Neutrophil Heterogeneity in Recurrent Severe Wheeze: A Signal Worth Following.

Neutrophils are key cells of the innate immune system. They are often the most numerous immune cell in the initial response, rapidly migrating and adapting to a diverse range of environments.1 Neutrophils are powerful effector cells, with prepackaged granules containing potent enzymes and antimicrobial factors that enable neutrophils to efficiently kill microbes and alter the tissue environment. However, this potent arsenal also means neutrophils can impart considerable damage to tissues. Neutrophils have been implicated with pathogenic roles across multiple respiratory diseases, including asthma.2 Here, people with severe asthma who have neutrophil-dominant inflammation in their airways typically present with steroid-resistant disease.3 In children with severe asthma that is refractory to high-dose inhaled corticosteroid treatment, those who have high airway neutrophil counts feature airway neutrophils with a high activation state, increased degranulation, and prolonged survival, when compared to those with lower neutrophil counts.4 Though long considered a homogenous population, research in the past decade has unveiled that, in fact, various subpopulations and/or states of neutrophil can be present in blood and tissue, and across health and disease states.5 In this issue, Bonner and colleagues set out to assess the immune composition of the lower airways in children with recurrent severe wheeze (RSW)6 through flow cytometry analysis. They particularly sought to advance their previous findings of differential cell count–based clusters in RSW7 by characterizing neutrophil heterogeneity, hypothesizing that in young children with RSW, distinct neutrophil subtypes would be observed. In this large and technical study, flow cytometry data were matched with bacteriological and viral assessments, sensitization status, prescribed treatments, and multiparameter measurements of soluble factors. Analysis of the collected data through a k-prototype clustering algorithm6 resulted in 3 defined clusters in their childhood RSW cohort. Key factors driving the separation of each cluster was both the differential composition of immune cells in the airway and the differential expression of CD62L on neutrophils (Figure 1). Cluster 1 featured airway infection with CD62Llo neutrophils, whereas cluster 2 displayed airway eosinophilia with neutrophils of the CD62Lhi phenotype. Cluster 3 consisted of children with neutrophilia but without eosinophilia or bacterial infection, where neutrophils also featured a CD62Lhi phenotype like cluster 2. Soluble CD62L levels in the lavage fluid also varied between clusters, inversely correlating with the cell surface measurements. Differential CD62L expression between the clusters was interpreted by the authors as suggesting alternative neutrophil activation states. A rolling and adhesion receptor shed by activated neutrophils, CD62L is a very common cell marker with broad commercial availability of antibodies. Although the study was unable to make observations regarding treatment or disease outcomes, CD62L would be a relatively straightforward marker to implement into routine testing of cells or fluid from lung samples. Key findings by Bonner et al. regarding neutrophil clusters in recurrent severe wheeze. Markers of interest for future study are also proposed for each cluster. Created in BioRender. Schofield, C. (2025) https://BioRender.com/ajce799. This study features several strengths. First, the authors recruited children aged 1 to 5 years with RSW undergoing bronchoscopy and performed several high-parameter analyses on the freshly collected samples, which is a significant logistical effort. Neutrophils are not amenable to long-term storage and performing these analyses fresh reduced any noise from changes to immune cells due to sample processing, thereby giving strength to their findings. Second, the flow cytometry results are robust, as the authors conducted fluorescence minus one controls. These relevant flow cytometry controls are important for accurately defining cell marker expression, particularly in airway samples where many cell populations may be autofluorescent. Performing these controls is impressive given the limited sample volume obtained from study participants. Third, the bronchoalveolar lavage and blood proteome was assessed with a large panel of 92 proteins plus 3 pertinent markers of neutrophil activity (soluble CD62L, myeloperoxidase, and double-stranded DNA). This proteomic assessment enhances the authors’ findings and provides biological signals to further understand the neutrophil states between the observed clusters. Finally, the authors followed up with analysis of an independent cohort to interrogate their clusters, establishing concordance between both cohorts. Although this large technical study by Bonner and colleagues provides some insights into how neutrophil phenotypes can underlie patient clusters in RSW, there were some limitations. First, the neutrophil specific flow cytometry assessments were ultimately completed on just 21 of the 106 participants, with measurements of the broader immune populations performed in 61 participants. This is not an uncommon challenge in young children in whom biological sample volumes can be minimal. Furthermore, there are several neutrophil subpopulations and phenotypes in the literature there were not assessed. Major markers of neutrophil subpopulations were restricted to the maturity markers CD10 and CD123. Ultimately, the only neutrophil marker that was different between clusters was CD62L. The authors proposed this, which indicates there were alternative neutrophil states present in childhood RSW, CD62L can also be reduced in both aged neutrophils,8 as well as immature banded neutrophils.9 Testing other neutrophil markers remains warranted and recently there have been multiple new, systematic flow cytometry approaches published that delve deeper into delineating neutrophil subpopulations.10-12 These approaches could be ideal strategies to further interrogate the clusters for neutrophils that are positive for CD49d, olfactomedin 4, CD177, or CD63, neutrophil subpopulations that have been related to allergy, neutrophil extracellular trap formation, migration, and degranulation.13,14 Finally, this was a cross-sectional study of children with severe RSW, so we cannot interpret any causation or how these clusters may relate to the larger childhood wheeze population. By identifying CD62L as a marker of neutrophil subtypes in early RSW that cluster independently of allergic sensitization and prescribed treatments, Bonner and colleagues provide an exciting hint that deeper exploration of neutrophils beyond differential cell counts could warrant new insights into RSW pathophysiology. With the increasing availability of spectral flow cytometry platforms that allow assessment of 2 to 3× more markers, plus improving cost and access to single-cell RNA sequencing technology, teasing out both the neutrophil subpopulations relevant to respiratory disease and their underlying regulatory factors is becoming possible, even in challenging pediatric samples. The larger neutrophil biology field is also making great strides to establish a roadmap for neutrophil classification across multiple analytical techniques15 to harmonize approaches and provide greater clarity on the biological roles of neutrophil subsets across tissues. As efforts are made to follow-up on neutrophils in childhood RSW and other respiratory disease, our field should embrace this neutrophil classification roadmap when designing analytical strategies to lavage and sputum. This will accelerate our understanding of neutrophil heterogeneity in the airways and ensure future findings can be translated into actionable strategies across respiratory diseases. Dr Garratt is supported by the Western Australian Future Health Research and Innovation Fund, which is an initiative of the WA State Government. Supplementary material is available at American Journal of Respiratory and Critical Care Medicine online. The authors declare no competing interests. Please see the ICMJE disclosure forms, which have been provided as supplementary material.