Neutrophilic asthma may not be a distinct endotype

Extract

Heterogeneity in asthma is well recognised, with established airway inflammatory endotypes that aim to characterise underlying pathobiological mechanisms driving clinical outcomes [1]. Sputum cytometry subdivides airway inflammatory phenotypes into eosinophilic, neutrophilic, mixed granulocytic and pauci-granulocytic [2]. Although there has been significant advancement in the treatment of asthma patients with a type 2 (T2) high signature (defined based on systemic biomarkers including elevated blood eosinophils (>300 per μL), fractional exhaled nitric oxide (>50 ppb) or immunoglobulin E (>30 IU·mL⁻¹)), limited treatment options are available for patients with a T2-low endotype (defined largely based on absence of systemic T2 biomarkers). This may be due to an imprecise definition of the T2-low endotype, which in most cases does not encompass airway eosinophilia (<2%), whether the eosinophilia is controlled by inhaled corticosteroids [3], or the presence of free eosinophil granules [4] or eosinophil proteins in airway macrophages [5].

Shareable abstract

Sputum neutrophilia must take into account total cell counts to differentiate trivial versus intense neutrophilia. Various aberrant cellular mechanisms may increase susceptibility to airway infections, which consequently lead to high neutrophil counts. https://bit.ly/44ZszWx

Heterogeneity in asthma is well recognised, with established airway inflammatory endotypes that aim to characterise underlying pathobiological mechanisms driving clinical outcomes [1]. Sputum cytometry subdivides airway inflammatory phenotypes into eosinophilic, neutrophilic, mixed granulocytic and pauci-granulocytic [2]. Although there has been significant advancement in the treatment of asthma patients with a type 2 (T2) high signature (defined based on systemic biomarkers including elevated blood eosinophils (>300 per μL), fractional exhaled nitric oxide (>50 ppb) or immunoglobulin E (>30 IU·mL⁻¹)), limited treatment options are available for patients with a T2-low endotype (defined largely based on absence of systemic T2 biomarkers). This may be due to an imprecise definition of the T2-low endotype, which in most cases does not encompass airway eosinophilia (<2%), whether the eosinophilia is controlled by inhaled corticosteroids [3], or the presence of free eosinophil granules [4] or eosinophil proteins in airway macrophages [5]. Additionally, in patients with neutrophilia (airway), there is increasing uncertainty regarding the precise role of neutrophils in asthma pathobiology and their clinical significance [6]. Moreover, failure of various clinical trials targeting key neutrophil recruitment pathways adds to the ever-growing scepticism [7]. Therefore, further studies evaluating clinical significance of airway neutrophilia, especially the activation, degranulation, release of extracellular traps, and subsequent epithelial damage by these cells, are desperately needed.

We read the recently published manuscript by Kuks et al. [8] in the ERJ Open Research with great interest. The study team utilised cross-sectional data from the large ATLANTIS prospective study, which was established to investigate the significance of small airways in asthma. In the study, the authors examined clinical characteristics associated with sputum and blood neutrophilia in patients with asthma (Global Initiative for Asthma (GINA) step 1–5). They highlighted that the prevalence of sputum neutrophil percentage and absolute counts was not significantly different in the asthma group compared to healthy controls. Furthermore, sputum neutrophilia was not associated with more severe asthma and did not predict worsening symptoms, airflow obstruction or exacerbations over a year. Conversely, blood neutrophilia was more common in asthma patients compared to healthy controls and was associated with elevated body mass index, female sex and oral corticosteroid use. Blood neutrophilia also did not predict airflow obstruction or exacerbations.

The study by Kuks et al. [8] utilised a large sample size with extensive clinical characterisation of patients, which adds to the robustness of the data. Furthermore, the authors also compared their findings to a subgroup of severe asthma patients (GINA 4 or 5) in the ATLANTIS cohort and an independent U-BIOPRED cohort, which confirmed the overall findings. One of the challenges in defining “neutrophilic asthma” is a lack of a consistent definition in existing asthma literature, where various sputum neutrophil cut-offs between 40% and 76% of total cell count (TCC) have been applied [9, 10]. In healthy subjects, the 90th percentile of neutrophil percentage is 64.4% [11]. Moreover, there is inconsistent use of TCC in combination with neutrophil percentage to differentiate between trivial neutrophilia (sputum neutrophils >64.4% and low TCC <9.7×10⁶ cells·g⁻¹) versus infective neutrophilia (sputum neutrophils >64.4% and high TCC >9.7×10⁶ cells·g⁻¹) (table 1) [7]. In this study, Kuks et al. [8] utilised two separate cut-off percentages (≥70.6% corresponding to upper 25th percentile, and ≥89.6% defined by upper 5th percentile) for sputum neutrophilia to strengthen their findings. Furthermore, there was a moderate correlation (r=0.51, p<0.01) between absolute and percent neutrophil counts, which adds to the accuracy of these results. Nonetheless, although absolute neutrophil counts were examined in the study, average TCCs reported were within normal range (median 3.55 (interquartile range 1.60–6.30)×10⁶ U·mL⁻¹), suggesting that the majority of asthma patients in this group probably had “trivial” neutrophilia (neutrophilia with normal TCC). Additionally, one of the inclusion criteria for the study was disease stability with no exacerbation 8 weeks prior to participation. As sputum collection was only done at baseline, a subset of patients with active airway infections (causing an exacerbation) and intense neutrophilia (neutrophilia with high TCC) may have been excluded from the study, thus underestimating the relationship between airway neutrophilia and clinical outcomes. Another limitation of the study is lack of data during exacerbations, whether the patients had an eosinophilic or neutrophilic inflammatory profile as well as prescribed treatment (corticosteroids, antibiotics or a combination of both). Further microbiome studies combined with total and neutrophil cell counts during exacerbations or stable disease would also be of interest to understand colonisation versus active infection. There is variability of sputum and blood neutrophil counts during clinically stable disease state and during exacerbations [1, 12]. Additional longitudinal assessment of sputum and blood neutrophils and their relationship with clinical characteristics over time would further provide more definitive answers to the role of neutrophilia in disease activity and progression. The ATLANTIS study excluded patients aged >65 years and with smoking history >10 pack-years. Since both ageing and smoking can cause airway neutrophilia, prevalence of neutrophilia in real-life asthma populations may be higher than reported in this cohort. Additionally, the authors highlighted that sputum neutrophilia was associated with a larger wall area as may be seen in bronchiectasis. Nonetheless, the impact of bronchiectasis-associated neutrophilia is unknown in this cohort as co-existing bronchiectasis was not specifically evaluated and was not an exclusion criterion in the study [6]. In clinical practice, trivial neutrophilia is often associated with irritants such as smoking or a consequence of inhaled corticosteroids [7]. The clinical significance of this is likely to be minimal as suggested by the results of this study. Given the inclusion criteria, this study does not address clinical outcomes in patients with asthma and intense neutrophilia due to acute airway infection.

TABLE 1.

Causes of sputum neutrophilia (>64.4%) in patients with asthma and their clinical implications

Intensity (sputum total cell count)	Causes	Clinical implications
<9.7×106 cells·g−1	Corticosteroids Smoking Age	Minimal clinical significance Long-term impact (>1 year) unknown
>9.7×106 cells·g−1	Infections:  Autoimmunity  Kinases  MicroRNAs Endogenous cellular mechanisms:  Inflammasomes  ILC2/3 trans-differentiation  IL-13, IL-22 activation  IL-6 trans-signalling  Mast cell	Investigate:  Other pulmonary diagnosis  Infection (sputum culture and sensitivity,  acid-fast bacillus, fungal, 16S RNA sequencing)  Immunodeficiency Treat:  Antimicrobials  Chronic azithromycin  Intravenous immunoglobulin  Hypertonic saline

ILC: innate lymphoid cell; IL: interleukin.

Several mechanisms are linked to neutrophilic inflammation, including CXC-chemokine receptor 2, cytokine signalling (including interleukin (IL)-6, IL-17, IL-23, tumour necrosis factor-α and interferon-γ), microRNAs, neutrophilic activation, inflammasomes (IL-1β and IL-18) and delayed resolution of inflammation [12]. Severe asthma patients with higher burden of neutrophil counts in sputum may have increased neutrophil extracellular traps (NETs) [13, 14] and autoantibodies [15], which are associated with infective exacerbations and worsening lung function [16]. NETs are large, extracellular web-like chromatic structures containing cytosolic and granule proteins, released reactive oxygen species, and mediators that may potentially damage airway epithelium, inducing alarmin release, and activating inflammasomes [17, 18]. Indeed, the SARP-3 cohort identified an “extracellular DNA high” population, where elevated sputum extracellular DNA was associated with neutrophilic inflammation, soluble NET components and increase in caspase 1 activity and IL-1β [17]. Although NETs may be triggered by endogenous signals such as immune complexes and damage-associated molecular patterns, microorganisms are the predominant driver of NET formation. Multi-omics analysis of sputum samples in the U-BIOPRED cohort has identified two distinct severe asthma clusters associated with neutrophilic inflammation: one cluster associated with microbial dysbiosis with predominant Moraxella catarrhalis and Haemophilus influenzae, and another associated with IL-22 cytokine activation [19]. Additional analysis from the U-BIOPRED cohort demonstrated that a neutrophilic transcriptome-associated cluster was associated with reduced microbial α-diversity and linked to inflammasome and neutrophil activation [20]. Further hierarchical clustering using relative dominant species identified a predominately H. influenzae cluster that was associated with worse asthma outcomes and sputum neutrophilia. Sputum transcriptomics of this cluster showed higher expression of NETosis, neutrophil activation and IL-6 trans-signalling pathways [21].

Aberrant cellular mechanisms may contribute to recurrent airway infections and persistent neutrophilic inflammation in a predominately T2 disease. Son et al. [15] demonstrated that 36% of patients with eosinophilic asthma have anti-MARCO (macrophage receptor with collagenous structure) IgG, which impairs bacterial uptake by macrophages and may contribute to recurrent airway infections. Patients with severe eosinophilic asthma with recurrent bacterial bronchitis were also noted to have an increased phosphoinositide 3-kinase (PI3K) activity (especially increased expression of the PIK3CD subunit), which was associated with reduced expression of MARCO (potentially increasing susceptibility to recurrent infections) and histone deacetylase (which may lead to steroid subsensitivity) [22]. PI3K is also a key mediator in promoting NETosis in response to microorganisms and endogenous immune complexes [23]. Additional roles of NETs in autoimmunity and sterile inflammation have been reviewed extensively [24]. Furthermore, IL-13 has a key pleiotropic effect in asthma and was recently also shown to be associated with neutrophilic inflammation and airway dysbiosis [25]. Recently, Ju et al. [26] also identified an “intermediate type 2 innate lymphoid cell (ILC2)” population, which displayed markers of both ILC2 (prostaglandin D2 receptor 2 (CRTH2), IL-5 and IL-13) and ILC3 (c-kit and IL-17A) and was associated with an increase in IL-1β and IL-18. Thus, ILC2-to-3 trans-differentiation may be an important mechanism driving mixed granulocytic inflammation. Furthermore, the role of neutrophilia associated with recurrent airway infections in patients with severe eosinophilic asthma is constantly evolving.

This study by Kuks et al. [8] sheds some evidence in the growing debate regarding clinical relevance and pathobiological significance of neutrophils in asthma. A key takeaway is that trivial neutrophilia probably does not have any clinical significance. However, we should proceed with caution, especially in patients with intense neutrophilia and recurrent infective exacerbations. Whether intense neutrophilia is related to altered airway microbiome or innate cellular mechanisms resulting in aseptic neutrophilic inflammation is yet to be answered.