Macrophage Polarization is Deregulated in Haemophilia

Macrophages make important contributions to inflammation and wound healing. We show here that macrophage polarization is deregulated in haemophilia in response to macrophage colony-stimulating factor (M-CSF) and partially in response to granulocyte-macrophage colony-stimulating factor (GM-CSF). As a result, haemophilia macrophages exhibit a specific impairment of M-CSF-mediated functions involved in wound healing such as clot invasion and phagocytosis. Haemophilia monocytes express reduced amounts of the receptors for M-CSF and GM-CSF, which correlates with a failure to express tumour necrosis factor α (TNFα) and CD163 in M-CSF-treated haemophilia macrophages and reduced expression of TNFα and CD206 after treatment with GM-CSF. Protein expression in response to M-CSF was regained with respect to CD163 and CD206 after embedding haemophilia monocytes in clotted plasma suggesting that a functioning coagulation system has positive effects on macrophage M2 polarization. Mimicking the functional deficits of haemophilia macrophages in normal macrophages was possible by adding leptin, which we found to be elevated in the blood of haemophilia patients, to a monocyte cell line. The increase of leptin occurred in conjunction with C-reactive protein in a body mass index-controlled cohort suggesting that haemophilia patients harbour chronic low-grade inflammation. Together, our data indicate that impaired clotting in haemophilia patients leads to increased inflammation and a deregulation in macrophage differentiation, which may explain the commonly observed deficits in wound healing and tissue regeneration

Cytotoxic Efficiency of Human CD8+ T Cell Memory Subtypes

Immunological memory is important to protect humans against recurring diseases. Memory CD8+ T cells are required for quick expansion into effector cells but also provide immediate cytotoxicity against their targets. Whereas many functions of the two main cytotoxic subtypes, effector memory CD8+ T cells (TEM) and central memory CD8+ T cells (TCM), are well defined, single TEM and TCM cell cytotoxicity has not been quantified. To quantify cytotoxic efficiency of TEM and TCM, we developed a FRET-based single cell fluorescent assay with NALM6 target cells which allows analysis of target cell apoptosis, secondary necrosis following apoptosis, and primary necrosis after TEM- or TCM-target cell contact. Both, single cell and population cytotoxicity assays reveal a higher cytotoxic efficiency of TEM compared to TCM, as quantified by target cell apoptosis and secondary necrosis. Perforin, granzyme B, FasL, but not TRAIL expression are higher in TEM compared to TCM. Higher perforin levels (likely in combination with higher granzyme levels) mediate higher cytotoxic efficiency of TEM compared to TCM. Both, TEM and TCM need the same time to find their targets, however contact time between CTL and target, time to induce apoptosis, and time to induce secondary necrosis are all shorter for TEM. In addition, immune synapse formation in TEM appears to be slightly more efficient than in TCM. Defining and quantifying single TEM and TCM cytotoxicity and the respective mechanisms is important to optimize future subset-based immune therapies

High-velocity collimated outflows in planetary nebulae: NGC 6337, He 2-186, and K 4-47

We have obtained narrow-band images and high-resolution spectra of the planetary nebulae NGC 6337, He 2-186, and K 4-47, with the aim of investigating the relation between their main morphological components and several low-ionization features present in these nebulae. The data suggest that NGC 6337 is a bipolar PN seen almost pole on, with polar velocities higher than 200 km/s. The bright inner ring of the nebula is interpreted to be the "equatorial" density enhancement. It contains a number of low-ionization knots and outward tails that we ascribe to dynamical instabilities leading to fragmentation of the ring or transient density enhancements due to the interaction of the ionization front with previous density fluctuations in the ISM. The lobes show a pronounced point-symmetric morphology and two peculiar low-ionization filaments whose nature remains unclear. The most notable characteristic of He 2-186 is the presence of two high-velocity (higher than 135 km/s) knots from which an S-shaped lane of emission departs toward the central star. K 4-47 is composed of a compact core and two high-velocity, low-ionization blobs. We interpret the substantial broadening of line emission from the blobs as a signature of bow shocks, and using the modeling of Hartigan, Raymond, & Hartman (1987), we derive a shock velocity of 150 km/s and a mild inclination of the outflow on the plane of the sky. We discuss possible scenarios for the formation of these nebulae and their low-ionization features. In particular, the morphology of K 4-47 hardly fits into any of the usually adopted mass-loss geometries for single AGB stars. Finally, we discuss the possibility that point-symmetric morphologies in the lobes of NGC 6337 and the knots of He 2-186 are the result of precessing outflows from the central stars.Comment: 16 pages plus 7 figures, ApJ accepted. Also available at http://www.iac.es/publicaciones/preprints.htm

Jets, knots and tails in planetary nebulae: NGC 3918, K 1-2 and Wray 17-1

We analyze optical images and high-resolution, long-slit spectra of three planetary nebulae which possess collimated, low-ionization features. NGC 3918 is composed of an inner, spindle-shaped shell mildly inclined with respect to the plane of the sky. Departing from the polar regions of this shell, we find a two-sided jet expanding with velocities which increase linearly with distance from 50 to 100 km/s. The jet is probably coeval with the inner shell (with the age of approximately 1000 D yr, where D is the distance in kpc), suggesting that its formation should be ascribed to the same dynamical processes which also shaped the main nebula, and not to a more recent mass loss episode. We discuss the formation of the aspherical shell and jet in the light of current hydrodynamical and magnetohydrodynamical theories. K 1-2 is a planetary nebula with a close binary nucleus which shows a collimated string of knots embedded in a diffuse, elliptical shell. The knots expand with a velocity similar to that of the elliptical nebula (25 km/s), except for an extended tail located out of the main nebula, which linearly accelerates up to 45 km/s. We estimate an inclination on the line of the sight of 40 degres for the string of knots; once the orientation of the orbit is also determined, this information will allow us to test the prediction of current theories of the occurrence of polar jets from close binary systems. Wray 17-1 has a complex morphology, showing two pairs of low-ionization structures located in almost perpendicular directions from the central star, and embedded in a large, diffuse nebula. The two pairs show notable similarities and differences, and their origin is very puzzling.Comment: 20 pages plus 10 figures. ApJ recently published (ApJ 523, 721 (1999)

High glucose distinctively regulates Ca2+ influx in cytotoxic T lymphocytes upon target recognition and thapsigargin stimulation

In CTLs: High glucose‐culture enhances thapsigargin‐induced SOCE but decreases target recognition‐induced Ca2+ influx. High glucose‐culture regulates expression of ORAIs and STIMs without affecting glucose uptake. More high glucose‐cultured CTLs are prone to necrosis after execution of killing. (...

Identification of molecular candidates which regulate calcium-dependent CD8+ T-cell cytotoxicity

Cytotoxic CD8+ T lymphocytes (CTL) eliminate infected cells or transformed tumor cells by releasing perforincontaining cytotoxic granules at the immunological synapse. The secretion of such granules depends on Ca2+- influx through store operated Ca2+ channels, formed by STIM (stromal interaction molecule)-activated Orai proteins. Whereas molecular mechanisms of the secretion machinery are well understood, much less is known about the molecular machinery that regulates the efficiency of Ca2+-dependent target cell killing. CTL killing efficiency is of high interest considering the number of studies on CD8+ T lymphocytes modified for clinical use. Here, we isolated total RNA from primary human cells: natural killer (NK) cells, non-stimulated CD8+ T-cells, and from Staphylococcus aureus enterotoxin A (SEA) stimulated CD8+ T-cells (SEA-CTL) and conducted whole genome expression profiling by microarray experiments. Based on differential expression analysis of the transcriptome data and analysis of master regulator genes, we identified 31 candidates which potentially regulate Ca2+-homeostasis in CTL. To investigate a putative function of these candidates in CTL cytotoxicity, we transfected either SEA-stimulated CTL (SEA-CTL) or antigen specific CD8+ T-cell clones (CTL-MART-1) with siRNAs specific against the identified candidates and analyzed the killing capacity using a real-time killing assay. In addition, we complemented the analysis by studying the effect of inhibitory substances acting on the candidate proteins if available. Finally, to unmask their involvement in Ca2+ dependent cytotoxicity, candidates were also analyzed under Ca2+-limiting conditions. Overall, we identified four hits, CCR5 (C-C chemokine receptor type five), KCNN4 (potassium calcium-activated channel subfamily N), RCAN3 (regulator of calcineurin) and BCL (Bcell lymphoma) 2 which clearly affect the efficiency of Ca2+ dependent cytotoxicity in CTL-MART-1 cells, CCR5, BCL2, and KCNN4 in a positive manner, and RCAN3 in a negative way

Targeting the Microtubule-Network Rescues CTL Killing Efficiency in Dense 3D Matrices

Efficacy of cytotoxic T lymphocyte (CTL)-based immunotherapy is still unsatisfactory against solid tumors, which are frequently characterized by condensed extracellular matrix. Here, using a unique 3D killing assay, we identify that the killing efficiency of primary human CTLs is substantially impaired in dense collagen matrices. Although the expression of cytotoxic proteins in CTLs remained intact in dense collagen, CTL motility was largely compromised. Using light-sheet microscopy, we found that persistence and velocity of CTL migration was influenced by the stiffness and porosity of the 3D matrix. Notably, 3D CTL velocity was strongly correlated with their nuclear deformability, which was enhanced by disruption of the microtubule network especially in dense matrices. Concomitantly, CTL migration, search efficiency, and killing efficiency in dense collagen were significantly increased in microtubule-perturbed CTLs. In addition, the chemotherapeutically used microtubule inhibitor vinblastine drastically enhanced CTL killing efficiency in dense collagen. Together, our findings suggest targeting the microtubule network as a promising strategy to enhance efficacy of CTL-based immunotherapy against solid tumors, especially stiff solid tumors

miR-34a: a new player in the regulation of T cell function by modulation of NF-κB signaling

NF-κB functions as modulator of T cell receptor-mediated signaling and transcriptional regulator of miR-34a. Our in silico analysis revealed that miR-34a impacts the NF-κB signalosome with miR-34a binding sites in 14 key members of the NF-κB signaling pathway. Functional analysis identified five target genes of miR-34a including PLCG1, CD3E, PIK3CB, TAB2, and NFΚBIA. Overexpression of miR-34a in CD4+ and CD8+ T cells led to a significant decrease of NFΚBIA as the most downstream cytoplasmic NF-κB member, a reduced cell surface abundance of TCRA and CD3E, and to a reduction of T cell killing capacity. Inhibition of miR-34a caused an increase of NFΚBIA, TCRA, and CD3E. Notably, activation of CD4+ and CD8+ T cells entrails a gradual increase of miR-34a. Our results lend further support to a model with miR-34a as a central NF-κB regulator in T cells

Differential Redox Regulation of ORAI Ion Channels: A Mechanism to Tune Cellular Calcium Signaling

Reactive oxygen species (ROS) are involved in many physiological and pathophysiological cellular processes. We used lymphocytes, which are exposed to highly oxidizing environments during inflammation, to study the influence of ROS on cellular function. Calcium ion (Ca2+) influx through Ca2+ release–activated Ca2+ (CRAC) channels composed of proteins of the ORAI family is essential for the activation, proliferation, and differentiation of T lymphocytes, but whether and how ROS affect ORAI channel function have been unclear. Here, we combined Ca2+ imaging, patch-clamp recordings and measurements of cell proliferation and cytokine secretion to determine the effects of hydrogen peroxide (H2O2) on ORAI channel activity and human T helper lymphocyte (TH cell) function. ORAI1, but not ORAI3, channels were inhibited by oxidation by H2O2. The differential redox sensitivity of ORAI1 and ORAI3 channels depended mainly on an extracellularly located reactive cysteine, which is absent in ORAI3. TH cells became progressively less redox-sensitive after differentiation into effector cells, a shift that would allow them to proliferate, differentiate, and secrete cytokines in oxidizing environments. The decreased redox sensitivity of effector TH cells correlated with increased expression of Orai3 and increased abundance of several cytosolic antioxidants. Knockdown of ORAI3 with small-interfering RNA rendered effector TH cells more redox-sensitive. The differential expression of Orai isoforms between naïve and effector TH cells may tune cellular responses under oxidative stress