*Review* **Neutrophils and Asthma**

**Akira Yamasaki \*, Ryota Okazaki and Tomoya Harada**

Department of Multidisciplinary Internal Medicine, Division of Respiratory Medicine and Rheumatology, Faculty of Medicine, Tottori University, Yonago 683-8504, Japan; okazaki0222@tottori-u.ac.jp (R.O.); tomo.h.308@tottori-u.ac.jp (T.H.)

**\*** Correspondence: yamasaki@tottori-u.ac.jp; Tel.: +81-859-38-6537

**Abstract:** Although eosinophilic inflammation is characteristic of asthma pathogenesis, neutrophilic inflammation is also marked, and eosinophils and neutrophils can coexist in some cases. Based on the proportion of sputum cell differentiation, asthma is classified into eosinophilic asthma, neutrophilic asthma, neutrophilic and eosinophilic asthma, and paucigranulocytic asthma. Classification by bronchoalveolar lavage is also performed. Eosinophilic asthma accounts for most severe asthma cases, but neutrophilic asthma or a mixture of the two types can also present a severe phenotype. Biomarkers for the diagnosis of neutrophilic asthma include sputum neutrophils, blood neutrophils, chitinase-3-like protein, and hydrogen sulfide in sputum and serum. Thymic stromal lymphoprotein (TSLP)/T-helper 17 pathways, bacterial colonization/microbiome, neutrophil extracellular traps, and activation of nucleotide-binding oligomerization domain-like receptor family, pyrin domaincontaining 3 pathways are involved in the pathophysiology of neutrophilic asthma and coexistence of obesity, gastroesophageal reflux disease, and habitual cigarette smoking have been associated with its pathogenesis. Thus, targeting neutrophilic asthma is important. Smoking cessation, neutrophiltargeting treatments, and biologics have been tested as treatments for severe asthma, but most clinical studies have not focused on neutrophilic asthma. Phosphodiesterase inhibitors, anti-TSLP antibodies, azithromycin, and anti-cholinergic agents are promising drugs for neutrophilic asthma. However, clinical research targeting neutrophilic inflammation is required to elucidate the optimal treatment.

**Keywords:** asthma; biomarkers; biologics; eosinophils; inflammation; neutrophils; treatment

#### **1. Introduction**

Asthma is a common chronic airway disease that affects about 350 million people worldwide and varies in prevalence from country to country. In Japan, the prevalence is 9–10% and the number of patients with asthma was 1,177,000 in 2014 [1,2]. Diagnosis of asthma is based on a history or current symptoms, such as chest tightness, wheezing, dyspnea, and cough, together with variable expiratory airway limitation assessed by peak expiratory flow or spirometry. Chronic airway inflammation is an important feature of asthma and is characterized by the presence of eosinophils, basophils, mast cells, neutrophils, T helper 2 (Th2) cells, type 2 innate lymphoid cells (ILC2), CD8+ T cells, B cells, and dendritic cells [3–5]. In the Japanese Guidelines for Adult Asthma, a diagnosis is based on: (I) repetitive symptoms, such as paroxysmal dyspnea, wheezing, chest tightness, and cough; (II) reversible airflow limitation; (III) airway hyper-responsiveness; (IV) airway inflammation; (V) an atopic state; and (VI) exclusion of other cardiopulmonary disease [2].

Asthma is a heterogenous airway disease, and since the 2000s, cluster analyses have identified several phenotypes [6–8]. The common phenotypes are allergic asthma; nonallergic asthma; adult-onset (late-onset) asthma; asthma with persistent airflow limitation, and asthma with obesity [9]. The Severe Asthma Research Program (SARP) identified five phenotypes in patients with severe and non-severe asthma [10]. Kuo et al. found three transcriptome-associated clusters (TACs) in patients with asthma. TAC1 is characterized by immune receptors and a sputum eosinophil increase, TAC2 is characterized by

**Citation:** Yamasaki, A.; Okazaki, R.; Harada, T. Neutrophils and Asthma. *Diagnostics* **2022**, *12*, 1175. https://doi.org/10.3390/ diagnostics12051175

Academic Editor: Koichi Nishimura

Received: 27 April 2022 Accepted: 5 May 2022 Published: 8 May 2022

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interferon-, tumor necrosis factor-, and inflammasome-associated genes and a sputum neutrophil increase, and TAC3 is characterized by genes associated with metabolic pathways, ubiquitination, and mitochondrial function, with no sputum increase [11].

Neutrophils are the most abundant cells in peripheral blood and are stored in pulmonary capillary beds [12]. These cells play important roles in the innate immune system by killing microbes, phagocytosis, granule release, and formation of neutrophil extracellular traps (NETs). The role of neutrophils in asthma has been studied, but there is much debate about the presence of neutrophilic asthma [13–16]. Since glucocorticoids enhance the survival of neutrophils, which constitutively express glucocorticoid receptor β (GRβ) [17,18], the elevation of neutrophil levels in the asthmatic airway is thought to be a consequence of corticosteroid treatment. However, neutrophils are also observed in steroid-naïve patients with asthma [19–22] and several studies have found evidence that neutrophilic inflammation is associated with severe asthma and with asthma exacerbation [23,24]. A cluster analysis has shown that sputum neutrophil counts were associated with more severe phenotypes [25]. Recently, Minchem et al. reviewed the pathology of chronic lung diseases, including asthma [26]. They described the heterogeneity of neutrophils and their interactions with several immune and structural cells, identifying anti-inflammatory, pro-resolving, and pro-repair functions via direct cell-to-cell communication as well as via soluble mediators [26]. Neutrophils also connect with other cells via exosomes and extracellular vesicles [27]. In chronic lung diseases, an overabundance of neutrophils may exacerbate inflammation and remodeling [26]. Therefore, neutrophilic inflammation is involved in the heterogeneity of asthma, and neutrophil-targeted treatment may be important for severe asthma. The pathogenesis, definition, and biomarkers of neutrophilic asthma and potential therapy for neutrophilic asthma are discussed in this review.

#### **2. Definition of Neutrophilic Asthma**

The phenotype of asthma is generally categorized by the cell profile of induced sputum. In a healthy person, this profile has 0.4 ± 0.9% eosinophils and 37.5 ± 20.5% neutrophils, with means plus 2SD and 90th percentiles of 2.2% and 1.1% for eosinophils, and 77.7% and 64.4% for neutrophils, respectively [28]. Eosinophilic asthma is defined as an increase in eosinophils to above 2% or 3% and neutrophilic asthma as an increase in neutrophils to above 60% or 76% in induced sputum [29]. Paucigranulocytic asthma is defined as neutrophils < 76% and eosinophils < 3%, and conversely, mixed granulocytic asthma is defined as neutrophils > 76% and eosinophils > 3% [30]. However, there is still no clear definition of neutrophilic asthma [13]. In children, neutrophil-predominant severe asthma is defined using a cut-off of ≥5% neutrophils in bronchial lavage fluid [31]. Alternative methods, such as nasal wash or nasal lavage, have also been used to evaluate neutrophilic asthma or non-eosinophilic asthma [32].

#### **3. Association of Eosinophils and Neutrophils**

Coexistence of neutrophils and eosinophils occurs in severe asthma [10,33,34], and recent studies have shown that patients with asthma with a mixture of neutrophilic and eosinophilic inflammation had accelerated decline of respiratory function [35–37]. In studies of the coexistence mechanism, Nagata et al. found that activation of neutrophils may induce migration of eosinophils through the basement membrane via interleukin-8 (IL-8) [38], and that leukotriene B4 (LTB4)-activated neutrophils which induced eosinophil migration and Toll-like receptor 4 (TLR4) expression on neutrophils may be involved in this mechanism [36,39]. Theophylline attenuates trans-basement membrane migration of eosinophils in vitro by suppressing superoxide anion generation [40]. Lavinskinene et al. showed that the sputum neutrophil counts after bronchial allergen challenge were related to peripheral blood neutrophil chemotaxis in patients with asthma [41].
