Activation of Eosinophils by NK cells.

<p>Autologous NK cells and eosinophils were cultured at the ratio NK cells: eosinophils (NK: Eos ratio) 0∶1 (i.e. eosinophils alone), 1∶1, 5∶1 and 10∶1 in the presence of interleukin-5 for 3 hours and 12 hours. (A) Gating strategy: live eosinophils were identified firstly on the basis of their size difference (FSC) and granularity (SSC) and secondly as Annexin V⁻ 7AAD⁻ cells. CD63 expression (black histogram) is shown on a representative experiment for the ratio NK cells: eosinophils (NK: Eos ratio) 0∶1 (i.e. eosinophils alone) and 10∶1. Isotypic control antibody is represented in dashed grey. Eosinophils were stained for CD63 (B), CD62L (C) and CD69 (D) expression. Results are shown for each donor and mean is indicated with the black line. One-way Anova (Friedman) tests were performed, followed by Dunn’s post tests. *p<0.05; ***p<0.001.</p

MAPK and PI3K pathways are involved in eosinophil degranulation.

<p>AnnexinV⁻ eosinophils (A) and NK cells (B), cultured alone or together at the 10∶1 NK:Eos ratio, were stained with anti-ERK or anti-phosphorylated ERK (pERK) antibodies. Eosinophils and NK cells were identified on the basis of their size difference (FSC) and granularity (SSC). Live eosinophils were defined as AnnexinV⁻ cells. Gray full lines and black dotted lines represent control antibody staining for each cell type alone or in co-culture, respectively. Gray plain histograms and black full lines represent staining with antibody of interest for each cell type alone or in co-culture, respectively. One representative experiment out of three is shown. (C) Eosinophils were pre-incubated with DMSO as control or with inhibitors of signaling pathways for 5 min at 37°C: PD 98059 25 μM an inhibitor of ERK pathway, SB 203580 1 μM an inhibitor of P38 MAPK, SP 600125 20 μM an inhibitor of JNK pathway and LY 294002 10 μM an inhibitor of PI3K pathway for the NK:Eos ratio 10∶1. Results show percentage of CD63⁺ eosinophils for each donor and mean is indicated with the black line. One-way Anova (Friedman) tests were performed, followed by Dunn’s post tests. *p<0.05, **p<0.01.</p

NK cell-induced eosinophil apoptosis involves mitochondrial reactive oxygen species (ROS).

<p>(A) After purification, NK cells and eosinophils were cultured in the presence of IL-5 for 12 hours at different NK:Eos ratios: 0∶1 (i.e. eosinophils alone); 1∶1; 5∶1; 10∶1. Eosinophils were identified on the basis of their size difference (FSC) and granularity (SSC). ROS staining was achieved using dihydroethidium (HE). Results are expressed as mean percentage of HE⁺ cells amongt the eosinophil population ± SEM. n = 12. Gaussian distributions were verified using D’Agostino and Pearson test, and One-way Anova test was performed, followed by Bonferroni’s post test. *p<0.05; **p<0.01. (B) Eosinophils were incubated with inhibitors of mitochondrial electron transport: rotenone and antimycin for 30 min at 37°C before 12 h co-culture with NK cells at the NK:Eos ratio of 10∶1. Results for each donor are shown and mean is indicated with the black line. Wilcoxon tests were performed. *p<0.05.</p

NK cells induce eosinophil apoptosis.

<p>After purification, NK cells and eosinophils were cultured in the presence of IL-5 for 3 and 12 h at different NK:Eos ratios: 0∶1 (i.e. eosinophils alone); 1∶1; 5∶1; 10∶1. Eosinophils were identified on the basis of their size difference (FSC) and granularity (SSC). Apoptosis and death of eosinophils was evaluated by Annexin/PI labeling. Dead eosinophils were defined as AnnexinV⁺ cells. Results are expressed as mean percentage of AnnexinV⁺ cells amongt the eosinophil population ± SEM. n = 16 (3 hours) or 18 (12 hours). Gaussian distributions were verified using D’Agostino and Pearson test, and One-way Anova tests were performed, followed by Bonferroni’s post tests. *p<0.05; **p<0.01; ***p<0.001.</p

Eosinophil degranulation and apoptosis are both abrogated after PFA-induced fixation of NK cells and separation of cell types by transwell.

<p>After purification, NK cells and eosinophils were cultured in the presence of IL-5 for 3 hours at the NK:Eos ratios: 0∶1 (i.e. eosinophils alone) and 10∶1. (A) Percentage of CD63 positive eosinophils cultured with NK cells at the 0∶1 (i.e. eosinophils alone) or 10∶1 NK:Eos ratios in the same culture well (direct contact) or in Transwell (separated with 3 μm filter). (B) Percentage of CD63 positive eosinophils cultured alone, with live NK cells or with PFA-fixed NK cells for the 10∶1 NK:Eos ratio. (C) Percentage of AnnexinV⁺ eosinophils cultured with NK cells at the 0∶1 (i.e. eosinophils alone) or 10∶1 NK:Eos ratios in the same culture well (direct contact) or in Transwell (separated with 3 μm filter). (D) Percentage of AnnexinV⁺ eosinophils cultured alone, with live NK cells or with PFA-fixed NK cells for the 10∶1 NK:Eos ratio. Results are shown for each donor and mean is indicated with the black line. One-way Anova (Friedman) tests were performed, followed by Dunn’s post tests. *p<0.05; **p<0.01; ***p<0.001.</p

Polycyclic Aromatic Hydrocarbons Reciprocally Regulate IL-22 and IL-17 Cytokines in Peripheral Blood Mononuclear Cells from Both Healthy and Asthmatic Subjects

<div><p>Pollution, including polycyclic aromatic hydrocarbons (PAH), may contribute to increased prevalence of asthma. PAH can bind to the Aryl hydrocarbon Receptor (AhR), a transcription factor involved in Th17/Th22 type polarization. These cells produce IL17A and IL-22, which allow neutrophil recruitment, airway smooth muscle proliferation and tissue repair and remodeling. Increased IL-17 and IL-22 productions have been associated with asthma. We hypothesized that PAH might affect, through their effects on AhR, IL-17 and IL-22 production in allergic asthmatics. Activated peripheral blood mononuclear cells (PBMCs) from 16 nonallergic nonasthmatic (NA) and 16 intermittent allergic asthmatic (AA) subjects were incubated with PAH, and IL-17 and IL-22 productions were assessed. At baseline, activated PBMCs from AA exhibited an increased IL-17/IL-22 profile compared with NA subjects. Diesel exhaust particle (DEP)-PAH and Benzo[a]Pyrene (B[a]P) stimulation further increased IL-22 but decreased IL-17A production in both groups. The PAH-induced IL-22 levels in asthmatic patients were significantly higher than in healthy subjects. Among PBMCs, PAH-induced IL-22 expression originated principally from single IL-22- but not from IL-17- expressing CD4 T cells. The Th17 transcription factors <i>RORA</i> and <i>RORC</i> were down regulated, whereas AhR target gene <i>CYP1A1</i> was upregulated. IL-22 induction by DEP-PAH was mainly dependent upon AhR whereas IL-22 induction by B[a]P was dependent upon activation of PI3K and JNK. Altogether, these data suggest that DEP-PAH and B[a]P may contribute to increased IL22 production in both healthy and asthmatic subjects through mechanisms involving both AhR -dependent and -independent pathways.</p></div

Transcript levels of genes involved in Th17/Th22 polarization.

<p>Activated PBMCs from nonallergic (NA) subjects (n = 8) and allergic asthmatic (AA) patients (n = 11) were incubated with or without PAH for 72hr, and gene mRNA level was assessed by Q-RT-PCR. Results are expressed as mean relative expression (RE) of 2^(-ΔCt) ± SEM, where the ΔCt value of the sample was determined by subtracting the Ct value of the target gene from the Ct value of the rs9 house keeping gene. *<i>P</i><.05,**<i>P</i><.01, ^#<i>P</i><.05 and ^##<i>P</i> <.01 AA versus NA subjects.</p

Cytokine profile of PAH-stimulated peripheral blood mononuclear cells in allergic asthmatics (AA) and nonallergic subjects (NA).

<p>IL-17A, IL-22 and IL-10 secretion by activated peripheral blood mononuclear cells from NA subjects (n = 10) and AA patients (n = 12) stimulated or not with different PAH for 72hr was determined by ELISA. Results are expressed as mean ± SEM. *<i>P</i><.05 and **<i>P</i><.01 versus control. ^#<i>P</i><.05 and ^##<i>P</i><.01 AA versus NA subjects.</p

Effects of AhR antagonist on IL-22 and IL-17A secretion by peripheral blood mononuclear cells from Allergic asthmatics (AA) and nonallergic subjects (NA).

<p>Activated peripheral blood mononuclear cells from NA subjects (n = 10) and AA patients (n = 12) were incubated or not with PAH, in the presence or not of AhR antagonist CH-223191 for 72hr, and cytokine production was assessed by ELISA in the supernatants. Results are expressed as mean ± SEM. *<i>P</i><.05, **<i>P</i><.01. The dotted line is set on the level of the antagonist-treated control cells.</p

Effects of kinase inhibitors on IL-22 and IL-17A secretion.

<p>Activated peripheral blood mononuclear cells from nonallergic (NA) subjects (n = 8) and allergic asthmatic (AA) patients (n = 8) were incubated or not with PAH and with or without inhibitors of kinases for 72hr, and cytokine production was evaluated by ELISA in the supernatants. Results are expressed as mean ± SEM. *<i>P</i><.05, **<i>P</i><.01 ***<i>P</i><.001. The dotted line is set on the level of inhibitor-treated control cells for IL-22 and of control cells for IL-17 and indicates the level to achieve for complete dependence upon the pathway evaluated.</p