Severe asthma patients contribute to 50-60% of asthma costs and are associated with poor quality of life and high mortality and morbidity . The unclear molecular mechanism and refractory response to traditional asthmatic therapies seen in these patients have been challenging for clinicians to treat this subtype of asthma. In this study, we used the DEGs between severe and mild asthma samples for the first time to construct a co-expression network by WGCNA and carried out a comprehensive analysis of key genes and pathological processes associated with asthma severity, hoping that the findings will provide ideas for the understanding and future treatment of severe asthma.
In total 6 modules were identified in this paper, of which 3 modules were positively related to asthma severity while 3 modules were negatively related to asthma severity. The brown module with the strongest relation to asthma severity and the significant MM-GS correlation was identified as the critical module. Enrichment analysis showed that genes in brown module were enriched in neutrophil degranulation and activation, leukocyte migration and chemotaxis, cytokine-cytokine receptor interaction, phagosome, chemokine signaling pathways. Then 10 hub genes in brown module were filtered and verified in another dataset, showing a moderate ability for discriminating severe asthma from mild-moderate asthma with AUC of 0.75.
Results of module-trait relationships showed modules positively related to asthma severity(brown, blue and green module) also positively related to ACQ score and GINA control grade while negatively related to FEV1 and FVC, which means the higher genes expression level in these modules, the worse asthma control and lung function. Besides, positive relationships were found between smoking status and high expression of genes in severe asthma. Previous studies showed that asthma patients exposed to smoke are typically steroid-refractory and result in uncontrolled asthma . One of the probable mechanisms has been linked with Th17 pathway ，which mediates neutrophilic activation and recruitment in airway. This is consistent with our enrichment analysis results that genes positively to smoking status were enriched in neutrophil degranulation, activation and migration. In addition, no modules were found to be linked with phenotype allergic rhinitis and nasal polyps.
For brown module genes, the significant enriched terms in GO and KEGG were as followings: neutrophil degranulation and activation, leukocyte migration and chemotaxis, cytokine-cytokine receptor interaction, phagosome, chemokine signaling pathway. Similar enrichment results were found when all modules positively related to asthma severity were considered. Asthma can be classified as “T-helper 2 (Th2)/type 2 asthma” and “non-Th2/type 2 asthma” based on the inflammatory pattern . Neutrophil inflammation, characterized by the lack of Th2-mediated inflammatory response and increasing airway neutrophils , has been linked with asthma severity [16, 17]. In fact, neutrophils contribute to the pathology of asthma, regardless of whether asthma is eosinophilic or non-eosinophilic [18, 19]. This is in accordance with the present enrichment analysis results that from mild to severe asthma, more neutrophils activated and migrated, as well as more cytokines produced and functioned. Besides, neutrophil inflammation is more prominent in patients fail to respond to inhaled corticosteroids, also referred to as severe asthma than other levels severity of asthma . Thus, novel treatments aiming at decreasing neutrophils may benefit patients with severe asthma.
Identified hub genes further provided biomarkers for severe asthma. CXCR1 and CXCR2 are chemokine receptors respond to IL-8, which is an essential chemokine enhances neutrophils migration into airways and contributes to asthma severity and lung damage . In this way, CXCR1/2 inhibition might be a rational therapeutic strategy for severe asthma treatment. For example, a selective CXCR2 antagonist named SCH527123 was reported to reduce sputum neutrophils and mild exacerbations . However, AZD5069, which is also an antagonist of CXCR2 was investigated and results showed it failed to reduce asthma exacerbations or improve lung function compared with placebo . Recently, a study suggested that KLF2, as a regulator of CXCR1/2, may represent an indicator of asthma severity when combined with CXCR1/2 . Thus provides another direction for the treatment of severe asthma target on CXCR1/2. Unlike CXCR1/2, CCR1 and CCR7 are chemokine receptors in Th2/type 2 pathway, which is supposed as the dominant inflammatory pathway underpinning severe asthma. CCR1 mainly expressed in eosinophils, macrophages, lymphocytes. It plays a role in the progression of asthma by promoting chemotaxis of leukocytes in the airway epithelium and probably by modulating the Th1 or Th2 cytokine balance . Biopsy of the airways has demonstrated elevated expression of CCR1 mRNA in mild-to-severe asthma . CCR7 involved in the migration and maturation of dendritic cells (DCs), which have been shown to facilitate the development of asthma [27-29]. CCR7 could also take part in the airway remodeling of severe asthma by enhancing fibrocytes transmigration . In addition, findings of CCR7 on immune tolerance in allergy‑induced asthmatic [31, 32] may provide ideas on the treatment of severe asthma.
Toll-like receptors (TLR) family is the first line of defense against invading microbes . They cause Th17 responses, leading to increased production of IL-8, IL-17, which can modify airway structures to contribute to the lower FEV1, remodeling, and airway obstruction seen in patients with severe neutrophilic asthma . This paper indicated TLR2 had increasing expression in severe asthma. In line with our results is the upregulation of TLR2 in sputum T cell of non-eosinophilic asthma and TLR2 pathway in severe asthma. However, recent study showed that TLR2 may reduce Th17 cytokines by suppressing a Th17 phenotype of Treg cells. That means TLR2 may induce remission of asthma . Another study in mouse also suggested appropriate stimulation of the TLR2/4 pathway may help to prevent asthma later in life . Thus, further studies are needed to reveal the effect of TLR2 on asthma and disease severity.
FPR1 is a powerful neutrophils chemotactic factor and have been linked to chronic inflammation diseases. Though FPR1 was reported to react to cigarette smoke [37, 38] and involve in anti-inflammatory activities of glucocorticoid , little was known about its effect on the process of asthma.
FCGR3B, FCGR2A and ITGAM are immune-related genes. FCGR2B and FCGR3A are associated with several immune-mediated diseases such as systemic lupus erythematosus and severe nephropathy. ITGAM is also known as the biomarker for systemic lupus erythematosus. PLEK is major protein kinase C substrate of platelets, monocytes, macrophages and lymphocytes. The exact function of these genes on asthma patients is not known.
For the first time, co-expression modules was built via WGCNA using DEGs of mild-severe asthmatics from bronchial epithelial brushings to discover mechanisms and hub genes in severe asthma. There are some limitations in our study. First, samples in this study are small, mainly due to the lack of related gene expression profiles with rich clinical phenotypes. Second, our research results are based on public online database information and future studies are needed to prove and it. Third, detailed mechanisms of identified hub genes required to be elucidated by more experiments.
In conclusion, we identified neutrophil degranulation and activation were key pathways in the development of asthma. Meanwhile, hub genes such as CXCR1, CXCR2, CCR1, CCR7, TLR2 and FPR1 were discovered act as core parts in asthma severity through either neutrophil inflammation pathway or T2 immune pathway. Our results can be useful to serve as potential immunotherapy targets and prognostic marker. Further mechanism studies are required to validate and elucidate our results.