Ten subjects with severe asthma (6 males and 4 females, mean age 33.3 ± 2.6) who met the criteria defined by ATS on refractory asthma 23 were recruited. To be classified as severe asthmatics, patients must have had high-dose inhaled corticosteroid: Budesonide 160 μg/twice a day (or equivalent) or daily anti-leukotriene for >50% of the last year, and at least 1 other add-on therapy on daily basis for the previous 12 months. They were also required to have two of the following criteria: daily short-acting β-agonist, persistent FEV1 <60% and FEV1/FVC <75% predicted, 1 urgent visit or at least 3 steroid bursts in the previous year, prompt deterioration with <25% steroid dose reduction, or previous near-fatal asthma within the last 3 years. Subject characteristics are summarized in Table  1. Exclusion criteria included smoking history or any other pulmonary diseases or co-existing medical conditions such as cardiac and renal diseases and uncontrolled hypertension. Ten normal control subjects (6 males and 4 females, mean age: 38.2 ± 3.4) were also recruited. All normal control subjects were non-smokers with normal lung function, no history or symptoms of allergy and respiratory diseases, and were not taking any medications for the preceding four weeks. The Ethics Committee of the King Khalid University Hospital in Riyadh reviewed and approved the study, and all subjects recruited signed written informed consent for the drawing of peripheral venous blood for the isolation of eosinophils.

Except for the medications indicated above, the patients were not receiving any other immunosupressive drugs. FEV ₁ forced expiratory volume; FVC forced vital capacity.

Peripheral venous blood were drawn from patients with severe asthma (120-180 ml) (60 ml every 3 months) and from normal control subjects (180-240 ml) (60 ml every 3 months). Eosinophils were isolated by negative selection using MACS Isolation Kit (Miltenyi Biotec, Auburn, CA, USA) as previously described 24 . Neutrophils, monocytes and T cells were labeled with anti-CD16, anti-CD14 and anti-CD3 Abs respectively bound to immunemagnetic beads and separated with MACS LD Separation column. Eosinophil purity was consistently >98% as evaluated by Hema3 (Fisher) staining and the viability of freshly isolated eosinophils was >99% as evaluated by Trypan blue dye exclusion. Isolated eosinophils were then cultured in RPMI + 10% FCS in the presence of 30 pg/ml IL-5 cytokine required for eosinophil survival in vitro 25 26 . Eosinophil viability ranged between 85 and 92% following stimulation and culture.

Eosinophils (2×10⁶ cells/ml cultured in 24 well plate) were stimulated with Th1 (IL-2, IFN-γ) (50 ng/ml), Th2 (IL-4, IL-5, IL-9, IL-13) (50 ng/ml), and Th17 (IL-17A, IL-17 F, IL-23) (10, 25, 50, or 100 ng/ml) cytokines (R&D Systems, Minneapolis, Minn., USA) for 24 hrs and supernatants were collected. In some experiments, eosinophils were treated with p38 mitogen-activated protein kinase (MAPK) inhibitors (SB 203580; 5 μM, Invivogen San Diego, CA, USA) or PI3K inhibitor (PI103; 5 μM, Cayman Chemical, Ann Arbor, Mich., USA) 2 hours prior to stimulation with IL-17. Levels of secreted TGF-β and IL-11 in supernatants were determined using ELISA assay (R&D Systems, Minneapolis, Minn., USA) according to the manufacturer instructions.

Eosinophils were stimulated with cytokines (Th1 (50 ng/ml), Th2 (50 ng/ml) or Th17 (50 ng/ml)) for 4 hours prior to cell harvest. In some experiments, eosinophils were treated with p38 MAPK inhibitors (SB 203580; 5 μM), or PI3K inhibitor (PI103; 5 μM) 2 hours prior to stimulation with IL-17. Cells were then harvested, total RNA extracted (levels of RNA extracted from 2×10⁶ eosinophils were 18.4 ± 4.7 μg for asthmatic eosinophils and 16.3 ± 3.9 μg for healthy controls) (RNeasy Mini kit, Qiagen, CA, USA) and modulations of the level of expression of TGF-β and IL-11 mRNA were determined using quantitative RT-PCR (Applied Biosystems, 7900 Fast RT-PCR system). Specific primers for TGF-β and IL-11 were as follows: TGF-β: Forward: 5-CTGGACACCCTAACCGTGAT-3, Reverse: 5-CTAGGCCGTGCTGCTGCT-3; IL-11: Forward: 5-GTGGCCAGATACAGCTGTCGC-3, Reverse: 5- GGTAGGACAGTAGGTCCGCTC-3. Relative expressions of TGF-β and IL-11 genes normalized with GAPDH were determined by the delta-delta Ct method 27 .

2×10⁶ eosinophil cells were starved using medium with 0.1% FBS for 18 hours. Cells were stimulated with 50 ng/mL IL-17A and IL-17 F for 0, 10, and 20 minutes and total proteins were extracted using lysis buffer (1% Triton X-100 containing protease and phosphatase inhibitor cocktails (Roche, Mannheim, Germany). Protein lysates (10 μg) were then resolved on 10% acrylamide SDS-PAGE gel and blots were probed with antibodies to phosphorylated p38 MAPK (Millipore) and total p38 MAPK (Millipore). Membranes were analyzed with an Odyssey IR scanner using Odyssey imaging software 3.0 (LI-COR Biosciences, Inc).

Data are presented as mean ± SD. Expression of pro-fibrotic cytokines was evaluated using ANOVA followed by Bonferroni-Dunn post hoc test. Non-parametric Mann–Whitney U test (Systat, version 7.0, SPSS, Chicago, IL) was used to evaluate significance in differential phosphorylation of MAPK. Values of p < 0.05 were considered statistically significant.

The patho-physiological characteristics of lung tissue inflammation during severe asthma differ significantly from those of the milder disease. While the airway tissues of mild asthmatics usually present preferential Th2 cytokine profile 28 , those from severe asthmatics show a Th17 lymphocyte infiltration and elevated cytokine levels, particularly Th1 cytokines (IFN-γ, IL-2), IL-17 and TGF-β 29 30 31 . Many T-helper cytokines were shown to play a significant role in regulating TGF-β expression and function in different types of cells 32 33 34 . However, their direct role in regulating eosinophil ability to produce pro-fibrotic cytokines was not studied. To investigate that, we first determined the basal expression levels of pro-fibrotic cytokines within peripheral blood eosinophils of 10 asthmatic and non-asthmatic individuals using real time RT-PCR. The levels of expression of TGF-β and IL-11 mRNA in eosinophils isolated from asthmatic individuals (ct values: TGF-β: 27.53 ± 0.21 IL-11: 28.80 ± 1.2) were comparable to those isolated from healthy controls (ct values: TGF-β: 27.70 ± 0.29 IL-11: 29.56 ± 0.86) (Figure  1A). Eosinophil supernatant IL-11 and TGF-β cytokines levels were also determined in the two groups using ELISA assay (Figure  1B). Similarly, no change in the secreted levels of these pro-fibrotic cytokines was detected between the two groups. We then investigated whether Th1 and Th2 cytokines play a role in regulating eosinophils pro-fibrotic cytokines production. To do that, we stimulated 2×10⁶ eosinophil cells isolated from 10 asthmatic as well as healthy individuals with Th1 (IL-2, IL-12, and IFN-γ), and Th2 (IL-4, IL-5, IL-9, and IL-13) cytokines as well as GM-CSF for 4 hrs. Total RNA was then extracted from stimulated eosinophils and the level of IL-11 and TGF-β was determined using real time RT-PCR. As shown in Figure 1C-D, stimulating asthmatic eosinophils with Th1 or Th2 cytokines did not affect TGF-β (ct values range: 27.63 ± 0.21 to 27.58 ± 0.79) (Figure  1C) or IL-11 (ct values range: 28.67 ± 0.84 to 28.76 ± 0.18) (Figure  1D) m-RNA levels. Similar results were obtained at higher concentrations of Th1 and Th2 cytokines (100 ng/ml) as well as for eosinophils isolated from healthy controls (data not shown). These results indicated that neither Th1 nor Th2 cytokines play a significant role in regulating expression of eosinophil derived pro-fibrotic cytokines.

IL-17A enhanced the production of IL-6 and IL-11 in bronchial fibroblasts 22 while IL-17 F was shown to induce the expression of TGF-β in human umbilical vein endothelial cells (HUVECs) 35 . IL-17A and IL-17 F were recently shown to be over expressed in bronchial lung tissue of asthmatic patients compared to healthy controls 29 and their level of expression was associated with the severity of the diseases. Interestingly, using FACS and western analysis, eosinophils were also shown to express receptors for Th17 cytokines 16 . We, therefore, hypothesised that Th17 cytokines may induce eosinophils to produce pro-fibrotic cytokines. To investigate that, we first determined the expression levels of IL-17R on eosinophils isolated from both groups. As indicated in Figure  2A, eosinophils from both healthy and asthmatic subjects express IL-17R. Although asthmatic eosinophils express higher levels of IL-17R, this increase did not reach significance. We next stimulated 2×10⁶ eosinophils, isolated from 10 severe asthmatic patients and 10 healthy controls, with IL-17A, IL17F, as well as IL-23, another Th17 cytokine for 4 hrs. Total RNA was then extracted and eosinophil expression of TGF-β and IL-11 mRNA was measured using real-time PCR. As shown in Figure  2B, contrary to stimulating eosinophils with IL-17A and IL-17 F alone, stimulation with a combination of IL-17A + F (ct value: 24.39 ± 0.17, P = 0.031; n = 10), or IL-23 (ct value: 24.42 ± 0.21, P = 0.025; n = 10) alone, induced a significant increase in the expression of eosinophil derived TGF-β. Further increase in TGF-β expression was observed when stimulating with double the amount of the combined cytokines IL-17A + F (ct value: 24.06 ± 0.4, P = 0.012; n = 10) and or IL-17A + F + IL-23 (ct values: 23.98 ± 0.16, P = 0.018; n = 10). Interestingly, this increase in TGF-β production was only observed within eosinophils isolated from asthmatic patients. Stimulation of eosinophils isolated from non-asthmatic individuals with Th17 cytokines had no effect on TGF-β production (ct values: IL-17A + F: 25.29 ± 0.15; IL-23: 25.22 ± 0.18; IL-17A + F (50 ng): 25.36 ± 0.14; IL-17A + F + 23 (50 ng): 25.78 ± 0.11, p = NS) (Figure  2B). Similarly, a combination of IL-17A and IL-17 F at different concentrations or IL-17A + F + IL-23 induced a significant increase in IL-11 mRNA expression within eosinophils isolated from asthmatics (ct values: IL-17A + F (25 ng): 24.61 ± 0.37, p = 0.029; IL-17A + F (50 ng): 23.71 ± 0.70, p = 0.009; IL-17A + F + 23 (50 ng): 23.80 ± 0.37, p = 0.014, n = 10) but not healthy subjects (ct values: IL-17A + F (25 ng): 25.18 ± 0.10; IL-17A + F (50 ng): 25.10 ± 0.11; IL-17A + F + 23 (50 ng): 25.00 ± 0.13, p = NS) (Figure  2C). To determine effective concentration inducing eosinophils release of TGF-β and IL-11 cytokines, a dose response effect of Th17 cytokines was performed. Eosinophils were treated with increasing concentration of Th17 cytokines and levels of TGF-β and IL-11 in their supernatant were determined using ELISA assay (Figure  3A). Although low concentrations of Th17 cytokines induced pro-fibrotic cytokine secretion, a significant enhancement of TGF-β and IL-11 release was only attained at 50 ng/ml and above. At this concentration, the level of eosinophil derived TGF-β was significantly increased following treatment with a combination of IL-17A + F (P = 0.032; n = 10), IL-23 (P = 0.049; n = 10) alone, or IL-17A + F + IL-23 (P = 0.043; n = 10) (Figure  3A). Similarly, IL-11 secreted levels were significantly upregulated following stimulation with a combination of IL-17A + F (P = 0.035; n = 10), IL-23 (P = 0.048; n = 10) alone, or IL-17A + F + IL-23 (P = 0.043; n = 10) (Figure  3B). This data suggest that, in an asthmatic environment, an additive effect of Th17 cytokines enhance the production of eosinophils derived pro-fibrotic cytokines.

P38 mitogen-activated protein kinase (MAPK), being at a critical junction of the IL-17 signaling pathways, has been shown by various reports to be a key regulator element for the activity of IL-17 cytokines 15 16 36 . To study the mechanism behind Th17 cytokines enhancement of eosinophil derived TGF-β production, eosinophils were isolated from peripheral blood of 10 asthmatic patients as described above. 2×10⁶ cells were treated, or not, with p38 MAPK or PI3K inhibitors (SB2035802 and PI103, respectively), or diluent control (DMSO) 2 hours prior to stimulation with IL-17. As shown in Figure  4, inhibiting phosphorylation of p38 MAPK significantly decreased the level of TGF-β (P = 0.011 (IL-17A + F); P = 0.015 (IL-17A + F + 23); n = 10) and IL-11 (P = 0.021 (IL-17A + F); P = 0.026 (IL-17A + F + 23); n = 10) secreted into eosinophil supernatants 24 hrs following Th17 cytokine stimulation (Figure  4A, B). This blocking effect was only specific to p38 MAPK as diluent control or inhibitor of another kinase (PI3K) did not affect the supernatant levels of TGF-β and IL-11 (P = NS; n = 10). This data indicated that p38 MAPK activation is critical for IL-17 induced eosinophil derived pro-fibrotic cytokine production. To confirm p38 MAPK phosphorylation following treatment with IL-17 cytokines, 2×10⁶ eosinophil cell were treated with IL-17A + F (50 ng/ml each) for 0, 10 and 20 minutes and the level of p38 MAPK phosphorylation was then determined using western analysis. As shown in Figure  4C, stimulating eosinophils with a combination of IL-17A and IL-17 F resulted in phosphorylation of p38 MAPK which seems to peak at 10 minutes (3.5 fold increase, p = 0.039). Inhibiting p38 MAPK, PI3K, or ERK1/2, however, did not interfere with the ability of IL-23 to stimulate eosinophil to produce pro-fibrotic cytokines. This indicated that IL-23 may use other mechanisms to stimulate pro-fibrotic cytokine release that need to be further investigated.