Involvement of oxidative stress and caspase 2-mediated intrinsic pathway signaling in age-related increase in muscle cell apoptosis in mice

Apoptosis has been implicated as a mechanism of loss of muscle cells in normal aging and plays an important role in age-related sarcopenia. To test the hypothesis that caspase 2 and c-Jun NH₂-terminal kinase (JNK)-mediated intrinsic pathway signaling contribute to skeletal muscle cell apoptosis in aging, we compared activation of caspase 2 and JNK and the in vivo expression of 4-hydroxynonenal protein adducts (4-HNE), inducible nitric oxide synthase (iNOS), glucose-6-phosphate dehydrogenase (G6PDH), B-cell lymphoma-2 (BCL-2), BAX, and phospho-BCL-2 in gastrocnemius muscles of young (5 months old) and old (25 months old) mice. A distinct age-related increase in 4-HNE and iNOS expression was readily detected in mice. Increased oxidative stress and iNOS induction were further accompanied by a decrease in G6PDH expression, activation of caspase 2 and JNK, and inactivation of BCL-2 through phosphorylation at serine 70, and caspase 9 activation. Regression analysis further revealed that increased muscle cell death in aging was significantly correlated with changes in the levels of these molecules. Taken together, our data indicate that caspase 2 and JNK-mediated intrinsic pathway signaling is one of the mechanisms involved in age-related increase in muscle cell apoptosis.     

Targeted deletion of the mitogen-activated protein kinase kinase 4 gene in the nervous system causes severe brain developmental defects and premature death

The c-Jun NH₂-terminal protein kinase (JNK) is a mitogen-activated protein kinase (MAPK) involved in the regulation of various physiological processes. Its activity is increased upon phosphorylation by the MAPK kinases MKK4 and MKK7. The early embryonic death of mice lacking an mkk4 or mkk7 gene has provided genetic evidence that MKK4 and MKK7 have nonredundant functions in vivo. To elucidate the physiological role of MKK4, we generated a novel mouse model in which the mkk4 gene could be specifically deleted in the brain. At birth, the mutant mice were indistinguishable from their control littermates, but they stopped growing a few days later and died prematurely, displaying severe neurological defects. Decreased JNK activity in the absence of MKK4 correlated with impaired phosphorylation of a subset of physiologically relevant JNK substrates and with altered gene expression. These defects resulted in the misalignment of the Purkinje cells in the cerebellum and delayed radial migration in the cerebral cortex. Together, our data demonstrate for the first time that MKK4 is an essential activator of JNK required for the normal development of the brain.     

Hepatocyte-specific deletion of cellular repressor of E1A-stimulated genes 1 exacerbates alcohol-induced liver injury by activating stress kinases

Alcohol-associated liver disease (ALD) encompasses a wide range of pathologies from simple steatosis to cirrhosis and hepatocellular carcinoma and is a global health problem. Currently, there are no effective pharmacological treatments for ALD. We have previously demonstrated that aging exacerbates the pathogenesis of ALD, but the underlying mechanisms are still poorly understood. Cellular repressor of E1A-stimulated genes 1 protein (CREG1) is a recently identified small glycoprotein that has been implicated in aging process by promoting cellular senescence and activating stress kinases. Thus, the current study aimed to explore the role of aging associated CREG1 in ALD pathogenesis and CREG1 as a potential therapeutic target. Hepatic and serum CREG1 protein levels were elevated in ALD patients. Elevation of hepatic CREG1 protein and mRNA was also observed in a mouse model of Gao-binge alcohol feeding. Genetic deletion of the Creg1 gene in hepatocytes (Creg1^∆hep) markedly exacerbated ethanol-induced liver injury, apoptosis, steatosis and inflammation. Compared to wild-type mice, Creg1^∆hep mice had increased phosphorylation of hepatic stress kinases such as apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK) and p38 but not TGF-β-activated kinase 1 (TAK1) or extracellular signal-regulated kinase (ERK) after alcohol feeding. In vitro, ethanol treatment elevated the phosphorylation of ASK1, JNK, and p38 in mouse hepatocyte AML-12 cells. This elevation was further enhanced by CREG1 knockdown but alleviated by CREG1 overexpression. Last, treatment with an ASK1 inhibitor abolished ethanol-induced liver injury and upregulated hepatic lipogenesis, proinflammatory genes and stress kinases in Creg1^∆hep mice. Taken together, our data suggest that CREG1 protects against alcoholic liver injury and inflammation by inhibiting the ASK1-JNK/p38 stress kinase pathway and that CREG1 is a potential therapeutic target for ALD.     

Evolutionary Conserved Role of c-Jun-N-Terminal Kinase in CO2-Induced Epithelial Dysfunction

Elevated CO₂ levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO₂ signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO₂ levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO₂ levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO₂-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO₂ signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO₂ signaling in mammals, diptera and nematodes.     

Inhibition of hepatic mitochondrial aldehyde dehydrogenase by carbon tetrachloride through JNK-mediated phosphorylation

The aim of this study was to investigate the mechanism of inhibition of mitochondrial aldehyde dehydrogenase (ALDH2) by carbon tetrachloride (CCl₄). CCl₄ administration caused marked hepatocyte ballooning and necrosis in the pericentral region. CCl₄ also inhibited hepatic ALDH2 activity in a time-dependent manner without altering the protein level, suggesting ALDH2 inhibition through covalent modifications such as phosphorylation by JNK. To demonstrate phosphorylation, the isoelectric point (pI) of ALDH2 in CCl₄-exposed rats was compared to that of untreated controls. Immunoblot analysis revealed that immunoreactive ALDH2 bands in CCl₄-exposed rats were shifted to acidic pI ranges on two-dimensional electrophoresis (2-DE) gels. Incubation with alkaline phosphatase significantly restored the suppressed ALDH2 activity with a concurrent alkaline pI shift of the ALDH2 spots. Both JNK and activated JNK were translocated to mitochondria after CCl₄ exposure. In addition, incubation with catalytically active JNK led to significant inhibition of ALDH2 activity, with an acidic pI shift on 2-DE gels. Furthermore, immunoprecipitation followed by immunoblot analysis with anti-phospho-Ser–Pro antibody revealed phosphorylation of a Ser residue(s) of ALDH2. These results collectively indicate a novel underlying mechanism by which CCl₄ exposure activates JNK, which translocates to mitochondria and phosphorylates ALDH2, contributing to inhibition of ALDH2 activity accompanied by decreased cellular defense capacity and increased lipid peroxidation.     

High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation

The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis, and cell adhesion and cell migration by phosphorylating paxillin and β-catenin. JNK phosphorylation downstream of AMP-activated protein kinase (AMPK) activation is required for high CO₂ (hypercapnia)-induced Na,K-ATPase endocytosis in alveolar epithelial cells. Here, we provide evidence that during hypercapnia, JNK promotes the phosphorylation of LMO7b, a scaffolding protein, in vitro and in intact cells. LMO7b phosphorylation was blocked by exposing the cells to the JNK inhibitor SP600125 and by infecting cells with dominant-negative JNK or AMPK adenovirus. The knockdown of the endogenous LMO7b or overexpression of mutated LMO7b with alanine substitutions of five potential JNK phosphorylation sites (LMO7b-5SA) or only Ser-1295 rescued both LMO7b phosphorylation and the hypercapnia-induced Na,K-ATPase endocytosis. Moreover, high CO₂ promoted the colocalization and interaction of LMO7b and the Na,K-ATPase α₁ subunit at the plasma membrane, which were prevented by SP600125 or by transfecting cells with LMO7b-5SA. Collectively, our data suggest that hypercapnia leads to JNK-induced LMO7b phosphorylation at Ser-1295, which facilitates the interaction of LMO7b with Na,K-ATPase at the plasma membrane promoting the endocytosis of Na,K-ATPase in alveolar epithelial cells.     

Caudatin induces cell cycle arrest and caspase-dependent apoptosis in HepG2 cell

In the present study, we investigate the anti-cancer activity and mechanism of caudatin, the C-21 steroidal glycosides, on human hepatoma cell line HepG2. The MTT assay and flow cytometry were used to evaluate HepG2 cell proliferation and cell cycle. Annexin-V/PI and DAPI staining were used to investigate cell apoptosis. Western blotting analysis was used to evaluate the expression levels of proteins. It is found that caudatin inhibits HepG2 cell growth and induces of G₀/G₁ phase arrest in a dose dependent manner, which is associated with a decreased in the expression of cyclinD₁ and increased the levels of p21 and p53. HepG2 cells dealing with caudatin showed typical characteristics of apoptosis. Western blotting analysis indicated that the levels of Bcl-2 were down-regulated after caudatin treatment, whereas the expression of Bax was up-regulated. Furthermore, caudatin-induced apoptosis was accompanied by activation of caspase-3, -9, and poly(ADP-Ribose) Polymerase (PARP). Treatment with caudatin also induced phosphorylation of extracellular-signal regulating kinase (ERK) and c-Jun N-terminal kinase (JNK). These results demonstrate that caudatin inhibits cell proliferation via DNA synthesis reduction and induces caspase-dependent apoptosis in HepG2 cell. Activation of ERK and JNK may be involved in caudatin-induced hepatoma cell apoptosis.     

Pyruvate dehydrogenase kinase activity of pig heart pyruvate dehydrogenase (E1 component of pyruvate dehydrogenase complex).

The pyruvate dehydrogenase (E1) and acetyltransferase (E2) components of pig heart and ox kidney pyruvate dehydrogenase (PDH) complex were separated and purified. The E1 component was phosphorylated (alpha-chain) and inactivated by MgATP. Phosphorylation was mainly confined to site 1. Addition of E2 accelerated phosphorylation of all three sites in E1 alpha and inactivation of E1. On the basis of histone H1 phosphorylation, E2 is presumed to contain PDH kinase, which was removed (greater than 98%) by treatment with p-hydroxymercuriphenylsulphonate. Stimulation of ATP-dependent inactivation of E1 by E2 was independent of histone H1 kinase activity of E2. The effect of E2 is attributed to conformational change(s) induced in E1 and/or E1-associated PDH kinase. PDH kinase activity associated with E1 could not be separated from it be gel filtration or DEAE-cellulose chromatography. Subunits of PDH kinase were not detected on sodium dodecyl sulphate/polyacrylamide gels of E1 or E2, presumably because of low concentration. The activity of pig heart PDH complex was increased by E2, but not by E1, indicating that E2 is rate-limiting in the holocomplex reaction. ATP-dependent inactivation of PDH complex was accelerated by E1 or by phosphorylated E1 plus associated PDH kinase, but not by E2 plus presumed PDH kinase. It is suggested that a substantial proportion of PDH kinase may accompany E1 when PDH complex is dissociated into its component enzymes. The possibility that E1 may possess intrinsic PDH kinase activity is considered unlikely, but may not have been fully excluded.     

AMP‐activated protein kinase deficiency exacerbates aging‐induced myocardial contractile dysfunction

Aging is associated with myocardial dysfunction although the underlying mechanism is unclear. AMPK, a key cellular fuel sensor for energy metabolism, is compromised with aging. This study examined the role of AMPK deficiency in aging‐associated myocardial dysfunction. Young or old wild‐type (WT) and transgenic mice with overexpression of a mutant AMPK α₂ subunit (kinase dead, KD) were used. AMPK α isoform activity, myocardial function and morphology were examined. DCF and JC‐1 fluorescence probes were employed to quantify reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm), respectively. KD mice displayed significantly reduced α₂ but not α₁ AMPK isoform activity at both ages with a greater effect at old age. Aging itself decreased α₁ isoform activity. Cardiomyocyte contractile function, intracellular Ca²⁺ handling, and SERCA2a levels were compromised with aging, the effects of which were exacerbated by AMPK deficiency. H&E staining revealed cardiomyocyte hypertrophy with aging, which was more pronounced in KD mice. TEM micrographs displayed severe disruption of mitochondrial ultrastructure characterized by swollen, irregular shape and disrupted cristae in aged KD compared with WT mice. Aging enhanced ROS production and reduced ΔΨm, the effects of which were accentuated by AMPK deficiency. Immunoblotting data depicted unchanged Akt phosphorylation and a significant decrease in mitochondrial biogenesis cofactor PGC‐1α in aged groups. AMPK deficiency but not aging decreased the phosphorylation of ACC and eNOS. Expression of membrane Glut4 and HSP90 was decreased in aged KD mice. Moreover, treatment of the AMPK activator metformin attenuated aging‐induced cardiomyocyte contractile defects. Collectively, our data suggest a role for AMPK deficiency in aging‐induced cardiac dysfunction possibly through disrupted mitochondrial function and ROS production.     

Reactive oxygen species control senescence‐associated matrix metalloproteinase‐1 through c‐Jun‐N‐terminal kinase

The lifetime exposure of organisms to oxidative stress influences many aging processes which involve the turnover of the extracellular matrix. In this study, we identify the redox‐responsive molecular signals that drive senescence‐associated (SA) matrix metalloproteinase‐1 (MMP‐1) expression. Precise biochemical monitoring revealed that senescent fibroblasts increase steady‐state (H₂O₂) 3.5‐fold (13.7–48.6 pM) relative to young cells. Restricting H₂O₂ production through low O₂ exposure or by antioxidant treatments prevented SA increases in MMP‐1 expression. The H₂O₂‐dependent control of SA MMP‐1 is attributed to sustained JNK activation and c‐jun recruitment to the MMP‐1 promoter. SA JNK activation corresponds to increases and decreases in the levels of its activating kinase (MKK‐4) and inhibitory phosphatase (MKP‐1), respectively. Enforced MKP‐1 expression negates SA increases in JNK phosphorylation and MMP‐1 production. Overall, these studies define redox‐sensitive signaling networks regulating SA MMP‐1 expression and link the free radical theory of aging to initiation of aberrant matrix turnover. J. Cell. Physiol. 225: 52–62, 2010. © 2010 Wiley‐Liss, Inc.     

JNK1 Phosphorylates SIRT1 and Promotes Its Enzymatic Activity

SIRT1 is a NAD-dependent deacetylase that regulates a variety of pathways including the stress protection pathway. SIRT1 deacetylates a number of protein substrates, including histones, FOXOs, PGC-1α, and p53, leading to cellular protection. We identified a functional interaction between cJUN N-terminal kinase (JNK1) and SIRT1 by coimmunoprecipitation of endogenous proteins. The interaction between JNK1 and SIRT1 was identified under conditions of oxidative stress and required activation of JNK1 via phosphorylation. Modulation of SIRT1 activity or protein levels using nicotinamide or RNAi did not modify JNK1 activity as measured by its ability to phosphorylate cJUN. In contrast, human SIRT1 was phosphorylated by JNK1 on three sites: Ser27, Ser47, and Thr530 and this phosphorylation of SIRT1 increased its nuclear localization and enzymatic activity. Surprisingly, JNK1 phosphorylation of SIRT1 showed substrate specificity resulting in deacetylation of histone H3, but not p53. These findings identify a mechanism for regulation of SIRT1 enzymatic activity in response to oxidative stress and shed new light on its role in the stress protection pathway.     

A selective small-molecule inhibitor of c-Jun N-terminal kinase 1

Indiscriminately suppressing total c-Jun N-terminal kinase (JNK) activity is not an appropriate strategy because each JNK appears to have a distinct function in cancer, asthma, diabetes, or Parkinson’s disease. Herein, we report that 7-(6-N-phenylaminohexyl)amino-2H-anthra[1,9-cd]pyrazol-6-one (AV-7) inhibited JNK1 activity, but not JNK2 or JNK3. We found that ultraviolet B (UVB) induced c-Jun phosphorylation and sub-G1 accumulation in JNK2^−/− murine embryonic fibroblasts, which contain an abundance of JNK1, but not JNK2. These results demonstrate that AV-7 is an isoform selective small-molecule inhibitor of JNK1 activity, which might be developed as a therapeutic against diabetes.

Structured summary

MINT-7148332: JNK3 (uniprotkb:P53779) phosphorylates (MI:0217) c-JUN (uniprotkb:P05412) by protein kinase assay (MI:0424)MINT-7148323: JNK2 (uniprotkb:P45984) phosphorylates (MI:0217) c-JUN (uniprotkb:P05412) by protein kinase assay (MI:0424)MINT-7148314: JNK1 (uniprotkb:P45983) phosphorylates (MI:0217) c-JUN (uniprotkb:P05412) by protein kinase assay (MI:0424)     

Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

Galectin-1 (gal-1), an endogenous β-galactoside-binding protein, triggers T-cell death through several mechanisms including the death receptor and the mitochondrial apoptotic pathway. In this study we first show that gal-1 initiates the activation of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 4 (MKK4), and MKK7 as upstream JNK activators in Jurkat T cells. Inhibition of JNK activation with sphingomyelinase inhibitors (20 μM desipramine, 20 μM imipramine), with the protein kinase C-δ (PKCδ) inhibitor rottlerin (10 μM), and with the specific PKCθ pseudosubstrate inhibitor (30 μM) indicates that ceramide and phosphorylation by PKCδ and PKCθ mediate gal-1-induced JNK activation. Downstream of JNK, we observed increased phosphorylation of c-Jun, enhanced activating protein-1 (AP-1) luciferase reporter, and AP-1/DNA-binding in response to gal-1. The pivotal role of the JNK/c-Jun/AP-1 pathway for gal-1-induced apoptosis was documented by reduction of DNA fragmentation after inhibition JNK by SP600125 (20 μM) or inhibition of AP-1 activation by curcumin (2 μM). Gal-1 failed to induce AP-1 activation and DNA fragmentation in CD3-deficient Jurkat 31-13 cells. In Jurkat E6.1 cells gal-1 induced a proapoptotic signal pattern as indicated by decreased antiapoptotic Bcl-2 expression, induction of proapoptotic Bad, and increased Bcl-2 phosphorylation. The results provide evidence that the JNK/c-Jun/AP-1 pathway plays a key role for T-cell death regulation in response to gal-1 stimulation.     

JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2

The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A₂ (cPLA₂α). This work dissects the pathway leading to cPLA₂α activation and LD biogenesis. Both processes were Ca²⁺-independent, as they took place after pharmacological blockade of Ca²⁺ transients elicited by serum or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). The single mutation D43N in cPLA₂α, which abrogates its Ca²⁺ binding capacity and translocation to membranes, did not affect enzyme activation and formation of LD. In contrast, the mutation S505A did not affect membrane relocation of the enzyme in response to Ca²⁺ but prevented its phosphorylation, activation, and the appearance of LD. Expression of specific activators of different mitogen-activated protein kinases showed that phosphorylation of cPLA₂α at Ser-505 is due to JNK. This was confirmed by pharmacological inhibition and expression of a dominant-negative form of the upstream activator MEKK1. LD biogenesis was accompanied by increased synthesis of ceramide 1-phosphate. Overexpression of its synthesizing enzyme ceramide kinase increased phosphorylation of cPLA₂α at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA₂α and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase.     

Silencing of nicotinamide nucleotide transhydrogenase impairs cellular redox homeostasis and energy metabolism in PC12 cells

Mitochondrial NADPH generation is largely dependent on the inner-membrane nicotinamide nucleotide transhydrogenase (NNT), which catalyzes the reduction of NADP(+) to NADPH utilizing the proton gradient as the driving force and NADH as the electron donor. Small interfering RNA (siRNA) silencing of NNT in PC12 cells results in decreased cellular NADPH levels, altered redox status of the cell in terms of decreased GSH/GSSG ratios and increased H(2)O(2) levels, thus leading to an increased redox potential (a more oxidized redox state). NNT knockdown results in a decrease of oxidative phosphorylation while anaerobic glycolysis levels remain unchanged. Decreased oxidative phosphorylation was associated with a) inhibition of mitochondrial pyruvate dehydrogenase (PDH) and succinyl-CoA:3-oxoacid CoA transferase (SCOT) activity; b) reduction of NADH availability, c) decline of mitochondrial membrane potential, and d) decrease of ATP levels. Moreover, the alteration of redox status actually precedes the impairment of mitochondrial bioenergetics. A possible mechanism could be that the activation of the redox-sensitive c-Jun N-terminal kinase (JNK) and its translocation to the mitochondrion leads to the inhibition of PDH (upon phosphorylation) and induction of intrinsic apoptosis, resulting in decreased cell viability. This study supports the notion that oxidized cellular redox state and decline in cellular bioenergetics - as a consequence of NNT knockdown - cannot be viewed as independent events, but rather as an interdependent relationship coordinated by the mitochondrial energy-redox axis. Disruption of electron flux from fuel substrates to redox components due to NNT suppression induces not only mitochondrial dysfunction but also cellular disorders through redox-sensitive signaling.     

Defects of protein phosphatase 2A causes corticosteroid insensitivity in severe asthma

Background

Corticosteroid insensitivity is a major barrier of treatment for some chronic inflammatory diseases, such as severe asthma, but the molecular mechanism of the insensitivity has not been fully elucidated. The object of this study is to investigate the role of protein phosphate 2A (PP2A), a serine/threonine phosphatase, on corticosteroid sensitivity in severe asthma.

Methodology/Principal Findings

Corticosteroid sensitivity was determined by the dexamethasone ability to inhibit TNFα-induced IL-8 or LPS-induced TNFα production. PP2A expression, glucocorticoid receptor (GR) nuclear translocation defined as the nuclear/cytoplasmic GR ratio and phosphorylation of GR-Ser²²⁶, c-Jun N-terminal kinase 1 (JNK1) and PP2A were analysed by Western-blotting. Phosphatase activity was measured by fluorescence-based assay. Okadaic acid (OA), a PP2A inhibitor, reduced corticosteroid sensitivity with reduced GR nuclear translocation and increased GR phosphorylation in U937 monocytic cells. PP2A knockdown by RNA interference showed similar effects. IL-2/IL-4 treatment to U937 reduced corticosteroid sensitivity, and PP2A expression/activity. In peripheral blood mononuclear cells (PBMCs) from severe asthma, the PP2A expression and activity were significantly reduced with concomitant enhancement of PP2A_C-Tyr³⁰⁷ phosphorylation compared with those in healthy volunteers. As the results, GR-Ser²²⁶ and JNK1 phosphorylation were increased. The expression and activity of PP2A were negatively correlated with phosphorylation levels of GR-Ser²²⁶. Furthermore, co-immunoprecipitation assay in U937 cells revealed that PP2A associated with GR and JNK1 and IL-2/IL-4 exposure caused dissociation of each molecule. Lastly, PP2A overexpression increased corticosteroid sensitivity in U937 cells.

Conclusions/Significance

PP2A regulates GR nuclear translocation and corticosteroid sensitivity possibly by dephosphorylation of GR-Ser²²⁶ via dephosphorylation of upstream JNK1. This novel mechanism will provide new insight for the development of new therapy for severe asthma.