Constance L. Atkins

Hyperresponsiveness to inhaled but not intravenous methacholine during acute respiratory syncytial virus infection in mice

BackgroundTo characterise the acute physiological and inflammatory changes induced by low-dose RSV infection in mice.MethodsBALB/c mice were infected as adults (8 wk) or weanlings (3 wk) with 1 × 105 pfu of RSV A2 or vehicle (intranasal, 30 μl). Inflammation, cytokines and inflammatory markers in bronchoalveolar lavage fluid (BALF) and airway and tissue responses to inhaled methacholine (MCh; 0.001 – 30 mg/ml) were measured 5, 7, 10 and 21 days post infection. Responsiveness to iv MCh (6 – 96 μg/min/kg) in vivo and to electrical field stimulation (EFS) and MCh in vitro were measured at 7 d. Epithelial permeability was measured by Evans Blue dye leakage into BALF at 7 d. Respiratory mechanics were measured using low frequency forced oscillation in tracheostomised and ventilated (450 bpm, flexiVent) mice. Low frequency impedance spectra were calculated (0.5 – 20 Hz) and a model, consisting of an airway compartment [airway resistance (Raw) and inertance (Iaw)] and a constant-phase tissue compartment [coefficients of tissue damping (G) and elastance (H)] was fitted to the data.ResultsInflammation in adult mouse BALF peaked at 7 d (RSV 15.6 (4.7 SE) vs. control 3.7 (0.7) × 104 cells/ml; p < 0.001), resolving by 21 d, with no increase in weanlings at any timepoint. RSV-infected mice were hyperresponsive to aerosolised MCh at 5 and 7 d (PC200 Raw adults: RSV 0.02 (0.005) vs. control 1.1 (0.41) mg/ml; p = 0.003) (PC200 Raw weanlings: RSV 0.19 (0.12) vs. control 10.2 (6.0) mg/ml MCh; p = 0.001). Increased responsiveness to aerosolised MCh was matched by elevated levels of cysLT at 5 d and elevated VEGF and PGE2 at 7 d in BALF from both adult and weanling mice. Responsiveness was not increased in response to iv MCh in vivo or EFS or MCh challenge in vitro. Increased epithelial permeability was not detected at 7 d.ConclusionInfection with 1 × 105 pfu RSV induced extreme hyperresponsiveness to aerosolised MCh during the acute phase of infection in adult and weanling mice. The route-specificity of hyperresponsiveness suggests that epithelial mechanisms were important in determining the physiological effects. Inflammatory changes were dissociated from physiological changes, particularly in weanling mice.

Respiratory syncytial virus infection alters surfactant protein A expression in human pulmonary epithelial cells by reducing translation efficiency

Infection of neonatal lung by respiratory syncytial virus (RSV) is a common cause of respiratory dysfunction. Lung alveolar type II and bronchiolar epithelial (Clara) cells secrete surfactant protein A (SP-A), a collectin that is an important component of the pulmonary innate immune system. SP-A binds to the virus, targeting the infectious agent for clearance by host defense mechanisms. We have previously shown that while the steady-state level of SP-A mRNA increases approximately threefold after RSV infection, steady-state levels of cellular and secreted SP-A protein decrease 40-60% in human type II cells in primary culture, suggesting a mechanism where the virus alters components of the innate immune response in infected cells. In these studies, we find that changes in SP-A mRNA and protein levels in RSV-infected NCI-H441 cells (a bronchiolar epithelial cell line) recapitulate the results in SP-A expression observed in primary lung cells. While SP-A protein is normally ubiquitinated, there is no change in the level of SP-A protein ubiquitination or proteasome activity during RSV infection, suggesting that the reduced levels of SP-A protein are not due to degradation by activated proteasomes. SP-A mRNA is appropriately processed and exported from the nucleus to the cytoplasm during RSV infection. As evidenced by polysome analysis of SP-A mRNA and pulse-chase analysis of newly synthesized SP-A protein, we find a decrease in translational efficiency that is specific for SP-A mRNA. Therefore, the decrease in SP-A protein levels observed after RSV infection of pulmonary bronchiolar epithelial cells results from a mechanism that affects SP-A mRNA translation efficiency.

Oral administration of surfactant protein-a reduces pathology in an experimental model of necrotizing enterocolitis.

Objectives: Necrotizing enterocolitis (NEC) frequently results in significant morbidity and mortality in premature infants. Others reported that mice deficient in pulmonary surfactant protein-A (SP-A) born and raised in a nonhygienic environment succumb to significant gastrointestinal tract pathology, and enteral administration of purified SP-A significantly reduced mortality. We hypothesized that oral administration of purified SP-A can ameliorate pathology in an experimental model of neonatal NEC. Methods: Experimental NEC was induced in newborn Sprague–Dawley rat pups by daily formula gavage and intermittent exposure to hypoxia. Purified human SP-A (5 &mgr;g/day) was administered by oral gavage. After 4 days, surviving pups were sacrificed, and intestinal pathology was assessed by histological examination of distal terminal ileal sections. Intestinal levels of inflammatory cytokines (IL-1&bgr;, IFN-&ggr;, and TNF-&agr;) were assessed by enzyme-linked immunosorbent assay and levels of Toll-like receptor 4 (TLR4) by Western analysis. Results: Sixty-one percent of the gavaged rat pups that survived to day 4 met the criteria for experimental NEC after hypoxia, whereas treatment with SP-A significantly reduced mortality and assessment of NEC. Intestinal levels of proinflammatory cytokines were significantly increased in pups exposed to hypoxia. Administration of SP-A to pups exposed to hypoxia significantly reduced IL-1&bgr; and TNF-&agr; levels, but had little effect on elevated levels of IFN-&ggr;. SP-A treatment of hypoxia-exposed pups significantly reduced expression of intestinal TLR4, key in NEC pathogenesis. Conclusions: In a rat model of experimental neonatal NEC, oral administration of SP-A reduces intestinal levels of proinflammatory cytokines and TLR4 protein and ameliorates adverse outcomes associated with gastrointestinal pathologies.

Functional and immune response to respiratory syncytial virus infection in aged BALB/c mice: A search for genes determining disease severity

ABSTRACT Background. Recent studies have demonstrated that respiratory syncytial virus (RSV) is a significant cause of morbidity and mortality in the elderly. The cellular mechanisms that determine the hosts susceptibility and severity of the disease are not well understood. In this study, we sought a mouse model of human respiratory disease by studying the functional and cellular response to RSV in aged animals. Methods. Aged BALB/c mice (>10 months of age) were infected with human RSV (strain A2) and compared with sham-infected mice. Clinical progress of the illness was monitored by daily assessment of weight changes and mortality. The animals were sacrificed four days postinfection. Lung pathology was obtained and viral titers were measured by plaque assay. Gene expression profiles were studied from lung tissue RNA using gene array. Results. RSV produced significant clinical illness in aged mice as evidenced by a 15% weight loss and a 10% mortality rate. Lung pathology revealed inflammatory changes with a predominance of neutrophils and diffuse alveolar damage. Microarray analysis revealed variable profiles of gene activation/downregulation at day 4 postinfection. RSV infection resulted in a proinflammatory response. Surprisingly, some of the genes involved in antigen-processing pathway were downregulated, specifically, genes implicated in the major histocompatibility complex (MHC) class II pathway. Conclusions. Our findings indicate that RSV infection produces profound functional and cellular changes in aged mice thus resembling the human disease described in the elderly. Further studies will be needed to understand the cellular mechanisms involved in the host response to RSV in aged mice.

Resistin deficiency in mice has no effect on pulmonary responses induced by acute ozone exposure

Acute exposure to ozone (O3), an air pollutant, causes pulmonary inflammation, airway epithelial desquamation, and airway hyperresponsiveness (AHR). Pro-inflammatory cytokines-including IL-6 and ligands of chemokine (C-X-C motif) receptor 2 [keratinocyte chemoattractant (KC) and macrophage inflammatory protein (MIP)-2], TNF receptor 1 and 2 (TNF), and type I IL-1 receptor (IL-1α and IL-1β)-promote these sequelae. Human resistin, a pleiotropic hormone and cytokine, induces expression of IL-1α, IL-1β, IL-6, IL-8 (the human ortholog of murine KC and MIP-2), and TNF. Functional differences exist between human and murine resistin; yet given the aforementioned observations, we hypothesized that murine resistin promotes O3-induced lung pathology by inducing expression of the same inflammatory cytokines as human resistin. Consequently, we examined indexes of O3-induced lung pathology in wild-type and resistin-deficient mice following acute exposure to either filtered room air or O3. In wild-type mice, O3 increased bronchoalveolar lavage fluid (BALF) resistin. Furthermore, O3 increased lung tissue or BALF IL-1α, IL-6, KC, TNF, macrophages, neutrophils, and epithelial cells in wild-type and resistin-deficient mice. With the exception of KC, which was significantly greater in resistin-deficient compared with wild-type mice, no genotype-related differences in the other indexes existed following O3 exposure. O3 caused AHR to acetyl-β-methylcholine chloride (methacholine) in wild-type and resistin-deficient mice. However, genotype-related differences in airway responsiveness to methacholine were nonexistent subsequent to O3 exposure. Taken together, these data demonstrate that murine resistin is increased in the lungs of wild-type mice following acute O3 exposure but does not promote O3-induced lung pathology.

Infant formula alters surfactant protein A (SP-A) and SP-B expression in pulmonary epithelial cells

Surfactant proteins A (SP‐A) and SP‐B are critical in the ability of pulmonary surfactant to reduce alveolar surface tension and provide innate immunity. Aspiration of infant milk formula can lead to lung dysfunction, but direct effects of aspirated formula on surfactant protein expression in pulmonary cells have not been described. The hypothesis that infant formula alters surfactant protein homeostasis was tested in vitro by assessing surfactant protein gene expression in cultured pulmonary epithelial cell lines expressing SP‐A and SP‐B that were transiently exposed (6 hr) to infant formula. Steady‐state levels of SP‐A protein and mRNA and SP‐B mRNA in human bronchiolar (NCI‐H441) and mouse alveolar (MLE15) epithelial cells were reduced in a dose‐dependent manner 18 hr after exposure to infant formula. SP‐A mRNA levels remained reduced 42 hr after exposure, but SP‐B mRNA levels increased 10‐fold. Neither soy formula nor non‐fat dry milk affected steady‐state SP‐A and SP‐B mRNA levels; suggesting a role of a component of infant formula derived from cow milk. These results indicate that infant formula has a direct, dose‐dependent effect to reduce surfactant protein gene expression. Ultimately, milk aspiration may potentially result in a reduced capacity of the lung to defend against environmental insults. Pediatr. Pulmonol. 2011; 46:903–912.

Plasminogen activator inhibitor‐1 does not contribute to the pulmonary pathology induced by acute exposure to ozone

Expression of plasminogen activator inhibitor (PAI)‐1, the major physiological inhibitor of fibrinolysis, is increased in the lung following inhalation of ozone (O3), a gaseous air pollutant. PAI‐1 regulates expression of interleukin (IL)‐6, keratinocyte chemoattractant (KC), and macrophage inflammatory protein (MIP)‐2, which are cytokines that promote lung injury, pulmonary inflammation, and/or airway hyperresponsiveness following acute exposure to O3. Given these observations, we hypothesized that PAI‐1 contributes to the severity of the aforementioned sequelae by regulating expression of IL‐6, KC, and MIP‐2 following acute exposure to O3. To test our hypothesis, wild‐type mice and mice genetically deficient in PAI‐1 (PAI‐1‐deficient mice) were acutely exposed to either filtered room air or O3 (2 ppm) for 3 h. Four and/or twenty‐four hours following cessation of exposure, indices of lung injury [bronchoalveolar lavage fluid (BALF) protein and epithelial cells], pulmonary inflammation (BALF IL‐6, KC, MIP‐2, macrophages, and neutrophils), and airway responsiveness to aerosolized acetyl‐β‐methylcholine chloride (respiratory system resistance) were measured in wild‐type and PAI‐1‐deficient mice. O3 significantly increased indices of lung injury, pulmonary inflammation, and airway responsiveness in wild‐type and PAI‐1‐deficient mice. With the exception of MIP‐2, which was significantly lower in PAI‐1‐deficient as compared to wild‐type mice 24 h following cessation of exposure to O3, no other genotype‐related differences occurred subsequent to O3 exposure. Thus, following acute exposure to O3, PAI‐1 neither regulates pulmonary expression of IL‐6 and KC nor functionally contributes to any of the pulmonary pathological sequelae that arise from the noxious effects of inhaled O3.

Role of prophylactic azithromycin to reduce airway inflammation and mortality in a RSV mouse infection model

Respiratory syncytial virus (RSV) infection is an important cause of morbidity and mortality in vulnerable populations. Macrolides have received considerable attention for their anti‐inflammatory actions beyond their antibacterial effect. We hypothesize that prophylactic azithromycin will be effective in reducing the severity of RSV infection in a mouse model.

Chemokine (C‐C Motif) Receptor‐Like 2 is not essential for lung injury, lung inflammation, or airway hyperresponsiveness induced by acute exposure to ozone

Inhalation of ozone (O3), a gaseous air pollutant, causes lung injury, lung inflammation, and airway hyperresponsiveness. Macrophages, mast cells, and neutrophils contribute to one or more of these sequelae induced by O3. Furthermore, each of these aforementioned cells express chemokine (C‐C motif) receptor‐like 2 (Ccrl2), an atypical chemokine receptor that facilitates leukocyte chemotaxis. Given that Ccrl2 is expressed by cells essential to the development of O3‐induced lung pathology and that chemerin, a Ccrl2 ligand, is increased in bronchoalveolar lavage fluid (BALF) by O3, we hypothesized that Ccrl2 contributes to the development of lung injury, lung inflammation, and airway hyperresponsiveness induced by O3. To that end, we measured indices of lung injury (BALF protein, BALF epithelial cells, and bronchiolar epithelial injury), lung inflammation (BALF cytokines and BALF leukocytes), and airway responsiveness to acetyl‐β‐methylcholine chloride (respiratory system resistance) in wild‐type and mice genetically deficient in Ccrl2 (Ccrl2‐deficient mice) 4 and/or 24 hours following cessation of acute exposure to either filtered room air (air) or O3. In air‐exposed mice, BALF chemerin was greater in Ccrl2‐deficient as compared to wild‐type mice. O3 increased BALF chemerin in mice of both genotypes, yet following O3 exposure, BALF chemerin was greater in Ccrl2‐deficient as compared to wild‐type mice. O3 increased indices of lung injury, lung inflammation, and airway responsiveness. Nevertheless, no indices were different between genotypes following O3 exposure. In conclusion, we demonstrate that Ccrl2 modulates chemerin levels in the epithelial lining fluid of the lungs but does not contribute to the development of O3‐induced lung pathology.

Effects of infant formula on cell homeostasis and cytokine levels in an in vivo and in vitro murine aspiration model

The role of infant formula aspiration in lung injury has not been studied extensively. We evaluated the effects of a single infant formula aspiration into the lungs of mice and the effect of infant formula exposure on cell lines representing murine alveolar macrophages and type II epithelial cells.