Richard Rabold

A Role for Immune Complexes in Enhanced Respiratory Syncytial Virus Disease

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and viral pneumonia in infants and young children. Administration of a formalin inactivated vaccine against RSV to children in the 1960s resulted in increased morbidity and mortality in vaccine recipients who subsequently contracted RSV. This incident precluded development of subunit RSV vaccines for infants for over 30 years, because the mechanism of illness was never clarified. An RSV vaccine for infants is still not available. Here, we demonstrate that enhanced RSV disease is mediated by immune complexes and abrogated in complement component C3 and B cell–deficient mice but not in controls. Further, we show correlation with the enhanced disease observed in children by providing evidence of complement activation in postmortem lung sections from children with enhanced RSV disease.

Particle Effects on Heart-Rate Regulation in Senescent Mice

Because epidemiology studies consistently identify the elderly at risk for air pollution-related morbidity and mortality, we developed a model of senescent-dependent susceptibility based on indices of physiological aging. In the current study, we hypothesized that heart-rate regulation during particulate matter (PM) exposure differs with senescence-dependent susceptibility owing to variation in autonomic nervous control. Heart rate (HR) and heart-rate variability (HRV) parameters were measured from 162 samples of 2-min electrocardiograph (ECG) recordings in age-matched healthy (n = 5) and terminally senescent (n = 3) AKR mice during 3-h exposures to filtered-air (FA, day 1) and carbon black (CB, day 4; < 200 μ g/m3). On day 1, HR was significantly (p <. 01) depressed during FA in terminally senescent mice. By day 4, HR was further slowed significantly (p <. 01) due to the effects of CB exposure for 3 days. The combined effects of terminal senescence and CB exposure acted to depress HR to an average (±SEM) 445 ± 40 bpm, or ∼ 80 bpm lower compared to healthy HR responses. The change in rMSSD, an HRV parameter corresponding to relative influences of parasympathetic tone on HR, was significantly (p <. 01) greater on day 1 and day 4 in terminally senescent mice compared to healthy mice. In contrast, the LF/HF ratio, an HRV parameter derived from spectral analysis indicating relative changes in cardiac sympathetic tone, was significantly (p <. 01) depressed in terminally senescent mice on day 1. By day 4, significant increases in LF/HF were evident in healthy mice during CB exposure, suggesting that HR regulation was associated with an increase in sympathetic tone. Alternatively, terminally senescent mice appeared to modulate a lower HR without change in LF/HF ratio during CB exposure, suggesting an absence of sympathetic tone. In conclusion, older healthy mice increase cardiac sympathetic tone during PM exposure while terminally senescent mice show a greater PM-induced parasympathetic tone in regulating HR. The significance of the current results suggest that PM-induced HR regulatory changes may ultimately depend on the degree of physiological aging.

Effects of leptin deficiency on postnatal lung development in mice

Leptin modulates energy metabolism and lung development. We hypothesize that the effects of leptin on postnatal lung development are volume dependent from 2 to 10 wk of age and are independent of hypometabolism associated with leptin deficiency. To test the hypotheses, effects of leptin deficiency on lung maturation were characterized in age groups of C57BL/6J mice with varying Lep(ob) genotypes. Quasi-static pressure-volume curves and respiratory impedance measurements were performed to profile differences in respiratory system mechanics. Morphometric analysis was conducted to estimate alveolar size and number. Oxygen consumption was measured to assess metabolic rate. Lung volume at 40-cmH(2)O airway pressure (V(40)) increased with age in each genotypic group, and V(40) was significantly (P < 0.05) lower in leptin-deficient (ob/ob) mice beginning at 2 wk. Differences were amplified through 7 wk of age relative to wild-type (+/+) mice. Morphometric analysis showed that alveolar surface area was lower in ob/ob compared with +/+ and heterozygote (ob/+) mice beginning at 2 wk. Unlike the other genotypic groups, alveolar size did not increase with age in ob/ob mice. In another experiment, ob/ob at 4 wk received leptin replacement (5 microg.g(-1) x day(-1)) for 8 days, and expression levels of the Col1a1, Col3a1, Col6a3, Mmp2, Tieg1, and Stat1 genes were significantly increased concomitantly with elevated V(40). Leptin-induced increases in V(40) corresponded with enlarged alveolar size and surface area. Gene expression suggested a remodeling event of lung parenchyma after exogenous leptin replacement. These data support the hypothesis that leptin is critical to postnatal lung remodeling, particularly related to increased V(40) and enlarged alveolar surface area.

Exposure to inhaled particulate matter impairs cardiac function in senescent mice

Daily exposure to particulate matter (PM) is known to adversely affect cardiac function and is also known to be exaggerated with senescence. This study tests the hypothesis that cardiac function is uniquely altered by PM exposure in senescent mice. A mechanism for PM-induced cardiac effects is also postulated by examining the activity of nitric oxide synthase (NOS) and the generation of reactive oxygen species (ROS) in heart tissue. Echocardiography is performed in awake 18- and 28-mo-old mice at baseline and immediately following 3-h exposures to either filtered air or carbon black (CB; approximately 400 microg/m3) on 4 days. At 28 mo, left ventricular diameter at end-systole and end-diastole is significantly (P < 0.05) elevated, and fractional shortening is significantly reduced (49 +/- 3% vs. 56 +/- 3%) with CB exposure. In vivo hemodynamic measurements at 28 mo also demonstrate significant (P < 0.05) reductions in ejection fraction and increases in right ventricular and pulmonary vascular pressures following CB exposure. Functional changes at 28 mo are associated with increased ROS production as suggested by enhanced luminol activity. This elevated ROS production with aging and CB exposure is attributable to NOS uncoupling. Measurements of natriuretic peptide (atrial and brain) transcription and matrix metalloproteinase (MMP2 and MMP9) activity in heart tissue are significantly (P < 0.05) amplified with senescence and exposure to CB, pointing to increased cardiac stress and remodeling. These results demonstrate that acute PM exposure reduces cardiac contractility in senescent mice, and this decline in function is associated with increased ROS production linked to NOS uncoupling.

Adverse cardiovascular effects with acute particulate matter and ozone exposures: interstrain variation in mice.

Objectives Increased ambient particulate matter (PM) levels are associated with cardiovascular morbidity and mortality, as shown by numerous epidemiology studies. Few studies have investigated the role of copollutants, such as ozone, in this association. Furthermore, the mechanisms by which PM affects cardiac function remain uncertain. We hypothesized that PM and O3 induce adverse cardiovascular effects in mice and that these effects are strain dependent. Study design After implanting radiotelemeters to measure heart rate (HR) and HR variability (HRV) parameters, we exposed C57Bl/6J (B6), C3H/HeJ (HeJ), and C3H/HeOuJ (OuJ) inbred mouse strains to three different daily exposures of filtered air (FA), carbon black particles (CB), or O3 and CB sequentially [O3CB; for CB, 536 ± 24 μg/m3; for O3, 584 ± 35 ppb (mean ± SE)]. Results We observed significant changes in HR and HRV in all strains due to O3CB exposure, but not due to sequential FA and CB exposure (FACB). The data suggest that primarily acute HR and HRV effects occur during O3CB exposure, especially in HeJ and OuJ mice. For example, HeJ and OuJ mice demonstrated dramatic increases in HRV parameters associated with marked brady-cardia during O3CB exposure. In contrast, depressed HR responses occurred in B6 mice without detectable changes in HRV parameters. Conclusions These findings demonstrate that important interstrain differences exist with respect to PM- and O3-induced cardiac effects. This interstrain variation suggests that genetic factors may modulate HR regulation in response to and recuperation from acute copollutant exposures.

Alterations of the lung methylome in allergic airway hyper‐responsiveness

Asthma is a chronic airway disorder characterized by recurrent attacks of breathlessness and wheezing, affecting 300 million people around the world (available at: www.who.int). To date, genetic factors associated with asthma susceptibility have been unable to explain the full etiology of asthma. Recent studies have demonstrated that the epigenetic disruption of gene expression plays an equally important role in the development of asthma through interaction with our environment. We sensitized 6‐week‐old C57BL/6J mice with house‐dust‐mite (HDM) extracts intraperitoneally followed by 5 weeks of exposure to HDM challenges (three times a week) intratracheally. HDM‐exposed mice showed an increase in airway hyper‐responsiveness (AHR) and inflammation together with structural remodeling of the airways. We applied methylated DNA immunoprecipitation‐next generation sequencing (MeDIP‐seq) for profiling of DNA methylation changes in the lungs in response to HDM. We observed about 20 million reads by a single‐run of massive parallel sequencing. We performed bioinformatics and pathway analysis on the raw sequencing data to identify differentially methylated candidate genes in HDM‐exposed mice. Specifically, we have revealed that the transforming growth factor beta signaling pathway is epigenetically modulated by chronic exposure to HDM. Here, we demonstrated that a specific allergen may play a role in AHR through an epigenetic mechanism by disrupting the expression of genes in lungs that might be involved in airway inflammation and remodeling. Our findings provide new insights into the potential mechanisms by which environmental allergens induce allergic asthma and such insights may assist in the development of novel preventive and therapeutic options for this debilitative disease. Environ. Mol. Mutagen. 55:244–255, 2014.

C5 Modulates Airway Hyperreactivity and Pulmonary Eosinophilia during Enhanced Respiratory Syncytial Virus Disease by Decreasing C3a Receptor Expression

ABSTRACT Enhanced respiratory syncytial virus disease, a serious pulmonary disorder that affected recipients of an inactivated vaccine against respiratory syncytial virus in the 1960s, has delayed the development of vaccines against the virus. The enhanced disease was characterized by immune complex-mediated airway hyperreactivity and a severe pneumonia associated with pulmonary eosinophilia. In this paper, we show that complement factors contribute to enhanced-disease phenotypes. Mice with a targeted disruption of complement component C5 affected by the enhanced disease displayed enhanced airway reactivity, lung eosinophilia, and mucus production compared to wild-type mice and C5-deficient mice reconstituted with C5. C3aR expression in bronchial epithelial and smooth muscle cells in the lungs of C5-deficient mice was enhanced compared to that in wild-type and reconstituted rodents. Treatment of C5-deficient mice with a C3aR antagonist significantly attenuated airway reactivity, eosinophilia, and mucus production. These results indicate that C5 plays a crucial role in modulating the enhanced-disease phenotype, by affecting expression of C3aR in the lungs. These findings reveal a novel autoregulatory mechanism for the complement cascade that affects the innate and adaptive immune responses.

Variation in Heart Rate Regulation and the Effects of Particle Exposure in Inbred Mice

Altered autonomic control of heart rate (HR) rhythm during exposure to particulate matter (PM) has been suggested in human and animal studies. Our lab has shown strain variation in HR regulation between quiescent C3H/HeJ (C3) and C57BL/6J (B6) mice: that is, C3 mice show a consistently higher HR by ∼ 80 bpm compared with B6 mice during a normal 24-h circadian cycle. In the current study, we hypothesize that the balance between sympathetic and parasympathetic control of HR during PM exposure varies between C3 and B6 mice. Radiotelemeters were implanted in C3 and B6 mice to measure HR responses and HR variability (HRV) parameters during successive 3-h exposures to filtered air (FA) or carbon black (CB, < 300 μg/m3). Exposures were repeated following administration of saline or parasympathetic (PS; atropine, 0.5 mg/kg ip) and sympathetic (S; propranolol, 1 mg/kg ip) blockade to study the autonomic regulation of HR during CB exposure. During FA exposure with saline, a significantly (p < .05) greater 3-h average HR response (bpm ± SEM) occurred in C3 compared with B6 mice (496 ± 22 vs. 427 ± 3). With PS blockade, the strain difference between C3 and B6 mice was not evident (485 ± 23 vs. 503 ± 61). With S blockade, the 3-h average HR responses for C3 mice were significantly (p < .05) reduced compared with saline (413 ± 18 vs. 392 ± 15 for B6). During CB exposure with saline, HR responses were again significantly (p < 0.05) elevated in C3 compared with B6 mice, but these HR responses were not different relative to FA exposure. With S blockade, HR was significantly (p < .05) elevated in B6 mice during CB relative to FA, but was unchanged in C3 mice. Collectively, these results suggest that strain variation in HR regulation is due to a robust PS tone evident in B6 mice and a predominant S tone in C3 mice. Furthermore, CB exposure alters HR regulation in B6 mice by modulating a withdrawal of PS tone. Finally, strain variation in HR between B6 and C3 mice in responding to acute PM exposure implies that robust genetic determinants modulate altered autonomic regulation in susceptible individuals.

Epigenetic Alterations by DNA Methylation in House Dust Mite–Induced Airway Hyperresponsiveness

Asthma is one of the most prevalent chronic lung diseases, affecting 235 million individuals around the world, with its related morbidity and mortality increasing steadily over the last 20 years. Exposure to the environmental allergen, house dust mite (HDM), results in airway inflammation with a variable degree of airway obstruction. Although there has been much experimental work in the past using HDM challenge models to understand mechanistic details in allergic inflammation and airway hyperresponsiveness (AHR), there has been no study on reprogramming of lung or airways mediated through epigenetic mechanisms in response to an acute HDM exposure. Male mice, 6 weeks of age, were administrated HDM extracts or saline at Days 1, 14, and 21. Exposure of mice to HDM extracts caused significant airway inflammation and increased AHR. These HDM-challenged mice also exhibited a change in global DNA methylation as compared with saline-exposed (control) mice. Next, by employing methylation-sensitive restriction fingerprinting, we identified a set of genes, showing aberrant methylation status, associated with the HDM-induced AHR. These candidate genes are known to be involved in cAMP signaling (pde4 d), Akt-signaling (akt1 s1), ion transport (tm6 sf1, pom121l2, and slc8a3), and fatty acid metabolism (acsl3). Slc8a3 and acsl3 were down-regulated, whereas pde4 d, akt1 s1, tm6 sf1, and pom121l2 were up-regulated in the mice exposed to HDM. Hence, our results suggest that HDM exposure induces a series of aberrant methylated genes that are potentially important for the development of allergic AHR.

Age-related changes in cardiac and respiratory adaptation to acute ozone and carbon black exposures: Interstrain variation in mice

Context: Epidemiological studies show positive associations between increased ambient air pollutant levels and adverse cardiopulmonary effects. These studies suggest that the elderly and those with certain genetic polymorphisms are susceptible to adverse air pollution-associated health events. Hypothesis/objective: We hypothesize that physiological responses to air pollutants vary with age and are genetically influenced. Materials and methods: To test this hypothesis, we exposed mice from three inbred strains (C57BL/6J, B6; C3H/HeJ, HeJ; C3H/HeOuJ, OuJ) to ozone (O3) and carbon black (CB) at two ages, (5 months, 12 months), for 3 consecutive days, to either filtered air (FA), CB particles, or O3 and CB sequentially (O3CB) (CB, 550 µg/m3; O3, 600 ppb). Heart rate (HR), HR variability (HRV), breathing, and core temperature (Tco) responses were analyzed. Results: We observed time-dependent physiological changes in response to O3CB exposure in each strain, relative to FA exposure for both age groups. Each mouse strain showed distinct adaptation profiles to repeated acute exposures to O3. In younger mice, several time-dependent effects (decreased HR and increased HRV) were prominent in HeJ and OuJ mice but not B6 mice. We also observed variability in adaptation in older mice. However, responses in older mice were generally attenuated when compared to the younger mice. In addition, cardiac–respiratory interactions were affected with CB and O3CB exposures albeit with patterns differing by age or exposure. Discussion/conclusion: Our results suggest that age considerably attenuates physiological responses to O3 and O3CB exposures. Age-related physiological changes such as increased oxidative stress in mouse tissue may be involved in this attenuation.