2D transwell chemotaxis and transendothelial migration are impaired in SIRPα^Δcyt phagocytes.

<p>A) C5a-induced 2D migration in transwell chemotaxis assay is regulated by SIRPα signaling. Data shown represents the difference between the maximum and the minimum fluorescent value reached within 1 h in BMDN and 2 h in BMDM. Values shown represent averages ± SEM of n = 4 independent experiments. Asterisk, p≤0,05. B) Transendothelial migration is deficient in SIRPα^<b>Δcyt</b> BMDN. WT and SIRPα^<b>Δcyt</b> BMDN were seeded over a mrTNFα-stimulated monolayer of bEnd5 cells. After 5’ a flow ratio of 0.9dyn/cm^<b>2</b> was applied and transendothelial migration was monitored by time lapse video microscopy using a phase-contrast lens. Data shown represent means ± SEM of 12 measurements done in 3 independent experiments. Asterisk, p≤0,05. C) BMDN and BMDM from SIRPα^<b>Δcyt</b> mice have similar levels of integrin expression. Cells were cultured as described in material and methods and stained with specific Abs against the indicated integrins. Gating was based on FCS and SSC. Histograms from representative experiments are shown for BMDN and BMDM. The dotted line represents the isotype control wile the continuous line represents WT (gray filled) or SIRPα^<b>Δcyt</b> (no filling) stainings.</p

Delayed recruitment of phagocytes to the peritoneal cavity in SIRPα^Δcyt mice.

<p>After i.p. injection of thioglycolate into WT and SIRPα^<b>Δcyt</b> mice, neutrophil and macrophage influx were determined in peritoneal lavages at the indicated time points. Total leukocytes were counted and cell populations were discriminated by FACS. Every time point is representative of at least 3 mice. Asterisk, p≤ 0,05. Note that there is a delay in the migration of both SIRPα^<b>Δcyt</b> phagocyte populations.</p

A comprehensive proteomics study on platelet concentrates: Platelet proteome, storage time and Mirasol pathogen reduction technology

<p>Platelet concentrates (PCs) represent a blood transfusion product with a major concern for safety as their storage temperature (20–24°C) allows bacterial growth, and their maximum storage time period (less than a week) precludes complete microbiological testing. Pathogen inactivation technologies (PITs) provide an additional layer of safety to the blood transfusion products from known and unknown pathogens such as bacteria, viruses, and parasites. In this context, PITs, such as Mirasol Pathogen Reduction Technology (PRT), have been developed and are implemented in many countries. However, several studies have shown <i>in vitro</i> that Mirasol PRT induces a certain level of platelet shape change, hyperactivation, basal degranulation, and increased oxidative damage during storage. It has been suggested that Mirasol PRT might accelerate what has been described as the platelet storage lesion (PSL), but supportive molecular signatures have not been obtained. We aimed at dissecting the influence of both variables, that is, Mirasol PRT and storage time, at the proteome level. We present comprehensive proteomics data analysis of Control PCs and PCs treated with Mirasol PRT at storage days 1, 2, 6, and 8. Our workflow was set to perform proteomics analysis using a gel-free and label-free quantification (LFQ) approach. Semi-quantification was based on LFQ signal intensities of identified proteins using MaxQuant/Perseus software platform. Data are available via ProteomeXchange with identifier PXD008119. We identified marginal differences between Mirasol PRT and Control PCs during storage. However, those significant changes at the proteome level were specifically related to the functional aspects previously described to affect platelets upon Mirasol PRT. In addition, the effect of Mirasol PRT on the platelet proteome appeared not to be exclusively due to an accelerated or enhanced PSL. In summary, semi-quantitative proteomics allows to discern between proteome changes due to Mirasol PRT or PSL, and proves to be a methodology suitable to phenotype platelets in an unbiased manner, in various physiological contexts.</p

The Guanine-Nucleotide Exchange Factor SGEF Plays a Crucial Role in the Formation of Atherosclerosis

<div><p>The passage of leukocytes across the endothelium and into arterial walls is a critical step in the development of atherosclerosis. Previously, we showed <em>in vitro</em> that the RhoG guanine nucleotide exchange factor SGEF (Arhgef26) contributes to the formation of ICAM-1-induced endothelial docking structures that facilitate leukocyte transendothelial migration. To further explore the <em>in vivo</em> role of this protein during inflammation, we generated SGEF-deficient mice. When crossed with <em>ApoE</em> null mice and fed a Western diet, mice lacking SGEF showed a significant decrease in the formation of atherosclerosis in multiple aortic areas. A fluorescent biosensor revealed local activation of RhoG around bead-clustered ICAM-1 in mouse aortic endothelial cells. Notably, this activation was decreased in cells from SGEF-deficient aortas compared to wild type. In addition, scanning electron microscopy of intimal surfaces of SGEF^−/− mouse aortas revealed reduced docking structures around beads that were coated with ICAM-1 antibody. Similarly, under conditions of flow, these beads adhered less stably to the luminal surface of carotid arteries from <em>SGEF</em>^−/− mice. Taken together, these results show for the first time that a Rho-GEF, namely SGEF, contributes to the formation of atherosclerosis by promoting endothelial docking structures and thereby retention of leukocytes at athero-prone sites of inflammation experiencing high shear flow. SGEF may therefore provide a novel therapeutic target for inhibiting the development of atherosclerosis.</p> </div

Gata1cKO^MKmice show alterations in the erythroid compartment.

<p>(a) Gating strategy to identify erythrocytes at consecutive stages of differentiation in the bone marrow and the spleen based on surface marker expression KIT, CD71 and Ter119. (b) Percentage of reticulocytes at consecutive stages of differentiation of live cells. Left graph depicts the bone marrow compartment, right the splenic compartment. (c) Photograph of representative spleens from Gata1cKO^MK and control mice shows the splenomegaly that Gata1cKO^MKdevelop.</p

Functional analysis of platelets from Gata1cKO^MK and SykcKO^MK mice show overlapping defects.

<p>(a) Flow cytometry-based platelet aggregation assay (FCA) shows the aggregation capacity of platelets when stimulated with different agonists. Gata1cKO^MK and WT^lox platelets were studied. PMA, phorbol myristate acid; Agg, aggretin; Botro. Botrocetin; Coll, collagen; CVX, convulxin. (b) MFI of receptors expressed on Gata1cKO^MKplatelets, relative expression of a given receptor in WT^lox platelets was set to 100. For clarification: CD61 (Itgb3), CD41 (Itga2b), CD42a (GPIX), CD42b (Gp1ba), CD42c (Gp1bb), CD49b (Itga2). (c) Flow cytometry-based platelet aggregation assay (FCA) shows the aggregation capacity of SykcKO^MK and WT^lox platelets when stimulated with various agonists as described above.</p

Membrane protrusions of intimal endothelial cells after ICAM-1 crosslinking.

<p><b>A:</b> Isolated aortas from SGEF^+/+ and SGEF^−/− mice were mounted with the intimal site facing up. After TNF-α treatment, the aortas were overlaid with anti-ICAM-1-Ab coated beads for 2 hours, before unbound beads were washed off and imaged by scanning electron microscopy. Representative scanning electron microscopy show attached anti-ICAM-1-Ab coated beads to the intimal surface of aortas from SGEF^+/+ and SGEF^−/− animals. Right panels show magnification of adherent bead. Scale bars represent 0.1 mm or 10 µm, as indicated. <b>B:</b> Quantification of antibody coated beads that induced membrane protrusions on aortas from SGEF^+/+ and SGEF^−/− animals. Anti-ICAM-1-Ab coated beads: SGEF^+/+: 53.3±14.8%; SGEF^−/−: 37.3±14.8%. Anti-VCAM-1-Ab coated beads: SGEF^+/+: 25.8±8.9%; SGEF^−/−: 24.2±10.0%. Anti-IgG1-Ab coated beads: SGEF^+/+: 8.2±7.0%; SGEF^−/−: 7.5±9.3%. Data are mean ±SEM. ***p<0.001. C: Quantification of total number of anti-ICAM-1-Ab or IgG1-Ab coated beads that adhered to the intimal surfaces of isolated and intact carotid arteries under flow conditions (5 dynes/cm²). Anti-ICAM-1-Ab coated beads: SGEF^+/+: 4.9±1.8; SGEF^−/−: 0.6±0.4. Anti-IgG1-Ab coated beads: SGEF^+/+: 1.0±1.1; SGEF^−/−: 0.8±0.6. Experiment was carried out at least 4 times. Data are mean ±SEM. ***p<0.001.</p

Gata1cKO^MK mice have a defect in the hematopoietic early precursor compartment

<p>(a) Flow cytometry analysis of the stem cell and committed progenitor compartment. LSK, Lin^-|Sca-1⁺|Kit⁺ cells; MP (Lin^-|Sca-1^-|Kit⁺), multipotent progenitors; CMP (MP gate—CD34⁺|CD16/CD32^mid), common myeloid progenitor; GMP (MP gate—CD34^-|CD16/CD32⁺), granulocyte-monocyte progenitor; MEP (MP gate—CD34^-|CD16/CD32^-), megakaryocyte-erythroid progenitor. (b) Percentage of the different hematopoietic progenitors. Absolute cell number of bone marrow megakaryocytes at consecutive stages of differentiation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154342#pone.0154342.ref031" target="_blank">31</a>]. The dot plot depicts the extra population, named II+ found exclusively in Gata1cKO^MK bone marrow. (c) Whisker/Box plot depicts plasma TPO levels from Gata1cKO^MK and WT^lox blood samples, as measured by ELISA. At least 5 mice were analyzed per genotype. (d) qPCR analysis of Pf4 mRNA expression levels in cultured bone marrow derived Gata1cKO^MK and WT^lox megakaryocytes.</p

Atherosclerotic plaque formation in mice fed a normal chow diet.

<p><b>A and B:</b> SGEF^+/+ApoE^−/− and SGEF^−/−ApoE^−/− mice were fed a chow diet for 16 weeks post weaning (weaning at 3 weeks of age), before aortas were isolated and the plaque area was determined by ORO staining (n = 11 for SGEF^+/+ApoE^−/− aortas; n = 9 for SGEF^−/−ApoE^−/− aortas; Data are mean ±SEM; N.S.: not significant; p-values represent 2-tailed Student's T-test with unequal variance). <b>A:</b> Quantification of ORO-stained area of the inner curvature of the aortic arch. The bar graphs at the left represent averaged measurements of all aortas. The right graph shows the measured ORO-stained area of individual aortas (represented by diamonds). SGEF^+/+ApoE^−/−: 4.8±2.0%; SGEF^−/−ApoE^−/−: 3.3±2.0%; p<0.11. <b>B:</b> Same as described under A for thoracic descending aorta: <b>B:</b> Descending thoracic aorta: SGEF^+/+ApoE^−/−: 0.8±0.4%; SGEF^−/−ApoE^−/−: 0.7±0.2%; p<0.43.</p

Defective activation of RhoG after ICAM-1 clustering on SGEF-deficient primary mouse aortic endothelial cells.

<p><b>A:</b> Molecular characterization of the ICAM-2-positive cell isolation. All cells (flow-through fraction (ICAM-2 negative) and ICAM-2 positive) were stimulated with 10 ng/ml TNF-α for 4 hours prior to lysis for Western blotting as indicated. The ICAM-2-positive cells expressed ICAM-1 and VE-cadherin, markers for endothelial cells. Also, SGEF expression was only measured in the isolates from wildtype animals and not from the knock-out. Hereafter, the ICAM-2-positive cells are referred to as mouse aortic endothelial cells (MAECs). <b>B:</b> Representative time-lapse imaging of ELMO-YFP recruitment to sites of adhesion of anti-ICAM-1-Ab coated beads on TNF-α-treated MAECs from SGEF^+/+ or SGEF^−/− animals. Arrowheads show bead location and only in the wildtype cells, ELMO recruitment is observed. Bar: 10 µm. <b>C</b> and <b>D:</b> Quantification of ELMO-YFP recruitment to adhered beads. Fluorescence intensity was measured in regions of adherent beads on MAECs, isolated from SGEF^+/+ and SGEF^−/− animals. Graph <b>C</b> shows measurements at each time point. Data represent pooled results from 3 independent experiments. Graph <b>D</b> shows averaged fluorescent intensity at time-points from 10 to 60 minutes after bead binding. Data represent pooled results from 3 independent experiments. Asterisks indicate ***p<0.05, **p<0.005 or *p<0.0005 according to Student T-test.</p