D105-24 Altered Nasal Neutrophil CD45 Expression Correlates With Clinical Severity in Infants Hospitalized for RSV Bronchiolitis

Rationale

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis, with disease severity closely related to dysfunction of the host immune response within the airway microenvironment. Comprehensive characterization of key immune cells, including neutrophils, in the nasal mucosa along with their maturation and activation states, is critical to elucidating their distinct roles in inflammation, tissue injury, and viral clearance. Hence, we investigated cell populations in the nasal mucosa of RSV-positive infants hospitalized with bronchiolitis.

Methods

Nasal wash (NW) samples were collected from 38 infants, 23 males (mean age 3.52 ± 2.77 months) and 15 females (mean age 2.93 ± 2.68 months), hospitalized for bronchiolitis during the 2023/2024 epidemic season at the Pediatric Emergency Department of Sapienza University Hospital. All specimens tested positive for RSV using the Xpert® Xpress CoV-2/Flu/RSV rapid molecular assay and/or in-house PCR-based assays. Flow cytometry was performed on fresh cell pellets, which were stained with fluorochrome-conjugated antibodies against CD45, CD14, CD16, CD66b, CD3, and CD326, as well as a viability dye, following standard protocols. Data were acquired using a CytoFLEX flow cytometer and analyzed with CytExpert software v2.4. At hospital discharge, we used an EXHALYZER® (EcoMedics, Switzerland) to calculate the time taken to achieve peak tidal expiratory flow as a percentage of total expiratory time (tPTEF/tE) from tidal breathing curves obtained during spontaneous sleep. We documented wheezing episodes occurring over 2 years follow-up (January 2024-January 2026).

Results

Neutrophils (CD45⁺CD16⁺CD66b⁺) represented the predominant immune population, with a median frequency of 85.90% within the gated viable CD45⁺ cell population, followed by non-classical monocytes (CD45⁺CD14⁻CD16⁺), which accounted for 50.84% of the gated viable CD45⁺ cells excluding neutrophils and T lymphocytes. Classical (CD45⁺CD14⁺CD16⁻) and intermediate (CD45⁺CD14⁺CD16⁺) monocytes were less common, at 1.12% and 0.21%, respectively, within the same parent population. Neutrophils were predominantly characterized by a CD16⁺CD66b⁺SSChigh phenotype, consistent with mature and highly granular cells. However, in infants with greater disease severity, a distinct shift in CD45 expression was observed within the neutrophil population, with a reduction in CD45high neutrophils and a concomitant expansion of CD45low subsets compared to infants with lower severity scores (p < 0.05). Infants with predominant CD45low neutrophils had slightly lower tPTEF/tE and more frequently recurrent wheezing.

Conclusions

The alteration observed in infants with greater disease severity may reflect a dysregulated neutrophil response associated with impaired maturation, aberrant activation, and increased inflammatory potential, thereby contributing to enhanced tissue damage and clinical severity of RSV-induced bronchiolitis and respiratory sequelae.

This abstract is funded by: Sapienza University of Rome