Stephane Esnault

The peptidyl-prolyl isomerase Pin1 regulates the stability of granulocyte-macrophage colony-stimulating factor mRNA in activated eosinophils

The infiltration, accumulation and degranulation of eosinophils in the lung represents a hallmark of active asthma. In vivo or in vitro eosinophil activation triggers the secretion of the antiapoptotic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). We now identify Pin1, a cis-trans isomerase, as an essential component of the ribonucleoprotein complex responsible for GM-CSF mRNA stabilization, cytokine secretion and the survival of activated eosinophils. Pin1 regulated the association of the AU-rich element–binding proteins AUF1 and hnRNP C with GM-CSF mRNA, accelerating or slowing decay, respectively. These data indicate Pin1 is a key mediator of GM-CSF production.

Y Box-Binding Factor Promotes Eosinophil Survival by Stabilizing Granulocyte-Macrophage Colony-Stimulating Factor mRNA

Short-lived peripheral blood eosinophils are recruited to the lungs of asthmatics after allergen challenge, where they become long-lived effector cells central to disease pathophysiology. GM-CSF is an important cytokine which promotes eosinophil differentiation, function, and survival after transit into the lung. In human eosinophils, GM-CSF production is controlled by regulated mRNA stability mediated by the 3′ untranslated region, AU-rich elements (ARE). We identified human Y box-binding factor 1 (YB-1) as a GM-CSF mRNA ARE-specific binding protein that is capable of enhancing GM-CSF-dependent survival of eosinophils. Using a transfection system that mimics GM-CSF metabolism in eosinophils, we have shown that transduced YB-1 stabilized GM-CSF mRNA in an ARE-dependent mechanism, causing increased GM-CSF production and enhanced in vitro survival. RNA EMSAs indicate that YB-1 interacts with the GM-CSF mRNA through its 3′ untranslated region ARE. In addition, endogenous GM-CSF mRNA coimmunoprecipitates with endogenous YB-1 protein in activated eosinophils but not resting cells. Thus, we propose a model whereby activation of eosinophils leads to YB-1 binding to and stabilization of GM-CSF mRNA, ultimately resulting in GM-CSF release and prolonged eosinophil survival.

Pin1 regulates TGF-β1 production by activated human and murine eosinophils and contributes to allergic lung fibrosis

Eosinophilic inflammation is a cornerstone of chronic asthma that often culminates in subepithelial fibrosis with variable airway obstruction. Pulmonary eosinophils (Eos) are a predominant source of TGF-beta1, which drives fibroblast proliferation and extracellular matrix deposition. We investigated the regulation of TGF-beta1 and show here that the peptidyl-prolyl isomerase (PPIase) Pin1 promoted the stability of TGF-beta1 mRNA in human Eos. In addition, Pin1 regulated cytokine production by both in vitro and in vivo activated human Eos. We found that Pin1 interacted with both PKC-alpha and protein phosphatase 2A, which together control Pin1 isomerase activity. Pharmacologic blockade of Pin1 in a rat asthma model selectively reduced eosinophilic pulmonary inflammation, TGF-beta1 and collagen expression, and airway remodeling. Furthermore, chronically challenged Pin1(-/-) mice showed reduced peribronchiolar collagen deposition compared with wild-type controls. These data suggest that pharmacologic suppression of Pin1 may be a novel therapeutic option to prevent airway fibrosis in individuals with chronic asthma.

The peptidyl-prolyl isomerase Pin1 facilitates cytokine-induced survival of eosinophils by suppressing Bax activation

The mechanisms by which cytokine signals prevent the activation and mitochondrial targeting of the proapoptotic protein Bax are unclear. Here we show, using primary human eosinophils, that in the absence of the prosurvival cytokines granulocyte-macrophage colony-stimulating factor and interleukin 5, Bax spontaneously underwent activation and initiated mitochondrial disruption. Inhibition of Bax resulted in less eosinophil apoptosis, even in the absence of cytokines. Granulocyte-macrophage colony-stimulating factor induced activation of the kinase Erk1/2, which phosphorylated Thr167 of Bax; this facilitated new interaction of Bax with the prolyl isomerase Pin1. Blockade of Pin1 led to cleavage and mitochondrial translocation of Bax and caspase activation, regardless of the presence of cytokines. Our findings indicate that Pin1 is a key mediator of prosurvival signaling and is a regulator of Bax function.

Hyaluronic Acid or TNF-α Plus Fibronectin Triggers Granulocyte Macrophage-Colony-Stimulating Factor mRNA Stabilization in Eosinophils Yet Engages Differential Intracellular Pathways and mRNA Binding Proteins

Eosinophils (Eos) accumulate in airways and lung parenchyma of active asthmatics. GM-CSF is a potent inhibitor of Eos apoptosis both in vitro and in vivo and is produced by activated fibroblasts, mast cells, T lymphocytes as well as Eos. Cytokine release by Eos is preceded by GM-CSF mRNA stabilization induced by TNF-α plus fibronectin. Hyaluronic acid (HA) is a major extracellular matrix proteoglycan, which also accumulates in the lung during asthma exacerbations. In this study we have analyzed the effects of HA on Eos survival and GM-CSF expression. We demonstrate that like TNF-α plus fibronectin, HA stabilizes GM-CSF mRNA, increases GM-CSF secretion, and prolongs in vitro Eos survival. GM-CSF mRNA stabilization accounts for most of the observed GM-CSF mRNA accumulation and protein production. Unlike TNF-α plus fibronectin, GM-CSF mRNA stabilization induction by HA requires continuous extracellular signal-regulated kinase phosphorylation. Finally, to identify potential protein regulators responsible for GM-CSF mRNA stabilization, immunoprecipitation-RT-PCR studies revealed increased GM-CSF mRNA associated with YB-1, HuR, and heterogeneous nuclear ribonucleoprotein (hnRNP) C after TNF-α plus fibronectin but only hnRNP C after HA. Thus, our data suggest that both TNF-α plus fibronectin and HA, which are relevant physiological effectors in asthma, contributes to long-term Eos survival in vivo by enhancing GM-CSF production through two different posttranscriptional regulatory pathways involving extracellular signal-regulated kinase phosphorylation and RNA binding proteins YB-1, HuR, and hnRNP C.

Ets-1 Regulates TNF-α-Induced Matrix Metalloproteinase-9 and Tenascin Expression in Primary Bronchial Fibroblasts

Increased subepithelial deposition of extracellular matrix proteins is a key feature in bronchial asthma. Matrix metalloproteinase-9 (MMP-9) is a proteolytic enzyme that degrades the extracellular matrix. Tenascin is an extracellular matrix glycoprotein that is abundant in thickened asthmatic subbasement membrane. The expression of MMP-9 and tenascin reflects disease activity in asthma and airway remodeling. The molecular mechanisms regulating the expression of these proteins remain unknown. Both MMP-9 and tenascin promoters contain an Ets binding site, suggesting control by Ets-1. Thus, we hypothesized that Ets-1 expression is increased in asthma and that it contributed to enhanced MMP-9 and tenascin expression. To test this hypothesis, we determined the expression of Ets-1 in bronchial biopsies obtained from asthmatic subjects and determined the expression of Ets-1, MMP-9, and tenascin by bronchial fibroblasts activated ex vivo. We observed that nuclear extracts from TNF-α-activated fibroblasts showed increased Ets-binding activity. In addition, TNF-α-activated fibroblasts had increased expression of Ets-1 mRNA and protein, which preceded an increase in MMP-9 and tenascin mRNA. Furthermore, treatment of fibroblasts with Ets-1 antisense oligonucleotides down-regulated TNF-α-induced Ets-1, MMP-9, and, to a lesser extent, tenascin protein expression or activity. Taken together, these data demonstrate that TNF-α increases MMP-9 and tenascin expression in bronchial fibroblasts via the transcription factor Ets-1, and suggest a role for Ets-1 in airway remodeling in asthma.

The Peptidyl-Prolyl Isomerase Pin1 Regulates Granulocyte-Macrophage Colony-Stimulating Factor mRNA Stability in T Lymphocytes

Cytokine production is associated with both the normal and pathologic inflammatory response to injury. Previous studies have shown that the immunosuppressants cyclosporin A or FK506, which interact with the peptidyl-propyl isomerases cyclophilin A and FK506-binding protein (FKBP12), respectively, block cytokine expression. A third member of the peptidyl-propyl isomerase family, Pin1 is expressed by immune and other cells. Pin1 has been implicated in cell cycle progression, is overexpressed in human tumors, and may rescue neurons from τ-associated degeneration. However, the role of Pin1 in the immune system remains largely unknown. In this study, we analyze the role of Pin1 in GM-CSF expression by human PBMC and CD4+ lymphocytes. We show that Pin1 isomerase activity is necessary for activation-dependent, GM-CSF mRNA stabilization, accumulation, and protein secretion, but not non-AU-rich elements containing cytokine mRNAs, including TGF-β and IL-4. Mechanistically, Pin1 mediated the association of the AU-rich element-binding protein, AUF1, with GM-CSF mRNA, which determined the rate of decay by the exosome.

Granulocyte Macrophage-Colony-Stimulating Factor mRNA Is Stabilized in Airway Eosinophils and Peripheral Blood Eosinophils Activated by TNF-α Plus Fibronectin

Airway eosinophils show prolonged in vitro survival compared with peripheral blood eosinophils (PBEos). Recent studies have shown that autocrine production and release of GM-CSF is responsible for enhanced survival, but the mechanisms controlling cytokine production remain obscure. We compared GM-CSF mRNA decay in eosinophils from bronchoalveolar lavage (BALEos) after allergen challenge or from PBEos. BALEos showed prolonged survival in vitro (60% at 4 days) and expressed GM-CSF mRNA. The enhanced survival of BALEos was 75% inhibited at 6 days by neutralizing anti-GM-CSF Ab. Based on transfection studies, GM-CSF mRNA was 2.5 times more stable in BALEos than in control PBEos. Treatment of PBEos with fibronectin and TNF-α increased their in vitro survival, GM-CSF mRNA expression, and GM-CSF mRNA stability to a comparable level as seen in BALEos. These data suggest that TNF-α plus fibronectin may increase eosinophil survival in vivo by controlling GM-CSF production at a posttranscriptional level.

Human eosinophils release IL‐1ß and increase expression of IL‐17A in activated CD4+ T lymphocytes

Differentiation and activation of CD4+ T cells is controlled by various cytokines produced by innate immune cells. We have shown that eosinophils (EOS) have the potential to influence Th1 and Th2 cytokine generation by CD4+ cells, but their influence on IL‐17A (IL‐17) has not been established.

Toll-like receptor 2 expression on human conjunctival epithelial cells: a pathway for Staphylococcus aureus involvement in chronic ocular proinflammatory responses

BACKGROUND Staphylococcus aureus colonization is common in atopic keratoconjunctivitis, potentially activating epithelial cells via toll-like receptor 2 (TLR-2) and the receptor for platelet-activating factor (PAFR). OBJECTIVES To examine human conjunctival epithelial cells for the expression of TLR-2 in vitro and in vivo and to evaluate the role of TLR-2 in S aureus-mediated activation of these cells. METHODS Conjunctival epithelial cells isolated from cadaveric tissues were stimulated with interferon gamma (IFN-gamma) or a commercial S aureus cell wall extract (Staphylococcus aureus-CWE) (with or without anti-TLR-2 blocking antibody or PAFR antagonist) and were analyzed for tumor necrosis factor alpha (TNF-alpha) and interleukin 8 (IL-8) release; surface expression of TLR-2, intercellular adhesion molecule-1, HLA, and CD14; and TLR-2 messenger RNA expression. Ocular surface cells collected via impression cytology were examined for TLR-2 expression via flow cytometry. RESULTS Expression of TLR-2 was up-regulated on conjunctival epithelial cells by IFN-gamma and Staphylococcus aureus-CWE. Expression of TLR-2 messenger RNA was increased by IFN-gamma. Staphylococcus aureus-CWE up-regulated intercellular adhesion molecule 1, HLA, and CD14 expression and increased TNF-alpha and IL-8 release in a dose-dependent manner. Anti-TLR-2 significantly inhibited TNF-alpha release, whereas PAFR antagonist significantly inhibited IL-8 release. Toll-like receptor 2 was expressed on conjunctival epithelial cells from 4 of 5 patients with atopic keratoconjunctivitis, 3 of 5 with seasonal allergies, and 0 of 3 without allergies. CONCLUSIONS Conjunctival epithelial cells express TLR-2 and may play an active role in the chronic ocular inflammatory response to S aureus through pathways that involve TLR-2 and PAFR.