The Role of Vitamin D and Vitamin D Receptor in Recurrent Wheezing in Children With Bronchiolitis: Immunological Association With T-lymphocyte Mitochondrial Mass

Bronchiolitis is one of the most common causes of hospitalization in infants, with a significant proportion of these children developing recurrent wheezing episodes or asthma later in life. The risk of wheezing appears to be influenced by the immune response, although the underlying mechanisms remain unclear.

Vitamin D and its receptor (VDR) regulate both T-cell function and mitochondrial activity, which is essential for T-cell activation. Mitochondrial mass (MM) is emerging as a novel marker of mitochondrial activity and immune cell metabolic dynamics. However, the study of MM in children with bronchiolitis is currently limited.

A prospective study will be conducted involving children under 2 years of age hospitalized for bronchiolitis during the period 2025-2026. Serum levels of 25-OH vitamin D (by chemiluminescence assay), VDR gene polymorphisms (through molecular techniques), and mitochondrial mass of T lymphocytes (by flow cytometry), will be determined in patients at the acute phase of bronchiolitis and in healthy controls. The children will be followed-up for 36 months, with systematic documentation of wheezing episodes and the prognostic value of the biomarkers mentioned above for the later development of recurrent wheezing will be assessed.

This study aims to explore the associations between vitamin D levels, VDR polymorphisms and T-cell mitochondrial mass with bronchiolitis severity and the subsequent risk of wheezing episodes. It is hoped that predictive biomarkers for wheezing risk following bronchiolitis and potential therapeutic targets will be identified.

Methods Study design: This is a prospective, non-interventional study that will be conducted at the "Aghia Sofia" General Children's Hospital.

Study Population: The study will include children under 2 years of age who are diagnosed with bronchiolitis and hospitalized to the Second Pediatric Department and the Neonatal Unit of the 1st Pediatric Department of the National and Kapodistrian University of Athens at "Aghia Sofia" Children's Hospital. Diagnosis will be established according to the 2021 NICE criteria for bronchiolitis.

The control group will consist of healthy children without bronchiolitis, matched to patients for gestational age, chronological age, sex, in a 1:4 control-to-case ratio. Controls will be recruited during the same period as the patients to ensure comparability of the two groups with respect to seasonality, which is known to affect serum vitamin D levels.

Study Duration: The study will be conducted from October 2025 to September 2028 (3 years).

Data Collection and Processing: Prior to data collection, parents will be informed for the purposes of the study, and written consent will be obtained. The parental consent form, covering both the purpose of the study and patient data management, will follow the standard WHO consent template and comply with current data protection regulations. At admission, a blood sample will be collected for routine laboratory tests (complete blood count, CRP, blood gases, and a serum biochemical profile including calcium, phosphorus, and alkaline phosphatase), as well as determination of serum 25-OH vitamin D levels, vitamin D receptor (VDR) polymorphisms, and the mitochondrial mass of T-lymphocytes. The etiology of bronchiolitis (RSV or non-RSV) will also be determined by viral detection in nasopharyngeal secretions.

In the control group, mitochondrial mass of T-lymphocytes, serum vitamin D levels, and VDR polymorphisms will also be assessed, along with routine laboratory tests.

Clinical and laboratory data of children with confirmed bronchiolitis and controls will be recorded in a dedicated SPSS database. Baseline information will include demographic characteristics, personal and family medical history, and known risk factors for bronchiolitis or severe disease.

A clinical examination will be performed, and bronchiolitis severity will be assessed using the Modified Tal Score (MTS). During hospitalization, disease progression will be recorded, including length of hospitalization, admission to NICU/PICU, blood gas parameters (pO₂, pCO₂, pH, HCO₃-), oxygen supplementation/FiO₂, ventilation type, pharmacological management, complications, and mortality. In cases of clinical deterioration, the timing and corresponding MTS will be recorded as an indicator of severity of the disease.

After discharge, children will be followed up until 3 years of age, with documentation of recurrent wheezing episodes, defined as ≥ two or more wheezing episodes during the first three years of life.

Laboratory Methods:

Serum 25-OH vitamin D levels will be measured using chemiluminescence immunoassay (CLIA), a CDC-approved method.

VDR gene polymorphisms will be determined using PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism). DNA will be isolated from peripheral blood. Using specific primers for VDR gene amplification, the polymorphisms of interest (FokI, BsmI, ApaI, and TaqI) will be identified. PCR products will undergo digestion with restriction endonucleases, and the resulting fragments will be analyzed by electrophoresis, enabling allele differentiation based on fragment length.

Mitochondrial mass of T-lymphocytes will be assessed by flow cytometry using fluorescent dyes that selectively bind to mitochondria. Peripheral blood T-lymphocytes will be isolated, incubated with the mitochondrial-specific fluorescent dye (reflecting total mitochondrial mass), and simultaneously stained with surface antibodies to identify T-cell subpopulations (CD4⁺, CD8⁺, CD14⁺). After washing and fixation, samples will be analyzed by flow cytometry, with fluorescence intensity reflecting relative mitochondrial mass.

Sample Size At least 100 samples from our population will be further analyzed. Statistical Analysis A descriptive analysis of the data will initially be performed. Subsequently, group comparisons will be conducted, and potential associations among variables of interest will be explored using univariate and multivariate analyses. Statistical analysis will be carried out with the Statistical Product and Service Solutions (SPSS) software. Τhe flow cytometry results will be analyzed using the Kaluza C Cytometry Analysis Software .

Consent and Ethics The study has been approved by the Ethics Committee of "Aghia Sophia" Children's Hospital (protocol number: 17253-09/07/2025). An informational leaflet will be provided to the participants' guardians, and written informed consent will be obtained prior to inclusion in the study. Clinical and epidemiological data will be used exclusively for research purposes. Patient information will remain strictly anonymous and all applicable personal data protection regulations will be fully applied.

Objectives The main research question is whether vitamin D levels and VDR polymorphisms are correlated with the mitochondrial mass of T-lymphocytes in infants with bronchiolitis, and whether they influence the risk of developing recurrent wheezing in later childhood.

The study 's additional aims are:

• To investigate the effect of serum 25-OH vitamin D levels on the severity of bronchiolitis and their association with the subsequent development of recurrent wheezing
• To study VDR polymorphisms (FokI, BsmI, ApaI and TaqI), their frequency and their association with vitamin D levels, bronchiolitis severity and the risk of developing recurrent wheezing
• To examine the mitochondrial mass of T-lymphocytes in bronchiolitis and its relationship with disease severity and the etiological agent causing bronchiolitis.
• To investigate whether the inclusion of maternal RSV vaccination in the National Immunization Program and/or the implementation of a monoclonal antibody prophylaxis against RSV in infants will alter the mitochondrial mass of T-lymphocytes in infants with RSV bronchiolitis.
• To study the epidemiology of bronchiolitis during the years 2025-2028 and assess potential changes following maternal RSV vaccination and/or the implementation of a monoclonal antibody program against RSV in infants.
• To evaluate the impact of new strategies for protecting infants against RSV infection (maternal vaccination, monoclonal antibody administration in infants) on the epidemiology of recurrent wheezing episodes over a 3-year period (2025-2028).
• To examine potential associations between T-cell mitochondrial mass and the occurrence of bronchiolitis complications.

Inclusion criteria

The study will include children under 2 years of age who are diagnosed with bronchiolitis and hospitalized to the Second Pediatric Department and the Neonatal Unit of the 1st Pediatric Department of the National and Kapodistrian University of Athens at "Aghia Sofia" Children's Hospital. Diagnosis will be established according to the 2021 NICE criteria for bronchiolitis.

The control group will consist of healthy children without bronchiolitis, matched to patients for gestational age, chronological age, and sex, at a 1:4 ratio. Controls will be recruited during the same period as the patients to ensure comparability of the two groups with respect to seasonality, which is known to affect serum vitamin D levels.

Εxclusion Criteria:

• Patients will be excluded from the study if they have a history of congenital heart disease, cystic fibrosis, bronchopulmonary dysplasia, immunodeficiency, use of immunosuppressive agents, prematurity with a gestational age of < 32 weeks, or if their guardians refuse their participation in the study.