Ciliary neurotrophic factor induces genes associated with inflammation and gliosis in the retina: a gene profiling study of flow-sorted, Müller cells

Abstract

Background: Ciliary neurotrophic factor (CNTF), a member of the interleukin-6 cytokine family, has been implicated in the development, differentiation and survival of retinal neurons. The mechanisms of CNTF action as well as its cellular targets in the retina are poorly understood. It has been postulated that some of the biological effects of CNTF are mediated through its action via retinal glial cells; however, molecular changes in retinal glia induced by CNTF have not been elucidated. We have, therefore, examined gene expression dynamics of purified Müller (glial) cells exposed to CNTF in vivo.

Methodology/principal findings: Müller cells were flow-sorted from mgfap-egfp transgenic mice one or three days after intravitreal injection of CNTF. Microarray analysis using RNA from purified Müller cells showed differential expression of almost 1,000 transcripts with two- to seventeen-fold change in response to CNTF. A comparison of transcriptional profiles from Müller cells at one or three days after CNTF treatment showed an increase in the number of transcribed genes as well as a change in the expression pattern. Ingenuity Pathway Analysis showed that the differentially regulated genes belong to distinct functional types such as cytokines, growth factors, G-protein coupled receptors, transporters and ion channels. Interestingly, many genes induced by CNTF were also highly expressed in reactive Müller cells from mice with inherited or experimentally induced retinal degeneration. Further analysis of gene profiles revealed 20-30% overlap in the transcription pattern among Müller cells, astrocytes and the RPE.

Conclusions/significance: Our studies provide novel molecular insights into biological functions of Müller glial cells in mediating cytokine response. We suggest that CNTF remodels the gene expression profile of Müller cells leading to induction of networks associated with transcription, cell cycle regulation and inflammatory response. CNTF also appears to function as an inducer of gliosis in the retina.

Figure 1. Flow-sorting Müller cells from mouse retina.

(a) Transverse section of retina from mgfap-egfp transgenic mice. Note GFP expression in radially-oriented, Müller cell processes. (b–c) Scatter plot of flow-sorted, dissociated cells from mgfap-egfp transgenic mouse retina. The cells used for RNA preparation were collected from the enclosed area in F indicated by the arrow. Dissociated cells from non-transgenic mouse retina showed no GFP-fluorescence.

Figure 2. Microarray data analysis.

(a) Hierarchical clustering showing 1261 probes that have a P-val ≤0.1 and a minimum 2-fold change between the CNTF and PBS samples at Day 1. Bright blue indicates lowest signal with increasing values indicated by yellow shading to bright red, representing peak signal. (b) Most significant 10 canonical pathways corresponding to Day 1. (c) Most significant biological functions for the same list of genes. (d) Hierarchical clustering showing 1541 probes that have a P-val ≤0.1 and a minimum 2-fold change between the CNTF and PBS samples at Day 3. (e) Most significant 10 canonical pathways corresponding to Day 3. (f) Most significant biological functions for the same list of genes.

Figure 3. qRT-PCR validation of selected genes using independent biological samples.

Predicted fold changes from microarray analysis and relative gene expression fold change from qRT-PCR for 3–6 independent biological replicates of flow-sorted Müller cells. Error bars indicate ±SEM.

Figure 4. Changes in pathways and processes induced by CNTF.

The diagram indicates potential biological processes and their inter-relationships in Müller cells at day 1 and day 3 after CNTF treatment. The processes and pathways were generated from IPA analysis.

Figure 5. The most prominently affected gene networks generated by Ingenuity Pathway Analysis.

(a) Day 1- Genes in this network are responsible for cellular development, connective tissue disorder and metabolic disease. (b) Day 3- Genes in this network are responsible for cellular function and maintenance, molecular transport and inflammatory response. Red color indicates induction, while green represents repression; color intensity correlates with fold change.

Figure 6. Overlapping genes in transcription profiles from Müller cells, astrocytes and retinal pigment epithelium (RPE).

Intensity signals for the top 2000 probes were used to generate sets of overlapping genes. The astrocyte and RPE transcriptome data are derived from published studies (56,57). Müller cell data represent signals from cells not exposed to CNTF. (a) The diagram shows that 381 transcripts were common to the three cell types, while 642 were shared between Müller cells and astrocytes, 565 between Müller cells and the RPE, and 604 between astrocytes and RPE. (b) Diagram showing overlap of transcripts among astrocytes, Muller cells and neurons. The neuron transcriptome data was obtained from a published study (56).

Figure 7. Gene networks common to Müller cells, astrocytes and RPE.

(a) Most significant canonical pathways; (b) Most significant biological functions for the same list of genes; and (c) Genes in this network are responsible mitochondrial function and metabolism.