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. 2020 Aug 20:156:176-189.
doi: 10.1016/j.freeradbiomed.2020.06.004. Epub 2020 Jul 4.

Retinal pigment epithelium transcriptome analysis in chronic smoking reveals a suppressed innate immune response and activation of differentiation pathways

Lei Wang  1 , Koray D Kaya  2 , Sujung Kim  3 , Matthew J Brooks  4 , Jie Wang  5 , Ying Xin  6 , Jiang Qian  7 , Anand Swaroop  8 , James T Handa  9
Affiliations

Retinal pigment epithelium transcriptome analysis in chronic smoking reveals a suppressed innate immune response and activation of differentiation pathways

Lei Wang et al. Free Radic Biol Med. .

Abstract

Cigarette smoking, a powerful mixture of chemical oxidants, is the strongest environmental risk factor for developing age-related macular degeneration (AMD), the most common cause of blindness among the elderly in western societies. Despite intensive study, the full impact of smoking on the retinal pigment epithelium (RPE), a central cell type involved in AMD pathobiology, remains unknown. The relative contribution of the known dysfunctional pathways to AMD, at what stage they are most pathogenic, or whether other processes are relevant, is poorly understood, and furthermore, whether smoking activates them, is unknown. We performed global RNA-sequencing of the RPE from C57BL/6J mice exposed to chronic cigarette smoke for 6 months to identify potential pathogenic and cytoprotective pathways. The RPE transcriptome induced by chronic cigarette smoking exhibited a mixed response of marked suppression of the innate immune response including type I and II interferons and upregulation of cell differentiation and morphogenic gene clusters, suggesting an attempt by the RPE to maintain its differentiated state despite smoke-induced injury. Given that mice exposed to chronic smoke develop early features of AMD, these novel findings are potentially relevant to the transition from aging to AMD.

Keywords: Age-related macular degeneration; Aging; Differentiation; Innate immunity; RNA sequencing; Smoking.

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Conflict of interest statement

Declaration of competing interest JTH received grant funding and royalties, and MC grant funding from Bayer Pharmaceutical, Inc.

Figures

Figure 1.
Figure 1.
Ultrastructural changes to the RPE after chronic smoking. A. Normal morphology and ultrastructure of the RPE from a C57BL6J mouse raised in air. Apical microvilli (MV) surround photoreceptor outer segments (POS). The basolateral (BL) RPE has normal infoldings (arrowheads). A variety of ultrastructural changes are seen in the RPE from C57BL6J mice exposed to smoke for 6 months. B. The microvilli are shortened, multiple vacuoles (V) are seen within the cell body, and the basal infoldings are shortened and widened (arrowheads). C. More severe mesenchymal shape to the RPE cell with similar changes to the microvilli and basal infoldings as in (B). Undigested POS (arrows) are seen in the basal region of the cell. D. Vacuoles with membranous debris are seen. BrM, Bruch’s membrane, CC, choriocapillaris. Bar = 5 μm.
Figure 2.
Figure 2.
Overview of differentially expressed genes. A. Principle Component Analysis for all expressed genes, totaling 15,559 genes. The samples clustered into two distinct groups of mice exposed to chronic cigarette smoking (CS) and air. B. Volcano plot of DEGs shows separation between air and CS treated mice. C. Gene Ontology enrichment of biological processes (BP) with chronic smoking exposure. Down-regulated genes from smoking are pooled in cluster1, and up-regulated genes in cluster2. The ratio of genes changed in each BP is reflected by the dot size. p-value is reflected by dot color, with red as the most confident change. D. Gene set enrichment analysis using the Hallmark Pathways. Pathways enriched in the down-regulated genes are indicated with a negative net enrichment score (light blue) and those enriched in the up-regulated genes have a positive enrichment score (red). Size indicates the number of genes in the pathway.
Figure 3.
Figure 3.
Enrichment plot of differentially expressed genes (DEGs) induced by chronic smoking in the RPE that are related to Epithelial mesenchymal transition. DEGs were ranked from positive to negative fold change and compared to the Broad Institute’s GSEA EMT 184 gene set. The enrichment score is plotted, which shows that the majority of DEGs were downregulated.
Figure 4.
Figure 4.
Signal transduction pathways involved in the major processes were identified by GO analysis and then String analysis, enrichment analysis by selecting the keyword “pathway” to identify the involved genes that were altered by chronic smoking. The signaling pathways with the strongest likelihood of involvement are indicated by the p values. The number of genes that were differentially expressed is also shown.
Figure 5.
Figure 5.
String analysis of STAT1 and STAT3 signaling after chronic smoking in the RPE/choroid. The network was built based on high confidence evidence from both experimental protein-protein interaction and curated databases. The thickness of the lines that represent interactions is proportional to the STRING combined probability score. A. 44 genes are connected to Stat1 with chronic smoking. B. 25 genes are connected to Stat3 with chronic smoking.
Figure 6.
Figure 6.
Reciprocal STAT1 and STAT3 signaling in the RPE/choroid after chronic smoking. A. Western blots of STAT1 and pSTAT1 from the RPE/choroid of air and smoke exposed mice. STAT1 and p-STAT1 were plotted as fold change of smoke to air treated mice. B. Western blots of STAT3 and pSTAT3 from the RPE/choroid of air and smoke exposed mice. STAT3 and p-STAT3 were plotted as fold change of smoke to air treated mice. C. Western blots of MMP3 and MMP14 from the RPE/choroid of air and smoke exposed mice. MMP3 and MMP14 were plotted as fold change of smoke to air treated mice. Data were normalized to β-actin. *p<0.05. **p<0.01, ***p<0.001.
Figure 7.
Figure 7.
STAT1 is decreased by cigarette smoke extract (CSE). A. RPE cells were treated with DMSO or 125 μg/ml CSE in the presence of 100 ng/ml IFN- α for 6 hrs. Western blots of STAT1 and pSTAT1 and their abundances were plotted as fold change. B. Cells were treated with DMSO or CSE for 24 hrs. Western blots of STAT3 and pSTAT3, and their abundances were plotted as fold change. *p<0.05. STAT3 silencing abrogates CSE-induced MMP14 production and MMP2 secretion. C. ARPE-19 cells were transfected with STAT3 siRNA and treated with 125 μg/ml CSE for 24 h. Western blot shows total STAT3, p-STAT3, and MMP14, and their abundances were plotted as fold change relative to DMSO-treated control siRNA treated cells. Data were normalized to β-actin. D. RPE cells were transfected with STAT3 siRNA, and treated with 125 μg/ml CSE for 24 h. Western blot shows total STAT3, p-STAT3, and MMP14 from cell lysates and MMP2 from the supernatant, and their abundances were plotted as fold change relative to DMSO-treated control siRNA treated cells. Data were normalized to β-actin. *p<0.05; **p<0.01.
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