IFN-γ In Bronchoalveolar Lavage Fluid Research Articles

MSC-EXO and tempol ameliorate bronchopulmonary dysplasia in newborn rats by activating HIF-1α.

Bronchopulmonary dysplasia (BPD) is a major complication of premature infants and an important cause of morbidity and mortality. This study investigates the effect of the combination of mesenchymal stem cells-derived exosomes (MSC-EXO) and tempol on BPD and analyzes its mechanism. MSC-EXO was extracted by centrifugation and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis, and western blot analysis(WB). Tidal volume (TV), minute ventilation (MV), peak inspiratory flow (PIF), and dynamic pulmonary compliance (Cdyn) of rats were measured by BuxCo pulmonary function experimental platform. Hematoxylin-eosin staining was performed to observe the lung morphology and radical alveolar count (RAC)and mean linear intercept (MLI) were assessed. Immunofluorescence (IF) was conducted to detect the expression of CD31 and α-SMA in pulmonary blood vessels. The kits were used to calculate malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (TAOC) concentration in lung tissue. Enzyme linked immunosorbent assaywas applied to detect the levels of IL-1β, IL-17, IL-6, and IFN-γ in bronchoalveolar lavage fluid. In addition, the expressions of HIF-1α, vascular endothelial growth factor(VEGF), p-PI3K, and p-AKT were analyzed by WB and IF. We successfully extracted and identified MSC-EXO. In BPD rats, TV, MV, PIF, and Cdyn decreased, alveoli were simplified, and the number of interalveoli small vessels, blood vessel density decreased. Moreover, RAC, CD31, TAOC, and SOD decreased, and MLI, α-SMA, MDA, IL-1β, IL-17, IL-6, and IFN-γ increased, which was reversed by the combination of MSC-EXO and tempol treatment after combined treatment. In addition, the expression levels of HIF-1α, VEGF, p-PI3K, and p-AKT were increased after combined treatment. Combined treatment could improve lung tissue injury, promote pulmonary vascular remodeling, restore lung function, and inhibit oxidative stress in BPD rats. These effects were achieved through activation of HIF-1α.

Salidroside Mitigates Airway Inflammation in Asthmatic Mice via the AMPK/Akt/GSK3β Signaling Pathway

Introduction: This study aimed to explore the effects and mechanisms of salidroside (SAL) in airway inflammation in asthmatic mice. Methods: Mice were sensitized with ovalbumin (OVA) to establish an asthma model. They were divided into the control group, OVA group, SAL low-dose group (SAL-L), SAL high-dose group (SAL-H), and dexamethasone (DXM) group. The airway reactivity of the mice was measured, and the total cells, neutrophils, eosinophils, and lymphocytes were counted, respectively. The levels of IL-4, IL-5, IL-13, and IFN-γ in bronchoalveolar lavage fluid (BALF) were detected by ELISA. Immunohistochemistry was used to detect the expression levels of p-AMPK, p-Akt, and p-GSK3β. Western blot was used to detect cytokine levels in lung tissue and p-AMPK, p-Akt, and p-GSK3β levels in LPS-induced 16HBE cells. Results: The airway hyperresponsiveness of asthmatic mice in the SAL-H group decreased (p < 0.05), and the total number of cells, neutrophils, eosinophils, and lymphocytes decreased significantly (p < 0.05). In addition, the airways of mice showed airway inflammatory infiltration and goblet cell proliferation, and the corresponding cellular inflammatory factors IL-4, IL-5, and IL-13 were significantly decreased. However, the expression of IFN-γ in BALF and lung tissues was increased (p < 0.05). Moreover, after the mice were treated with SAL, the phosphorylation level of AMPK was significantly increased, which further reduced the phosphorylation levels of Akt and GSK3β (p < 0.05). Both SAL and AMPK inhibitors exerted effects on LPS-induced 16HBE cells, consistent with in vivo results. Conclusion: SAL can inhibit bronchial hyperresponsiveness and reduce tracheal inflammation by increasing AMPK phosphorylation and inhibiting Akt and GSK3β signaling pathways.

The Cholinergic Drug Pyridostigmine Alleviates Inflammation During LPS-Induced Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a critical illness complication that is associated with high mortality. ARDS is documented in severe cases of COVID-19. No effective pharmacological treatments for ARDS are currently available. Dysfunctional immune responses and pulmonary and systemic inflammation are characteristic features of ARDS pathogenesis. Recent advances in our understanding of the regulation of inflammation point to an important role of the vagus-nerve-mediated inflammatory reflex and neural cholinergic signaling. We examined whether pharmacological cholinergic activation using a clinically approved (for myasthenia gravis) cholinergic drug, the acetylcholinesterase inhibitor pyridostigmine alters pulmonary and systemic inflammation in mice with lipopolysaccharide (LPS)-induced ARDS. Male C57Bl/6 mice received one intratracheal instillation of LPS or were sham manipulated (control). Both groups were treated with either vehicle or pyridostigmine (1.5 mg/kg twice daily, 3 mg/day) administered by oral gavage starting at 1 h post-LPS and euthanized 24 h after LPS administration. Other groups were either sham manipulated or received LPS for 3 days and were treated with vehicle or pyridostigmine and euthanized at 72 h. Pyridostigmine treatment reduced the increased total number of cells and neutrophils in the bronchoalveolar lavage fluid (BALF) in mice with ARDS at 24 and 72 h. Pyridostigmine also reduced the number of macrophages and lymphocytes at 72 h. In addition, pyridostigmine suppressed the levels of TNF, IL-1β, IL-6, and IFN-γ in BALF and plasma at 24 and 72 h. However, this cholinergic agent did not significantly altered BALF and plasma levels of the anti-inflammatory cytokine IL-10. Neither LPS nor pyridostigmine affected BALF IFN-γ and IL-10 levels at 24 h post-LPS. In conclusion, treatments with the cholinergic agent pyridostigmine ameliorate pulmonary and systemic inflammatory responses in mice with endotoxin-induced ARDS. Considering that pyridostigmine is a clinically approved drug, these findings are of substantial interest for implementing pyridostigmine in therapeutic strategies for ARDS.

Butyrate mediates the amelioration of Staphylococcal Enterotoxin B-induced Acute Respiratory Distress Syndrome by downregulating the expression of Histone Deacetylases

Abstract Despite rigorous global containment and quarantine efforts that have been applied globally to control SARS-CoV-2, the spreading of this contagious disease has accelerated all over the world. In our study, we used staphylococcal enterotoxin B (SEB), which is a superantigen can lead to toxic shock syndrome in C57BL/6 murine model and thus, mimics COVID19. In the current study, we investigated the suppressive effect of sodium butyrate (BUT) on a murine model of SEB-induced Acute Respiratory Distress Syndrome (ARDS). The data shows that supplementation with BUT at a dose of 200 mg/kg orally reduced overall ARDS severity. Plethysmography analysis data indicated that sodium butyrate improves the clinical functions of the lungs. In addition, our flow cytometry data indicated that BUT mediated the suppression of lung mononuclear cells such as CD4+ T cells, CD8+ T cells, VB8+ T cells and NK-T cells while at the same time increasing T regulatory cells. Cytokine storm is one of the most important challenge in COVID19 cases, in our study, we have found that the proinflammatory cytokines such as IL-2, TNFα and IFNγ in broncho-alveolar lavage fluid (BALF) significantly decreased in SEB+BUT compared to SEB+VEH as detected by ELISA. Additionally, histopathological analysis of H &amp; E stained lungs indicated that BUT decreased the level of infiltration of inflammatory cells in the lungs of SEB+BUT compared to SEB+VEH mice. Additionally, we investigated the role of BUT on the expression of histone deacetylases (HDACs). Collectively, our data suggested that BUT ameliorated SEB-induced ARDS by downregulating the expression of class I and Class II HDACs which mediate the increase in T regulatory cells that suppress the inflammatory response in SEB-induced ARDS.

Childhood co-exposure of cold stress and PM2.5 aggravates the susceptibility and severity of asthma in adulthood of mice.

Both cold stress and ambient fine particle particulate matter (PM2.5 ) has been reported to aggravate and induce respiratory problems like asthma, but the mechanism involved in that has not been fully understood. Therefore, the present study is to explore the mechanism involved in the increased susceptibility and severity of asthma caused by cold stress and PM2.5 exposure. Urban PM2.5 of Shanghai was concentrated to simulate a PM2.5 -polluted environment with an average concentration of 400 μg/m3 , where 1-month young C57BL/6J mice were exposed for 2 months under cold stress (2°C). Co-exposure of cold stress and PM2.5 in childhood of mice led to significant infiltration of inflammatory cells in the peribronchial region or airspaces and the thickening or fibrosis of alveolar septum, increased OVA-specific IgE in serum and total cells, eosinophil cells, and the levels of inflammatory cytokines including IL-4, IL-8, IL-1β, IL-5, IL-13, and IFN-γ in bronchoalveolar lavage fluid (BALF) of asthma mice. Moreover, mice in co-exposure group presented a significantly high cough feature, reduced catalase (CAT), glutathione (GSH), superoxide dismutase (SOD), and elevated malonaldehyde (MDA) elevated in BALF; increased ratio of Th2/Th1 and the markable inhibition of Th17 differentiation toward Treg cells in the adulthood of asthma mice. Cold stress and PM2.5 co-exposure in childhood may promote the deterioration of asthma symptoms in adulthood of mice by increasing inflammatory cytokines, ROS formation, Th2/Th1 imbalance, and suppressing the differentiation of Th17 toward Treg cells, which will help to provide experimental references when making some therapeutic strategies in allergic diseases through focusing on some natural solutions.

Inhibitory effect of Xiaochuan pill on obstinate cough and its role in regulation of TLR4-MyD88-NF-κBp65 signaling pathway.

Cough variant asthma (CVA) is the main cause of obstinate cough. This study aimed to observe the therapeutic effect of Xiaochuan pill on CVA in a rat model, and to explore the mechanisms. The rats were sensitized and challenged with 4% ovaibumin (OA) and 2% Al(OH)3 to establish the CVA models. They were treated with Xiaochuan pill (at the dose of 0.9, 1.8, 3.6 g/kg) or montelukast sodium once a day for 14 days. After 7 and 14 days of intervention, 5 and 10 rats were randomly selected from each group to collect bronchoalveolar lavage fluid (BALF), trachea, and lungs. The number of white blood cells (WBC) and eosinophils (EOS), and the levels of IL-1β, TNF- α, and IFN-γ in BALF were detected. Histopathological examination of lung tissue was performed to observe the histomorphological changes. The expressions of TLR4, MyD88, NF-κBp65, and p-p65 in lung tissue were detected by Western blotting. The numbers of WBC and EOS in BALF of CVA rats were significantly decreased by Xiaochuan pill (P<0.05 or P<0.01). The hyperplasia of tracheal, bronchial mucosa and the infiltration of inflammatory cells in lung were alleviated obviously. After 14 d of intervention, high dose of Xiaochuan pill significantly increased the level of IFN- γ (P<0.01), reduced the levels of IL-1β (P<0.05) and TNF-α (P<0.05), and decreased the expressions of TLR4, MyD88, p65, and p-p65 (P<0.05 or P<0.01). Xiaochuan pill exerts the significant therapeutic effect on obstinate cough in rats. The mechanism of action may be related to the regulation of TLR4-MyD88-NF-κBp65 signaling pathway as well as the inflammation and immune response.

Effects of Low Concentration Hydrogen Inhalation on Asthma and Sleep Function in Mice

This study was designed to investigate the effects of low concentration hydrogen inhalation on asthma and sleep function in mice and the potential mechanism. In the asthma experiment, BALB/c mice were randomly divided into normal control group, asthma model group and hydrogen treatment group. After establishing ovalbumin (OVA)-induced asthma model, the hydrogen treatment group mice were treated by inhalation of hydrogen (24-26 mL/L per day) for 7 consecutive days, and the normal control group and asthma model group mice received similar treatment by inhalation of air. The levels of interleukin (IL)-4, IL-13, and interferon-γ (IFN-γ) in bronchoalveolar lavage fluid (BALF) were measured by commercially available ELISA kits. The levels of malondialdehyde (MDA) and glutathione (GSH), as well as the activity of superoxide dismutase (SOD) in lung tissue were detected by colorimetric assays. The pathological changes in lung tissue were assessed by HE staining. In the sleep experiment, ICR mice were randomly divided into blank control group and 1 d, 3 d, 5 d hydrogen treatment groups and diazepam group. The effects of inhalation of 24-26 mL/L per day hydrogen on the sleep duration induced by intraperitoneal injection of upper-threshold dose of sodium pentobarbital and the sleep latency in response to subthreshold dose were evaluated. In the asthma experiment, the asthma model group showed higher levels of IL-4 and IL-13 ( P<0.05) and lower levels of IFN-γ ( P<0.001) in BALF, as compared to the normal control group. The content of MDA in lung tissue was also significantly increased ( P<0.01), companied by a decreased GSH concentration ( P <0.05) and a mildly reduced SOD activity ( P>0.05). Compared to the asthma model group, treatment with hydrogen significantly decreased the levels of IL-4 and IL-13 and increased the level of IFN-γ in BALF ( P<0.05). Moreover, without alteration of the MDA production ( P>0.05), hydrogen inhalation greatly increased GSH level and restored the SOD activity ( P<0.05) in lung tissue. Additionally, the HE staining data showed that the hydrogen treatment attenuated the pulmonary histopathological changes. In the sleep experiment, compared with the blank control group, the sleep latency was significantly shorter ( P<0.05) and the sleep duration was longer ( P<0.001) in all the hydrogen treatment groups after receiving an upper-threshold dose of sodium pentobarbital. Meanwhile, in all the hydrogen treatment groups, the sleep latency was significantly longer ( P<0.001) and the sleep duration was shorter ( P<0.001) when compared to the diazepam group. Compared with the blank control group, after intraperitoneal injection of a subthreshold dose of sodium pentobarbital, the sleep latency was significantly increased in both 1 d and 5 d hydrogen treatment groups, and there was no significant difference as compared to the diazepam group. In the 3 d hydrogen treatment group, the sleep latency was only slightly increased ( P>0.05), which was significantly lower than that of the diazepam group ( P<0.05). Low concentration hydrogen inhalation could alleviate OVA-induced asthma in mice, and the mechanism might be related to the anti-oxidative and anti-inflammatory effects of hydrogen. Also, low concentration hydrogen inhalation could improve sleep function in mice.

Inhibition of the PI3K/AKT Signaling Pathway or Overexpression of Beclin1 Blocks Reinfection of Streptococcus pneumoniae After Infection of Influenza A Virus in Severe Community-Acquired Pneumonia.

Streptococcus pneumoniae (S. pneumoniae) and viruses are considered as primary risks of community-acquired pneumonia (CAP), and the effects of co-infection bacterial and virus in the prognosis of patients with severe CAP (SCAP) are poorly described. Therefore, this study is conducted to investigate the regulation of Beclin1-PI3K/AKT axis in reinfection of S. pneumoniae after influenza A virus in mice model of bronchoalveolar lavage fluid (BALF). Samples of sputum and BALF were collected from patients with SCAP for etiological detection. The expression of each gene was determined by RT-qPCR and western blot analysis. Influenza A/PR/8/34 and S. pneumoniae were used to establish the mice model of reinfection pneumonia. The virus quantity, expression levels of inflammatory factors, bacterial load, and myeloperoxidase (MPO) activity were tested. HE staining was applied to observe histopathology of lung tissue. The expression of Beclin1 was downregulated and the PI3K/AKT pathway was activated in viral pneumonia. In vivo experiment, the reinfection of S. pneumoniae following influenza A virus infection increased the number of S. pneumoniae population, the activity of MPO, and the expression of TNF-α, IL-6, and IFN-γ in BALF of mice. In contrast, inhibition of the PI3K/AKT pathway or overexpression of Beclin1 reduced the number of S. pneumoniae population, the activity of MPO, and the expression of TNF-α, IL-6, and IFN-γ in BALF of mice reinfected with S. pneumoniae after influenza A virus infection. Collectively, our study demonstrates that inhibition of the PI3K/AKT signaling pathway or overexpressed Beclin1 alleviates reinfection of S. pneumoniae after influenza A virus infection in SCAP.

Abstract 4076: Finding the optimal strategy of incorporating immune checkpoint inhibitor inALK-positive non-small-cell lung cancer usingEML4-ALKtransgenic mouse model

Abstract Introduction: EML4-ALK is a distinct molecular entity that is highly sensitive to ALK tyrosine kinase inhibitors (TKIs). Although the role of oncogenic and native ALK in modulating the immune responses has been suggested, immune checkpoint inhibitors (ICIs) have not proved efficacy in ALK-positive non-small cell lung cancer (NSCLC) so far. In this study, we evaluated comprehensive immunomodulatory effect of ALK TKI and aimed to suggest optimal method of incorporating ICIs in ALK-positive NSCLC using an EML4-ALK transgenic mice model. Methods: Ceritinib or anti-PD-1 was treated in EML4-ALK transgenic mice, and tumor response was evaluated using magnetic resonance imaging. Progression-free survival (PFS) and overall-survival (OS) were measured to compare the efficacy in mice. To examine the dynamics of immune response, flow cytometry of tumor region, cytokine-ELISA of bronchoalveolar lavage (BAL) fluid were performed at baseline and at the time of disease progression. Sequencing of ALK kinase domain was performed to identify acquired ALK mutations. Results: Upfront ceritinib was clearly more efficacious than anti-PD-1 upfront, as shown by the median PFS was 139 days vs.13 days (P &amp;lt; 0.05). Use of ceritinib in the first line resulted in a prolonged OS compared to the use in the second line (OS 203 days vs. 135 days, P&amp;lt; 0.05). The efficacy of ceritinib and anti-PD-1 combination was not more effective than ceritinib alone in the first line. Of note, we identified 1 mouse which showed a complete response to anti-PD-1 after progression on 1st line ceritinib. Tumors which progressed on ceritinib showed increased proportion of CD8+ tumor infiltrating T cells among total CD3+ T cells, and increased expression of IFN-γ in BAL fluid, representing Th1-dominant immune phenotype. In tumors which acquired resistance to ceritinib, we identified known ALK kinase domain mutations such as G1202R with a concomitant increased tumor mutational burden compared to ceritinib-naïve tumors. Ceritinib-resistant tumors also had significantly increased memory cytotoxic T cells and PD-1-expressing helper T cells in total CD3+ population. On the contrary, we noted decreased expressions of central memory T cells, effector memory T cells, IFN-γ in tumors which progressed after ceritinib and anti-PD-1 combination therapy. Conclusion: Our study confirmed that combination of ALK inhibitor and ICI is not efficacious, as evidenced by low infiltrating T cells and memory T cells. We concluded that tumor progressing on ceritinib could be sensitive to anti-PD-1 due to increased neoantigens derived from acquired ALK mutations, increased tumor mutational burden and increased number of memory T cells and PD-1-expressing helper T cells. Citation Format: Kyoung-Ho Pyo, Sun Min Lim, Ha Ni Jo, Jae Seok Cho, Jae Hwan Kim, Ji Min Lee, Chun-Feng Xin, Sung Eun Kim, Chae Won Park, Wongeun Lee, Hye Ryun Kim, Byoung Chul Cho. Finding the optimal strategy of incorporating immune checkpoint inhibitor inALK-positive non-small-cell lung cancer usingEML4-ALKtransgenic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4076.

The association between Cathepsin S and pulmonary function and CT phenotypes in patients with chronic obstructive pulmonary disease

Objective: To explore the value of cathepsin S in the bronchoalveolar lavage fluid (BALF) of patients with chronic obstructive pulmonary disease (COPD) in the evaluation of pulmonary function and CT phenotypes. Method: From April 2014 to April 2017, 46 patients with stable COPD were enrolled, and 29 healthy volunteers served as the control group. The patients were divided into 4 subgroups: GOLD Ⅰ(n=12), GOLD Ⅱ(n=6), GOLD Ⅲ(n=14), GOLD Ⅳ(n=14). The levels of cathepsin S and IFN-γ in BALF were determined by enzyme-linked immunosorbent assay (ELISA). The percentage ratio of low attenuation area to total lung area (LAA%), two times the ratio of airway wall thickness to outer diameter(2T/D), and the ratio of wall area to total cross-sectional area (WA) were measured by HRCT. Results: There were significant differences in the levels of cathepsin S in BALF between the groups (F=6.639, P=0.000). BALF cathepsin S levels were as follows: GOLD Ⅳ grou P>GOLD Ⅲ grou P>GOLD Ⅱ grou P>GOLD group Ⅰ >healthy control group (P value were all<0.05); LAA grade 3>LAA grade 2>LAA grade 1>LAA grade 0 (P value were all<0.05). Correlation analysis showed that BALF cathepsin S levels were correlated negatively with FEV(1)/FVC, FEV(1)% predicted, and DLCO% (r value was -0.065、-0.576、-0.392, respectively, P value were all<0.05), and but positively with RV/TLC%, LAA%, 2T/D, WA and IFN-γ(r value was 0.695, 0.497, 0.142, 0.309, 0.148, respectively, P value were all<0.05). Conclusion: The levels of cathepsin S were associated with the degree of airflow limitation and emphysema phenotype in COPD.

Effect of tangeretin on ovalbumin-provoked allergic respiratory asthma in Swiss albino mice

Purpose: To evaluate the effect of tangeretin on ovalbumin (OVA)-induced allergic respiratory asthma in Swiss albino mice. Methods: Mice were injected intraperitoneally with 50 μg OVA dissolved in water and 1 mg of aluminium hydroxide (alum) on the first day, and on day 12. On days 22, 26 and 30, the mice were exposed to 1 % OVA for 30 min. Mice in the asthma control group received saline via the same route. Tangeretin (50 or 100 mg/kg body weight) was given orally from day 1 to day 30. On the day of sensitization and challenge, tangeretin (50 or 100 mg/kg body weight) was injected 30 min prior to the treatment. Bronchial function, degree of inflammation, broncho-alveolar lavage fluid (BALF), levels of IL-4, IL-5, IL-13 and IFN-γ in BALF, serum total IgE, OVA-specific IgE, IgG1 and IgG2a, as well as histopathology of lungs were determined. Results: Tangeretin administration led to significant reduction in airway sensitivity, and significant decreases in leukocyte, macrophage, and eosinophil counts (p < 0.05). It also brought about significant decreases in the levels of the pro-inflammatory cytokines (IL-4, IL-5 and IL-13) in both BALF and serum (p < 0.05). In addition, tangeretin reversed the OVA-induced histopathological anomalies in the lungs. Conclusion: These results demonstrate that tangeritin exerts protective effects against OVA-induced allergic respiratory asthma in Swiss albino mice, and that the drug can potentially be developed for the management of allergic asthma. Keywords: Allergic asthma, Inflammation, Cytokines, Ovalbumin, Tangeretin

Antiasthmatic Effects of Sanglong Pingchuan Decoction through Inducing a Balanced Th1/Th2 Immune Response.

Objective To investigate the antiasthmatic effects of Sanglong pingchuan decoction (SLPCD) and to explore its mechanisms of action. Methods The serum, bronchoalveolar lavage fluid (BALF), and lung tissues from OVA-induced allergic asthma mice were collected 24 h after the last administration. Lung pathological changes were observed by H&E staining. The inflammatory cells in BALF were counted by flow cytometry. The levels of total IgE in serum and cytokines in BALF were determined by ELISA. The expression levels of cytokine mRNA in lung were assayed by qRT-PCR. Results SLPCD significantly inhibited airway inflammation, reduced inflammatory cells in BALF, reduced the levels of total IgE in serum and Th2 cytokines (IL-10 and IL-13) in BALF, and downregulated the mRNA expression levels of Th2 cytokines (IL-4, IL-5, IL-10, and IL-13) in lung of asthmatic mice. However, SLPCD remarkably elevated the level of Th1 cytokine IFN-γ in BALF and upregulated the mRNA expression levels of Th1 cytokines (IL-2 and IFN-γ) in lung of asthmatic mice. Conclusion SLPCD could attenuate airway inflammation and alleviate the pathogenesis in asthma mice through inducing a balanced Th1/Th2 response and could act as an effective drug for treatment of asthma.

Elevated expression of miR-146, miR-139 and miR-340 involved in regulating Th1/Th2 balance with acute exposure of fine particulate matter in mice

Airborne fine particulate matter (PM2.5) is detrimental to human health, and frequently leads to a variety of lung diseases. Recently, IARC conclude that particulate matter is carcinogenic to humans (level one). However, the pulmonary toxicological mechanism induced by PM2.5 remains obscure. Our previous studies confirmed that PM2.5 hurt the human immune system by means of causing the imbalance of Th1/Th2 lymphocytes. MicroRNAs (miRNAs) are post-transcriptional gene suppressors and potential mediators of environmental effects, which play an important role in the regulation of CD4+T lymphocyte differentiation. In order to further understand the roles of microRNAs in regulating the imbalance of Th1/Th2 differentiation triggered by PM2.5, mice were subjected to intratracheal instillation of 2.5, 10, or 20mg/kg PM2.5 in this study. Mice were euthanized on the 1st, 7th and 14th day to screen out differential miRNAs in lung tissue by employing the miRNA microarray. The expression levels of IL-4 and IFN-γ in bronchoalveolar lavage fluid (BALF) were quantified by ELISA and their mRNA expressions in lung tissue were detected by qRT-PCR. The experiment demonstrated that 10 differential miRNAs (miR-146a, -146b, -139, -129, -340, -691, -181a, -155, -21-3p, and -21-5p) were up-regulated. IL-4 levels were found decreased, nevertheless, IFN-γ levels were increased, and the IL-4/IFN-γ ratio was inclined to Th1 shifting. Besides that, we also found that miRNA-691, -181a, -146a, -146b, -21a-3p, -21a-5p, and -340 had a positive linear correlation with BALF IFN-γ, while a negative linear correlation between microRNAs (miR-146, -139, -340, -21, and -181a) and the IL-4/IFN-γ level of BALF was observed. In conclusion, elevated microRNAs profiles correlated with T lymphocyte immune imbalance, driving a Th1-biased immune response after acute PM2.5 exposure. These findings improve our understanding of the toxicological pathways of PM2.5 exposure.

Effects of inhaled corticosteroids on the expression of TNF family molecules in murine model of allergic asthma

ABSTRACTBackground: The tumor necrosis factor superfamily member LIGHT (the official gene symbol approved by NCBI Gene Database), an inflammatory factor secreted by T cells after allergen exposure, recently discovered to play crucial roles in asthmatic airway remodeling. However, it is unclear whether LIGHT could be controlled by inhaled corticosteroids, a key component of asthma management. This study was to investigate the effects and potential mechanisms of inhaled budesonide on the expressions of LIGHT and its receptors (LTβR and HVEM) of lung tissues in ovalbumin-sensitized mice. Materials and Methods: Thirty-three BALB/c mice were randomly divided into the control, asthma model, and budesonide treatment groups (11 in each group). Mice were sensitized and challenged by OVA to develop mouse model of chronic asthma, and treated with aerosolized budesonide before OVA challenge. Bronchoalveolar lavage fluid (BALF) and lungs were obtained after the final OVA challenge. Protein and mRNA Levels of LIGHT, LTβR, and HVEM in the lungs were investigated by immunohistochemistry, image analysis, and real-time PCR. Expressions of IL-6 and IFN-γ in BALF were measured by ELISA. Results: Inhaled budesonide significantly reduced protein and mRNA levels of lung LIGHT, LTβR, and HVEM in asthmatic mice. Correspondingly, the number of eosinophils and neutrophils and IL-6 levels in BALF after budesonide treatment were found to be decreased, whereas the IFN-γ levels in BALF were increased. Moreover, the expressions of LIGHT and HVEM mRNA showed positive correlation with IL-6 levels in the treatment group. Conclusions: Inhaled budesonide can down-regulate the expressions of LIGHT, LTβR, and HVEM in the lungs of asthmatic mice, and LIGHT/LTβR/HVEM interactions may be a potentially key target for asthma treatment.

Oral administration of Lactobacillus paracasei L9 attenuates PM2.5-induced enhancement of airway hyperresponsiveness and allergic airway response in murine model of asthma.

This study investigated allergy immunotherapy potential of Lactobacillus paracasei L9 to prevent or mitigate the particulate matter 2.5 (PM2.5) enhanced pre-existing asthma in mice. Firstly, we used a mouse model of asthma (a 21-day ovalbumin (OVA) sensitization and challenge model) followed by PM2.5 exposure twice on the same day of the last challenge. PM2.5 was collected from the urban area of Beijing and underwent analysis for metals and polycyclic aromatic hydrocarbon contents. The results showed that PM2.5 exposure enhanced airway hyper-responsiveness (AHR) and lead to a mixed Th2/ IL-17 response in asthmatic mice. Secondly, the PM2.5 exposed asthmatic mice were orally administered with L9 (4×107, 4×109 CFU/mouse, day) from the day of first sensitization to the endpoint, for 20 days, to investigate the potential mitigative effect of L9 on asthma. The results showed that L9 ameliorated PM2.5 exposure enhanced AHR with an approximate 50% decrease in total airway resistance response to methacholine (48 mg/ml). L9 also prevented the exacerbated eosinophil and neutrophil infiltration in bronchoalveolar lavage fluid (BALF), and decreased the serum level of total IgE and OVA-specific IgG1 by 0.44-fold and 0.3-fold, respectively. Additionally, cytokine production showed that L9 significantly decreased T-helper cell type 2 (Th2)–related cytokines (IL-4, -5, -13) and elevated levels of Th1 related IFN-γ in BALF. L9 also reduced the level of IL-17A and increased the level of TGF-β. Taken together, these results indicate that L9 may exert the anti-allergic benefit, possibly through rebalancing Th1/Th2 immune response and modulating IL-17 pro-inflammatory immune response. Thus, L9 is a promising candidate for preventing PM exposure enhanced pre-existing asthma.

Correction: Picroside II Attenuates Airway Inflammation by Downregulating the Transcription Factor GATA3 and Th2-Related Cytokines in a Mouse Model of HDM-Induced Allergic Asthma

[This corrects the article DOI: 10.1371/journal.pone.0167098.].

Picroside II Attenuates Airway Inflammation by Downregulating the Transcription Factor GATA3 and Th2-Related Cytokines in a Mouse Model of HDM-Induced Allergic Asthma.

Picroside II isolated from Pseudolysimachion rotundum var. subintegrum has been used as traditional medicine to treat inflammatory diseases. In this study, we assessed whether picroside II has inhibitory effects on airway inflammation in a mouse model of house dust mite (HDM)-induced asthma. In the HDM-induced asthmatic model, picroside II significantly reduced inflammatory cell counts in the bronchoalveolar lavage fluid (BALF), the levels of total immunoglobulin (Ig) E and HDM-specific IgE and IgG1 in serum, airway inflammation, and mucus hypersecretion in the lung tissues. ELISA analysis showed that picroside II down-regulated the levels of Th2-related cytokines (including IL-4, IL-5, and IL-13) and asthma-related mediators, but it up-regulated Th1-related cytokine, IFNγ in BALF. Picroside II also inhibited the expression of Th2 type cytokine genes and the transcription factor GATA3 in the lung tissues of HDM-induced mice. Finally, we demonstrated that picroside II significantly decreased the expression of GATA3 and Th2 cytokines in developing Th2 cells, consistent with in vivo results. Taken together, these results indicate that picroside II has protective effects on allergic asthma by reducing GATA3 expression and Th2 cytokine bias.

Evaluation of amniotic mesenchymal cell derivatives on cytokine production in equine alveolar macrophages: an in vitro approach to lung inflammation.

BackgroundData obtained in both animal models and clinical trials suggest that cell-based therapies represent a potential therapeutic strategy for lung repair and remodeling. Recently, new therapeutic approaches based on the use of stem cell derivatives (e.g., conditioned medium (CM) and microvesicles (MVs)) to regenerate tissues and improve their functions were proposed. The aim of this study was to investigate the immunomodulatory effects of equine amniotic mesenchymal cell derivatives on lipopolysaccharide (LPS)-induced cytokine production in equine alveolar macrophages, which may be beneficial in lung inflammatory disorders such as recurrent airway obstruction (RAO) in horses. RAO shares many features with human asthma, including an increased number of cells expressing mRNA for interleukin (IL)-4 and IL-5 and a decreased expression of IFN-γ in bronchoalveolar lavage fluid (BALF) of affected horses.MethodsThe release of TNF-α, IL-6, and TGF-β1 at different time points (1, 24, 48, and 72 h) was measured in equine alveolar macrophages stimulated or not with LPS (10 and 100 ng/mL) in the presence or absence of 10 % CM or 50 × 106 MVs/mL.Cytokines were measured using commercially available ELISA kits. For multiple comparisons, analysis of variance was used with Tukey post-hoc test. Differences were considered significant at p ≤ 0.05.ResultsSignificant modulatory effects of CM on LPS-induced TNF-α release at 24 h, and of both CM and MVs on TNF-α release at 48 h were observed. A trend toward a modulatory effect of both CM and MVs on the release of TGF-β and possibly IL-6 was visible over time.ConclusionsResults support the potential use of CM and MVs in lung regenerative medicine, especially in situations in which TGF-β may be detrimental, such as respiratory allergy. Further studies should evaluate the potential clinical applications of CM and MVs in equine lung diseases, such as RAO and other inflammatory disorders.

Effectiveness of methylprednisolone in treatment of children with refractory Mycoplasma pneumoniae pneumonia and its relationship with bronchoalveolar lavage cytokine levels

To investigate cytokine level in bronchoalveolar lavage fluid (BALF) in children with refractory Mycoplasma pneumoniae pneumonia (RMPP) and the effects of methylprednisolone on RMPP. Sixty cases with RMPP and 20 cases with bronchial foreign body with no respiratory tract infection as control group hospitalized in Department of Pulmonary Diseases, the Children's Hospital Affiliated to Medical School of Zhejiang University from February 2012 to February 2013 were enrolled. The RMPP patients were divided into two groups randomly (30 cases in each). Steroid group were given methylprednisolone 2 mg/(kg·d) intravenously for 3 days, and the cases in non steroid group were not given steroid therapy. Patients whose fever relieved after steroid treatment were classified as defervesced group while the others were classified as non defervesced group. Each patient was examined with fiberoptic bronchoscopy and bronchoalveolar lavage 3 days after admission and cytokine level in BALF of each patient was detected. (1) In steroid group, the proportion of patients whose fever disappeared within 3 days after steroid therapy was 9/30 cases (30%), and in non steroid group no one responded within 3 days after medication, showing statistically significant difference (χ² = 14.073, P=0.002), at the same time, the duration of cough in steroid group was significantly shorter than that in non steroid group (5.1 d vs. 7.0 d, t=-2.276, P=0.027). The total fever time of steroid group was 4.7 days, which as compaired with non steroid group (6.7 days) was shorter, but the difference was not significant (t=-1.351, P=0.134). (2) IL-1 β, IL-4, IL-6, IL-8, IL-10, IFN-γ in BALF of steroid group and non steroid group were both significantly higher than that of control group. But the same comparison between steroid group and non steroid group showed no significant difference. (3) In steroid group, IL-2 and IL-8 in BALF of patient whose fever disappeared after steroid therapy were both significantly lower than that of patients who still had fever (t=2.771, 2.054, P=0.010, 0.049) , but no significant difference was found between the two groups in BALF IL-1 β, IL-4, IL-6, IL-10, IFN-γ levels (P>0.05). (1) Three days of 2 mg/(kg·d) methylprednisolone therapy had the antipyretic effect in children with RMPP, and could shorten the length of cough. (2) Incresed BALF IL-1 β, IL-4, IL-6, IL-8, IL-10, IFN-γ levels were observed in RMPP and high level of BALF IL-2 and IL-8 might have some relevance with persistent fever of RMPP in children.

The upregulated expression of OX40/OX40L and their promotion of T cells proliferation in the murine model of asthma.

To investigate whether the expression of OX40/OX40 ligand (OX40L) was upregulated in a murine model of asthma and their significance in the pathogenesis of asthma. After an ovalbumin-sensitized/challenged murine model of asthma was established, the expressions of OX40, OX40L in peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage fluid (BALF) cell pellets were measured. Then T cell proliferation was analyzed by cell counting kit-8 (CCK8), and the protein levels of OX40 and OX40L in the lungs were determined by immunohistochemistry. The concentrations of IL-4 and IFN-γ in BALF and T cell culture supernatant were evaluated by ELISA. The percentages of CD4(+)OX40(+), CD19(+)OX40L(+), F4/80(+)OX40L(+) in PBMCs and BALF cell pellets were higher in asthma group than in control group (all P<0.01). The proliferation capacity of T cells in asthma group was higher than that in control group (P<0.05). In asthma group, stimulation of OX40 by anti-OX40 mAb obviously promoted T cell proliferation and secretion of IL-4 and IFN-γ. Immunohistochemistry assay showed that OX40 and OX40L protein levels were higher in asthma group than those in control group (all P<0.05). The expressions of OX40 and OX40L were upregulated in the murine asthmatic model. The upregulation of OX40/OX40L signals could induce the proliferation and cytokines secretion of T cells in asthmatic mice, indicating that OX40/OX40L signal was involved in the pathogenesis of asthma.