Pharyngeal microflora disruption by antibiotics promotes airway hyperresponsiveness after respiratory syncytial virus infection

Background

Regulatory T cells (Treg cells), which are essential for regulation of immune response to respiratory syncytial virus (RSV) infection, are promoted by pharyngeal commensal pneumococcus. The effects of pharyngeal microflora disruption by antibiotics on airway responsiveness and relative immune responses after RSV infection have not been clarified.

Methods

Female BALB/c mice (aged 3 weeks) were infected with RSV and then treated with either oral antibiotics or oral double distilled water (ddH₂O) from 1 d post infection (pi). Changes in pharyngeal microflora were analyzed after antibiotic treatment for 7 d and 14 d. At 8 d pi and 15 d pi, the inflammatory cells in bronchoalveolar lavage fluid (BALF) were investigated in combination with tests of pulmonary histopathology, airway hyperresponsiveness (AHR), pulmonary and splenic Treg cells responses. Pulmonary Foxp3 mRNA expression, IL-10 and TGF-β1 in BALF and lung homogenate were investigated at 15 d pi. Ovalbumin (OVA) challenge was used to induce AHR after RSV infection.

Results

The predominant pharyngeal commensal, Streptococcus, was cleared by antibiotic treatment for 7 d. Same change also existed after antibiotic treatment for 14 d. After RSV infection, AHR was promoted by antibiotic treatment at 15 d pi. Synchronous decreases of pulmonary Treg cells, Foxp3 mRNA and TGF-β1 were detected. Similar results were observed under OVA challenge.

Conclusions

After RSV infection, antibiotic treatment cleared pharyngeal commensal bacteria such as Streptococcus, which consequently, might induce AHR and decrease pulmonary Treg cells.     

Decreased expression of the NF-κB family member RelB in lung fibroblasts from Smokers with and without COPD potentiates cigarette smoke-induced COX-2 expression

Background

Heightened inflammation, including expression of COX-2, is associated with COPD pathogenesis. RelB is an NF-κB family member that attenuates COX-2 in response to cigarette smoke by a mechanism that may involve the miRNA miR-146a. There is no information on the expression of RelB in COPD or if RelB prevents COX-2 expression through miR-146a.

Methods

RelB, Cox-2 and miR-146a levels were evaluated in lung fibroblasts and blood samples derived from non-smokers (Normal) and smokers (At Risk) with and without COPD by qRT-PCR. RelB and COX-2 protein levels were evaluated by western blot. Human lung fibroblasts from Normal subjects and smokers with and without COPD, along with RelB knock-down (siRNA) in Normal cells, were exposed to cigarette smoke extract (CSE) in vitro and COX-2 mRNA/protein and miR-146a levels assessed.

Results

Basal expression of RelB mRNA and protein were significantly lower in lung cells derived from smokers with and without COPD, the latter of which expressed more Cox-2 mRNA and protein in response to CSE. Knock-down of RelB in Normal fibroblasts increased Cox-2 mRNA and protein induction by CSE. Basal miR-146a levels were not different between the three groups, and only Normal fibroblasts increased miR-146a expression in response to smoke. There was a positive correlation between systemic RelB and Cox-2 mRNA levels and circulating miR-146a levels were higher only in GOLD stage I subjects.

Conclusions

Our data indicate that RelB attenuates COX-2 expression in lung structural cells, such that loss of pulmonary RelB may be an important determinant in the aberrant, heightened inflammation associated with COPD pathogenesis.     

Pulmonary arterial dysfunction in insulin resistant obese Zucker rats

Background

Insulin resistance and obesity are strongly associated with systemic cardiovascular diseases. Recent reports have also suggested a link between insulin resistance with pulmonary arterial hypertension. The aim of this study was to analyze pulmonary vascular function in the insulin resistant obese Zucker rat.

Methods

Large and small pulmonary arteries from obese Zucker rat and their lean counterparts were mounted for isometric tension recording. mRNA and protein expression was measured by RT-PCR or Western blot, respectively. K_V currents were recorded in isolated pulmonary artery smooth muscle cells using the patch clamp technique.

Results

Right ventricular wall thickness was similar in obese and lean Zucker rats. Lung BMPR2, K_V1.5 and 5-HT_2A receptor mRNA and protein expression and K_V current density were also similar in the two rat strains. In conductance and resistance pulmonary arteries, the similar relaxant responses to acetylcholine and nitroprusside and unchanged lung eNOS expression revealed a preserved endothelial function. However, in resistance (but not in conductance) pulmonary arteries from obese rats a reduced response to several vasoconstrictor agents (hypoxia, phenylephrine and 5-HT) was observed. The hyporesponsiveness to vasoconstrictors was reversed by L-NAME and prevented by the iNOS inhibitor 1400W.

Conclusions

In contrast to rat models of type 1 diabetes or other mice models of insulin resistance, the obese Zucker rats did not show any of the characteristic features of pulmonary hypertension but rather a reduced vasoconstrictor response which could be prevented by inhibition of iNOS.     

FABP4 Dynamics in Obesity: Discrepancies in Adipose Tissue and Liver Expression Regarding Circulating Plasma Levels

Background

FABP4 is predominantly expressed in adipose tissue, and its circulating levels are linked with obesity and a poor atherogenic profile.

Objective

In patients with a wide BMI range, we analyze FABP4 expression in adipose and hepatic tissues in the settings of obesity and insulin resistance. Associations between FABP4 expression in adipose tissue and the FABP4 plasma level as well as the main adipogenic and lipolytic genes expressed in adipose tissue were also analyzed.

Methods

The expression of several lipogenic, lipolytic, PPAR family and FABP family genes was analyzed by real time PCR. FABP4 protein expression in total adipose tissues and its fractions were determined by western blot.

Results

In obesity FABP4 expression was down-regulated (at both mRNA and protein levels), with its levels mainly predicted by ATGL and inversely by the HOMA-IR index. The BMI appeared as the only determinant of the FABP4 variation in both adipose tissue depots. FABP4 plasma levels showed a significant progressive increase according to BMI but no association was detected between FABP4 circulating levels and SAT or VAT FABP4 gene expression. The gene expression of FABP1, FABP4 and FABP5 in hepatic tissue was significantly higher in tissue from the obese IR patients compared to the non-IR group.

Conclusion

The inverse pattern in FABP4 expression between adipose and hepatic tissue observed in morbid obese patients, regarding the IR context, suggests that both tissues may act in a balanced manner. These differences may help us to understand the discrepancies between circulating plasma levels and adipose tissue expression in obesity.     

Up-regulation of hexokinase1 in the right ventricle of monocrotaline induced pulmonary hypertension

Background

Pulmonary arterial hypertension (PAH) is a proliferative arteriopathy associated with a glycolytic shift during heart metabolism. An increase in glycolytic metabolism can be detected in the right ventricle during PAH. Expression levels of glycolysis genes in the right ventricle during glycolysis that occur in monocrotaline (MCT)-induced pulmonary hypertension (PH) remain unknown.

Methods

PH was induced by a single subcutaneous injection of MCT (50 mg/kg) into rats, eventually causing right heart failure. Concurrently, a control group was injected with normal saline. The MCT-PH rats were randomly divided into three groups according to MCT treatment: MCT-2 week, 3 week, and 4 week groups (MCT-2w, 3w, 4w). At the end of the study, hemodynamics and right ventricular hypertrophy were compared among experimental groups. Expression of key glycolytic candidate genes was screened in the right ventricle.

Results

We observed an increase in mean pulmonary arterial pressure, right ventricular systolic pressure and right ventricular hypertrophy index three weeks following MCT injection. Alterations in the morphology and structure of right ventricular myocardial cells, as well as the pulmonary vasculature were observed. Expression of hexokinase 1 (HK1) mRNA began to increase in the right ventricle of the MCT-3w group and MCT-4w group, while the expression of lactate dehydrogenase A (LDHA) was elevated in the right ventricle of the MCT-4w group. Hexokinase 2(HK2), pyruvate dehydrogenase complex α1 (PDHα1), and LDHA mRNA expression showed no changes in the right ventricle. HK1 mRNA expression was further confirmed by HK1 protein expression and immunohistochemical analyses. All findings underlie the glycolytic phenotype in the right ventricle.

Conclusions

There was an increase in the protein and mRNA expression of hexokinase-1 (HK1) three and four weeks after the injection of monocrotaline in the right ventricle, intervention of HK1 may be amenable to therapeutic intervention.     

Circulating Progenitor Cells and Vascular Dysfunction in Chronic Obstructive Pulmonary Disease

Background

In chronic obstructive pulmonary disease (COPD), decreased progenitor cells and impairment of systemic vascular function have been suggested to confer higher cardiovascular risk. The origin of these changes and their relationship with alterations in the pulmonary circulation are unknown.

Objectives

To investigate whether changes in the number of circulating hematopoietic progenitor cells are associated with pulmonary hypertension or changes in endothelial function.

Methods

62 COPD patients and 35 controls (18 non-smokers and 17 smokers) without cardiovascular risk factors other than cigarette smoking were studied. The number of circulating progenitors was measured as CD45⁺CD34⁺CD133⁺ labeled cells by flow cytometry. Endothelial function was assessed by flow-mediated dilation. Markers of inflammation and angiogenesis were also measured in all subjects.

Results

Compared with controls, the number of circulating progenitor cells was reduced in COPD patients. Progenitor cells did not differ between control smokers and non-smokers. COPD patients with pulmonary hypertension showed greater number of progenitor cells than those without pulmonary hypertension. Systemic endothelial function was worse in both control smokers and COPD patients. Interleukin-6, fibrinogen, high sensitivity C-reactive protein, vascular endothelial growth factor and tumor necrosis factor were increased in COPD. In COPD patients, the number of circulating progenitor cells was inversely related to the flow-mediated dilation of systemic arteries.

Conclusions

Pulmonary and systemic vascular impairment in COPD is associated with cigarette smoking but not with the reduced number of circulating hematopoietic progenitors. The latter appears to be a consequence of the disease itself not related to smoking habit.     

Serum Amyloid A Induces Toll-Like Receptor 2-Dependent Inflammatory Cytokine Expression and Atrophy in C2C12 Skeletal Muscle Myotubes

Background

Skeletal muscle wasting is an important comorbidity of Chronic Obstructive Pulmonary Disease (COPD) and is strongly correlated with morbidity and mortality. Patients who experience frequent acute exacerbations of COPD (AECOPD) have more severe muscle wasting and reduced recovery of muscle mass and function after each exacerbation. Serum levels of the pro-inflammatory acute phase protein Serum Amyloid A (SAA) can rise more than 1000-fold in AECOPD and are predictively correlated with exacerbation severity. The direct effects of SAA on skeletal muscle are poorly understood. Here we have examined SAA effects on pro-inflammatory cachectic cytokine expression (IL-6 and TNFα) and atrophy in C2C12 myotubes.

Results

SAA increased IL-6 (31-fold) and TNFα (6.5-fold) mRNA levels compared to control untreated cells after 3h of SAA treatment, and increased secreted IL-6 protein at 24h. OxPAPC, a dual TLR2 and TLR4 inhibitor, reduced the response to SAA by approximately 84% compared to SAA alone, and the TLR2 neutralising antibody T2.5 abolished SAA-induced expression of IL-6, indicating that SAA signalling in C2C12 myotubes is primarily via TLR2. SAA also reduced myotube width by 10–13% and induced a 2.5-fold increase in the expression of the muscle atrophy gene Atrogin-1, suggesting direct effects of SAA on muscle wasting. Blocking of TLR2 inhibited the SAA-induced decrease in myotube width and Atrogin-1 gene expression, indicating that SAA induces atrophy through TLR2.

Conclusions

These data demonstrate that SAA stimulates a robust pro-inflammatory response in skeletal muscle myotubes via the TLR2-dependent release of IL-6 and TNFα. Furthermore, the observed atrophy effects indicate that SAA could also be directly contributing to the wasting and poor recovery of muscle mass. Therapeutic strategies targeting this SAA-TLR2 axis may therefore ameliorate muscle wasting in AECOPD and a range of other inflammatory conditions associated with loss of muscle mass.     

Comprehensive characterisation of pulmonary and serum surfactant protein D in COPD

Background

Pulmonary surfactant protein D (SP-D) is considered as a candidate biomarker for the functional integrity of the lung and for disease progression, which can be detected in serum. The origin of SP-D in serum and how serum concentrations are related to pulmonary concentrations under inflammatory conditions is still unclear.

Methods

In a cross-sectional study comprising non-smokers (n = 10), young - (n = 10), elderly smokers (n = 20), and smokers with COPD (n = 20) we simultaneously analysed pulmonary and serum SP-D levels with regard to pulmonary function, exercise, repeatability and its quaternary structure by native gel electrophoresis. Statistical comparisons were conducted by ANOVA and post-hoc testing for multiple comparisons; repeatability was assessed by Bland-Altman analysis.

Results

In COPD, median (IQR) pulmonary SP-D levels were lower (129(68) ng/ml) compared to smokers (young: 299(190), elderly: 296(158) ng/ml; p < 0.01) and non-smokers (967(708) ng/ml; p < 0.001). The opposite was observed in serum, with higher concentrations in COPD (140(89) ng/ml) as compared to non-smokers (76(47) ng/ml; p < 0.01). SP-D levels were reproducible and correlated with the degree of airway obstruction in all smokers. In addition, smoking lead to disruption of the quaternary structure.

Conclusions

Pulmonary and serum SP-D levels are stable markers influenced by smoking and related to airflow obstruction and disease state. Smaller subunits of pulmonary SP-D and the rapid increase of serum SP-D levels in COPD due to exercise support the translocation hypothesis and its use as a COPD biomarker.

Trial registration

no interventional trial     

Direct phenotypical and functional dysregulation of primary human B cells by human immunodeficiency virus (HIV) type 1 in vitro

Background

Human immunodeficiency virus type 1 (HIV-1) induces a general dysregulation of immune system. Dysregulation of B cell compartment is generally thought to be induced by HIV-related immune activation and lymphopenia. However, a direct influence of HIV-1 particles on B cells was recently proposed as the third pathway of B cells dysregulation.

Methods/Principal Findings

We evaluated the direct and specific consequences of HIV-1 contact on activation, survival, proliferation and phenotype of primary B cells in vitro. Moreover, we examined expression of activation-induced cytidine deaminase (AID) mRNA that is responsible for class switch recombination (CSR) and somatic hypermutation (SHM). Here, we report that changes observed in cellular proliferation, phenotypes and activation of B cells could be caused by direct contact between HIV-1 particles and primary B cells in vitro. Finally, direct HIV-1-derived B cells activation led to the increase of AID mRNA expression and its subsequent CSR function was detected in vitro.

Conclusion/Significance

We showed that HIV-1 could directly induce primary B cells dysregulation triggering phenotypical and functional abilities of B cells in vitro that could explain in some extent early B-cell abnormalities in HIV disease.     

ANG-1 TIE-2 and BMPR signalling defects are not seen in the nitrofen model of pulmonary hypertension and congenital diaphragmatic hernia

Background

Pulmonary hypertension (PH) is a lethal disease that is associated with characteristic histological abnormalities of the lung vasculature and defects of angiopoetin-1 (ANG-1), TIE-2 and bone morphogenetic protein receptor (BMPR)-related signalling. We hypothesized that if these signalling defects cause PH generically, they will be readily identifiable perinatally in congenital diaphragmatic hernia (CDH), where the typical pulmonary vascular changes are present before birth and are accompanied by PH after birth.

Methods

CDH (predominantly left-sided, LCDH) was created in Sprague-Dawley rat pups by e9.5 maternal nitrofen administration. Left lungs from normal and LCDH pups were compared at fetal and postnatal time points for ANG-1, TIE-2, phosphorylated-TIE-2, phosphorylated-SMAD1/5/8 and phosphorylated-ERK1/2 by immunoprecipitation and Western blotting of lung protein extracts and by immunohistochemistry on lung sections.

Results

In normal lung, pulmonary ANG-1 protein levels fall between fetal and postnatal life, while TIE-2 levels increase. Over the corresponding time period, LCDH lung retained normal expression of ANG-1, TIE-2, phosphorylated-TIE-2 and, downstream of BMPR, phosphorylated-SMAD1/5/8 and phosphorylated-p44/42.

Conclusion

In PH and CDH defects of ANG-1/TIE-2/BMPR-related signalling are not essential for the lethal vasculopathy.     

The Role of Catalase in Pulmonary Fibrosis

Background

Catalase is preferentially expressed in bronchiolar and alveolar epithelial cells, and acts as an endogenous antioxidant enzyme in normal lungs. We thus postulated epithelial damage would be associated with a functional deficiency of catalase during the development of lung fibrosis.

Methods

The present study evaluates the expression of catalase mRNA and protein in human interstitial pneumonias and in mouse bleomycin-induced lung injury. We examined the degree of bleomycin-induced inflammation and fibrosis in the mice with lowered catalase activity.

Results

In humans, catalase was decreased at the levels of activity, protein content and mRNA expression in fibrotic lungs (n = 12) compared to control lungs (n = 10). Immunohistochemistry revealed a decrease in catalase in bronchiolar epithelium and abnormal re-epithelialization in fibrotic areas. In C57BL/6J mice, catalase activity was suppressed along with downregulation of catalase mRNA in whole lung homogenates after bleomycin administration. In acatalasemic mice, neutrophilic inflammation was prolonged until 14 days, and there was a higher degree of lung fibrosis in association with a higher level of transforming growth factor-β expression and total collagen content following bleomycin treatment compared to wild-type mice.

Conclusions

Taken together, these findings demonstrate diminished catalase expression and activity in human pulmonary fibrosis and suggest the protective role of catalase against bleomycin-induced inflammation and subsequent fibrosis.     

Cigarette smoke-induced pulmonary emphysema in scid-mice. Is the acquired immune system required?

Background

Chronic obstructive pulmonary disease is associated with a chronic inflammatory response of the host to chronic exposure to inhaled toxic gases and particles. Although inflammatory cells of both the innate and adaptive immune system infiltrate the lungs in pulmonary emphysema and form lymphoid follicles around the small airways, the exact role of the acquired immune system in the pathogenesis of emphysema is not known.

Methods

In this study, wild type Balb/c mice and immunodeficient scid mice – which lack functional B- and T-cells – were exposed to mainstream cigarette smoke (CS) for 5 weeks or 6 months.

Results

Subacute CS-exposure for 5 weeks significantly increased innate inflammatory cells (neutrophils, macrophages and dendritic cells) in the bronchoalveolar lavage (BAL) fluid of wild type mice and scid mice, which correlated with the CS-induced upregulation of the chemokines Monocyte Chemotactic Protein-1, Macrophage Inflammatory Protein-3α and KC (= mouse Interleukin-8). Chronic CS-exposure for 6 months significantly increased the number of neutrophils, macrophages, dendritic cells, CD4⁺ and CD8⁺ T-lymphocytes in BAL fluid and lungs of wild type mice compared to air-exposed littermates, and augmented the size and number of peribronchial lymphoid follicles. In contrast, neither B-lymphocytes, nor T-lymphocytes, nor lymphoid follicles could be discerned in the lungs of air- or CS-exposed scid mice. Importantly, chronic CS-exposure induced pulmonary emphysema in both wild type animals and scid mice, as evidenced by a significant increase in the mean linear intercept and the destructive index of CS-exposed versus air-exposed animals. The CS-induced emphysema was associated with increased mRNA expression of matrix metalloproteinase-12 in the lungs and increased protein levels of Tumor Necrosis Factor-α in the BAL fluid of CS-exposed Balb/c and scid mice compared to air-exposed littermates.

Conclusion

This study suggests that the adaptive immune system is not required per se to develop pulmonary emphysema in response to chronic CS-exposure, since emphysema can be induced in scid mice, which lack lymphoid follicles as well as functional B- and T-cells.     

Creation of Lung-Targeted Dexamethasone Immunoliposome and Its Therapeutic Effect on Bleomycin-Induced Lung Injury in Rats

Objective

Acute lung injury (ALI), is a major cause of morbidity and mortality, which is routinely treated with the administration of systemic glucocorticoids. The current study investigated the distribution and therapeutic effect of a dexamethasone(DXM)-loaded immunoliposome (NLP) functionalized with pulmonary surfactant protein A (SP-A) antibody (SPA-DXM-NLP) in an animal model.

Methods

DXM-NLP was prepared using film dispersion combined with extrusion techniques. SP-A antibody was used as the lung targeting agent. Tissue distribution of SPA-DXM-NLP was investigated in liver, spleen, kidney and lung tissue. The efficacy of SPA-DXM-NLP against lung injury was assessed in a rat model of bleomycin-induced acute lung injury.

Results

The SPA-DXM-NLP complex was successfully synthesized and the particles were stable at 4°C. Pulmonary dexamethasone levels were 40 times higher with SPA-DXM-NLP than conventional dexamethasone injection. Administration of SPA-DXM-NLP significantly attenuated lung injury and inflammation, decreased incidence of infection, and increased survival in animal models.

Conclusions

The administration of SPA-DXM-NLP to animal models resulted in increased levels of DXM in the lungs, indicating active targeting. The efficacy against ALI of the immunoliposomes was shown to be superior to conventional dexamethasone administration. These results demonstrate the potential of actively targeted glucocorticoid therapy in the treatment of lung disease in clinical practice.     

Alveolar macrophage-epithelial cell interaction following exposure to atmospheric particles induces the release of mediators involved in monocyte mobilization and recruitment

Background

Studies from our laboratory have shown that human alveolar macrophages (AM) and bronchial epithelial cells (HBEC) exposed to ambient particles (PM₁₀) in vitro increase their production of inflammatory mediators and that supernatants from PM₁₀-exposed cells shorten the transit time of monocytes through the bone marrow and promote their release into the circulation.

Methods

The present study concerns co-culture of AM and HBEC exposed to PM₁₀ (EHC-93) and the production of mediators involved in monocyte kinetics measured at both the mRNA and protein levels. The experiments were also designed to determine the role of the adhesive interaction between these cells via the intercellular adhesion molecule (ICAM)-1 in the production of these mediators.

Results

AM/HBEC co-cultures exposed to 100 μg/ml of PM₁₀ for 2 or 24 h increased their levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, macrophage inflammatory protein (MIP)-1β, monocyte chemotactic protein (MCP)-1, interleukin (IL)-6 and ICAM-1 mRNA, compared to exposed AM or HBEC mono-cultures, or control non-exposed co-cultures. The levels of GM-CSF, M-CSF, MIP-1β and IL-6 increased in co-cultured supernatants collected after 24 h exposure compared to control cells (p < 0.05). There was synergy between AM and HBEC in the production of GM-CSF, MIP-1β and IL-6. But neither pretreatment of HBEC with blocking antibodies against ICAM-1 nor cross-linking of ICAM-1 on HBEC blocked the PM₁₀-induced increase in co-culture mRNA expression.

Conclusion

We conclude that an ICAM-1 independent interaction between AM and HBEC, lung cells that process inhaled particles, increases the production and release of mediators that enhance bone marrow turnover of monocytes and their recruitment into tissues. We speculate that this interaction amplifies PM₁₀-induced lung inflammation and contributes to both the pulmonary and systemic morbidity associated with exposure to air pollution.