Involvement of Janus kinase-dependent Bcl-xL overexpression in steroid resistance of group 2 innate lymphoid cells in asthma.

The mechanisms underlying the development of steroid resistance in asthma remain unclear. To establish whether as well as the mechanisms by which the activation of Janus kinases (JAKs) is involved in the development of steroid resistance in asthma, murine steroid-resistant models of the proliferation of group 2 innate lymphoid cells (ILC2s) in vitro and asthmatic airway inflammation in vivo were analysed. ILC2s in the lungs of BALB/c mice were sorted and then incubated with IL-33, thymic stromal lymphopoietin (TSLP), and/or IL-7 with or without dexamethasone (10 nM), the pan-JAK inhibitor, delgocitinib (1-10 000 nM), and/or the Bcl-xL inhibitor, navitoclax (1-100 nM), followed by the detection of viable and apoptotic cells. The anti-apoptotic factor, Bcl-xL was detected in ILC2s by flow cytometry. As a steroid-resistant asthma model, ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at a high dose of 500 µg four times. Dexamethasone (1 mg/kg, i.p.), delgocitinib (3-30 mg/kg, p.o.), or navitoclax (30 mg/kg, p.o.) was administered during the challenges. Cellular infiltration into the lungs was analysed by flow cytometry. Airway remodelling was histologically evaluated. The following results were obtained. (1) Cell proliferation concomitant with a decrease in apoptotic cells was induced when ILC2s were cultured with TSLP and/or IL-7, and was potently inhibited by dexamethasone. In contrast, when the culture with TSLP and IL-7 was performed in the presence of IL-33, the proliferative response exhibited steroid resistance. Steroid-resistant ILC2 proliferation was suppressed by delgocitinib in a concentration-dependent manner. (2) The culture with IL-33, TSLP, and IL-7 induced the overexpression of Bcl-xL, which was clearly inhibited by delgocitinib, but not by dexamethasone. When ILC2s were treated with navitoclax, insensitivity to dexamethasone was significantly cancelled. (3) The development of airway remodelling and the infiltration of ILC2s into the lungs in the asthma model were not suppressed by dexamethasone, but were dose-dependently inhibited by delgocitinib. Combination treatment with dexamethasone and either delgocitinib or navitoclax synergistically suppressed these responses. Therefore, JAKs appear to play significant roles in the induction of steroid resistance by up-regulating Bcl-xL in ILC2s. The inhibition of JAKs and Bcl-xL has potential as pharmacotherapy for steroid-resistant asthma, particularly that mediated by ILC2s.

Involvement of CCR5 on interstitial macrophages in the development of lung fibrosis in severe asthma.

CCR5 may be involved in the pathogenesis of asthma; however, the underlying mechanisms remain unclear. In comparison with a mild asthma model, subepithelial fibrosis was more severe and CCR5 gene expression in the lungs was significantly higher in our recently developed murine model of steroid-resistant severe asthma. Treatment with the CCR5 antagonist, maraviroc, significantly suppressed the development of subepithelial fibrosis in bronchi, whereas dexamethasone did not. On the other hand, increases in leukocytes related to type 2 inflammation, eosinophils, Th2 cells, and group 2 innate lymphoid cells in the lungs were not affected by the treatment with maraviroc. Increases in neutrophils and total macrophages were also not affected by the CCR5 antagonist. However, increases in transforming growth factor (TGF)-ß-producing interstitial macrophages (IMs) were significantly reduced by maraviroc. The present results confirmed increases in CCR5-expressing IMs in the lungs of the severe asthma model. In conclusion, CCR5 on IMs plays significant roles in the development of subepithelial fibrosis in severe asthma through TGF-ß production in the lungs.

Steroid-Insensitive Gene Expression of Extracellular Matrix Components and Pro-fibrotic Factors in the Lung Associated with Airway Hyperresponsiveness in Murine Asthma.

Between 5 and 10% of asthma patients do not respond to glucocorticoid therapy. Experimental animal models are indispensable for investigating the pathogenesis of steroid-resistant asthma; however, the majority of murine asthma models respond well to glucocorticoids. We previously reported that multiple intratracheal administration of ovalbumin (OVA) at a high dose (500 µg/animal) induced steroid-insensitive airway eosinophilia and remodeling with lung fibrosis, whereas a low dose (5 µg/animal) caused steroid-sensitive responses. The aims of the present study were as follows: 1) to clarify whether airway hyperresponsiveness (AHR) in the two models is also insensitive and sensitive to a glucocorticoid, respectively, and 2) to identify steroid-insensitive genes encoding extracellular matrix (ECM) components and pro-fibrotic factors in the lung. In comparisons with non-challenged group, the 5- and 500-µg OVA groups both exhibited AHR to methacholine. Daily intraperitoneal treatment with dexamethasone (1 mg/kg) significantly suppressed the development of AHR in the 5-µg OVA group, but not in the 500-µg OVA group. Among genes encoding ECM components and pro-fibrotic factors, increased gene expressions of fibronectin and collagen types I, III, and IV as ECM components as well as 7 matrix metalloproteinases, tissue inhibitor of metalloproteinase-1, transforming growth factor-ß1, and activin A/B as pro-fibrotic factors were insensitive to dexamethasone in the 500-µg OVA group, but were sensitive in the 5-µg OVA group. In conclusion, steroid-insensitive AHR developed in the 500-µg OVA group and steroid-insensitive genes encoding ECM components and pro-fibrotic factors were identified. Drugs targeting these molecules have potential in the treatment of steroid-resistant asthma.

Ceramide Nanoliposomes as Potential Therapeutic Reagents for Asthma.

Ceramides are an emerging class of anti-inflammatory lipids, and nanoscale ceramide-delivery systems are potential therapeutic strategies for inflammatory diseases. This study investigated the therapeutic effects of ceramide nanoliposomes (CNL) on type 2 inflammation-based asthma, induced by repeated ovalbumin (OVA) challenges. Asthmatic mice intratracheally treated with ceramide-free liposomes (Ghost) displayed typical airway remodeling including mucosal accumulation and subepithelial fibrosis, whereas, in CNL-treated mice, the degree of airway remodeling was significantly decreased. Compared to the Ghost group, CNL treatment unexpectedly failed to significantly influence formation of type 2 cytokines, including IL-5 and IL-13, known to facilitate pathogenic production of airway mucus predominantly comprising MUC5AC mucin. Interestingly, CNL treatment suppressed OVA-evoked hyperplasia of MUC5AC-generating goblet cells in the airways. This suggests that CNL suppressed goblet cell hyperplasia and airway mucosal accumulation independently of type 2 cytokine formation. Mechanistically, CNL treatment suppressed cell growth and EGF-induced activation of Akt, but not ERK1/2, in a human lung epithelial cell culture system recapitulating airway goblet cell hyperplasia. Taken together, CNL is suggested to have therapeutic effects on airway remodeling in allergic asthma by targeting goblet cell hyperplasia. These findings raise the potential of ceramide-based therapies for airway diseases, such as asthma.

Local IL-10 replacement therapy was effective for steroid-insensitive asthma in mice.

Subgroups of patients with severe asthma showing marked increases in sputum eosinophils and/or neutrophils are insensitive to corticosteroids. Previous reports have shown that exogenous administration of an anti-inflammatory cytokine, interleukin (IL)-10 negatively regulated both eosinophilic and neutrophilic migration into tissues. The objective of this study was to elucidate whether intratracheal IL-10 administration suppresses asthmatic responses in a steroid-insensitive model of mice. Ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at 500 µg/animal four times. Dexamethasone (1 mg/kg, intraperitoneal) or IL-10 (25 ng/mouse, intratracheal) was administered during the multiple challenges. The number of leukocytes, expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and IL-10 receptor in the lung, and the development of airway remodeling and hyperresponsiveness were evaluated after the fourth challenge. Consistent with our previous study, dexamethasone hardly suppressed the development of airway remodeling and hyperresponsiveness. Although intratracheal IL-10 administration did not affect the development of airway remodeling, the infiltration of eosinophils and neutrophils, and the development of airway hyperresponsiveness were significantly inhibited. Moreover, IL-10 administration significantly decreased the numbers of ICAM-1+ and VCAM-1+ pulmonary vascular endothelial cells, which express IL-10 receptor 1, even though neither production of eosinophilic nor neutrophilic cytokines in the lung was inhibited. Therefore, IL-10 can suppress eosinophil and neutrophil infiltration by inhibiting the proliferation of ICAM-1+ and VCAM-1+ pulmonary vascular endothelial cells, resulting in inhibition of airway hyperresponsiveness in steroid-insensitive asthmatic mice. IL-10 replacement therapy may be clinically useful for the treatment of steroid-insensitive asthma.

Pathogenic changes in group 2 innate lymphoid cells (ILC2s) in a steroid-insensitive asthma model of mice.

A certain population of asthma patients is resistant to steroid therapy, whereas the mechanisms remain unclear. One of characteristic features of steroid-resistant asthma patients is severe airway eosinophilia based on type-2 inflammation. Aims of this study were: 1) to develop a murine model of steroid-resistant asthma, 2) to elucidate that predominant cellular source of a type-2 cytokine, IL-5 was group 2 innate lymphoid cells (ILC2s), 3) to analyze pathogenic alteration of ILC2s in the severe asthma, and 4) to evaluate therapeutic potential of anti-IL-5 monoclonal antibody (mAb) on the steroid-resistant asthma. Ovalbumin (OVA)-sensitized BALB/c mice were intratracheally challenged with OVA at 5 or 500 µg/animal 4 times. Development of airway eosinophilia and remodeling in 5-µg OVA model were significantly suppressed by 1 mg/kg dexamethasone, whereas those in 500-µg OVA model were relatively insensitive to the dose of dexamethasone. ILC2s isolated from the lung of the steroid-insensitive model (500-µg OVA) produced significantly larger amounts of IL-5 in response to IL-33/TSLP than ILC2s from the steroid-sensitive model (5-µg OVA). Interestingly, TSLP receptor expression on ILC2s was up-regulated in the steroid-insensitive model. Treatment with anti-IL-5 mAb in combination with dexamethasone significantly suppressed the airway remodeling of the steroid-insensitive model. In conclusion, multiple intratracheal administration of a high dose of antigen induced steroid-insensitive asthma in sensitized mice. IL-5 was mainly produced from ILC2s, phenotype of which had been pathogenically altered probably through the up-regulation of TSLP receptors. IL-5 blockage could be a useful therapeutic strategy for steroid-resistant asthma.

Sublingual immunotherapy for 4 years increased the number of Foxp3+ Treg cells, which correlated with clinical effects.

OBJECTIVE: At least 3 years of sublingual immunotherapy (SLIT) is required to achieve long-term clinical tolerance for allergens. However, immunological changes with more than 3 years of SLIT have not yet been elucidated in detail. The present study investigated whether the numbers of regulatory T (Treg) cells and regulatory B (Breg) cells increased with 4 years of SLIT and if these increases correlated with clinical effects for pollinosis. METHODS: Seven Japanese cedar pollinosis patients received SLIT in 2014 or 2015 and continued treatment until May 2019. In May 2017 and May 2019, peripheral blood mononuclear cells (PBMCs) were collected from the patients, and analyzed by flow cytometer. RESULTS: (1) The visual analogue scale (VAS) was significantly higher in 2019 than in 2017. (2) The percentages of Foxp3+ Treg cells, type 1 regulatory T (Tr1) cells, and Breg cells in PBMCs were significantly higher in 2019 than in 2017. (3) The percentage of Foxp3+ Treg cells in PBMCs positively correlated with VAS, whereas those of Tr1 cells and Breg cells did not. CONCLUSIONS: These results suggest that 4 years of SLIT is needed to achieve sustained increases in Foxp3+ Treg cells, which are closely associated with the efficacy of SLIT.