Figure 1. PTX3 is released by NBh cells expressing an immune activation–related gene signature. (A) Microarray analysis of human NC and NBh cells. The Venn diagram summarizes comparably and differentially expressed genes, whereas the heat map provides a global view of differentially expressed genes. Colors correspond to significant fold change (FC) expression. Red, high expression; blue, low expression. (B) Gene ontology analysis showing the top 10 biological processes up-regulated (red) and down-regulated (blue) in human NBh cells compared with NC cells. Numbers indicate genes. (C) Scatter plot illustrating gene expression signal intensity in human NC and NBh cells. Circles indicate some of the top differentially expressed genes. (D) GSEA comparing genes expressed by NBh cells and GM-CSF–activated NC cells. Normalized enrichment score (NES) indicates correlation between individual gene sets. Positive correlation, NES >0 (red bars); negative correlation, NES <0 (blue bars). The top 10 genes similarly up-regulated in NBh cells and GM-CSF–treated NC cells are listed. FDR, false discovery rate. (E) qRT-PCR of mRNAs for EGR-1, IκBζ (NFKBIZ), TNFAIP3, and EDN-1 in human NC and NBh cells. Results are normalized to mRNA for β-actin and presented as relative expression (RE) compared with a reference set of NC cells. (F) qRT-PCR of PTX3 mRNA (top) and ELISA of PTX3 protein (bottom) from human NC cells cultured with or without GM-CSF and/or LPS for 1 d. PTX3 mRNA is normalized to mRNA for β-actin and presented as relative expression compared with NC cells cultured with medium alone (Ctrl). (G and H) IFA of human spleens stained for elastase (ELA; green), PTX3 (red), and IgD or LPS (blue). FO, follicle; PFZ, perifollicular zone. Bars: (G) 200 µm; (H) 40 µm; (H, right inset) 6 µm. (I and J) qRT-PCR of PTX3 mRNA (I) and ELISA of PTX3 protein (J) from human NC and NBh cells. PTX3 mRNA is normalized to β-actin mRNA and shown as relative expressions compared with NC cells. ELISA was performed after 2 d of culture in medium alone. Data show one experiment with six biological replicates for each cell type (A–D), one representative experiment of at least four with similar results (E–H), or two experiments with at least three samples in each experimental group (I and J). Error bars, SEM. *, P < 0.05; **, P < 0.01 (two-tailed Student’s t test).
Figure 1.

PTX3 is released by NBh cells expressing an immune activation–related gene signature. (A) Microarray analysis of human NC and NBh cells. The Venn diagram summarizes comparably and differentially expressed genes, whereas the heat map provides a global view of differentially expressed genes. Colors correspond to significant fold change (FC) expression. Red, high expression; blue, low expression. (B) Gene ontology analysis showing the top 10 biological processes up-regulated (red) and down-regulated (blue) in human NBh cells compared with NC cells. Numbers indicate genes. (C) Scatter plot illustrating gene expression signal intensity in human NC and NBh cells. Circles indicate some of the top differentially expressed genes. (D) GSEA comparing genes expressed by NBh cells and GM-CSF–activated NC cells. Normalized enrichment score (NES) indicates correlation between individual gene sets. Positive correlation, NES >0 (red bars); negative correlation, NES <0 (blue bars). The top 10 genes similarly up-regulated in NBh cells and GM-CSF–treated NC cells are listed. FDR, false discovery rate. (E) qRT-PCR of mRNAs for EGR-1, IκBζ (NFKBIZ), TNFAIP3, and EDN-1 in human NC and NBh cells. Results are normalized to mRNA for β-actin and presented as relative expression (RE) compared with a reference set of NC cells. (F) qRT-PCR of PTX3 mRNA (top) and ELISA of PTX3 protein (bottom) from human NC cells cultured with or without GM-CSF and/or LPS for 1 d. PTX3 mRNA is normalized to mRNA for β-actin and presented as relative expression compared with NC cells cultured with medium alone (Ctrl). (G and H) IFA of human spleens stained for elastase (ELA; green), PTX3 (red), and IgD or LPS (blue). FO, follicle; PFZ, perifollicular zone. Bars: (G) 200 µm; (H) 40 µm; (H, right inset) 6 µm. (I and J) qRT-PCR of PTX3 mRNA (I) and ELISA of PTX3 protein (J) from human NC and NBh cells. PTX3 mRNA is normalized to β-actin mRNA and shown as relative expressions compared with NC cells. ELISA was performed after 2 d of culture in medium alone. Data show one experiment with six biological replicates for each cell type (A–D), one representative experiment of at least four with similar results (E–H), or two experiments with at least three samples in each experimental group (I and J). Error bars, SEM. *, P < 0.05; **, P < 0.01 (two-tailed Student’s t test).

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