A Distinct Inhibitory Function for miR-18a in Th17 Cell Differentiation.

Th17 cell responses orchestrate immunity against extracellular pathogens but also underlie autoimmune disease pathogenesis. In this study, we uncovered a distinct and critical role for miR-18a in limiting Th17 cell differentiation. miR-18a was the most dynamically upregulated microRNA of the miR-17-92 cluster in activated T cells. miR-18a deficiency enhanced CCR6+ RAR-related orphan receptor (ROR)γt+ Th17 cell differentiation in vitro and increased the number of tissue Th17 cells expressing CCR6, RORγt, and IL-17A in airway inflammation models in vivo. Sequence-specific miR-18 inhibitors increased CCR6 and RORγt expression in mouse and human CD4+ T cells, revealing functional conservation. miR-18a directly targeted Smad4, Hif1a, and Rora, all key transcription factors in the Th17 cell gene-expression program. These findings indicate that activating signals influence the outcome of Th cell differentiation via differential regulation of mature microRNAs within a common cluster.

Erratum: Discovery of a highly potent glucocorticoid for asthma treatment.

[This corrects the article DOI: 10.1038/celldisc.2015.35.].

α8ß1 integrin regulates nutrient absorption through an Mfge8-PTEN dependent mechanism.

Coordinated gastrointestinal smooth muscle contraction is critical for proper nutrient absorption and is altered in a number of medical disorders. In this work, we demonstrate a critical role for the RGD-binding integrin α8ß1 in promoting nutrient absorption through regulation of gastrointestinal motility. Smooth muscle-specific deletion and antibody blockade of α8 in mice result in enhanced gastric antral smooth muscle contraction, more rapid gastric emptying, and more rapid transit of food through the small intestine leading to malabsorption of dietary fats and carbohydrates as well as protection from weight gain in a diet-induced model of obesity. Mechanistically, ligation of α8ß1 by the milk protein Mfge8 reduces antral smooth muscle contractile force by preventing RhoA activation through a PTEN-dependent mechanism. Collectively, our results identify a role for α8ß1 in regulating gastrointestinal motility and identify α8 as a potential target for disorders characterized by hypo- or hyper-motility.

Discovery of a highly potent glucocorticoid for asthma treatment.

Glucocorticoids are the most effective treatment for asthma. However, their clinical applications are limited by low efficacy in severe asthma and by undesired side effects associated with high dose or prolonged use. The most successful approach to overcome these limitations has been the development of highly potent glucocorticoids that can be delivered to the lungs by inhalation to achieve local efficacy with minimal systemic effects. On the basis of our previous structural studies, we designed and developed a highly potent glucocorticoid, VSGC12, which showed an improved anti-inflammation activity in both cell-based reporter assays and cytokine inhibition experiments, as well as in a gene expression profiling of mouse macrophage RAW264.7 cells. In a mouse asthma model, VSGC12 delivered a higher efficacy than fluticasone furoate, a leading clinical compound, in many categories including histology and the number of differentiated immune cells. VSGC12 also showed a higher potency than fluticasone furoate in repressing most asthma symptoms. Finally, VSGC12 showed a better side effect profile than fluticasone furoate at their respective effective doses, including better insulin response and less bone loss in an animal model. The excellent therapeutic and side effect properties of VSGC12 provide a promising perspective for developing this potent glucocorticoid as a new effective drug for asthma.

Effective treatment of mouse sepsis with an inhibitory antibody targeting integrin αvß5.

OBJECTIVE: Integrin αvß5 has been identified as a regulator of vascular leak and endothelial permeability. We hypothesized that targeting αvß5 could represent a viable treatment strategy for sepsis. DESIGN: Integrin ß5 subunit knockout and wild-type 129/svJae mice and wild-type mice treated with αvß5 blocking or control antibodies were tested in models of intraperitoneal lipopolysaccharide and cecal ligation and puncture. Human umbilical vein endothelial cell and human lung microvascular endothelial cell monolayers were treated with αvß5 antibodies to assess for effects on lipopolysaccharide-induced changes in transendothelial resistance and on patterns of cytoskeletal reorganization. SETTING: Laboratory-based research. SUBJECTS: Mice and endothelial cell monolayers. INTERVENTIONS, MEASUREMENTS, AND MAIN RESULTS: Measurements taken after intraperitoneal lipopolysaccharide and/or cecal ligation and puncture included mortality, vascular leak, hematocrit, quantification of a panel of serum cytokines/chemokines, and assessment of thioglyccolate-induced leukocyte migration. ß5 knockout mice had decreased mortality after intraperitoneal lipopolysaccharide and cecal ligation and puncture and decreased vascular leak, as measured by extravasation of an I-labeled intravascular tracer. Treating clinically ill mice with αvß5 antibodies, up to 20 hrs after intraperitoneal lipopolysaccharide and cecal ligation and puncture, also resulted in decreased mortality. αvß5 antibodies attenuated lipopolysaccharide-induced transendothelial resistance changes and cytoskeletal stress fiber formation in both human umbilical vein endothelial cell and human lung microvascular endothelial cell monolayers. αvß5 antibodies had no effect on cytokine/chemokine serum levels after cecal ligation and puncture. ß5 knockout mice and wild-type controls did not exhibit differences in thioglyccolate-induced leukocyte migration. CONCLUSIONS: Our studies suggest that αvß5 is an important regulator of the vascular endothelial leak response in sepsis and that αvß5 blockade may provide a novel approach to treating this devastating disease syndrome.

Absence of integrin αvß3 enhances vascular leak in mice by inhibiting endothelial cortical actin formation.

RATIONALE: Sepsis and acute lung injury (ALI) have devastatingly high mortality rates. Both are associated with increased vascular leak, a process regulated by complex molecular mechanisms. OBJECTIVES: We hypothesized that integrin αvß3 could be an important determinant of vascular leak and endothelial permeability in sepsis and ALI. METHODS: ß3 subunit knockout mice were tested for lung vascular leak after endotracheal LPS, and systemic vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. Possible contributory effects of ß3 deficiency in platelets and other hematopoietic cells were excluded by bone marrow reconstitution experiments. Endothelial cells treated with αvß3 antibodies were evaluated for sphingosine-1 phosphate (S1P)­mediated alterations in barrier function, cytoskeletal arrangement, and integrin localization. MEASUREMENTS AND MAIN RESULTS: ß3 knockout mice had increased vascular leak and pulmonary edema formation after endotracheal LPS, and increased vascular leak and mortality after intraperitoneal LPS and cecal ligation and puncture. In endothelial cells, αvß3 antibodies inhibited barrier-enhancing and cortical actin responses to S1P. Furthermore, S1P induced translocation of αvß3 from discrete focal adhesions to cortically distributed sites through Gi- and Rac1-mediated pathways. Cortical αvß3 localization after S1P was decreased by αvß3 antibodies, suggesting that ligation of the αvß3 with its extracellular matrix ligands is required to stabilize cortical αvß3 focal adhesions. CONCLUSIONS: Our studies identify a novel mechanism by which αvß3 mitigates increased vascular leak, a pathophysiologic function central to sepsis and ALI. These studies suggest that drugs designed to block αvß3 may have the unexpected side effect of intensifying sepsis- and ALI-associated vascular endothelial leak.

Integrin alphavbeta5 regulates lung vascular permeability and pulmonary endothelial barrier function.

Increased lung vascular permeability is an important contributor to respiratory failure in acute lung injury (ALI). We found that a function-blocking antibody against the integrin alphavbeta5 prevented development of lung vascular permeability in two different models of ALI: ischemia-reperfusion in rats (mediated by vascular endothelial growth factor [VEGF]) and ventilation-induced lung injury (VILI) in mice (mediated, at least in part, by transforming growth factor-beta [TGF-beta]). Knockout mice homozygous for a null mutation of the integrin beta5 subunit were also protected from lung vascular permeability in VILI. In pulmonary endothelial cells, both the genetic absence and blocking of alphavbeta5 prevented increases in monolayer permeability induced by VEGF, TGF-beta, and thrombin. Furthermore, actin stress fiber formation induced by each of these agonists was attenuated by blocking alphavbeta5, suggesting that alphavbeta5 regulates induced pulmonary endothelial permeability by facilitating interactions with the actin cytoskeleton. These results identify integrin alphavbeta5 as a central regulator of increased pulmonary vascular permeability and a potentially attractive therapeutic target in ALI.

Akt phosphorylation is not sufficient for insulin-like growth factor-stimulated myogenin expression but must be accompanied by down-regulation of mitogen-activated protein kinase/extracellular signal-regulated kinase phosphorylation.

IGF-I has a unique biphasic effect on skeletal muscle differentiation. Initially, IGF-I inhibits expression of myogenin, a skeletal muscle-specific regulatory factor essential for myogenesis. Subsequently, IGF-I switches to stimulating expression of myogenin. The mechanisms that mediate this switch in IGF action are incompletely understood. Several laboratories have demonstrated that the phosphatidylinositol-3-kinase/Akt signaling pathway is essential for myogenic differentiation and have suggested that this pathway mediates IGF-I stimulation of myogenin mRNA expression, an early critical step in the differentiation process. These studies, however, did not address concurrent Akt and MAPK/ERK1/2 phosphorylation, the latter of which is also known to regulate myogenic differentiation. In the present study in rat L6E9 muscle cells, we have manipulated ERK1/2 phosphorylation with either an upstream inhibitor or activator and examined concurrent levels of Akt and ERK1/2 phosphorylation and of myogenin mRNA expression in response to treatment with IGF-I. We find that even in the presence of phosphorylated Akt, it is only when ERK1/2 phosphorylation is inhibited that IGF-I can stimulate myogenin mRNA expression. Thus, although Akt phosphorylation may be necessary, it is not sufficient for induction of myogenic differentiation by IGF-I and must be accompanied by a decrease in ERK1/2 phosphorylation.

Early stimulation and late inhibition of extracellular signal-regulated kinase 1/2 phosphorylation by IGF-I: a potential mechanism mediating the switch in IGF-I action on skeletal muscle cell differentiation.

IGF-I has a unique biphasic effect on skeletal muscle cell differentiation. Initially, IGF-I inhibits differentiation and promotes proliferation of skeletal myoblasts. Subsequently, IGF-I switches to stimulating differentiation of these cells. The mechanisms responsible for this switch in IGF action remain unknown. We have examined the role of extracellular signal-regulated kinase (Erk)1/2 signaling in mediating the early inhibitory and late stimulatory effects of IGF-I on the gene expression of myogenin, a skeletal muscle-specific transcription factor essential for myogenic differentiation. We find that, concurrent with its early inhibitory and late stimulatory effects on myogenin mRNA, IGF-I has a biphasic but opposite effect on phosphorylation of Erk1/2: initially, IGF-I increases and subsequently decreases the phosphorylation of Erk1/2 in comparison to untreated cells. Cotreatment with an inhibitor of Erk1/2 activation prevents the early IGF-I-stimulation of Erk1/2 phosphorylation and partially reverses IGF-I-inhibition of myogenin mRNA. Conversely, preventing the late IGF-I-induced decrease in Erk1/2 phosphorylation blocks IGF-I-stimulation of myogenin mRNA. Our data indicate that the time-dependent, opposing effects of IGF-I on skeletal muscle cell differentiation are mediated, at least in part, by biphasic but opposite effects on activation of the Erk1/2 MAPK signaling pathway.