Figure 1.
Identification of the IL-4 targets in CLL and in NBC.
(A) Scheme of the strategy used to identify the IL-4 targets in patients and in controls. (B) Heat maps for expression of IL-4 targets which had: top panel, similar changes in CLL and NBC and above 3-fold change for both; center panel, significantly higher changes in CLL and above 3-fold change in CLL; bottom panel, significantly higher changes in NBC and above 3-fold change in NBC. IL-4 targets are ordered alphabetically. In the event that several probes represent the same gene, only one is shown. Relative expression levels are depicted according to the shown log2 color scale.
Table 1.
Top 50 IL4 targets in CLL and NBC.
Figure 2.
Validation of IL-4 targets in CLL and NBC by qPCR.
Box whiskers representations of qPCR validations of 14 IL-4 targets representative of (A) restricted to CLL; (B) restricted to NBC; and (C) common to CLL and NBC. QPCR data are expressed as −ΔΔCt. IL-4 targets are ordered alphabetically.
Figure 3.
Pearson correlation analysis of the IL-4 upregulated targets compared between themselves and with ZAP-70.
Triangular heat map representing the pairwise correlation coefficients (R) of the IL-4 upregulated targets between themselves. The IL-4 targets are ordered according to their correlation coefficients with ZAP-70, which are represented at the left and bottom sides. Cut-off values for positive or negative correlations with ZAP-70 were set at 0.4 and −0.4, respectively. In the event that several probes represent the same gene, only one is shown. Correlation coefficients are depicted according to the shown color scale. Inset shows Pearson correlation analysis between ZAP-70 levels by microarray (expressed as log2 ratios) and by qPCR (expressed as –ΔΔCt ratios).
Figure 4.
Pathways and networks differently affected according to MetaCore analysis of IL-4 targets.
(A) Pathways and networks differently affected in CLL and NBC. (B) Pathways and networks differently affected in ZAP-70 positive and negative patients. Relevant genes for these functions within each group are specified.
Figure 5.
Effect of an NFκB inhibitor on apoptosis and gene expression response to IL-4 in CLL.
(A) Apoptosis of NBC and CLL cells cultured for 18 h in the absence or presence of IL-4, and CLL cells cultured with IL-4 plus an NFκB activation inhibitor (NFκBi) at 1 µM and 10 µM. CLL are represented together and also separated in ZAP-70 positive and negative. Apoptosis was measured as the percentage of cells labelled with Annexin V (top panel). The bottom panel represents results in CLL patients after subtracting the percentage of apoptotic cells of the Ctrl samples to the IL-4, IL-4 plus 1 µM NFκBi, and IL-4 plus 10 µM NFκBi samples. T tests were used to compare the levels of apoptosis between cell types and conditions. When differences were significant the p-values are indicated as follows: *, p<0.05; **, p<0.01; ***, p<0.001. (B) Expression of the ZAP-70Pos IL-4 targets SUSD2 and CCR2, and (C) of the ZAP-70Neg IL-4 targets AUH, LY75, NFIL3, and BCL6), measured by qPCR, in 7 ZAP-70 positive and 8 ZAP-70 negative patients. The ratios for expression of IL-4 targets following treatment with IL-4 alone, IL-4 plus NFκBi at 1 µM, and IL-4 plus NFκBi at 10 µM, compared to Ctrl, are represented. P-values are depicted as in A.
Table 2.
IL-4 targets correlated with protection by IL-4 in CLL.
Figure 6.
Map of the IL-4 signaling pathway and the potential role of ZAP-70.
Adaptation of the pathway map entitled “Immune response_IL-4 – anti-apoptotic action” from MetaCore from Thomson Reuters. The map has been simplified leaving the minimal elements for activation of transcription factors essential in regulation of gene expression. ZAP-70 and its interactions with members of the pathway reported in the MetaCore database have been added to suggest its potential involvement in the pathway, and a link has been added for the reported interaction between NFκB and STAT6 [21]. Red arrows indicate that IL4R, SOCS1, RPS6AK2, and NFKBIZ are components of the pathway identified as IL-4 upregulated targets in this study. Other genes of the pathway were significantly regulated by cell culture, such as AKT3 (up) and GRB2 (down).