Functional immunophenotyping of blood neutrophils identifies novel endotypes of viral response in preschool children with recurrent wheezing

Abstract

Background: Preschool children with recurrent wheezing are heterogeneous, with differing responses to respiratory viral infections. Although neutrophils are crucial for host defense, their function has not been studied in this population.

Objective: We performed functional immunophenotyping on isolated blood neutrophils from 52 preschool children with recurrent wheezing (aeroallergen sensitization, n = 16; no sensitization, n = 36).

Methods: Blood neutrophils were purified and cultured overnight with polyinosinic:polycytidylic acid [poly(I:C)] as a viral analog stimulus. Neutrophils underwent next-generation sequencing with Reactome pathway analysis and were analyzed for cytokine secretion, apoptosis, myeloperoxidase, and extracellular DNA release. CD14⁺ monocytes were also exposed to neutrophil culture supernatant and analyzed for markers of M1 and M2 activation.

Results: A total of 495 genes, related largely to the innate immune system and neutrophil degranulation, were differently expressed in children with versus without aeroallergen sensitization. Functional experiments identified more neutrophil degranulation and extracellular trap formation (ie, more myeloperoxidase and extracellular DNA) and less neutrophil proinflammatory cytokine secretion in children with aeroallergen sensitization. Neutrophils also shifted CD14⁺ monocytes to a more anti-inflammatory (ie, M2) phenotype in sensitized children and a more proinflammatory (ie, M1) phenotype in nonsensitized children. Although both groups experienced viral exacerbations, annualized exacerbation rates prompting unscheduled health care were also higher in children without aeroallergen sensitization after enrollment.

Conclusions: Systemic neutrophil responses to viral infection differ by allergic phenotype and may be less effective in preschool children without allergic inflammation. Further studies of neutrophil function are needed in this population, which often has less favorable therapeutic responses to inhaled corticosteroids and other therapies directed at type 2-high inflammation.

Keywords: Asthma; RNA-Seq; endotype; innate immunity; neutrophil; neutrophil extracellular trap; next generation sequencing; phenotype; respiratory infection; sensitization; virus; wheezing.