The impact of the cytoskeleton dynamics on immune responses

Immune cells are susceptible to a variety of external stimuli that triggers cell surface receptors, leading to activation of intracellular proteins and changes in the actin cytoskeleton. The Rho-GTPases are a family of proteins that regulate several aspects of actin polymerization. They take part in an intricate network of other proteins and can therefore have a direct or indirect role in several cell functions. In this study I aimed to define the role of the Rho-GTPases Cdc42 and Rac2, as well as the effector proteins WASp and N-WASp in the adaptive immune system. In paper I the aim was to dissect the particular contribution of WASp-deficiency to the induction of skin pathology in Wiskott-Aldrich syndrome patients, using mouse models. Our study showed that WASp-deficient dendritic cells accumulate in the skin; were able to induce an increase in cross-presentation of exogenous antigen and therefore increased CD8+ T cell proliferation and activation in vivo and in vitro. This was caused by increased expression of Rac2 in the cytoplasm, which regulates the phagosomal pH and promotes loading of antigen on MHC class I molecules. In paper II we aimed to understand the role of Cdc42 for B cell activation using a mouse where we deleted Cdc42 conditionally in mature B cells. Mice with Cdc42-deficient B cells showed reduced marginal zone and follicular B cell numbers. Immunized mice had reduced germinal center B cells associated with reduced specific antibody titers in serum. In vitro assays revealed less spreading on antibody-coated surfaces and a skewed CD4+ T cell activation towards more production of IFN and less IL-2. In paper III we studied the combined contribution of WASp and N-WASp deficiency in B cells. A WASp KO mouse where N-WASp was conditionally deleted only in B cells (cDKO) was used. Compared to WT and WASp KO mice, the cDKO mouse had a large reduction in marginal zone B cells as well as decreased marginal zone B cell precursors and follicular B cells. B cells in cDKO mice also failed to mount a specific antibody response to both T- dependent and T-independent antigens with reduced specific antibody titers, together with decreased antigen uptake by marginal zone B cells and transportation to the follicle. In paper IV we investigated the effect of the hyperactive mutated WASp, found in X-linked neutropenia patients, on murine B and T cells. XLN-WASp mutations induced increased polymerized actin in both T and B cells. We detected reduced spreading but normal chemotaxis toward specific chemokines by both cell types. We also observed reduced proliferation of B cells but not T cells and increased apoptosis of both cell types in vitro. The latter is probably due to increased genomic instability in B and T cells as observed by a FISH-telomere assay. Altogether, this study shows that Rho GTPases and their effector proteins are crucial for correct cell function and the cellular and humoral immune responses. Notably, the phenotypic severity can be increased by depletion of closely related intracellular proteins suggesting the existence of compensatory mechanisms and overlapping roles. The characterization of the Rho GTPases function may contribute to more precise interventions and therapeutic success

Nuclear Wiskott–Aldrich syndrome protein co-regulates T cell factor 1-mediated transcription in T cells

Background: The Wiskott–Aldrich syndrome protein (WASp) family of actin-nucleating factors are present in the cytoplasm and in the nucleus. The role of nuclear WASp for T cell development remains incompletely defined. Methods: We performed WASp chromatin immunoprecipitation and deep sequencing (ChIP-seq) in thymocytes and spleen CD4+ T cells. Results: WASp was enriched at genic and intergenic regions and associated with the transcription start sites of protein-coding genes. Thymocytes and spleen CD4+ T cells showed 15 common WASp-interacting genes, including the gene encoding T cell factor (TCF)12. WASp KO thymocytes had reduced nuclear TCF12 whereas thymocytes expressing constitutively active WASpL272P and WASpI296T had increased nuclear TCF12, suggesting that regulated WASp activity controlled nuclear TCF12. We identify a putative DNA element enriched in WASp ChIP-seq samples identical to a TCF1-binding site and we show that WASp directly interacted with TCF1 in the nucleus. Conclusions: These data place nuclear WASp in proximity with TCF1 and TCF12, essential factors for T cell development. Electronic supplementary material The online version of this article (doi:10.1186/s13073-017-0481-6) contains supplementary material, which is available to authorized users

A breakthrough on Amanita phalloides poisoning: an effective antidotal effect by polymyxin B

Amanita phalloides is responsible for more than 90 % of mushroom-related fatalities, and no effective antidote is available. a-Amanitin, the main toxin of A. phalloides, inhibits RNA polymerase II (RNAP II), causing hepatic and kidney failure. In silico studies included docking and molecular dynamics simulation coupled to molecular mechanics with generalized Born and surface area method energy decomposition on RNAP II. They were performed with a clinical drug that shares chemical similarities to a-amanitin, polymyxin B. The results show that polymyxin B potentially binds to RNAP II in the same interface of a-amanitin, preventing the toxin from binding to RNAP II. In vivo, the inhibition of the mRNA transcripts elicited by a-amanitin was efficiently reverted by polymyxin B in the kidneys. Moreover, polymyxin B significantly decreased the hepatic and renal a-amanitin-induced injury as seen by the histology and hepatic aminotransferases plasma data. In the survival assay, all animals exposed to a-amanitin died within 5 days, whereas 50 % survived up to 30 days when polymyxin B was administered 4, 8, and 12 h post-a-amanitin. Moreover, a single dose of polymyxin B administered concomitantly with a-amanitin was able to guarantee 100 % survival. Polymyxin B protects RNAP II from inactivation leading to an effective prevention of organ damage and increasing survival in a-amanitin-treated animals. The present use of clinically relevant concentrations of an already human-use-approved drug prompts the use of polymyxin B as an antidote for A. phalloides poisoning in humans.Juliana Garcia, Vera Marisa Costa, Ricardo Dinis-Oliveira and Ricardo Silvestre thank FCT-Foundation for Science and Technology-for their PhD grant (SFRH/BD/74979/2010), Post-doc grants (SFRH/BPD/63746/2009 and SFRH/BPD/110001/2015) and Investigator grants (IF/01147/2013) and (IF/00021/2014), respectively. This work was supported by the Fundacao para a Ciencia e Tecnologia (FCT) - project PTDC/DTPFTO/4973/2014 - and the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundacao para a Ciencia e Tecnologia) through project Pest-C/EQB/LA0006/2013

Heterogeneity in age-related white matter changes

White matter changes occur endemically in routine magnetic resonance imaging (MRI) scans of elderly persons. MRI appearance and histopathological correlates of white matter changes are heterogeneous. Smooth periventricular hyperintensities, including caps around the ventricular horns, periventricular lining and halos are likely to be of non-vascular origin. They relate to a disruption of the ependymal lining with subependymal widening of the extracellular space and have to be differentiated from subcortical and deep white matter abnormalities. For the latter a distinction needs to be made between punctate, early confluent and confluent types. Although punctate white matter lesions often represent widened perivascular spaces without substantial ischemic tissue damage, early confluent and confluent lesions correspond to incomplete ischemic destruction. Punctate abnormalities on MRI show a low tendency for progression, while early confluent and confluent changes progress rapidly. The causative and modifying pathways involved in the occurrence of sporadic age-related white matter changes are still incompletely understood, but recent microarray and genome-wide association approaches increased the notion of pathways that might be considered as targets for therapeutic intervention. The majority of differentially regulated transcripts in white matter lesions encode genes associated with immune function, cell cycle, proteolysis, and ion transport. Genome-wide association studies identified six SNPs mapping to a locus on chromosome 17q25 to be related to white matter lesion load in the general population. We also report first and preliminary data that demonstrate apolipoprotein E (ApoE) immunoreactivity in white matter lesions and support epidemiological findings indicating that ApoE is another factor possibly related to white matter lesion occurrence. Further insights come from modern MRI techniques, such as diffusion tensor and magnetization transfer imaging, as they provide tools for the characterization of normal-appearing brain tissue beyond what can be expected from standard MRI scans. There is a need for additional pre- and postmortem studies in humans, including these new imaging techniques

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) wide-spread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications

Deletion of Dock10 in B Cells Results in Normal Development but a Mild Deficiency upon In Vivo and In Vitro Stimulations

We sought to identify genes necessary to induce cytoskeletal change in B cells. Using gene expression microarray, we compared B cells stimulated with interleukin-4 (IL-4) and anti-CD40 antibodies that induce B cell spreading, cell motility, tight aggregates, and extensive microvilli with B cells stimulated with lipopolysaccharide that lack these cytoskeletal changes. We identified 84 genes with 10-fold or greater expression in anti-CD40 + IL-4 stimulated B cells, one of these encoded the guanine nucleotide exchange factor (GEF) dedicator of cytokinesis 10 (Dock10). IL-4 selectively induced Dock10 expression in B cells. Using lacZ expression to monitor Dock10 promoter activity, we found that Dock10 was expressed at all stages during B cell development. However, specific deletion of Dock10 in B cells was associated with a mild phenotype with normal B cell development and normal B cell spreading, polarization, motility, chemotaxis, aggregation, and Ig class switching. Dock10-deficient B cells showed lower proliferation in response to anti-CD40 and IL-4 stimulation. Moreover, the IgG response to soluble antigen in vivo was lower when Dock10 was specifically deleted in B cells. Together, we found that most B cell responses were intact in the absence of Dock10. However, specific deletion of Dock10 in B cells was associated with a mild reduction in B cell activation in vitro and in vivo

Insulin Influences Autophagy Response Distinctively in Macrophages of Different Compartments

Background/Aims: Diabetes mellitus (DM) is characterized by hyperglycemia, associated to a lack or inefficiency of the insulin to regulate glucose metabolism. DM is also marked by alterations in a diversity of cellular processes that need to be further unraveled. In this study, we examined the autophagy pathway in diabetic rat macrophages before and after treatment with insulin. Methods: Bone marrow-derived macrophages (BMM), bronchoalveolar lavage (BAL) and splenic tissue of diabetic male Wistar rats (alloxan, 42 mg/kg, i.v., 10 days) and control rats (physiological saline, i.v.). Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 8 h before experiments. For characterization of the model and evaluation of the effect of insulin on the autophagic process, the following analyzes were performed: (a) concentrations of cytokines: interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-4, IL-10, cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-2 in the BAL supernatant was measured by ELISA; (b) characterization of alveolar macrophage (AM) of the BAL as surface antigens (MHCII, pan-macrophage KiM2R, CD11b) and autophagic markers (protein microtubule-associated light chain (LC)3, autophagy protein (Atg)12 by flow cytometry and confocal microscopy (c) study of macrophages differentiated from the bone marrow by flow cytometry and confocal microscopy (d) histology of the spleen by immunohistochemistry associated with confocal microscopy. Results: Interestingly, insulin exerted antagonistic effects on macrophages from different tissues. Macrophages from bronchoalveolar lavage (BAL) enhanced their LC3 autophagosome bound content after treatment with insulin whereas splenic macrophages from red pulp in diabetic rats failed to enhance their Atg 12 levels compared to control animals. Insulin treatment in diabetic rats did not change LC3 content in bone marrow derived macrophages (BMM). M1 and M2 macrophages behaved accordingly to the host they were derived from. Diabetic M1 BMM had their LC3 vesicle-bound content diminished and M2 BMM enhanced their LC3 levels and insulin treatment failed to rescue autophagy to control levels. Insulin normalizes CINC-2 level but does not modulate autophagy markers. Conclusion: Taking these results together, diabetic macrophages derived from different compartments show different levels of autophagy markers compared to healthy animals, therefore, they suffer distinctively in the absence of insulin

Concatemeric Broccoli reduces mRNA stability and induces aggregates.

Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol