Fig 1: Inhibition of TNFa blocks increase in number of TH+ monocytes and amount of TH per monocyte.a and b Acutely isolated monocytes from three healthy donors were seeded at 2 million cells per well in duplicate ultra-low-adherence plates, and treated with TNFa (17 ng/mL), XPro1595 (50 ng/mL), IL6 (17 ng/mL) or combinations thereof as indicated. c In samples assayed by flow cytometry, using counting beads as a reference value to quantify the number of TH+ cells, co-incubation with TNFa and XPro1595 significantly reduced the number of TH+ monocytes relative to TNFa treatment alone. Treatment with IL6 or IL6 + XPro1595 resulted in no significant change in the number of TH+ monocytes. Values are represented as fold change relative to vehicle (n = 3 per group, one way ANOVA, *p < 0.05, **p < 0.01). d TH concentration (picogram per milligram total protein) significantly increases upon TNFa treatment, and is reduced significantly to baseline levels following co-incubation with TNFa and XPro1595. Neither IL6 nor IL6 + XPro1595 significantly increased TH quantity (n = 3 per group, one way ANOVA, *p < 0.05). Data are shown as ±SEM.
Fig 2: Signal transducer and activator of transcription 3 (STAT3) phosphorylation is decreased by dopamine in human monocytes. Fresh blood samples obtained from healthy donors were unstimulated or pre-incubated with dopamine (DA) in the presence or absence of interleukin (IL)-6 for 15 min. Afterwards, the extent of phosphorylated STAT3 (pSTAT3) was evaluated by intracellular immunostaining in the CD14+ population and analysed by flow cytometry using the gating strategy indicated in Figure S7 in Supplementary Material. (A) Representative histograms for pSTAT3 in peripheral blood CD14+ cells unstimulated (black lines) or treated with 1 µM DA (green lines) in the absence (left panel) or in the presence of IL-6 (right panel). The extent of pSTAT3 was also determined in peripheral blood CD14+ cells stimulated with IL-6 alone (red line). (B) The extent of pSTAT3 in unstimulated (white symbols) or IL-6 treated (black symbols) CD14+ in the presence of increasing DA concentrations was quantified as the mean fluorescence intensity (MFI) associated with pSTAT3-immunostaining (left panel) or as the ratio of the pSTAT3-associated MFI of stimulated cells to the pSTAT3-associated MFI of unstimulated cells (in-fold, right panel). Values represent mean ± SEM, n = 5. *p < 0.05 by unpaired two-tailed Student’s t-test.
Fig 3: S703I JAK1 Confers Constitutively Active and Hyperresponsive STAT Signaling(A) Localization of the S703I (red) mutation to the pseudokinase domain (blue) of JAK1, as represented in the linear sequence and the predicted structure, as modeled on TYK2.(B) Immunoblotting for STAT phosphorylation in U4C Cells (JAK1−/−) reconstituted with lentiviruses for vector control (luciferase), WT JAK1, or S703I JAK1 and stimulated for 15 min with indicated doses of IFN-α (IU/mL).(C) qPCR from transduced U4C cells for IFN-stimulated gene expression (MX1 and RSAD2) at baseline and following 100 IU/mL of IFN-α for 8 h (n = 3). Columns represent means and error bars represent standard deviations. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, 2-tailed Student’s t test with Welch’s correction.(D and E) Stimulation of transduced U4C cells with IFN-γ (0.1 ng/mL) (D) or IL-6 (25 ng/mL) (E) for 15 min.(F and G) Derivation of B-EBV cells and isolation of JAK1 WT/WT and JAK1 S703I/WT cells from patient blood, followed by stimulation with IFN-α (100 IU/mL) (F) or IFN-γ (0.1 ng/mL) (G) for 15 min. All of the data are representative of at least 3 independent experiments.See also Figure S2.
Fig 4: Analysis of the expression of purinergic ectoenzymes in CD8 T-cells stimulated in vitro with recombinant IL-6. Frequency of CD39+CD73-CD8+ T-cells (A) unstimulated (medium) or stimulated with (B) 20 ng/mL or (C) 20 pg/mL of IL-6. Frequency of CD39-CD73+ CD8+ T-cells (D) unstimulated (medium) or stimulated with (E) 20 ng/mL or (F) 20 pg/mL of IL-6. Frequency of CD39+CD73+CD8+ T-cells (G) unstimulated (medium) or stimulated with (H) 20 ng/mL and (I) 20 pg/mL of IL-6 for 48 h (n = 7) (* p < 0.05; ** p < 0.01; *** p < 0.001).
Fig 5: Correlation analysis between IL-6 plasma levels and subpopulations of NO+ monocytes. (A) Frequency of CD14+CD16+ monocytes producing NO at admission (n=18) and (B, C) NO production in different monocyte subsets at hospital discharge (n=20) vs. IL-6 plasma concentrations. Dotted lines represent the confidence interval for the correlation coefficient.
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