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. 2020 Sep 18;11:2129. doi: 10.3389/fimmu.2020.02129

FIGURE 3.

FIGURE 3

CCL2 neutralization triggers an NF-κB/miR-155 regulatory network to regulate immune responses. (A) Predicted interaction network showing the regulatory relationships of miR-155, NF-κB, and their target genes in clusters 1 and 2. TFs, gene, and miR are represented as rectangular, oval, and hexagonal nodes, respectively. The edge colors represent different relationships: blue for the repression of miR-155 and FOS to genes or NFKB1, respectively; green for the regulation of NFKB1 to genes or miR-155; and black for the regulation of other TFs to genes or TFs. Nodes in yellow correspond to NFKB1 targets, and nodes in blue correspond to miR-155 targets. (B) Analysis by qPCR of miR-155 levels in MDMs treated or not with anti-CCL2 Ab for 4 h. The results obtained with three different donors are shown. (C) Effect of NF-κB inhibition on miR-155 level. MDMs were treated with BMS-345541 (10 μM) for 1 h prior to anti-CCL2 Ab (2.5 μg/mL) exposure for 4 h. Total RNA was then extracted and retrotranscribed, and miR-155 was amplified by qPCR. Data represent mean values (+SE) of the results obtained with three donors. **p < 0.01 (anti-CCL2 Ab vs. control; BMS-345541 + anti-CCL2 vs. anti-CCL2 Ab). (D) Venn diagram showing the overlay between NFKB1 target genes in clusters 1 and 2 (dataset 1) with dataset 2. (E) Box plots of DESeq2 normalized counts of NFKB1, NFKBIA, STAT1, IRF1, IRF7, CCR5, CCL3, CCL4, and CCL5 transcripts following 4-h exposure to anti-CCL2 Ab. Each condition has five biological replicates (datasets 1 and 2). (F) Analysis by qPCR of the levels of NFKB1, NFKBIA, STAT1, IRF1, IRF7, CCR5, CCL3, CCL4, and CCL5 in MDMs treated or not with anti-CCL2 Ab for 4 h. The y axis represents the log2(fold change) values derived from RNA-seq (blue) and qPCR (red). (G) Correlation between qPCR and RNA-seq data for the genes reported in panel F. The correlation of the fold changes was calculated by the Pearson correlation coefficient. Results of panels (F,G) are based on RNA-seq data from dataset 1 and qPCR data from four donors.