hypoxia; leukocyte larceny; red blood cell; white blood cell; platelet; pseudohypoxemia
Ambient ozone has a significant impact on human health. We have made considerable progress in understanding the fundamental mechanisms that regulate the biological response to ozone. It is increasingly clear that genes of innate immunity play a central role in both infectious and non-infectious lung disease. The biological response to ambient ozone provides a clinically relevant environmental exposure that allows us to better understand the role of innate immunity in non-infectious airways disease. In this brief review, we focus on: (1) specific cell types in the lung modified by ozone; (2) ozone and oxidative stress; (3) the relationship between genes of innate immunity and ozone; (4) the role of extracellular matrix in reactive airways disease; and (5) the effect of ozone on the adaptive immune system. We summarize recent advances in understanding the mechanisms that ozone contributes to environmental airways disease.
ozone; oxidative stress; innate immunity; environment; surfactant; toll-like receptor; asthma; extracellular matrix; mindin; hyaluronan
The posttranscriptional mechanisms by which RNA binding proteins (RBPs) regulate T-cell differentiation and cytokine production in vivo remain unclear. The RBP HuR binds to labile mRNAs, usually leading to increases in mRNA stability and/or translation. Previous work demonstrated that HuR binds to the mRNAs encoding the Th2 transcription factor trans-acting T-cell–specific transcription factor (GATA-3) and Th2 cytokines interleukin (IL)-4 and IL-13, thereby regulating their expression. By using a novel conditional HuR knockout (KO) mouse in which HuR is deleted in activated T cells, we show that Th2-polarized cells from heterozygous HuR conditional (OX40-Cre HuRfl/+) KO mice had decreased steady-state levels of Gata3, Il4 and Il13 mRNAs with little changes at the protein level. Surprisingly, Th2-polarized cells from homozygous HuR conditional (OX40-Cre HuRfl/fl) KO mice showed increased Il2, Il4 and Il13 mRNA and protein via different mechanisms. Specifically, Il4 was transcriptionally upregulated in HuR KO T cells, whereas Il2 and Il13 mRNA stabilities increased. Additionally, when using the standard ovalbumin model of allergic airway inflammation, HuR conditional KO mice mounted a robust inflammatory response similar to mice with wild-type HuR levels. These results reveal a complex differential posttranscriptional regulation of cytokines by HuR in which gene dosage plays an important role. These findings may have significant implications in allergies and asthma, as well as autoimmune diseases and infection.
Ambient ozone is a criteria air pollutant that impacts both human morbidity and mortality. The effect of ozone inhalation includes both toxicity to lung tissue and alteration of the host immunologic response. The innate immune system facilitates immediate recognition of both foreign pathogens and tissue damage. Emerging evidence supports that ozone can modify the host innate immune response and that this response to inhaled ozone is dependent on genes of innate immunity. Improved understanding of the complex interaction between environmental ozone and host innate immunity will provide fundamental insight into the pathogenesis of inflammatory airways disease. We review the current evidence supporting that environmental ozone inhalation: (1) modifies cell types required for intact innate immunity, (2) is partially dependent on genes of innate immunity, (3) primes pulmonary innate immune responses to LPS, and (4) contributes to innate-adaptive immune system cross-talk.
environmental; toll-like receptor; TLR4; CD44; hyaluronan; asthma; genetic; gene x environment
Severe respiratory failure is a well recognized complication of pandemic H1N1 influenza infection. Limited data regarding the efficacy of rescue therapies including high frequency oscillatory ventilation (HFOV) and extracorporeal membranous oxygenation (ECMO) have been previously reported in the setting of H1N1 influenza infection in the United States.
Retrospective, single center cohort study.
Pediatric, cardiac, surgical, and medical intensive care units in a single tertiary care center in the United States.
127 consecutive patients with confirmed Influenza A infection requiring hospitalization between April 1, 2009 and October 31, 2009.
Electronic medical records were reviewed for demographic and clinical data.
Measurements and main results
The number of ICU admissions appears inversely related to age with 69% of admissions less than 20 years of age. Median duration of ICU care was 10.0 days [4.0, 24.0], and median duration of mechanical ventilation was 8.0 days [0.0, 23.5]. Rescue therapy (HFOV or ECMO) was utilized in 36% (12/33) of ICU patients. The severity of respiratory impairment was determined by PaO2/FiO2 ratio (P/F) and oxygenation index (OI). HFOV at 24-hours resulted in improvements in median P/F (71 [58, 93] vs. 145 [126, 185]; P<0.001), OI (27 [20, 30] vs. 18 [12, 25]; P=0.016), and FiO2 (100 [70, 100] vs. 45 [40, 55]; P<0.001). ECMO resulted in anticipated improvement in parameters of oxygenation at both 2- hours and 24-hours after initiation of therapy. Despite the severity of oxygenation impairment, overall survival for both rescue therapies was 75% (9/12), 80% (4/5) for HFOV alone, and 71% (5/7) for HFOV + ECMO.
In critically ill adult and pediatric patients with H1N1 infection and severe lung injury, the utilization of HFOV and ECMO can result in significant improvements in P/F ratio, OI, and FiO2. However, the impact on mortality is less certain.
influenza; acute respiratory distress syndrome; H1N1; mechanical ventilation; high frequency oscillatory ventilation; extracorporeal membrane oxygenation (ECMO); hypoxemia; respiratory failure; lung injury
Hyaluronan is a high molecular weight component of pulmonary extracelluar matrix and lung injury can generate low molecular weight hyaluronan fragment (HA) that functions as endogenous ligand to cell surface receptors CD44 and toll like receptor 4 (TLR4). This leads to activation of intracellular NFκB signaling and pro-inflammatory cytokine production. Based on previous information that ozone exposure causes increased HA in bronchial alveolar lavage fluid (BALF) and ozone pre-exposure primes immune response to inhaled LPS, we hypothesized that HA production during ozone exposure augments the inflammatory response to LPS. We demonstrate that acute ozone exposure at 1ppm for 3 hours primes the immune response to low dose aerosolized LPS in C57BL/6J mice, resulting in increased neutrophil recruitment into the airspaces, increased levels of protein and pro-inflammatory cytokines in the BALF, and increased airway hyperresponsiveness (AHR). Intratracheal instillation of endotoxin-free HA (25 μg) enhances the biological response to inhaled LPS in a manner similar to ozone pre-exposure. In vitro studies using bone marrow-derived macrophages indicate that HA enhances LPS responses measured by TNFα production while immunofluorescence staining of murine alveolar macrophages demonstrates that HA induces TLR4 peripheralization and lipid raft co-localization. Collectively, our observations support that ozone primes macrophage responsiveness to low dose LPS, in part, due to hyaluronan-induced TLR4 peripheralization in lung macrophages.
environmental; ozone; lipopolysaccharide; hyaluronan; TLR4; toll-like receptor; innate immunity; macrophage; lung injury
Asthma remains an important cause of morbidity and mortality with an incidence that continues to rise. Despite the importance of this disease, the mechanisms by which the host develops allergic airways disease remain poorly understood. The development of allergic airways disease appears to be contingent on activation of both the innate and adaptive immune system, but little is known about the cross-talk between these two systems. The extracellular matrix protein mindin (Spondin 2) has been previously demonstrated to have functional roles in both the innate and adaptive immunological responses. Previous work supports that pulmonary challenge with fungal-associated allergenic proteinase (FAP) induces an innate allergic response. We hypothesized that mindin would modify the biological response to FAP. Saline or FAP was administered by oropharyngeal aspiration to C57BL/6 wild type or mindin-null mice every 4 days for a total of five exposures. FAP exposed C57BL/6 mice developed enhanced airway hyperresponsiveness (AHR) to methacholine challenge and increased neutrophils and eosinophils in the bronchoalveolar lavage as compared to saline exposed controls. These responses were significantly reduced in mindin-null mice exposed to FAP. FAP challenge was associated with a broad induction of cytokines (IL-1β, TNFα, Th1, Th2, and IL-17), chemokines, and growth factors, which were reduced in mindin-null mice exposed to FAP. RNA expression in lung monocytes for representative M1 and M2 activation markers were increased by FAP, but were independent of mindin. Our observations support that challenge with FAP results in activation of both innate and adaptive immune signaling pathways in a manner partially dependent on mindin. These findings suggest a potential role for the extracellular matrix protein mindin in cross-talk between the innate and adaptive immune systems.
Environment; Asthma; Reactive airways disease; Extracellular matrix; Allergy; Aspergillus
Emphysema is currently a leading cause of mortality with no known effective therapy to attenuate progressive loss of lung function. Previous work support that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is protective to the lung through induction of hundreds of antioxidant genes. In models of lung injury, the expression of NAD(P)H:quinine oxidoreductase 1 (NQO1) is upregulated in a manner dependent on Nrf2 and human emphysema is associated with reduced levels of NQO1. However, the functional role of NQO1 in emphysema remains unknown. In this study, we demonstrate the protective role of NQO1 in the development of emphysema using mouse models. NQO1 deficient animals demonstrate premature age-related emphysema and were more susceptible to both elastase and inhaled lipopolysaccharide (LPS) models of emphysema. The absence of NQO1 was associated with enhanced markers of oxidant stress. Treatment of NQO1 deficient animals with the antioxidant N-acetyl cysteine reversed the NQO1-dependent emphysematous changes. In vitro studies utilizing either inhibition or induction of NQO1 demonstrate a potent antioxidant role of NQO1 in macrophages, suggesting a role of macrophage-derived oxidants in the pathogenesis of emphysema. These novel findings support a functional role of NQO1 in protecting the lung from development of emphysema.
Rationale: Obesity is associated with increased prevalence and severity of asthma. Adipose tissue macrophages can contribute to the systemic proinflammatory state associated with obesity. However, it remains unknown whether alveolar macrophages have a unique phenotype in overweight/obese patients with asthma.
Objectives: We hypothesized that leptin levels would be increased in the bronchoalveolar lavage fluid from overweight/obese subjects and, furthermore, that leptin would alter the response of alveolar macrophages to bacterial LPS.
Methods: Forty-two subjects with asthma and 46 healthy control subjects underwent research bronchoscopy. Bronchoalveolar lavage fluid from 66 was analyzed for the level of cellular inflammation, cytokines, and soluble leptin. Cultured primary macrophages from 22 subjects were exposed to LPS, leptin, or leptin plus LPS. Cytokines were measured in the supernatants.
Measurements and Main Results: Leptin levels were increased in overweight/obese subjects, regardless of asthma status (P = 0.013), but were significantly higher in overweight/obese subjects with asthma. Observed levels of tumor necrosis factor-α were highest in overweight/obese subjects with asthma. Ex vivo studies of primary alveolar macrophages indicated that the response to LPS was most robust in alveolar macrophages from overweight/obese subjects with asthma and that preexposure to high-dose leptin enhanced the proinflammatory response. Leptin alone was sufficient to induce production of proinflammatory cytokines from macrophages derived from overweight/obese subjects with asthma.
Conclusions: Ex vivo studies indicate that alveolar macrophages derived from overweight/obese subjects with asthma are uniquely sensitive to leptin. This macrophage phenotype, in the context of higher levels of soluble leptin, may contribute to the pathogenesis of airway disease associated with obesity.
tumor necrosis factor-α; leptin; innate immunity; lipopolysaccharide; environmental lung disease
Inhalation of ambient ozone alters populations of lung macrophages. However, the impact of altered lung macrophage populations on the pathobiology of ozone is poorly understood. We hypothesized that sub-populations of macrophages modulate the response to ozone. We exposed C57BL/6 mice to ozone (2 ppm × 3h) or filtered air. 24 h after the exposure, the lungs were harvested and digested and the cells underwent flow cytometry. Analysis revealed a novel macrophage subset present in ozone exposed mice, which were distinct from resident alveolar macrophages (AM) and identified by enhanced Gr-1+ expression (Gr-1 Macs). Further analysis identified that Gr-1+ Macs exhibited high expression of MARCO, CX3CR1, and NQO1. Gr-1+ Macs were present in the absence of CCR2, suggesting that they were not derived from a CCR2-dependent circulating intermediate. Using PKH26-PCL to label resident phagocytic cells, we demonstrated that Gr-1 Macs were derived from resident lung cells. This new subset was diminished in the absence of CX3CR1. Interestingly, CX3CR1-null mice exhibited enhanced responses to ozone, including increased airway hyperresponsiveness (AHR), exacerbated neutrophil influx, accumulation of 8-isoprostanes and protein carbonyls, and increased expression of cytokines (CXCL2, IL-1β, IL-6, CCL2, and TNF-α). Our results identify a novel subset of lung macrophages, which are derived from a resident intermediate, dependent upon CX3CR1, and appear to protect the host from the biological response to ozone.
Rationale: Previously, we demonstrated a candidate region for susceptibility to airspace enlargement on mouse chromosome 5. However, the specific candidate genes within this region accounting for emphysema-like changes remain unrecognized. c-Kit is a receptor tyrosine kinase within this candidate gene region that has previously been recognized to contribute to the survival, proliferation, and differentiation of hematopoietic stem cells. Increases in the percentage of cells expressing c-Kit have previously been associated with protection against injury-induced emphysema.
Objectives: Determine whether genetic variants of c-Kit are associated with spontaneous airspace enlargement.
Methods: Perform single-nucleotide polymorphism association studies in the mouse strains at the extremes of airspace enlargement phenotype for variants in c-Kit tyrosine kinase. Characterize mice bearing functional variants of c-Kit compared with wild-type controls for the development of spontaneous airspace enlargement. Epithelial cell proliferation was measured in culture.
Measurements and Main Results: Upstream regulatory single-nucleotide polymorphisms in the divergent mouse strains were associated with the lung compliance difference observed between the extreme strains. c-Kit mutant mice (KitW-sh/W-sh), when compared with genetic controls, developed altered lung histology, increased total lung capacity, increased residual volume, and increased lung compliance that persist into adulthood. c-Kit inhibition with imatinib attenuated in vitro proliferation of cells expressing epithelial cell adhesion molecule.
Conclusions: Our findings indicate that c-Kit sustains and/or maintains normal alveolar architecture in the lungs of mice. In vitro data suggest that c-Kit can regulate epithelial cell clonal expansion. The precise mechanisms that c-Kit contributes to the development of airspace enlargement and increased lung compliance remain unclear and warrants further investigation.
genetic; tyrosine kinase; SASH; chronic obstructive pulmonary disease; aging
Background: The role of the Nlrp3 inflammasome in nonallergic airway hyperresponsiveness (AHR) has not previously been reported. Recent evidence supports both interleukin (IL) 1β and short fragments of hyaluronan (HA) as contributors to the biological response to inhaled ozone.
Objective: Because extracellular secretion of IL-1β requires activation of the inflammasome, we investigated the role of the inflammasome proteins ASC, caspase1, and Nlrp3 in the biological response to ozone and HA.
Methods: C57BL/6J wild-type mice and mice deficient in ASC, caspase1, or Nlrp3 were exposed to ozone (1 ppm for 3 hr) or HA followed by analysis of airway resistance, cellular inflammation, and total protein and cytokines in bronchoalveolar lavage fluid (BALF). Transcription levels of IL-1β and IL-18 were determined in two populations of lung macrophages. In addition, we examined levels of cleaved caspase1 and cleaved IL-1β as markers of inflammasome activation in isolated alveolar macrophages harvested from BALF from HA-treated mice.
Results: We observed that genes of the Nlrp3 inflammasome were required for development of AHR following exposure to either ozone or HA fragments. These genes are partially required for the cellular inflammatory response to ozone. The expression of IL-1β mRNA in alveolar macrophages was up-regulated after either ozone or HA challenge and was not dependent on the Nlrp3 inflammasome. However, soluble levels of IL-1β protein were dependent on the inflammasome after challenge with either ozone or HA. HA challenge resulted in cleavage of macrophage-derived caspase1 and IL-1β, suggesting a role for alveolar macrophages in Nlrp3-dependent AHR.
Conclusions: The Nlrp3 inflammasome is required for the development of ozone-induced reactive airways disease.
asthma; environment; extracellular matrix; innate immunity; ozone; toll-like receptor
Rationale: Ozone is a common environmental air pollutant that contributes to hospitalizations for respiratory illness. The mechanisms, which regulate ozone-induced airway hyperresponsiveness, remain poorly understood. We have previously reported that toll-like receptor 4 (TLR4)–deficient animals are protected against ozone-induced airway hyperresponsiveness (AHR) and that hyaluronan (HA) mediates ozone-induced AHR. However, the relation between TLR4 and hyaluronan in the airway response to ozone remains unexplored.
Objectives: We hypothesized that HA acts as an endogenous TLR4 ligand for the development of AHR after ozone-induced environmental airway injury.
Methods: TLR4-deficient and wild-type C57BL/6 mice were exposed to either inhaled ozone or intratracheal HA and the inflammatory and AHR response was measured.
Measurements and Main Results: TLR4-deficient mice have similar levels of cellular inflammation, lung injury, and soluble HA levels as those of C57BL/6 mice after inhaled ozone exposure. However, TLR4-deficient mice are partially protected from AHR after ozone exposure as well as after direct intratracheal instillation of endotoxin-free low molecular weight HA. Similar patterns of TLR4-dependent cytokines were observed in the bronchial alveolar lavage fluid after exposure to either ozone or HA. Exposure to ozone increased immunohistological staining of TLR4 on lung macrophages. Furthermore, in vitro HA exposure of bone marrow–derived macrophages induced NF-κB and production of a similar pattern of proinflammatory cytokines in a manner dependent on TLR4.
Conclusions: Our observations support the observation that extracellular matrix HA contributes to ozone-induced airways disease. Furthermore, our results support that TLR4 contributes to the biological response to HA by mediating both the production of proinflammatory cytokines and the development of ozone-induced AHR.
environmental airways injury; asthma; toll-like receptor; macrophage; TNF-α
Background: Exposure to ozone has been associated with adverse health effects, including premature mortality and cardiopulmonary and respiratory morbidity. In 2008, the U.S. Environmental Protection Agency (EPA) lowered the primary (health-based) National Ambient Air Quality Standard (NAAQS) for ozone to 75 ppb, expressed as the fourth-highest daily maximum 8-hr average over a 24-hr period. Based on recent monitoring data, U.S. ozone levels still exceed this standard in numerous locations, resulting in avoidable adverse health consequences.
Objectives: We sought to quantify the potential human health benefits from achieving the current primary NAAQS standard of 75 ppb and two alternative standard levels, 70 and 60 ppb, which represent the range recommended by the U.S. EPA Clean Air Scientific Advisory Committee (CASAC).
Methods: We applied health impact assessment methodology to estimate numbers of deaths and other adverse health outcomes that would have been avoided during 2005, 2006, and 2007 if the current (or lower) NAAQS ozone standards had been met. Estimated reductions in ozone concentrations were interpolated according to geographic area and year, and concentration–response functions were obtained or derived from the epidemiological literature.
Results: We estimated that annual numbers of avoided ozone-related premature deaths would have ranged from 1,410 to 2,480 at 75 ppb to 2,450 to 4,130 at 70 ppb, and 5,210 to 7,990 at 60 ppb. Acute respiratory symptoms would have been reduced by 3 million cases and school-loss days by 1 million cases annually if the current 75-ppb standard had been attained. Substantially greater health benefits would have resulted if the CASAC-recommended range of standards (70–60 ppb) had been met.
Conclusions: Attaining a more stringent primary ozone standard would significantly reduce ozone-related premature mortality and morbidity.
health benefits; health impact assessment; ozone; standards
Acute exacerbations of pulmonary fibrosis are characterized by rapid decrements in lung function. Environmental factors that may contribute to acute exacerbations remain poorly understood. We have previously demonstrated that exposure to inhaled lipopolysaccharide (LPS) induces expression of genes associated with fibrosis. To address whether exposure to LPS could exacerbate fibrosis, we exposed male C57BL/6 mice to crystalline silica, or vehicle, followed 28 days later by LPS or saline inhalation. We observed that mice receiving both silica and LPS had significantly more total inflammatory cells, more whole lung lavage MCP-1, MIP-2, KC and IL-1β, more evidence of oxidative stress and more total lung hydroxyproline than mice receiving either LPS alone, or silica alone. Blocking oxidative stress with N-acetylcysteine attenuated whole lung inflammation but had no effect on total lung hydroxyproline. These observations suggest that exposure to innate immune stimuli, such as LPS in the environment, may exacerbate stable pulmonary fibrosis via mechanisms that are independent of inflammation and oxidative stress.
Surfactant protein-A (SP-A) has well-established functions in reducing bacterial and viral infections but its role in chronic lung diseases such as asthma is unclear. Mycoplasma pneumoniae (Mp) frequently colonizes the airways of chronic asthmatics and is thought to contribute to exacerbations of asthma. Our lab has previously reported that during Mp infection of non-allergic airways, SP-A aides in maintaining airway homeostasis by inhibiting an overzealous TNF-alpha mediated response and, in allergic mice, SP-A regulates eosinophilic infiltration and inflammation of the airway. In the current study, we used an in vivo model with wild type (WT) and SP-A−/− allergic mice challenged with the model antigen ovalbumin (Ova) that were concurrently infected with Mp (Ova+Mp) to test the hypothesis that SP-A ameliorates Mp-induced stimulation of eosinophils. Thus, SP-A could protect allergic airways from injury due to release of eosinophil inflammatory products. SP-A deficient mice exhibit significant increases in inflammatory cells, mucus production and lung damage during concurrent allergic airway disease and infection (Ova+Mp) as compared to the WT mice of the same treatment group. In contrast, SP-A deficient mice have significantly decreased Mp burden compared to WT mice. The eosinophil specific factor, eosinophil peroxidase (EPO), which has been implicated in pathogen killing and also in epithelial dysfunction due to oxidative damage of resident lung proteins, is enhanced in samples from allergic/infected SP-A−/− mice as compared to WT mice. In vitro experiments using purified eosinophils and human SP-A suggest that SP-A limits the release of EPO from Mp-stimulated eosinophils thereby reducing their killing capacity. These findings are the first to demonstrate that although SP-A interferes with eosinophil-mediated biologic clearance of Mp by mediating the interaction of Mp with eosinophils, SP-A simultaneously benefits the airway by limiting inflammation and damage.
Asthma is a complex heritable disease that is increasing in prevalence and severity, particularly in developed countries such as the United States, where 11% of the population is affected. The contribution of environmental and genetic factors to this growing epidemic is currently not well understood. We developed the hypothesis, based on previous literature, that changes in DNA methylation resulting in aberrant gene transcription may enhance the risk of developing allergic airway disease. Our findings indicate that in mice, a maternal diet supplemented with methyl donors enhanced the severity of allergic airway disease that was inherited transgenerationally. Using a genomic approach, we discovered 82 gene-associated loci that were differentially methylated after in utero supplementation with a methyl-rich diet. These methylation changes were associated with decreased transcriptional activity and increased disease severity. Runt-related transcription factor 3 (Runx3), a gene known to negatively regulate allergic airway disease, was found to be excessively methylated, and Runx3 mRNA and protein levels were suppressed in progeny exposed in utero to a high-methylation diet. Moreover, treatment with a demethylating agent increased Runx3 gene transcription, further supporting our claim that a methyl-rich diet can affect methylation status and consequent transcriptional regulation. Our findings indicate that dietary factors can modify the heritable risk of allergic airway disease through epigenetic mechanisms during a vulnerable period of fetal development in mice.
Ambient ozone (O3) is a commonly encountered environmental air pollutant with considerable impact on public health. Many other inhaled environmental toxicants can substantially affect pulmonary immune responses. Therefore, it is of considerable interest to better understand the complex interaction between environmental airway irritants and immunologically based human disease. The innate immune system represents the first line of defense against microbial pathogens. Intact innate immunity requires maintenance of an intact barrier to interface with the external environment, effective phagocytosis of microbial pathogens, and precise detection of pathogen-associated molecular patterns. We use ambient O3 as a model to highlight the importance of understanding the role of exposure to ubiquitous air toxins and regulation of basic immune function. Inhalation of O3 is associated with impaired antibacterial host defense, in part related to disruption of epithelial barrier and effective phagocytosis of pathogens. The functional response to ambient O3 seems to be dependent on many components of the innate immune signaling. In this article, we review the complex interaction between inhalation of O3 and pulmonary innate immunity.
Toll-like receptor; tlr4; environmental airways injury; macrophage; epithelia
Ozone exposure is associated with exacerbation of reactive airways disease. We have previously reported that the damage-associated molecular pattern, hyaluronan, is required for the complete biological response to ambient ozone and that hyaluronan fragments signal through toll-like receptor 4 (TLR4). In this study, we further investigated the role of TLR4 adaptors in ozone–induced airway hyperresponsiveness (AHR) and the direct response to hyaluronan fragments (HA). Using a murine model of AHR, C57BL/6J, TLR4−/−, MyD88−/−, and TIRAP−/− mice were characterized for AHR after exposure to either ozone (1 ppm×3 h) or HA fragments. Animals were characterized for AHR with methacholine challenge, cellular inflammation, lung injury, and production of pro-inflammatory cytokines. Ozone-exposed C57BL/6J mice developed cellular inflammation, lung injury, pro-inflammatory cytokines, and AHR, while mice deficient in TLR4, MyD88 or TIRAP demonstrated both reduced AHR and reduced levels of pro-inflammatory cytokines including TNFα, IL-1β, MCP-1, IL-6 and KC. The level of hyaluronan was increased after inhalation of ozone in each strain of mice. Direct challenge of mice to hyaluronan resulted in AHR in C57BL/6J mice, but not in TLR4−/−, MyD88−/−, or TIRAP−/− mice. HA-induced cytokine production in wild-type mice was significantly reduced in TLR4−/−, MyD88−/−, or TIRAP−/− mice. In conclusion, our findings support that ozone-induced airway hyperresponsiveness is dependent on the HA-TLR4-MyD88-TIRAP signaling pathway.
Background: Our previous work demonstrated that the extracellular matrix protein mindin contributes to allergic airways disease. However, the role of mindin in nonallergic airways disease has not previously been explored.
Objectives: We hypothesized that mindin would contribute to airways disease after inhalation of either lipopolysaccharide (LPS) or ozone.
Methods: We exposed C57BL/6J and mindin-deficient (–/–) mice to aerosolized LPS (0.9 μg/m3 for 2.5 hr), saline, ozone (1 ppm for 3 hr), or filtered air (FA). All mice were evaluated 4 hr after LPS/saline
exposure or 24 hr after ozone/FA exposure. We characterized the physiological and biological responses by analysis of airway hyperresponsiveness (AHR) with a computer-controlled small-animal ventilator (FlexiVent), inflammatory cellular recruitment, total protein in bronchoalveolar lavage fluid (BALF), proinflammatory cytokine profiling, and ex vivo bronchial ring studies.
Results: After inhalation of LPS, mindin–/– mice demonstrated significantly reduced total cell and neutrophil recruitment into the airspace compared with their wild-type counterparts. Mindin–/– mice also exhibited reduced proinflammatory cytokine production and lower AHR to methacholine challenge by FlexiVent. After inhalation of ozone, mice had no detectible differences in cellular inflammation or total BALF protein dependent on mindin. However, mindin–/– mice were protected from increased proinflammatory cytokine production and AHR compared with their C57BL/6J counterparts. After ozone exposure, bronchial rings derived from mindin–/– mice demonstrated reduced constriction in response to carbachol.
Conclusions: These data demonstrate that the extracellular matrix protein mindin modifies the airway response to both LPS and ozone. Our data support a conserved role of mindin in production of proinflammatory cytokines and the development of AHR in two divergent models of reactive airways disease, as well as a role of mindin in airway smooth muscle contractility after exposure to ozone.
airway smooth muscle; endotoxin; innate immunity; lipopolysaccharide; LPS; lung; mindin; ozone; Tlr4; toll-like receptor
Activation of the host antibacterial defenses by the toll-like receptors (TLR) also selectively activates energy-sensing and metabolic pathways, but the mechanisms are poorly understood. This includes the metabolic and mitochondrial biogenesis master co-activators, Ppargc1a (PGC-1α) and Ppargc1b (PGC-1β) in Staphylococcus aureus (S. aureus) sepsis. The expression of these genes in the liver is markedly attenuated inTLR2−/− mice and markedly accentuated in TLR4−/− mice compared with wild type (WT) mice. We sought to explain this difference by using specific TLR-pathway knockout mice to test the hypothesis that these co-activator genes are directly regulated through TLR2 signaling. By comparing their responses to S. aureus with WT mice, we found that MyD88-deficient and MAL-deficient mice expressed hepatic Ppargc1a and Ppargc1b normally, but that neither gene was activated in TRAM-deficient mice. Ppargc1a/b activation did not require NF-kβ, but did require an interferon response factor (IRF), because neither gene was activated in IRF-3/7 double-knockout mice in sepsis, but both were activated normally in Unc93b1-deficient (3d) mice. Nuclear IRF-7 levels in TLR2−/− and TLR4−/− mice decreased and increased respectively post-inoculation and IRF-7 DNA-binding at the Ppargc1a promoter was demonstrated by chromatin immunoprecipitation. Also, a TLR2-TLR4-TRAM native hepatic protein complex was detected by immunoprecipitation within 6 h of S. aureus inoculation that could support MyD88-independent signaling to Ppargc1a/b. Overall, these findings disclose a novel MyD88-independent pathway in S. aureus sepsis that links TLR2 and TLR4 signaling in innate immunity to Ppargc1a/b gene regulation in a critical metabolic organ, the liver, by means of TRAM, TRIF, and IRF-7.
Allergic asthma, a major cause of morbidity and leading cause of hospitalizations, is an inflammatory disease orchestrated by T helper cells and characterized by the lung migration of eosinophils, which are important asthma effector cells. Lung migration of inflammatory cells requires, among other events, the chemokine receptor transduction of lung-produced inflammatory chemokines. Despite the widespread prevalence of this disease, the molecular mechanisms regulating chemokine production and receptor regulation in asthma are poorly understood. Previous work from our laboratory demonstrated that β-arrestin−2 positively regulates the development of allergic airway disease in a mouse model, partly through positive regulation of T-lymphocyte chemotaxis to the lung. However, β-arrestin−2 is expressed in many cell types, including other hematopoietic cells and lung structural cells, which are involved in the development and manifestation of allergic airway disease. To determine the cell types required for β-arrestin–2–dependent allergic inflammation, we generated bone marrow chimera mice. Using the ovalbumin murine model of allergic airway disease, we show that eosinophilic and lymphocytic inflammation is restored in chimeric mice, with expression of β-arrestin−2 exclusively on hematopoietic-derived cell types. In contrast, airway hyperresponsiveness is dependent on the expression of β-arrestin−2 in structural cells. Our data demonstrate that the expression of β-arrestin−2 in at least two divergent cell types contributes to the pathogenesis of allergic airway disease.
asthma; bone marrow transplant; β-arrestin-2; airway hyperresponsiveness
Human exposure to ozone is associated with increased prevalence of allergic asthma. Here, we demonstrate that ozone increases levels of activated dendritic cells in thoracic lymph nodes and promotes allergic sensitization through a TLR4-dependent pathway.
ozone; toll-like receptor; asthma; dendritic cells; sensitization; adjuvant
Chronic lung diseases are marked by excessive inflammation and epithelial remodeling. Reduced Clara cell secretory function and corresponding decreases in the abundance of the major Clara cell secretory protein (CCSP) are characteristically seen in these disease states. We sought to define the impact of Clara cell and CCSP depletion on regulation of the lung inflammatory response. We used chemical and genetic mouse models of Clara cell and CCSP deficiency (CCSP−/−) coupled with Pseudomonas aeruginosa LPS elicited inflammation. Exposure of Clara cell–depleted or CCSP−/− mice to LPS resulted in augmented inflammation as assessed by polymorphonuclear leukocyte recruitment to the airspace. Gene expression analysis and pathway modeling of the CCSP−/− inflammatory response implicated increased TNF-α signaling. Consistent with this model was the demonstration of significantly elevated TNF-α in airway fluid of LPS-stimulated CCSP−/− mice compared with similarly exposed wild-type mice. Increased LPS-elicited TNF-α production was also observed in cultured lung macrophages from CCSP−/− mice compared with wild-type mice. We demonstrate that macrophages from Clara cell–depleted and CCSP−/− mice displayed increased Toll-like receptor 4 surface expression. Our results provide evidence that Clara cells can attenuate inflammation through regulation of macrophage behavior, and suggest that epithelial remodeling leading to reduced Clara cell secretory function is an important factor that increases the intensity of lung inflammation in chronic lung disease.
Clara cell; Clara cell secretory protein; inflammation; LPS; macrophage