Figure 4.

CD127–STAT5 axis constrains inflammatory gene expression in human monocytes. (A) CD14⁺ monocytes were pretreated with or without LPS for 6 h, followed by various doses of human IL-7 (from 1 pg/ml to 10 ng/ml) for 30 min. STAT5 activation was detected by Western blotting. β-Actin was used as a loading control. One representative result from three independent experiments is shown (left). The protein level of p-STAT5(Y694) was quantified by densitometry, normalized to total STAT5 protein, and expressed relative to untreated (without LPS and IL-7) sample (right). *, P < 0.05; paired t test. Data are shown as the mean ± SD of three independent experiments. (B) CD14⁺ monocytes were transfected with negative control or IL7R-specific siRNA (siControl or siIL7R). 2 d after transfection, cells were stimulated with LPS (10 ng/ml) for 3 h, and mRNA of IL7R, IL6, and TNF were measured by qPCR. Relative expression was normalized to an internal control (GAPDH) and expressed relative to LPS untreated siControl sample. *, P < 0.05; paired t test. Each pair of data points indicates an independent experiment. IL7R, n = 4; IL6, n = 4; TNF, n = 3. (C) With CD127-based FACS sorting for 6-h LPS-treated monocytes, ATAC-seq datasets were generated for CD127^high and C127^low monocytes from three donors separately. Heat maps of pooled ATAC-seq signals around the total identified OCRs were shown in CD127^high and C127^low monocytes. Each row indicates one OCR, and the rows were sorted by the decreasing ATAC-seq signals in OCRs. (D) Volcano plot shows the differentially opened OCRs by ATAC-seq between CD127^high and CD127^low monocytes (P < 0.05; fold change ≥2 or ≤0.5). Highly opened OCRs in CD127^high population (CD127^high feature OCRs) and highly opened OCRs in CD127^low population (CD127^low feature OCRs) are indicated in red and in blue, respectively. Fold changes are shown as the mean value of CD127^high/CD127^low ratio from three donors. (E) Pie graph shows the percentages of CD127^high and C127^low feature OCRs in total identified OCRs in LPS-activated monocytes. (F) Heat maps (left) show the ATAC-seq signals around the CD127^high feature and CD127^low feature OCRs in each pair of CD127^high and CD127^low monocytes from three donors and corresponding ATAC-seq signal fold changes (CD127^high/CD127^low; right) were log₂ transformed and expressed by color from blue (negative) to red (positive). (G) Top five results from motif enrichment analysis in CD127^high monocyte featured OCRs are shown according to the decreasing −log₁₀(P value) for enriched motifs. P value by binomial distribution. (H) CD14⁺ monocytes were pretreated with STAT5 inhibitor (100 µM) for 2 h and then were stimulated with LPS (10 ng/ml). mRNA levels of IL6 (LPS 6 h) and TNF (LPS 3 h) were measured using qPCR. *, P < 0.05 by paired t test. Each pair of data points represents an independent experiment (n = 6). Independent experiments in A, B, and H were performed with cells from one healthy donor for each experiment.