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.

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.

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.

Adoptive transfer of type 1 regulatory T cells suppressed the development of airway hyperresponsiveness in ovalbumin-induced airway inflammation model mice.

Type 1 regulatory T (Tr1) cells are CD4+ T cells that produce a large amount of IL-10, an anti-inflammatory cytokine. However, it has not been fully elucidated whether Tr1 cells suppress allergic asthma. In this study, the effects of adoptive transfer of in vitro-induced Tr1 cells on allergic asthma were evaluated. Splenocytes from ovalbumin (OVA)-sensitized BALB/c mice were cultured with OVA, IL-21, IL-27, and TGF-ß. After culture, IL-10-producing CD4+ T cells were isolated by Dynabeads mouse CD4 and IL-10 secretion assay, and analyzed by flow cytometry. Purified Tr1 cells (IL-10+ CD4+ T cells) were intravenously injected into OVA-sensitized BALB/c mice. The recipient mice were intratracheally challenged with OVA. Airway hyperresponsiveness to methacholine was assessed by the forced oscillation technique, followed by bronchoalveolar lavage (BAL). Almost all of the induced IL-10-producing CD4+ T cells were negative for interferon-γ, IL-4, IL-17A, and forkhead box P3, suggesting that the cells were Tr1 cells. The adoptive transfer of Tr1 cells significantly suppressed the development of airway hyperresponsiveness, and increases in IL-5, eosinophils, and neutrophils in BAL fluid. In conclusion, we demonstrated that Tr1 cells suppressed allergic asthma in mice.

Phenotype analyses of IL-10-producing Foxp3- CD4+ T cells increased by subcutaneous immunotherapy in allergic airway inflammation.

INTRODUCTION: The mechanisms of allergen immunotherapy are not fully elucidated. Here, we sought to develop a murine model to demonstrate the effectiveness of subcutaneous immunotherapy (SCIT) for allergic responses. As excessive antigen dosages may induce immune tolerance in sensitized mice, the effects of SCIT were assessed by varying the antigen dosage. The mechanisms of SCIT were analyzed by focusing on the induction of Foxp3+ Treg cells and IL-10-producing Foxp3- CD4+ T cells, as well as on the phenotype of the latter cells. METHODS: Ovalbumin (OVA) + Al(OH)3-sensitized mice received subcutaneous dosages of OVA at 0.01, 0.1 or 1 mg/animal for SCIT, followed by intratracheal challenges with OVA at 5, 50 or 500 µg/animal. RESULTS: The maximum effects of SCIT were observed with 1 mg/animal of OVA for airway inflammation induced by 5 µg/animal of OVA, in which airway eosinophilia and Th2 cytokine production were markedly suppressed. The increase in the OVA-specific IgE level was significantly suppressed by SCIT. The development of bronchial epithelial thickening and mucus accumulation were also suppressed by SCIT. Concomitantly, IL-10-producing Foxp3- CD4+ T cells were increased in the lungs by SCIT, but Foxp3+ Treg cells were not. Most of the induced IL-10-producing Foxp3- CD4+ T cells were negative for either IL-5 or LAG-3, but positive for CD49b. CONCLUSION: We successfully developed an airway allergic model for SCIT. It was suggested that most of IL-10-producing Foxp3- CD4+ regulatory T cells increased by SCIT in the lungs were CD49b+ CD4+ regulatory T cells, but neither Th2 cells nor Tr1 cells.

Differences in Gene Regulation by Dual Ligands of Nuclear Receptors Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR) in HepG2 Cells Stably Expressing CAR/PXR.

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate various genes involved in xenobiotics and drug metabolism. In many cases, CAR/PXR share ligands termed dual ligands of CAR/PXR. It is difficult to investigate the effect of CAR/PXR dual ligands in cell lines because CAR and PXR expression is scarcely detected in cultured cell lines. Here, we established a tetracycline-inducible human CAR and stably human PXR-overexpressing HepG2 cell line (HepTR/hCAR/hPXR) to examine CAR/PXR dual ligands. In the present study, we investigated the regulation of CYP2B6, CYP2C9, CYP3A4, and UDP-glucuronosyl transferase, which are target genes of CAR/PXR, by dual ligands of CAR/PXR in two transfectants. Activation of CAR and PXR in cells treated with a high dose of CITCO [6-(4-chlorophenyl)-imidazo(2,1-b)thiazole-5-carbaldehyde] or cotreated with rifampicin and tetracycline resulted in synergistic enhancement of CYP3A4, but not CYP2B6, CYP2C9, or UGT1A1, mRNA expression in HepTR/hCAR/hPXR cells. In contrast, this synergistic effect was not observed in HepTR/hCAR cells. These observations were also demonstrated in human primary hepatocytes. Taken together, our results suggest that dual ligands of CAR/PXR show distinct gene regulation patterns by cross-talk between CAR and PXR. Furthermore, the two newly established cell lines are useful tools to investigate dual ligands of CAR/PXR.

Characterization and antimicrobial susceptibility of Dysgonomonas capnocytophagoides isolated from human blood sample.

Dysgonomonas capnocytophagoides belongs to a group of facultative anaerobic Gram-negative coccobacilli that was formerly designated CDC group DF-3. We evaluated the characteristics of this microbe and its susceptibility to antimicrobial agents. In this study, D. capnocytophagoides was isolated by anaerobic blood cultures from a 78-year-old male with pancreatic cancer, ischemic heart disease, and diabetes mellitus, who also showed symptoms of cholangitis. The isolated strain demonstrated resistance to various beta-lactams, erythromycin, aminoglycosides, and fluoroquinolones, but was susceptible to sulfamethoxazole-trimethoprim, clindamycin, minocycline, and chloramphenicol. The results of all biochemical tests and the homology of the 16S rRNA gene were consistent with previous reports of D. capnocytophagoides.