Allergy Asthma Immunol Res.  2016 May;8(3):206-215. 10.4168/aair.2016.8.3.206.

Flagellin Modulates the Function of Invariant NKT Cells From Patients With Asthma via Dendritic Cells

• Shim JU
• Rhee JH
• Jeong JU
• Koh YI

• KMID: 2391040
• DOI: http://doi.org/10.4168/aair.2016.8.3.206

Abstract

PURPOSE
Invariant natural killer T (iNKT) cells play a critical role in the pathogenesis of asthma. We previously reported the association between circulating Th2-like iNKT cells and lung function in asthma patients and the suppressive effect of Toll-like receptor 5 ligand flagellin B (FlaB) on asthmatic in a mouse model. Thus, we investigated whether FlaB modulates the function of circulating iNKT cells in asthmatic patients.
METHODS
Peripheral blood mononuclear cells (PBMCs) were treated with FlaB, and the secreted and intracellular cytokines of iNKT cells were evaluated by using ELISA and flow cytometry, respectively, following stimulation with alpha-galactosylceramide. Foxp3+ iNKT cells were also measured. To determine the effect of FlaB-treated dendritic cells (DCs) on iNKT cells, we co-cultured CD14+ monocyte-derived DCs and T cells from patients with house dust mite-sensitive asthma and analyzed intracellular cytokines in iNKT cells.
RESULTS
A reduction of IL-4 and IL-17 production by iNKT cells in PBMCs after FlaB treatment was alleviated following blocking of IL-10 signaling. A decrease in the frequencies of IL-4+ and IL-17+ iNKT cells by FlaB-treated DCs was reversed after blocking of IL-10 signaling. Simultaneously, an increase in Foxp3+ iNKT cells induced by FlaB treatment disappeared after blocking of IL-10.
CONCLUSIONS
FlaB may inhibit Th2- and Th17-like iNKT cells and induce Foxp3+ iNKT cells by DCs via an IL-10-dependent mechanism in asthmatic patients. In patients with a specific asthma phenotype associated with iNKT cells, FlaB may be an effective immunomodulator for iNKT cell-targeted immunotherapy.

Keyword

Asthma; flagellin; natural killer T cell; Toll-like receptor 5

MeSH Terms

Animals
Asthma*
Cytokines
Dendritic Cells*
Dust
Enzyme-Linked Immunosorbent Assay
Flagellin*
Flow Cytometry
Humans
Immunotherapy
Interleukin-10
Interleukin-17
Interleukin-4
Lung
Mice
Natural Killer T-Cells*
Phenotype
T-Lymphocytes
Toll-Like Receptor 5
Cytokines
Dust
Flagellin
Interleukin-10
Interleukin-17
Interleukin-4
Toll-Like Receptor 5

Figure

• 

  Fig. 1 Protocol for the measurement of cytokines of α-GalCer-stimulated iNKT cells in FlaB-treated PBMC cultures. PBMCs were treated with FlaB for 48 hours. To stimulate iNKT cells, α-GalCer was added 24 hours before cytokine measurements. Secreted and intracellular cytokines were measured using ELISA and flow cytometry, respectively. In some experiments, IL-10R or IL-12 mAbs were added 6 hours before FlaB treatment to determine IL-10- or IL-12-dependent effect of FlaB on iNKT cell function.

• 

  Fig. 2 Representative examples of flow cytometric analysis. (A) Intracellular cytokines of iNKT cells in PBMCs. Lymphocytes and Vα24+ and Vβ11+ iNKT cells were gated. IL-4+, IFN-γ+ or IL-17+ cells were measured. Histograms show cytokine-positive iNKT cells (colored) and isotype antibodies (blank). (B) Appropriate measurement time point for each intracellular cytokine following α-GalCer stimulation in PBMCs. IL-4+, IFN-γ+, or IL-17+ iNKT cells were determined at baseline and 6, 12, and 24 hours after α-GalCer stimulation. Histograms show cytokine-positive iNKT cells (colored) and isotype antibodies (blank).

• 

  Fig. 3 IL-4, IFN-γ, and IL-17 production from α-GalCer-stimulated iNKT cells following FlaB treatment of PBMC cultures from asthma patients and healthy controls. FlaB treatment and α-GalCer stimulation were performed as described in Fig. 1. Cytokines in supernatants were measured by using ELISA. (A) Cytokine production in cells from asthma patients (n=10). (B) Cytokine production in cells from healthy controls (n=10). A Wilcoxon test was performed. Horizontal bars represent the median.

• 

  Fig. 4 Intracellular IL-4, IFN-γ, IL-17, and IL-10 production of α-GalCer-stimulated iNKT cells following FlaB treatment in PBMC cultures from asthma patients and healthy controls. FlaB treatment and α-GalCer stimulation were performed as described in Fig. 1. Intracellular cytokines were measured using flow cytometry. (A) Intracellular cytokine production in cells from asthma patients (n=10). (B) Intracellular cytokine production in cells from healthy controls (n=10). A Wilcoxon test was performed. Horizontal bars represent the median. NS, not significant.

• 

  Fig. 5 IL-10-dependent effect of FlaB on IL-4 and IL-17 production in iNKT cells from asthma patients (n=6) and IL-12-dependent effect of FlaB on IL-4 and IL-17 production in iNKT cells from normal controls (n=5). IL-10R or IL-12 mAbs were added before FlaB treatment as described in the Fig. 1 protocol. Secreted and intracellular cytokines were measured using ELISA (A) and flow cytometry (B) in cells from asthma patients, respectively, and using ELISA (C) and flow cytometry (D) in cells from normal controls, respectively. A Wilcoxon test was performed. Horizontal bars represent the median. NS, not significant.

• 

  Fig. 6 Effect of FlaB-treated DCs on intracellular IL-4 and IL-17 production of α-GalCer-stimulated iNKT cells following FlaB treatment of PBMCs from asthma patients. CD14+ monocyte-derived DCs were generated and treated for 48 hours. (A) IL-10 and IL-12 production from FlaB-treated DCs in asthma patients (n=4) and normal controls (n=3) measured by ELISA. In HDM-sensitive asthma patients (Table 2), FlaB-treated DCs and autologous CD3+ T cells were co-cultured with IL-10R or IL-12 mAbs in the presence of HDM extracts for 6 days. Intracellular cytokines of α-GalCer-stimulated iNKT cells were determined by flow cytometry. (B) Characteristics of each experimental group in the co-cultures. (C) IL-10-dependent inhibition by FlaB-treated DCs on IL-4+ iNKT cells (n=3). (D) IL-10-dependent inhibition by FlaB-treated DCs on IL-17+ iNKT cells (n=3). A Wilcoxon test was performed. Data represent the mean±SEM. For Fig. 6C and D, *P<0.05 and **P<0.01 compared to α-GalCer group. NS, not significant.

• 

  Fig. 7 IL-10-dependent induction of Foxp3+ iNKT cells following FlaB treatment of PBMCs from asthma patients. FlaB treatment and α-GalCer stimulation were performed as described in Fig. 1. IL-10R mAb was added before FlaB treatment, and intracellular cytokines were measured by flow cytometry. (A) A representative example for analyzing Foxp3+ iNKT cells. (B) IL-10-dependent induction of Foxp3+ iNKT cells (n=3). A Wilcoxon test was performed. Data represent the mean±SEM.

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