STAT3 Regulates Monocyte TNF-Alpha Production in Systemic Inflammation Caused by Cardiac Surgery with Cardiopulmonary Bypass

BACKGROUND: Cardiopulmonary bypass (CPB) surgery initiates a controlled systemic inflammatory response characterized by a cytokine storm, monocytosis and transient monocyte activation. However, the responsiveness of monocytes to Toll-like receptor (TLR)-mediated activation decreases throughout the postoperative course. The purpose of this study was to identify the major signaling pathway involved in plasma-mediated inhibition of LPS-induced tumor necrosis factor (TNF)-α production by monocytes. METHODOLOGY/PRINCIPAL FINDINGS: Pediatric patients that underwent CPB-assisted surgical correction of simple congenital heart defects were enrolled (n = 38). Peripheral blood mononuclear cells (PBMC) and plasma samples were isolated at consecutive time points. Patient plasma samples were added back to monocytes obtained pre-operatively for ex vivo LPS stimulations and TNF-α and IL-6 production was measured by flow cytometry. LPS-induced p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation by patient plasma was assessed by Western blotting. A cell-permeable peptide inhibitor was used to block STAT3 signaling. We found that plasma samples obtained 4 h after surgery, regardless of pre-operative dexamethasone treatment, potently inhibited LPS-induced TNF-α but not IL-6 synthesis by monocytes. This was not associated with attenuation of p38 MAPK activation or IκB-α degradation. However, abrogation of the IL-10/STAT3 pathway restored LPS-induced TNF-α production in the presence of suppressive patient plasma. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that STAT3 signaling plays a crucial role in the downregulation of TNF-α synthesis by human monocytes in the course of systemic inflammation in vivo. Thus, STAT3 might be a potential molecular target for pharmacological intervention in clinical syndromes characterized by systemic inflammation

Zur sprachlichen und stilistischen Würdigung der altkirchenslavischen Vita Constantini

Overdruk uit: Südost-Forschungen, p. 74-10

Neonatal thymectomy reveals differentiation and plasticity within human naive T cells

The generation of naive T cells is dependent on thymic output, but in adults, the naive T cell pool is primarily maintained by peripheral proliferation. Naive T cells have long been regarded as relatively quiescent cells; however, it was recently shown that IL-8 production is a signatory effector function of naive T cells, at least in newborns. How this functional signature relates to naive T cell dynamics and aging is unknown. Using a cohort of children and adolescents who underwent neonatal thymectomy, we demonstrate that the naive CD4+ T cell compartment in healthy humans is functionally heterogeneous and that this functional diversity is lost after neonatal thymectomy. Thymic tissue regeneration later in life resulted in functional restoration of the naive T cell compartment, implicating the thymus as having functional regenerative capacity. Together, these data shed further light on functional differentiation within the naive T cell compartment and the importance of the thymus in human naive T cell homeostasis and premature aging. In addition, these results affect and alter our current understanDing on the identification of truly naive T cells and recent thymic emigrants

Post-perfusion plasma suppresses LPS-induced TNF-α production by monocytes.

<p><b>A</b>. Percentage of TNF-α producing cells in the monocyte population after <i>ex vivo</i> LPS stimulation (100 ng/mL) of patient PBMC isolated at various time points (n = 4). <b>B</b>. Reduced TNF-α synthesis by monocytes after LPS (10 ng/mL) stimulation in whole blood assays with patient samples obtained at the indicated time points (n = 5). <b>C</b>. Experimental setup for experiments shown in D,E,G-I. In short, patient PBMC obtained before surgery (Pre-op) were mixed with control (pooled AB plasma from healthy donors) or autologous patient plasma samples obtained at indicated time points, followed by LPS (100 ng/mL) stimulation for 4 h. Monocyte populations (CD14/SSC gate) were then analyzed for intracellular TNF-α and IL-6 synthesis. <b>D</b>. Significantly reduced production of TNF-α by monocytes after LPS stimulation in the presence of plasma samples from different sources (n = 13). Shown are percentages of TNF-α producing monocytes relative to control (100%). *<i>P</i><0.05, **<i>P</i><0.001 vs. control (ANOVA). <b>E</b>. Percentages of IL-6 producing monocytes as in D. **<i>P</i><0.001 vs. control (ANOVA). <b>F</b>. Dexamethasone levels in patient plasma samples as measured by radio-immunoassay (n = 9). Median ± interquartile range. *<i>P</i><0.05 vs. pre-op (ANOVA). <b>G</b>. Production of TNF-α and IL-6 by monocytes after LPS stimulation in the presence of dexamethasone-free plasma samples (n = 4). *<i>P</i><0.05 vs. control (ANOVA). <b>H</b>. Mean fluorescence intensities (MFI) of TNF-α and IL-6 in monocytes after LPS stimulation in different plasma milieus (n = 7). *<i>P</i><0.05, **<i>P</i><0.001 vs. control (ANOVA). <b>I</b>. Representative flow cytometry results (contour plots) of the LPS-induced TNF-α production by monocytes in the presence of control or patient plasma (Pre-op, End-CPB, 4 h or 24 h post-perfusion plasma from a No-dexamethasone patient). Isotype control: mouse IgG1. Data represented as mean ± SEM, unless otherwise indicated.</p

STAT3 signaling is required for the suppressive effects of post-perfusion plasma on TNF-α production.

<p><b>A</b>. Pre-treatment of 4 h post-surgery plasma samples with anti-IL-10 partially restored TNF-α production by patient monocytes in response to LPS (n = 10). Control: plasma from healthy donors. <b>B</b>. Activation of STAT3 in monocytes by incubation with suppressive (4 h post-perfusion) but not control (24 h post-perfusion) plasma. Cells were incubated in the absence or presence of LPS to match the experimental setup as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035070#pone-0035070-g002" target="_blank">Fig. 2</a>. <b>C</b>. Pre-treatment of patient PBMC with active STAT3 inhibitor (pY-STAT3i) but not control peptide (STAT3i) before LPS stimulation in the presence of post-surgery plasma restored TNF-α synthesis (left panel), in contrast to IL-6 (right panel). Shown are percentages of TNF-α and IL-6 producing monocytes normalized to control (24 h post-surgery) plasma (n = 8). <b>D</b>. TNF-α and IL-6 levels measured in supernatants of LPS-stimulated mononuclear cells after pre-treatment with STAT3 inhibitor or control peptide, in the presence of 4 h post-surgery plasma (n = 8). Cytokine levels were normalized to LPS stimulation in control plasma from healthy donors due to interassay variability. All results are depicted as mean ± SEM. *<i>P</i><0.05 vs. control condition (ANOVA), ns: not significant.</p