Background
Wheezing is a common problem in preschool children and contributes to substantial morbidity, parental stress, and healthcare costs [
1‐
3]. Recurrent wheezing is a common symptom of asthma during the preschool period (< 6 years); however, most children who experience wheezing episodes early in life do not go on to develop childhood asthma [
4]. Early identification of preschool children with persistent wheezing or asthma is important to prevent morbidity associated with chronic asthma, such as long-term lung function decline [
5,
6].
Various predictive tools, such as the modified Asthma Predictive Index (mAPI), have been developed based on readily obtainable clinical variables to identify young children at high risk of persistent wheezing or asthma [
7,
8]. However, the sensitivity (ranging from 15 to 75%) and positive clinical predictive value (ranging from 12 to 74%) of these tools are low [
7]. Recent studies have shown that combining clinical parameters and biomarkers, such as exhaled breath biomarkers and/or specific gene expression signatures, may improve prognosis prediction of preschool recurrent wheezing [
9,
10]. Furthermore, accumulating evidence suggests that dysregulation of iron homeostasis is associated with childhood wheezing and asthma. Serum and exhaled breath condensate iron concentrations are significantly reduced in childhood asthma patients compared to healthy controls [
11,
12]. A longitudinal study of mothers and children reported that lower umbilical cord iron status is associated with increased occurrence of wheezing [
13]. However, it remains unclear whether iron homeostasis dysregulation is present in the airways of preschoolers with recurrent wheezing and whether iron homeostasis indices can add value to clinical parameters in prognosis prediction in preschool children with recurrent wheezing.
In the present study, we aimed to: (1) evaluate airway iron homeostasis in preschool children with recurrent wheezing by determining the expression levels of iron metabolism-related genes and iron levels in bronchoalveolar lavage (BAL) samples, and (2) investigate the value of adding iron homeostasis indices to clinical information in predicting persistent wheezing in preschoolers.
Discussion
We investigated iron homeostasis in the airways of recurrent wheezing preschoolers and assessed the predictive value of iron-related predictors when added to mAPI or clinical predictors of persistent wheezing. Results suggested that the iron export molecule SLC40A1 was greatly reduced, while the iron intake factor TFR1 and iron storage factors FTH and FTL were increased in the airways of recurrent wheezing patients, consistent with the markedly lower iron levels in the BAL supernatants compared with the controls. Importantly, adding iron-related predictor data (mRNA expression of SLC40A1 and FTL) significantly improved the predictive ability of mAPI and clinical predictors of persistent wheezing, with the combined model of both iron and clinical predictors achieving the highest AUC (0.84).
We found that preschoolers with recurrent wheezing had reduced iron levels in their airways, significantly lower expression of SLC40A1, and higher expression of TFR1, FTH, and FTL. Most cells acquire iron by importing transferrin-bound iron from the blood via TFR1 [
27], and ferritin, consisting of 24 heavy (FTH) and light (FTL) chain subunits, is the main iron storage protein [
28]. Ferroportin (encoded by SLC40A1), is the only recognized mammalian iron exporter protein [
27]. Thus, decreased expression of SLC40A1 and increased expression of TFR1, FTH, and FTL may explain the decrease in extracellular iron and possible accumulation of intracellular or tissue iron in the airways of patients with recurrent wheezing. Animal studies have demonstrated significant accumulation of iron in lung tissue in a house dust mite-induced murine asthma model, with pulmonary iron accumulation resulting in key features of asthma, including type 2 cytokine (interleukin-13) production, airway hyperresponsiveness, and airway fibrosis [
29]. However, the precise role of iron metabolism disruption in the pathogenesis of recurrent wheezing is not yet clear, and thus further research is warranted.
ROC analysis showed that iron predictors alone had moderate predictive value for persistent wheezing (AUC = 0.75, Table
2). However, when iron-related predictors (SLC40A1 and FTL) were added to the mAPI or clinical predictors, the prediction of persistent wheezing was significantly improved. Although mAPI is the most commonly used index in predicting prognosis in preschoolers with recurrent wheezing [
30], our results showed that mAPI alone performed poorly in predicting persistent wheezing (AUC = 0.67, Table
2), primarily due to its low sensitivity (0.54, Table
2), consistent with previous reports [
14,
30,
31]. However, the combination of mAPI and iron predictors led to a substantial increase in sensitivity (0.98, Table
2) and predictive ability (AUC = 0.82, Table
2), while losing specificity (0.56, Table
2).
The present study indicated that shorter duration of breastfeeding, earlier age of first wheezing episode, and aeroallergen sensitivity were risk factors of persistent wheezing, consistent with previous studies showing that these factors are associated with the development of asthma in preschool-aged children with recurrent wheezing [
8,
14,
32]. The model consisting of the three clinical predictors had a moderate predictive capacity for recurrent wheezing prognosis (AUC = 0.72, Table
2) but relatively poor specificity (0.61, Table
2). Interestingly, adding iron predictor information not only markedly increased the potential (AUC = 0.84, Table
2) to predict persistent wheezing but also increased the sensitivity (0.81, Table
2).
Our findings support previous studies showing that the addition of biomarkers and/or specific gene expression signatures to clinical parameters can help improve prognosis assessment in preschoolers with recurrent wheezing [
9,
10]. Klaassen et al. [
10] followed 202 preschoolers with recurrent wheezing and identified those who developed asthma at the age of 6 years. They found that adding information on nine exhaled volatile organic compounds and expression of six inflammatory genes to the asthma predictive index can significantly improve prediction (AUC = 0.95) of asthma in preschoolers with recurrent wheezing [
10]. However, this model is somewhat complicated by the combination of multiple biomarkers and clinical parameters, making routine clinical screening and generalization in clinical practice difficult. More recently, in a longitudinal study of 135 preschool recurrent wheezers, Kreißl et al. [
9] found that exhaled breath condensate pH combined with the asthma predictive index, serum allergen-specific immunoglobulin E (IgE), and several other clinical parameters can improve prediction (AUC = 0.94) for early detection of preschool recurrent wheezers with an increased risk of developing asthma. These and our findings highlight the need for integrated assessment of clinical and biomarker information and the need to develop more reliable prognostic biomarkers for young children with recurrent wheezing.
The present study has several limitations. First, the study population was restricted to hospitalized children and the measurements of iron-related indices were invasive and may be difficult to perform in the general pediatric population. Therefore, our results may have limited external validity to broader samples of children, such as outpatients. Additionally, the prognostic value of circulating iron parameters for preschool asthma patients as more accessible and less invasive indicators of iron metabolism deserves further exploration. Our results are also limited by the lack of long-term follow-up. Second, although we used a method to minimize overfitting and performed internal validation, this was a single-center study with a small sample size, and potential selection bias and unknown discrepancies among participants may have confounded study findings. External validation with independent samples is necessary to further evaluate the predictive performance of iron indices in other populations. Finally, while our preliminary study implicated iron metabolism dysregulation in the airways of preschoolers with recurrent wheezing and several iron-related indices as possible predictors of persistent wheezing, the underlying signaling pathways and exact roles of disordered iron metabolism in recurrent wheezing remain to be elucidated.
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