Membrane trafficking as an active regulator of constitutively secreted cytokines

Item does not contain fulltextImmune-cell activation by inflammatory stimuli triggers the transcription and translation of large amounts of cytokines. The transport of newly synthesized cytokines to the plasma membrane by vesicular trafficking can be rate-limiting for the production of these cytokines, and immune cells upregulate their exocytic machinery concomitantly with increased cytokine expression in order to cope with the increasing demand for trafficking. Whereas it is logical that trafficking is rate-limiting for regulated secretion where an intracellular pool of molecules is waiting to be released, the reason for this is not obvious for constitutively secreted cytokines, such as interleukin-6 (IL-6), interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha). These constitutively secreted cytokines are primarily regulated at the transcriptional and/or translational level but mounting evidence presented here shows that cells might also increase or decrease the rate of post-Golgi cytokine trafficking to modulate their production. Therefore, in this Hypothesis, we ask the question: why is there a need to limit the trafficking of constitutively secreted cytokines? We propose a model where cells monitor and adjust their production rate of cytokines by sensing the intracellular level of cytokines while they are in transit to the plasma membrane. This self-regulation of cytokine production could prevent an overshooting response of acute-phase cytokines, such as IL-6, IL-12 and TNF-alpha, upon acute infection

Reverse Signaling by MHC-I Molecules in Immune and Non-Immune Cell Types

Major histocompatibility complex (MHC) molecules are well-known for their role in antigen (cross-) presentation, thereby functioning as key players in the communication between immune cells, for example dendritic cells (DCs) and T cells, or immune cells and their targets, such as T cells and virus-infected or tumor cells. However, much less appreciated is the fact that MHC molecules can also act as signaling receptors. In this process, here referred to as reverse MHC class I (MHC-I) signaling, ligation of MHC molecules can lead to signal-transduction and cell regulatory effects in the antigen presenting cell. In the case of MHC-I, reverse signaling can have several outcomes, including apoptosis, migration, induced or reduced proliferation and cytotoxicity towards target cells. Here, we provide an overview of studies showing the signaling pathways and cell outcomes upon MHC-I stimulation in various immune and non-immune cells. Signaling molecules like RAC-alpha serine/threonine-protein kinase (Akt1), extracellular signal-regulated kinases 1/2 (ERK1/2), and nuclear factor-kappaB (NF-kappaB) were common signaling molecules activated upon MHC-I ligation in multiple cell types. For endothelial and smooth muscle cells, the in vivo relevance of reverse MHC-I signaling has been established, namely in the context of adverse effects after tissue transplantation. For other cell types, the role of reverse MHC-I signaling is less clear, since aspects like the in vivo relevance, natural MHC-I ligands and the extended downstream pathways are not fully known.The existing evidence, however, suggests that reverse MHC-I signaling is involved in the regulation of the defense against bacterial and viral infections and against malignancies. Thereby, reverse MHC-I signaling is a potential target for therapies against viral and bacterial infections, cancer immunotherapies and management of organ transplantation outcomes

Novel and conventional inhibitors of canonical autophagy differently affect LC3-associated phagocytosis

In autophagy, LC3-positive autophagophores fuse and encapsulate the autophagic cargo in a double-membrane structure. In contrast, lipidated LC3 (LC3-II) is directly formed at the phagosomal membrane in LC3-associated phagocytosis (LAP). In this study, we dissected the effects of autophagy inhibitors on LAP. SAR405, an inhibitor of VPS34, reduced levels of LC3-II and inhibited LAP. In contrast, the inhibitors of endosomal acidification bafilomycin A1 and chloroquine increased levels of LC3-II, due to reduced degradation in acidic lysosomes. However, while bafilomycin A1 inhibited LAP, chloroquine did not. Finally, EACC, which inhibits the fusion of autophagosomes with lysosomes, promoted LC3 degradation possibly by the proteasome. Targeting LAP with small molecule inhibitors is important given its emerging role in infectious and autoimmune diseases

Congenital disorder of glycosylation caused by starting site-specific variant in syntaxin-5

Mutations in genes critical for proper intra-Golgi transport can cause human syndromes due to defects in glycosylation of proteins. Here, the authors identify a human variant of Syntaxin-5 that causes fatal multisystem disease and mislocalization of glycosyltransferases due to altered Golgi transport

Guía de práctica clínica para la prevención, diagnóstico, tratamiento y rehabilitación de la falla cardiaca en población mayor de 18 años, clasificación B, C y D

La falla cardíaca es un síndrome clínico caracterizado por síntomas y signos típicos de insuficiencia cardíaca, adicional a la evidencia objetiva de una anomalía estructural o funcional del corazón. Guía completa 2016. Guía No. 53Población mayor de 18 añosN/

3D reconstructions confirm the observations derived from single IHC sections.

<p>3D reconstructions of resting (<b>A</b>), stimulated (<b>B</b>) and recovered IHCs (<b>C</b> and <b>D</b>). Endocytotic organelles are shown in purple. Note the presence of tubular organelles both before and after stimulation. Most organelles, including the tubular ones, are replaced by small vesicles during the recovery periods. Insets show magnified regions from the four different cell region (cuticular plate, top, nuclear and basal regions). Note the increased number of endosome-like organelles at the base of the cell after stimulation and during recovery.</p

FM 1-43 penetrates rapidly into IHCs.

<p>(<b>A</b>) Scheme of the organ of Corti, indicating the areas of the IHCs that were imaged. (<b>B</b>) FM 1-43 (10 µM) labeling of IHCs was imaged at room temperature at four cellular levels: stereocilia bundle, top, nuclear and basal. FM 1-43 reaches the cytoplasm, diffusing in an apex-to-base fashion (right panel). This observation is typical for 16 independent experiments.</p

FM 1-43 photo-oxidation suggests that synaptic vesicles recycle at the base of IHCs.

<p>(<b>A</b>)–(<b>D</b>) The electron micrographs show organelles containing the photo-oxidation product (DAB precipitate) at rest (<b>A</b>), during stimulation (<b>B</b>), or after a 5-minute (<b>C</b>) or 30-minute (<b>D</b>) recovery period. Some typical organelles are indicated by red arrowheads. Large tubular organelles are indicated by the dashed red traces. Mitochondria are indicated by white asterisks. Note that the basal region contains few labeled organelles at rest; their number increases after stimulation. Note also that the tubular organelles from the top and nuclear regions, found both at rest and during stimulation, disappear during the recovery periods. Scale bars, 200 nm. (<b>E</b>–<b>H</b>) Analysis of organelle labeling under the different experimental conditions. The percentage of the cell volume occupied by labeled organelles was measured in four different cellular regions: cuticular plate (<b>E</b>), top (<b>F</b>), nuclear (<b>G</b>) and basal (<b>H</b>). The graphs show averages performed for electron micrographs from 14 (resting), 12 (stimulation), 4 (5 minute recovery) and 4 (30 minute recovery) independent experiments. We used one-way ANOVA tests to check whether stimulation produced any changes in the different cellular regions. No significant differences were found for the cuticular plate region (<b>E</b>), for the top region (<b>F</b>), and for the nuclear region (<b>G</b>); all <i>P</i> values were higher than 0.05. In contrast, the ANOVA test revealed significant differences for the basal region (<b>H</b>); <i>P</i><0.05. A <i>post hoc</i> Bonferroni test indicated that stimulation increases the amount of label significantly in this region, compared to the resting control (<i>P</i><0.001).</p

The vesicles forming during recovery in the top region of the IHCs are significantly larger than synaptic vesicles.

<p>(A) Analysis of the organelle numbers from the 3D reconstructions presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088353#pone-0088353-g005" target="_blank">Fig. 5</a>. Organelles containing the photo-oxidation product were classified in three categories: tubules (large elongated or flattened organelles), cisterns (round or elliptic organelles) and small vesicles. Tubules dominate at rest. Cisterns are induced to form during stimulation. Almost all organelles were processed to small vesicles during recovery. Images show representative organelles for each of the categories. Scale bar, 200 nm. (B) Quantification of the size of small vesicles (<120 nm) at the top and the base of the cell, after a 5-minute recovery. The latter are significantly smaller, and very similar to <i>bona fide</i> synaptic vesicles from the efferent boutons. The bars show means ± SEM, from 260, 690 and 219 vesicles (top, base and efferent, respectively). A one-way ANOVA test indicated that the vesicle populations were significantly different. A <i>post hoc</i> Bonferroni test revealed that the vesicles at the top of the IHCs were significantly larger than those in the basal region, and than those from the efferent boutons (<i>P</i><0.0001). The latter were not significantly different from each other (<i>P</i>>0.05). The analysis included all small vesicles found in the relevant IHC regions in the 3D reconstructions presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088353#pone-0088353-g005" target="_blank">Fig. 5</a>. The data points were sufficiently numerous to allow us to verify that the distributions were bell-shaped (using histograms of vesicle size). The variance was similar between the data groups: approximately 72, 190 and 143 units for top, base and efferent, respectively. The graphs show the entire data set we obtained in the 3D reconstructions; each 3D reconstruction is derived from one representative experiment.</p

Most commercial fluorescent markers fail in reporting endocytosis in IHCs.

<p>(<b>A</b>) FM 1-43 labels IHCs strongly in conditions where endocytosis is strongly reduced by low temperature (on ice, middle panel) or abolished by fixation (right panel). The labeling pattern is identical to that obtained at room temperature (RT, left panel). (<b>B</b>) Styryl dyes from the FM family (FM 1-84, FM 4-64, FM 4-64FX, AM 1-43), ranging in sizes between 450 and 470 Da, permeated IHCs in a similar fashion to FM 1-43 at room temperature. In contrast, the larger FM 3-25 (843 Da) did not penetrate into the tissue, producing a very faint labeling. The membrane-binding dye 5-dodecanoylaminofluorescein (DCF, 530 Da) had a similar effect. (<b>C</b>) An additional membrane dye, Di-2-ANEPEQ (549 Da), labeled IHCs in the same fashion as FM 1-43. Soluble compounds, such as calcein (623 Da) and 3000 Da Dextran coupled to fluorescein, labeled the intercellular spaces throughout the organ of Corti, but were not taken up efficiently by the cells. All dyes were used at a concentration of 10 µM, with the exception of Di-2-ANEPEQ, which was used at 100 µM, and DCF, used at 180 µM. Scale bars 10 µm. (<b>D</b>) Analysis of labeling intensity for all dyes that appeared to penetrate into the IHCs. The fluorescence intensity of the cells (excluding the nucleus) was normalized to the background intensity, and is expressed as fold over background. The black bars show results from cells incubated with the different dyes at room temperature. The gray bars show cells incubated on ice, at 2–4°C. The following numbers of IHCs were analyzed. FM 1-43: 26 at room temperature, 19 on ice; FM 1-84: 7, 27; FM 4-64: 13, 23; FM 4-64FX: 8, 20; AM 1-43: 20, 16; DCF: 8, 15; Di-2-ANEPEQ: 30, 35.</p