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

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

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

Cytokine profile and cell expression of PAH-stimulated peripheral blood mononuclear cells cultured in Th17 conditions.

<p><b>A</b> IL-17A and IL-22 production by activated peripheral blood mononuclear cells from 6 nonallergic (NA) subjects and 6 allergic asthmatic (AA) patients stimulated or not with SRM or B[a]P for 6 days was determined by ELISA. Results are expressed as mean ± SEM. *<i>P</i><.05 versus corresponding control. ^#<i>P</i><.05 AA versus NA subjects. <b>B</b> Activated peripheral blood mononuclear cells were stimulated or not with SRM and B[a]P, stained with antibodies against cytokine and cell surface markers, and evaluated by flow cytometry. Results are expressed as mean ± SEM percentage of cytokine positive cell populations for n = 7–11 subjects. *<i>P</i><.05 versus corresponding control, ^##<i>P</i><.01, ^###<i>P</i><.001.</p

Th17/Th22 type profile of anti-CD3/CD28-activated peripheral blood mononuclear cells.

<p><b>A</b> Cytokine secretion by activated peripheral blood mononuclear cells from nonallergic (NA) subjects (n = 10) and allergic asthmatic (AA) patients (n = 12), was evaluated at 72hr by ELISA and expressed as mean ± SEM ng/ml. <b>B</b> Gene mRNA level in activated peripheral blood mononuclear cells from NA subjects (n = 8) and AA patients (n = 11), was evaluated at 72hr by Q-RT-PCR and 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, and ***<i>P</i><.001.</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