Nicotinic alpha 7 receptor expression and modulation of the lung epithelial response to lipopolysaccharide
Fig 5
RNA-Seq results reveal α7-impact on cell-specific changes in transcription following LPS challenge.
A) CD45- interstitial cells were isolated and RNA-Seq performed. Transcript data were converted to CDS values and these were then compared as labeled between α7-genotypes following challenge with i.n. saline or i.n. LPS as indicated. The lines indicate a 2-fold threshold difference in expression between genotypes and the number (N) of gene transcripts exceeding the 200 average read depth minimal cut-off (see S1 Table). Genes achieving a 4-fold or greater are colored, and some of these genes that exhibit among the greatest change in relative expression between control (Saline) and i.n. LPS (LPS) treatments are identified by their gene name. B) GeneMANIA derived plots [23,24] based upon gene transcript read averages between α7-genotypes in response to i.n. LPS. Gene clusters to the left include transcripts exceeding a genotype-based average read depth of 2-fold or 4-fold expression for the gene clusters to the right. Diagrams were generated using the default settings (Max resultant genes and attributes were set to zero). Subsets of key functional gene groupings as defined by GeneMANIA are indicated. In the α7G control the i.n. LPS response is dominated by two highly significant functional groups inclusive of ‘innate immune response’ and ‘regulators of cytokine production’ gene sets. The ‘innate immune response’ groups are retained when the 4-fold stringency cut off analysis was applied. In contrast, the same analysis of the i.n. LPS response enhanced specifically in the α7E260A:G lung epithelium reveals three different gene groups. These include genes of ‘extracellular matrix’, ‘inorganic substance response’ and ‘lung epithelium secretions’ of which the ‘extracellular matrix’ genes, and ‘lung epithelial secretions’ are retained after increasing stringency to greater than 4-fold. C) Quantitative average CDS read depth measures for each α7 genotype and treatment group are compared for some of the major genes defined both in the GeneMANIA analysis as epithelial secretions and from the plot in (A). The inverse relationship between gene expression in response to i.n. LPS that is related to α7G-genotypes is apparent and highly significant (** = p>0.01; *** = p>0.0001). D) Genes were subgrouped into defined cell-specific transcripts for Club (blue), ciliated (red), ATI (violet) and ATII (green) cells (Table 3 and S1 Table). The relative shift in α7E260A:G expression after LPS is shown and some genes are identified that exhibit particularly robust shifts in expression by Club and ATII cells (relative increase by α7E260A:G to the control) versus genes transcripts that were increased in ciliated cells for the same comparisons but relatively unchanged for ATI cells as shown by their grouping around 1.0 when compared to the expression differences in saline exposed (control tissues). E) Polar plots of the same cell-specific genes plotted in order of the difference in expression between saline and LPS exhibits the most dysregulation in Club cell gene expression followed by ciliated cells and ATII cells. ATI cells, which exhibit no α7-expression or number differences between genotypes, were again grouped around the expected 1.0 coordinates indicating no change in expression.