Soluble CD83 Alleviates Experimental Autoimmune Uveitis by Inhibiting Filamentous Actin-Dependent Calcium Release in Dendritic Cells

Soluble CD83 (sCD83) is the extracellular domain of the membrane-bound CD83 molecule, and known for its immunoregulatory functions. Whether and how sCD83 participates in the pathogenesis of uveitis, a serious inflammatory disease of the eye that can cause visual disability and blindness, is unknown. By flow cytometry and imaging studies, we show that sCD83 alleviates experimental autoimmune uveitis (EAU) through a novel mechanism. During onset and recovery of EAU, the level of sCD83 rises in the serum and aqueous humor, and CD83+ leukocytes infiltrate the inflamed eye. Systemic or topical application of sCD83 exerts a protective effect by decreasing inflammatory cytokine expression, reducing ocular and splenic leukocyte including CD4+ T cells and dendritic cells (DCs). Mechanistically, sCD83 induces tolerogenic DCs by decreasing the synaptic expression of co-stimulatory molecules and hampering the calcium response in DCs. These changes are caused by a disruption of the cytoskeletal rearrangements at the DC–T cell contact zone, leading to altered localization of calcium microdomains and suppressed T-cell activation. Thus, the ability of sCD83 to modulate DC-mediated inflammation in the eye could be harnessed to develop new immunosuppressive therapeutics for autoimmune uveitis

Planet Formation Imager (PFI): science vision and key requirements

The Planet Formation Imager (PFI) project aims to provide a strong scientific vision for ground-based optical astronomy beyond the upcoming generation of Extremely Large Telescopes. We make the case that a breakthrough in angular resolution imaging capabilities is required in order to unravel the processes involved in planet formation. PFI will be optimised to provide a complete census of the protoplanet population at all stellocentric radii and over the age range from 0.1 to ~100 Myr. Within this age period, planetary systems undergo dramatic changes and the final architecture of planetary systems is determined. Our goal is to study the planetary birth on the natural spatial scale where the material is assembled, which is the "Hill Sphere" of the forming planet, and to characterise the protoplanetary cores by measuring their masses and physical properties. Our science working group has investigated the observational characteristics of these young protoplanets as well as the migration mechanisms that might alter the system architecture. We simulated the imprints that the planets leave in the disk and study how PFI could revolutionise areas ranging from exoplanet to extragalactic science. In this contribution we outline the key science drivers of PFI and discuss the requirements that will guide the technology choices, the site selection, and potential science/technology tradeoffs.S.K. acknowledges support from an STFC Rutherford Fellowship (ST/J004030/1) and Philip Leverhulme Prize (PLP-2013-110). Part of this work was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration

Mikrovilläre Positionierung von L-Selektin und der Einfluß auf die Adhäsion

During inflammation and immune surveillance, initial contacts (tethering) between free-flowing leukocytes and the endothelium are vitally dependent on the presentation of the adhesion receptor L-selectin on leukocyte microvilli. Determinants that regulate receptor targeting to microvilli are, however, largely elusive. Therefore, we systematically swapped the extracellular (EC), transmembrane (TM) and intracellular (IC) domains of L-selectin and CD44, a hyaluronan receptor expressed on the cell body and excluded from microvilli. Electron microscopy of transfected human myeloid K562 cells showed that the highly conserved TM domains are responsible for surface positioning. The TM segment of L-selectin forced chimeric molecules to microvilli and the CD44 TM domain evoked expression on the cell body, while the IC and EC domains hardly influenced surface localization. Transfectants with microvillus-based chimeras showed a significantly higher adhesion rate to a physiological ligand under flow but not under static conditions as compared to cells with cell body- expressed receptors. Substitution of the IC domain of L-selectin by that of CD44 caused diminished tethering, but no change in surface distribution indicating that both microvillus positioning and cytoskeletal anchoring contribute to leukocyte tethering. These findings demonstrate that TM domains of L-selectin and CD44 play a crucial role in cell adhesion under flow by targeting receptors to microvilli or the cell body, respectively.Das angeborene und erworbene Immunsystem umfasst verschiedene Zelltypen, die zur Ausübung ihrer Funktion die Blutbahn in Richtung Gewebe verlassen müssen. Zur Kommunikation der Zellen mit dem Gefäßendothel dienen Oberflächenrezeptoren, deren sequentieller Einsatz zur Extravasation führt. In Lymphozyten ist der initiale Schritt abhängig von L-Selektin (CD62L). Es ist bekannt, dass lymphozytäres L-Selektin fast ausschliesslich auf membranären Protrusionen, den Mikrovilli, exprimiert ist während der planare Zellkörper weitgehend ausgespart bleibt. Auch eine Vielzahl anderer Membranproteine werden aktiv in diesen Mikrodomänen gruppiert. Da diese besondere Oberflächentopologie eine funktionelle Bedeutung für die zelluläre Adhäsionsrate hat, ist die Entschlüsselung der biochemischen Mechanismen, die an der Kompartmentalisierung von Rezeptoren auf der Zelloberfläche beteiligt sind, von hoher Bedeutung. In der vorliegenden Arbeit wurde der Einfluß der extrazellulären (EC), transmembranären (TM) und intrazellulären (IC) Domäne von CD44, einem Protein mit Lokalisierung ausschliesslich auf dem planaren Zellkörper, und L-Selektin auf die Zelloberflächentopologie beider Proteine untersucht. Für diese Untersuchungen wurden chimäre Formen beider Adhäsionsrezeptoren konstruiert, in denen eine oder mehrere Domänen gezielt ausgetauscht wurden. Die chimären Proteine wurden in einem Modellsystem mit humanen K562 Zellen exprimiert und die Rezeptortopologie mittels Elektronenmikroskopie qualitativ und quantitiv ausgewertet. Mittels Flusskammerexperimenten konnte anschliessend die funktionelle Bedeutung der Oberflächenlokalisation beider Adhäsionsproteine eruiert werden. Es zeigte sich, daß die TM Domäne alleine die Lokalisation des Rezeptors auf der Zelloberfläche bestimmt. Der TM Abschnitt von CD44 führte unabhängig von der IC und EC Komponente zur Expression auf dem Zellkörper mit Aussparung der Mikrovilli. Entsprechend bewirkte der TM Abschnitt von L-Selektin eine ausschliesslich mikrovilläre Lokalisation. Die IC Verankerung von L-Selektin oder CD44 hatte keinen spezifischen Einfluß. Funktionell zeigten mikrovillär exprimierte Rezeptoren eine höhere Adhäsionsrate unter Fluss, aber nicht unter statischen Bedingungen. Wurde in CD44 die TM Domöne durch die von L-Selektin ersetzt, führte dies zu einer deutlichen Steigerung der Adhäsionsfähigkeit der transfizierten Zellen auf Hyaluronsäure unter Fluss-bedingungen. Austausch der IC Domäne von L-Selektin durch die von CD44 führte zu geringerer Adhäsion trotz gleichbleibender Membranpositionierung, was die Bedeutung der Verankerung von L-Selektin an das Zytoskelett für seine Funktionalität unterstreicht. Insgesamt zeigt diese Arbeit einen neuen Mechanismus der Kompart-mentalisierung von Oberflächenrezeptoren in unpolarisierten Zellen, der alleine durch transmembranäre Signale gesteuert wird und relevante Auswirkungen auf die zelluläre Adhäsion von Zellen des Immunsystems hat

Patrolling Mechanics of Non-Classical Monocytes in Vascular Inflammation

Non-classical monocytes have emerged as the preeminent vascular housekeepers. Continuous intravascular screening is enabled by slow patrolling on the endothelium and allows a rapid response to local perturbations. Intravital imaging has been crucial to elucidate the molecular mechanisms and migratory phenotype of patrolling. In this review, we discuss technical requirements of intravital microscopy such as imaging modalities, labeling strategies, and data analysis. We further focus on patrolling kinetics and adhesion receptors in different organs and vascular beds including arteries during homeostasis and vascular inflammation and define pertinent questions in the field

C4d Deposition after Allogeneic Renal Transplantation in Rats Is Involved in Initial Apoptotic Cell Clearance

In the context of transplantation, complement activation is associated with poor prognosis and outcome. While complement activation in antibody-mediated rejection is well-known, less is known about complement activation in acute T cell-mediated rejection (TCMR). There is increasing evidence that complement contributes to the clearance of apoptotic debris and tissue repair. In this regard, we have analysed published human kidney biopsy transcriptome data clearly showing upregulated expression of complement factors in TCMR. To clarify whether and how the complement system is activated early during acute TCMR, experimental syngeneic and allogeneic renal transplantations were performed. Using an allogeneic rat renal transplant model, we also observed upregulation of complement factors in TCMR in contrast to healthy kidneys and isograft controls. While staining for C4d was positive, staining with a C3d antibody showed no C3d deposition. FACS analysis of blood showed the absence of alloantibodies that could have explained the C4d deposition. Gene expression pathway analysis showed upregulation of pro-apoptotic factors in TCMR, and apoptotic endothelial cells were detected by ultrastructural analysis. Monocytes/macrophages were found to bind to and phagocytise these apoptotic cells. Therefore, we conclude that early C4d deposition in TCMR may be relevant to the clearance of apoptotic cells

The TWEAK/Fn14 pathway is required for calcineurin inhibitor toxicity of the kidneys.

Calcineurin inhibitor toxicity (CNT) is a frequent occurrence in transplanted renal grafts and autochthone kidneys from patients undergoing long-term treatment with calcineurin inhibitors, notably cyclosporin A (CsA) and tacrolimus. Here, we show an indispensable role of the tumor necrosis factor superfamily (TNFS) molecule TNF-related weak inducer of apoptosis (TWEAK) (TNFSF12) in the pathogenesis of acute CNT lesions in mice. A deficiency in TWEAK resulted in limited tubulotoxicity after CsA exposure, which correlated with diminished expression of inflammatory cytokines and reduced intraparenchymal infiltration with immune cells. We further identified tubular epithelial cells of the kidney as major targets of CsA activity and found that Fn14 (tumor necrosis factor receptor superfamily 12A), the receptor for TWEAK, is a highly CsA-inducible gene in these cells. Correlating with this, CsA pretreatment sensitized tubular epithelial cells specifically to the pro-inflammatory activities of recombinant TWEAK in vitro. Moreover, injection of rTWEAK alone into mice induced moderate disease similar to CsA, and rTWEAK combined with CsA resulted in synergistic nephrotoxicity. These findings support the importance of tubular epithelial cells as cellular targets of CsA toxicity and introduce TWEAK as a critical contributor to CNT pathogenesis

Endothelial protective monocyte patrolling in large arteries intensified by western diet and atherosclerosis

Non-classical mouse monocyte (CX3CR1, Ly-6C) patrolling along the vessels of the microcirculation is critical for endothelial homeostasis and inflammation. Due to technical challenges it is currently not established how patrolling occurs in large arteries. OBJECTIVE:: This study was undertaken to elucidate the molecular, migratory and functional phenotype of patrolling monocytes in the high shear and pulsatile environment of large arteries in healthy, hyperlipidemic and atherosclerotic conditions. METHODS AND RESULTS:: Applying a new method for stable, long-term two-photon intravital microscopy of unrestrained large arteries in live CX3CR1-GFP mice, we show that non-classical monocytes patrol inside healthy carotid arteries at a velocity of 36 μm/min, three times faster than in microvessels. The tracks are less straight but lead preferentially downstream. The number of patrolling monocytes is increased 9-fold by feeding wildtype mice a western diet or by applying topical TLR7/8 agonists. A similar increase is seen in CX3CR1/apoE mice on chow diet, with a further 2-3-fold increase on western diet (22-fold over healthy). In plaque conditions, monocytes are readily captured onto the endothelium from free flow. Stable patrolling is unaffected in CX3CR1-deficient mice and involves the contribution of LFA-1 and α4 integrins. The endothelial damage in atherosclerotic carotid arteries was assessed by electron microscopy and correlates with the number of intraluminal patrollers. Abolishing patrolling monocytes in Nr4a1 apoE mice leads to pronounced endothelial apoptosis. CONCLUSIONS:: Arterial patrolling is a prominent new feature of non-classical monocytes with unique molecular and kinetic properties. It is highly upregulated in hyperlipidemia and atherosclerosis in an CX3CR1-independent fashion, and plays a potential role in endothelial protection.Circulation Research is published on behalf of the American Heart Association, Inc., by Wolters Kluwer. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made.Fil: Quintar, Amado Alfredo. La Jolla Institute for Allergy and Immunology; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Ciencias de la Salud. Universidad Nacional de Córdoba. Instituto de Investigaciones en Ciencias de la Salud; ArgentinaFil: McArdle, Sara. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Wolf, Dennis. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Marki, Alex. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Ehinger, Erik. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Vassallo, Melanie. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Miller, Jacqueline. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Mikulski, Zbigniew. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Ley, Klaus Friedrich. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Buscher, Konrad. La Jolla Institute for Allergy and Immunology; Estados Unido

Protection from septic peritonitis by rapid neutrophil recruitment through omental high endothelial venules

Acute peritonitis is a frequent medical condition that can trigger severe sepsis as a life-threatening complication. Neutrophils are first-responders in infection but recruitment mechanisms to the abdominal cavity remain poorly defined. Here, we demonstrate that high endothelial venules (HEVs) of the greater omentum constitute a main entry pathway in TNFα-, Escherichia coli (E. coli)- and caecal ligation and puncture-induced models of inflammation. Neutrophil transmigration across HEVs is faster than across conventional postcapillary venules and requires a unique set of adhesion receptors including peripheral node addressin, E-, L-selectin and Mac-1 but not P-selectin or LFA-1. Omental milky spots readily concentrate intra-abdominal E. coli where macrophages and recruited neutrophils collaborate in phagocytosis and killing. Inhibition of the omental neutrophil response exacerbates septic progression of peritonitis. This data identifies HEVs as a clinically relevant vascular recruitment site for neutrophils in acute peritonitis that is indispensable for host defence against early systemic bacterial spread and sepsis