Effects of chitinase-1 inhibitor in obesity-induced and -aggravated asthma in a murine model.

AIMS: Despite recent investigations on the role of chitinase in asthma, its role in obesity-induced asthma has not been evaluated. Therefore, we investigated the roles of chitin, chitinase-1, and a chitinase-1 inhibitor (compound X, CPX) in a murine model. MAIN METHODS: We assigned C57BL/6 mice to the ovalbumin (OVA) model or obesity model group. In the OVA model, mice received intraperitoneal OVA twice within a 2-week interval and intranasal OVA for 3 consecutive days. Additionally, chitin was intranasally administered for 3 consecutive days, and CPX was intraperitoneally injected three times over 5 days. In the obesity model, a high-fat diet (HFD) was maintained for 13 weeks, and CPX was intraperitoneally injected eight times over 4 weeks. KEY FINDINGS: In the OVA model, chitin aggravated OVA-induced airway hyper-responsiveness (AHR), increased bronchoalveolar lavage fluid (BALF) cell proliferation, increased fibrosis, and increased the levels of various inflammatory cytokines (including chitinase-1, TGF-ß, TNF-α, IL-1 ß, IL-6, IL-4, and IL-13). CPX treatment significantly ameliorated these effects. In the obesity model, HFD significantly increased AHR, BALF cell proliferation, fibrosis, and the levels of various inflammatory cytokines. Particularly, compared to the control group, the mRNA expression of chitinase, chitinase-like molecules, and other molecules associated with inflammation and the immune system was significantly upregulated in the HFD and HFD/OVA groups. Immunofluorescence analysis also showed increased chitinase-1 expression in these groups. CPX significantly ameliorated all these effects in this model. SIGNIFICANCE: This study showed that CPX can be an effective therapeutic agent in asthma, especially, obesity-induced and -aggravated asthma to protect against the progression to airway remodeling and fibrosis.

Particulate matter10-induced airway inflammation and fibrosis can be regulated by chitinase-1 suppression.

BACKGROUND: Particulate matter10 (PM10) can induce airway inflammation and fibrosis. Recently, chitinase-1 has been shown to play key roles in inflammation and fibrosis. We aimed to investigate the effects of chitinase-1 inhibitor in PM10-treated murine mice models. METHODS: In female BALB/c mice, PM10 was intranasally administered six times over 3 weeks, and ovalbumin (OVA) was intraperitoneally injected and then intranasally administered. Chitinase-1 inhibitor (CPX) 6 times over 3 weeks or dexamethasone 3 times in the last week were intraperitoneally administered. Two days after the last challenges, mice were euthanized. Messenger RNA sequencing using lung homogenates was conducted to evaluate signaling pathways. RESULTS: PM10 and/or OVA-induced airway inflammation and fibrosis murine models were established. CPX and dexamethasone ameliorated PM10 or PM10/OVA-induced airway hyper-responsiveness, airway inflammation, and fibrosis. CPX and dexamethasone also reduced levels of various inflammatory markers in lung homogenates. PM10 and OVA also induced changes in mRNA expression across an extreme range of genes. CPX and dexamethasone decreased levels of mRNA expression especially associated with inflammation and immune regulation. They also significantly regulated asthma and asthma-related pathways, including the JACK-STAT signaling pathway. CONCLUSIONS: Chitinase-1 suppression by CPX can regulate PM10- and OVA-induced and aggravated airway inflammation and fibrosis via an asthma-related signaling pathway.

House dust mite-induced Akt-ERK1/2-C/EBP beta pathway triggers CCL20-mediated inflammation and epithelial-mesenchymal transition for airway remodeling.

House dust mite (HDM) allergens cause inflammatory responses and chronic allergic diseases such as bronchial asthma and atopic dermatitis. Here, we investigate the mechanism by which HDM induces C-C chemokine ligand 20 (CCL20) expression to promote chronic inflammation and airway remodeling in an HDM-induced bronchial asthma mouse model. We showed that HDM increased CCL20 levels via the Akt-ERK1/2-C/EBPß pathway. To investigate the role of CCL20 in chronic airway inflammation and remodeling, we made a mouse model of CCL20-induced bronchial asthma. Treatment of anti-CCL20Ab in this mouse model showed the reduced airway hyper-responsiveness and inflammatory cell infiltration into peribronchial region by neutralizing CCL20. In addition, CCL20 induced the Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation through NLRP3 deubiquitination and transcriptional upregulation in BEAS-2B cells. As expected, anti-CCL20Ab markedly suppressed NLRP3 activation induced by CCL20. Moreover, HDM-induced CCL20 leads to epithelial-mesenchymal transition in the lung epithelium which appears to be an important regulator of airway remodeling in allergic asthma. We also found that anti-CCL20Ab attenuates airway inflammation and remodeling in an HDM-induced mouse model of bronchial asthma. Taken together, our results suggest that HDM-induced CCL20 is required for chronic inflammation that contributes airway remodeling in a mouse model of asthma.

Obesity-induced Vitamin D Deficiency Contributes to Lung Fibrosis and Airway Hyperresponsiveness.

Vitamin D (VitD) has pleiotropic effects. VitD deficiency is closely involved with obesity and may contribute to the development of lung fibrosis and aggravation of airway hyperresponsiveness (AHR). We evaluated the causal relationship between VitD deficiency and the lung pathologies associated with obesity. In vivo effects of VitD supplementation were analyzed using high-fat diet (HFD)-induced obese mice and TGF-ß1 (transforming growth factor-ß1) triple transgenic mice. Effects of VitD supplementation were also evaluated in both BEAS-2B and primary lung cells from the transgenic mice. Obese mice had decreased 25-OH VitD and VitD receptor expressions with increases of insulin resistance, renin and angiotensin-2 system (RAS) activity, and leptin. In addition, lung pathologies such as a modest increase in macrophages, enhanced TGF-ß1, IL-1ß, and IL-6 expression, lung fibrosis, and AHR were found. VitD supplementation to HFD-induced obese mice recovered these findings. TGF-ß1-overexpressing transgenic mice enhanced macrophages in BAL fluid, lung expression of RAS, epithelial-mesenchymal transition markers, AHR, and lung fibrosis. VitD supplementation also attenuated these findings in addition to the attenuation of the expressions of TGF-ß1, and phosphorylated Smad-2/3 in lung. Supplementing in vitro-stimulated BEAS-2B and primary lung cells with VitD inhibited TGF-ß1 expression, supporting the suppressive effect of VitD for TGF-ß1 expression. These results suggest that obesity leads to VitD deficiency and worsens insulin resistance while enhancing the expression of leptin, RAS, TGF-ß1, and proinflammatory cytokines. These changes may contribute to the development of lung fibrosis and AHR. VitD supplementation rescues these changes and may have therapeutic potential for asthma with obesity.

Effects of Particulate Matter 10 Inhalation on Lung Tissue RNA expression in a Murine Model.

BACKGROUND: Particulate matter 10 (PM10; airborne particles <10 µm) inhalation has been demonstrated to induce airway and lung diseases. In this study, we investigate the effects of PM10 inhalation on RNA expression in lung tissues using a murine model. METHODS: Female BALB/c mice were affected with PM10, ovalbumin (OVA), or both OVA and PM10. PM10 was administered intranasally while OVA was both intraperitoneally injected and intranasally administered. Treatments occurred 4 times over a 2-week period. Two days after the final challenges, mice were sacrificed. Full RNA sequencing using lung homogenates was conducted. RESULTS: While PM10 did not induce cell proliferation in bronchoalveolar fluid or lead to airway hyper-responsiveness, it did cause airway inflammation and lung fibrosis. Levels of interleukin 1ß, tumor necrosis factor-α, and transforming growth factor-ß in lung homogenates were significantly elevated in the PM10-treated group, compared to the control group. The PM10 group also showed increased RNA expression of Rn45a, Snord22, Atp6v0c-ps2, Snora28, Snord15b, Snora70, and Mmp12. Generally, genes associated with RNA splicing, DNA repair, the inflammatory response, the immune response, cell death, and apoptotic processes were highly expressed in the PM10-treated group. The OVA/PM10 treatment did not produce greater effects than OVA alone. However, the OVA/PM10-treated group did show increased RNA expression of Clca1, Snord22, Retnla, Prg2, Tff2, Atp6v0c-ps2, and Fcgbp when compared to the control groups. These genes are associated with RNA splicing, DNA repair, the inflammatory response, and the immune response. CONCLUSION: Inhalation of PM10 extensively altered RNA expression while also inducing cellular inflammation, fibrosis, and increased inflammatory cytokines in this murine mouse model.

Efficacy of transdermal immunotherapy with biodegradable microneedle patches in a murine asthma model.

BACKGROUND: House dust mite (HDM) is a well-known cause of asthma. Allergen-specific immunotherapy (AIT) can only modify the natural course of the disease. Conventional routes of HDM AIT are subcutaneous or sublingual. Subcutaneous immunotherapy (SCIT) has a disadvantage of systemic hypersensitive reaction, and the sublingual immunotherapy has a disadvantage of local allergic reaction and low drug adherence. OBJECTIVE: To overcome the weak points of conventional AIT, we developed a HDM loaded biodegradable microneedle patch (MNP) for transdermal immunotherapy (TDIT). We aim to demonstrate the efficacy of TDIT in murine asthma model triggered by HDM compared with conventional SCIT. METHODS: To make HDM asthma mouse model, 5-week-old BALB/c female mice were sensitized and challenged by intranasal administration of HDM. The mice were divided into 5 groups: sham, asthma, low (10 µg) and high dose (100 µg) SCIT, and TDIT (10 µg). To make HDM loaded MNP, droplet-born air blowing method was used. Airway hyperresponsiveness and allergic inflammation markers were analysed by bronchoalveolar lavage fluid, immunohistochemistry, serum immunoglobulin (Ig) analysis, and lung cytokine assays. RESULTS: Airway hyperresponsiveness was ameliorated by TDIT. Eosinophilic inflammation in bronchoalveolar lavage was improved without adverse reactions. Reduction of Th2 (IL-4, IL-5, and IL-13) cytokines, and HDM-specific IgE, induction of Treg (IL-10, TGF-ß), Th1 (IFN-γ) cytokines were observed. Eosinophilic infiltration, goblet cell hyperplasia, and subepithelial fibrosis were also alleviated by TDIT. These changes were more significant in the TDIT group than in subcutaneous AIT group. CONCLUSION: In conclusion, HDM loaded biodegradable TDIT is a novel treatment option to treat asthma which showed more effectiveness and may have better safety profiles than conventional SCIT.

Soluble CD93 in allergic asthma.

CD93 has been shown critical roles in inflammatory and immune diseases. However, in allergic asthma, the potential roles of soluble CD93 (sCD93) have not been well studied. We conducted house dust mite (HDM) stimulation with Der p 1 in BEAS-2B and U937 cells, followed by treatment with dexamethasone or small interfering RNA against CD93. A HDM-induced murine allergic asthma model was also established. We estimated the power of sCD93 to predict allergic asthma in a retrospective post-hoc analysis containing 96 human samples. HDM-stimulated BEAS-2B cells showed increased mRNA expression levels of IL-6, IL-8, IL-33, TSLP, and CD93. The CD93 level in culture supernatants steadily increased for 24 h after allergen stimulation, which was significantly suppressed by both dexamethasone and CD93 silencing. CD93 silencing increased IL-6 and TSLP, but not IL-33 levels in culture supernatants. HDM-induced asthma mice showed significant airway hyperresponsiveness and inflammation with Th2 cytokine activation, along with decreased CD93 expression in bronchial epithelial cells and lung homogenates but increased serum CD93 levels. The sCD93 level in asthma patients was significantly higher than that in healthy controls and could predict asthma diagnosis with moderate sensitivity (71.4%) and specificity (82.4%) (AUC = 0.787, P < 0.001). The level of sCD93 which has potential role to predict asthma significantly increased after HDM stimulation via IL-6 and TSLP in vitro and in vivo.

Empagliflozin and Dulaglutide are Effective against Obesity-induced Airway Hyperresponsiveness and Fibrosis in A Murine Model.

Patients with asthma with obesity experience severe symptoms, are unresponsive to conventional asthma treatment, and lack proper pharmacotherapy. Empagliflozin and dulaglutide, developed for diabetes, reduce weight, decrease insulin resistance, and exert additive effects. We evaluated the efficacy of empagliflozin, dulaglutide, and their combination on obesity-induced airway hyperresponsiveness (AHR) and lung fibrosis using a murine model. We assigned C57BL/6J mice to five groups: control, high-fat diet (HFD), and HFD with empagliflozin, dulaglutide, or both. Mice received a 12-week HFD, empagliflozin (5 days/week, oral gavage), and dulaglutide (once weekly, intraperitoneally). Both drugs significantly attenuated HFD-induced weight increase, abnormal glucose metabolism, and abnormal serum levels of leptin and insulin, and co-treatment was more effective. Both drugs significantly alleviated HFD-induced AHR, increased macrophages in bronchoalveolar lavage fluid (BALF), and co-treatment was more effective on AHR. HFD-induced lung fibrosis was decreased by both drugs alone and combined. HFD induced interleukin (IL)-17, transforming growth factor (TGF)-ß1, and IL-1ß mRNA and protein expression, which was significantly reduced by empagliflozin, dulaglutide, and their combination. Tumour necrosis factor (TNF)-α and IL-6 showed similar patterns without significant differences. HFD-enhanced T helper (Th) 1 and Th17 cell differentiation was improved by both drugs. Empagliflozin and dulaglutide could be a promising therapy for obesity-induced asthma and showed additive effects in combination.

Insulin resistance mediates high-fat diet-induced pulmonary fibrosis and airway hyperresponsiveness through the TGF-ß1 pathway.

Prior studies have reported the presence of lung fibrosis and enhanced airway hyperresponsiveness (AHR) in mice with high-fat-diet (HFD)-induced obesity. This study evaluated the role of TGF-ß1 in HFD-induced AHR and lung fibrosis in a murine model. We generated HFD-induced obesity mice and performed glucose and insulin tolerance tests. HFD mice with or without ovalbumin sensitization and challenge were also treated with an anti-TGF-ß1 neutralizing antibody. AHR to methacholine, inflammatory cells in the bronchoalveolar lavage fluid (BALF), and histological features were evaluated. Insulin was intranasally administered to normal diet (ND) mice, and in vitro insulin stimulation of BEAS-2b cells was performed. HFD-induced obesity mice had increased insulin resistance, enhanced AHR, peribronchial and perivascular fibrosis, and increased numbers of macrophages in the BALF. However, they did not have meaningful eosinophilic or neutrophilic inflammation in the lungs compared with ND mice. The HFD enhanced TGF-ß1 expression in the bronchial epithelium, but we found no differences in the expression of interleukin (IL)-4 or IL-5 in lung homogenates. Administration of the anti-TGF-ß1 antibody attenuated HFD-induced AHR and lung fibrosis. It also attenuated goblet cell hyperplasia, but did not affect the AHR and inflammatory cell infiltration induced by OVA challenge. The intranasal administration of insulin enhanced TGF-ß1 expression in the bronchial epithelium and lung fibrosis. Stimulating BEAS-2b cells with insulin also increased TGF-ß1 production by 24 h. We concluded that HFD-induced obesity-associated insulin resistance enhances TGF-ß1 expression in the bronchial epithelium, which may play an important role in the development of lung fibrosis and AHR in obesity.

Soluble CD93 in Serum as a Marker of Allergic Inflammation.

PURPOSE: CD93 is receiving renewed attention as a biomarker of inflammation. We aimed to evaluate the potential for serum sCD93 to serve as a novel biomarker for allergic inflammation. MATERIALS AND METHODS: We enrolled 348 subjects with an allergic disease [allergic rhinitis (AR), chronic spontaneous urticaria (CSU), or bronchial asthma (BA)], including 14 steroid-naïve BA patients who were serially followed-up. RESULTS: The serum sCD93 levels (ng/mL) in patients with exacerbated AR (mean±standard deviation, 153.1±58.4) were significantly higher than in patients without AR (132.2±49.0) or with stable AR (122.3±42.1). Serum sCD93 levels in exacerbated CSU (169.5±42.8) were also significantly higher than those in non-CSU (132.4±51.6) and stable CSU (122.8±36.2). This trend was also seen in BA. Serum levels in patients with ICS-naïve BA (161.4±53.1) were significantly higher than those in healthy controls without BA (112.2±30.8), low- and medium-dose ICS users. Serum sCD93 levels in high-dose ICS users (72.2±20.6) were significantly lower than those in low- and medium-dose users. The serum sCD93 levels in steroid-naïve patients with BA (195.1±72.7) decreased after ICS use for 4 weeks (134.4±42.8) and 8 weeks (100.7±13.4), serially. CONCLUSION: Elevated serum sCD93 levels reflected exacerbated status of allergic diseases, including CSU, AR, and asthma. ICS use significantly diminished serum sCD93 levels in steroid-naïve patients with BA. This result may suggest sCD93 in serum as a therapeutic marker for allergic inflammation.

Toxic and adjuvant effects of silica nanoparticles on ovalbumin-induced allergic airway inflammation in mice.

BACKGROUND: Silica nanoparticles (SNPs) can easily enter in respiratory system via inhalation because of their low molecular weight and ease of dispersion. Toxicity and adverse effects of SNPs vary according to the physical characteristics of the particle. METHODS: To evaluate the toxic and adjuvant effects of 3 types of SNPs in the airway system, six-week-old female BALB/c mice were intranasally administered 3 types of SNPs (spherical [S-SNP], mesoporous [M-SNP], and polyethylene glycol-conjugated [P-SNP]) alone or SNPs/ovalbumin (OVA), three times weekly for 2 weeks. Airway hyper-responsiveness (AHR), bronchoalveolar lavage fluid (BALF), cytokine levels, and histology of the lungs were analyzed. RESULTS: The S-SNPs/OVA group and M-SNPs/OVA group showed significant AHR, compared to the control group. Among all SNP-treated groups, the group administered SNPs/OVA showed greater inflammatory cell infiltration in BALF, extensive pathological changes, and higher cytokine levels (IL-5, IL-13, IL-1ß, and IFN-γ) than those administered SNPs alone or saline/OVA. CONCLUSION: Exposure to SNPs alone and SNPs/OVA induced toxicity in the respiratory system. SNPs alone showed significant toxic effects on the airway system. Meanwhile, SNPs/OVA exerted adjuvant effects to OVA of inducing allergic airway inflammation. In particular, M-SNPs showed the most severe airway inflammation in both direct toxicity and adjuvant effect assays. P-SNPs induced less inflammation than the other types of SNPs in both models.

Roflumilast Ameliorates Airway Hyperresponsiveness Caused by Diet-Induced Obesity in a Murine Model.

Obese patients with asthma respond poorly to conventional asthma medications, resulting in severe symptoms and poor prognosis. Roflumilast, a phosphodiesterase-4 inhibitor that lowers the levels of various substances that are implicated in obese subjects with asthma, may be effective in the treatment of those subjects. We evaluated the potential of roflumilast as a novel therapeutic agent for obese subjects with asthma. We designed three models: diet-induced obesity (DIO); DIO with ovalbumin (OVA); and OVA. We fed C57BL/6J mice a high-fat diet for 3 months with or without OVA sensitization and challenge. Roflumilast or dexamethasone was administered orally three times at 2-day intervals in the last experimental week. Airway hyperresponsiveness resulting from DIO significantly improved in the roflumilast-treated group compared with the dexamethasone-treated groups. Although DIO did not affect the cell proliferation in bronchoalveolar lavage fluid, increased fibrosis was seen in the DIO group, which significantly improved from treatment with roflumilast. DIO-induced changes in adiponectin and leptin levels were improved by roflumilast, whereas dexamethasone aggravated them. mRNA levels and proteins of TNF-α, transforming growth factor-ß, IL-1ß, and IFN-γ increased in the DIO group and decreased with roflumilast. The reactive oxygen species levels were also increased in the DIO group and decreased by roflumilast. In the DIO plus OVA and OVA models, roflumilast improved Th1 and Th2 cell activation to a greater extent than dexamethasone. Roflumilast is significantly more effective than dexamethasone against airway hyperresponsiveness caused by DIO in the murine model. Roflumilast may represent a promising therapeutic agent for the treatment of obese patients with asthma.

Acute exposure to silica nanoparticles aggravate airway inflammation: different effects according to surface characteristics.

Silica nanoparticles (SNPs) are widely used in many scientific and industrial fields despite the lack of proper evaluation of their potential toxicity. This study examined the effects of acute exposure to SNPs, either alone or in conjunction with ovalbumin (OVA), by studying the respiratory systems in exposed mouse models. Three types of SNPs were used: spherical SNPs (S-SNPs), mesoporous SNPs (M-SNPs), and PEGylated SNPs (P-SNPs). In the acute SNP exposure model performed, 6-week-old BALB/c female mice were intranasally inoculated with SNPs for 3 consecutive days. In the OVA/SNPs asthma model, the mice were sensitized two times via the peritoneal route with OVA. Additionally, the mice endured OVA with or without SNP challenges intranasally. Acute SNP exposure induced significant airway inflammation and airway hyper-responsiveness, particularly in the S-SNP group. In OVA/SNPs asthma models, OVA with SNP-treated group showed significant airway inflammation, more than those treated with only OVA and without SNPs. In these models, the P-SNP group induced lower levels of inflammation on airways than both the S-SNP or M-SNP groups. Interleukin (IL)-5, IL-13, IL-1ß and interferon-γ levels correlated with airway inflammation in the tested models, without statistical significance. In the mouse models studied, increased airway inflammation was associated with acute SNPs exposure, whether exposed solely to SNPs or SNPs in conjunction with OVA. P-SNPs appear to be relatively safer for clinical use than S-SNPs and M-SNPs, as determined by lower observed toxicity and airway system inflammation.