The online version of this article (doi:10.1186/s13601-017-0143-1) contains supplementary material, which is available to authorized users.
Anna Maria Riccio, Pierluigi Mauri contributed equally to this manuscript.
Bronchial asthma is a heterogeneous disease characterized by three cardinal features: chronic inflammation, variable airflow obstruction, and airway hyperresponsiveness. Asthma has traditionally been defined using nonspecific clinical and physiologic variables that encompass multiple phenotypes and are treated with nonspecific anti-inflammatory therapies. Based on the modulation of airway remodeling after 12 months of anti-immunoglobulin E (IgE) treatment, we identified two phenotypes (omalizumab responder, OR; and non-omalizumab responder, NOR) and performed morphometric analysis of bronchial biopsy specimens. We also found that these two phenotypes were correlated with the presence/absence of galectin-3 (Gal-3) at baseline (i.e., before treatment). The aims of the present study were to investigate the histological and molecular effects of long-term treatment (36 months) with anti-IgE and to analyze the behavior of OR and NOR patients.
All patients were treated with the monoclonal antibody anti-IgE omalizumab for 36 months. The bronchial biopsy specimens were evaluated using morphometric, eosinophilic, and proteomic analysis (MudPIT). New data were compared with previous data, and unsupervised cluster analysis of protein profiles was performed.
After 36 months of treatment with omalizumab, reduction of reticular basement membrane (RBM) thickness was confirmed in OR patients (Gal-3-positive at baseline); similarly, the protein profiles (over 500 proteins identified) revealed that, in the OR group, levels of proteins specifically related to fibrosis and inflammation (e.g., smooth muscle and extracellular matrix proteins (including periostin), Gal-3, and keratins decreased by between 5- and 50-fold. Eosinophil levels were consistent with molecular data and decreased by about tenfold less in ORs and increased by twofold to tenfold more in NORs. This tendency was confirmed (p < 0.05) based on both fold change and DAVE algorithms, thus indicating a clear response to anti-IgE treatment in Gal-3-positive patients.
Our results showed that omalizumab can be considered a disease-modifying treatment in OR. The proteomic signatures confirmed the presence of Gal-3 at baseline to be a biomarker of long-term reduction in bronchial RBM thickness, eosinophilic inflammation, and muscular and fibrotic components in omalizumab-treated patients with severe asthma. Our findings suggest a possible relationship between Gal-3 positivity and improved pulmonary function.
Additional file 1. Natural logarithm (ln) of fold change between baseline (T0) and long term anti-IgE treatment (T36) for each subject. Significant values (ln[Fold Change]>|0.6| are are reported in red or blue: positive ( blue) and negative ( red) values indicate increase and decrease at T36, respectively. Specifically, for ln(Fold Change) >0.6 ( blue) increase at T36; on the contrary, if it is <-0.6 ( red) decrease at T36. SMPs: smooth muscle proteins. aActual Fold Change was 0 (0/n, or 0/0, see Table 2); for avoiding invalid logarithm it was put at 1/n or 1/1; bActual Fold Change ∞ (n/0); for avoiding invalid logarithm it was put at n/1. * p-value; T-test2 is without NOR2 subject, because at T36 its behaviour is similar to ORs (se Fig. 6); in bold significant T-tests; ** #/mm2 ratio; *** score ratio.
Additional file 2. Complete list of identified proteins in OR (Responders) and NOR (Non-Responders) bronchial biopsies collected after 36 months of anti-IgE treatment. Accession number NCBI, Uniprot entry, Protein name, pI and MW, Spectral Count (SpC) and Score were reported for each protein. The symbol (*) was used to indicate the smooth muscle proteins. The symbol (†) was used to indicate the periostin. The symbol (°) was used to indicate the keratins.
Additional file 3. Virtual 2D map in logarithmic scale of FFPE bronchial biopsies, generated using MAProMa software (546 proteins). Proteins are plotted according to their theoretical pI and MW. A color/shape code is assigned to each protein according to SEQUEST score value. Proteins with score ≤15 are reported as yellow/ triangle, proteins with score ≥35 are reported as red/ circle, and proteins in the range 15–35 are reported as blue/ square. The protein lists are reported in Additional file 2.
Additional file 4. Changes of abundance levels, calculated by DAVE algorithm from MAPROMA software [ 9], for eosinoplis, smooth muscle proteins, periostin, keratins and Gal-3 in OR and NOR patients at baseline (T0) and after 36 months (T36) of anti-IgE treatment. Negative value indicates decrease at T36; on the contrary, positive value indicates increase at T36. OR and NOR classification is related to reduction (OR) or not (NOR) of RBM thickness after 12 months of anti-IgE treatment.
Additional file 5. Changes of abundance levels, expressed as natural logarithm of score fold change (ln[T36/T0]), for Gal-3 in OR and NOR patients at baseline (T0) and after 36 months (T36) of anti-IgE treatment. Negative value indicates decrease of Gal-3 at T36; on the contrary, positive value indicates increase of Gal-3 at T36 (see Additional file 1). OR and NOR classification is related to reduction (OR) or not (NOR) of RBM thickness after 12 months of anti-IgE treatment.
Additional file 6. Differential Analysis (DAVE index) between baseline (T0) and long term anti-IgE treatment (T36) for each subject, using DAVE index from MAPROMA software [ 9]. Significant values (DAVE > |0.4|) are reported in red or blue: positive ( blue) and negative ( red) values indicate increase and decrease at T36, respectively. SMPs: smooth muscle proteins. Of note, DAVE algorithm, tpical of proteomics evaluation, was also applied to eosinophils; the obtained values resulted in good agreement with evaluation obtained by ln[T36/T0] (see also Additional file 1 and compare Fig. 2 and Additional file 4). * p-value; T-test2 is without NOR2 subject, because at T36 its behaviour is similar to ORs (see Fig. 6); in bold significant T-tests.
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- Galectin-3: an early predictive biomarker of modulation of airway remodeling in patients with severe asthma treated with omalizumab for 36 months
Anna Maria Riccio
Laura De Ferrari
Dario Di Silvestre
Roberto Walter Dal Negro
Giorgio Walter Canonica
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