Downregulation of IRS-1 expression causes inhibition of corneal angiogenesis.

PURPOSE: The antiangiogenic effect of an antisense oligodeoxynucleotide (ODN) targeting insulin receptor substrate (IRS)-1 was evaluated on rat corneal neovascularization. METHODS: Eyes with neovessels were treated with subconjunctival injections of IRS-1 antisense oligonucleotide (ASODN), IRS-1 sense ODN (SODN), or PBS. At 8 and 24 hours after the first subconjunctival injection, the expression of IRS-1, VEGF, and IL-1beta mRNA was evaluated. IRS-1 protein levels were also measured at 8 hours by Western blot analysis (n = 4/group). On day 10, corneal neovascularization was quantified in flatmount corneas of rats treated daily from days 4 to 9. RESULTS: On day 10, new vessels covered 95.5% +/- 4% of the corneal area in PBS-treated eyes, 92% +/- 7% in SODN-treated eyes and 59% +/- 20% in ASODN-treated eyes (P < 0.001). In the ASODN-treated group, the expression and synthesis of IRS-1 were significantly downregulated when compared with the control groups. ASODN did not significantly affect the expression of VEGF but significantly decreased the expression of IL-1beta at 24 hours (P = 0.04). CONCLUSIONS: Subconjunctival injections of IRS-1 antisense ODN significantly inhibit rat corneal neovascularization. This effect may be mediated by a downregulation of IL-1beta. IRS-1 proteins may be interesting targets for the regulation of angiogenesis mediated by insulin, hypoxia, or inflammation.

Downregulation of cannabinoid receptor 1 from neuropeptide Y interneurons in the basal ganglia of patients with Huntington's disease and mouse models.

Cannabinoid receptor 1 (CB(1) receptor) controls several neuronal functions, including neurotransmitter release, synaptic plasticity, gene expression and neuronal viability. Downregulation of CB(1) expression in the basal ganglia of patients with Huntington's disease (HD) and animal models represents one of the earliest molecular events induced by mutant huntingtin (mHtt). This early disruption of neuronal CB(1) signaling is thought to contribute to HD symptoms and neurodegeneration. Here we determined whether CB(1) downregulation measured in patients with HD and mouse models was ubiquitous or restricted to specific striatal neuronal subpopulations. Using unbiased semi-quantitative immunohistochemistry, we confirmed previous studies showing that CB(1) expression is downregulated in medium spiny neurons of the indirect pathway, and found that CB(1) is also downregulated in neuropeptide Y (NPY)/neuronal nitric oxide synthase (nNOS)-expressing interneurons while remaining unchanged in parvalbumin- and calretinin-expressing interneurons. CB(1) downregulation in striatal NPY/nNOS-expressing interneurons occurs in R6/2 mice, Hdh(Q150/Q150) mice and the caudate nucleus of patients with HD. In R6/2 mice, CB(1) downregulation in NPY/nNOS-expressing interneurons correlates with diffuse expression of mHtt in the soma. This downregulation also occludes the ability of cannabinoid agonists to activate the pro-survival signaling molecule cAMP response element-binding protein in NPY/nNOS-expressing interneurons. Loss of CB(1) signaling in NPY/nNOS-expressing interneurons could contribute to the impairment of basal ganglia functions linked to HD.

Cutting edge: cyclic polypeptide and aminoglycoside antibiotics trigger IL-1β secretion by activating the NLRP3 inflammasome.

Clinical use of antibiotics is based on their capacity to inhibit bacterial growth via bacteriostatic or bacteriocidal effects. In this article, we show that the aminoglycoside antibiotic neomycin, the cyclic lipopeptide antibiotic polymyxin B, and the cyclic peptide antibiotics gramicidin and tyrothricin can induce IL-1β secretion in bone marrow dendritic cells and macrophages. LPS priming was required to trigger the transcription and translation of pro-IL-1β but was independent of TNFR or IL-1R signaling. All four antibiotics required the NLRP3 inflammasome, the adaptor ASC, and caspase-1 activation to secrete IL-1β, a process that depended on potassium efflux but was independent of P2X7 receptor. All four antibiotics induced neutrophil influx into the peritoneal cavity of mice, which required NLRP3 only in the case of polymyxin B. Together, certain antibiotics have the potential to directly activate innate immunity of the host.

Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells.

IL-2 immunotherapy is an attractive treatment option for certain metastatic cancers. However, administration of IL-2 to patients can lead, by ill-defined mechanisms, to toxic adverse effects including severe pulmonary edema. Here, we show that IL-2-induced pulmonary edema is caused by direct interaction of IL-2 with functional IL-2 receptors (IL-2R) on lung endothelial cells in vivo. Treatment of mice with high-dose IL-2 led to efficient expansion of effector immune cells expressing high levels of IL-2Rbetagamma, including CD8(+) T cells and natural killer cells, which resulted in a considerable antitumor response against s.c. and pulmonary B16 melanoma nodules. However, high-dose IL-2 treatment also affected immune cell lineage marker-negative CD31(+) pulmonary endothelial cells via binding to functional alphabetagamma IL-2Rs, expressed at low to intermediate levels on these cells, thus causing pulmonary edema. Notably, IL-2-mediated pulmonary edema was abrogated by a blocking antibody to IL-2Ralpha (CD25), genetic disruption of CD25, or the use of IL-2Rbetagamma-directed IL-2/anti-IL-2 antibody complexes, thereby interfering with IL-2 binding to IL-2Ralphabetagamma(+) pulmonary endothelial cells. Moreover, IL-2/anti-IL-2 antibody complexes led to vigorous activation of IL-2Rbetagamma(+) effector immune cells, which generated a dramatic antitumor response. Thus, IL-2/anti-IL-2 antibody complexes might improve current strategies of IL-2-based tumor immunotherapy.

Constitutively active mutants of the beta1-adrenergic receptor.

We provide the first evidence that point mutations can constitutively activate the beta(1)-adrenergic receptor (AR). Leucine 322 of the beta(1)-AR in the C-terminal portion of its third intracellular loop was replaced with seven amino acids (I, T, E, F, C, A and K) differing in their physico-chemical properties. The beta(1)-AR mutants expressed in HEK-293 cells displayed various levels of constitutive activity which could be partially inhibited by some beta-blockers. The results of this study might have interesting implications for future studies aiming at elucidating the activation process of the beta(1)-AR as well as the mechanism of action of beta-blockers.

Type I IFN-mediated regulation of IL-1 production in inflammatory disorders.

Although contributing to inflammatory responses and to the development of certain autoimmune pathologies, type I interferons (IFNs) are used for the treatment of viral, malignant, and even inflammatory diseases. Interleukin-1 (IL-1) is a strongly pyrogenic cytokine and its importance in the development of several inflammatory diseases is clearly established. While the therapeutic use of IL-1 blocking agents is particularly successful in the treatment of innate-driven inflammatory disorders, IFN treatment has mostly been appreciated in the management of multiple sclerosis. Interestingly, type I IFNs exert multifaceted immunomodulatory effects, including the reduction of IL-1 production, an outcome that could contribute to its efficacy in the treatment of inflammatory diseases. In this review, we summarize the current knowledge on IL-1 and IFN effects in different inflammatory disorders, the influence of IFNs on IL-1 production, and discuss possible therapeutic avenues based on these observations.

Interferon-γ Induces Expression of MHC Class II on Intestinal Epithelial Cells and Protects Mice from Colitis

Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD) and involve CD4(+) T cells, which are activated by major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells (APCs). However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC) affects CD4(+) T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL)-10 receptor-blocking antibodies (anti-IL10R mAb). To assess the role of interferon (IFN)-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+) T-helper type (Th)1 cells - but not group 3 innate lymphoid cells (ILCs) or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+) T cells and forkhead box P3 (FoxP3)(+) regulatory T (Treg) cells. IFN-γ produced mainly by CD4(+) T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.

P2Y1 receptor-evoked glutamate exocytosis from astrocytes: control by tumor necrosis factor-alpha and prostaglandins.

ATP, released by both neurons and glia, is an important mediator of brain intercellular communication. We find that selective activation of purinergic P2Y1 receptors (P2Y1R) in cultured astrocytes triggers glutamate release. By total internal fluorescence reflection imaging of fluorescence-labeled glutamatergic vesicles, we document that such release occurs by regulated exocytosis. The stimulus-secretion coupling mechanism involves Ca2+ release from internal stores and is controlled by additional transductive events mediated by tumor necrosis factor-alpha (TNFalpha) and prostaglandins (PG). P2Y1R activation induces release of both TNFalpha and PGE2 and blocking either one significantly reduces glutamate release. Accordingly, astrocytes from TNFalpha-deficient (TNF(-/-)) or TNF type 1 receptor-deficient (TNFR1(-/-)) mice display altered P2Y1R-dependent Ca2+ signaling and deficient glutamate release. In mixed hippocampal cultures, the P2Y1R-evoked process occurs in astrocytes but not in neurons or microglia. P2Y1R stimulation induces Ca2+ -dependent glutamate release also from acute hippocampal slices. The process in situ displays characteristics resembling those in cultured astrocytes and is distinctly different from synaptic glutamate release evoked by high K+ stimulation as follows: (a) it is sensitive to cyclooxygenase inhibitors; (b) it is deficient in preparations from TNF(-/-) and TNFR1(-/-) mice; and (c) it is inhibited by the exocytosis blocker bafilomycin A1 with a different time course. No glutamate release is evoked by P2Y1R-dependent stimulation of hippocampal synaptosomes. Taken together, our data identify the coupling of purinergic P2Y1R to glutamate exocytosis and its peculiar TNFalpha- and PG-dependent control, and we strongly suggest that this cascade operates selectively in astrocytes. The identified pathway may play physiological roles in glial-glial and glial-neuronal communication.

Physiology of GLP-1--lessons from glucoincretin receptor knockout mice.

GLP-1 has both peripheral and central actions, as this hormone is secreted by gut endocrine cells and brainstem neurons projecting into the hypothalamus and other brain regions. GLP-1 has multiple regulatory functions participating in the control of glucose homeostasis, beta-cell proliferation and differentiation, food intake, heart rate and even learning. GLP-1 action depends on binding to a specific G-coupled receptor linked to activation of the adenylyl cyclase pathway. Analysis of mice with inactivation of the GLP-1 receptor gene has provided evidence that absence of GLP-1 action in the mouse, despite this hormone potent physiological effects when administered in vivo, only leads to mild abnormalities in glucose homeostasis without any change in body weight. However, a critical role for this hormone and its receptor was demonstrated in the function of the hepatoportal vein glucose sensor, in contrast to that of the pancreatic beta-cells, although absence of both GLP-1 and GIP receptors leads to a more severe phenotype characterized by a beta-cell-autonomous defect in glucose-stimulated insulin secretion. Together, the studies of these glucoincretin receptor knockout mice provide evidence that these hormones are part of complex regulatory systems where multiple redundant signals are involved.

NY-ESO-1-specific circulating CD4+ T cells in ovarian cancer patients are prevalently T(H)1 type cells undetectable in the CD25+ FOXP3+ Treg compartment.

Spontaneous CD4(+) T-cell responses to the tumor-specific antigen NY-ESO-1 (ESO) are frequently found in patients with epithelial ovarian cancer (EOC). If these responses are of effector or/and Treg type, however, has remained unclear. Here, we have used functional approaches together with recently developed MHC class II/ESO tetramers to assess the frequency, phenotype and function of ESO-specific cells in circulating lymphocytes from EOC patients. We found that circulating ESO-specific CD4(+) T cells in EOC patients with spontaneous immune responses to the antigen are prevalently T(H)1 type cells secreting IFN-γ but no IL-17 or IL-10 and are not suppressive. We detected tetramer(+) cells ex vivo, at an average frequency of 1:25,000 memory cells, that is, significantly lower than in patients immunized with an ESO vaccine. ESO tetramer(+) cells were mostly effector memory cells at advanced stages of differentiation and were not detected in circulating CD25(+)FOXP3(+)Treg. Thus, spontaneous CD4(+) T-cell responses to ESO in cancer patients are prevalently of T(H)1 type and not Treg. Their relatively low frequency and advanced differentiation stage, however, may limit their efficacy, that may be boosted by immunogenic ESO vaccines.

Targeting IL-1β and IL-17A driven inflammation during influenza-induced exacerbations of chronic lung inflammation.

For patients with chronic lung diseases, such as chronic obstructive pulmonary disease (COPD), exacerbations are life-threatening events causing acute respiratory distress that can even lead to hospitalization and death. Although a great deal of effort has been put into research of exacerbations and potential treatment options, the exact underlying mechanisms are yet to be deciphered and no therapy that effectively targets the excessive inflammation is available. In this study, we report that interleukin-1β (IL-1β) and interleukin-17A (IL-17A) are key mediators of neutrophilic inflammation in influenza-induced exacerbations of chronic lung inflammation. Using a mouse model of disease, our data shows a role for IL-1β in mediating lung dysfunction, and in driving neutrophilic inflammation during the whole phase of viral infection. We further report a role for IL-17A as a mediator of IL-1β induced neutrophilia at early time points during influenza-induced exacerbations. Blocking of IL-17A or IL-1 resulted in a significant abrogation of neutrophil recruitment to the airways in the initial phase of infection or at the peak of viral replication, respectively. Therefore, IL-17A and IL-1β are potential targets for therapeutic treatment of viral exacerbations of chronic lung inflammation.

Angiotensin receptor blockers are not associated with reduced inflammatory markers in the general population.

OBJECTIVE: Angiotensin receptor blockers (ARBs) have been suggested to reduce inflammation in randomized controlled trials. We assessed the association between ARBs and inflammatory markers in a general population setting. METHODS: This is a population-based prospective study conducted in Lausanne, Switzerland. Baseline data from 933 participants on antihypertensive drugs (424 on ARBs) was collected in 2003-2006. Follow-up data from 1120 participants (572 on ARBs) was collected in 2009-2012. C-reactive protein (CRP), interleukins 1β and 6 and tumor necrosis factor alpha (TNF-α) were assessed and categorized in quartiles. RESULTS: At baseline, no differences were found between participants taking or not taking ARBs for all inflammatory markers studied, and this association persisted after multivariate adjustment: odds ratios (ORs) and (95% confidence interval) for being in the highest quartile of interleukin-1β, interleukin-6, TNF-α and CRP for participants on ARB compared to participants not on ARB were 1.23 (0.89-1.70), 1.26 (0.93-1.70), 1.14 (0.85-1.53) and 1.27 (0.96-1.69) respectively (P > 0.05). These findings were further replicated in the follow-up study: OR and (95% CI) of 1.10 (0.78-1.55), 0.87 (0.64-1.19), 0.83 (0.61-1.14) and 0.91 (0.68-1.22) for interleukin-1β, interleukin-6, TNF-α and CRP respectively (P > 0.05). Finally, no effect of ARBs was found when comparing participants who received ARBs throughout the 5.4-year follow-up with participants on other antihypertensive drugs: OR and (95% CI) of 0.93 (0.61-1.42), 0.80 (0.54-1.17), 0.86 (0.59-1.25) and 0.95 (0.67-1.35) for interleukin-1β, interleukin-6, TNF-α and CRP respectively (P > 0.05). CONCLUSION: ARBs are not associated with reduced levels of inflammatory markers in the general population.

Interleukin-15 as a potential regulator of the innate immune response

Interleukin-15 (IL-15) is a newly-discovered cytokine that is produced by activated monocytes early in the course of the innate immune response. IL-15 is able to bind to components of the interleukin-2 receptor (IL-2R) despite the fact that it has no sequence homology with IL-2. IL-15 stimulates human natural killer cell proliferation, cytotoxicity, and cytokine production and can substitute for IL-2 under most conditions. In vitro studies indicate that monocyte-derived IL-15 may be an important determinant of IFN-gamma production by NK cells. In addition, IL-15 is able to promote the survival of natural killer cells under serum-free conditions. The IL-15 receptor is a heterotrimeric complex which is composed of the IL-2Rß and g chains in combination with a unique alpha chain (IL-15a). The IL-15Ra chain has strong sequence homology to the IL-2Ra chain and confers high affinity binding to the IL-15R. In contrast to IL-2, transcript for IL-15 and IL-15a is expressed in a number of tissues and indicates that IL-15 may be an important ligand for cells that express components of the IL-2R

Ox-LDL increases OX40L in endothelial cells through a LOX-1-dependent mechanism

Oxidative low-density lipoprotein (Ox-LDL) is a key risk factor for the development of atherosclerosis, and it can stimulate the expression of a variety of inflammatory signals. As a new and highly sensitive inflammation index, OX40L may be a key to understanding the mechanisms that regulate interactions between cells within the vessel wall and inflammatory mediators during the development of atherosclerosis. To investigate whether Ox-LDL regulates OX40L expression through an oxidized LDL-1 receptor (LOX-1)-mediated mechanism, we investigated the effect of different concentrations of Ox-LDL (50, 100, 150 µg/mL) on endothelial cell proliferation and apoptosis. Stimulation with Ox-LDL increased OX40L protein 1.44-fold and mRNA 4.0-fold in endothelial cells, and these effects were inhibited by blocking LOX-1. These results indicate that LOX-1 plays an important role in the chronic inflammatory process in blood vessel walls. Inhibiting LOX-1 may reduce blood vessel inflammation and provide a therapeutic option to limit atherosclerosis progression.

Évaluation pré-clinique et clinique de deux nouvelles stratégies dans le traitement des maladies cardiovasculaires : activité anti-thrombotique de l'inhibition du récepteur P2Y1 et activité anti-inflammatoire de l'inhibition du récepteur PAR-1.

Alors que la plaquette a un rôle prépondérant dans le maintient de l'hémostase, son implication dans la formation de la thrombose est également incontestée. Il existe à présent des traitements antiplaquettaires mais ceux-ci présentent quelques failles. Le but de ma maîtrise a donc été de caractériser de nouvelles voies d'inhibition de l'activité plaquettaire. Les deux études qui ont été réalisées concernent l'inhibition de récepteurs couplés aux protéines G, soit le récepteur à l'ADP P2Y1 et le récepteur à la thrombine PAR-1. Dans un premier temps, l'étude du récepteur P2Y1 suggère que l'inhibition de celui-ci seul ou en combinaison avec l'inhibition du récepteur P2Y12 présente un potentiel thérapeutique chez des patients coronariens stables. Dans un deuxième temps, l'étude d'un nouvel antiplaquettaire qui est présentement en phase III de son développement clinique, soit le SCH 530348, a été effectuée. Étant un antagoniste du récepteur PAR-1, le SCH 530348 a des effets qui se répercutent à la fois chez la plaquette et les leucocytes qui possèdent eux aussi ce récepteur. Cette deuxième étude suggère que complémentairement à son effet chez la plaquette, cet inhibiteur diminue les marqueurs de l'inflammation systémique.