Bronchial asthma in children is a kind of chronic airway inflammation caused by immune regulation disorder(14). In the past, Th1 cells were considered to be the protective factor of bronchial asthma. Among many pathological mechanisms of asthma, the imbalance of Th1/Th2 cells plays a major role, which is considered to be the most classic pathogenesis of asthma(15, 16). However, studies have found that the imbalance theory is different from the experimental results. Blocking Th2 cytokines cannot effectively alleviate asthma, so it cannot fully explain the pathological mechanism of bronchial asthma(17). Th9 cell is a new type of CD4+ T cell subtype, which is named because it mainly secretes IL-9. For a long time, IL-9 has been regarded as a related factor secreted by Th2 cells. Until 2008, Th9 cells were discovered as a new type of CD4+ T cells(18). Although Th2 cells can also secrete IL-9, the amount of IL-9 secreted by Th2 cells is far less than that of Th9 cells. It was proved that IL-9 in peripheral blood of asthmatic children mainly came from Th9(5). Also, studies have further confirmed that the CD4+ T cells producing IL-9 showed completely different Th cell subtypes, that is Th9: ①Compared with Th2, Th9 secretes more IL-9, while other cytokines related to Th2, such as IL-4, IL-5 and IL-13, secrete less; ②Th9 cells did not express GATA binding protein 3 (GATA-3), orphan associated receptor (ROR-γT) and forkhead box protein P3 (Foxp3), the key transcription factors of Th2, Th17 and Treg, respectively(19).
A large number of studies have shown that Th9 is involved in the pathogenesis of asthma. In human asthma, Neurath et al have found that Th9 and IL-9 in the peripheral blood of adult asthma patients were higher than those of healthy people(20). IL-9 protein was found in the sputum of asthmatic patients, and the expression of IL-9 increased with the aggravation of the disease(21). A similar phenomenon was also found in asthmatic children. Jia et al have found that IL-9 level is significantly elevated in allergic asthma children, along with the existence of antigen-specific Th9 cells, suggesting that Th9 cells may be the major source of IL-9 in children with allergic asthma(5). In addition, animal experiments showed that not only the proportion of Th9 cells in peripheral blood and the level of IL-9 in asthma model mice were significantly higher than those in control group, but also the level of IL-9 mRNA in lung tissue was significantly increased(22). Our study observed IL-9 level in lung tissue and BALF and the proportion of CD4+ Th9 cells in the PBMCs of asthmatic children were significantly higher than that of non-asthmatic children, which was consistent with the existing reporters.
Different cytokines can affect the differentiation of primitive CD4+ T cells into different subtypes. TGF-β and IL-4 can promote Th cells to differentiate into Th9 cells. According to Veldhoen’s study, in the absence of IL-6, the synergistic effect of TGF-β 1 and IL-4 can promote the highly polarized primitive Th cells to differentiate into Th9 cells(6). In another experiment, when Dardalhon studied the original CD4+ foxp32−CD62l+ T cells from mice, he also found that under the combined effect of TGF-β 1 and IL-4, the original cells differentiated into Foxp3− effector T cells that produced IL-9 and IL-10, namely Th9 cells(18). Herein, we successfully induced CD4+ T cells to differentiate into Th9 cell with TGF-β and IL-4. Studies have shown that the development of Th9 and IL-9-producing T cells requires a unique transcription factor, such as IRF4(23), BATF3(24) and IRF1(25), which have been proved to be transcription factors of IL-9 gene and regulate the transcription of IL-9 gene by binding to the promoter during Th9 cell differentiation. FOXO1 can bind to IL-9 and IRF4 promoters and promote IL-9 production by T cells. Inhibition of FOXO1 can inhibit IL-9 secretion by T cells(13). In this study, we also found significantly upregulated IRF4, FOXO1 and IL-9 in lung tissues of asthma mice, and these mRNA and protein expression were all increased under the condition of inducing Th9 cell differentiation, which was in agreement with the result reported before.
Abnormal miRNA expression has been extensively reported and considered as a marker of human diseases. By searching PubMed, a large number of miRNAs have been reported to be involved in the pathogenesis of asthma(26, 27). MiR-493-5p is indicated to be a tumor suppressor gene, but its expression and role in asthma are unknown. In this study, PBMCs were isolated from peripheral blood of asthmatic children, and high throughput sequencing was used to screen different expression of miRNAs. The informatics analysis results showed that miR-493-5p, which was related to Th cell differentiation, was downregulated in PBMCs of asthmatic children. Further studies showed that the expression of miR-493-5p was downregulated in lung tissue and BALF of asthmatic mice, and miR-493-5p could negatively regulate the differentiation of Th9 cells in vitro. Animal experiments have further shown that miR-493-5p agomiR attenuated allergic airway inflammation and airway hyperreactivity. In addition, using a dual-luciferase reporter system, we have found that FOXO1 is a direct target of miR-493-5p. The subsequent rescue experiments proved miR-493-5p regulating the Th9 cells differentiation by targeting FOXO1.
Actually, our mice models are not obtained through gene editing, but OVA sensitive asthma model. Asthma is a polygenic disease in humans, but in view of ethical issues and current technical limitations, we have only done relevant experiments in model mice, but not in human. In addition, our experiments were carried out to explore the pathological mechanisms of human asthma, and this study might benefit more from some confirmation with human cells. Perhaps there might be a difference between human and mouse cells, which needs our further consummation.
In conclusion, this study demonstrated that miR-493-5p inhibits airway inflammation in asthma through suppressed FOXO1 expression to reduce Th9 cell differentiation, and decreased IL-9 secretion. MiR-493-5p/FOXO1 may serve as a potential therapeutic target in the development of asthma.