Fig. 2
From: NFκB signaling drives pro-granulocytic astroglial responses to neuromyelitis optica patient IgG

NMO IgG induces expression of inflammatory and reactive astrocyte genes in mouse astroglia. a–f Gene expression was assessed by microarray analysis of astroglia after 24 h of stimulation with 100 μg/mL NMO IgG (NMO) or control IgG (CON). Changes in expression were calculated by comparison to untreated cultures. a A heatmap reveals robust up- and downregulation of numerous genes only in cells stimulated with NMO IgG. Of 22640 genes detected on the microarray, 3628 differed between NMO and CON IgG stimulation at p < 0.05. Fold changes for these genes are mapped on a log2 scale, with values downregulated to <−0.5-fold in green and values upregulated to >+0.5 shown in red. Note that because only significantly changed genes are mapped, there is a discontinuity between the upregulated and downregulated genes. b A subset of chemokine and cytokine genes are shown on a log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. White represents zerofold change relative to untreated samples. c A subset of genes encoding canonical NFκB-dependent factors are shown on a log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. d A subset of NFκB-dependent stress response genes sorted by gene name on log2 scale, with downregulation <−2-fold in green and upregulation >+2-fold shown in red. White represents zerofold change relative to untreated samples. e A published reactive astrocyte transcriptional response pattern (“reactive”) [18] was compared to the changes induced by astroglial stimulation with NMO IgG or CON IgG. These factors were mapped on a log2 scale with <0-fold change shown in white and >+5-fold induction shown in red. f Published data reporting the astrocyte transcriptional response to LPS, middle cerebral artery occlusion (“MCAO”), or PBS [18] were compared to our data for NMO IgG or CON IgG stimulation. The heatmap shows all genes detected on our array; genes with fold change values between −0.26 and +0.26 on a log2 scale following NMO IgG stimulation are excluded from the figure (discontinuity in the NMO lane). Genes downregulated <−2-fold are in green, unchanged genes are black, and genes upregulated >+2-fold are shown in red. A hierarchical cluster analysis showing Euclidean distance and average linkage score was performed in Gitools. The published data used for these comparisons were accessed via the GEO database at NCBI. g The NFκB canonical pathway was identified as a top response pathway (p = 4.14E-07) using the Ingenuity Pathway Analysis package. Top upstream regulators in this pathway were identified as Stat1 (z = 5.530), MyD88 (z = 5.603), Ripk2 (z = 2.486), and IRF3 (z = 2.804). Likewise, IFNγ (z = 9.203), IFNβ1 (z = 2.412), CSF2 (z = 2.789), and TNFα (z = 2.121) were identified as top response factors possibly involved in NFκB activation following NMO IgG stimulation. The microarray data were generated in two separate experiments performed with triplicate samples; the purified IgG used for these two experiments were derived from separate patient serum pools (Additional file 1: Table S1). The initial inclusion criteria for detection on the microarray were based on Illumina Beadchip significance calls. Genes exhibiting significant differences between NMO IgG- and CON IgG-stimulated samples were identified using Storey’s positive false-discovery rate for multiple hypothesis testing