The Transcriptional Programme of Human Heart Valves Reveals the Natural History of Infective Endocarditis

Infective endocarditis (IE) is an infectious disease that is mainly caused by Staphylococcus aureus and Streptococcus sp. It usually leads to valvular destruction and vegetation formation. Its pathophysiology is badly understood and likely involves immune and coagulation systems with close interactions with the microorganism. Our objective was to evaluate host response by comparing transcriptional profiles of cardiac valves from IE patients with controls. Hierarchical clustering revealed a signature of IE consisting of 146 genes. Among the 89 up-regulated genes, we identified two genes strongly associated with IE: metalloproteinase 12 (MMP-12) and aquaporin-9, a member of the aquaglyceroporin membrane channel family. The up-regulation of MMP-12 gene is strengthened by the down-modulation of the gene encoding its inhibitor TIMP3. In addition, MMP-12 was expressed in macrophages infiltrating EI valves. We also found that aquaporin-9 was expressed in endothelial cells lining neo-vessel lumen, suggesting that aquaporin-9 might be associated with neovascularization of infected valves leading to tissue oedema secondary to the inflammatory process. The Gene Ontology annotation and the resulting functional classification showed that most up-regulated genes account for recruitment of inflammatory cells in vegetations, angiogenesis and remodelling of endocardium tissue. A network analysis confirmed the involvement of molecules related to the remodelling of endocardium tissue and angiogenesis in IE. It also evidenced the role of caspases, especially that of caspase-9 and intrinsic apoptotic pathway in IE. Based on this study we propose a scenario for the natural history of IE in humans. Some parameters identified in this work could become tools for measuring the disease activity and should be tested as biomarkers for diagnosis or prognosis assessment in future studies

New Microbicidal Functions of Tracheal Glands: Defective Anti-Infectious Response to Pseudomonas aeruginosa in Cystic Fibrosis

Tracheal glands (TG) may play a specific role in the pathogenesis of cystic fibrosis (CF), a disease due to mutations in the cftr gene and characterized by airway inflammation and Pseudomonas aeruginosa infection. We compared the gene expression of wild-type TG cells and TG cells with the cftr ΔF508 mutation (CF-TG cells) using microarrays covering the whole human genome. In the absence of infection, CF-TG cells constitutively exhibited an inflammatory signature, including genes that encode molecules such as IL-1α, IL-β, IL-32, TNFSF14, LIF, CXCL1 and PLAU. In response to P. aeruginosa, genes associated with IFN-γ response to infection (CXCL10, IL-24, IFNγR2) and other mediators of anti-infectious responses (CSF2, MMP1, MMP3, TLR2, S100 calcium-binding proteins A) were markedly up-regulated in wild-type TG cells. This microbicidal signature was silent in CF-TG cells. The deficiency of genes associated with IFN-γ response was accompanied by the defective membrane expression of IFNγR2 and altered response of CF-TG cells to exogenous IFN-γ. In addition, CF-TG cells were unable to secrete CXCL10, IL-24 and S100A8/S100A9 in response to P. aeruginosa. The differences between wild-type TG and CF-TG cells were due to the cftr mutation since gene expression was similar in wild-type TG cells and CF-TG cells transfected with a plasmid containing a functional cftr gene. Finally, we reported an altered sphingolipid metabolism in CF-TG cells, which may account for their inflammatory signature. This first comprehensive analysis of gene expression in TG cells proposes a protective role of wild-type TG against airborne pathogens and reveals an original program in which anti-infectious response was deficient in TG cells with a cftr mutation. This defective response may explain why host response does not contribute to protection against P. aeruginosa in CF

Effet d'un contexte inflammatoire sur le transfert de gènes dans les cellules trachéales glandulaires mucoviscidosiques

La mucoviscidose, maladie monogénique létale, est causée par une mutation dans le gène cftr. Les essais cliniques de thérapie génique, visant à délivrer par aérosol le gène CFTR normal humain dans les poumons, conduisent à une expression du gène très faible et transitoire. La mucoviscidose se caractérise, au niveau pulmonaire, par une infection chronique et une inflammation sévère. Nous nous sommes demandés si ce contexte inflammatoire pouvait être en partie responsable des échecs observés de la thérapie génique de la mucoviscidose. Nous avons montré que l'inflammation non spécifique, et en particulier les deux cytokines impliquées dans la réponse Th1/Th2, le TNFa. et l'IL-4, n'inhibait pas l'efficacité de transfert de gène, mais inhibait l'expression du gène transféré dans les cellules trachéales glandulaires mucoviscidosiques. L'étude des voies de signalisation induites par ces deux cytokines a montré que l'inhibition de l'expression du transgène était prévenue par les glucocorticoïdes mais pas par les AINS. Nous avons également montré que le système de transcytose des IgA était fonctionnel et efficace dans ces cellules, et que la présence d'un vecteur était capable d'augmenter l'expression du récepteur aux IgA dans les cellules. Nous proposons alors une nouvelle orientation de recherche sur la thérapie génique, qui ne peut prétendre à l'heure actuelle soigner la mucoviscidose, en tenant compte de l'inflammation perpétuelle dans les poumons des patients. Il nous semble également important de trouver de nouvelles possibilités d'administration du vecteur, afin de ne pas se heurter au système de défense humoral.NANCY1-SCD Medecine (545472101) / SudocSudocFranceF

Direct evidence for the interaction of stathmin along the length and the plus end of microtubules in cells: Stathmin binds to microtubules in cells

International audienceStathmin is a prominent destabilizer of microtubules (MTs). Extensive in vitro studies suggest strongly that stathmin could act by sequestering tubulin and/or by binding to the MT tips. In cells, the molecular mechanisms of stathmin binding to tubulin and/or MTs and its implications for the MT dynamics remain unexplored. Using immunofluorescence resonance energy transfer and fluorescence recovery after photobleaching, we analysed the ability of stathmin and its phospho-forms (on Ser16, 25, 38 and 63) to interact with tubulin and MTs in A549 cells. Consistent with in vitro studies, we detected stathmin-tubulin interactions at the MT plus-ends and in the cytosol. Interestingly, we also observed a novel pool of stathmin bound along the MT. The expression of truncated stathmin and the use of MT-stabilizing taxol further showed that the C-terminal domain of stathmin is the main contributor to this binding, and that the phosphorylation state of stathmin plays a role in its binding along the MT wall. Our findings demonstrate that stathmin binds directly along the MT wall. This pool of stathmin would be readily available to participate in protofilament dissociation when the moving plus-end of a depolymerizing MT reaches the stathmin molecules

Tau regulates the microtubule-dependent migration of glioblastoma cells via the Rho-ROCK signaling pathway

International audienceThe pathological significance of Tau in mechanisms driving cell migration in glioblastoma is unclear. Using shRNA approach to deplete microtubule-stabilizing Tau in U87 cells, we determined its impact on cytoskeletal coordination during migration. We demonstrated here that the motility of Tau-down-expressing cells (shTau) was significantly 36% lower than control cells. The shTau cells displayed weakly changed motility in the presence of nocodazole inhibiting microtubule formation. Such reduced motility of shTau cells was characterized by a 28% lower amount of microtubules bundles upon non-adhesive edges of the tails. In accord with Tau-stabilized microtubules required for cell movement, measurements of the front, body and rear section displacements of cells showed inefficient tail retraction in shTau cells. The tail retraction was restored by Y27632 inhibitor of the Rho-ROCK signaling. Moreover, we clearly identified that shTau cells displayed relocation of the active phosphoforms of p190-RhoGAP (inhibiting Rho-ROCK) and focal adhesion kinase (FAK) in cell bodies. In conclusion, our findings indicate that Tau governs the remodeling of microtubule and actin networks in the phase of retraction of the tail of cells necessary for effective migration

<i>Sb</i>S blocked αvβ5 integrin interaction with vitronectin.

<p>(A) Vitronectin- or Fibronectin-coated plates were incubated in the absence (Vitronectin and Fibronectin) or presence of <i>Sb</i>S (vitronectin+ <i>Sb</i>S and fibronectin+ <i>Sb</i>S) prior to addition of cells. Isolated HCT-8/E11 cells incubated without (light bars) or with <i>Sb</i>S (dark bars) were then seeded on plates. Cell adhesion to ECM was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s2" target="_blank">Methods</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s3" target="_blank">Results</a> are expressed as the percentage of cell adhesion compared to untreated cells (Ctrl). Data represent the mean + SD of 3 separate experiments. (B) Isolated HT29-D4 cells were treated with or without <i>Sb</i>S (dilution 1/8) and plated on vitronectin at the indicated concentrations. Cell-vitronectin adhesion was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s2" target="_blank">Methods</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s3" target="_blank">Results</a> are expressed as the percentage of cell adhesion. Data represent the mean+SD of 3 separate experiments. *** P<0.001.</p

<em>Saccharomyces boulardii</em> Improves Intestinal Epithelial Cell Restitution by Inhibiting αvβ5 Integrin Activation State

<div><p>Intestinal epithelial cell damage is frequently seen in the mucosal lesions of infectious or inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. Complete remission of these diseases requires both the disappearance of inflammation and the repair of damaged epithelium. <em>Saccharomyces boulardii</em> (<em>Sb</em>, Biocodex) is a non-pathogenic yeast widely used as a preventive and therapeutic probiotic for the prevention and treatment of diarrhea and other gastrointestinal disorders. We recently showed that it enhances the repair of intestinal epithelium through activation of α2β1 integrin collagen receptors. In the present study, we demonstrated that α2β1 integrin is not the sole cell-extracellular matrix receptor involved during <em>Sb</em>-mediated intestinal restitution. Indeed, by using cell adhesion assays, we showed that <em>Sb</em> supernatant contains heat sensitive molecule(s), with a molecular weight higher than 9 kDa, which decreased αvβ5 integrin-mediated adhesion to vitronectin by competing with the integrin. Moreover, <em>Sb</em>-mediated changes in cell adhesion to vitronectin resulted in a reduction of the αvβ5signaling pathway. We used a monolayer wounding assay that mimics <em>in vivo</em> cell restitution to demonstrate that down-modulation of the αvβ5 integrin-vitronectin interaction is related to <em>Sb</em>-induced cell migration. We therefore postulated that <em>Sb</em> supernatant contains motogenic factors that enhance cell restitution through multiple pathways, including the dynamic fine regulation of αvβ5 integrin binding activity. This could be of major importance in diseases characterized by severe mucosal injury, such as inflammatory and infectious bowel diseases.</p> </div

αvβ5 integrin is required for HCT-8/E11 cell interaction with vitronectin.

<p>(A) Isolated HCT-8/E11 cells were incubated without (Ctrl) or with <i>Sb</i>S (<i>Sb</i>S) in the absence (-) or presence of either anti-αv or -α5 integrin mAbs. Cells were then seeded on vitronectin (3,15 µg/ml). Cell adhesion was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s2" target="_blank">Methods</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s3" target="_blank">Results</a> are expressed as the percentage of cell adhesion compared to untreated cells (Ctrl). Data represent the mean+SD of 3 separate experiments. (B) HCT-8/E11 cells were transfected with anti -αv-integrin siRNAs (SiRNAαv) or scramble oligos (SiRNACtrl) for 48 h. Cells were then plated on vitronectin for 2 h. Cell-ECM adhesion was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s2" target="_blank">Methods</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045047#s3" target="_blank">Results</a> are expressed as the percentage of cell adhesion. Data represent the mean+SD of 3 separate experiments. ***P<0.001.</p