Time course of TLR-induced production of TNF-α and IL-12 in circulating mDCs from patients with aneurysmal subarachnoid hemorrhage.

<p>Intracellular cytokine measurement was performed in circulating mDCs from SAH patients (N = 21) on days 2, 5 and 10 and from HC (N = 11). The percentages of mDCs expressing TNF-α or IL-12 were assessed after a 3.5-hour <i>ex vivo</i> stimulation with (<b>A</b>) polyIC (TLR-3 agonist), (<b>B</b>) lipopolysaccharide (TLR-4 agonist) or (<b>C</b>) CL097 (TLR-7/8 agonist). The percentage of positive DCs without TLR-stimulation was below 1% (data not shown). The results are presented as percentages of mDCs expressing TNF-α (%TNF-α⁺) or IL-12 (% IL-12⁺). Plots represent median (Interquartile ranges). HC: healthy controls. mDCs: myeloid dendritic cells. SAH: aneurysmal subarachnoid hemorrhage. TNF-α: tumour necrosis factor -α. IL-12: intreleukin-12. * <i>P</i><0.05.</p

Time course of TLR-induced productions of TNF-α and IFN-α in circulating pDCs from patients with aneurysmal subarachnoid hemorrhage.

<p>Intracellular cytokine measurement was performed in circulating pDCs from SAH patients (N = 21) on days 2, 5 and 10 after brain injury and from HC (N = 11). The percentages of pDCs expressing TNF-α or IFN-α were assessed after a 3.5-hour <i>ex vivo</i> stimulation with (<b>A</b>) CL097 (TLR-7/8 agonist) or (<b>B</b>) CpG (TLR-9 agonist). The percentage of positive pDCs without TLR-stimulation was below 1% (data not shown). The results are presented as percentages of pDCs expressing TNF-α (%TNF-α⁺) or IFN-α (%IFN-α⁺). Plots represent median (Interquartile ranges). HC: healthy controls. IFN-α: interferon. pDCs: plasmacytoid dendritic cells. SAH: aneurysmal subarachnoid hemorrhage. TNF-α: tumour necrosis factor -α. *<i>P</i><0.05.</p

Gating strategy used to identify blood DC subsets and intracellular cytokines production in DCs in whole blood stimulated with TLR ligands.

<p>Whole blood samples were incubated with TLR3, 4, 7/8 or 9 ligands for 3.5-hour and then stained for identification of myeloid DC (HLA-DR+, Lin-, CD11c+, CD123-) and plasmacytoid DC (HLA-DR+, lin−, CD11c−, CD123+) together with intracellular cytokine production (TNFα, IL-12, IFNα).</p

Time course of circulating mDCs and pDCs numbers in patients with aneurysmal subarachnoid hemorrhage.

<p>Comparison of circulating (A) myeloid DC and (B) plasmacytoid DC counts in SAH patients (N = 21) on days 2, 5 and 10 compared with HC (N = 11). Plots represent median (Interquartile ranges). HC: healthy controls. SAH: aneurysmal subarachnoid hemorrhage. DCs: dendritic cells. * <i>P</i><0.05, ** <i>P</i><0.01.</p

Exploratory comparison of mDC and pDC status on day 2 in survivors and non-survivors.

<p>(<b>A</b>) The number of circulating myeloid DCs and plasmacytoid DCs were compared on day 2 between 13 survivors and 8 non-survivors. On day 2, mDCs and pDCs were stimulated <i>ex vivo</i> with polyIC (TLR-3 agonist), lipopolysaccharide (TLR-4 agonist), CL097 (TLR-7/8 agonist) and CpG (TLR-9 agonist) for 3.5 hours. (B) The percentages of mDCs expressing TNF-α or IL-12 and (C) the percentages of pDCs expressing TNF-α or IFN-α were compared between survivors and non-survivors. The percentage of positive DCs without TLR stimulation was below 1% (data not shown). The results are presented as percentages of DCs expressing TNF-α (%TNF-α⁺), IL-12 (%IL-12⁺) or IFN-α (%IFN-α⁺). Plots represent median (Interquartile ranges). * <i>P</i><0.05.</p

Characteristics of the study population.

<p>ARDS: acute respiratory distress syndrome, ICU: intensive care unit, SAH: subarachnoid hemorrhage, SAPS: simplified acute physiological score, WFNS: World Federation of Neurological Surgeons.</p

Suppressive function of circulating T regulatory cells.

<p><b>A</b>, Gating strategy for Treg sorting and typical results after sorting. <b>B</b>, Proliferative responses of Treg, effector responder T cells and Treg:Tresp co-cultures (ratio1/1) upon CD3+CD28 stimulation. Histograms are expressed as mean value +/− SD of cpm for healthy controls (HC ; n = 18), remission patients (RP ; n = 15), and active patients (PA ; n = 19). <b>C</b>, The percentage of suppression of responding cells was determined as 1−(proliferation of co-culture/proliferation of responder population alone)×100. Data are presented as boxplots with whiskers from minimum to maximum. <b>D</b>, A suppressive assay mixing Treg and effector T cells from and AP patient and a healthy volunteer was performed. Black bars represent suppression with HC Treg, and white bars with patient Treg. The results of one experiment out of three with similar results are shown.</p

Concentration of IL-17 in co-culture supernatants.

<p>IL-17 was quantified in supernatant using a multiplex fluorescent bead immunoassay: the levels of IL-17 produced by Treg, responder T cells and co-culture were not statistically different between the 3 groups.</p

Frequency, number and phenotype of Treg in patients vs. controls.

<p>Histograms represent: <b>A</b>, Proportion of CD4⁺CD25^highCD127^low/− T cells within CD4⁺CD3⁺ T cells (as gated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018734#pone-0018734-g002" target="_blank">figure 2A</a>) for HC (mean, 6.8%, <i>n</i> = 18), remission, (mean, 6.9%, <i>n</i> = 15), and acute (mean, 6.7%, <i>n</i> = 19) AAV patients. No significant difference was observed for percentage of Treg cells between the 3 groups. <b>B</b>, Absolute numbers of CD4⁺CD25^highCD127^low/− T cells in whole blood for HC (mean, 60.3 cells/µL), remission (mean, 47.5 cells/µL), and acute (mean, 40.1cells/µL) AAV patients. <b>C</b>, MFI of FOX-P3 in gated Treg and <b>D</b>, Mean percentage of CD39⁺ cells within Treg. Data are presented as boxplots with whiskers from minimum to maximum.</p

Clinical features of patients.

<p>Clinical features of patients.</p