IL-9 pathway in asthma: New therapeutic targets for allergic inflammatory disorders

doi:10.1016/S0091-6749(99)70165-X

Journal of Allergy and Clinical Immunology

Volume 103, Issue 5, Supplement, May 1999, Pages S485-S491

https://doi.org/10.1016/S0091-6749(99)70165-X Get rights and content

Abstract

Background: Asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of allergic inflammation and bronchial hyperresponsiveness (BHR). The incidence of asthma continues to rise in industrialized countries despite advances in the identification of cellular and molecular mediators that are associated with the disease. Because of its importance in human health, additional research and alternative therapeutic strategies are justified to create more effective treatments for this debilitating disease. Objective: Studies use recombinant inbred mice to demonstrate that BHR in mouse models of asthma is associated with a genetic alteration at the IL-9 locus, where IL-9 expression in lung is strongly associated with bronchial responsiveness. We have investigated the ability of intratracheal instilled IL-9 to induce asthmatic-like responses in naive C57BL/6 (B6) mice, which express very low levels of IL-9. Methods: IL-9 or vehicle was intratracheal instilled in naive B6 mice for 10 days. Mice were analyzed for effects on BHR, lung eosinophilia, and serum total IgE levels. Results: Phenotypic effects of B6 mice instilled with IL-9 were increased eosinophils in the bronchoalveolar lavage and significantly elevated serum total IgE. Moreover, IL-9 was found to induce IL-5Rα in vivo and in vitro, suggesting a potential mechanism for the novel actions described for IL-9 on eosinophils. Conclusion: Increased levels of IL-9 in the airway of naive B6 mice induced lung eosinophilia and serum total IgE levels, which are 2 clinical features of asthma. These data support a central role for the IL-9 pathway in the complex pathogenesis of allergic inflammation. (J Allergy Clin Immunol 1999;103:485-91.)

Section snippets

Primary cell cultures and cell lines

KG-1 cells were obtained from the American Type Culture Collection and cultured according to the supplier. Mo7e cells were maintained in RPMI-1640 supplemented with 10% FCS and 30 ng/mL of recombinant human GM-CSF. TS1 cells are a murine T_H2 -like cell line that was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS and 25 ng/mL rmIL-9. One hundred thousand cells were plated in 96-well plates, washed 3 times, and incubated overnight in serum-free media before recombinant

Genetic variability in bronchial responsiveness

Significant differences in bronchial responsiveness were observed between the hyperresponsive D2 mice and hyporesponsive B6 mice and the inbred (B6D2)F₁ offspring, which were intermediate in phenotype (Fig 1).

. Genetic variability in bronchial responsiveness. Bronchial responsiveness (airway pressure time index [APTI ]) to atracurium 20 mg/kg, intravenously, is shown for the hyporesponsive C57BL/6J and hyperresponsive DBA/2J strains and for the (B6D2)F₁ mice that demonstrate an intermediate

DISCUSSION

We have previously identified the IL-9 gene as a candidate in allergic asthma on the basis of linkage homologic features between humans and mice for bronchial responsiveness.⁹ Moreover, these data demonstrated that allelic variation at the IL-9 locus is responsible, in part, for biologic variability in baseline bronchial responsiveness between inbred strains of mice. The bronchial hyporesponsive B6 strain was found to express very low levels of IL-9 in the lung in comparison with the

Acknowledgements

The authors thank Jacques Van Snick and Jean Christophe Renauld for their suggestions and advice.

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Previous studies have demonstrated that inflammation is closely related to the occurrence and development of heart failure (HF). As an inflammation-related cytokine, interleukin (IL)-9 has been reported to be involved in the development of cardiovascular diseases. However, the role of IL-9 in HF in response to isoproterenol (ISO) stimulation has barely been explored. Thus, this study aimed to investigate whether IL-9 participates in HF and the possible associated mechanisms.
Chronic ISO infusion was used to establish an HF model, and the IL-9 levels in mice and isolated cardiomyocytes were measured. In addition, ISO-treated mice received an injection of recombinant mouse IL-9 (rIL-9) or an antimouse IL-9 neutralizing monoclonal antibody (mAb) to investigate the effects of IL-9 on cardiac function, hypertrophy, and fibrosis.
IL-9 levels were significantly increased in mice and isolated cardiomyocytes after ISO treatment. Treatment with rIL-9 resulted in aggravated cardiac dysfunction and amplified cardiac hypertrophy and fibrosis, whereas treatment with the anti-IL-9 neutralizing mAb ameliorated cardiac dysfunction and reduced cardiac hypertrophy and fibrosis in ISO-treated mice. In addition, ISO infusion–induced cardiac inflammation and cardiomyocyte apoptosis was aggravated by rIL-9 but prevented by the anti-IL-9 mAb. IL-9 did not activate signal transducer and activator of transcription (STAT)1 or STAT5 but induced STAT3 phosphorylation in ISO-induced HF. Moreover, S31-201, a specific STAT3 inhibitor, nearly abolished rIL-9-induced increases in cardiac dysfunction, hypertrophy, and fibrosis in response to ISO stimulation.
IL-9 aggravated cardiac dysfunction and amplified cardiac hypertrophy and fibrosis in the ISO-induced HF model by activating STAT3 signalling. These data indicate that blocking IL-9 may be an attractive pharmacotherapeutic strategy for the treatment of cardiac hypertrophy and fibrosis induced by chronic β-adrenergic receptor activation to limit the progression of HF.
Des études ont démontré qu’il existe un lien étroit entre l’inflammation et l’apparition d’une insuffisance cardiaque (IC). L’interleukine-9 (IL-9), une cytokine inflammatoire, a notamment été associée à l’apparition de maladies cardiovasculaires. Or, le rôle de l’IL-9 dans l’IC en réponse à une stimulation par l’isoprotérénol (ISO) a été très peu étudié. Ainsi, cette étude visait à déterminer si l’IL-9 contribue à l’IC et à examiner les mécanismes possiblement associés à cette contribution.
Un modèle d’IC a été créé à partir d’une perfusion prolongée d’ISO, puis les taux d’IL-9 chez des souris et dans des cardiomyocytes isolés ont été mesurés. Les souris auxquelles on a administré de l’ISO ont aussi reçu une injection d’IL-9 recombinante (IL-9r) de souris ou d’anticorps monoclonal (AcM) neutralisant anti-IL-9 de souris. Le but était d’étudier les effets de l’IL-9 sur la fonction, l’hypertrophie et la fibrose cardiaques.
Les taux d’IL-9 ont augmenté de manière significative chez les souris et dans les cardiomyocytes isolés exposés à l’ISO. Chez les souris, l’administration d’IL-9r a aggravé la dysfonction cardiaque et amplifié l’hypertrophie et la fibrose cardiaques, tandis que le traitement par l’AcM neutralisant anti-IL-9 a atténué la dysfonction cardiaque et réduit l’hypertrophie et la fibrose cardiaques. En outre, l’IL-9r a intensifié l’inflammation cardiaque et l’apoptose des cardiomyocytes induites par la perfusion d’ISO, tandis que l’AcM anti-IL-9 les a empêchées. L’IL-9 n’a pas activé le signal transducteur et activateur de la transcription STAT1 ni le STAT5, mais a provoqué la phosphorylation de STAT3 en cas d’IC induite par l’ISO. Par ailleurs, l’inhibiteur S31-201, spécifique de STAT3, a presque éliminé l’accentuation de la dysfonction, de l’hypertrophie et de la fibrose cardiaques induite par l’IL-9r en réponse à la stimulation par l’ISO.
Dans le modèle d’IC induite par l’ISO, l’IL-9 a aggravé la dysfonction cardiaque et amplifié l’hypertrophie et la fibrose cardiaques en activant la voie de signalisation STAT3. Ces données montrent que l’inhibition de l’IL-9 pourrait être une stratégie pharmacothérapeutique intéressante pour traiter l’hypertrophie et la fibrose cardiaques induites par l’activation chronique des récepteurs bêta-adrénergiques et limiter l’évolution de l’IC.
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Abdominal aortic aneurysm (AAA) is a degenerative inflammatory disease with unknown etiology. AAA is characterized by abdominal aortic dilatation more than 3 cm and is often asymptomatic, but the rupture of aneurysm can lead to death. Age, smoking and male sex are major predisposing factors of AAA.
This study compares the effect of Helicobacter (H.) pylori and Lactobacillus (L.) acidophilus on the cytokine profile of PBMCs of 5 men with abdominal aortic aneurysm (AAA) and 5 men with normal/insignificant angiography, CT-Scan and ultrasonography results in the single-culture and in the co-culture with HUVECs. IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17 F, IL-21, IL-22, IFN-γ and TNF-α were measured in culture supernatants using a commercial fluorescent-labeled-bead assay.
In general, CagA+ H. pylori-extract induced higher production of IFN-γ, IL-13 and IL-21 by PBMCs. Treatment of patients’ PBMCs with CagA⁺ H. pylori-extract induced Th2 cytokines while treatment of controls’ PBMCs with CagA⁺ H. pylori-extract increased Th1 cytokines. In the co-culture, however, patients’ PBMCs produced Th1 cytokines irrespective of extract treatment, while controls’ PBMCs produced Th2 cytokines and decreased IL-10. CagA+ H. pylori- as well as L. acidophilus-extract induced higher levels of IL-9 by controls’ PBMCs in co-culture with HUVECs than patients (P = 0.05 and P = 0.01).
The cytokine pattern of PBMCs induced by CagA+ H. pylori- and L. acidophilus-extracts in the co-culture with HUVECs shows differences in AAA patients and in comparison to controls. Decreased secretion of IL-9, IL-21 and IL-22 by PBMCs of patients treated with CagA+ H. pylori extract in co-culture, as opposed to non-AAA controls may indicate the active role ECs play in AAA. Simultaneous production of IL-10 and Th1 cytokines in patients and pronounced Th2 cytokines in controls in response to both bacteria may point to the inherent differences between patients and controls, which need further investigation.
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Increased activity of STAT6 predisposes toward allergic disease, while a decreased activity greatly reduces the severity of a number of diseases, such as food allergies and eosinophilic esophagitis [268–272]. Blocking the SH2 domain of STAT6 and other strategies that inhibit its phosphorylation and further downstream signaling are therefore under investigation and may be useful in maintaining allergic diseases [273,274]. Given the fact that STAT proteins pass over the information of dozens of cytokines like IFNs, ILs and colony stimulating factors, it is evident that they are now recognized as one of the most important pathways involved in the development and regulation of cells that mediate innate and adaptive immunity [275,276].
The Signal Transducer and Activator of Transcription (STAT) family of proteins consists of transcription factors that play a complex and essential role in the regulation of physiologic cell processes, such as proliferation, differentiation, apoptosis and angiogenesis, and serves to organize the epigenetic landscape of immune cells. To date, seven STAT genes have been identified in the human genome; STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6. They all account for diverse effects in response to extracellular signaling proteins, mainly by altering gene transcription in the effector cells. Members of the STAT family have been implicated in human cancer development, progression, metastasis, survival and resistance to treatment. Particularly STAT3 and STAT5 are of interest in cancer biology. They are currently considered as oncogenes, but their signaling is embedded into a complex and delicate balance between different (counteracting) transcription factors, and thus, in some contexts they can have a tumor suppressive role. Assessing STAT signaling mutations as well as screening for aberrant STAT pathway activation may have a role to predict sensitivity to immunotherapy and targeted STAT inhibition. In the present comprehensive review of the literature, we discuss in-depth the role of each STAT family member in cancer, assemble cutting-edge information on the use of these molecules as potential biomarkers and targets for treatment, and address why their clinical implementation is controversy.
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Citation Excerpt :
Regarding the contribution of IL-9 to the development of experimental allergic airway disease, a consistent picture does not yet exist. Intratracheal application of IL-9 [7,6] as well as lung-specific overexpression of IL-9 [8,12] promote eosinophilia, mucus production, and AHR. Production of mucus appears to depend mainly on IL-9-induced IL-13 [12].
Cylindromatosis (CYLD) is a ubiquitously expressed deubiquitinating enzyme which removes activating ubiquitin residues from important signaling molecules of the NF-κB pathway. In CYLD^ex7/8 transgenic mice, a naturally occurring short isoform (sCYLD) is overexpressed in the absence of full length CYLD, leading to excessive NF-κB activity.
Herein, we investigated the impact of the CYLD^ex7/8 mutation selectively in T cells on the development of experimental allergic airway disease induced by sensitization and challenge with ovalbumin. Compared with their wildtype littermates, mice bearing the T cell-specific mutation (CD4⁺CYLD^ex7/8) display stronger eosinophilia and mucus production in the lungs and higher IgE serum levels. The reason for these observations is excessive production of T cell-derived IL-9, a cytokine to whom allergy-promoting properties were ascribed. Consequently, blockade of IL-9 in CD4⁺CYLD^ex7/8 mice alleviates the development of disease symptoms. Thus, by polarization of the T cell cytokine response, sCYLD can favor the development of allergic airway disease.
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Recent studies have highlighted a crucial regulatory role of the cytokine IL-9 in driving immune responses in chronic inflammatory and autoimmune diseases at mucosal surfaces. IL-9 activates various types of immune and non-immune cells carrying the membrane bound IL-9R. IL-9 signaling plays a pivotal role in controlling the differentiation and activation of these cells by inducing the Jak/STAT pathway. In particular, IL-9 induces activation of T helper cells and affects the function of various tissue resident cells such as mast cells and epithelial cells in the mucosa. Importantly, recent findings suggest that blockade of IL-9 signaling is effective in treating experimental models of autoimmune and chronic inflammatory diseases such as inflammatory bowel diseases, allergic disorders such as food allergy and asthma. Thus, blockade of IL-9 and IL-9R signaling emerges as potentially novel approach for therapy of inflammatory diseases in the mucosal immune system.
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The secretion of specific cytokines distinguishes T helper cell subsets and confers the ability of the T cell to promote distinct types of inflammation. The restricted nature of cytokine production among these subsets makes them attractive therapeutic targets. The interleukin (IL)-9-secreting subset termed Th9 cells are observed in patients and in mouse models associated with immunity to parasite infections, autoimmunity, tumor immunity, and allergic inflammation. The conservation of Th9 phenotype and function across evolution suggests that Th9 cells play a prominent role in effective immunity. In this chapter we outline preclinical mouse studies and human studies that demonstrate a role for Th9/IL-9 in the pathogenesis of atopic disease, colitis, central nervous system paralysis, and protection against helminthic parasite infection and tumor progression. We further outline current therapeutics that might be used to modulate Th9 activity in a clinical setting.

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^☆: Reprint requests: Roy Clifford Levitt, MD, Magainin Institute of Molecular Medicine, Magainin Pharmaceuticals, 5110 Campus Dr, Plymouth Meeting, PA 19462.

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IL-9 pathway in asthma: New therapeutic targets for allergic inflammatory disorders☆,☆☆

Abstract

Section snippets

Primary cell cultures and cell lines

Genetic variability in bronchial responsiveness

DISCUSSION

Acknowledgements

Genomics

Genomics

Genomics

Immunobiology

J Allergy Clin Immunol

Asthma

N Engl J Med

Genetic and environmental influences on airway inflammation in asthma

Int Arch Allergy Immunol

Genetic susceptibility to asthma–bronchial hyperresponsiveness coinherited with a major gene for atopy

N Engl J Med

A genome–wide search for asthma susceptibility loci in ethnically diverse populations

Nature Genetics

A genome-wide search for quantitative trait loci underlying asthma

Nature

Linkage analysis of IL4 and other chromosome 5q31.1 markers and total serum immunoglobulin E concentrations

Science

Allelic association of gene markers on chromosomes 5q and 11q with atopy and bronchial hyperresponsiveness

Am J Respir Crit Care Med

Interleukin–9: a candidate gene for asthma

Proc Natl Acad Sci

Expression of airway hyperreactivity to acetylcholine as a simple autosomal recessive trait in mice

FASEB J

Respiratory system mechanics in mice measured by end-inflation occlusion

J Appl Physiol

A genetic model for evaluation of susceptibility to ozone-induced inflammation

Am J Physiol