Level of Fatty Acid Binding Protein 5 (FABP5) Is Increased in Sputum of Allergic Asthmatics and Links to Airway Remodeling and Inflammation

<div><p>Background</p><p>The inflammatory processes in the upper and lower airways in allergic rhinitis and asthma are similar. Induced sputum and nasal lavage fluid provide a non-invasive way to examine proteins involved in airway inflammation in these conditions.</p><p>Objectives</p><p>We conducted proteomic analyses of sputum and nasal lavage fluid samples to reveal differences in protein abundances and compositions between the asthma and rhinitis patients and to investigate potential underlying mechanisms.</p><p>Methods</p><p>Induced sputum and nasal lavage fluid samples were collected from 172 subjects with 1) allergic rhinitis, 2) asthma combined with allergic rhinitis, 3) nonallergic rhinitis and 4) healthy controls. Proteome changes in 21 sputum samples were analysed with two-dimensional difference gel electrophoresis (2D-DIGE), and the found differentially regulated proteins identified with mass spectrometry. Immunological validation of identified proteins in the sputum and nasal lavage fluid samples was performed with Western blot and ELISA.</p><p>Results</p><p>Altogether 31 different proteins were identified in the sputum proteome analysis, most of these were found also in the nasal lavage fluid. Fatty acid binding protein 5 (FABP5) was up-regulated in the sputum of asthmatics. Immunological validation in the whole study population confirmed the higher abundance levels of FABP5 in asthmatic subjects in both the sputum and nasal lavage fluid samples. In addition, the vascular endothelial growth factor (VEGF) level was increased in the nasal lavage fluid of asthmatics and there were positive correlations between FABP5 and VEGF levels (r=0.660, p<0.001) and concentrations of FABP5 and cysteinyl leukotriene (CysLT) (r=0.535, p<0.001) in the nasal lavage fluid.</p><p>Conclusions</p><p>FABP5 may contribute to the airway remodeling and inflammation in asthma by fine-tuning the levels of CysLTs, which induce VEGF production.</p></div

False colour two-dimensional differential gel electrophoresis (2D-DIGE) image of identified protein spots in sputum.

<p>False colour image of the identified protein spots (circled and named) on a 2-D DIGE-gel of the sputum fluid phase samples of 21 subjects subdivided as follows: asthma with allergic rhinitis, allergic rhinitis, nonallergic rhinitis, and healthy controls. Samples of the subjects with asthma with allergic rhinitis (reddish up-regulated) and with nonallergic rhinitis (green up-regulated), as well as the internal standard are shown. The identified proteins are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127003#pone.0127003.s005" target="_blank">S1 Table</a>.</p

Immunological confirmation of findings.

<p>2D-DIGE gel spot abundances for the FABP5 of the 21 induced sputum samples (A). Western blot analysis for 138 induced sputum samples (B) and for 158 nasal lavage fluid samples (C) with FABP5 antibody. ELISA results for vascular endothelial growth factor (VEGF) (D) and cysteinyl leukotriene (CysLT) (E) measurements from 90 nasal lavage fluid samples. The figures show mean with standard error of the mean. * p < 0.05, **p < 0.01. AR = allergic rhinitis, Asthma+AR = asthma and allergic rhinitis, NAR = nonallergic rhinitis.</p

Heatmap of up- and down-regulated protein gel spots in sputum (A) and in nasal lavage fluid (B).

<p>Hierarchical clustering reveals the differences between the protein regulation of the groups, indicating dissimilarities between asthma (Asthma+AR), allergic rhinitis (AR) and healthy controls, whereas the nonallergic rhinitis (NAR) group shows the least differences to the up- or down-regulation of the healthy controls.</p

Characteristics of study subjects.

<p>*Patients with a negative control wheal of ≥ 2mm were excluded from skin prick test (SPT) analysis n = 166. IgE, immunoglobulin E; FEV1, forced expiratory volume in one second; FVC, forced vital capacity, FeNO, exhaled nitric oxide</p><p>Characteristics of study subjects.</p

Study flow chart.

<p>Study flow chart.</p

Relationships between differently abundant sputum proteins in study groups.

<p>Average ratios of statistically significant (p-value ≤ 0.05) up- and down-regulated sputum proteins are presented for the allergic rhinitis (AR), asthma and allergic rhinitis (asthma+AR) and non-allergic rhinitis (NAR) groups in comparison to the control group, and for AR and NAR groups in comparison to the asthma+AR group. More thorough identification parameters from the Mascot protein search can be found in supplement <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127003#pone.0127003.s005" target="_blank">S1 Table</a>.</p><p>Relationships between differently abundant sputum proteins in study groups.</p

Characteristics of subjects selected for proteomic analysis.

<p>* One subject had concurrent atopic exzema and total IgE 2713 kU/l: Total IgE varied between 30 and 210 kU/l in the other subjects in the allergic rhinitis group. SPT, skin prick test; IgE, immunoglobulin E; FVC, forced vital capacity; FEV1, forced expiratory volume in one second; FeNO, exhaled nitric oxide</p><p>Characteristics of subjects selected for proteomic analysis.</p