Introduction

Bronchiolitis is the most frequent cause of lower respiratory tract infection in infants under the age of 1 year, with the highest incidence between 3 and 6 months1. The respiratory syncytial virus (RSV) is the most common cause, but other viruses such as human rhinovirus, human bocavirus, human metapneumovirus and coronaviruses can also be responsible2.

The innate immune system plays a crucial role in the initial response to infection, especially during the first months of life when the adaptive immune system is not yet fully developed3,4. Neutrophils are key players in the cell-mediated immune response, contributing to inflammation and representing the predominant cell type in the airways of infants with bronchiolitis5. However, in some cases, this response can become excessive, leading to counterproductive hyperinflammation6 that can be associated with subsequent lung function impairment7.

Lipocalin-2 (LCN-2, also known as NGAL – Neutrophil Gelatinase-Associated Lipocalin) is a protein belonging to the lipocalin family, involved in various physiological and pathological biological processes8,9. Neutrophils and LCN-2 are closely linked, as LCN-2 is released by neutrophils during the immune response. However, it is also produced by epithelial cells, kidney cells, and the liver under conditions of stress or inflammation10,11.

LCN-2 plays a role in the innate immune response of the respiratory tract through several mechanisms, including iron sequestration, regulation of the inflammatory response, and protection of lung tissue by counteracting oxidative stress caused by excessive neutrophil activation12.

LCN-2 is implicated in various respiratory diseases, both acute and chronic13,14,15. In patients with severe asthma, particularly those with neutrophilic inflammation, LCN-2 levels are elevated. LCN-2 may contribute to corticosteroid resistance, a common issue in severe asthma16,17. Research suggests that LCN-2 could serve as a potential biomarker for distinguishing different asthma phenotypes. Bronchiolitis, especially if severe and caused by RSV or rhinovirus, can increase the risk of respiratory sequelae such as recurrent wheezing and asthma18,19,20. LCN-2 may exist not only in its free form, but also as a complex with matrix metalloproteinase-9 (the MMP-9/LCN-2 complex), thereby supporting the extracellular matrix-degrading activity of the MMP-9 enzyme. Matrix metalloproteinase-9 (MMP-9) has been associated with the pathogenesis of RSV-induced bronchiolitis, as infection with RSV enhances MMP-9 secretion by human bronchial epithelial cells21. Notably, evidence from preclinical models indicates that MMP-9 also exhibits antiviral activity, contributing to the suppression of viral replication in RSV-infected mice22.

Some studies have shown that in children with severe RSV-induced bronchiolitis, the inflammation is dominated by neutrophils23, resembling the pattern observed in some forms of steroid-resistant severe asthma24,25. In the contest of pulmonary disease, only one study reported the circulating levels of LCN-2 as free form and in its conjugated (MMP-9/LCN-2 complex) in adult patients affected by chronic obstructive pulmonary disease (COPD), disclosing an association with the state of systemic inflammation in COPD related to cigarette smoking26.

To our knowledge no studies investigated concomitant presence of LCN-2, MMP-9 and MMP-9/LCN-2 complex in the serum of infants with bronchiolitis.

For this reason, the current study aimed to measure the serum levels of LCN-2, MMP-9, and the MMP-9/LCN-2 complex in infants hospitalized with bronchiolitis, and to correlate these levels with clinical and hematological parameters in order to evaluate their potential as biomarkers of disease severity.

Materials and methods

Patient’s recruitment and sampling

Fifty-six infants under one year of age admitted to the Policlinico Umberto I hospital at the Department of Maternal Infantile and Urological Sciences at “Sapienza” University of Rome, Italy, with a diagnosis of bronchiolitis were recruited for this prospective observational cohort study. Based on power analysis calculator for the sample size in a pilot study, we obtained that with 56 total sample size we could guarantee an actual power of 0,95. (for additional details see Supplementary material and results).

Exclusion criteria comprised:

  • prior specific drug treatments (including anti-inflammatory or corticosteroids, or antibiotic therapy within 15 days before enrollment).

  • severe underlying conditions (such as cardiovascular, prematurity, chronic pulmonary, or autoimmune disorders).

During hospitalization a severity score ranging from 0 to 8 was assigned to infants every day, as previously described27. The score at admission and the most severe score were recorded. The score includes four parameters: respiratory frequency (0 = < 45 acts/min; 1 = 45–60 acts/min; 2 = >60 acts/min), oxygen saturation (0 = >95%, 1 = 90%–95%; 2 = < 90%), retractions (0 = none, 1 = slight, 2 = severe or nasal flares) and reduction of food intake (0 = normal; 1 = reduced; 2 = fluid therapy required), as previously described. Supplemental oxygen (O₂) was administered when oxygen saturation persistently dropped below 92% on room air. The need for oxygen therapy was used as an indicator of disease severity.

Peripheral blood samples (0.5 mL) were collected in BD VacutainerTM Serum Separation MicroTubes and centrifuged at 3000 rpm for 15 min to separate the serum from blood cells. The serum was then stored at − 80 ◦C until the day of analysis.

Respiratory viruses’ identification

A nasopharyngeal aspirate was collected at the admission by injecting 1 mL of 0.9% physiological solution into each nostril. This sample was used for the isolation and amplification of viral nucleic acid through Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) or nested PCR methods. Fourteen respiratory viruses were tested for, including RSV, Influenza A/B, Coronavirus (OC43, 229E, NL-63, HUK1), Adenovirus, Rhinovirus, Parainfluenza (types 1–3), Metapneumovirus, and Bocavirus, in the laboratory of the Department of Molecular Medicine at Sapienza University of Rome, as previously described28.

Serum LCN-2, MMP-9, MMP-9/LCN-2 complex analysis

Human serum LCN-2 (Cat. No. DY8556), MMP-9 (Cat. No. DY911), MMP-9/LCN-2 complex (Cat. No. DY1757) were measured using a sandwich enzyme-linked-immunosorbent assay (ELISA) kits (DuoSet ELISA, R&D Systems, Minneapolis, MN, USA), according to the protocols provided by the manufacturer. Briefly, 100 uL of standard curve and unknown samples were added in duplicate to each well and incubated for 2 h at room temperature. The optical density of each well was measured using a microplate reader (NeoBiotech, Seoul, Republic of Korea) set to 450 nm. A standard curve for each ELISA test has been created, by using a software able to generate a four-parameter logistic (4-PL) curve-fit. Final concentration of unknown samples has been extrapolated by using a data analysis tool (MyAssays; https://www.myassays.com/), considering the dilution factor (for LCN-2, 1:200 – for MMP-9, 1:500 – for MMP-9/LCN-2 complex, 1:10). Data has been represented as ng/mL29,30.

Statistical analysis

Statistical analysis was performed using GraphPad Prism Version 9.5.1 software for macOS (Boston, MA, USA). Data were presented as mean ± standard error mean (SEM). Student t-test or the two-way ANOVA test have been used as appropriate. Receiver operating characteristics (ROC) analyses were performed with the standard parameters in Prism 9, using Wilson/Brown method for confidence interval calculation. The Spearman correlation test was performed to investigate the association between serum LCN-2, MMP-9, and MMP-9/LCN-2 complex levels with the clinical data. For the statistical significance and the measure of significance testing, we calculated the probability value concept (p-value) with a confidence interval (CI) of 95%. A p-value of 0.05 was used as the cutoff for significance.

Ethical statement

All clinical investigations were conducted according to the Declaration of Helsinki principles. This study population belongs to an Italian cohort of term, healthy infants hospitalized with bronchiolitis at the Department of Maternal Infantile and Urological Sciences, “Sapienza” University of Rome (BROME- Bronchiolitis in Rome-cohort). Written informed consent to participate in the study was obtained from the parents or legal guardians of all participants. The trial was conducted in accordance with the Helsinki Declaration. The study was approved by the Ethics Committee of the “Policlinico Umberto I” Hospital (No. 2377/2012).

Results

Description of the enrolled infants

The group of enrolled infants included fifty-six infants with a mean age of 0.29 ± 0.03 years, distributed as 39% females and 61% males. Table 1 summarizes the main characteristic of the recruited patients.

Table 1 Anamnestic, clinical, and biochemical data of the recruited infants hospitalized with bronchiolitis.
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Serum LCN-2, MMP-9, MMP-9/LCN-2 complex

Table 2 reported serum values of LCN-2, MMP-9 and MMP-9/LCN-2 complex in infant hospitalized with bronchiolitis.

Table 2 Descriptive statistics of LCN-2, MMP-9 and MMP-9/LCN-2 complex serum levels in recruited patients.
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Upon stratification by oxygen therapy requirement during hospitalization, infants receiving oxygen flow had significantly higher levels of LCN-2, MMP-9, and the MMP-9/LCN-2 complex compared to infants who did not require oxygen supplementation (Fig. 1, panels A, B and C respectively) (LCN-2: t = 2.202, df = 50, p = 0.032; MMP-9: t = 2.523, df = 48, p = 0.015; MMP-9/LCN-2 complex: t = 2.651, df = 52, p = 0.012).

Notably, when stratified by sex, free LCN-2 (p < 0.039) and its conjugated form (MMP-9/LCN-2 complex, p < 0.027), but not MMP-9 (p = 0.073) alone, were significantly higher in males requiring oxygen supplementation compared to females (Fig. 1, panels A’, B’ and C’).

Fig. 1
figure 1

Scatter plots showing circulating levels of LCN-2, MMP-9, and the MMP-9/LCN-2 complex in patients stratified based on oxygen supplementation (panels A, B and C). Gender effect on serum level of the three biomarkers has been shown in panels A’, B’ and C’. Student’s t test (panel A, B, C), two-way ANOVA (panel A’, B’, C’) * p < 0.05.

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Table 1S (see supplementary materials and results) illustrates effect size for Student’s t-test.

The observed effect size (Cohen’s d) for all the three biomarkers indicates a moderate effect. While the standard error is relatively high, the estimate remains consistent with a potentially relevant difference, supporting the value of additional research in this area.

ROC analyses, summarized in Table 3, were performed to assess diagnostic performance of the biomarkers in predicting need for oxygen therapy. Overall, the analysis shows that among the three markers considered in this pilot study, LCN-2 appears to be the most promising in differentiating between the two patient groups.

Table 3 ROC curves for Circulating levels of LCN-2, MMP-9, and the MMP-9/LCN-2 complex in infants receiving oxygen supplementation versus those not receiving it. For each marker, the following parameters are reported: area under the curve, standard error, 95% confidence interval, and p-values.
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When evaluating the etiology of bronchiolitis in relation to oxygen requirements (as a measure of disease severity), no significant differences were observed in the circulating levels of LCN-2, MMP-9, or the MMP-9/LCN-2 complex (Table 2S in Supplementary Materials and Results).

Finally, logistic regression analysis confirmed that despite the presence of potential confounding factors (gestational age, RSV status, days since symptom onset, and sex), biomarker levels were still associated with the need for oxygen supplementation (for detais see Supplementary Material and Results).

Spearman’s correlation

Table 4 summarizes the significant correlations between LCN-2, MMP-9, the MMP-9/LCN-2 complex, and hematological parameters in infants with bronchiolitis. As shown, LCN-2, MMP-9, and the MMP-9/LCN-2 complex were positively correlated with each other. Both LCN-2 and MMP-9 showed a positive correlation with neutrophils, while only LCN-2 was positively correlated with total white blood cells. Finally, MMP-9 was negatively correlated with eosinophils..

Following this new stratification and focusing on the most severe score, we observed a pronounced—though not consistently statistically significant—trend suggesting an association between elevated levels of the three biomarkers and increased severity scores (see Table 3S in Supplementary materials and results).

Table 4 Significant spearman’s correlations between LCN-2, MMP-9, MMP-9/LCN-2 complex and hematological parameters in recruited infants.
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Discussion

In the current study, the presence of LCN-2, MMP-9, and the MMP-9/LCN-2 complex was detected for the first time in the serum of infants less than one year old, hospitalized with bronchiolitis. Furthermore, all three biomarkers were found to be overexpressed in patients requiring oxygen therapy compared to those who did not need oxygen supplementation. Analysis of the correlation revealed a positive relationship between LCN-2 and MMP-9 with white blood cells and neutrophils, key factors in the context of respiratory diseases. Stratifying patients based on the highest severity score recorded during hospitalization revealed that elevated levels of the three biomarkers were associated with greater clinical severity.

LCN-2 is a member of the lipocalin family, is secreted by various cell types, including neutrophils, macrophages, and epithelial cells in response to pro-inflammatory stimuli9,31. During acute inflammation, particularly in the presence of bacterial infections or tissue injury, LCN-2 levels have been found elevated in several conditions32.

LCN-2 plays a crucial role in the immune response, particularly in relation to neutrophil activity, making it an important biomarker and mediator also in respiratory diseases9. In this context, several studies, primarily focused on the adult population, have examined the presence of LCN-2 in various conditions involving altered respiratory function. In these conditions, LCN-2, either alone or in combination with MMP-9 (whose enzymatic activity can be enhanced through the formation of the MMP-9/LCN-2 complex), may contribute to both the acute and chronic phases of these pathologies, potentially offering predictive and/or prognostic clinical value.

In adult patients with lower respiratory tract infections, LCN-2 levels were elevated compared to healthy controls and showed higher sensitivity and specificity than other biomarkers, including IL-6, in distinguishing disease states13. LCN-2 also differentiated between restrictive allograft syndrome (RAS) and bronchiolitis obliterans syndrome, with higher levels and local expression observed in RAS patients14. In severe acute respiratory distress syndrome (ARDS), LCN-2 levels in serum and bronchoalveolar lavage fluid had prognostic value for mortality and ventilator need, possibly indicating alveolar epithelial injury33. Similarly, in idiopathic pulmonary fibrosis, elevated LCN-2 levels correlated negatively with lung function34.

Among the pathologies related to the respiratory system, asthma is of particular interest due to its potential connection with infant bronchiolitis. The association between bronchiolitis and asthma has been the subject of scientific debate, as it involves multiple complex aspects35. Bronchiolitis is not only considered a potential trigger for the onset of asthma but is also regarded as a clinical indicator of an inherent susceptibility to the disease36. This association is more accurately characterized as the result of an interplay between genetic or environmental predispositions—such as a family history of asthma, bronchopulmonary dysplasia, specific metabolites, or genetic loci—and external triggering factors, including respiratory syncytial virus (RSV) and rhinoviruses37,38. Moreover the severity of bronchiolitis could be a major risk factor for developing wheezing before 6 years of age35. To date investigations on asthma prediction tools for infants with severe bronchiolitis are object of current research39.

Emerging scientific evidence has highlighted the involvement of the lipocalin system in the pathophysiology of neutrophilic asthma. Adult patients affected by severe stable asthma exhibited serum LCN-2 levels positively correlated with the duration of the pathology, but not with indicators of Th2 response (eosinophils and IgE)40. Pediatric patients with asthma displayed elevated levels of LCN-2 in sputum supernatants compared to healthy controls, reflecting neutrophil activation in the airways and highlighting LCN-2 as an appropriate indicator for assessing asthma severity in pediatric patients15.

In the present study it has been disclosed the up-regulation of the LCN-2 system (both the free form and its conjugated complex with MMP-9) in infants requiring oxygen supplementation, making it a promising biomarker candidate for bronchiolitis severity. Male infants exhibit elevated levels of LCN-2 and its conjugated form, the MMP-9/LCN-2 complex, in severe bronchiolitis cases requiring oxygen support, compared to their female counterparts. Interesting, this observation aligns with epidemiological data indicating that male infants are more frequently diagnosed with bronchiolitis and tend to experience more severe disease manifestations than females41.

The role of the MMP-9/LCN-2 complex in respiratory function remains poorly investigated. The MMP-9/LCN-2 complex may have a distinct functional significance compared to the free form of LCN-2. It is well established that the conjugated complex enhances the proteolytic activity of the MMP-9 enzyme, making it a key player in the context of tissue remodeling following severe inflammatory processes42. Patients with community-acquired pneumonia exhibited elevated circulating levels of both the MMP-9/LCN-2 complex and free LCN-243, as well as MMP-944, which decreased following antibiotic treatment. A direct correlation between LCN-2 and the MMP-9/LCN-2 complex was observed, consistent with the findings of the current clinical study. This proportional increase in the two forms of LCN-2 (free and conjugated) suggests the existence of distinct functional roles for each. In this regard, a study conducted on patients with chronic obstructive pulmonary disease (COPD) demonstrated that MMP-9 and the proMMP-9/LCN-2 complex could distinguish between COPD smokers and healthy smokers, whereas LCN-2 appeared to serve as a marker of smoking status, independent of disease presence26. It can be speculated that MMP-9, particularly in its enhanced form when bound to LCN-2, plays a crucial role in airway tissue remodeling in smokers with COPD.

The presence of higher levels of MMP-9/LCN-2 complex in infants with bronchiolitis requiring oxygen supplementation, respect to those with no needs, induces reflection on its potential clinical relevance. Severe forms of bronchiolitis are characterized by neutrophilic inflammation, mucus hypersecretion, and airway epithelial cell sloughing that could predispose to defect respiratory function7,45,46. Although direct evidence on the MMP-9/LCN-2 complex in bronchiolitis/asthma is currently limited, both MMP-9 and LCN-2 have been individually associated with airway inflammation and remodeling, suggesting a potential role of their complex in the pathophysiology of respiratory defects disease. Further research is warranted to clarify its clinical relevance and biomarker potential.

One of the main challenges encountered in this study was the difficulty in collecting blood samples from healthy infants, primarily due to ethical and practical concerns. In very young children, obtaining parental consent for such procedures is often problematic, which significantly limited the feasibility of including a control group. This absence of controls represents a relevant limitation, as it prevents a broader comparison of LCN-2, MMP-9, and MMP-9/LCN-2 complex levels, hindering a clearer understanding of their potential role as biomarkers for the clinical course and prognosis of bronchiolitis. Additionally, the study’s sample size was small and uneven, a factor that may have introduced bias. However, this was largely a consequence of the narrow inclusion and exclusion criteria and the single-center nature of the research.

Conclusion and perspective

Increased levels of LCN-2 and MMP-9, particularly in their complexed form, were associated with more severe cases of bronchiolitis, suggesting a link with neutrophil-driven inflammation. These findings indicate that these biomarkers may help stratify disease severity and could have potential relevance in predicting long-term respiratory outcomes.

Ethics approval and consent to participate

All clinical investigations were conducted according to the Declaration of Helsinki principles. This study population belongs to an Italian cohort of term, healthy infants hospitalized with bronchiolitis at the Department of Maternal Infantile and Urological Sciences, “Sapienza” University of Rome (BROME- Bronchiolitis in Rome-cohort). Written informed consent to participate in the study was obtained from the parents or legal guardians of all participants. The trial was conducted in accordance with the Helsinki Declaration. The study was approved by the Ethics Committee of the “Policlinico Umberto I” Hospital (No. 2377/2012).