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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 gene expression and the secretion of angiogenic growth factors. The mRNA levels (B, C, E) 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. The levels of VEGF-A165 and bFGF proteins (D) were determined with ELISA in the cultured media of cells stimulated for 6 and 24 h, and are expressed in percent of unstimulated controls (100%). A: Expression of growth 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, D: Near-confluent cultures were stimulated with poly(I:C; 500 µg/ml). C. Dose-dependent effect of poly(I:C). Confluent cultures were stimulated with poly(I:C; 100 and 500 µg/ml, respectively). E: Near-confluent cultures were stimulated with CpG-ODN (500 nM). 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. Significant difference versus unstimulated controls: *p<0.05. ●, p<0.05.
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f4: Effects of viral RNA and viral/bacterial DNA on gene expression and the secretion of angiogenic growth factors. The mRNA levels (B, C, E) 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. The levels of VEGF-A165 and bFGF proteins (D) were determined with ELISA in the cultured media of cells stimulated for 6 and 24 h, and are expressed in percent of unstimulated controls (100%). A: Expression of growth 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, D: Near-confluent cultures were stimulated with poly(I:C; 500 µg/ml). C. Dose-dependent effect of poly(I:C). Confluent cultures were stimulated with poly(I:C; 100 and 500 µg/ml, respectively). E: Near-confluent cultures were stimulated with CpG-ODN (500 nM). 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. Significant difference versus unstimulated controls: *p<0.05. ●, p<0.05.

Mentions: Progression of AMD to the neovascular stage is driven by angiogenic growth factors such as VEGF, bFGF, and HB-EGF, which are produced, for example, by RPE cells [29-31]. We compared the effects of viral RNA and viral/bacterial DNA on the gene expression of these factors in RPE cells (Figure 4A). As shown in Figure 4B,C, the synthetic analog of viral RNA poly(I:C) induced a significant (p<0.05) increase in the expression of the bFGF gene in RPE cells and had no effect on the expression of the VEGF gene. Poly(I:C) also induced a moderate increase in the expression of the HB-EGF gene in non-confluent cultures (Figure 4B). The administration of poly(I:C) also induced a significant (p<0.05) increase in the secretion of the bFGF protein from RPE cells, while it had no effect on the secretion of VEGF protein (Figure 4D). We did not find detectable amounts of soluble HB-EGF protein with ELISA in the media of cells cultured for 6 or 24 h in the absence and presence of poly(I:C; 500 µM; not shown). Stimulation of RPE cells with the synthetic analog of viral/bacterial DNA, CpG-ODN, did not alter the cellular levels of VEGF, bFGF, and HB-EGF mRNAs (Figure 4E). The data suggest that viral RNA induces expression and secretion of bFGF.


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 gene expression and the secretion of angiogenic growth factors. The mRNA levels (B, C, E) 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. The levels of VEGF-A165 and bFGF proteins (D) were determined with ELISA in the cultured media of cells stimulated for 6 and 24 h, and are expressed in percent of unstimulated controls (100%). A: Expression of growth 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, D: Near-confluent cultures were stimulated with poly(I:C; 500 µg/ml). C. Dose-dependent effect of poly(I:C). Confluent cultures were stimulated with poly(I:C; 100 and 500 µg/ml, respectively). E: Near-confluent cultures were stimulated with CpG-ODN (500 nM). 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. Significant difference versus unstimulated controls: *p<0.05. ●, p<0.05.
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f4: Effects of viral RNA and viral/bacterial DNA on gene expression and the secretion of angiogenic growth factors. The mRNA levels (B, C, E) 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. The levels of VEGF-A165 and bFGF proteins (D) were determined with ELISA in the cultured media of cells stimulated for 6 and 24 h, and are expressed in percent of unstimulated controls (100%). A: Expression of growth 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, D: Near-confluent cultures were stimulated with poly(I:C; 500 µg/ml). C. Dose-dependent effect of poly(I:C). Confluent cultures were stimulated with poly(I:C; 100 and 500 µg/ml, respectively). E: Near-confluent cultures were stimulated with CpG-ODN (500 nM). 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. Significant difference versus unstimulated controls: *p<0.05. ●, p<0.05.
Mentions: Progression of AMD to the neovascular stage is driven by angiogenic growth factors such as VEGF, bFGF, and HB-EGF, which are produced, for example, by RPE cells [29-31]. We compared the effects of viral RNA and viral/bacterial DNA on the gene expression of these factors in RPE cells (Figure 4A). As shown in Figure 4B,C, the synthetic analog of viral RNA poly(I:C) induced a significant (p<0.05) increase in the expression of the bFGF gene in RPE cells and had no effect on the expression of the VEGF gene. Poly(I:C) also induced a moderate increase in the expression of the HB-EGF gene in non-confluent cultures (Figure 4B). The administration of poly(I:C) also induced a significant (p<0.05) increase in the secretion of the bFGF protein from RPE cells, while it had no effect on the secretion of VEGF protein (Figure 4D). We did not find detectable amounts of soluble HB-EGF protein with ELISA in the media of cells cultured for 6 or 24 h in the absence and presence of poly(I:C; 500 µM; not shown). Stimulation of RPE cells with the synthetic analog of viral/bacterial DNA, CpG-ODN, did not alter the cellular levels of VEGF, bFGF, and HB-EGF mRNAs (Figure 4E). The data suggest that viral RNA induces expression and secretion of bFGF.

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