Arisa Igarashi

and 2 more

To the Editor: Bronchial asthma is characterized by restricted airflow due to chronic airway inflammation, and frequent lower respiratory viral infections in early life are a significant risk factor for development of the disease. Previous studies demonstrated that anti-viral interferon (IFN) production, including of IFN-α, IFN-β and IFN-λ, by leukocytes and bronchial epithelial cells can be impaired in asthma patients.1 An epidemiological study found that allergic sensitization precedes wheeze during asthma development in children, suggesting that Type 2 (T2) conditions play a key role in the impaired anti-viral IFN production. Furthermore, a prospective cohort study showed that, regardless of the type of virus, each successive lower respiratory viral infection with wheeze increases the risk of asthma by about 1.5 fold.2 However, we still don’t have a full understanding of the precise mechanism(s) of how respiratory viral infections under T2 conditions lead to development of asthma.A meta-analysis of large-scale genome-wide association studies revealed that both IL-33 and its receptor, IL-33 receptor(IL-33R ; also known as ST2 ), are closely associated with asthma development.3 Indeed, IL-33 expression was reportedly increased in rhinovirus-infected bronchial epithelial cells and correlated significantly with the disease severity of asthma.4 This suggests that virus-induced IL-33 in the airway may be fundamentally involved in the mechanistic links between viral infection and development and/or exacerbation of asthma. In addition, impairment of anti-viral IFN production was reported to cause necrosis—but not apoptosis—of the virus-infected epithelium,5 which results in release of bioactive IL-33.MicroRNAs (miRNAs) are small, non-coding RNA molecules (containing about 22 nucleotides) that are found in diverse organisms. miRNAs regulate expression of a broad spectrum of target genes through RNA silencing and/or post-transcriptional regulation. Among them, microRNA-29a (miR-29a) was induced by respiratory syncytial virus (RSV) infection in a human lung adenocarcinoma cell line, A549, and suppressed expression of IFN (α, β and ω) receptor 1 (IFNAR1).6 Furthermore, miR-29a regulated the expression of soluble ST2 (sST2), a decoy receptor for IL-33, in human tenocytes.7 Based on those earlier findings, we focused on miR-29 in the present study. We hypothesized that T2 cytokine induces miR-29 expression in bronchial epithelial cells, leading to suppression of both sST2 release and IFNAR1 expression by epithelial cells, and culminating in asthma development and/or exacerbation.Based on that hypothesis, we first examined whether T2 cytokine and inflammatory cytokine induced sST2 production in a human bronchial epithelial cell line, BEAS-2B. The detailed methods are described in Supporting Information. Specific ELISA showed that IL-4 and TNF-α synergistically induced sST2 release from BEAS-2B, in a dose-dependent manner (Figure 1A). Next, to examine the effects of miR-29 overexpression or inhibition on that cytokine-induced sST2 release, BEAS-2B cells were first transfected with miR-29 mimics or inhibitors for 24 hours and then stimulated with a combination of IL-4 and TNF-α for 48 hours.The human miR-29 family consists of three mature members, i.e., miR-29a, miR-29b, and miR-29c. These miR-29s are encoded by the miR-29a/b-1 cluster on chromosome 7q32.3 and the miR-29c/b-2 cluster on chromosome 1q32.2, respectively (Figure 1B).8 The three family members share an identical seed sequence (Figure 1B), and their functional properties are thought to be similar. We examined the effects of miR-29a and miR-29b in this study. ELISA of the culture supernatants showed that inhibition of miR-29a or miR-29b significantly enhanced cytokine-induced sST2 release (Figure 1C). In contrast, overexpression of miR-29a or miR-29b almost completely inhibited that release, indicating that these miR-29s regulate sST2 release from bronchial epithelial cells under T2 conditions. Of note, neither inhibition nor overexpression of miR-29a or miR-29b had any effects on the protein levels of the ST2 receptor in the BEAS-2B cells (Figure 1D, upper panel). These results suggest that T2 cytokine-induced miR-29 plays a critical role in IL-33-dependent allergic inflammation through regulation of sST2 release from bronchial epithelial cells.Furthermore, transfection of either miR-29a or miR-29b inhibitors significantly enhanced IFNAR1 protein expression in the BEAS-2B cells (Figure 1D, middle panel), which is consistent with earlier findings for miR-29a in A549 cells.6 Conversely, transfection of miR-29 mimics resulted in reduced IFNAR1 expression in BEAS-2B cells, suggesting that overproduction of miR-29s in bronchial epithelial cells may lead to suppression of antiviral responses by IFNs. Thus, we found that miR-29s simultaneously regulate the expression of both sST2 and IFNAR1 in bronchial epithelial cells. Our findings suggest the possibility that T2 cytokine-induced miR-29s in airway epithelial cells are key players in the development and/or exacerbation of asthma triggered by respiratory viral infections through both decreasing IFN-regulated antiviral activities and exacerbating IL-33-dependent allergic inflammation.miRNAs are released from cells into the extracellular environment via exosomes, which can then fuse with target cells. This process can deliver various proteins and nucleic acids, including miRNAs, into even distant target/receiving cells.9 We, therefore, examined whether exosomes similarly export miR-29s from bronchial epithelial cells. BEAS-2B cells were stimulated with a combination of IL-4 and TNF-α for 48 hours, and exosomal fractions were collected from the culture supernatants. Although qPCR detected both miR-29a and miR-29b in the exosomes even without that T2 cytokine stimulation (control), both of their copy numbers were significantly increased by that stimulation (Figure 2A). Furthermore, Western blot analysis also found that expression of CD81, an exosome marker, was enhanced by the cytokine stimulation (Figure 2B). These results suggest that T2 cytokine-stimulated epithelial cells release more exosomes containing more miR-29s than unstimulated cells.This study has several limitations. First, no functional experiments were performed in this study to confirm the effects of the changes in sST2 release or IFNAR1 expression. In addition, we did not measure the expression levels of miR-29s in clinical samples.Figure S1 summarizes our findings as a schematic illustration of bronchial epithelial cells. Based on those findings, we hypothesize that elevated nasal, bronchial and/or exosomal levels of miR-29s in infancy may be useful biomarker(s) for predicting later development of asthma, and further studies are needed. Our data suggest a new perspective that miRNAs are crucially involved in the association between viral infection and asthma development. We believe that our research has great significance in pointing to a novel direction for further studies and the existence of a new key player, i.e., miRNAs, in the relationship between viral infections and asthma development.