VE-PTP maintains the endothelial barrier via plakoglobin and becomes dissociated from VE-cadherin by leukocytes and by VEGF

We have shown recently that vascular endothelial protein tyrosine phosphatase (VE-PTP), an endothelial-specific membrane protein, associates with vascular endothelial (VE)–cadherin and enhances VE-cadherin function in transfected cells (Nawroth, R., G. Poell, A. Ranft, U. Samulowitz, G. Fachinger, M. Golding, D.T. Shima, U. Deutsch, and D. Vestweber. 2002. EMBO J. 21:4885–4895). We show that VE-PTP is indeed required for endothelial cell contact integrity, because down-regulation of its expression enhanced endothelial cell permeability, augmented leukocyte transmigration, and inhibited VE-cadherin–mediated adhesion. Binding of neutrophils as well as lymphocytes to endothelial cells triggered rapid (5 min) dissociation of VE-PTP from VE-cadherin. This dissociation was only seen with tumor necrosis factor α–activated, but not resting, endothelial cells. Besides leukocytes, vascular endothelial growth factor also rapidly dissociated VE-PTP from VE-cadherin, indicative of a more general role of VE-PTP in the regulation of endothelial cell contacts. Dissociation of VE-PTP and VE-cadherin in endothelial cells was accompanied by tyrosine phoshorylation of VE-cadherin, β-catenin, and plakoglobin. Surprisingly, only plakoglobin but not β-catenin was necessary for VE-PTP to support VE-cadherin adhesion in endothelial cells. In addition, inhibiting the expression of VE-PTP preferentially increased tyrosine phosphorylation of plakoglobin but not β-catenin. In conclusion, leukocytes interacting with endothelial cells rapidly dissociate VE-PTP from VE-cadherin, weakening endothelial cell contacts via a mechanism that requires plakoglobin but not β-catenin

VE-PTP controls blood vessel development by balancing Tie-2 activity

Vascular endothelial protein tyrosine phosphatase (VE-PTP) is an endothelial-specific receptor-type tyrosine phosphatase that associates with Tie-2 and VE-cadherin. VE-PTP gene disruption leads to embryonic lethality, vascular remodeling defects, and enlargement of vascular structures in extraembryonic tissues. We show here that antibodies against the extracellular part of VE-PTP mimic the effects of VE-PTP gene disruption exemplified by vessel enlargement in allantois explants. These effects require the presence of the angiopoietin receptor Tie-2. Analyzing the mechanism we found that anti–VE-PTP antibodies trigger endocytosis and selectively affect Tie-2–associated, but not VE-cadherin–associated VE-PTP. Dissociation of VE-PTP triggers the activation of Tie-2, leading to enhanced endothelial cell proliferation and enlargement of vascular structures through activation of Erk1/2. Importantly, the antibody effect on vessel enlargement is also observed in newborn mice. We conclude that VE-PTP is required to balance Tie-2 activity and endothelial cell proliferation, thereby controlling blood vessel development and vessel size

BACH2 immunodeficiency illustrates an association between super-enhancers and haploinsufficiency.

The transcriptional programs that guide lymphocyte differentiation depend on the precise expression and timing of transcription factors (TFs). The TF BACH2 is essential for T and B lymphocytes and is associated with an archetypal super-enhancer (SE). Single-nucleotide variants in the BACH2 locus are associated with several autoimmune diseases, but BACH2 mutations that cause Mendelian monogenic primary immunodeficiency have not previously been identified. Here we describe a syndrome of BACH2-related immunodeficiency and autoimmunity (BRIDA) that results from BACH2 haploinsufficiency. Affected subjects had lymphocyte-maturation defects that caused immunoglobulin deficiency and intestinal inflammation. The mutations disrupted protein stability by interfering with homodimerization or by causing aggregation. We observed analogous lymphocyte defects in Bach2-heterozygous mice. More generally, we observed that genes that cause monogenic haploinsufficient diseases were substantially enriched for TFs and SE architecture. These findings reveal a previously unrecognized feature of SE architecture in Mendelian diseases of immunity: heterozygous mutations in SE-regulated genes identified by whole-exome/genome sequencing may have greater significance than previously recognized

Resistance to Inhibitors of Cholinesterase (Ric)-8A and Gα_i Contribute to Cytokinesis Abscission by Controlling Vacuolar Protein-Sorting (Vps)34 Activity

<div><p>Resistance to inhibitors of cholinesterase (Ric)-8A is a guanine nucleotide exchange factor for Gα_i, Gα_q, and Gα_12/13, which is implicated in cell signaling and as a molecular chaperone required for the initial association of nascent Gα subunits with cellular membranes. Ric-8A, Gα_i subunits, and their regulators are localized at the midbody prior to abscission and linked to the final stages of cell division. Here, we identify a molecular mechanism by which Ric-8A affects cytokinesis and abscission by controlling Vps34 activity. We showed that Ric-8A protein expression is post-transcriptionally controlled during the cell cycle reaching its maximum levels at mitosis. A FRET biosensor created to measure conformational changes in Ric-8A by FLIM (Fluorescence Lifetime Imaging Microscopy) revealed that Ric-8A was in a close-state during mitosis and particularly so at cytokinesis. Lowering Ric-8A expression delayed the abscission time of dividing cells, which correlated with increased intercellular bridge length and multinucleation. During cytokinesis, Ric-8A co-localized with Vps34 at the midbody along with Gα_i and LGN, where these proteins functioned to regulate Vps34 phosphatidylinositol 3-kinase activity.</p></div

Ric-8A inhibition increases the length of the intercellular bridge, delays abscission time and promotes multinucleation.

<p>(<b>A</b>) Immunoblot of Ric-8A and Gα_i3 protein expressions after treatment of HeLa cells with siRNA control or siRNA directed at Ric-8A or at Gα_i subunit 1–3. (<b>B</b>) Panels show a representative deconvolution of z-stacks and 3D reconstruction of siRNA control (top panel) or Ric-8A siRNA (bottom panel) treated HeLa cells. Scale bar is 5 µm. Magnification of midbody region and Ric-8A localization is shown in the white squares. (<b>C</b>) Intercellular bridge length quantified from 3 independent experiments (*, p<0.05) by immunostaining with an anti-α-tubulin antibody. (<b>D</b>) Distribution and the average abscission time of HeLa cells transiently transfected with siRNA-Ric-8A or siRNA-control and the photo-activable GFP plasmid. Results from 12 cells were acquired in 4 independent experiments (*, p<0.05). (<b>E</b>) Histogram represents the percentage of total of cells with a mitotic phenotype (prophase to cytokinesis) and the number of cells showing an intercellular bridge. At least 500 cells were analyzed in 3 independent experiments, (n.s. stands for non-significant). (<b>F–G</b>) HeLa cells transiently transfected with siRNA-Ric-8A or siRNA-control were analyzed for their phospho histone H3-ser10 levels either by western blotting technique (<b>F</b>) or by flow cytometry technique (<b>G</b>) focusing on G2-M positive cells. (<b>H</b>) The percentage of multinucleated HeLa cells among cells transiently transfected with siRNA Control, siRNA Ric-8A, siRNA RGS14, siRNA GRK2 or siRNA LGN was assessed by flow cytometry 48 h after second siRNA transfection, (*, p<0.05, n = 3). The percentage in white indicates the amount of reduction in mRNA content for the targeted gene.</p

Ric-8A conformational change occurs during mitosis.

<p>(<b>A</b>) HeLa cells were transiently transfected with GFP-Ric-8A-mCherry. Focusing on metaphase cells using transmission light imaging, FRET by FLIM measurements were done on live cells. The panel shows a representative lifetime imaging and an electronic magnification of the interchromosome area (square 1) containing the midbody is displayed (<b>B</b>) Results of the average lifetime in whole cells at different stages of the cell cycle. Each point represents a single cell. (<b>C</b>) Results of the average lifetime in the different sub-cellular compartments at different stages of the cell cycle. The data were acquired from 4 independent transfections and from 6–15 cells for each bar.</p

Locally Produced IL-10 Limits Cutaneous Vaccinia Virus Spread.

Skin infection with the poxvirus vaccinia (VV) elicits a powerful, inflammatory cellular response that clears virus infection in a coordinated, spatially organized manner. Given the high concentration of pro-inflammatory effectors at areas of viral infection, it is unclear how tissue pathology is limited while virus-infected cells are being eliminated. To better understand the spatial dynamics of the anti-inflammatory response to a cutaneous viral infection, we first screened cytokine mRNA expression levels after epicutaneous (ec.) VV infection and found a large increase the anti-inflammatory cytokine IL-10. Ex vivo analyses revealed that T cells in the skin were the primary IL-10-producing cells. To understand the distribution of IL-10-producing T cells in vivo, we performed multiphoton intravital microscopy (MPM) of VV-infected mice, assessing the location and dynamic behavior of IL-10 producing cells. Although virus-specific T cells were distributed throughout areas of the inflamed skin lacking overt virus-infection, IL-10+ cells closely associated with large keratinocytic foci of virus replication where they exhibited similar motility patterns to bulk antigen-specific CD8+ T cells. Paradoxically, neutralizing secreted IL-10 in vivo with an anti-IL-10 antibody increased viral lesion size and viral replication. Additional analyses demonstrated that IL-10 antibody administration decreased recruitment of CCR2+ inflammatory monocytes, which were important for reducing viral burden in the infected skin. Based upon these findings, we conclude that spatially concentrated IL-10 production limits cutaneous viral replication and dissemination, likely through modulation of the innate immune repertoire at the site of viral growth

Ric-8A is phosphorylated on S501 during G2/M phase.

<p>Asynchronous or nocodazole G2/M arrested HeLa cells were treated with cyclin dependent kinase inhibitors (Roscovitine, 3 h, 1 µM or Olomoucine, 3 h, 1 µM) prior to be assessed as indicated below. (<b>A</b>) A representative Ric-8A and actin immunoblots of anti- phospho-p190 antibody immunoprecipitates prepared from HeLa cells (<b>B</b>) Graph represents the percentage of phosphorylated Ric-8A as assessed in A. Results are expressed as a percentage of the nocodazole treated condition from 4 independent experiments (*, p<0.05). (<b>D</b>) Lysates from nocodazole G2/M enriched GFP, GFP-Ric-8A wt, GFP-Ric-8A S88A, GFP-Ric-8A S155A or GFP-Ric-8A S501A expressing HeLa cells were immunoprecipitated using anti-phospho-p190 antibody, resolved on a Tris-glycine gel, and immunoblotted for Ric-8A.</p

Ric-8A protein expression is increased during G2/M phase.

<p>(<b>A</b>) HeLa cells were blocked at the G1/S boundary by thymidine block and then released for the indicated times. Asynchronous or G2/M arrested cells (nocodazole 1 µM, 16 h) were used as controls. Ric-8A expression was detected by immunoblot analysis. Synchronization efficiency was verified by CyclinB1 immunoblotting. (<b>B</b>) Ric-8A and actin immunoblots of lysates prepared from HeLa cells enriched in G1, S or G2/M phase cells by cell sorting according to their DNA content. Numbers are the fold increase in Ric-8A expression normalized to actin expression using the level found in G1 phase as a baseline. (<b>C</b>) Quantification of Ric-8A and LGN mRNA expression detected by quantitative RT-PCR in nocodazole G2-M enriched HeLa cells. mRNA expression was normalized to β-actin mRNA expression, (*, p<0.05, n = 3).</p

The role of dermis resident macrophages and their interaction with neutrophils in the early establishment of Leishmania major infection transmitted by sand fly bite.

There is substantial experimental evidence to indicate that Leishmania infections that are transmitted naturally by the bites of infected sand flies differ in fundamental ways from those initiated by needle inocula. We have used flow cytometry and intravital microscopy (IVM) to reveal the heterogeneity of sand fly transmission sites with respect to the subsets of phagocytes in the skin that harbor L. major within the first hours and days after infection. By flow cytometry analysis, dermis resident macrophages (TRMs) were on average the predominant infected cell type at 1 hr and 24 hr. By confocal IVM, the co-localization of L. major and neutrophils varied depending on the proximity of deposited parasites to the presumed site of vascular damage, defined by the highly localized swarming of neutrophils. Some of the dermal TRMs could be visualized acquiring their infections via transfer from or efferocytosis of parasitized neutrophils, providing direct evidence for the "Trojan Horse" model. The role of neutrophil engulfment by dermal TRMs and the involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these interactions and in sustaining the anti-inflammatory program of dermal TRMs was supported by the effects observed in neutrophil depleted and in Axl-/-Mertk-/- mice. The Axl-/-Mertk-/- mice also displayed reduced parasite burdens but more severe pathology following L. major infection transmitted by sand fly bite