Leukocytes In Bronchoalveolar Lavage Fluid Research Articles

A novel peptide binding to the C-terminal domain of connective tissue growth factor for the treatment of bleomycin-induced pulmonary fibrosis

Using phage display technology, 810A was developed as a novel peptide, which binds to the C-terminal domain of connective tissue growth factor (CTGF). The present study investigated whether this peptide could neutralize CTGF and block its biological activity as a specific strategy to treat pulmonary fibrosis in vitro and in vivo. Human bronchial epithelial (16HBE) cells were treated with CTGF to evaluate the anti-CTGF capability of 810A using MTT testing, cell migration assays, and Western blotting. The action of 810A in mouse models of bleomycin (BLM)-induced pulmonary fibrosis was evaluated according to leukocyte counts in bronchoalveolar lavage fluid (BALF), hydroxyproline content, and pathological analysis. Treatment with 810A resulted in inhibition of cell proliferation, migration, and increases in transforming growth factor-beta (TGF-β) and α-Smooth muscle actin (α-SMA) caused by CTGF treatment in vitro. In vivo, 810A significantly alleviated fibrosis in the pulmonary index. Inflammation was inhibited, as inferred by the reduced number of leukocytes in BALF. Pathophysiological analysis indicated that hydroxyproline content, alveolar enlargement, interstitial inflammatory infiltration, and collagen deposition were reduced. Collectively, these data suggest that 810A may potentially be a CTGF-specific, small-molecule antagonist, providing a new method for prevention and treatment of pulmonary fibrosis.

The protective effects of Thalictrum minus L. on lipopolysaccharide-induced acute lung injury.

Thalictrum minus L., a Mongolian folk medicinal plant, was applied for the treatment of bacterial and fungal infection, tuberculosis and lung inflammation. The present work aims to elucidate the protective effects of Thalictrum minus L.(TML) against lipopolysaccharide (LPS)-induced acute lung injury and the underlying mechanisms. The mice model of acute lung injury was induced by LPS via endotracheal drip, and TML (10, 20, 40 mg/kg) were administered orally 1 h prior to LPS. The efficacy and molecular mechanisms in the presence or absence of TML were investigated. We demonstrated that treatment with TML aqueous extract protected the mice from acute lung injury induced by LPS administration. TML significantly inhibited weight loss in mice, decreased the lung wet to dry weight (W/D) ratios and attenuated lung histopathological changes, such as infiltration of inflammatory cells and coagulation, pulmonary edema. Furthermore, we found that TML markedly reduced the LPS-induced inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), decreased nitric oxide (NO), and increased superoxide dismutase (SOD) in bronchoalveolar lavage fluid (BALF), and effectively ameliorated LPS-induced increased total protein, leukocyte and macrophages in BALF. In addition, TML pronouncedly suppressed the activation of the MAPKs p38-NLRP3/caspase-1 and COX2, increased the expression of p-AMPK-Nrf2, and suppressed the expression of KEAP, apoptotic-related protein as well as autophagy. These results suggested that TML ameliorated LPS-induced acute lung injury by inhibiting the release of inflammatory cytokines and reducing oxidative damage associated with the MAPKs p38-NLRP3/caspase-1 and COX2 signaling pathways, AMPK-Nrf2/KEAP signaling pathways, as well as apoptosis and autophagy.

Damage-Associated Molecular Patterns and Myeloid-Derived Suppressor Cells in Bronchoalveolar Lavage Fluid in Chronic Obstructive Pulmonary Disease Patients.

Myeloid-derived suppressor cells (MDSCs) are present in the human lung microenvironment, and they may be involved in the local inflammatory process in chronic obstructive pulmonary disease (COPD). Chronic inflammation in COPD may induce immunogenic cell death of structural airway cells, causing the release of damage-associated molecular patterns (DAMPs). DAMPs may activate the innate and adaptive immune system. The relationship between MDSCs and DAMPs in COPD is poorly described in the available literature. Objectives. (1) Assessment of MDSC percentage and DAMP concentration in bronchoalveolar lavage fluid (BALF) and peripheral blood. (2) Analysis of the relationship between MDSC percentage and chosen DAMPs. Patients and Methods. 30 COPD patients were included. Using monoclonal antibodies directly conjugated with fluorochromes in flow cytometry, MDSCs were assessed in BALF and peripheral blood. The concentration of DAMPs was estimated using sandwich ELISA. Using the Bradford method, the total protein concentrations were evaluated. Results. The percentage of MDSCs among MC in BALF correlated well with the concentration of defensin and heat shock protein 27. Assessing the percentage of MDSCs among all leukocytes in BALF, we revealed a significant correlation with the concentration of defensin, hyaluronic acid, and surfactant protein A. No dependencies occurred between DAMPs and MDSCs in peripheral blood. Conclusion. MDSCs and DAMPs occur in the COPD patient lung microenvironment. Significant correlations between them found in BALF may indicate their influence on the local inflammatory process in COPD. These relationships allow better understanding of the inflammatory process in COPD.

Protective Effects of Morin Against Bleomycin-Induced Pulmonary Fibrosis in Mice

Background: Idiopathic pulmonary fibrosis (IPF) is a fatal and incurable lung disease. Morin, a natural product with antioxidant and anti-inflammatory activity, could reduce lung inflammation induced by lipopolysaccharide (LPS) and liver fibrosis in previous studies. Objectives: The aim of this study was to evaluate the antifibrotic activity of morin in the pulmonary fibrosis induced by bleomycin in mice. Methods: Pulmonary fibrosis was induced by the intratracheal instillation of bleomycin (3 mg/kg) in C57Bl/6J mice. Morin (10, 20, and 40 mg/kg, p.o.) was given to mice from day 0 to 21 after bleomycin administration. The mice were sacrificed on day 21 to measure the total number of cells, the percentage of leukocytes in bronchoalveolar lavage fluid (BALF), lung hydroxyproline content, lung index, and oxidative stress markers. Histopathological changes were evaluated by the microscopic examination of sections stained with hematoxylin-eosin and Masson’s trichrome. Results: Our data showed that treatment with morin significantly attenuated the infiltration of inflammatory cells, hydroxyproline content, lung index, and oxidative stress that were elevated in fibrotic lungs. In addition, morin could reduce the pathological changes induced by bleomycin. Conclusions: Based on the study, morin, probably by its anti-inflammatory and antioxidant activities, can attenuate pulmonary fibrosis induced by bleomycin in mice.

Protective effects of ethyl gallate and pentagalloylglucose, the active components of Qingwen Baidu Decoction, against lipopolysaccharide-induced acute lung injury in rats.

BackgroundThe aim of this study was to investigate the bioactive constituents of Qingwen Baidu Decoction (QBD) in a rat model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Our previous studies showed that ethyl gallate (EG) and pentagalloylglucose (PGG) were the active components of QBD for the treatment of ALI.Materials and methodsWe isolated two compounds and identified the structures of them by nuclear magnetic resonance and mass spectrometer. Lung injury was induced by intratracheal instillation with LPS (5 mg/kg). Rats were randomly divided into six groups: Control group; LPS group; 5 mL/kg DEX + LPS group; 6.6 g/kg QBD extract + LPS group; 17.16 mg/kg PGG + LPS group; 7.26 mg/kg EG + LPS group. The effects of compounds on LPS-induced the number of total cells, neutrophils influx, protein leakage, W/D weight ratio and pulmonary histological changes were examined.ResultsThe results demonstrated that pretreatment with EG and PGG could notably inhibit lung edema and attenuate the pulmonary histological changes (P<0.05). The pretreatment also decreased the number of total cells and polymorphonuclear leukocytes in bronchoalveolar lavage fluid (BALF) (P<0.05).ConclusionEthyl gallate and pentagalloylglucose of QBD played a protective role in preventing LPS-induced ALI. The results supported further study of EG and PGG as potential candidates for preventing ALI.

Taurine treatment decreases inflammation and oxidative stress in lungs of adult mice exposed to cigarette smoke

Taurine is the major free amino acid found in mammalian cells and is known to be an antioxidant and membrane-stabilizing agent. This study aimed to evaluate the effects of taurine on oxidative stress and inflammatory response in the lungs of mice exposed to cigarette smoke. Fifty male C57BL/6 mice were divided into 5 groups: control group (CG), vehicle group (VG), taurine group (TG), cigarette smoke group (CSG), and cigarette smoke + taurine group (CSTG). For five consecutive days, CSG and CSTG were exposed to 4 cigarettes 3 times a day. Taurine administration was able to reduce total leukocytes in bronchoalveolar lavage fluid in CSTG compared to CSG. There was an increase in antioxidant superoxide dismutase and catalase activity in CSG compared to that in CG and TG, and a decrease in CSTG compared to CSG. There was an increase in the concentration of TNF and IL-17 in CSG and CSTG compared to CG and TG. There was an increase in the concentration of IL-22 in CSG compared to CG and TG, and a decrease in CSTG compared to CSG. The administration of taurine has been shown to reduce the inflammation and oxidative stress induced by short-term exposure to cigarette smoke.

The protective effect of lidocaine on lipopolysaccharide-induced acute lung injury in rats through NF-κB and p38 MAPK signaling pathway and excessive inflammatory responses.

Acute lung injury is a severe disease with a high rate of mortality, leading to more important illness. We aimed at exploring the protective role and potential mechanisms of lidocaine on lipopolysaccharide (LPS)-induced acute lung injury (ALI). Sprague Dawley (SD) rats were randomly assigned to control group receiving 0.9% saline solution, LPS group treated with 4 mg/kg LPS i.p., LPS + lidocaine(treated with 4 mg/kg LPS i.p. followed by giving 1 mg/kg, 3 mg/kg, 5 mg/kg of lidocaine i.v.). Lung specimens and the bronchoalveolar lavage fluid (BALF) were collected for histopathological examination and biochemical analyze 12 h after LPS induction. The cytokines expression of TNF-α, IL-6 and MCP-1 was measured by ELISA. In addition, the malondialdehyde (MDA) content, the activities of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) in lung tissues were also detected using ELISA. The protein expressions of p38, p-p38, p65, p-p65 and IκB were analyzed by Western blot. The results indicated that after lidocaine treatment was able to decrease significantly wet-to-dry (W/D) ratio and ameliorate the histopathologic damage. Additionally, total protein content and the number of leukocytes in BALF significantly decreased. ELISA result indicated that the levels of TNF-α, IL-6 and MCP-1 in BALF were markedly suppressed. Meanwhile, the activities of T-AOC and SOD in lung tissues significantly increased, while the content of MDA significantly decreased after treatment with lidocaine. Moreover, Western blot suggested that lidocaine inhibited phosphorylation of NF-κB p65 and p38 MAPK. Therefore, lidocaine could ameliorate the LPS-induced lung injury via NF-κB/p38 MAPK signaling and excessive inflammatory responses, providing a potential for becoming the anti-inflammatory agent against lung injury.

Deficiency of Leukocyte‐Specific Protein 1 (LSP1) Alleviates Asthma in a Mouse Model

Asthma is one of the most serious forms of respiratory disease, causing economic losses, high mortality, and morbidity globally. However, its etiology is still ambiguous. Many inflammatory cells, including T lymphocytes, eosinophils, mast cells, macrophages, and neutrophils, are recruited into the airway of asthma lung patients. In the previous study, we reported that LSP1 deficiency reduces neutrophil migration and acute lung inflammation, but its role in asthma remains unknown. Therefore, in this study, we developed an asthma‐like disease in LSP1‐deficient (Lsp1−/−) and wild‐type (WT) 129/SvJ mice (n=6 each treatment group) through two intraperitoneal (IP) injections of 2μg OVA/2mg alum two weeks apart, followed by three aerosol challenges with 1% OVA two days apart after two weeks of the last IP injection. We hypothesized that the absence of LSP1 inhibited airway inflammation in this OVA‐induced asthma mouse model due to inhibition of immune cell migration.Immunohistochemistry staining and western blot data showed that the level of LSP1 increased in asthma mouse lungs, compared with normal healthy lungs. Results from immuno‐gold as well as immunofluorescent staining illustrated that LSP1 was expressed on endothelium, bronchiolar epithelium, neutrophils, eosinophils, lymphocytes, alveolar macrophages, and pulmonary intravascular macrophages. There was no significant difference in peripheral leukocyte numbers between normal and asthmatic WT and Lsp1−/− mice. However, compared to Lsp1−/− asthma mice, WT asthma mice had higher levels leukocytes in broncho‐alveolar lavage fluid, higher myeloperoxidase, a marker of neutrophils, and eosiperoxidase levels in lungs (P&lt;0.05). The level of OVA‐specific IgE, but not IgA and IgG1, in the serum of WT asthma mice was higher than that of Lsp1−/− asthma mice (P&lt;0.05). Other research reported that IL‐4, IL‐5, IL‐6, IL‐13, and KC are associated with eosinophilia and neutrophil migration. We found that the absence of the LSP1 gene significantly reduced the levels of IL‐4, IL‐5, IL‐6, IL‐13, and KC (P&lt;0.05) but not the levels of IL‐17, eotaxin, IFN‐gamma, and total protein in broncho‐alveolar lavage fluid. The airway hyper‐responsiveness response to methacholine in Lsp1−/− asthma mice was ameliorated compared to WT asthma mice (P&lt;0.05). Histopathology evaluation findings showed more recruited inflammatory cells, as well as airway and blood vessel wall thickening, in the lungs of WT mice than in those of Lsp1−/− mice. These data indicate that LSP1 promotes airway inflammation associated with asthma in this mouse model. LSP1 may be a potential target for therapeutic intervention in allergic lung disease or asthma in humans.Support or Funding InformationWe thank to the Natural Science and Engineering Research Council (NSERC), Graduate Student Scholarship from Integrated Training Program in Infectious Disease, Food Safety and Public Policy (ITraP), Devolved Graduate Scholarship from Department of Veterinary Biomedical Sciences, and Graduate Student Scholarship from Western College of Veterinary Medicine, University of Saskatchewan, Canada for supporting this research. We thank Ms. LaRhonda Sobchishin for the technical support provided for the electron microscope technique and Ms. Eiko Kawamura for technical support in using a confocal microscope.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Thymic Stromal Lymphopoietin Signaling Pathway Inhibition Attenuates Airway Inflammation and Remodeling in Rats with Asthma

Background/Aims: Thymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in the regulation of immune responses. However, a detailed understanding of the TSLP signaling pathway in asthma remains elusive. In this study, we aimed to investigate the role of the TSLP signaling pathway in asthma and its effect on airway inflammation and remodeling. Methods: Forty Sprague Dawley (SD) rats were evenly classified into control, asthma, IgG2a mAb and anti-TSLP mAb groups. Ovalbumin (OVA)-induced asthma models were successfully established. Blood, bronchoalveolar lavage fluid (BALF) and lung tissue samples were prepared. Total BALF leukocytes were counted, and the proportions of different leukocyte types were determined. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry were performed to determine the mRNA and protein levels of TSLP, OX40L, α-smooth muscle actin (α-SMA, a marker of airway remodeling in asthma) and collagen I in the plasma. Enzyme-linked immunosorbent assay (ELISA) was carried out to measure the concentrations of TSLP, OX40L, and other inflammatory factors, such as interferon (IFN)-γ, interleukin (IL)-4, IL-5 and IL-13, in the plasma. Results: Compared with the control group, there were more leukocytes, increased EOS and LYM proportions, higher Underwood and PAS scores, increased WT_t, WT_m, WA_t/A₀, WA_m/WA_t, WT_t/R₀, WT_m/WT_t, TSLP, OX40L, a-SMA and collagen I mRNA and protein levels, and higher SLP, OX40L, IL-4, IL-5 and IL-13 levels, but lower MON proportions and IFN-γ levels in the asthma and IgG2a mAb groups. Compared with the asthma and IgG2a mAb groups, there were less leukocytes, decreased EOS and LYM proportions, lower Underwood and PAS scores, decreased WT_t, WT_m, WA_t/A₀, WA_m/WA_t, WT_t/R₀, WT_m/WT_t, TSLP, OX40L, a-SMA and Collagen I mRNA and protein levels, and lower levels of SLP, OX40L, IL-4, IL-5 and IL-13, but higher MON proportions and IFN-γ levels in the anti-TSLP mAb group. WT_m and WT_t were positively associated with the TSLP, OX40L, α-SMA and collagen-I levels in the rat lung tissues. Conclusion: The results indicate that TSLP may be an important contributor for asthma development as TSLP signaling blockade attenuates airway inflammation and remodeling in asthmatic rats.

The administration of surfactant decreased oxidative stress in lungs of mice exposed to cigarette smoke

The alveolar surfactant, which composition consists of a unique and complex mixture of lipids and proteins, has immunomodulatory action. This study aimed to evaluate the effects of exogenous surfactant on pulmonary inflammatory response in mice exposed to cigarette smoke (CS). Twenty-four mice C57BL/6 were divided into four groups: control group exposed to ambient air (CG); surfactant treated group (SG); CS exposed group (CSG) and CS exposed group treated with surfactant (CSSG). For five days, CSG and CSSG were exposed to 12 commercial cigarettes/day and SG and CSSG received the surfactant by intranasal instillation. At the end of the experiment, the animals were euthanatized for the collection of bronchoalveolar lavage fluid (BALF) and lungs. The total number of leukocytes in BALF increased in CSG compared to CG, however, there was a decrease in CSSG compared to CSG. There was an increase in lipid peroxidation in SG and CSG compared to CG while there was a decrease in CSSG compared to CSG. Regarding the antioxidant enzymes, the catalase (CAT) activity increased in all groups compared to CG and the superoxide dismutase (SOD) activity decreased in CSG compared to the CG and SG. There was an increase in TNF in SG, CSG and CSSG compared to CG. There was an increase in IL-17 in CSSG compared to CG. There was an increase in CCL5 in SG and CSSG compared to CG. Therefore, our results demonstrated that the administration of exogenous surfactant was able to decrease the oxidative processes in the lungs of mice induced by short-term exposure to CS.

Effect of interferon-γ on airway inflammation following respiratory syncytial virus reinfection in mice

Objective: To identify the role of interferon (IFN)-γ during respiratory syncytial virus (RSV) re-infection in mice. Method: Female wild type C57BL/6 mice and IFN-γ knockout mice (IFN-γ(-/-) mice) at the age of 6 to 8 weeks were randomly divided into two groups: control group and RSV group, according to random number table.Each group was further divided into primary infection group and re-infection group.There were 8 groups.Mice were sacrificed on days 5, 7, 14 to collect samples.There were 5-8 mice in each group at each time point.And experiment was repeated twice. Leukocytes in bronchoalveolar lavage fluid (BALF) were counted, left lung tissues were stained with HE and histopathological scoring (HPS) was performed.The concentrations of IFN-γ, IL-5, IL-13 were determined with ELISA.T test or single factor analysis of variance was used to compare between groups. Result: (1) Mice infected or reinfected with RSV showed pale hair, weight loss, decreased activity and anorexia.(2) IFN-γ levels significantly increased on days 5 and 7 following RSV primary infection and reinfection as compared to control groups in wild type mice ((192±44) vs.(36±8) and (531±161) vs.(23±4) pg/ml on day 5, (100±23) vs.(36±8) and (862±186) vs.(23±4) pg/ml on day 7, t=2.654, 2.513, 2.654, 3.968, all P<0.05). (3) Compared to the RSV-reinfected IFN-γ(-/-) mice, RSV-reinfected wild type mice had less body weight loss ((13.6±2.6)% vs.(22.7±2.9)% on day 5, (18.0±3.1)% vs.(26.5±1.8)% on day 7, t=2.314, 2.308, both P<0.05), lower lung tissue histopathological score ((1.50±0.09) vs.(2.07±0.11) on day 5, (1.53±0.11) vs.(2.08±0.09) on day 7, (1.10±0.06) vs.(1.59±0.08) on day 14, t=3.916, 3.890, 4.837, all P<0.01), less BALF inflammatory cells count ((11.6±2.0) vs.(44.2±10.6)×10(5)/ml on day 5, (18.2±3.9) vs.(38.3±2.2)×10(5)/ml on day 7, t=2.818, 4.786, both P<0.05), and lower levels of IL-5 and IL-13 ((24±3) vs.(148±23), (23±4) vs.(169±26) pg/ml on day 5, (30±8) vs.(233±44), (20±5) vs.(182±19) pg/ml on day 7, (91±6) vs.(129±19), (62±8) vs.(132±5) pg/ml on day 14, t=5.252, 5.445, 4.517, 7.326, 3.816, 7.577, all P<0.01). Conclusion: IFN-γ can alleviate airway inflammation following RSV reinfection in mice.

The Nonantibiotic Macrolide EM703 Improves Survival in a Model of Quinolone-Treated Pseudomonas aeruginosa Airway Infection

Macrolide antibiotics are used as anti-inflammatory agents, e.g., for prevention of exacerbations in chronic obstructive pulmonary disease and cystic fibrosis. Several studies have shown improved outcomes after the addition of macrolides to β-lactam antibiotics for treatment of severe community-acquired pneumonia. However, a beneficial effect of macrolides in treating Gram-negative bacterial airway infections, e.g., those caused by Pseudomonas aeruginosa, remains to be shown. Macrolide antibiotics have significant side effects, in particular, motility-stimulating activity in the gastrointestinal tract and promotion of bacterial resistance. In this study, EM703, a modified macrolide lacking antibiotic and motility-stimulating activities but with retained anti-inflammatory properties, was used as an adjunct treatment for experimental P. aeruginosa lung infection, in combination with a conventional antibiotic. Airway infections in BALB/cJRj mice were induced by nasal instillation of P. aeruginosa; this was followed by treatment with the quinolone levofloxacin in the absence or presence of EM703. Survival, inflammatory responses, and cellular influx to the airways were monitored. Both pretreatment and simultaneous administration of EM703 dramatically improved survival in levofloxacin-treated mice with P. aeruginosa airway infections. In addition, EM703 reduced the levels of proinflammatory cytokines, increased the numbers of leukocytes in bronchoalveolar lavage fluid, and reduced the numbers of neutrophils present in lung tissue. In summary, the findings of this study show that the immunomodulatory properties of the modified macrolide EM703 can be important when treating Gram-negative pneumonia, as exemplified by P. aeruginosa infection in this study.

From the Cover: Lung-Specific Overexpression of Constitutively Active IKK2 Induces Pulmonary and Systemic Inflammations but Not Hypothalamic Inflammation and Glucose Intolerance.

The lung is constantly exposed to ambient pollutants such as ambient fine particulate matter (PM2.5), making it one of the most frequent locations of inflammation in the body. Given the establishment of crucial role of inflammation in the pathogenesis of cardiometabolic diseases, pulmonary inflammation is thus widely believed to be an important risk factor for cardiometabolic diseases. However, the causality between them has not yet been well established. To determine if pulmonary inflammation is sufficient to cause adverse cardiometabolic effects, SFTPC-rtTA+/-tetO-cre+/-pROSA-inhibitor κB kinase 2(IKK2)ca+/- (LungIKK2ca) and littermate SFTPC-rtTA+/-tetO-cre-/-pROSA-IKK2ca+/- wildtype (WT) mice were fed with doxycycline diet to induce constitutively active Ikk2 (Ikk2ca) overexpression in the lung and their pulmonary, systemic, adipose, and hypothalamic inflammations, vascular function, and glucose homeostasis were assessed. Feeding with doxycycline diet resulted in IKK2ca overexpression in the lungs of LungIKK2ca but not WT mice. This induction of IKK2ca was accompanied by marked pulmonary inflammation as evidenced by significant increases in bronchoalveolar lavage fluid leukocytes, pulmonary macrophage infiltration, and pulmonary mRNA expression of tumor necrosis factor α (Tnfα) and interleukin-6 (Il-6). This pulmonary inflammation due to lung-specific overexpression of IKK2ca was sufficient to increase circulating TNFα and IL-6 levels, adipose expression of Tnfα and Il-6 mRNA, aortic endothelial dysfunction, and systemic insulin resistance. Unexpectedly, no significant alteration in hypothalamic expression of Tnfα and Il-6 mRNA and glucose intolerance were observed in these mice. Pulmonary inflammation is sufficient to induce systemic inflammation, endothelial dysfunction, and insulin resistance, but not hypothalamic inflammation and glucose intolerance.

Role of PIM2 in allergic asthma.

T cell-associated inflammation, particularly type 2 inflammation, has an important role in asthma pathogenesis, which is suppressed by regulatory T cells (Tregs). Proviral integration site for Moloney murine leukemia virus 2 (PIM2), a member off the serine/threonine kinase family, promotes the growth and survival of T cells and influences the function of Treg cells. However, whether PIM2 affects asthma pathogenesis remains unclear. Peripheral blood mononuclear cells and Treg cells from asthmatic and healthy subjects were obtained, and the expression level of PIM2 was measured by reverse transcription-quantitative polymerase chain reaction and immunocytochemistry. In addition, BALB/c female mice sensitized and challenged by ovalbumin were used as an asthma model, and PIM2 inhibitor was injected during the challenge period to observe the effect of PIM2 on asthma. The asthma symptoms were recorded, and airway hyper-responsiveness (AHR), expression levels of cytokines in the serum or bronchoalveolar lavage fluid (BALF), and the number of BALF leukocytes were evaluated. In addition, hematoxylin and eosin staining and immunohistochemistry of lung tissues was performed. The results demonstrated that PIM2 was overexpressed in patients with asthma in natural Treg cells. Inhibition of PIM2 attenuated asthma symptoms, and improved AHR and airway inflammation compared with asthmatic mice without inhibition of PIM2. In addition, expression levels of interleukin (IL)-10 and forkhead box protein 3 (FOXP3) in BALF were increased following PIM2 inhibition (IL-10, 470.3±21.78 vs. 533.7±25.55 pg/ml, P<0.05; FOXP3, 259±4.68 vs. 279.3±3.68 pg/ml; asthma and PIM2 inhibition groups, respectively; P<0.05). In conclusion, PIM2 may exhibit an important role in asthma pathogenesis and exacerbate AHR, airway inflammation and asthma symptoms. These effects of PIM2 may be dependent on Treg cells and the secretion of IL-10 by Tregs. The results of the present study suggest that PIM2 may be a potential target molecule for asthma treatment.

Exacerbating effects of PM2.5 in OVA-sensitized and challenged mice and the expression of TRPA1 and TRPV1 proteins in lungs

ABSTRACTObjective: To investigate the effects of particulate matter ≤ 2.5 microns (PM2.5) on asthma-related phenotypes and on lung expression of TRPA1 and TRPV1 proteins in a mouse model of asthma. Methods: Female BALB/c mice were utilized to establish 28- and 42-day asthma models. Mice were sensitized with ovalbumin (OVA) and challenged with OVA, OVA plus normal saline (NS), or OVA plus PM2.5 at two doses, 1.6 or 8.0 mg kg−1. PM2.5 was instilled intratracheally without anesthesia. After the final OVA challenge was performed, 24 hours later, the changes in airway resistance (RI) and lung dynamic compliance (Cdyn) in response to acetylcholine chloride (ACH) were evaluated, and blood, bronchoalveolar lavage fluid (BALF) and lung tissue were taken at that time. The number of eosinophils in blood and various leukocytes in BALF were determined. Lung protein was extracted and probed for TRPA1 and TRPV1 expression. Interleukin (IL)-13, substance P (SP), prostaglandin D2 (PGD2) and nerve growth factor (NGF) in BALF were measured by enzyme-linked immunosorbent assay. Results: PM2.5 treated mice showed significantly greater changes in the number of inflammatory cells in blood and BALF, in RI and Cdyn in response to ACH, and in lung histopathology, indicated by inflammatory cell infiltration, thickened bronchial smooth muscles and bronchial mucosa damage, compared to controls. In addition, higher expression of TRPA1 and TRPV1 in lung and IL-13, SP, PGD2 and NGF in BALF were seen in mice exposed to PM2.5. All effects were most pronounced in mice in the 42-day model. Conclusions: PM2.5 exacerbates effects of asthma in this model, possibly by regulating TRPA1 and TRPV1 and the relevant neurokines.

The time-dependent health and biochemical effects in rats exposed to stainless steel welding dust and its soluble form

ABSTRACTWelding processes that generate fumes containing toxic metals, such as hexavalent chromium (Cr(VI)), manganese (Mn), and nickel (Ni), have been implicated in lung injury, inflammation, and lung tumor promotion in animal models. The principal objective of this study was to determine the dynamics of toxic effects of inhalation exposure to morphologically rated welding dust from stainless steel welding and its soluble form in TSE System with a dynamic airflow. We assessed the pulmonary toxicity of welding dust in Wistar rats exposed to 60.0 mg/m3 of respirable-size welding dust (mean diameter 1.17 µm) for 2 weeks (6 h/day, 5 days/week); the aerosols were generated in the nose-only exposure chambers (NOEC). An additional aim included the study of the effect of betaine supplementation on oxidative deterioration in rat lung during 2 weeks of exposure to welding dust or water-soluble dust form. The animals were divided into eight groups (n = 8 per group): control, dust, betaine, betaine + dust, soluble-form dust, soluble-form dust + betaine, saline and saline + betaine groups. Rats were euthanized 1 or 2 weeks after the last exposure for assessment of pulmonary toxicity. Differential cell counts, total protein concentrations and cellular enzyme (lactate dehydrogenase—LDH) activities were determined in bronchoalveolar lavage (BAL) fluid, and corticosterone and thiobarbituric acid reactive substances (TBARS) concentrations were assessed in serum. The increase in polymorphonuclear (PMN) leukocytes in BAL fluid (a cytological index of inflammatory responses of the lung) is believed to reflect pulmonary toxicity of heavy metals. Biomarkers of toxicity assessed in bronchoalveolar fluids indicate that the level of the toxic effect depends mainly on the solubility of studied metal compounds; biomarkers that showed treatment effects included: total cell, neutrophil and lymphocyte counts, total protein concentrations, and cellular enzyme (lactate dehydrogenase) activity. Betaine supplementation at 250 mg/kg/day in all study rats groups attenuated stress indices, and corticosterone and TBARS serum levels, and simultaneously stimulated increase of polymorphonuclear cells in BALF of rats. The study confirmed deleterious effect of transitory metals and particles during experimental inhalation exposure to welding dusts, evidenced in the lungs and brain by increased levels of total protein, higher cellular influx, rise of LDH in BALF, elevated TBARS and increased corticosterone in serum of rats. Our result confirm also the hypothesis about the effect of the welding dusts on the oxidative stress responsible for disturbed systemic homeostasis and impairment of calcium regulation.

Bronchoalveolar lavage for the diagnosis of Pulmonary Langerhans cell histiocytosis

BackgroundThe histologic diagnosis of Pulmonary Langerhans cell histiocytosis (PLCH) is invasive and can cause complications. To confirm the diagnosis of PLCH, guidelines therefore recommend measuring CD1a-positive bronchoalveolar lavage fluid (BALF) cells despite its poor sensitivity and specificity. Thus, an improved diagnostic accuracy of BALF cell analysis would be desirable. MethodsUsing four-colour flow cytometry, plasmacytoid and myeloid dendritic cells (DCs) were analysed in BALF of 10 newly diagnosed, untreated, smoking patients with PLCH, and compared with BALF DCs from 40 asymptomatic smokers and 21 never-smokers. ResultsCompared with controls, myeloid DCs (median: 0.79% of BALF leukocytes) and their subpopulation of Langerhans cells (median: 0.44% of BALF leukocytes) were not increased in PLCH. Patients with PLCH displayed a normal expression of the maturity marker CD83 on BALF myeloid DCs. However, the expression of the co-signaling molecule CD80 on BALF myeloid DCs was significantly lower than in both control groups, with the lowest expression found in more severe disease (presence of cysts > 2 cm in diameter). Based on receiver operating characteristic (ROC) curve analysis, a cut-off of 53% CD80-positive BALF myeloid DCs was optimal for the diagnosis of PLCH, yielding a sensitivity of 0.90 and a specificity of 0.90. ConclusionsBALF Langerhans cells are not increased in PLCH. However, PLCH is characterised by a low expression of CD80 on BALF myeloid DCs. Due to its considerably higher sensitivity and specificity, this marker appears to be more appropriate to diagnose PLCH than the currently recommended marker CD1a.

Impact of Dietary Tomato Juice on Changes in Pulmonary Oxidative Stress, Inflammation and Structure Induced by Neonatal Hyperoxia in Mice (Mus musculus).

Many preterm infants require hyperoxic gas for survival, although it can contribute to lung injury. Experimentally, neonatal hyperoxia leads to persistent alterations in lung structure and increases leukocytes in bronchoalveolar lavage fluid (BALF). These effects of hyperoxia on the lungs are considered to be caused, at least in part, by increased oxidative stress. Our objective was to determine if dietary supplementation with a known source of antioxidants (tomato juice, TJ) could protect the developing lung from injury caused by breathing hyperoxic gas. Neonatal mice (C57BL6/J) breathed either 65% O2 (hyperoxia) or room air from birth until postnatal day 7 (P7d); some underwent necropsy at P7d and others were raised in room air until adulthood (P56d). In subsets of both groups, drinking water was replaced with TJ (diluted 50:50 in water) from late gestation to necropsy. At P7d and P56d, we analyzed total antioxidant capacity (TAC), markers of oxidative stress (nitrotyrosine and heme oxygenase-1 expression), inflammation (interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) expression), collagen (COL) and smooth muscle in the lungs; we also assessed lung structure. We quantified macrophages in lung tissue (at P7d) and leukocytes in BALF (at P56d). At P7d, TJ increased pulmonary TAC and COL1α1 expression and attenuated the hyperoxia-induced increase in nitrotyrosine and macrophage influx; however, changes in lung structure were not affected. At P56d, TJ increased TAC, decreased oxidative stress and reversed the hyperoxia-induced increase in bronchiolar smooth muscle. Additionally, TJ alone decreased IL-1β expression, but following hyperoxia TJ increased TNF-α expression and did not alter the hyperoxia-induced increase in leukocyte number. We conclude that TJ supplementation during and after neonatal exposure to hyperoxia protects the lung from some but not all aspects of hyperoxia-induced injury, but may also have adverse side-effects. The effects of TJ are likely due to elevation of circulating antioxidant concentrations.

NK cells contribute to persistent airway inflammation and AHR during the later stage of RSV infection in mice.

RSV can lead to persistent airway inflammation and AHR and is intimately associated with childhood recurrent wheezing and asthma, but the underlying mechanisms remain unclear. There are high numbers of NK cells in the lung, which not only play important roles in the acute stage of RSV infection, but also are pivotal in regulating the pathogenesis of asthma. Therefore, in this study, we assumed that NK cells might contribute to persistent airway disease during the later stage of RSV infection. Mice were killed at serial time points after RSV infection to collect samples. Leukocytes in bronchoalveolar lavage fluid (BALF) were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. Cytokines were detected by ELISA, and NK cells were determined by flow cytometry. Rabbit anti-mouse asialo-GM-1 antibodies and resveratrol were used to deplete or suppress NK cells. Inflammatory cells in BALF, lung tissue damage and AHR were persistent for 60days post-RSV infection. Type 2 cytokines and NK cells were significantly increased during the later stage of infection. When NK cells were decreased by the antibodies or resveratrol, type 2 cytokines, the persistent airway inflammation and AHR were all markedly reduced. NK cells can contribute to the RSV-associated persistent airway inflammation and AHR at least partially by promoting type 2 cytokines. Therefore, therapeutic targeting of NK cells may provide a novel approach to alleviating the recurrent wheezing subsequent to RSV infection.

Regulatory T Cell Depletion Abolishes the Protective Effect of Dietary Galacto-Oligosaccharides on Eosinophilic Airway Inflammation in House Dust Mite–Induced Asthma in Mice

Background: In a murine model for house dust mite (HDM)–induced asthma, dietary galacto-oligosaccharides have been shown to suppress allergic symptoms. Previously, CD25+ regulatory T cells (Tregs) induced by nondigestible oligosaccharides were found to protect against allergy development.Objective: The aim of the current study was to examine the effect of anti–CD25-induced Treg depletion in a murine HDM-induced asthma model and to study the contribution of Tregs in the protective effect of dietary intervention with galacto-oligosaccharides.Methods: Male BALB/c mice (aged 6–8 wk) were intranasally sensitized and challenged with phosphate-buffered saline (PBS) or HDM. Two weeks before sensitization and throughout the whole experiment, mice were fed a control or 1% w/w galacto-oligosaccharide diet. Tregs were depleted by anti-mouse CD25 antibody (intraperitoneally injected). On day 14, T helper cell subtypes in lung and spleen were analyzed and cytokines were measured. Leukocyte subtypes were analyzed in the bronchoalveolar lavage fluid, and interleukin (IL)–33 and chemokines were measured in lung homogenate supernatants.Results: Anti-CD25 treatment depleted CD25+ Forkhead box P3+ Tregs in the lung and spleen of control and HDM-allergic mice (P < 0.0001) by >70% while increasing the percentage of activated T helper cells (P < 0.05) and type 2 T helper cells (P < 0.05). This was associated with increased IL-10, IL-4, and IL-13 concentrations in supernatants of ex vivo restimulated lung cells (P < 0.01). Bronchoalveolar lavage fluid leukocyte numbers and percentages of eosinophils and lymphocytes were greater in HDM-allergic mice compared with PBS mice (P < 0.01) but remained unaffected by the anti-CD25 treatment. Galacto-oligosaccharides decreased airway eosinophilia compared with HDM-allergic mice fed the control diet (from 47.8% ± 6.7% to 26.6% ± 8.5%, P < 0.01). This protective effect was lost in anti–CD25-treated mice (P < 0.05). In lung homogenates of HDM-allergic mice, IL-33 was increased compared with PBS mice (from 2.8 ± 0.3 to 5.4 ± 0.6 ng protein/mg, P < 0.01). Galacto-oligosaccharides abrogated the increase in IL-33 compared with HDM-allergic mice fed the control diet (3.0 ± 0.6 ng protein/mg, P < 0.05), which was abolished by the anti-CD25 treatment (P < 0.01).Conclusions: Treg depletion enhances pulmonary type 2 T helper cell frequency and cytokine release in HDM-induced asthma in mice. Galacto-oligosaccharides decreased airway eosinophilia and IL-33 concentrations in the lung, which was abrogated by Treg depletion. This indicates that galacto-oligosaccharides have a beneficial effect in the prevention of HDM-induced allergic asthma by supporting pulmonary Treg function.