Discussion
To our knowledge, this is the first study that establishes the metabolic signatures and underlying molecular pathways of different inflammatory asthma phenotypes in induced sputum sample by combing untargeted and targeted metabolomics. This study demonstrated that the unique metabolic profiles that existed between different inflammatory asthma phenotypes and healthy subjects with 77 differential metabolites identified. Pathway topology analysis uncovered that 5 pathways could be involved in the pathogenesis of different asthma phenotypes. Further, 24 targeted quantification metabolites were validated significantly differentially expressed between asthma inflammatory phenotypes, which significantly correlated to clinical and inflammatory profiles. Finally, adenosine 5’-monophosphate, allantoin and nicotinamide were shown to predict rate ratios of severe asthma exacerbations. These findings indicated novel immunometabolic mechanisms in different asthma phenotypes, with providing more phenotypic and therapeutic implications.
There have been many metabolomics studies on the difference between asthmatics and healthy subjects or on distinct asthma severity have been performed extensively10, 26. As for the metabolome research on infammatory phenotype, some studies have shown that inflammatory asthma phenotypes can be discriminated by an electronic nose breath analyzer12. Brinkman et al27 further demonstrated that inflammatory phenotypes of severe asthma could be identified by unbiased clustering of exhaled breath profiles using eNose technology. However, eNose technology seems to be suitable for the noninvasive identification of asthma inflammatory phenotypes but is principally unable to identify the individual metabolic pathway and metabolites driving the distinction between the subgroups.
This current study unraveled that histidine metabolism, glycerophospholipid metabolism, nicotinate and nicotinamide metabolism, linoleic acid metabolism, and phenylalanine, tyrosine and tryptophan biosynthesis were involved in the pathogenesis of different asthma phenotypes. In fact, previous studies supported our results as histidine metabolism was identified as implicated pathway between asthma and healthy subjects28, and glycerophospholipid metabolism was also participated in the pathogenesis of asthma, especially EA29, 30. In addition, linoleic acid metabolism was the most significant pathway between NA and EA or PGA (all impact value =1, P < 0.05), which indicated that the important role of linoleic acid metabolism in the NA. Panda et al. 31had proved that linoleic acid metabolite leads to steroid resistant asthma features partially through NF-κB. Therefore, it is speculated that the development of targeted treatment of linoleic acid metabolism has important implications for the individualized treatment of NA.
Notably, it found that differential metabolites correlated to clinical and inflammatory profiles. Adenosine-5’-monophosphate was related with poor asthma control and severe asthma exacerbation. The discovery that adenosine-5’-monophosphate levels are increased in the bronchoalveolar lavage fluid of patients with asthma and increase further after allergen challenge raises the possibility that the adenosine generated in asthmatic airways itself contributes to the pathogenesis of asthma32, 33. It is possible that the recruitment and activation of inflammatory cells and subsequent smooth muscle contraction that occur during asthma exacerbation leads to an increase in oxygen and energy demand, thus leading to an increase in the level of adenosine in the airway in asthma34. We also demonstrated for the first time that allantoin and nicotinamide were involved in severe asthma exacerbation. The roles of these metabolites in asthma pathogenesis and clinical outcomes of asthma remain unknown and further studies are required to their involvement in asthma.
As for the inflammation markers, histamine was significantly related to FeNO and IL-5. Histamine has been shown to play a key role in the pathogenesis of a variety of allergic diseases, including allergic asthma35. Except for the partial expression of histamine by immune cells such as basophil36, mast cells are the main source of histamine expression and express a large number of different receptors on their cell surface, such as,FcεR1, FcγRI, complement receptors (C3AR and C5AR), and ligand receptors such as neurotrophic factor, substance P, vasoactive intestinal peptide, and adenosine phosphate37-40. These ligand and allergens can activate mast cells to release the pro-inflammatory mediators including histamine. Furthermore, histamine has been shown to promote TH cell differentiation and the release of type 2 inflammatory factors such as IL-5. Consistently, antihistmine has been demonstrated to significantly reduce the risk of emergency visit and hospitalization for asthma, especially in asthmatic patients with other allergic diseases, such as allergic rhinitis 41, 42.Therefore, antihistamine therapy may be considered as an additive treatment for eosinophilic asthma, as it is often associated with allergic diseases such as allergic rhinitis, as also found in current study.
Various amino acids and amino acid dipeptides such as taurine, alanyl-leucine were significantly correlated to TNF-α, IL-1β, IL-8 and IL-17, which indicated that these differential metabolites may play a very important role in the development of NA. For example, Taurine, a β-amino acid that is not integrated into proteins, is highly expressed in the intracellular chambers of most tissues. Taurine has been reported to be increased in bronchoalveolar lavage fluid in asthmatic patients43, 44. I has been shown shown that plasma taurine is formed with arachidonic acid in asthmatic patients45 Animal studies have also shown that branching-chain amino acids valine, leucine and isoleucine mediated asthma-related airway inflammation through lipid oxidation pathways46. Notably, this study also found a number of amino dipeptides that had not been reported in previous studies, and their differential expression were closely related to neutrophilic asthma. Because induced sputum specimens are biological specimens of open airway, they do not simply reflect the changes of human metabolism, but also may reflect the changes of local airway microenvironment, including local microbiota and human host. In fact, recent studies have shown a significant correlation between neutrophilic asthma and airway microbiology47.Therefore, this study illustrated important differences of these amino acid dipeptides may reflect the metabolic characteristics of airway microorganisms of NA, and the analysis combined with microbiology may provide us with further scientific evidence.
This study has several limitations to be addressed. First, an external validation in a new cohort was lacking. However, the differential metabolites were validated by exploring the relationships of differential metabolites with clinical and inflammation profiles of asthma in an independent cohort population would strengthen the results. Second, as for the unavailability of reference standards, some of the identified metabolites in the discovery set are undetected, which somewhat limits the exploring of the potential metabolic signature.
In conclusion, different inflammatory asthma phenotypes have specific metabolic profiles with 77 differential metabolic signatures and 5 underlying molecular pathways identified. These metabolic pathways identified involve the histidine metabolism, glycerophospholipid metabolism, nicotinate and nicotinamide metabolism, linoleic acid metabolism, phenylalanine, tyrosine and tryptophan biosynthesis. Differential metabolites identified correlate to clinical and inflammatory profiles of asthma, which may serve as potential therapeutic target in different inflammatory asthma phenotypes.