Increased frequency of dual-positive TH2/TH17 cells in bronchoalveolar lavage fluid characterizes a population of patients with severe asthma

doi:10.1016/j.jaci.2014.05.038

Journal of Allergy and Clinical Immunology

Volume 134, Issue 5, November 2014, Pages 1175-1186.e7

https://doi.org/10.1016/j.jaci.2014.05.038 Get rights and content

Background

T_H2 cells can further differentiate into dual-positive T_H2/T_H17 cells. The presence of dual-positive T_H2/T_H17 cells in the airways and their effect on asthma severity are unknown.

Objective

We sought to study dual-positive T_H2/T_H17 cells in bronchoalveolar lavage (BAL) fluid from asthmatic patients, examine their response to glucocorticoids, and define their relevance for disease severity.

Methods

Bronchoscopy and lavage were performed in 52 asthmatic patients and 25 disease control subjects. T_H2 and T_H2/T_H17 cells were analyzed by using multicolor flow cytometry and confocal immunofluorescence microscopy. Cytokines were assayed by means of ELISA.

Results

Dual-positive T_H2/T_H17 cells were present at a higher frequency in BAL fluid from asthmatic patients compared with numbers seen in disease control subjects. High-level IL-4 production was typically accompanied by high-level IL-17 production and coexpression of GATA3 and retinoic acid receptor–related orphan receptor γt. Increased presence of T_H2/T_H17 cells was associated with increased IL-17 production in lavage fluid. T_H2/T_H17 cell counts and IL-17 production correlated with PC₂₀ for methacholine, eosinophil counts, and FEV₁. T_H2/T_H17 cells, unlike T_H2 cells, were resistant to dexamethasone-induced cell death. They expressed higher levels of mitogen-activated protein–extracellular signal-regulated kinase kinase 1, a molecule that induces glucocorticoid resistance. On the basis of the dominance of BAL fluid T_H2 or T_H2/T_H17 cells, we identified 3 subgroups of asthma: T_H2^predominant, T_H2/T_H17^predominant, and T_H2/T_H17^low. The T_H2/T_H17^predominant subgroup manifested the most severe form of asthma, whereas the T_H2/T_H17^low subgroup had the mildest asthma.

Conclusion

Asthma is associated with a higher frequency of dual-positive T_H2/T_H17 cells in BAL fluid. The T_H2/T_H17^predominant subgroup of asthmatic patients manifested glucocorticoid resistance in vitro. They also had the greatest airway obstruction and hyperreactivity compared with the T_H2^predominant and T_H2/T_H17^low subgroups.

Section snippets

Human subjects

The study subjects were recruited from the outpatient clinics of National Jewish Health. The study protocol for bronchoscopy and BAL was approved by the institutional review board. Informed consent was obtained from study subjects. Patients were allowed to continue their routine medication.

Processing of BAL fluid cells and flow cytometry

Bronchoscopy and BAL were performed, as described previously.²³ BAL fluid was processed immediately. Cells were isolated by means of centrifugation. Supernatant fluid was placed into aliquots in small samples

Detection of single T_H2 and T_H17 cells and dual T_H2/T_H17 cells in BAL fluid from asthmatic patients

We studied BAL cells from 52 asthmatic patients and 25 disease control subjects. Most of the patients were referred to National Jewish Health for diagnosis and management of refractory asthma. Others were referred for routine asthma care. We use the term refractory in this article to indicate uncontrolled asthma, which could be moderate or severe, as determined based on the Expert Panel Report 3 criteria. The clinical characteristics of the study subjects are shown in Table I. Bronchoscopy and

Discussion

The presence of allergic sensitivity (skin test result positivity or IgE-specific antibody in serum) can be detected in a vast majority, but not all, asthmatic patients.³⁴ In addition to IgE, mild-to-moderate eosinophilia is a characteristic feature of asthma. Interestingly, some nonallergic asthmatic patients also have blood eosinophilia. Both IgE antibody and eosinophilia are driven by a T_H2-type immune response. Thus the presence of T_H2 cells in lung tissue and BAL fluid is anticipated and

References (62)

X.O. Yang et al.
Molecular antagonism and plasticity of regulatory and inflammatory T cell programs
Immunity
(2008)
H.J. Koenen et al.
Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells
Blood
(2008)
Y.K. Lee et al.
Late developmental plasticity in the T helper 17 lineage
Immunity
(2009)
G. Wei et al.
Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells
Immunity
(2009)
L. Zhou et al.
Plasticity of CD4+ T cell lineage differentiation
Immunity
(2009)
A. Barczyk et al.
Interleukin-17 in sputum correlates with airway hyperresponsiveness to methacholine
Respir Med
(2003)
S. Molet et al.
IL-17 is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines
J Allergy Clin Immunol
(2001)
W. Al-Ramli et al.
T(H)17-associated cytokines (IL-17A and IL-17F) in severe asthma
J Allergy Clin Immunol
(2009)
I. Agache et al.
Increased serum IL-17 is an independent risk factor for severe asthma
Respir Med
(2010)
J.T. Good et al.
Refractory asthma: importance of bronchoscopy to identify phenotypes and direct therapy
Chest
(2012)

X.O. Yang et al.

STAT3 regulates cytokine-mediated generation of inflammatory helper T cells

J Biol Chem

(2007)

P.R. Mittelstadt et al.

Inhibition of AP-1 by the glucocorticoid-inducible protein GILZ

J Biol Chem

(2001)

D. Robinson et al.

Activation of CD4+ T cells, increased TH2-type cytokine mRNA expression, and eosinophil recruitment in bronchoalveolar lavage after allergen inhalation challenge in patients with atopic asthma

J Allergy Clin Immunol

(1993)

C.E. Brightling et al.

TH2 cytokine expression in bronchoalveolar lavage fluid T lymphocytes and bronchial submucosa is a feature of asthma and eosinophilic bronchitis

J Allergy Clin Immunol

(2002)

C.J. Bossley et al.

Pediatric severe asthma is characterized by eosinophilia and remodeling without T(H)2 cytokines

J Allergy Clin Immunol

(2012)

M. Ciofani et al.

A validated regulatory network for Th17 cell specification

Cell

(2012)

X. Li et al.

MEK is a key regulator of gliogenesis in the developing brain

Neuron

(2012)

E. Irusen et al.

p38 Mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces its activity: role in steroid-insensitive asthma

J Allergy Clin Immunol

(2002)

D.J. Jackson et al.

Asthma exacerbations: origin, effect, and prevention

J Allergy Clin Immunol

(2011)

D.S. Robinson et al.

Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma

N Engl J Med

(1992)

B. Burrows et al.

Association of asthma with serum IgE levels and skin-test reactivity to allergens

N Engl J Med

(1989)

R.C. Strunk et al.

Omalizumab for asthma

N Engl J Med

(2006)

R.L. Coffman et al.

Reversal of polarized T helper 1 and T helper 2 cell populations in murine leishmaniasis

Ciba Found Symp

(1995)

S. Mocci et al.

Induction of a Th2 population from a polarized Leishmania-specific Th1 population by in vitro culture with IL-4

J Immunol

(1995)

L. Cosmi et al.

Identification of a novel subset of human circulating memory CD4(+) T cells that produce both IL-17A and IL-4

J Allergy Clin Immunol

(2010)

Y.H. Wang et al.

A novel subset of CD4(+) T(H)2 memory/effector cells that produce inflammatory IL-17 cytokine and promote the exacerbation of chronic allergic asthma

J Exp Med

(2010)

C. Malmhall et al.

Immunophenotyping of circulating T helper cells argues for multiple functions and plasticity of T cells in vivo in humans—possible role in asthma

PLoS One

(2012)

L. McKinley et al.

TH17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice

J Immunol

(2008)

J. Pene et al.

Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes

J Immunol

(2008)

D.M. Bullens et al.

IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx?

Respir Res

(2006)

C.K. Wong et al.

Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-gamma, IL-4, IL-10 and IL-13) in patients with allergic asthma

Clin Exp Immunol

(2001)

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: Supported by National Institutes of Health (NIH) grants RO1 AI091614 and N01 HHSN272200700048C to R.A. M.M.G. is supported by career development grants from CCTSI (NIH/NCATS Colorado CTSI KL2 TR000156) and Children's Environmental Center grant from the National Institute of Environmental Health Sciences (NIEHS PO1 ES-018181/EPA GAD 834515010).

: Disclosure of potential conflict of interest: J. Good has provided chart reviews for Lincare–Wheeler Trigg O'Donnell LLP, Faraci Wolanske LLC, and Pryor Johnson Carney Karr Nixon PC; is employed by National Jewish Health; has received research support from MedImmune; has received payment for lectures from Merck and Genentech; has received payment for development of educational presentations from QuantiaMD; and has received travel expenses from the Aspen Lung Conference. M. M. Gorska has received research support from the Denver Children's Environmental Health Center Faculty Development Investigator Award, the National Institutes of Health (NIH)/National Center for Advancing Translational Sciences Colorado CTSI KL2 TR000156 Award, and the Sheldon C. Siegel–Asthma and Allergy Foundation of America Investigator Grant Award. R. J. Martin has received honoraria for consultancy and travel from Teva Pharmaceuticals and Boehringer Ingelheim; has received research support from MedImmune; and has received royalties from UpToDate. R. Alam has received research support from the National Institutes of Health. The rest of the authors declare that they have no relevant conflicts of interest.

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Mechanisms of allergy and clinical immunologyIncreased frequency of dual-positive TH2/TH17 cells in bronchoalveolar lavage fluid characterizes a population of patients with severe asthma

Background

Objective

Methods

Results

Conclusion

Section snippets

Human subjects

Processing of BAL fluid cells and flow cytometry

Detection of single TH2 and TH17 cells and dual TH2/TH17 cells in BAL fluid from asthmatic patients

Discussion

Immunity

Blood

Immunity

Immunity

Immunity

Respir Med

J Allergy Clin Immunol

J Allergy Clin Immunol

Respir Med

Chest

J Biol Chem

J Biol Chem

J Allergy Clin Immunol

J Allergy Clin Immunol

J Allergy Clin Immunol

Cell

Neuron

J Allergy Clin Immunol

J Allergy Clin Immunol

Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma

N Engl J Med

Association of asthma with serum IgE levels and skin-test reactivity to allergens

N Engl J Med

Omalizumab for asthma

N Engl J Med

Reversal of polarized T helper 1 and T helper 2 cell populations in murine leishmaniasis

Ciba Found Symp

Induction of a Th2 population from a polarized Leishmania-specific Th1 population by in vitro culture with IL-4

J Immunol

Identification of a novel subset of human circulating memory CD4(+) T cells that produce both IL-17A and IL-4

J Allergy Clin Immunol

A novel subset of CD4(+) T(H)2 memory/effector cells that produce inflammatory IL-17 cytokine and promote the exacerbation of chronic allergic asthma

J Exp Med

Immunophenotyping of circulating T helper cells argues for multiple functions and plasticity of T cells in vivo in humans—possible role in asthma

PLoS One

TH17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice

J Immunol

Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes

J Immunol

IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx?

Respir Res

Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-gamma, IL-4, IL-10 and IL-13) in patients with allergic asthma

Clin Exp Immunol

Mechanisms of allergy and clinical immunology
Increased frequency of dual-positive T_H2/T_H17 cells in bronchoalveolar lavage fluid characterizes a population of patients with severe asthma

Detection of single T_H2 and T_H17 cells and dual T_H2/T_H17 cells in BAL fluid from asthmatic patients