Perea et al. Respiratory Research (2020) 21:21 https://doi.org/10.1186/s12931-020-1278-5 RESEARCH Open Access Reduced airway levels of fatty-acid binding protein 4 in COPD: relationship with airway infection and disease severity Lídia Perea1, Ana Rodrigo-Troyano2, Elisabet Cantó1, Marisol Domínguez-Álvarez3,4, Jordi Giner2, Ferran Sanchez-Reus5, Judit Villar-García6, Sara Quero4,7, Marian García-Núñez4,7,8, Alicia Marín4,8, Eduard Monsó4,7, Rosa Faner4,9, Alvar Agustí4,9, Silvia Vidal1† and Oriol Sibila2*† Abstract Background: For still unclear reasons, chronic airway infection often occurs in patients with Chronic Obstructive Pulmonary Disease (COPD), particularly in those with more severe airflow limitation. Fatty-acid binding protein 4 (FABP4) is an adipokine involved in the innate immune response against infection produced by alveolar macrophages (Mɸ). We hypothesized that airway levels of FABP4 may be altered in COPD patients with chronic airway infection. Methods: In this prospective and controlled study we: (1) compared airway FABP4 levels (ELISA) in induced sputum, bronchoalveolar lavage fluid (BALF) and plasma samples in 52 clinically stable COPD patients (65.2 ± 7.9 years, FEV1 59 ± 16% predicted) and 29 healthy volunteers (55.0 ± 12.3 years, FEV1 97 ± 16% predicted); (2) explored their relationship with the presence of bacterial airway infection, defined by the presence of potentially pathogenic bacteria (PPB) at ≥103 colony-forming units/ml in BALF; (3) investigated their relationship with the quantity and proportion of Mɸ in BALF (flow cytometry); and, (4) studied their relationship with the severity of airflow limitation (FEV1), GOLD grade and level of symptoms (CAT questionnaire). Results: We found that: (1) airway levels of FABP4 (but not plasma ones) were reduced in COPD patients vs. controls [219.2 (96.0–319.6) vs. 273.4 (203.1–426.7) (pg/ml)/protein, p = 0.03 in BALF]; (2) COPD patients with airway infection had lower sputum FABP4 levels [0.73 (0.35–15.3) vs. 15.6 (2.0–29.4) ng/ml, p = 0.02]; (3) in COPD patients, the number and proportion of Mɸ were positively related with FABP4 levels in BALF; (4) BALF and sputum FABP4 levels were positively related with FEV1, negatively with the CAT score, and lowest in GOLD grade D patients. Conclusions: Airway FABP4 levels are reduced in COPD patients, especially in those with airway infection and more severe disease. The relationship observed between Mɸ and airway FABP4 levels supports a role for FABP4 in the pathogenesis of airway infection and disease severity in COPD. Keywords: FABP4, Chronic obstructive pulmonary disease, Macrophages, Bronchoalveolar lavage fluid * Correspondence: osibila@santpau.cat † Silvia Vidal and Oriol Sibila share the senior authorship 2 Respiratory Department, Hospital de la Santa Creu i Sant Pau, Biomedical Research Institute Sant Pau (IIB Sant Pau), Autonomous University of Barcelona, Barcelona, Spain Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Perea et al. Respiratory Research (2020) 21:21 Page 2 of 8 Introduction Participants Chronic Obstructive Pulmonary Disease (COPD) cur- The diagnosis of COPD was established according to the rently is the third leading cause of mortality worldwide Global Initiative for Chronic Obstructive Lung Disease [1]. Chronic airway bacterial infection often occurs in (GOLD) guidelines [12]. The inclusion criteria were: 8 COPD patients, particularly in those with more severe weeks of clinical stability (defined by the absence of an airflow limitation [2, 3]. The presence of airway infection exacerbation that required oral corticosteroids and/or increases the economic impact of the disease and antibiotic treatment), age between 40 and 75 years, and worsens clinical outcomes including increased mortality FEV1 between 20 and 70% predicted. All patients under- [4]. Several studies have demonstrated that innate im- went a computerized tomography scan, and those with munity alterations favor airway infection in COPD [5, 6]. bronchiectasis, lung cancer, pneumonia, and/or intersti- However, the underlying biological mechanisms leading tial lung diseases were excluded. Other exclusion criteria to chronic airway infection in COPD have not been yet were patients with active malignant disease and/or any fully elucidated. type of immunosuppression, drug addiction or alcohol Alveolar macrophages (Mɸ) are a central component abuse. As controls, we included adult volunteers without of the innate immune response against airway infection. respiratory diseases and with normal spirometry re- Several alterations of Mɸ in marker expression and func- cruited in these same centers [13]. tions have been previously described in COPD [7, 8]. Among many other functions, Mɸ produce fatty acid- Clinical assessment binding protein 4 (FABP4), also known as adipocyte A- Demographic data, the number of exacerbations in the FABP or aP2. FABP4 is a member of the FABP family of previous year, the time from last exacerbation, relevant small-molecular weight intracellular lipid chaperones that comorbid conditions and current treatments were re- functions as a secreted adipokine and plays a role in air- corded at inclusion using standardized questionnaires. All way defense against infection [9, 10]. For instance, in an patients underwent spirometry (Datospir-600; Sibelmed experimental model of Pseudomonas aeruginosa infection SA, Barcelona, Spain) following international recommen- in mice, the presence of FABP4 protected against airway dations. Reference values were those of Mediterranean infection [11]. However, limited data on COPD and its re- populations [14]. lationship with disease severity and airway infections are available. We hypothesized that airway FABP4 levels may be re- Samples collection and processing duced in COPD patients, especially in those with the more Bronchoalveolar lavage fluid (BALF), induced sputum severe disease and with potentially more dysfunctional and plasma were obtained from all participants and were Mɸ. Accordingly, this study sought to: (1) compare airway processed immediately. BALF samples were recovered (and plasma) FABP4 levels in COPD and healthy volun- using 150 ml saline lavage with the bronchoscope teers and, (2) study their relationship with presence of air- wedged in the right middle lobe. BALF samples were way infection, quantity and proportion of Mɸ and several centrifuged at 800 xg for 10 min to obtain the cellular clinical markers of disease severity, such as the severity of pellet and the supernatant. Induced sputum was col- airflow limitation (FEV1), the GOLD classification and the lected just before the bronchoscopy as previously de- level of symptoms (CAT questionnaire). scribed [15]. Sputum samples were disaggregated using dithiothreitol (Oxoid Ltd., Hampshire, United Kingdom) for 15 min and were centrifuged at 600 xg for 6 min to ob- Methods tain the supernatant. Proteases inhibitors (Calbiochem, Study design and ethics San Diego, CA) were added to the supernatants during This was a prospective, multicenter, cross-sectional thawing. Plasma samples were obtained from blood col- study that included clinically stable COPD patients and lected in ethylenediamine tetra-acetic acid (EDTA) tube healthy volunteers with normal lung function, who and centrifuged at 850 xg for 10 min. BALF and sputum served as controls. Participants were recruited from five supernatants and plasma were stored immediately at − university tertiary hospitals [Hospital de la Santa Creu i 80 °C until analysis. Sant Pau (Barcelona, Spain), Hospital del Mar (Barce- lona, Spain), Hospital Universitari Parc Taulí (Sabadell, Microbiological study Spain), Hospital Germans Trias i Pujol (Barcelona, Samples were processed for qualitative and quantitative Spain) and Hospital Clínic (Barcelona Spain)]. The study bacteriology, as previously described [16]. Airway infec- protocol was approved by the local institutional review tion was defined as the presence of potentially patho- board (IIBSP-MIC-2015-57) and all subjects gave signed genic bacteria (PPB) at ≥103 colony-forming units/ml in informed consent. BALF [17] in clinically stable patients. Perea et al. Respiratory Research (2020) 21:21 Page 3 of 8 FABP4 measurement Table 1 Demographics and clinical characteristics among FABP4 levels were measured by validated, commercially controls and COPD patients available ELISA kit (RayBiotech, Peachtree Corners, GA) Controls (n = 29) COPD (n = 52) P value according to the manufacturer’s instructions. The limit Age 55.0 ± 12.3 65.2 ± 7.9 0.0002 of kit detection was 38 pg/ml. The dilutions used were Male, n (%) 22 (75.9) 34 (65.4) 0.6 1/7 for BALF supernatants, 1/5 for sputum supernatants BMI (kg/m2) 28.7 ± 9.7 26.3 ± 4.7 0.7 and 1/75 for plasma. BALF FABP4 levels were adjusted Smoking status, n (%) to the total protein content quantified using Qubit fluorometer (Invitrogen, Carlsbad, CA). Never 5 (17.2) 0 (0.0) < 0.0001 Former 10 (34.5) 41 (78.8) BALF flow cytometry Current 14 (48.3) 11 (21.1) In COPD patients, cellular pellet obtained from BALF Pack-years 26.0 ± 19.0 43.5 ± 19.2 0.002 samples was lysed to avoid red blood cells contamin- FEV1 (% pred) 97 ± 16 59 ± 16 < 0.0001 ation (RBC lysing solution; BioLegend, San Diego, CA). FVC (% pred) 92 ± 15 85 ± 16 0.07 Cells were resuspended in one ml of PBS supplemented with 2% bovine serum albumin (BSA; Roche Diagnostics FEV1/FVC 0.79 ± 0.05 0.52 ± 0.14 < 0.0001 GmbH, Mannheim, Germany) to quantify the number of Data is presented as mean ± SD unless otherwise indicated total cells in a MACSQuant cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were adjusted to patients (40%) were frequent exacerbators, defined as 1x106cells/ml and stained for 15 min at room temperature those patients who suffered from 2 or more exacerba- in dark with viability dye (Zombie NIR; BioLegend), CD45- tions during the previous year to the inclusion. FITC, CD14-APC (Immunotools GmbH, Friesoythe, Twelve COPD patients (23%) had airway infection. Germany) and CD15-PE (Biolegend, San Diego, CA). Patients with airway infection were predominantly males Aggregated and non-viable cells were excluded from the (92%) and older than non-infected ones (Table 2). analysis. Mɸ were gated according to CD45 positive popu- Haemophilus influenzae was the most common PPB iso- lation, CD15 negative, CD14 positive and high side scatter lated (n = 9, 75%), followed by Streptococcus pneumoniae (SSC) parameter. (n = 2, 17%) and Moraxella catharralis (n = 1, 8%). Statistical analyses Airway and systemic FABP4 levels in patients and Results of continuous variables are presented as mean controls and standard deviation (SD) or median and interquartile Airway FABP4 levels were lower in COPD patients than range [25th – 75th percentile IQR], according to the in controls, reaching statistical significant differences in Kolmogorov-Smirnov test of normality distribution, BALF [219.2 (96.0–319.6) vs. 273.4 (203.1–426.7) (pg/ whereas categorical variables are presented as frequen- ml)/protein, p = 0.03], but not in sputum [12.2 (0.7– cies. Groups were compared using Student t-test, 27.5) vs. 14.5 (0.5–45.6) ng/ml, p = 0.4] (Fig. 1A, C). Yet, ANOVA test or their corresponding non-parametrical we observed a positive significant correlation between test when required. Correlations were analyzed using BALF and sputum FABP4 levels in the entire population Spearman’s Rho due to the variables did not present a (rho = 0.31, p = 0.01) and in COPD patients (rho = 0.37, normal distribution. A p-value < 0.05 was considered sig- p = 0.01). On the other hand, plasma FABP4 levels were nificant. Statistical analyses were performed using SPSS similar in patients and controls [26.6 (19.3–39.5) vs. 25.2 version 22 and Graph Pad Prism 7 software. (18.3–36.5) ng/ml, p = 0.4)] and no significant correl- ation was observed between systemic and airway FABP4 Results levels. Characteristics of participants Fifty-two COPD patients and 29 controls were included. FABP4 levels and airway infection in COPD patients Table 1 shows their demographic and clinical character- Airway FABP4 levels were lower in COPD patients with istics. COPD patients were older (65.2 ± 7.9 vs. 55.0 ± airway infection vs. those without it, reaching statistical 12.3, p = 0.0002) than controls but gender (75.9 vs. significant differences in sputum [0.73 (0.35–15.3) vs. 65.4% males, p = 0.6) and BMI (26.3 ± 4.7 vs. 28.7 ± 9.7, 15.6 (2.0–29.4) ng/ml, p = 0.02] but not in BALF [181.5 p = 0.7) were similar in both groups. Airflow limitation (28.7–395.5) vs. 228.5 (106.3–319.1) (pg/ml)/protein, in patients with COPD ranged from mild to severe p = 0.7) (Fig. 1B, D). No differences in plasma FABP4 (FEV1 of 59 ± 15% of predicted) whereas spirometry was levels were observed between COPD patients with and normal in controls by design. Twenty-four patients without airway infection [29.8 (20.4–45.0) vs. 26.1 (19.3– (46%) were classified as GOLD grade C and D, and 21 37.9) ng/ml, p = 0.6]. Perea et al. Respiratory Research (2020) 21:21 Page 4 of 8 Table 2 Patient demographics, clinical characteristics and prior treatments among non-infected and infected patients Non-infected (n = 40) Infected (n = 12) P value Age 63.7 ± 7.6 69.9 ± 6.9 0.02 Male, n (%) 23 (57.5) 11 (91.7) 0.04 Smoking status, n (%) Never 0 (0) 0 (0) 0.7 Former 31 (77.5) 10 (83.3) Current 9 (22.5) 2 (16.7) Pack-years 42.3 ± 20.0 47.8 ± 16.1 0.2 Comorbid conditions, n (%) Cardiovascular 13 (32.5) 6 (50) 0.3 Hypertension 15 (37.5) 6 (50) 0.4 Diabetes 0 (0) 0 (0) 1 Gastroesophageal reflux 14 (35) 6 (50) 0.3 Treatment, n (%) ICS 22 (55) 6 (50) 0.5 LABA 32 (80) 8 (66.7) 0.3 LAMA 32 (80) 11 (91.7) 0.3 FEV1 (% pred) 58 ± 169 61 ± 17 0.6 FVC (% pred) 87 ± 16 76 ± 17 0.04 2 BMI (kg/m ) 26.4 ± 4.8 26.0 ± 4.4 0.8 GOLD stage, n (%) A 13 (32.5) 6 (50) 0.4 B 8 (20) 1 (8.3) C 9 (22.5) 1 (8.3) D 10 (25) 4 (33.4) Prior exacerbations, n (%) 0 16 (40) 4 (33.3) 0.9 1 8 (20) 3 (25) ≥2 16 (40) 5 (41.7) Weeks from last exacerbation 26.2 ± 18.9 27.3 ± 19.1 0.6 CAT questionnaire 11.2 ± 6.5 10.5 ± 7.6 0.6 Data is presented as mean ± SD unless otherwise indicated FABP4 levels and disease severity FABP4 and alveolar Mɸ Airway (but not plasma) FABP4 levels were related with Alveolar Mɸ represented 49.9 (26.4–76.8) % of cells in several measures of disease severity, including GOLD BALF of COPD patients, corresponding to an absolute stage, airway limitation and quality of life. Patient with number of 140 (48.9–311) Mɸ per μl. Both proportion and GOLD D had the lowest values both in BALF (Fig. 2A) absolute number of alveolar Mɸ were significantly related and sputum (Fig. 2B). In addition, a positive correlation with BALF FABP4 levels [proportion (rho = 0.54, p = among airway FABP4 levels and FEV1 (% predicted) 0.0003) and absolute number (rho = 0.52, p = 0.0006)] (Fig. 3A, B) and a negative correlation with levels of (Fig. 4). No significant differences among infected and non- symptoms measured in CAT questionnaire (Fig. 3C, D) infected patients and related to GOLD stage were found. were observed. No differences in airway FABP4 levels were observed Discussion regarding age and smoking status. Patients using inhaled The main results of this study show that, as hypothe- corticosteroid (ICS) had lower values in BAL compared sized: (1) airway (but not plasma) levels of FABP4 are re- with those without ICS [(pg/ml)/protein, 123.6 (12.6– duced in COPD patients, particularly in those with 306.4) vs 250 (171.6–344.7), p = 0.03)]. airway infection and more severe disease; and, (2) BALF Perea et al. Respiratory Research (2020) 21:21 Page 5 of 8 Fig. 1 Airway FABP4 levels in COPD. A BALF FABP4 levels in controls and COPD patients and (B) in non-infected and infected patients. C Sputum FABP4 levels in controls and COPD patients and (D) in non-infected and infected patients. P-values were obtained by Mann-Whitney test. *p- value < 0.05. Data is represented as median with interquartile range Fig. 2 Association of airway FABP4 levels and disease severity. A BALF and (B) Sputum FABP4 levels and GOLD stages classified in mild (GOLD A), moderate (GOLD B), severe (GOLD C) and very severe (GOLD D). P-values were obtained by Mann-Whitney test. *p-value < 0.05 and **p-value < 0.01. Data is represented as median with interquartile range Perea et al. Respiratory Research (2020) 21:21 Page 6 of 8 Fig. 3 Relationship between FEV1 and (A) BALF FABP4 and (B) sputum FABP4. Relationship between CAT score and (C) BALF FABP4 and (D) sputum FABP4. Data was obtained using Spearman correlation FABP4 levels are related to the number and proportion airway inflammation [19]. Likewise, a dysregulation of of alveolar Mɸ. Collectively, these results suggest that re- airway FABP4 has been previously described in duced production of FABP4 by alveolar Mɸ is associated asthma [20] where it has been shown in experimental with the presence of airway infection in COPD. models that FABP4 participates in the recruitment and activation of eosinophils [21]. In patients with Previous studies COPD, a dysregulation of systemic FABP4 compared FABP4 is an adipokine widely studied in metabolic with controls has been suggested [22], which is at and cardiovascular diseases [18]. Its role in chronic variance with our findings here. Differences may be respiratory diseases is poorly understood, although it related to distinct inclusion criteria. Whereas Zhang has been recognized that FABP4 is associated with et al excluded patients with metabolic and vascular Fig. 4 Relationship between BALF FABP4 levels and (A) proportion and (B) absolute number of BALF Mɸ. Data was obtained using Spearman correlation Perea et al. Respiratory Research (2020) 21:21 Page 7 of 8 comorbidities [22], known to be associated with ele- controls in phenotype [29] and in the ability to phago- vated systemic FABP4 levels [23, 24], we did not cytose bacteria and apoptotic cells [30, 31]. Forth, we because cardiovascular comorbidities are highly preva- have not determined FABP4 in infected patients without lent in patients with COPD (74% in our cohort) and COPD and it would be of great interest to better under- we did not want to bias our study population. In any stand its role in the pathogenesis or airway infection. case, to our knowledge, our study is the first to inves- Finally, we did not obtain follow-up samples, so we can- tigate airway FABP4 levels, and their relationship with not infer if any therapeutic intervention (e.g., treatment airway infection and disease severity in COPD. with low dose azithromycin) can restore airway FABP4 levels and what clinical consequences that may have. Interpretation of novel findings Bacterial airway infections are relevant in the natural his- Conclusions tory of COPD [3] because it worsens clinical outcomes, in- FABP4 airway levels (but not plasma ones) are reduced cluding mortality and costs [4]. The molecular and in COPD patients, especially in those with chronic air- cellular mechanisms that favor bacterial infection in some way infection and more severe disease, in relation to a COPD patients are, however, not yet fully elucidated. We reduced number of alveolar Mɸ. These observations observed that airway FABP4 levels were reduced in COPD may be relevant for a better understanding of the patho- patients, particularly in those with evidence of airway in- genesis (and eventual prevention or treatment) of fection, and we found a relationship between BALF Mɸ chronic airway infection in these patients. (absolute number and proportion) and FABP4 levels in Abbreviations COPD. Given that alveolar Mɸ produce FABP4 [9], these APC: Allophycocyanin; BALF: Bronchoalveolar lavage fluid; BSA: Bovine serum observations support a pathogenic role for a locally defect- albumin; CAT: COPD assessment test; COPD: Chronic obstructive pulmonary ive Mɸ production of FABP4. In support of this interpret- disease; EDTA: Ethylenediamine tetraacetic acid; ELISA: Enzyme-linked immunosorbent assay; FABP4: Fatty-acid binding protein 4; FEV1: Forced ation is the fact that FABP4 facilitates the interaction expiratory volume in one second; FITC: Fluorescein isothiocyanate; between Mɸ and neutrophils through the regulation of GOLD: Global Initiative for chronic obstructive lung disease; IQR: Interquartile CXCL1, a chemokine secreted by Mɸ to recruit neutro- range; PE: Phycoerythrin; PPB: Potentially pathogenic bacteria; SD: Standard deviation; SSC: Side scatter phils to the site of infection [11, 25]. Further works study- ing Mɸ subpopulations would help to better understand Acknowledgments the immunological mechanism related to FABP4 produc- Authors thank all participants in the study for the willingness to contribute to medical research, and all field workers of the different participating tion by Mɸ. centers for their dedication and quality of their daily work. On the other hand, we found that patients with severe COPD showed the lowest airway FABP4 levels. We also Authors’ contributions Study design: OS, SV, AA, RF and EM. Patient recruitment and data collection: found that there was a direct relationship between OS, AR, MD, JG, FS, JV, SQ, MG, AM, RF. Performed experiments and sample airway FABP4 concentration and FEV1 levels and an in- processing: LP, AR, EC, SV. Writing the manuscript: LP, OS, SV, AA, EM. verse relationship between the former and CAT score Revising of the manuscript and approval of submission: all authors. Responsible for the overall content as guarantor: OS. (i.e. worse health status). In keeping with these observa- tions, some previous studies had demonstrated that pa- Funding tients with severe COPD have altered airway innate This study is supported by Fundació Ramon Pla i Armengol, Instituto de Salud Carlos III, Fondos FEDER (PI15/02042, PI15/00167 and PI18/00311). RF is immunity [16, 26, 27] that leads to chronic airway in- recipient of a MS research contract (CP16/000039). OS is supported by PERIS. flammation and dysregulation of normal alveolar Mɸ function [28]. Yet, because our study is cross-sectional, Availability of data and materials All data generated or analyzed during this study are included in this we cannot infer what is the cause and what is the conse- published article. quence, this is, what comes first. Ethics approval and consent to participate The study protocol was approved by the local institutional review board Potential limitations (IIBSP-MIC-2015-57) and all subjects gave signed informed consent. Our study has some limitations that deserve comment. First, controls were younger than COPD patients, but Consent for publication Obtained. we did not observe any relationship between airway FABP4 levels and the age of the participants. Second, in- Competing interests duced sputum was collected just before the bronchos- The authors declare that they have no competing interests. copy so we cannot discard any interaction with BALF Author details samples, although this procedure had been applied to all 1 Inflammatory Diseases, Institut de Recerca de l’Hospital de la Santa Creu i the participants. Third, we did not perform flow cytome- Sant Pau, Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona, Spain. 2Respiratory Department, Hospital de la Santa Creu i Sant Pau, try in BALF from controls, but it is well reported in the Biomedical Research Institute Sant Pau (IIB Sant Pau), Autonomous University literature that COPD alveolar Mɸ differ from healthy of Barcelona, Barcelona, Spain. 3Pneumology Department, Hospital del Mar, Perea et al. 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