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,
,
Son équipe travaille sur le polynucléaire neutrophile et plus spécifiquement sur les interaction entre les pathogènes et l'environnement sur cette cellule ,
, Dans cette étude princeps, l'idée était d'évaluer l'état d'oxygénation des cellules dans le plasma
, Université de Bordeaux, IBGCUMR 5095, 1 rue Camille Saint Saëns, 33077 Bordeaux Cedex, France. 3 UMR-CNRS UMR -5164 Immunoconcept, 146 rue Léon Saignat, 33076 Bordeaux, France. 4 Faculty of Health and Medical Sciences, du sang veineux de 12 donneurs sains non-fumeurs a été prélevé, vol.9002, p.67000
, Blood samples were collected either in commercial collection tubes (BD Vacutainer K2E (EDTA), ref 368,861) or in Hypoxytubes developed in collaboration with the Greiner Bio One (GBO) company, containing a limited amount of O 2 . (tubes were sealed under a nitrogen atmosphere). Internal pO 2 was quantified in commercial tubes and in Hypoxytubes using, (0123456789) Scientific RepoRtS | (2020) 10:10659 |
, All participants gave written informed consent and all the study procedures were carried out in accordance with the Declaration of Helsinki principles. Human blood was collected from healthy patients at the ICAReB service of the Pasteur Institut (authorization No. 2020_0120), cell culture. HEK293T (ATCC CRL-1573) and Hep-G2 (ATCC HB-8065) were cultured in DMEM + 8%
, White blood cells (WBCs) were purified form whole blood in an anoxic chamber by the addition of a 6% dextran solution (30 min, RT). The WBC-containing supernatant was collected and resuspended in RPMI 1,640 (Thermofisher); remaining red blood cells were eliminated with a lysis buffer. Cells were fixed in paraformaldehyde (PFA) 3.3% for immunofluorescent labelling or labeled with fluorescent marker for flow cytometry analysis, as previously described 16 . plasma po 2 measurement and components' dosage. Immediately after blood collection, the plasma pO 2 was measured directly in the blood collection tube using an oximeter with a standardized microsensor equipped with a steel needle (Unisense), as previously described 17 . Following centrifugation for 5 min at 2,000×g, the plasma was acidified with an equal volume of 10% (w/v) metaphosphoric acid (MPA) containing 2 mmol/L of disodium-EDTA. Ascorbate concentration was quantified by high-performance liquid chromatography with coulometric detection, as described previously 18 . Likewise, using high-performance liquid chromatography with coulometric detection, ?-and ?-tocopherol were analyzed as described by Sattler et al. 19 , and ubiquinone and ubiquinol as described elsewhere 20, SVF. Cells were seeded onto 24-well plates and incubated 24 h at 37 °C at 0% (anoxic cabinet) or 21% O 2
, Immediately after blood collection, samples were centrifuged, and plasma fractions were loaded in closed cuves (2 mL). Oxygen consumption fluxes were assessed when reaching constant values. Experiments were conducted with fresh plasma and after oxidation (exposure to atmospheric air: at least 30 min on a rotator mixer, Plasma oxygen reduction rate quantification. Plasma oxygen consumption rate was measured with an oximeter (Oroboros O2k-FluoRespirometer)
, Invitrogen); nuclei with DAPI. Cell imaging was performed with a confocal microscope, Leica DM5500 TCS SPE). Flow cytometry. Cells were resuspended in PBS + 2 mM EDTA, labeled with 100 nM TMRM (T5428, Sigma-Aldrich) and analyzed with FACSCcalibur (BD Biosciences, vol.124, p.366
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Recombinant sOX40L does not induce Treg cell death and Purified CD14+ 766 monocytes cultured in the presence of SLE sera (SLE-DC) or ex-vivo SLE-mDC expressed ,
, Sorted CD4 + CD25 + CD127 -Tregs cells were cultured with or without sOX40L (100 ng/mL) and
/PI staining was performed after 3 days of culture for the assessment of cell death, p.771 ,
, Annexin-V + /PI + cells were 772 considered as dead cells. (B), Cumulative data showing the percentage of dead cells after 3 days of 773 Tregs culture, Representative dot plot of Treg Annexin-V/PI staining after 3 days of culture
, Purified CD14 + monocytes from healthy donors were cultured with GM-CSF+IL-4 or SLE Sera (SLE
, Cumulative results for OX40L-expression (as expressed by MFI) in both 777 conditions, GM-CSF+IL-4 DC (n=8), SLE-DC (n=21). Cumulative data are shown with S.E.M and P 778 value < 0.01 (**). (E-F) Ex-vivo OX40L expression level was assessed within circulating APCs such as
SLEDAI < 6) SLE) APCs were analyzed by 781 flow cytometry. The figure shows a representative staining and cumulative data represented as mean 782 with S.E.M. Data are compared using non parametric Mann-Whitney test, CD14+ monocytes and CD19+ B lymphocytes cells. 7 healthy donors and 15 SLE 780 patients (including 9 active (SLEDAI ? 6 and 7 quiescent ,
A) Effect of sOX40L on the proliferative capacity of Effector CD4+ T cells following activation with anti-789 CD3 and anti-CD28 at different concentrations. Purified CD4 T cells were stained with CFSE and 790 stimulated with or without agonist soluble OX40L (100ng/ml) for 4 days with different concentration of 791 pre-coated anti-CD3 and soluble anti-CD28, Effect of sOX40L on effector CD4 T cells proliferation alone 787, vol.788 ,
, Effect of soluble OX40L (100ng/ml) co-stimulation on effector CD4 T cells proliferation stimulated by 793 anti-CD3 and anti-CD28 micro-beads
, Dose effect of soluble OX40L co-stimulation (100ng/ml) on purified effector CD4 T cells stimulated 795 by pre-coated anti-CD3 (1ug/ml) and soluble anti-CD28 (3ug/ml) or anti-CD3+anti-CD28 beads for 4 796 days, vol.4
Blood CD4 + CD25 high Tregs and serum concentration of sOX40L in healthy donors and 813 SLE patients ,
, A) Serum concentration of sOX40L (in ng/ml) from HD (n=15), iSLE (n=14) and aSLE patients (n=11)
, M, and compared using non-parametric Kruskall-Wallis 817 test with Dunn's correction for multiple comparisons, p.818
, Peripheral Effector CD4 T cells, Tregs, and B cells were investigated for OX40 820 expression by flow cytometry. PBMCs were isolated and stained for OX40 expression from Healthy 821 donors (n=8) and SLE patients (n=9). (C) Representative staining and (D) OX40 MFI values with S.E.M 822 are shown and compared using one-way ANOVA, Correlation between sOX40L concentration in serum and SLE activity (SLEDAI), using Spearman rank 819 correlation test. (C-D)
, 01 (**), p<0.0001 (****). (E), sOX40L impacts Foxp3 expression in different Treg subset
, ml) supplemented by soluble anti-CD28 (3ug/ml) in 96 wells plate for 3 days. Intra-nuclear Foxp3 826 expression was assessed by flow cytometry on different Treg sub-population based on CD25 and 827 CD45RA cell surface expression: active Tregs (CD45RA -CD25 high ), resting Tregs (CD45RA + CD25 + ) and 828 non-functional Tregs (CD45RA -CD25 + ) subset. 5 different donors were studied in 2 independent 829 experiments. Individual data are shown with mean and S.E.M, and compared using non-parametric two-830 tailed Mann-Whitney test, Frequency of blood CD4 + CD25 high Tregs in HD 831 (n=10) and SLE patients with inactive (iSLE, n=25)
, Kruskall-Wallis test with Dunn's correction for multiple comparisons
, Results are 835 expressed in Giga/L and are compared using non-parametric Kruskall-Wallis test with Dunn's correction 836 for multiple comparisons. iSLE, patients with inactive disease (SLEDAI<6); aSLE, CD4 + CD25 high Tregs cell in blood of HD (n=10) and iSLE (n=25) and aSLE patients (n=24)
Tregs intranuclear Helios expression following OX40L co-stimulation ,
, Intranuclear Helios expression level was evaluated 877 by flow cytometry. 9 independents experiments using 9 different Tregs donors were realized. Data are 878 shown as mean MFI and compared using two-tailed paired non-parametric Wilcoxon test, p.5