related macular degeneration (AMD) is associated with systemic and local inflammation. Various studies suggested that viral or bacterial infection may aggravate retinal inflammation in the aged retina. We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells.

Methods: Cultured human RPE cells were stimulated with poly(I:C; 500 µg/ml) or CpG-ODN (500 nM). Alterations in gene expression and protein secretion were determined with real-time RT-PCR and ELISA, respectively. Phosphorylation of signal transduction molecules was revealed by western blotting.

Results: Poly(I:C) induced gene expression of the pattern recognition receptor TLR3, transcription factors (HIF-1α, p65/NF-κB), the angiogenic factor bFGF, inflammatory factors (IL-1β, IL-6, TNFα, MCP-1, MIP-2), and complement factors (C5, C9, CFB). Poly(I:C) also induced phosphorylation of ERK1/2 and p38 MAPK proteins, and the secretion of bFGF and TNFα from the cells. CpG-ODN induced moderate gene expression of transcription factors (p65/NF-κB, NFAT5) and complement factors (C5, C9), while it had no effect on the expression of various TLR, angiogenic factor, and inflammatory factor genes. The activities of various signal transduction pathways and transcription factors were differentially involved in mediating the poly(I:C)-induced transcriptional activation of distinct genes.

Conclusions: The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina. The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit viral retinal inflammation.

No MeSH data available.


Related in: MedlinePlus