Acute Responsive Elevation of Serum Secretory Acid Sphingomyelinase Activity in Infants with Respiratory Syncytial Virus Bronchiolitis

Background: Respiratory syncytial virus (RSV) bronchiolitis is the leading cause of hospitalization in infants. Biomarkers of disease severity may be signicant in improving management. Blood secretory acid sphingomyelinase (S-ASM) activity has been recognized as a biomarker of cytokine release, inammation, oxidative stress, and damage to the vascular endothelium in various diseases. The present study aimed to evaluate whether serum S-ASM activity is increased and correlates with disease severity in infants with RSV bronchiolitis. Methods: Serum S-ASM activity was determined in 31 infants with RSV bronchiolitis, nine infants with RSV-negative febrile infection, and eight healthy infants. Laboratory data including two cytokines, interferon-γ (IFN-γ) and interleukin-18, and clinical observatory ndings, including clinical severity score specic for bronchiolitis, were analyzed for correlation with the levels of serum S-ASM activity. Results: Serum S-ASM activity was signicantly higher among the 31 infants with RSV bronchiolitis (9.5 ± 5.4 nmol/mL/h) than in those in each of the control groups (RSV-negative febrile infection patients, 4.0 ± 1.4 nmol/mL/h, p<0.005; healthy controls, 4.3 ± 1.9 nmol/mL/h, p<0.005). Serum S-ASM activity negatively correlated with IFN-γ levels (rho=−0.448, p=0.0129), but were not correlated with any other outcomes analyzed in this study. Conclusion: Serum S-ASM activity was signicantly higher in infants with RSV bronchiolitis; however, its clinical signicance requires further study.

Conclusion: Serum S-ASM activity was signi cantly higher in infants with RSV bronchiolitis; however, its clinical signi cance requires further study.

Background
Bronchiolitis is the most common lower respiratory infection and one of the leading causes of hospitalization in young children 1 . Bronchiolitis is predominantly a viral disease caused by respiratory syncytial virus (RSV) in more than 70% of cases, followed by rhinovirus in descending order of frequency 2 . Despite the acute phase of bronchiolitis often requiring oxygen administration or admission into intensive care units, no effective treatment strategies have been established other than supportive care 1 . In addition, an association between viral bronchiolitis and the development of recurrent wheezing and asthma as its long-term sequelae in later childhood has been demonstrated in various studies 3,4 . From the perspective of disease management, the prediction of disease severity and long-term prognosis may be signi cant in this disease.
To clarify the underlying immunological mechanisms and identify immune biomarkers predicting disease severity, numerous studies have evaluated immune biomarker concentrations with respect to disease severity in the blood of infants with bronchiolitis 5 . In a systematic review, less than two dozen biomarkers, including cytokines and chemokines, immune cell subsets, and gene variants, could signi cantly predict and stratify disease severity, but all such biomarkers had comparable studies that reported opposite results each other 5 , suggesting that there are no speci c immune biomarkers of disease severity in infants with bronchiolitis.
Acid sphingomyelinase (ASM) is a key lysosomal enzyme in sphingolipid metabolism that hydrolyzes sphingomyelin, an important cellular membrane component, into ceramide and plays a role in normal membrane turnover 6 . On the other hand, ceramide functions as a second intracellular messenger implicated in a variety of cellular functions, and ASM activation has been reported as a proactive cellular response in many biological functions and pathological reactions in human conditions 6 . The sphingomyelin phosphodiesterase 1 gene that encodes ASM rst leads to a common precursor protein, pre-pro-ASM, which is cleaved into pro-ASM and processed into the two types of ASM, lysosomal ASM (L-ASM) and S-ASM, through N-and C-terminal modi cations and differential glycosylation inside Golgi 7 . L-ASM is transported into the lysosome, exposed to cellular pools of Zn 2+ , and then saturated with Zn 2+ as a metallophosphodiesterase 7 . In contrast, S-ASM is secreted into the extracellular space and is located extracellularly without Zn 2+ saturation 7 . Consequently, while L-ASM needs no additional Zn 2+ ions for activation, S-ASM needs Zn 2+ ions for activation and S-ASM activity can be measured using an assay buffer containing Zn 2+ in various body uids, including blood 8 ; ASM activation has been demonstrated to be linked with the extracellular secretion of ASM, which can be detected as an increase in S-ASM activity.
However, the precise mechanisms of extracellular secretion of ASM remain to be elucidated.
More than 20 clinical studies have been conducted to measure the blood S-ASM activity in human diseases, resulting in a signi cant increase in S-ASM activity in various speci c diseases, such as heart disease, diabetes mellitus 2, Alzheimer's disease, alcohol dependence, and in ammatory diseases including sepsis, systemic in ammatory response syndrome, hepatitis C, systemic vasculitis, in ammatory renal disease, and lymphohistiocytosis 7 . The results suggest that the increase in S-ASM activity might re ect cytokine release, in ammation, oxidative stress, or damage to the vascular endothelium, which is thought to be an abundant source of S-ASM 7 .
In our recent study, serum S-ASM activity was measured in children with acute common illness 9 . Fifty children, as a study subject, consisted of ve groups based on clinical diagnoses: RSV bronchiolitis, adenovirus infection, streptococcal infection, asthma, and other infections due to unknown origin. As a result, the serum S-ASM activity was signi cantly elevated in a group of six patients with RSV bronchiolitis at more than two times that in healthy children and the other illness groups. The study preliminarily suggested an association between ASM activation and RSV bronchiolitis in infants.
In this study, serum S-ASM activity was studied in another cohort of 31 patients with RSV bronchiolitis to reveal the clinical signi cance of serum S-ASM activity.

Study population
Thirty-one infants, aged 0-11 months, diagnosed with RSV bronchiolitis were enrolled in this study. None of them were born prematurely (at or before 35 weeks of gestation) or had any history of diseases, including bronchopulmonary dysplasia, hemodynamically signi cant congenital heart diseases, neuromuscular disorders, immunocompromise, or Down syndrome. They had never received palivizumab injection before admission. The patients' demographic characteristics are presented in Table 1. They were admitted to the Akita Kousei Medical Center between August 2018 and January 2020, and their diagnosis of RSV infection was made performing antigen detection tests on nasopharyngeal discharges (ImunoAce RSV Neo, Shizuoka, Japan). The clinical diagnosis of RSV bronchiolitis was made based on criteria that comprised cough, increase in respiratory rate, chest retraction, prolongation of expiratory time, sibilant rhonchi, and hyperin ated lungs on chest X-ray 10 . Blood was collected on admission and serum was stored frozen at <−20°C.
Eight healthy infants and the nine other RSV-negative hospitalized febrile infants, including three diagnoses of acute bronchitis, two of adenovirus infections, one of hand-foot-and-mouth disease, one of febrile convulsion, one of mycoplasma infection, and one of otitis media, all of whom presented with fever and respiratory symptoms, were also included as controls in this study. RSV-negative infections were con rmed using an antigen detection test.
Patients with RSV bronchiolitis were scored from 0 to 3 points for each of four factors (oxygen saturation of peripheral artery, respiratory rate, wheezing, and depressed breathing) for a maximum of 12 points, every day during hospitalization. We modi ed a previously reported scoring system ( Table 2) 11,12 . Medical records were accessed for age, sex, clinical symptoms, complications, need for oxygen administration, and laboratory data including white blood cell count, platelet count, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, hemoglobin, and C-reactive protein (CRP) levels.
Levels of serum INF-γ and IL-18 were determined using the Human IFN gamma ELISA Kit (Thermo Fisher Scienti c, Waltham, MA, USA) and Human IL-18 ELISA Kit (MBL Inc., Tokyo, Japan), respectively.

Statistical Analysis
Data were analyzed using the IBM SPSS statistics 26.0 software package, and the results are presented as the mean ± standard deviation. The Kruskal-Wallis test was used to compare the mean differences between the two groups. Spearman's rank correlation coe cient was used to examine the correlation between serum S-ASM levels with the clinical study measures. Statistical signi cance was set at p < 0.05.

Results
The patients with RSV bronchiolitis, patients with RSV-negative febrile infection, and healthy controls are summarized in Table 1. Healthy controls were comparable to patients with RSV bronchitis in terms of age, sex, gestational age, and birth weight.
Levels of serum S-ASM, IFN-γ, and IL-18 in acute RSV bronchiolitis (Fig. 1) The mean serum S-ASM activity was 9.5 ± 5.4 nmol/mL/h in the 31 patients with RSV bronchiolitis, whereas the nine patients with RSV-negative febrile infection and the eight healthy infants had mean levels of 4.3 ± 1.9 nmol/mL/h and 4.0 ± 1.4 nmol/mL/h, respectively. The serum S-ASM activity was signi cantly higher among the patients with RSV bronchiolitis than in the patients with RSV-negative febrile infection (p<0.005) and healthy controls (p<0.005).
The mean serum INF-γ was 8.8 ± 11.4 pg/mL in the 31 patients with RSV bronchiolitis, whereas the nine patients with RSV-negative febrile infection and eight healthy infants had mean levels of 14.0 ± 23.7 pg/mL and 0.0 ± 0.0 pg/mL, respectively. The serum IFN-γ level was signi cantly higher among the patients with RSV bronchiolitis (p<0.005) and among the patients with RSV-negative febrile infection (p<0.05) than in the healthy controls.
The mean serum IL-18 was 431.3 ± 276.3 pg/mL in the 31 patients with RSV bronchiolitis, whereas the nine patients with RSV-negative febrile infection and eight healthy infants had levels of 260.9 ± 77.5 pg/mL and 240.8 ± 137.8 pg/mL, respectively. No signi cant difference in IL-18 levels was observed between the three groups of infants.
Relationship between S-ASM activity and clinical severity score, laboratory ndings, and clinical observatory ndings in RSV bronchiolitis The relationship between S-ASM activity and clinical severity score on admission was not statistically signi cant) (Fig.2). Spearman's rho correlations between S-ASM activity and age, sex, clinical severity score on admission, days from onset of fever, oxygen saturation, demand for oxygen therapy, duration of administration, administration of antibiotics, administration of steroids, white blood cells, CRP, and IL-18 were not statistically signi cant (Table 3). Spearman's rho correlation between S-ASM activity and IFN-γ (rho=−0.448, p=0.012) was statistically signi cant (Table 3).

Discussion
The serum S-ASM activity was signi cantly higher among 31 infants with RSV bronchiolitis than in controls, which included healthy controls and RSV-negative febrile patients. This result con rmed our previous study that found higher serum S-ASM activity in six hospitalized infants with RSV bronchiolitis than that in ve patients with adenovirus infection, nine patients with streptococcal infection, six patients with asthma attacks, and 24 patients with other infections 9 . Both results collectively suggest that elevated serum S-ASM activity may be a characteristic pathological reaction in infants with RSV bronchiolitis. During the last two decades, blood S-ASM activity has been studied and shown to be elevated in various speci c human diseases 7 ; however, this is the rst study con rming that elevation of serum S-ASM activity is a characteristic pathological reaction in the acute phase of viral infection.
There has been only one study describing the relationship between viral infection and elevation of serum S-ASM activity 14 . In this study, signi cantly higher S-ASM activity was observed in the serum of patients with chronic hepatitis C virus than that in healthy individuals. Especially in chronic hepatitis C infection, S-ASM activity correlated signi cantly with markers of hepatic injury and showed high discriminative power. They concluded that chronic hepatitis C virus infection and non-alcoholic fatty liver disease induces upregulation of serum acid sphingomyelinase, which appears to be a novel biomarker in hepatopathies. In our patients with RSV bronchiolitis, none of them had any liver dysfunction related to hepatic injury; thus, elevation of serum S-ASM activity may originate from a different mechanism than that due to hepatitis C virus infection.
Until now, increased S-ASM activity has been related to the enhanced release of the enzyme and enhanced activation via oxidative stress, in ammation, cytokine release, or damage to the vascular endothelium, and was, therefore, regarded as a state marker of these conditions 7 . In addition, S-ASM was speci cally demonstrated to be a cytokine-responsive enzyme that increases in activity upon stimulation with interleukin-1β or tumor necrosis factor-α (TNF-α) 15 . As is well known, acute bronchiolitis caused by RSV triggers an in ammatory response with the production and release of several pro-in ammatory cytokines 5,16 . The two common cytokines are TNF-α and INF-γ 17 . In this study, we did not measure the levels of TNF-α in our patients, but numerous studies measured the levels of TNF-α in patients with RSV bronchiolitis, reporting various associations between TNF-α levels and bronchiolitis severity. Thus, the elevation of S-ASM may originate from the cellular reaction to the release of various cytokines in RSV bronchiolitis.
In addition, the relationship between S-ASM activity and laboratory data and clinical ndings, including clinical severity score, was statistically analyzed in this study. No positive correlation was found and only serum INF-γ was found to be negatively correlated. INF-γ has been shown to be one of the various cytokines released in patients with RSV bronchiolitis. Numerous reports have attempted to clarify how the level of IFN-γ is associated with clinical severity or clinical ndings. However, comparable studies have reported con icting results 5,17 . Therefore, we could not speculate on the clinical and pathological signi cance of the negative correlation between S-ASM and INF-γ in our study. Further study of S-ASM activity might be needed to clarify the original site and mechanism of ASM secretion in lower respiratory tract infections.

Conclusion
The present study demonstrated that blood S-ASM was signi cantly elevated in infants with RSV bronchiolitis, suggesting an association between ASM activation and RSV bronchiolitis in infants. In addition, blood levels of S-ASM might be a possible biomarker for clinical utility; however, its clinical signi cance requires further study.

Declarations
Ethics approval and consent to participate Ethical approval was obtained from the Ethics Committee of Akita Kousei Medical Center and Akita University, Graduate School of Medicine, Akita, Japan. Written informed consent was obtained from the parents or guardians of patients and controls in accordance with the Declaration of Helsinki.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.