Effect of Biologicals on Alternative Functions of Eosinophils in Severe Asthma

Severe asthma is observed in 3 to 5% of the asthmatic population and is characterized by a higher rate of exacerbations, poorly controlled asthma and/or a poor lung function. During the last ten years, anti- Immunoglobulin E (IgE) and anti-IL5 (and anti-IL5R) have considerably improved severe asthma status. Treatment with chronic or oral course of corticosteroids is indeed responsible for a lot of side effects. There is however a lack of biomarkers for the prediction of responders to anti-IL5 and anti-IgE.

The investigators have previously found that sputum eosinophil counts are good predictors for improvement in lung function of severe asthmatics using anti-IL5. Sputum cells might not be the only relevant sputum marker for responders to biologics.

The objective is to evaluate the ability of sputum cytokines (protein levels and genes) (IL-3, IL-4, IL-5, IL-13, IL-33 and GM-CSF) to predict the response of severe asthmatics after 6 months and 1 year of treatment with biologics in terms of reduction in exacerbations and corticosteroid use, improvement in FEV1, in asthma control, in asthma quality of life and the effect on sputum and blood inflammation.

Severe asthmatics seen in the Asthma Clinic of CHU of Liege are very well characterized using baseline measure of blood samples, induced sputum, lung function, fractional exhaled nitric oxide (FeNO), asthma control questionnaires (ACT - ACQ) and asthma quality of life questionnaires (AQLQ).

Data on exacerbations during the previous year and current inhaled and oral treatment is also recorded. If FEV1 value is upper than 70% of the predicted value, severe asthmatics also perform a methacholine challenge to evaluate bronchial hyperresponsiveness.

All these measures are repeated after 6 months and 1 year of treatment with anti-IgE and anti-IL5 or anti-IL5R. All the sputum supernatants are stored at -80°C and RNA from sputum cells is also available.

Target sample size: 50 patients

The investigators plan to measure different cytokines in the sputum (IL3, GM-CSF, IL5, IL-13, IL-33, IL-4…) and to evaluate their ability to predict the response after 6 months and 1 year of treatment with different biologics in terms of reduction in exacerbations and corticosteroid use, improvement in FEV1 (+200ml), in asthma control (ACQ decrease >0.5, ACT increase >3), in asthma quality of life (increase in AQLQ score > 0.5) and the effect on sputum and blood inflammation.

Asthmatic patients are recruited through the outpatient clinic and pulmonary rehabilitation centre (CHU, Sart-Tilman, Liege). Asthma is diagnosed as described in the GINA guidelines.

Sputum induction and processing. The sputum is induced and processed as follow: The whole sputum is collected in a plastic container, weighed, and homogenized with three volumes of phosphate-buffered saline (PBS), vortexed for 30 s, and centrifuged at 800g for 10 min at 4° C. The supernatant is separated from the cell pellet by filtration through 2 layers of sterile gauze. A mucolysis is performed by adding an equal volume of 6.5 mM dithiothreitol and the suspension is rocked during 20 min. After a centrifugation of 10 min at 550g, the squamous cells and total cell counts as well as the cell viability are checked by trypan blue exclusion with a manual hemocytometer. The differential leukocyte count is performed on cytospins stained with May-Grünwald-Giemsa on 500 cells.

If the quality criteria (< 30% of squamous cells and viability > 50%) are respected, the cell pellet is mixed with 5 volumes of RNAprotect cell reagent (Qiagen, Hilden, Germany) and kept at -80 °C until RNA extraction.

In total, 50 patient with successful induced sputum supernatant samples before anti-IL-5, anti-IL5R or anti-IgE therapy as well as 6 months and 1 year after will be analyzed. Regarding the sputum cells, patients with good quality samples before and after therapy will allow gene expression analysis.

Immunoassays: The concentrations of the mediators contained in the sputum supernatant are assessed by ELISA Luminex performance assay (R and D systems, Minneapolis, USA) according to the manufacturer's instructions. Spiking experiments of cytokines in sputum supernatants are performed to check that the recovery is between 80% and 120% for all the analytes.

The mediator protein levels will be obtained using Luminex performance XL discovery kit (R and D systems, Minneapolis, USA) for IL-3 and IL-4 (detection limit : 0.09 pg/ml for both). A Luminex high sensitivity performance kit will be used for IL-5, IL-13, IL-33 and GM-CSF. Detection limits are 0.4, 5.1, 1.8 and 1.6 pg/ml respectively.

RNA extraction and RT-qPCR methods Regarding the mediator gene expression levels, primers and probes will all be obtained from IDT (Integrated DNA Technologies, Skokie, IL, USA). PCR efficiencies will be calculated using qbase+ qPCR analysis software (Biogazelle, Zwijnaarde, Belgium). The same program will be used to obtain relative quantitation in gene expression using the 2-∆∆Ct method. HPRT1 and GNB2L1 will be used as reference genes. The patients will be compared with a cohort of 7 healthy subjects.

The Taqman PCR step is performed in 96-well plates and for each sample, 2 µl of cDNA (diluted 1:4) is used in a total reaction volume of 12.5 µl including 500 nM forward primer, 500 nM reverse primer and 250 nM probe for all the tested genes. Every qPCR are realized in duplicate and included non-template controls for each gene. Amplification is performed on the LightCycler 480 Real-Time PCR (Roche, Pleasanton, CA, USA) during 45 cycles as follows: initial activation step: 95 °C for 15 min; denaturation stage: 94 °C for 15 s; annealing and elongation stage: 60 °C for 1 min. All the procedure is done according to the MIQE guidelines for the minimum information required for a qPCR experiment.

Cytokines will also be measured in the blood of a subset of patients.

Study Endpoints

Primary endpoint:

To identify predictors of reduction in exacerbations and in oral corticosteroids dose.

Secondary endpoint:

To identify predictors of improvement in ACQ (-0.5), ACT (+3pts), AQLQ (+ 0.5), sputum eosinophils (<3%), FEV1 (+ 200ml).

Statistical Plan or Data analysis The results will be expressed as mean¡SD or mean¡SEM for continuous variables; median (range) for skewed distributions. For categorical variables, the number of observations and percentages will be given in each category. Comparisons between different subgroups will be performed with a Kruskal-Wallis test or paired t-test / Wilcoxon signed rank test. The Spearman correlation coefficient will be used to measure the association between clinical parameters.

Power calculations indicated a required total sample size of 50 subjects to confirm a change in outcomes.