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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The PGC family of transcriptional co-activators (PGC-1α [Ppargc1a], PGC-1β [Ppargc1b], and PRC [Pprc]) coordinates the upregulation of mitochondrial biogenesis, and Ppargc1a is known to be activated in response to mitochondrial damage in sepsis. Therefore, we postulated that the PGC family is regulated by the innate immune system. We investigated whether mitochondrial biogenesis and PGC gene expression are disrupted in an established model of Staphylococcus aureus sepsis both in mice with impaired innate immune function (TLR2−/− and TLR4−/−) and in wild-type controls. We found an early up-regulation of Ppargc1a and Ppargc1b post-infection (at 6 h) in WT mice, but the expression of both genes was concordantly dysregulated in TLR2−/− mice (no increase at 6 h) and in TLR4−/− mice (amplified at 6 h). However, the third family member, PRC, was regulated differently, and its expression increased significantly at 24 h in all three mouse strains (WT, TLR2−/−, and TLR4−/−). In silico analyses showed that Ppargc1a and Ppargc1b share binding sites for microRNA mmu-mir-202-3p. Thus, miRNA-mediated post-transcriptional mRNA degradation could account for the failure to increase the expression of both genes in TLR2−/− mice. The expression of mmu-mir-202-3p was measured by real-time PCR and found to be significantly increased in TLR2−/− but not in WT or TLR4−/− mice. In addition, it was found that mir-202-3p functionally decreases Ppargc1a mRNA in vitro. Thus, both innate immune signaling through the TLRs and mir-202-3p-mediated mRNA degradation are implicated in the co-regulation of Ppargc1a and Ppargc1b during inflammation. Moreover, the identification of mir-202-3p as a potential factor for Ppargc1a and Ppargc1b repression in acute inflammation may open new avenues for mitochondrial research and, potentially, therapy.

Mitochondrial biogenesis protects metabolism from mitochondrial dysfunction produced by activation of innate immunity by lipopolysaccharide (LPS) or other bacterial products. Here we tested the hypothesis in mouse heart that activation of toll-like receptor-4 (TLR4), which induces early-phase genes that damage mitochondria, also activates mitochondrial biogenesis through induction of nitric oxide synthase (NOS2). We compared three strains of mice: wild type (Wt) C57BL/6J, TLR4−/−, and NOS2−/− for cardiac mitochondrial damage and mitochondrial biogenesis by real-time RT-PCR, Western analysis, immunochemistry, and isoform analysis of myosin heavy chain (MHC) after sub-lethal heat-killed E. coli (HkEC). After HkEC, Wt mice displayed significant myocardial mtDNA depletion along with enhanced TLR4 and NOS2 gene and protein expression that normalized in 72h. HkEC generated less cytokine stress in TLR4−/− and NOS2−/− than Wt mice, and NOS2−/− mice had mtDNA damage comparable to Wt, both knockout strains failed to restore mtDNA copy number because of mitochondrial transcriptosome dysfunction. Wt mice also showed the largest β-MHC isoform switch, but MHC recovery lagged in the NOS2−/− and TLR4−/− strains. The NOS2−/− mice also unexpectedly revealed the co-dependency of TLR4 expression on NOS2. These findings demonstrate the decisive participation of NOS2 induction by TLR4 in optimization of mitochondrial biogenesis and MHC expression after gram negative challenge.

Rationale: The etiology and pathogenesis of angiogenesis in idiopathic pulmonary fibrosis (IPF) is poorly understood. Inter-α-trypsin inhibitor (IaI) is a serum protein that can bind to hyaluronan (HA) and may contribute to the angiogenic response to tissue injury.

Objectives: To determine whether IaI promotes HA-mediated angiogenesis in tissue injury.

Methods: An examination was undertaken of angiogenesis in IaI-sufficient and -deficient mice in the bleomycin model of pulmonary fibrosis and in angiogenesis assays in vivo and in vitro. IaI and HA in patients with IPF were examined.

Measurements and Main Results: IaI significantly enhances the angiogenic response to short-fragment HA in vivo and in vitro. lal deficiency Ieads to decreased angiogenesis in the matrigel model, and decreases lung angiogenesis after bleomycin exposure in mice. IaI is found in fibroblastic foci in IPF, where it colocalizes with HA. The colocalization is particularly strong in vascular areas around fibroblastic foci. Serum levels of IaI and HA are significantly elevated in patients with IPF compared with control subjects. High serum IaI and HA levels are associated with decreased lung diffusing capacity, but not FVC.

Conclusions: Our findings indicate that serum IaI interacts with HA, and promotes angiogenesis in lung injury. IaI appears to contribute to the vascular response to lung injury and may lead to aberrant angiogenesis.

Clinical trial registered with www.clinicaltrials.gov (NCT00016627).

Lipopolysaccharide (LPS) is ubiquitous in the environment. Recent epidemiologic data suggest that occupational exposure to inhaled LPS can contribute to the progression of chronic obstructive pulmonary disease. To address the hypothesis that inhaled LPS can cause emphysema-like changes in mouse pulmonary parenchyma, we exposed C57BL/6 mice to aerosolized LPS daily for 4 weeks. By 3 days after the end of the 4-week exposure, LPS-exposed mice developed enlarged airspaces that persisted in the 4-week recovered mice. These architectural alterations in the lung are associated with enhanced type I, III, and IV procollagen mRNA as well as elevated levels of matrix metalloproteinase (MMP)-9 mRNA, all of which have been previously associated with human emphysema. Interestingly, MMP-9–deficient mice were not protected from the development of LPS-induced emphysema. However, we demonstrate that LPS-induced airspace enlargement was associated with apoptosis within the lung parenchyma, as shown by prominent TUNEL staining and elevated cleaved caspase 3 immunoreactivity. Antineutrophil antiserum-treated mice were partially protected from the lung destruction caused by chronic inhalation of LPS. Taken together, these findings demonstrate that inhaled LPS can cause neutrophil-dependent emphysematous changes in lung architecture that are associated with apoptosis and that these changes may be occurring through mechanisms different than those induced by cigarette smoke.

Background

The study of genome-wide DNA methylation changes has become more accessible with the development of various array-based technologies though when studying species other than human the choice of applications are limited and not always within reach. In this study, we adapted and tested the applicability of Methylation Specific Digital Karyotyping (MSDK), a non-array based method, for the prospective analysis of epigenetic changes after perinatal nutritional modifications in a mouse model of allergic airway disease. MSDK is a sequenced based method that allows a comprehensive and unbiased methylation profiling. The method generates 21 base pairs long sequence tags derived from specific locations in the genome. The resulting tag frequencies determine in a quantitative manner the methylation level of the corresponding loci.

Results

Genomic DNA from whole lung was isolated and subjected to MSDK analysis using the methylation-sensitive enzyme Not I as the mapping enzyme and Nla III as the fragmenting enzyme. In a pair wise comparison of the generated mouse MSDK libraries we identified 158 loci that are significantly differentially methylated (P-value = 0.05) after perinatal dietary changes in our mouse model. Quantitative methylation specific PCR and sequence analysis of bisulfate modified genomic DNA confirmed changes in methylation at specific loci. Differences in genomic MSDK tag counts for a selected set of genes, correlated well with changes in transcription levels as measured by real-time PCR. Furthermore serial analysis of gene expression profiling demonstrated a dramatic difference in expressed transcripts in mice exposed to perinatal nutritional changes.

Conclusion

The genome-wide methylation survey applied in this study allowed for an unbiased methylation profiling revealing subtle changes in DNA methylation in mice maternally exposed to dietary changes in methyl-donor content. The MSDK method is applicable for mouse models of complex human diseases in a mixed cell population and might be a valuable technology to determine whether environmental exposures can lead to epigenetic changes.

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.

LPS from bacteria is ubiquitous in the environment and can cause airway disease and modify allergic asthma. Identification of gene products that modulate the biologic response to inhaled LPS will improve our understanding of inflammatory airways disease. Previous work has identified quantitative trait loci for the response to inhaled LPS on chromosomes 2 and 11. In these regions, 28 genes had altered RNA expression after inhalation of LPS, including CD44, which was associated with differences in both TNF-α levels and neutrophil recruitment into the lung. It has previously been shown that CD44 can modulate macrophage recruitment in response to Mycobacterium tuberculosis, as well as clearance of neutrophils after lung injury with both bleomycin and live Escherichia coli bacteria. In this study, we demonstrate that the biologic response to inhaled LPS is modified by CD44. Macrophages failed to be recruited to the lungs of CD44-deficient animals at all time points after LPS exposure. CD44-deficient macrophages showed reduced motility in a Transwell migration assay, reduced ability to secrete the proinflammatory cytokine TNF-α, reduced in vivo migration in response to monocyte chemotactic protein-1, and diminished adhesion to vascular endothelia in the presence of TNF-α. In addition, CD44-deficient animals had 150% fewer neutrophils at 24 h and 50% greater neutrophils 48 h after LPS exposure. These results support the role of CD44 in modulating the biologic response to inhaled LPS.

Rationale: Idiopathic interstitial pneumonia (IIP) and its familial variants are progressive and largely untreatable disorders with poorly understood molecular mechanisms. Both the genetics and the histologic type of IIP play a role in the etiology and pathogenesis of interstitial lung disease, but transcriptional signatures of these subtypes are unknown.

Objectives: To evaluate gene expression in the lung tissue of patients with usual interstitial pneumonia or nonspecific interstitial pneumonia that was either familial or nonfamilial in origin, and to compare it with gene expression in normal lung parenchyma.

Methods: We profiled RNA from the lungs of 16 patients with sporadic IIP, 10 with familial IIP, and 9 normal control subjects on a whole human genome oligonucleotide microarray.

Results: Significant transcriptional differences exist in familial and sporadic IIPs. The genes distinguishing the genetic subtypes belong to the same functional categories as transcripts that distinguish IIP from normal samples. Relevant categories include chemokines and growth factors and their receptors, complement components, genes associated with cell proliferation and death, and genes in the Wnt pathway. The role of the chemokine CXCL12 in disease pathogenesis was confirmed in the murine bleomycin model of lung injury, with C57BL/6CXCR4+/− mice demonstrating significantly less collagen deposition than C57BL/6CXCR4+/+ mice. Whereas substantial differences exist between familial and sporadic IIPs, we identified only minor gene expression changes between usual interstitial pneumonia and nonspecific interstitial pneumonia.

Conclusions: Taken together, our findings indicate that differences in gene expression profiles between familial and sporadic IIPs may provide clues to the etiology and pathogenesis of IIP.

Endotoxin (LPS), a Gram-negative cell wall component, has potent proinflammatory properties. Acute LPS exposure causes airway inflammation; chronic exposure causes airway hyperreactivity and remodeling. IL-10 is an important antiinflammatory cytokine, which is decreased in patients with airway disease, such as asthma and cystic fibrosis. To examine the physiologic and therapeutic role of IL-10 in acute and chronic LPS-induced airway disease. Mice were exposed to aerosolized LPS once or daily for 4 wk. Endpoints were airway inflammation, airway reactivity to methacholine, extracellular matrix protein expression, and histologic analysis. IL-10–deficient mice developed significantly enhanced airway cellularity and remodeling when compared with C57BL/6 mice after chronic LPS inhalation. However they demonstrated less airway hyperreactivity associated with higher inducible nitric oxide synthase (iNOS), endothelial NOS (eNOS), and lung lavage fluid nitrite levels. In a bone marrow transplantation model, the IL-10 antiinflammatory effect was dependent on the hematopoietic but not on the parenchymal IL-10 expression. Induced epithelial human IL-10 expression protected from the LPS effects and led to decreased collagen production. IL-10 attenuates chronic LPS-induced airway inflammation and remodeling. Physiologically, the antiinflammatory effect of IL-10 is mediated by hematopoietic cells. Therapeutically, adenovirus-driven expression of human IL-10 in airway epithelia is sufficient for its protective effect on inflammation and remodeling. The role of IL-10 on airway hyperreactivity is complex: IL-10 deficiency protects against LPS-induced hyperreactivity, and is associated with higher eNOS, iNOS, and airway nitrate levels.

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.

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.

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.