**2.1 Cellular biomarkers**

EOS in peripheral blood were considered as an important biomarker for asthma, and can predict the treatment response [6]. A small prospective cohort study of hospitalized infants with asthma demonstrated that elevated EOS in convalescence can predict an increased risk of asthma in the future [7, 8] Neutrophils (NEU) in peripheral blood can assess asthma control and prognosis, the counts of NEU over 5000/ul means that asthma symptoms were poorly controlled and likely to get worse [9]. Basophils contains cytoplasmic secretory granules, and was consisted by proteoglycans and histamine [10]. Basophil activation test (BAT) was a useful method for marking CD63 and CD203c, which were the most common surface markers of basophil activation. The detection of CD63 and CD203c implied that basophil degranulation and may led to histamine release, which provide crucial information for the diagnosis of allergic asthma [11].

Mast cell (MC) also played an important role in allergic inflammation. A study suggested that interactions between mast cells and airway smooth muscle cells were critical for the development of the disordered airway physiology in asthma [12]. Therefore, mast cell activation test can be used as a diagnostic method of asthma.

Innate lymphoid cells (ILC), which was different from T cells and B cells, are located on the mucosal surface of the intestine and played an important role in enhancing the immune response, maintaining mucosal integrity and promoting the formation of lymphoid organs. According to the cytokine expression profile, ILC can be divided into three groups: ILC1, ILC2 and ILC3, among which ILC2 can produce a large number of T2 cytokines, such as IL5 and IL13 [13], which can promote EOS and airway hyperresponsiveness (AHR), led to exacerbating the symptoms of asthma. The level of activated ILC2s in blood, bronchoalveolar lavage fluid (BALF), and sputum of asthmatic patients were increasing compared with healthy controls [14]. Thus, ILC2 can be regard as an important biomarker for the assessment of asthma.

T helper (Th2) and non Th2 were phenotypes of asthma and have been determined by CD4+T cells [15]. Th2 asthma was characterized by elevated EOS and high levels of interleukin (IL)4, IL5 and IL13 [16]. In contrast, non Th2 asthma was characterized by NEU infiltration and high levels of IFNγ and IL17 [15]. Since the progression pattern and treatment plan of asthma depend on the differentiation of CD4+T cells, clarifying the biological role of CD4+T cells in the pathogenesis of asthma was very important to develop effective treatment and predict the prognosis of asthma patients [17].

Forkhead box P3 (Foxp3)+ regulatory T (Treg) cells were a special subgroup of CD4+T cells, which played a key role in maintaining immune tolerance and inhibiting immune response to antigens [18]. In patients with severe asthma, the number of Treg cells in blood, BALF and sputum was decreased [19, 20], which concluded that Treg cells can be used to assess asthma severity.

Macrophages were account for about 70% of the immune cells in the asllergic asthma, and played an important role in airway inflammation [20]. A study has shown that the impaired function of alveolar macrophages always be presented in children with poorly controlled asthma which were, characterized by decreased phagocytosis and increased apoptosis [21]. Therefore, macrophages also play an important role in assessment of asthma administration.

## **2.2 Antibody biomarkers**

Mucosal IgA neutralizes bacteria and viruses by interfering with epithelial adhesion and improving the characteristics of mucus capture and antigen removal [22]. One report have shown that infants with low IgA levels have more common asthma and more severe allergic symptoms. In addition, infants born to allergic parents were more prone to deficiency of salivary IgA [23]. Another report shows that serum IgA levels in adult patients with asthma are associated with asthma severity [24]. Therefore, IgA level has certain guiding significance for the severity of asthma symptoms.

The amount of total IgE (tIgE) in serum and the presence of allergenspecific IgE(sIgE) antibodies are important biomarkers to assess the phenotype and symptoms of asthma patients. The level of sIgE in serum may also be helpful to predict persistent wheezing. Furthermore, tIgE was associated with asthma and can be considered as a supplementary indicator for the severity of asthma [25]. One study investigated that in the HDM sensitized children, the ratio of sIgG to sIgE in asthma children was significantly lower than that of nonasthma children, and was the lowest among the children with the most severe asthmatic symptoms, which speculated that sIgG may play a certain inhibitory role in the pathogenesis of asthma [26]. Thus, sIgG/sIgE has been used as a biomarker for more accurate evaluation of asthma than single sIgE.

## **2.3 Cytokine markers**

Allergic asthma was driven by Thelper type 2 (Th2) cells, inducing the production of inflammatory cytokines such as IL4, IL5 and IL13. IL4 and IL13 are key drivers of a variety of atopic diseases [27]. In addition to Th2 cells, other lymphocytes include γδT cell subsets, natural killer T (NKT) cells, T follicular helper cells (Tfh) cells and type 2 innate lymphoid cells (ILC2s) can also produce IL4 and/or IL13 [28]. IL4 was a differentiation factor that polarizes naive CD4+T cells to Th2 phenotype [29]. It was essential in inducing local Th2 response and the development of pulmonary eosinophilic inflammation [30], but didn't have direct effect on mucus production [31].

IL5 can increase expression of CC chemokine receptor 3 (CCR3) by mature EOS [32], it was also conducive to the recruitment and activation of EOS in asthma patients [33]. Although the activation of Th2 cells in allergic asthma lead to the increase of some cytokines, such as IL13, IL4, IL5, and granulocytemacrophage colonystimulating factor (GMCSF) [34], the predominant cytokine associated with antigeninduced eosinophilic inflammation still was IL5 [35]. In brief, IL5 palyed an important role in the evaluation of eosinophilic inflammation in asthma.

IL13 can induce B cells to synthesize IgG4 and IgE, which provided pivotal signal in allergic disease [36]. As aT2 inflammatory cytokins, IL13 can be produced by CD4+T, EOS, MC, basophils, and NKT [37]. IL13 had various roles in asthma, for example, it can switched antibody synthesis of plasma cell and produced IgE, and promoted the migration of EOS to the lungs. Because of the EOS synthesis and the upregulation of adhesion molecules bound to EOS, goblet cell proliferation and mucus production would increased, which lead to increased sputum and AHR [38].

Asthma patients have a higher levels of serum IL4, IL5 and IL13 compared with healthy controls these cytokines were also increased in acute asthma [39]. A clinical study has shown that blocking both IL4 and IL13 signaling can significantly reduce the exacerbation of severe asthma [40], and after antiIL5 treatment, 83% of patients with severe asthma had a favorable responses [41].

CD4+T cells, particularly activated Th2 cells, have been found to represent a major cellular source for IL31 [42]. Polymorphisms in IL31 is associated with IgE production in asthma patients [43]. at the same time, IL31 promoted the occurrence of chemokines and proinflammatory cytokines in human bronchial epithelial cells (HBECs), and could lead to a Th2dominant inflammation in asthma [44]. The levels of IL31 in serum and BALF were increased in asthma patients and IL31 also was positively correlated with Th2 cytokines (IL5, IL13, TSLP) and the severity of asthma [45].

Th17 related cytokines such as IL17A, IL17F, IL21 and IL22 were secreted by Th17 cells. In the mice model of allergic asthma, the impairment of IL17R signal delayed the recruitment of neutrophils to the alveolar cavity [46]. IL17 also activated airway NEU by increasing elastase and myeloperoxidase activities, and promoted exacerbation of asthma [46]. It has shown that IL17 may play an indirect role in airway remodeling of asthma, the increased concentration of IL17 in PBMCs and plasma always implied that the asthmatic symptoms prone to more severe [47, 48]

IL9 can be produced by a variety of cells including Th2 cells, Th9 cells, EOS and NEU [16], and Th9 cells were the main source of IL9. Th9 cells promoted mast cell accumulation and activation in mice model of allergic pulmonary inflammation [49], while IL9 can inhibit the production of IFNγ and promote secretion of mucus and IgE [50, 51]. A study have found that both Th9 cell and IL9 of peripheral blood increased in allergic asthma patients [52], which means that IL9 can be regarded as a biomarker of asthma.

IL25, IL33 and TSLP derived from airway epithelium and played an important role in the pathogenesis of asthma [53]. Among them, IL25 not only targeted innate immune cells to produce Th2 cytokines, but also guided the translation of naive Th cells to Th2 cells [54]. Overexpression of IL25 in lung epithelium induced epithelial cell proliferation, increased mucus secretion, airway infiltration of eosinophils and macrophages, and upregulated the chemokines related to Th2 cells [55]. Plasma IL25 levels were also associated with epithelial IL25 expression and may be useful for predicting responses to asthma therapy [56].

Genome wide and candidate gene association studies have identified that common single nucleotide polymorphisms (SNPs) in IL33 and IL1 receptor like 1 (IL1RL1) loci associated with asthma, especially pediatric asthma [57]. IL33 activated a large number of immune cells and structural cells by binding to IL33 receptor complex, which can promote occurrence and exacerbation of asthma [58]. The IL33/ST2 (suppression of tumorigenicity 2) axis triggered the release of several proinflammatory mediators, such as chemokines and cytokines, and induced systemic T2 inflammation in vivo [59]. IL33/ST2 pathway also contributed to allergen induced airway

inflammation and hyperresponsiveness [60]. Compared with healthy individuals, the concentration of IL33 in plasma was higher in asthma patients [61].

To some extent, AHR, mucus overproduction and airway remodeling, were considered to be drived by TSLP through its downstream proinflammatory effect [62]. Stimulation of basophils with TSLP can increase the percentage of IL25 receptor (IL17RB) and ST2, suggesting that TSLP can enhance the responsiveness of basophils to other alarmin cytokines [63]. The levels of plasma TSLP in asthma patients were higher than that in healthy controls, Airway submucosal EOS would be reduced by blocking TSLP in patients with moderatetosevere uncontrolled asthma compared with placebo [64].
