The Division directs basic and clinical research programs related to asthma, cystic fibrosis and bronchopulmonary dysplasia. Faculty research is currently funded by the National Institutes of Health and Cystic Fibrosis Foundation. Areas of study include:
- viral-induced exacerbations of asthma and other chronic airways diseases
- signal transduction mechanisms regulating airway smooth muscle hypertrophy
- role of mesenchymal stem cells in lung injury and repair
- role of viral and bacterial co-infection in the pathogenesis of cystic fibrosis lung disease
- interactive effects of diesel exhaust and respiratory viral infection on asthmatic children
- role of adipose-tissue macrophages in the pathogenesis of obesity-associated diseases such as metabolic syndrome and diabetes
- use of high resolution computerized tomography of the chest to evaluate cystic fibrosis lung disease
- use of forced oscillometry as an outcome measure in preschool children with cystic fibrosis
- lung function in children and adolescents with sickle cell disease and its correlation with pulmonary hypertension.
For more information about the research of individual faculty members, see below
Manuel Arteta, M.D.
J. Kelley Bentley, Ph.D.
Amy Goldstein Filbrun, M.D., M.S.
Marc B. Hershenson, M.D.
Toby C. Lewis, M.D., M.P.H.
Carey N. Lumeng, M.D., Ph.D.
Samya Z. Nasr, M.D.
Umadevi Sajjan, Ph.D.
Manuel Arteta, M.D.
Dr. Arteta’s research interests include: 1) pulmonary function in children and adolescents with sickle cell disease and its correlation with pulmonary hypertension; and 2) pediatric asthma case management.
The Pulmonary Hypertension and Hypoxic Response in Sickle Cell Disease (PUSH) study is an ongoing prospective, observational multicenter study initiated in 2006 to study the prevalence, risk factors and clinical consequences of pulmonary hypertension in children and adolescents with sickle cell disease. Dr. Arteta is the participating pediatric pulmonologist at the University of Michigan center.
Pulmonary Function and Tricuspid Regurgitant Velocity in Pediatric Sickle Cell Disease. Am J Respir Crit Care Med 2008, 177: A262
J. Kelley Bentley, Ph.D.
Dr. Bentley is interested in the biochemical and cellular mechanisms underlying airway remodeling in asthma. His current research interest focuses on the potential contribution of mesenchymal stem cells to this process. This project is described below
Mesenchymal Stem Cells in a Mouse Model of Asthma
Patients with asthma have increased airway smooth muscle airway smooth muscle mass. Increased mass could be due to abnormal smooth muscle cell proliferation, hypertrophy or migration of progenitor cells from other compartments to the airway wall. Our pilot studies employing confocal microscopy and flow cytometry indicate that ovalbumin (OVA) sensitization and challenge, a model of allergic asthma, increases the number of lung cells expressing markers typically found on mesenchymal stem cells (MSCs). Our hypothesis is that airway remodeling includes the differentiation of smooth muscle from undifferentiated progenitor cells. To this hypothesis, we propose two Specific Aims:
1. Determine whether ovalbumin sensitization and challenge increases the number of MSCs in the lung. We hypothesize that OVA treatment increases the number of lung and airway smooth muscle MSCs.
To test this hypothesis, lung mesenchymal stem cells will be identified by confocal immunofluorescence microscopy and flow cytometry. Pure populations of mesenchymal stem cells will be isolated using cell sorting and differential adherence. The ability of these cells to differentiate into adipocytes, chondrocytes, osteocytes and smooth muscle will be tested in culture.
2. Determine the origin of airway MSCs, the factors which draw ASM progenitors into sensitized airways, and the factors which differentiate them into smooth muscle. We hypothesize that: 1) lung MSCs originate from the bone marrow; and 2) CCL12 (the murine homolog of MCP-1/CCL2) and bFGF are responsible for homing of MSCs to the airways; 3) transforming growth factor (TGF)-b is responsible for differentiating MSCs into smooth muscle.
To test these hypotheses, a bone marrow chimeric mouse will be generated by introducing bone marrow cells from green fluorescent protein-expressing CByJ.B6-Tg (UBC-GFP) 30 Scha/J mice into congenic BALB/cByJ mice. Chimeric animals will subsequently undergo OVA sensitization and challenge. Homing of MSCs to the lung and differentiation to smooth muscle will be monitored by confocal microscopy and flow cytometry. MCP-1, bFGF, TGF-b and their receptors will be localized with MSCs and airway smooth muscle in the lungs of OVA-sensitized and treated mice using confocal immunofluorescent microscopy. MCP-1, bFGF and TGF-b mRNA and protein expression will be assessed by enzyme linked immunosorbent assay (ELISA) and quantitative reverse-transcriptase polymerase chain reaction (qPCR) of lavage and lung extracts. The chemotactic activity of MCP-1 and bFGF for mouse MSCs will be tested in a Boyden chamber. The requirement of MCP-1, bFGF and TGF-b for MSC homing to the lung and differentiation to smooth muscle will be assessed using neutralizing antibodies.
Amy Goldstein Filbrun, M.D., M.S.
Dr. Filbrun is interested in the measurement of pulmonary function in infants and pre-school children. Measurement of pulmonary function is necessary not only to monitor disease progression, but to assess the effects of treatment.
She is currently performing a longitudinal study of lung function in infants with a history of prematurity and bronchopulmonary dysplasia.
Other research projects include:
“A Phase IV, Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial of Pulmozyme in 3-5 Year Old Patients with Cystic Fibrosis.” This study is designed to evaluate the effect of Pulmozyme on pulmonary function, health related quality of life and respiratory symptoms in 3- to 5-year-old children with CF. This study will also serve as a pilot study to assess whether forced oscillometry, a newer lung function test that is easier for preschool children to perform, is useful as an outcome measure.
“Infant Study of Inhaled Saline in Cystic Fibrosis.” This study is a randomized, controlled trial to assess the efficacy and safety of inhaled 7% hypertonic saline twice daily for 48 weeks among infants and young children with CF, 4-60 months of age at enrollment. The primary hypothesis is that, compared to isotonic saline, hypertonic saline will decrease the rate of pulmonary exacerbations during the 48 week treatment period. The results of the proposed trial may for the first time provide evidence that early initiation of hypertonic saline, by improving mucociliary clearance, may delay or hinder the cycle of infection and inflammation responsible for progressive airway damage in CF lung disease.
Marc B. Hershenson, M.D.
The Hershenson laboratory studies cellular and molecular mechanisms underlying chronic airways diseases such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis and bronchopulmonary dysplasia. This work is funded by the National Institutes of Health.
At this time, the laboratory is focusing on three main projects:
Mechanisms by which rhinovirus, a common cold virus, induces exacerbations of asthma and other chronic airways diseases
Rhinovirus (RV) infection accounts for a large fraction of asthma exacerbations. Airway neutrophils and IL-8 levels are increased in RV-induced exacerbations, suggesting that RV stimulates exacerbations by inducing epithelial cell expression of (ELR)+ C-X-C chemokines, leading to an exaggerated inflammatory response.
In pilot studies, we have shown that RV39 induces IL-8, ENA-78 and GRO-a expression in primary, mucociliary-differentiated human tracheal epithelial cells. In 16HBE14o- cells, RV39 infection activates Src, PI 3-kinase, Akt and ERK minutes after infection, and activation of these kinases is required for IL-8 expression. RV increases C-X-C chemokine expression induced by two pro-asthmatic cytokines, IL-13 and TNFa. Finally, RV1B infection of C57/BL6 mice increases airway neutrophils and levels of MIP-2, a murine ELR(+) C-X-C chemokine. We therefore hypothesize that RV is sufficient to activate biochemical signaling pathways involved in the asthmatic response, providing a mechanism for RV-induced asthma exacerbations.
Specific Aim 1: Characterize upstream activators and downstream effectors of PI 3-kinase required for RV-induced ELR(+) C-X-C chemokine expression. We hypothesize that: 1) RV colocalizes with Src, PI 3-kinase, Akt and Grb2 in lipid rafts; 2) Src is required for activation of the PI 3-kinase/Akt pathway; 3) Class IA, II and III PI 3-kinases are required for maximal RV-induced expression of IL-8, ENA-78 and GROa; and 4) maximal NF-kB activation requires PI 3-kinase-dependent activation of NADPH oxidase.
Specific Aim 2: Determine the biochemical signaling mechanisms responsible for cooperative effects of RV and pro-asthmatic cytokines on airway epithelial cell IL-8 expression. We hypothesize that: 1) ERK and JNK regulate IL-8 expression via activation of the AP-1 promoter site, which functions as a basal level enhancer; 2) additive effects of RV39 and TNFa are mediated by increased p65 RelA phosphorylation and NF-kB transactivation; 3) synergistic effects of RV39 and IL-13 are mediated by increased AP-1 transactivation.
Specific Aim 3: Determine the steps in the viral life cycle required or sufficient for RV-induced signaling and chemokine responses and, conversely, determine the requirement of host cell signal transduction for viral infection. We hypothesize that: 1) ICAM1 ligation is required and sufficient for activation of Src, PI 3-kinase, Akt, ERK and JNK; 2) viral replication is not required for activation of these signaling intermediates; and 3) PI 3-kinase activation is required for RV39 internalization.
Specific Aim 4: Determine the requirements of PI 3-kinase signaling and ELR(+) C-X-C chemokines for RV-induced responses in vivo. We hypothesize that: 1) RV1B infection is sufficient for airway inflammation and epithelial cell signaling in vivo; 2) PI 3-kinase is required for RV1B-induced airway inflammation in vivo; and 3) C-X-C chemokine receptor (CXCR)-2 regulates RV1B-induced airway inflammation in vivo.
Understanding RV-induced asthma exacerbations will lead to improvements in the treatment of this disease
Biochemical signaling mechanisms by which airway smooth muscle cells increase their size (hypertrophy) and contractile protein expression
Increased airway smooth muscle mass, due either to hypertrophy or hyperplasia, is present in patients with asthma. While the biochemical pathways regulating cell proliferation have been well studied, little is known about the biochemical pathways regulating airway smooth muscle protein synthesis, cell size or the accumulation of contractile apparatus proteins. Recent data from cell culture models and patients with asthma suggest that airway smooth muscle contractile protein expression may be regulated in a post-transcriptional manner. In this revised application, we propose that airway smooth muscle hypertrophy requires the activation of specific translational control pathways. To test this, we will study 1) a cell culture model of hypertrophy in which primary human bronchial smooth muscle cells are treated with transforming growth factor (TGF)-b; 2) two mouse models of asthma; and 3) biopsy samples from human asthmatics. We propose three Specific Aims:
Specific Aim 1. Determine the contributions of contractile apparatus transcription and translation in the development of human airway smooth muscle hypertrophy. We hypothesize that: 1) TGFb promotes airway smooth muscle hypertrophy by increasing both gene transcription and translational efficiency; and 2) murine airway remodeling is characterized in part by smooth muscle hypertrophy.
Specific Aim 2. Examine the contribution of cap-dependent protein synthesis to human airway smooth muscle hypertrophy. We hypothesize that: 1) 4E-BP phosphorylation, which increases the availability of eIF4E for eIF4F complex formation, is required for TGFb-induced airway smooth muscle hypertrophy; 2) eiF4E phosphorylation by MAP kinase signal integrating kinase (MNK)-1 is required for airway smooth muscle hypertrophy; and 3) airway remodeling is characterized in part by increased phosphorylation of airway smooth muscle 4E-BP and eIF4E.
Specific Aim 3. Examine the contribution of cap-independent protein synthesis to human airway smooth muscle hypertrophy. We hypothesize that: 1) p70 ribosomal S6 kinase (S6 kinase) is neither required nor sufficient for airway smooth muscle hypertrophy; 2) GSK3b phosphorylation and inactivation is required and sufficient for airway smooth muscle hypertrophy; and 3) airway remodeling is characterized in part by increased phosphorylation of airway smooth muscle GSK3-b.
Understanding biochemical mechanisms of airway remodeling in asthma will lead to improvements in the treatment of this disease.
Potential role of alveolar mesenchymal cells in lung injury and repair
We have obtained pilot data showing that tracheal aspirates from week-old premature infants undergoing mechanical ventilation for respiratory distress syndrome contain fibroblast-like cells with surface markers and differentiation potential typically found in mesenchymal stem cells. The cells are positive for Stro-1, CD73, CD90, CD105 and CD166, but negative for CD34 and CD45, indicating that they are of stromal but not hematopoietic origin. Further, they exhibit ample proliferative capacity and are capable of differentiation into osteocytes, adipocytes, myofibroblasts and epithelial cells. The conditioned medium from these cells holds mitogenic activity for airway epithelial cells. Finally, we have typically identified these cells only in the tracheal aspirates of premature infants who subsequently develop chronic lung disease, i.e., broncho-pulmonary dysplasia (BPD).
We therefore hypothesize that multipotent lung mesenchymal cells participate in neonatal lung repair, and that these cells are a biomarker for the development of BPD. To test this general hypothesis, we propose the following Specific Aims.
Specific Aim 1. Further characterize multipotent lung mesenchymal cells from premature infants, and determine mechanisms by which these cells are recruited to the airspaces. We hypothesize that: 1) lung mesenchymal cell cultures contain one or more individual clones of multipotent cells; 2) epithelial injury promotes proliferation of lung mesenchymal cells and their migration to the airspaces via induction of bFGF, monocyte chemoattractant protein (MCP-1) and stromal cell-derived factor (SDF)-1a expression.
Specific Aim 2. Characterize potential mechanisms by which multipotent lung mesenchymal cells from premature infants participate in lung repair. We hypothesize that: 1) lung mesenchymal cells produce trophic factors capable of promoting respiratory epithelial wound repair and angiogenesis; 2) when stimulated by transforming growth factor (TGF)-b, lung mesenchymal cells differentiate into myofibroblasts, thereby promoting fibrogenesis.
Specific Aim 3. Correlate the presence of mesenchymal stem cells in premature infants with the development of BPD. We hypothesize that the presence of lung mesenchymal stem cells is a biomarker for the development of BPD. We will prospectively compare the clinical outcomes of premature infants from whom multipotent lung mesenchymal cells have been isolated with those from whom cells are not isolated, focusing on days of oxygen supplementation and the development of BPD. Infant lung function, including lung volumes, forced expiratory flows and respiratory system compliance, will also be assessed.
Understanding the role of multipotent lung mesenchymal cells in the pathogenesis of BPD will lead to improvements in the treatment of this disease.
Toby Crowe Lewis, M.D., M.P.H.
Interaction between air pollution and allergy on the respiratory health of asthmatic children.
Indoor environmental exposures play a key role in asthma aggravation. Strong associations have been found with indoor allergens, particularly to cockroach and dust mite, to which a child is sensitized, but also with exposure to indoor tobacco smoke, indoor fungi, and other irritants. Increased ambient air pollution levels, particularly respirable particulates (PM2.5 and PM10) and ozone, have been reported to precipitate symptoms of asthma and to increase emergency department visits and hospitalizations for asthma. The role of outdoor ambient pollutants in the potentiation of the inflammatory effects of subsequent exposure to allergens has been examined in animal and human experimental models, but there is limited data on the interaction between these pro-inflammatory stimuli in the “real life” of asthmatics.
As part of her work at the Michigan Center for the Environment and Children’s Health, Dr. Lewis is measuring asthma-related health outcomes over a two-year period among 300 asthmatic children in Detroit, MI. She is analyzing the effect of simultaneous exposure to air pollution, and household cockroach allergen levels among sensitized asthmatics. Preliminary results indicate that there is a significant reduction in pulmonary function with exposure to high levels of ozone among children with allergy to cockroach, but not among those who are not sensitized. This project is also being conducted using a CBPR (community-based participatory research) approach. Future studies will investigate the role of tobacco smoke and other allergens in this enhanced response to air pollution.
Impact of a lay environmental outreach worker on family behavior change and asthma morbidity reduction among urban African-American and Hispanic children with asthma.
Urban children, particularly children of color, bear a disproportionate burden of the morbidity associated with the asthma epidemic that has been occurring in the US over the last three decades. This phenomenon has been attributed to multiple factors, including reduced access to healthcare and increased environmental exposures associated with poor housing stock and residence in areas with high concentration of air pollution, and is clearly compounded by the social environmental stress of inner-city living.
In collaboration with colleagues at the Michigan Center for the Environment and Children’s Health (MCECH), Dr. Lewis is conducting a randomized trial of a community-based intervention consisting of two years of home visits by a lay environmental outreach worker with the goal of modifying the home indoor environment to reduce an asthmatic child’s exposure to triggering agents. This study takes a comprehensive approach to health; taking into account the interplay between the individual, the physical environment, and the social environment. It is conducted with a community-based participatory research (CBPR) strategy, where community leaders serve as co-investigators and are involved with all aspects of research design, implementation, and assessment.
Interactive Effects of Diesel Exhaust and Respiratory Viruses on Asthmatic Children
Traffic-associated air pollution and respiratory viral infections (URIs) are well-documented, independent stimulants of asthma symptoms, and both are associated with significant asthma morbidity. There is growing laboratory and epidemiologic evidence to suggest that air pollution and URIs have interactive effects on the respiratory system. We have shown, in a prior epidemiologic study, that asthmatics with cold symptoms show more changes in lung function associated with prior exposure to high pollution than those without colds. However, the independent and interactive effects of pollutants attributable to diesel and other vehicular
emissions and URIs on asthma exacerbation in children have not been extensively studied.
This project will test the general hypothesis that traffic-associated air pollution, specifically diesel exhaust, and respiratory viral infections combine to induce exaggerated airway responses in children with asthma. Two potential mechanisms of interaction will be evaluated: a) traffic-associated air pollution increases susceptibility to viral URI, and b) combined traffic pollutant exposure and URI elicits augmented inflammatory and oxidative stress responses. To test this hypothesis of interaction, we propose two Specific Aims:
1. Compare asthmatic children living near highways with diesel truck traffic to those living distant from high volume roads for a) the frequency of symptomatic viral URIs and b) changes in nasal expression of viral adhesion proteins and interferons; and
2. Prospectively evaluate the interactions between air pollution and viral upper respiratory infections on a) asthma morbidity, and b) markers of inflammation and oxidative stress.
Recent publications (last five years)
Carey N. Lumeng, M.D., Ph.D.
The major goal of the laboratory is to understand the links between obesity and inflammation. The low grade chronic inflammation induced by obesity is a major contributor to the development of obesity-associated diseases such as cardiovascular disease and Type 2 diabetes. Adipose tissue is a major source of inflammatory factors in obese humans and animal models and it has recently been showed that macrophages infiltrate adipose tissue in obesity and generate the majority of these proinflammatory factors. Interestingly, these adipose tissue macrophages (ATMs) have been shown to be both necessary and sufficient to generate insulin resistance in obesity models.
We have shown that pro-inflammatory macrophages directly alter adipocyte insulin signaling and that ATMs can assume different activation states. M2 or alternatively activated ATMs dominate in lean adipose tissue. With high fat diet, a new population of M1 or classically activated macrophages invade adipose tissue and contribute to adipocyte dysfunction.
Our laboratory is interested in understanding the factors that regulate the switch from M2 to M1 ATMs in adipose tissue, how ATMs are trafficked into fat with different dietary exposure, how ATMs may relate to adipogenesis and angiogenesis in the fat pad, how ATMs differ between fat depots, and the molecular factors that govern the inflammatory activation of ATMs with obesity.
To answer these questions we employ a range of genetic, cell biological, microscopic and molecular techniques to examine the function of ATMs in obese mice as well as from human samples. Understanding how ATMs function can provide novel insights into the pathogenesis of obesity-induced inflammation and can potentially identify novel ways to block these inflammatory changes as possible treatments for obesity-associated diseases such as diabetes.
Samya Z. Nasr, M.D.
Dr Nasr has been focused on clinical research in cystic fibrosis. Through her work, patients at the University of Michigan are among the first to receive new treatments for CF. Dr. Nasr is holder of a Cystic Fibrosis Foundation-sponsored “Therapeutics Development Center” grant. She is currently responsible for the following studies:
“An Open Label, Multi-Center Study to Assess the Safety and Tolerability of Pancrelipase Delayed Release Capsules in Infants and Children Less than Seven Years of Age with Pancreatic Exocrine Insufficiency Due to Cystic Fibrosis”
“A randomized, double-blind, placebo-controlled parallel group study to investigate the safety and efficacy of two doses of tiotropium bromide (2.5 µg and 5 µg) administered once daily via the Respimat® device for 12 weeks in patients with cystic fibrosis”
“A Double-Blind, Randomized, Multi-Center, Placebo-Controlled, Cross-Over Study to Assess the efficacy and safety of Pancrelipase Delayed Release 12,000 Unit Capsules in Subjects Ages 7-11 with Pancreatic Exocrine Insufficiency Due to Cystic Fibrosis”
“Randomized, Double-Blind, Placebo-Controlled, Multicenter Study to Evaluate the Safety and Efficacy of Inhaled Ciprofloxacin to Placebo in Subjects with Cystic Fibrosis”
“A Double-Blind, Multicenter, Multinational, Randomized, Placebo-Controlled Trial Evaluating Aztreonam Lysine for Inhalation in Patients with Cystic Fibrosis, Mild Lung Disease, and P. aeruginosa”
“A Phase 2, Multi-Center, Randomized, Double-Blind, Placebo-Controlled Study to Evaluate the Safety, Tolerability and Efficacy of Three Dosage Regimens of MP-376 Solution for Inhalation Given for 28 days to Stable Cystic Fibrosis Patients”
“A Phase 3, International, Multi-Center, Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Efficacy and Safety Study of Denufosol Tetrasodium Inhalation Solution in Patients with Cystic Fibrosis Lung Disease and FEV1 ≥ 75% Predicted”
“Multi-center Trial to Validate Protein Biomarkers of a Pulmonary Exacerbation in Cystic Fibrosis”
“An Open-Label Clinical Study Evaluating the Long-Term Safety of ALTU-135 for the Treatment of Patients with Cystic Fibrosis-Related Exocrine Pancreatic Insufficiency”
“Multi-center, Multi-national, Randomized, Placebo-Controlled Trial of Azithromycin in Subjects with Cystic Fibrosis 6-18 years old, Culture negative for Pseudomonas aeruginosa”
“A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial of Pulmozyme® Withdrawal on Exercise Tolerance in Cystic Fibrosis Subjects with Severe Lung Disease”
“A Randomized, Double-blind, within dose, placebo-controlled, Study to Investigate the Safety, Tolerability, and Pharmacokinetics of Increasing Single and Multiple Doses (28-day Dosing) of Tiotropium Bromide Administered once daily via the Respimat® Device in Cystic Fibrosis Patients”
“A Randomized, Open-label, Multicenter, Phase 3 Trial to Assess the Safety of Tobramycin Inhalation Powder Compared to TOBI in Cystic Fibrosis Subjects”
“Efficacy and Safety of Intermittent Antimicrobial Therapy for the Treatment of the New Onset Pseudomonas Aeruginosa Airway Infection in Young Patients with Cystic Fibrosis”
“Longitudinal Assessment of Risk Factors/Impact of Pseudomonas Aeruginosa Acquisition and Early Antipseudomonal Treatment in Children with Cystic Fibrosis”
Finally, Dr. Nasr conducts research into the use of high-resolution CT scanning of the chest to monitor the progression of CF lung disease, as well as the response to treatment.
Umadevi Sajjan, Ph.D.
Dr. Sajjan is an independent investigator in the field of host-pathogen interactions. Her research is funded by the National Institutes of Health and Cystic Fibrosis Foundation. Dr. Sajjan’s current research projects include:
Quercetin and Innate Immune Responses in COPD
Quercetin (3,3',4',5,7-pentahydroxyflavone) is the major flavonoid in the human diet. It is found in onions, broccoli, apples, berries and tea, and present in extracts from Ginko biloba and St. Johns Wort, both popular health supplements. Flavonoids share a common chemical structure consisting of two phenol rings linked through three carbons. Quercetin has potent antioxidant effects, combining with free radical species to form considerably less reactive phenoxy radicals. Quercetin also has inhibitory effects on several lipid, protein tyrosine and serine/threonine kinases, including phosphatidylinositol (PI) 3-kinase.
Chronic obstructive pulmonary disease (COPD) is characterized by airways inflammation with goblet cell hyperplasia, irreversible airway obstruction, chronic bacterial infection of the lower airways, reduced mucociliary clearance, emphysema; and impaired innate immunity. Flavonoids with antioxidant and anti-inflammatory properties may influence chronic inflammatory diseases such as COPD. Dietary intake of flavonols (a subclass of flavonoids including quercetin and kaempferol) has been positively associated with pulmonary function (FEV1) and inversely associated with chronic cough and breathlessness in Dutch COPD patients. Further, intake of polyphenol-containing fruit was inversely correlated with 20-yr COPD mortality in three European countries. Together these studies suggest that quercetin and other flavanoids may positively influence outcome in COPD.
We have shown that quercetin, administered by gavage needle, blocks airways hyperresponsiveness and monocyte chemoattractant protein (MCP-1/CCL2) expression in a mouse model of asthma by attenuating signaling through a PI 3-kinase/Akt/nuclear factor (NF)-kB pathway. In the latter study, quercetin also increased airway epithelial cell eukaryotic initiation factor (eIF)-2a phosphorylation, a key event in the antiviral response which limits viral protein synthesis and replication. In addition, results from our pilot studies suggest that quercetin inhibits interleukin (IL)-8/CXCL8 expression from airway epithelial cells and human monocyte-derived macrophages in response to P. aeruginosa infection. Quercetin also decreased invasion of epithelial cells by P. aeruginosa. Quercetin reduced lung inflammation and loss of elastic recoil in lipopolysaccharide (LPS) and elastase-treated mice with physiologic and histologic changes typical of COPD, as well as the inflammatory response of these “COPD” mice to non-typeable H. influenzae. Finally, quercetin inhibited internalization of rhinovirus (RV) in cultured airway epithelial cells and reduced RV-induced neutrophilic inflammation in vivo. Based on these observations, we offer the general hypothesis that quercetin modulates innate immune responses in COPD. To test this hypothesis, we propose the following Specific Aims.
Specific Aim 1: Determine the effects of quercetin on airway inflammation and tissue destruction in a mouse model of COPD. We hypothesize that: 1) quercetin attenuates airways inflammation and emphysematous changes caused by elastase and LPS, a constituent of cigarette smoke; 2) quercetin reduces LPS-induced inflammatory responses in alveolar macrophages by inhibiting recruitment of CD14, extracellular signal regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase to lipid rafts; 3) reduction of LPS-induced tumor necrosis factor (TNF)-a expression, in turn, attenuates epithelial cell expression of C-X-C chemokines and mucin glycoproteins; and 4) quercetin prevents LPS-induced oxidative stress in macrophages and airway epithelial cells.
Specific Aim 2: Determine the effects of quercetin on the innate response to bacterial infection in a mouse model of COPD. We hypothesize that: 1) after infection with H. influenzae or P. aeruginosa, quercetin inhibits lung inflammation in LPS and elastase-treated “COPD mice;” 2) quercetin inhibits Toll-like receptor (TLR)-mediated pro-inflammatory responses in airway epithelial cells; 3) quercetin attenuates PI 3-kinase-dependent bacterial uptake in airway epithelial cells; and 4) quercetin reduces cytotoxic effects by inhibiting bacteria-induced oxidative stress in epithelial cells.
Specific Aim 3: Determine the effects of quercetin on the innate response to rhinoviral infection in a mouse model of COPD. We have developed an animal model of RV infection using RV1B, a minor group RV which binds to the low-density lipoprotein receptor (LDL-R). We hypothesize that: 1) quercetin inhibits neutrophilic airway inflammation in RV-infected LPS- and elastase-treated “COPD mice;” 2) quercetin inhibits internalization of RV in airway epithelial cells; 3) quercetin decreases airway epithelial cell C-X-C chemokine expression; and 4) quercetin increases the expression of interferon (IFN)- in mononuclear cells and increases the phosphorylation of eukaryotic initiation factor (eIF)-2 in airway epithelial cells, thereby reducing viral load.
Understanding the basic mechanisms by which quercetin modulates inflammation in a mouse model of COPD may lead to a new treatment for this devastating disease.
Cooperativity of rhinovirus and P. aeruginosa in CF lung disease
Progressive obstructive lung disease is the major cause of mortality in individuals with cystic fibrosis (CF), with chronic Pseudomonas aeruginosa (PA) infection playing an important role. In addition, intermittent acute viral infections have been associated with declines in lung function, acquisition of new bacterial infections and raised anti-Pseudomonal antibodies, implicating an important role for viruses in the development and progression of lung disease. By a sensitive
polymerase chain reaction method, rhinovirus (RV) was detected in 41-58% of CF patients with virus-related acute respiratory complaints, suggesting that RV is an important cause of CF respiratory exacerbations, as it is in patients with asthma and chronic obstructive pulmonary disease (COPD). Further, though the incidence of viral infections seems to be equal in children with CF and controls, CF children have more episodes of lower airway symptoms with colds, suggesting a greater impact of respiratory viral infections in clinical status of CF patients.
Results from our pilot experiments indicate that CF mice infected with mucoid PA (MPA) followed by RV develop severe lung inflammation and show persistence of both bacteria and virus up to 7 days. Further, welldifferentiated CF cell cultures produce higher amounts of IL-8 in response to co-infection with non-mucoid PA (NPA) and RV, and show higher viral and bacterial loads than cells infected with either RV or PA alone. Finally, RV infection reduces transepithelial resistance and increases transmigration of bacteria across polarized epithelial cells. Accordingly, we offer the general hypothesis that RV and PA cooperate in causing airway inflammation in CF. To address this hypothesis, we propose the following Specific Aims.
1. Determine the combined effects of RV and MPA in vivo. We hypothesize that RV infection of mice with existing MPA lung infection synergistically increases lung inflammation due to increased bacterial and viral load and causes a decline in pulmonary function.
2. Determine the mechanisms by which RV infection increases the pro-inflammatory potential of PA in vitro. We hypothesize that RV with PA-infected CF epithelial cells potentiates production of proinflammatory cytokines by (i) causing dispersal and blooming of planktonic bacteria from biofilm matrix; (ii) increasing planktonic bacterial binding to epithelial cells by increasing exposure or expression of bacterial receptors; and (iii) promote transmigration of bacteria across polarized epithelium by disrupting barrier function.
3. Determine whether prior infection of CF airway epithelial cell cultures with PA increases binding and replication of RV. We hypothesize that infection with PA increases the expression
Co-infections of rhinovirus and bacteria in chronic lung disorders
In patients with chronic obstructive pulmonary disease (COPD), which is characterized by bacterial colonization of the airways, co-infection with respiratory viruses further impairs host defenses, leading to bacterial overgrowth and infection, as well as disease exacerbation.
Our pilot studies indicate that pre-infection of well-differentiated airway epithelial cell cultures with rhinovirus (RV), the virus responsible for most respiratory tract infections, increases binding of both nontypeable Hemophilus influenzae (NTHI) and Pseudomonas aeruginosa (PA), as well as bacteria-induced chemokine expression. Further, we have found that RV infection of polarized airway epithelial cells induces the expression of new receptors for PA. Finally, we have developed two new mouse models which will allow us to test whether pre-infection with RV increases the susceptibility to bacterial infection in vivo. First, inoculation with RV1B, a minor group RV which binds to the low-density lipoprotein receptor, induces neutrophilic airway inflammation and hyperresponsiveness in C57/BL6 mice. Second, we have developed a
murine model of COPD by sequential intranasal treatment with elastase and lipopolysaccharide. Our pilot studies indicate that elastase/LPS-treated “COPD mice” pre-infected with RV are more susceptible to infection with NTHI.
In this project, we propose the general hypothesis that RV increases airway epithelial cell
expression of bacterial receptors, thereby predisposing the epithelium to bacterial infection. To address this, we propose the following Specific Aims:
1. Determine RV-induced changes in the airway epithelial cell membrane that potentiate bacterial adherence and/or internalization. We hypothesize that RV infection of airway epithelial cells increases the expression of new bacterial receptors.
2. Determine the effects of co-infection with RV and bacteria in vivo. We hypothesize that: (i) preinfection of mouse airways with RV1B increases persistence of bacteria in infected mice; (ii) RV infection potentiates bacteria-induced inflammation in normal and elastase/LPS-treated “COPD mice;” and (iii) RV increases the abundance of receptors for bacteria in vivo.
Understanding the basic mechanisms by which viruses predispose the airways to secondary bacterial infection in COPD will improve existing preventive and therapeutic strategies for this disease.