To the EditorAsthma is characterized by recurrent airflow limitation with airway hyperresponsiveness (AHR) due to chronic inflammation.1 Various types of cells, cytokines, chemokines and mediators are involved in the pathogenesis of asthma.2 The mechanisms underlying AHR have attracted considerable attention in recent decades. AHR reportedly did not develop in several asthma models, e.g., mice lacking certain cell types—such as mast cells or eosinophils—or not expressing certain genes, such as for type 2 cytokines or FcεRI. However, T2 inflammation itself is also impaired in some of these genetically-engineered mice. In addition, these models do not completely recapitulate human asthma. Therefore, the specific molecular pathway(s) regulating human AHR remains unknown.3In humans, mechanisms of AHR have been partially identified in ex vivo models using lung, trachea, or bronchi,4,5 but the in vivo mechanisms remain unclear. Recently, biologics have been approved for steroid-refractory asthma and provide strong evidence of roles for certain molecular pathways in the pathogenesis of asthma.6,7 For example, an anti-IgE monoclonal antibody (omalizumab) did not alleviate AHR even after the symptoms were significantly reduced.8 However, no studies have investigated AHR after treatment with anti-IL-5, anti-IL-5R, or anti-IL-4Rα antibodies.Here, we describe our experience using dupilumab, an anti-IL-4Rα antibody, to treat a 12-year-old boy with refractory atopic asthma.An 8-year-old boy who had suffered from bronchial asthma since he was 3 years old was admitted to a tertiary-care hospital, Yawatahama City General Hospital, due to a severe asthma exacerbation in May 2016. Following discharge, he visited the allergy unit of the hospital. He was treated with an oral leukotriene receptor antagonist, a β2 agonist and slow-release theophylline (SRT), as well as with inhaled high-dose corticosteroid and a long-acting β2 agonist. However, his asthma symptoms did not fully resolve. In Spring of 2019, he developed pertussis, with repeated chronic coughing and wheezing episodes, and his respiratory function subsequently worsened. He became unable to exercise normally at school due to exercise-induced bronchoconstriction. In September 2019, he was subcutaneously injected with 600 mg of dupilumab, followed by 300 mg every 2 weeks.AHR, respiratory function, serum total and specific IgE levels, and peripheral eosinophil counts were evaluated before and at 4 and 6 months after starting dupilumab. AHR was assessed by the standard acetylcholine inhalation test (AcIT) in accordance with the guideline of the Japanese Society of Allergology. That is, he inhaled acetylcholine chloride solutions diluted with saline from low to high concentrations (0, 39, 78, 156, 313, 625, 1250, 2500, 5000, 10000, 20000 µg/mL) for two minutes, followed by a pulmonary function test; this was repeated until there was a 20% decline in his forced expiratory volume in one second (FEV1). The last concentration of inhaled solution was defined as the threshold of the AcIT, and the cut-off concentration between asthma patients and normal subjects was 10000 µg/ml. All medications except SRT were stopped 48 hours before the AcIT, and SRT was stopped 18 hours before the AcIT.His subjective symptoms gradually improved during the first 4 months of dupilumab treatment. Surprisingly, his AHR induced by acetylcholine inhalation completely disappeared at that point, and that status continued for two months. SRT and other oral and inhaled bronchodilators were discontinued 7 months after dupilumab was started. Since then, he has experienced no asthma symptoms, such as cough, wheeze or dyspnea, even after daily exercise, although his respiratory function has not yet fully recovered.Table 1 shows the following data that were determined before and at 4 and 6 months after starting dupilumab: respiratory function data, serum theophylline level, AcIT threshold concentration, serum total IgE level,Dermatophagoides pteronyssinus -specific IgE level, and peripheral eosinophil count. After starting dupilumab, the AcIT threshold concentration increased dramatically, from 313 to over 20000 µg/mL, and the total and specific IgE levels decreased, but the peripheral eosinophil count decreased only by half.We administered dupilumab, an anti-IL-4Rα antibody, to a 12-year-old boy with severe atopic asthma, and his asthmatic symptoms disappeared, with drastic improvement of AHR. His total and specific IgE levels decreased markedly, but his peripheral eosinophil count decreased only by half. On the other hand, an earlier study found that omalizumab, an anti-IgE biologic, improved the clinical symptoms and decreased the peripheral eosinophil count, but AHR remained unchanged.8 Taken together, those findings imply that blockade of IL-4 and IL-13 is involved in the causation of AHR.IL-4 and IL-13, but not IL-5 or IL-17A, were reported to induce hyperresponsiveness to histamine by enhancing expression of histamine H1 receptor and cysteinyl leukotriene receptor 1 in isolated human small airway tissue.5 On the other hand, dupilumab treatment abrogated those effects of IL-4 and IL-13.5 The clinical course of our case is in good agreement with those earlier findings.This report has a limitation, since it reports a single case. Nevertheless, we believe that this is the first study showing that dupilumab therapy directly improved AHR in an atopic asthma patient. In the present patient, the peripheral eosinophil count after dupilumab treatment decreased only by half. That is presumably because IL-5, the critical cytokine for eosinophil development, activation and survival, was not inhibited by dupilumab. The dupilumab treatment also reduced his asthma symptoms and improved his quality of life, although his respiratory function, especially the maximal mid-expiratory flow, was not normalized, presumably due to the presence of airway remodeling. We expect that his respiratory function will improve later, and further observation and assessment are thus needed.In conclusion, IL-4R signaling is likely involved in development of AHR in atopic asthma patients.

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.

Background: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE cell-mediated food allergy characterized by repetitive vomiting and other gastrointestinal symptoms. Although little is known about FPIES pathophysiology, some cytokines have been reported to be involved. Since one of the main symptoms is vomiting, which is common to other diseases, it is difficult to distinguish acute FPIES from other conditions such as infectious enterocolitis. Thus, specific biomarkers are required for differential diagnosis. We aimed to identify potential biomarkers distinguishing acute FPIES from infectious enterocolitis and IgE-mediated anaphylaxis, which also cause vomiting. Methods: Seven patients with acute FPIES, nine with IgE-mediated anaphylaxis, and six with infectious enterocolitis were enrolled. The serum concentrations of interleukins (IL)-2, -4, -6, -8, -10, interferon-γ, and tumor necrosis factor-α were measured and compared among the three groups of patients. The serum concentrations of IL-2 and IL-10 were also compared between the symptomatic and asymptomatic stages. Alterations in serum cytokine levels were evaluated in acute FPIES during an oral food challenge test. Results: Serum IL-2 and IL-10 levels were significantly higher in acute FPIES patients than in patients with infectious enterocolitis and IgE-mediated anaphylaxis, whereas no significant differences were detectable in the serum levels of the other cytokines. The IL-2 and IL-10 elevation was only observed in the symptomatic stage of acute FPIES. Conclusion: The elevation in serum levels of IL-2 and IL-10 was specifically observed in symptomatic acute FPIES cases, suggesting that the measurement of IL-2 and IL-10 could be employed for differential diagnosis.