LETTER:
To the Editor;
The receptor for advanced glycation end products (RAGE) is a protein member of the immunoglobulin superfamily. It is constitutively expressed in the lungs and localized in the basal membrane of type 1 and 2 alveolar epithelial cells. Also, the RAGE has been described in vascular smooth muscle cells, endothelial cells, and immune cells.
Due to its main expression in the lungs the has been studied in several respiratory diseasesdescribed as an important inflammatory mediator in allergic airway inflammation, asthma, pulmonary fibrosis, lung cancer, chronic obstructive pulmonary disease, acute lung injury, pneumonia, cystic fibrosis, bronchopulmonary dysplasia, and COVID-191.
The RAGE-axis is activated through the presence of a wide variety of endogenous ligands like the advanced glycation end products (AGEs), S100/calgranulin proteins or high mobility group box 1 protein (HMGB1). Those RAGE-ligands, expressed in pro-inflammatory states, upregulate the RAGE expression and induce the upregulation of soluble isoforms. The soluble RAGE or sRAGE is the predominant RAGE isoform in plasma. It is derived from membrane-bound RAGE or full-length RAGE (mRAGE or fl-RAGE) by proteolytic cleavage2. Others soluble RAGE forms are consequence of RNA alternative splicing, its main example is the endogenous secretory RAGE (esRAGE)3. The function of those soluble isoforms is not completely understood, and it has been hypothesized that they have an anti-inflamatory role as RAGE decoy receptor. Because of its similarity with mRAGE, the sRAGE may capture and inactivate circulating ligands. This may prevent RAGE-axis activation and therefore, stop or downregulate the proinflammatory status4.
Cystic fibrosis (CF) is the most common life-threatening genetic condition in Caucasian population. It is associated with bronchial infection and airway inflammation due to an impaired mucociliary clearance. Different inflammatory pathways play a critical role in CF lung disease progression, making it an attractive area of research and important therapeutic target.
Few studies have explored the RAGE-axis in CF. These studies have been focused on CF related diabetes (CFRD) and have shown contradictory results. Mulrennan et al5 assessed RAGE in healthy controls, CF patients, CFRD patients and diabetics no-CF. They concluded that sRAGE did not differ significantly among any group. In the other side, sputum sRAGE level were significantly lower in CF and CFRD patients compared to healthy and diabetics subjects, despite a markedly overexpressed of mRAGE mRNA in CF and CFRD sputum. In addition, they found a significantly positive correlation with sputum sRAGE level and forced expiratory volume in 1 second percent predicted (FEV1pp). In contrast, Hunt et al6 did not find any significant relationships between plasma sRAGE levels and absolute values of FEV1. But the plasma advanced glycation end products (AGEs) were significantly elevated in CFRD and correlate negatively with FEV1. This report found that CFRD patients had high plasma levels of AGEs and S100A12, but sRAGE was not significantly different among CF, CFRD and healthy controls.
Based on previously explained, our group present this preliminary study. The first objective was to describe the plasma sRAGE levels in children with cystic CF (CFp). Later we compare these values with the sRAGE levels in a healthy cohort. Finally, we study its possible correlation with CFp baseline clinical features.
We conducted a single-center, cross-sectional observational study in the Pediatric CF Unit of a tertiary hospital in Madrid (Spain). The study was carried out according to the principles of the Declaration of Helsinki and current legislation and approved by the ethic committee. We included consecutive clinically stable CF patients (aged < 18 years) since November 2018 to October 2019. Blood tests and clinical data collection were performed at the same time as the annual review. We include clinical and demographic data such age, sex, pancreatic status, CF liver disease (CFLD), diabetes related to CF (CFRD), airway colonization-infection, pulmonary function test, CFTR mutations, body mass index, current treatments, respiratory support, history of hemoptysis, history of allergic bronchopulmonary aspergillosis (ABPA). Plasma sRAGE level were measured by Human RAGE Quantikine ELISA Kit (R&D Systems Inc., Minneapolis, MN), using an enzyme-linked polyclonal antibody specific for human RAGE (extracellular domain). We collected 3 ml of whole blood via venipuncture in EDTA blood tube and spun at 1500 rpm for 10 min. After that, plasma was stored at −80 °C until use with the commercial kits. Serum sRAGE level was compared to a historical cohort of healthy controls.
Thirty-five CFp were included, their clinical characteristics are provided in Table 1. The healthy cohort differs significantly from our CFp in age, with a median age of 9 and 12,04 years respectively (p = 0.003) (Figure 1). The median plasma sRAGE level in CFp was 1494,75 pg/ml [interquartile range (IQR) 708,75pg/ml], compared with 714,20 pg/ml (IQR) in the historical cohort of healthy controls, being statistically significantly different (Wilcoxon-Mann-Whitney test, p < 0,001). Univariate analyzes were performed (Spearman’s Rho) to correlate clinical characteristics with plasma sRAGE levels. A positive correlation was found between plasma sRAGE level and the following spirometry values: forced expiratory volume in 1 second/forced vital capacity ratio (FEV1/FVC) (0.488, p 0,004) and forced expiratory flow between 25% and 75% (FEF25%-75%) (0.38, p 0,032).
In this preliminary study, the plasma sRAGE level were higher in CFp than in healthy controls. To our knowledge, our study is the largest to describe plasma sRAGE values ​​in CFp and the only one carried out in pediatric CF population. Also, in contrast with previous papers, we described a positive correlation between FEV1/FVC and FEF25%-75% and plasma sRAGE5,6.
As said, the sRAGE levels were higher in CFp than in healthy controls. This observation may be due to a proinflammatory status in the respiratory airway of CFp. The sRAGE cleavaged from lung cells may act as a decoy receptor for the different RAGE ligands. Related with this observation we also described a positive correlation between FEV1/FVC and FEF25%-75% and plasma sRAGE. Both spirometric values inform about airway obstruction and early lung damage in CF. In our cohort, those children with higher sRAGE levels showed lower values. The possible sRAGE protective role in these children should be studied and confirmed in future studies. Finally, we did not observe correlation between sRAGE and FEV1. Most our patients have a normal FEV1 baseline value and this may influence our results which are similar to the observations done by Hunt et al6.
Our preliminary study has several limitations. It was cross-sectional and do not allow to study sRAGE temporal dynamic in the children included. Related to the comparison with healthy children we observed that they were younger than our CFp group. We compared the plasma sRAGE level with an historical cohort, not matched controls. It may affect the results, but we found a large difference in the absolute value of sRAGE, hardly explainable only by age. About the RAGE-axis, we didn’t assess RAGE-ligands or sRAGE levels from the respiratory airway so we cannot evaluate if sRAGE showed intrinsic respiratory airway upregulation.
In conclusion, our pilot study shown that CFp have higher plasma sRAGE level than healthy controls. Also, plasma sRAGE level is positively correlated with spirometry less obstruction. Further multicenter studies are needed to determine the true role of sRAGE in CFp with a larger population and more clinical and molecular variables.