Figure 6. Pathway analysis of RNA-Seq differential expression profiles reveals age-related inflammatory priming in eyes of A/J mice.

(A) Pathway analysis with Ingenuity software unveiled an aberrant inflammatory network in A/J mice characterized by priming of IFN at 1 month of age, as evidenced by increased activation of Irf7 and Stat1, coordinated increased expression of Stat1-induced secondary response genes, induction of positive regulatory loop genes in the inflammatory process, and expression of genes involved in immune cell activation. Numbers at left of each gene represent the RNA-Seq FPKM values from A/J (top; red) and B6 (bottom; black) eyes; fold differences are indicated next to the vertical arrows. (B) RT-PCR of Gbp1, Irf7, and Stat1 (shaded red in A) showed that this inflammatory priming network was preferentially exacerbated in older mice, with pronounced increases in gene product expression from 1 to 8 months of age in A/J relative to B6 mice. (C) BALB/c mice also exhibited features of inflammatory priming at 1 month of age when the same pathways were examined, but these changes were less pronounced than those in A/J mice. Numbers at left of each gene represent the RNA-Seq FPKM values from BALB/c (top; blue) and B6 (bottom; black) eyes; fold differences are indicated next to the vertical arrows. (D) RT-PCR of Gbp1, Irf7, and Stat1 (shaded blue in C) showed that in BALB/c mice, inflammatory priming was not exacerbated from 1 to 8 months of age, similar to findings in B6 mice.