DNA-dependent protein kinase inhibition blocks asthma in mice and modulates human endothelial and CD4⁺ T-cell function without causing severe combined immunodeficiency - 05/02/15

Doi : 10.1016/j.jaci.2014.09.005

Mohamed A. Ghonim, MS ^a,^c,^{^{∗
These authors contributed equally to this work.}}, Kusma Pyakurel, BS ^a,^{^{∗
These authors contributed equally to this work.}}, Jihang Ju, PhD ^a,^{^{∗
These authors contributed equally to this work.}}, Paulo C. Rodriguez, PhD ^a, Matthew R. Lammi, MD ^b, Christian Davis ^a, Mohammad Q. Abughazleh, BS ^a, Moselhy S. Mansy, PhD ^c, Amarjit S. Naura, PhD ^a,^⁎,^{^{‡
Dr Naura is currently affiliated with the Department of Biochemistry, Panjab University, Chandigarh, India.}} , A. Hamid Boulares, PhD ^a,^⁎
^a Stanley S. Scott Cancer Center, LSU Health Sciences Center, New Orleans, La
^b Pulmonary and Critical Care Section, LSU Health Sciences Center, New Orleans, La
^c Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt

^∗Corresponding authors: A. Hamid Boulares, PhD, Or: Amarjit S. Naura, PhD, 1700 Tulane Ave, New Orleans, LA 70112.

Abstract

Background

We reported that DNA-dependent protein kinase (DNA-PK) is critical for the expression of nuclear factor κB–dependent genes in TNF-α–treated glioblastoma cells, suggesting an involvement in inflammatory diseases.

Objective

We sought to investigate the role of DNA-PK in asthma.

Methods

Cell culture and ovalbumin (OVA)– or house dust mite–based murine asthma models were used in this study.

Results

DNA-PK was essential for monocyte adhesion to TNF-α–treated endothelial cells. Administration of the DNA-PK inhibitor NU7441 reduced airway eosinophilia, mucus hypersecretion, airway hyperresponsiveness, and OVA-specific IgE production in mice prechallenged with OVA. Such effects correlated with a marked reduction in lung vascular cell adhesion molecule 1 expression and production of several cytokines, including IL-4, IL-5, IL-13, eotaxin, IL-2, and IL-12 and the chemokines monocyte chemoattractant protein 1 and keratinocyte-derived chemokine, with a negligible effect on IL-10/IFN-γ production. DNA-PK inhibition by gene heterozygosity of the 450-kDa catalytic subunit of the kinase (DNA-PKcs^+/−) also prevented manifestation of asthma-like traits. These results were confirmed in a chronic model of asthma by using house dust mite, a human allergen. Remarkably, such protection occurred without causing severe combined immunodeficiency. Adoptive transfer of T_H2-skewed OT-II wild-type CD4⁺ T cells reversed IgE and T_H2 cytokine production but not airway hyperresponsiveness in OVA-challenged DNA-PKcs^+/− mice. DNA-PK inhibition reduced IL-4, IL-5, IL-13, eotaxin, IL-8, and monocyte chemoattractant protein 1 production without affecting IL-2, IL-12, IFN-γ, and interferon-inducible protein 10 production in CD3/CD28-stimulated human CD4⁺ T cells, potentially by blocking expression of Gata3. These effects occurred without significant reductions in T-cell proliferation. In mouse CD4⁺ T cells in vitro DNA-PK inhibition severely blocked CD3/CD28-induced Gata3 and T-bet expression in CD4⁺ T cells and prevented differentiation of T_H1 and T_H2 cells under respective T_H1- and T_H2-skewing conditions.

Conclusion

Our results suggest DNA-PK as a novel determinant of asthma and a potential target for the treatment of the disease.

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Key words : DNA-dependent protein kinase, asthma, GATA-3, T_H2 cytokines, adhesion molecules, eosinophilia, severe combined immunodeficiency disease, T-cell receptor

Abbreviations used : AHR, BAL, DNA-PK, DNA-PKcs, FACS, HDM, HUVEC, LSMC, LSUHSC, MCP-1, NF-κB, NHEJ, OVA, SCID, TCR, VCAM-1, WT

Plan

Methods

Animals

OVA sensitization and challenge, house dust mite extract challenge, and airway hyperresponsiveness measurement

Organ recovery, tissue processing, cell staining, T_H2 cytokine and OVA-specific IgE assessments, fluorescence-activated cell sorting analysis, and immunohistochemistry

Cell culture, treatments, immunoblot analysis, and cell adhesion assay

Human blood collection, mouse splenocyte preparation, CD4⁺ T-cell purification, T-cell receptor stimulation, and RT-PCR

T_H cell skewing, T_H1 and T_H2 cytokine assessment, and adoptive transfer

Data analysis

Results

DNA-PK protein level and function are critical for VCAM-1 expression and it is required for adhesion of inflammatory cells to endothelial cells on TNF-α treatment

DNA-PK is involved in allergic lung inflammation, and its inhibition reduces eosinophilia, mucus hypersecretion, AHR, and IgE production in a mouse model of asthma

DNA-PK inhibition blocks OVA-induced asthma-like traits by reducing production of T_H2 cytokines in a mouse model of asthma

DNA-PKcs gene heterozygosity prevents eosinophilia, mucus hypersecretion, production of T_H2 cytokines, increase in OVA-specific IgE levels, and AHR in an animal model of asthma

DNA-PK inhibition pharmacologically or by gene heterozygosity blocks asthma-like manifestation in an HDM asthma model

Adoptive transfer of in vitro T_H2-skewed CD4⁺ T cells partially reverses IgE and T_H2 cytokine production, but not AHR, in OVA-exposed DNA-PKcs^+/− mice

DNA-PK inhibition by gene heterozygosity or pharmacologic drugs does not cause a severe combined immunodeficiency phenotype, alter the fate of plasma B cells, or change overall spleen weight

Differential roles of DNA-PK in the production of T_H2 and T_H1 cytokines on T-cell receptor stimulation in human CD4⁺ T cells without a prominent effect on their proliferation

DNA-PK inhibition severely blocks CD3/CD28-induced Gata3 expression in naive mouse or human CD4⁺ T cells and prevents in vitro differentiation of mouse T_H1 and T_H2 cells under respective T_H1- and T_H2-skewing conditions

Discussion

Supported in part by grant HL072889 from the National Institutes of Health (NIH) and funds from the Louisiana Cancer Research Center (New Orleans, La; to A.H.B.), by the Egyptian Cultural and Educational Bureau (Egypt), by P20GM103501 from the NIH (to A.S.N. [program director: Dr A. Ochoa]), by postdoctoral fellowship 11POST8000008 (to J.J.) and predoctoral fellowship 14PRE19630012 (to K.P.) from the American Heart Association, and by a NIH/U54 GM104940 (LACaTS) Scholar grant to M.R.L.

Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

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Vol 135 - N° 2

P. 425-440 - février 2015 Retour au numéro

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DNA-dependent protein kinase inhibition blocks asthma in mice and modulates human endothelial and CD4⁺ T-cell function without causing severe combined immunodeficiency - 05/02/15

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