Fig. 2.

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