Epithelial Responses in Radiation-Induced Lung Injury (RILI) Allow Chronic Inflammation and Fibrogenesis.

Radiation models, such as whole thorax lung irradiation (WTLI) or partial-body irradiation (PBI) with bone-marrow sparing, have shown that affected lung tissue displays a continual progression of injury, often for months after the initial insult. Undoubtably, a variety of resident and infiltrating cell types either contribute to or fail to resolve this type of progressive injury, which in lung tissue, often develops into lethal and irreversible radiation-induced pulmonary fibrosis (RIPF), indicating a failure of the lung to return to a homeostatic state. Resident pulmonary epithelium, which are present at the time of irradiation and persist long after the initial insult, play a key role in the maintenance of homeostatic conditions in the lung and have often been described as contributing to the progression of radiation-induced lung injury (RILI). In this study, we took an unbiased approach through RNA sequencing to determine the in vivo response of the lung epithelium in the progression of RIPF. In our methodology, we isolated CD326+ epithelium from the lungs of 12.5 Gy WTLI C57BL/6J female mice (aged 8-10 weeks and sacrificed at regular intervals) and compared irradiated and non-irradiated CD326+ cells and whole lung tissue. We subsequently verified our findings by qPCR and immunohistochemistry. Transcripts associated with epithelial regulation of immune responses and fibroblast activation were significantly reduced in irradiated animals at 4 weeks postirradiation. Additionally, alveolar type-2 epithelial cells (AEC2) appeared to be significantly reduced in number at 4 weeks and thereafter based on the diminished expression of pro-surfactant protein C (pro-SPC). This change is associated with a reduction of Cd200 and cyclooxygenase 2 (COX2), which are expressed within the CD326 populations of cells and function to suppress macrophage and fibroblast activation under steady-state conditions, respectively. These data indicate that either preventing epithelial cell loss that occurs after irradiation or replacing important mediators of immune and fibroblast activity produced by the epithelium are potentially important strategies for preventing or treating this unique injury.

Epithelial Responses in Radiation-Induced Lung Injury (RILI) Allow Chronic Inflammation and Fibrogenesis.

Radiation models, such as whole thorax lung irradiation (WTLI) or partial-body irradiation (PBI) with bone-marrow sparing, have shown that affected lung tissue displays a continual progression of injury, often for months after the initial insult. Undoubtably, a variety of resident and infiltrating cell types either contribute to or fail to resolve this type of progressive injury, which in lung tissue, often develops into lethal and irreversible radiation-induced pulmonary fibrosis (RIPF), indicating a failure of the lung to return to a homeostatic state. Resident pulmonary epithelium, which are present at the time of irradiation and persist long after the initial insult, play a key role in the maintenance of homeostatic conditions in the lung and have often been described as contributing to the progression of radiation-induced lung injury (RILI). In this study, we took an unbiased approach through RNA sequencing to determine the in vivo response of the lung epithelium in the progression of RIPF. In our methodology, we isolated CD326+ epithelium from the lungs of 12.5 Gy WTLI C57BL/6J female mice (aged 8-10 weeks and sacrificed at regular intervals) and compared irradiated and non-irradiated CD326+ cells and whole lung tissue. We subsequently verified our findings by qPCR and immunohistochemistry. Transcripts associated with epithelial regulation of immune responses and fibroblast activation were significantly reduced in irradiated animals at 4 weeks postirradiation. Additionally, alveolar type-2 epithelial cells (AEC2) appeared to be significantly reduced in number at 4 weeks and thereafter based on the diminished expression of pro-surfactant protein C (pro-SPC). This change is associated with a reduction of Cd200 and cyclooxygenase 2 (COX2), which are expressed within the CD326 populations of cells and function to suppress macrophage and fibroblast activation under steady-state conditions, respectively. These data indicate that either preventing epithelial cell loss that occurs after irradiation or replacing important mediators of immune and fibroblast activity produced by the epithelium are potentially important strategies for preventing or treating this unique injury.

Airway basal cell injury after acute diacetyl (2,3-butanedione) vapor exposure.

RATIONALE: Diacetyl (DA; 2,3-butanedione) is a chemical found commonly in foods and e-cigarettes. When inhaled, DA causes epithelial injury, though the mechanism of repair remain poorly understood. The objective of this study was to evaluate airway basal cell repair after DA vapor exposure. METHODS: Primary human bronchial epithelial cells were exposed to DA or PBS for 1 h. Lactate dehydrogenase, cleaved caspase 3/7 and trans-epithelial electrical resistance were measured prior to and following exposure. Exposed cultures were analyzed for the airway basal cell markers keratin 5 and p63 as well as ubiquitin and proteasome activity. Cultures were also treated with a proteasome inhibitor (MG132). RESULTS: DA vapor exposure caused a transient decrease in trans-epithelial electrical resistance in all DA-exposed cultures. Supernatant lactate dehydrogenase and cleaved caspase 3/7 increased significantly at the highest DA concentration but not at lower DA concentrations. Increased keratin 5 ubiquitination occurred after DA exposure but resolved by day 3. Damage to airway basal cells persisted at day 3 in the presence of MG132. CONCLUSIONS: Diacetyl exposure results in airway basal cell injury with keratin 5 ubiquitination and decreased p63 expression. The ubiquitin-proteasome-pathway partially mediates airway basal cell repair after acute DA exposure.

Modeling radiation-induced lung injury: lessons learned from whole thorax irradiation.

Models of thoracic irradiation have been developed as clinicians and scientists have attempted to decipher the events that led up to the pulmonary toxicity seen in human subjects following radiation treatment. The most common model is that of whole thorax irradiation (WTI), applied in a single dose. Mice, particularly the C57BL/6J strain, has been frequently used in these investigations, and has greatly informed our current understanding of the initiation and progression of radiation-induced lung injury (RILI). In this review, we highlight the sequential progression and dynamic nature of RILI, focusing primarily on the vast array of information that has been gleaned from the murine model. Ample evidence indicates a wide array of biological responses that can be seen following irradiation, including DNA damage, oxidative stress, cellular senescence and inflammation, all triggered by the initial exposure to ionizing radiation (IR) and heterogeneously maintained throughout the temporal progression of injury, which manifests as acute pneumonitis and later fibrosis. It appears that the early responses of specific cell types may promote further injury, disrupting the microenvironment and preventing a return to homeostasis, although the exact mechanisms driving these responses remains somewhat unclear. Attempts to either prevent or treat RILI in preclinical models have shown some success by targeting these disparate radiobiological processes. As our understanding of the dynamic cellular responses to radiation improves through the use of such models, so does the likelihood of preventing or treating RILI.

Apoptosis Resistance in Fibroblasts Precedes Progressive Scarring in Pulmonary Fibrosis and Is Partially Mediated by Toll-Like Receptor 4 Activation.

Inhalation of environmental toxicants such as cigarette smoke, metal or wood dust, silica, or asbestos is associated with increased risk for idiopathic pulmonary fibrosis (IPF). IPF involves progressive scarring of lung tissue, which interferes with normal respiration and is ultimately fatal; however, the complex cellular mechanisms of IPF pathogenesis remain unclear. Fibroblast apoptosis is essential in normal wound healing but is dysregulated in IPF. Recent studies suggest that Toll-like receptor 4 (TLR4) is key in the onset of IPF. Here, radiation-induced PF was used as a model for IPF because it very closely mimics the progressive and intractable nature of IPF. Female C57BL/6J (C57) and C57BL/6J TLR4-/- mice were exposed to a single dose of 13 Gy whole-thorax ionizing radiation. Although both strains showed similar levels of immediate radiation-induced damage, C57 mice exhibited more extensive fibrosis at 22-week postirradiation (PI) than TLR4-/- mice. Isolated C57 primary 1° MLFs showed decreased apoptosis susceptibility as early as 8-week postirradiation, a phenotype that persisted for the remainder of the radiation response. TLR4-/- 1° mouse lung fibroblasts did not exhibit significant apoptosis resistance at any point. Systemic release of high mobility group box 1, a TLR4 agonist, during the pneumonitis phase of the radiation response may act through TLR4 to contribute to fibroblast apoptosis resistance and thus interfere with wound resolution. These findings demonstrate that apoptosis resistance occurs earlier in pulmonary fibrosis pathogenesis than previously assumed, and that TLR4 signaling is a key mediator in this process.

Recurrent DNA damage is associated with persistent injury in progressive radiation-induced pulmonary fibrosis.

PURPOSE: Radiation-induced lung injuries (RILI), namely radiation pneumonitis and/or fibrosis, are dose-limiting outcomes following treatment for thoracic cancers. As part of a search for mitigation targets, we sought to determine if persistent DNA damage is a characteristic of this progressive injury. METHODS: C57BL/6J female mice were sacrificed at 24 h, 1, 4, 12, 16, 24 and 32 weeks following a single dose of 12.5 Gy thorax only gamma radiation; their lungs were compared to age-matched unirradiated animals. Tissues were examined for DNA double-strand breaks (DSBs) (γ-H2A.X and p53bp1), cellular senescence (senescence-associated beta-galactosidase and p21) and oxidative stress (malondialdehyde). RESULTS: Data revealed consistently higher numbers of DSBs compared to age-matched controls, with increases in γ-H2A.X positivity beyond 24 h post-exposure, particularly during the pathological phases, suggesting periods of recurrent DNA damage. Additional intermittent increases in both cellular senescence and oxidative stress also appeared to coincide with pneumonitis and fibrosis. CONCLUSIONS: These novel, long-term data indicate (a) increased and persistent levels of DSBs, oxidative stress and cellular senescence may serve as bioindicators of RILI, and (b) prevention of genotoxicity, via mitigation of free radical production, continues to be a potential strategy for the prevention of pulmonary radiation injury.

A Potential Biomarker for Predicting the Risk of Radiation-Induced Fibrosis in the Lung.

Biomarkers could play an essential role during triage in the aftermath of a radiological event, where exposure to radiation will be heterogeneous and complicated by concurrent trauma. Used alongside biodosimetry, biomarkers can identify victims in need of treatment for acute radiation effects, and might also provide valuable information on later developing consequences that need to be addressed as part of a treatment strategy. Indeed, because the lung is particularly sensitive to radiation and resultant late effects not only affect quality of life, but can also lead to morbidity, the risk of developing downstream pulmonary complications in exposed individuals requires assessment. In this study, analyses of changes in pulmonary and circulating content of club cell secretory protein (CCSP) and surfactant protein D (SP-D), expressed by epithelial club cells and type II pneumocytes in the lung, respectively, were used to evaluate pulmonary epithelial damage in several lung injury models. Using a combined radiation exposure model, fibrosis-susceptible C57BL/6J (C57) and alveolitis-prone C3H/HeJ (C3H) mice received 5 Gy total-body irradiation plus 2.5-10 Gy whole-lung irradiation, and lung and plasma samples were collected throughout the course of the radiation response, at time points ranging from 24 h to 26 weeks postirradiation. Radiation significantly reduced bronchiole CCSP coverage in C57 mice at 26 weeks, a response that varied in extent among animals, but correlated with the severity of fibrosis in each animal. Interestingly, plasma CCSP content was elevated in C57 mice at multiple time points preceding and during the fibrotic period; this response that was not observed in C3H mice. Circulating CCSP/SP-D ratios, calculated as an index of lung integrity, were similarly increased throughout the time course in C57, but not C3H, mice. Furthermore, when the thoracic doses were reduced to subthreshold levels for fibrosis induction (2.5 or 7.5 Gy), although the CCSP/SP-D ratio in lung homogenates demonstrated dose-responsive changes, this was not reflected in the plasma ratios at acute and late time points. Importantly, plasma CCSP/SP-D ratios also were not significantly altered in C57 mice exposed to LPS, and only transiently decreased in influenza-exposed mice, demonstrating a level of specificity for radiation-induced lung injury. These results indicate that the CCSP/SP-D ratio, measured in plasma, is sensitive to individual variation in radiation sensitivity, correlates with fibrosis development, can be detected early after exposure and is specific to radiation-induced injury. This suggests that the CCSP/SP-D ratio may be useful as a biomarker of radiation-induced pulmonary fibrosis.

Role of Infiltrating Monocytes in the Development of Radiation-Induced Pulmonary Fibrosis.

Lung exposure to radiation induces an injury response that includes the release of cytokines and chemotactic mediators; these signals recruit immune cells to execute inflammatory and wound-healing processes. However, radiation alters the pulmonary microenvironment, dysregulating the immune responses and preventing a return to homeostasis. Importantly, dysregulation is observed as a chronic inflammation, which can progress into pneumonitis and promote pulmonary fibrosis; inflammatory monocytes, which are bone marrow derived and express CCR2, have been shown to migrate into the lung after radiation exposure. Although the extent to which recruited inflammatory monocytes contribute to radiation-induced pulmonary fibrosis has not been fully investigated, we hypothesize that its pathogenesis is reliant on this population. The CC chemokine ligand, CCL2, is a chemotactic mediator responsible for trafficking of CCR2+ inflammatory cells into the lung. Therefore, the contribution of this mediator to fibrosis development was analyzed. Interleukin (IL)-1ß, a potent pro-inflammatory cytokine expressed during the radiation response, and its receptor, IL-1R1, were also evaluated. To this end, CCR2-/-, IL-1ß-/- and IL-1R1-/- chimeric mice were generated and exposed to 12.5 Gy thoracic radiation, and their response was compared to wild-type (C57BL/6) syngeneic controls. Fibrotic foci were observed in the periphery of the lungs of C57 syngeneic mice and CCR2-/- recipient mice that received C57 bone marrow (C57 > CCR2-/-) by 16 and 12 weeks after irradiation, respectively. In contrast, in the mice that had received bone marrow lacking CCR2 (CCR2-/- > C57 and CCR2-/- syngeneic mice), no pulmonary fibrosis was observed at 22 weeks postirradiation. This observation correlated with decreased numbers of infiltrating and interstitial macrophages compared to controls, as well as reduced proportions of pro-inflammatory Ly6C+ macrophages observed at 12-18 weeks postirradiation, suggesting that CCR2+ macrophages contribute to radiation-induced pulmonary fibrosis. Interestingly, reduced proportions of CD206+ lung macrophages were also present at these time points in CCR2-/- chimeric mice, regardless of donor bone marrow type, suggesting that the phenotype of resident subsets may be influenced by CCR2. Furthermore, chimeras, in which either IL-1ß was ablated from infiltrating cells or IL-1R1 from lung tissues, were also protected from fibrosis development, correlating with attenuated CCL2 production; these data suggest that IL-1ß may influence chemotactic signaling after irradiation. Overall, our data suggest that CCR2+ infiltrating monocyte-derived macrophages may play a critical role in the development of radiation-induced pulmonary fibrosis.

Radiation induced pulmonary fibrosis as a model of progressive fibrosis: Contributions of DNA damage, inflammatory response and cellular senescence genes.

Purpose/Aim of Study: Studies of pulmonary fibrosis (PF) have resulted in DNA damage, inflammatory response, and cellular senescence being widely hypothesized to play a role in the progression of the disease. Utilizing these aforementioned terms, genomics databases were interrogated along with the term, "pulmonary fibrosis," to identify genes common among all 4 search terms. Findings were compared to data derived from a model of radiation-induced progressive pulmonary fibrosis (RIPF) to verify that these genes are similarly expressed, supporting the use of radiation as a model for diseases involving PF, such as human idiopathic pulmonary fibrosis (IPF). MATERIALS AND METHODS: In an established model of RIPF, C57BL/6J mice were exposed to 12.5 Gy thorax irradiation and sacrificed at 24 hours, 1, 4, 12, and 32 weeks following exposure, and lung tissue was compared to age-matched controls by RNA sequencing. RESULTS: Of 176 PF associated gene transcripts identified by database interrogation, 146 (>82%) were present in our experimental model, throughout the progression of RIPF. Analysis revealed that nearly 85% of PF gene transcripts were associated with at least 1 other search term. Furthermore, of 22 genes common to all four terms, 16 were present experimentally in RIPF. CONCLUSIONS: This illustrates the validity of RIPF as a model of progressive PF/IPF based on the numbers of transcripts reported in both literature and observed experimentally. Well characterized genes and proteins are implicated in this model, supporting the hypotheses that DNA damage, inflammatory response and cellular senescence are associated with the pathogenesis of PF.

Effects of IL-4 on pulmonary fibrosis and the accumulation and phenotype of macrophage subpopulations following thoracic irradiation.

PURPOSE: Thoracic irradiation injures lung parenchyma, triggering inflammation and immune cell activation, leading to pneumonitis and fibrosis. Macrophage polarization contributes to these processes. Since IL-4 promotes pro-fibrotic macrophage activation, its role in radiation-induced lung injury was investigated. MATERIALS AND METHODS: Lung macrophage subpopulations were characterized from 3-26 weeks following exposure of WT and IL-4-/- mice to 0 or 12.5 Gray single dose thoracic irradiation. RESULTS: Loss of IL-4 did not prevent fibrosis, but blunted macrophage accumulation within the parenchyma. At 3 weeks following exposure, cell numbers and expression of F4/80 and CD206, an alternative activation marker, decreased in alveolar macrophages but increased in infiltrating macrophages in WT mice. Loss of IL-4 impaired recovery of these markers in alveolar macrophages and blunted expansion of these populations in infiltrating macrophages. CD206+ cells were evident in fibrotic regions of WT mice only, however Arg-1+ cells increased in fibrotic regions in IL-4-/- mice only. Radiation-induced proinflammatory Ly6C expression was more apparent in alveolar and interstitial macrophages from IL-4-/- mice. CONCLUSIONS: IL-4 loss did not prevent alternative macrophage activation and fibrosis in irradiated mice. Instead, a role is indicated for IL-4 in maintenance of macrophage populations in the lung following high single dose thoracic irradiation.

Whole-Lung Irradiation Results in Pulmonary Macrophage Alterations that are Subpopulation and Strain Specific.

Exposure of the lung to radiation produces injury and inflammatory responses that result in microenvironmental alterations, which can promote the development of pneumonitis and/or pulmonary fibrosis. It has been shown that after other toxic insults, macrophages become phenotypically polarized in response to microenvironmental signals, orchestrating the downstream inflammatory responses. However, their contribution to the development of the late consequences of pulmonary radiation exposure remains unclear. To address this issue, fibrosis-prone C57BL/6J mice or pneumonitis-prone C3H/HeJ mice were whole-lung irradiated with 0 or 12.5 Gy and lung digests were collected between 3 and 26 weeks after radiation exposure. CD45(+) leukocytes were isolated and characterized by flow cytometry, and alveolar, interstitial and infiltrating macrophages were also detected. Ly6C, expressed by pro-inflammatory monocytes and macrophages, and mannose receptor (CD206), a marker of alternative activation, were assessed in each subpopulation. While the total number of pulmonary macrophages was depleted at 3 weeks after lung irradiation relative to age-matched controls in both C57 and C3H mice, identification of discrete subpopulations showed that this loss in cell number occurred in the alveolar, but not the interstitial or infiltrating, subsets. In the alveolar macrophages of both C57 and C3H mice, this correlated with a loss in the proportion of cells that expressed CD206 and F4/80. In contrast, in interstitial and infiltrating macrophages, the proportion of cells expressing these markers was increased at several time points after irradiation, with this response generally more pronounced in C3H mice. Radiation exposure was also associated with elevations in the proportion of alveolar and interstitial macrophage subpopulations expressing Ly6C and F4/80, with this response occurring at earlier time points in C57 mice. Although the radiation dose used in this study was not isoeffective for the inflammatory response in the two strains, the differences observed in the responses of these discrete macrophage populations between the fibrosis-prone versus pneumonitis-prone mice nonetheless suggest a possible role for these cells in the development of long-term consequences of pulmonary radiation exposure.

Long-Lasting Impact of Neonatal Exposure to Total Body Gamma Radiation on Secondary Lymphoid Organ Structure and Function.

The acute period after total body irradiation (TBI) is associated with an increased risk of infection, principally resulting from the loss of hematopoietic stem cells, as well as disruption of mucosal epithelial barriers. Although there is a return to baseline infection control coinciding with the apparent progressive recovery of hematopoietic cell populations, late susceptibility to infection in radiation-sensitive organs such as lung and kidney is known to occur. Indeed, pulmonary infections are particularly prevalent in hematopoietic cell transplant (HCT) survivors, in both adult and pediatric patient populations. Preclinical studies investigating late outcomes from localized thoracic irradiation have indicated that the mechanisms underlying pulmonary delayed effects are multifactorial, including exacerbated and persistent production of pro-inflammatory molecules and abnormal cross-talk among parenchymal and infiltrating immune and inflammatory cell populations. However, in the context of low-dose TBI, it is not clear whether the observed exacerbated response to infection remains contingent on these same mechanisms. It is possible instead, that after systemic radiation-induced injury, the susceptibility to infection may be independently related to defects in alternative organs that are revealed only through the challenge itself; indeed, we have hypothesized that this defect may be due to radiation-induced chronic effects in the structure and function of secondary lymphoid organs (SLO). In this study, we investigated the molecular and cellular alterations in SLO (i.e., spleen, mediastinal, inguinal and mesenteric lymph nodes) after TBI, and the time points when there appears to be immune competence. Furthermore, due to the high incidence of pulmonary infections in the late post-transplantation period of bone marrow transplant survivors, particularly in children, we focused on outcomes in mice irradiated as neonates, which served as a model for a pediatric population, and used the induction of adaptive immunity against influenza virus as a functional end point. We demonstrated that, in adult animals irradiated as neonates, high endothelial venule (HEV) expansion, generation of follicular helper T cells (TFH) and formation of splenic germinal centers (GC) were rapidly and, more importantly, persistently impaired in SLO, suggesting that the early-life exposure to sublethal radiation had long-lasting effects on the induction of humoral immunity. Although the neonatal TBI did not affect the overall outcome from influenza infection in the adults at the earlier time points assessed, we believe that they nonetheless contribute significantly to the increased mortality observed at subsequent late time points. Furthermore, we speculate that the detrimental and persistent impact on the induction of CD4 T- and B-cell responses in the mediastinal lymph nodes will decrease the animals' ability to respond to other aerial pathogens. Since many of these pathogens are normally cleared by antibodies, our findings provide an explanation for the susceptibility of survivors of childhood HCT to life-threatening respiratory tract infections. These findings have implications regarding the need for increased monitoring in pediatric hematopoietic cell transplant patients, since they indicate that there are ongoing and cumulative defects in SLO, which, importantly, develop during the immediate and early postirradiation period when patients may appear immunologically competent. The identification of changes in immune-related signals may offer bioindicators of progressive dysfunction, and of potential mechanisms that could be targeted so as to reduce the risk of infection from extracellular pathogens. Furthermore, these results support the potential susceptibility of the pediatric population to infection after sublethal irradiation in the context of a nuclear or radiological event.

Examining the Effects of External or Internal Radiation Exposure of Juvenile Mice on Late Morbidity after Infection with Influenza A.

A number of investigators have suggested that exposure to low-dose radiation may pose a potentially serious health risk. However, the majority of these studies have focused on the short-term rather than long-term effects of exposure to fixed source radiation, and few have examined the effects of internal contamination. Additionally, very few studies have focused on exposure in juveniles, when organs are still developing and could be more sensitive to the toxic effects of radiation. To specifically address whether early-life radiation injury may affect long-term immune competence, we studied 14-day-old juvenile pups that were either 5 Gy total-body irradiated or injected internally with 50 µCi soluble (137)Cs, then infected with influenza A virus at 26 weeks after exposure. After influenza infection, all groups demonstrated immediate weight loss. We found that externally irradiated, infected animals failed to recover weight relative to age-matched infected controls, but internally (137)Cs contaminated and infected animals had a weight recovery with a similar rate and degree as controls. Externally and internally irradiated mice demonstrated reduced levels of club cell secretory protein (CCSP) message in their lungs after influenza infection. The externally irradiated group did not recover CCSP expression even at the two-week time point after infection. Although the antibody response and viral titers did not appear to be affected by either radiation modality, there was a slight increase in monocyte chemoattractant protein (MCP)-1 expression in the lungs of externally irradiated animals 14 days after influenza infection, with increased cellular infiltration present. Notably, an increase in the number of regulatory T cells was seen in the mediastinal lymph nodes of irradiated mice relative to uninfected mice. These data confirm the hypothesis that early-life irradiation may have long-term consequences on the immune system, leading to an altered antiviral response.

Critical role of non-muscle myosin light chain kinase in thrombin-induced endothelial cell inflammation and lung PMN infiltration.

The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response. In this study, we developed a new mouse model that permits investigation of lung inflammation associated with intravascular coagulation. Using this mouse model and in vitro approaches, we addressed the role of non-muscle myosin light chain kinase (nmMLCK) in thrombin-induced endothelial cell (EC) inflammation and lung neutrophil (PMN) infiltration. Our in vitro experiments revealed a key role of nmMLCK in ICAM-1 expression by its ability to control nuclear translocation and transcriptional capacity of RelA/p65 in EC. When subjected to intraperitoneal thrombin challenge, wild type mice showed a marked increase in lung PMN infiltration via expression of ICAM-1. However, these responses were markedly attenuated in mice deficient in nmMLCK. These results provide mechanistic insight into lung inflammatory response associated with intravascular coagulation and identify nmMLCK as a critical target for modulation of lung inflammation.

Exacerbation of lung radiation injury by viral infection: the role of Clara cells and Clara cell secretory protein.

Viral infections have been associated with exacerbation of disease in human cases of idiopathic pulmonary fibrosis. Since pulmonary fibrosis is a common outcome after irradiation to the lung, we hypothesized that viral infection after radiation exposure would exacerbate radiation-induced lung injury. Epithelial injury, a frequent outcome after infection, has been hypothesized to contribute to the pathogenesis of pulmonary fibrosis and bronchiolar epithelial Clara cells participate in epithelial repair. Therefore, it was further hypothesized that altered responses after irradiation involve the bronchiolar epithelial Clara cells. C57BL/6J or CCSP(-/-) mice were irradiated with 0 (sham), 5, 10 or 15 Gy to the whole thorax. At ten weeks post-irradiation, animals were mock infected or infected with influenza A virus and body weight and survival were monitored. Pulmonary function was assessed by whole-body plethysmography. The Clara cell markers, CCSP and Cyp2f2, were measured in the lung by qRT-PCR, and protein expression was visualized in the lung by immunofluorescence. Following pulmonary function tests, mice were sacrificed and tissues were collected for pathological analysis. In 15 Gy irradiated animals infected with influenza A virus, accelerated respiratory rates, reduced pulmonary function, and exacerbated lung pathology occurred earlier post-irradiation than previously observed after irradiation alone, suggesting infection accelerates the development of radiation injury. After irradiation alone, CCSP and Cyp2f2 mRNA levels were reduced, correlating with reductions in the number of Clara cells lining the airways. When combined with infection, these markers further declined and an apparent delay in recovery of mRNA expression was observed, suggesting that radiation injury leads to a chronic reduction in the number of Clara cells that may potentiate the epithelial injury observed after influenza A virus infection. This novel finding may have considerable therapeutic implications with respect to both thoracic tumor patients and recipients of bone marrow transplants.

Cationic nanoparticles disrupt cellular signaling in a cholesterol dependent manner.

In this study, we investigate the interaction of charged polystyrene particles with respiratory epithelial cells. Experiments were designed to reveal the effect of cellular interaction with particles of varying size and charge and how such interactions could alter cytokine induced cellular processes. A549 cells, containing a stably transfected Interleukin-8 (IL-8) Luciferase promoter construct, were cultured in the presence of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) and polystyrene particles for 4.5h. Several endpoints were measured: IL-8 gene expression, IL-6 protein, IL-8 protein, and NF-кB translocation. At 4.5h, cellular viability was maintained for the particle exposed cells. Interestingly, we found that only the cationic nanoparticles have the ability to disrupt the TNF-α stimulated cellular signaling by reducing the IL-8 promoter activity, IL-6 and IL-8 protein. Cationic nanoparticles reduce these processes through interfering with NF-κB translocation. However, the activity of the cationic nanoparticles on the IL-8 promoter and NF-κB translocation is lost when cholesterol is depleted from A549 cells before particle exposure, which suggests that cationic nanoparticles rely on membrane cholesterol integrity to disrupt cellular signaling.

Lung irradiation increases mortality after influenza A virus challenge occurring late after exposure.

PURPOSE: To address whether irradiation-induced changes in the lung environment alter responses to a viral challenge delivered late after exposure but before the appearance of late lung radiation injury. METHODS AND MATERIALS: C57BL/6J mice received either lung alone or combined lung and whole-body irradiation (0-15 Gy). At 10 weeks after irradiation, animals were infected with 120 HAU influenza virus strain A/HKx31. Innate and adaptive immune cell recruitment was determined using flow cytometry. Cytokine and chemokine production and protein leakage into the lung after infection were assessed. RESULTS: Prior irradiation led to a dose-dependent failure to regain body weight after infection and exacerbated mortality, but it did not affect virus-specific immune responses or virus clearance. Surviving irradiated animals displayed a persistent increase in total protein in bronchoalveolar lavage fluid and edema. CONCLUSIONS: Lung irradiation increased susceptibility to death after infection with influenza virus and impaired the ability to complete recovery. This altered response does not seem to be due to a radiation effect on the immune response, but it may possibly be an effect on epithelial repair.

Neonatal oxygen increases sensitivity to influenza A virus infection in adult mice by suppressing epithelial expression of Ear1.

Oxygen exposure in premature infants is a major risk factor for bronchopulmonary dysplasia and can impair the host response to respiratory viral infections later in life. Similarly, adult mice exposed to hyperoxia as neonates display alveolar simplification associated with a reduced number of alveolar epithelial type II cells and exhibit persistent inflammation, fibrosis, and mortality when infected with influenza A virus. Because type II cells participate in innate immunity and alveolar repair, their loss may contribute to oxygen-mediated sensitivity to viral infection. A genomewide screening of type II cells identified eosinophil-associated RNase 1 (Ear1). Ear1 was also detected in airway epithelium and was reduced in lungs of mice exposed to neonatal hyperoxia. Electroporation-mediated gene delivery of Ear1 to the lung before infection successfully reduced viral replication and leukocyte recruitment during infection. It also diminished the enhanced morbidity and mortality attributed to neonatal hyperoxia. These findings demonstrate that novel epithelial expression of Ear1 functions to limit influenza A virus infection, and its loss contributes to oxygen-associated epithelial injury and fibrosis after infection. People born prematurely may have defects in epithelial innate immunity that increase their risk for respiratory viral infections.

Micronutrient levels in children exposed to secondhand tobacco smoke.

INTRODUCTION: Antioxidant micronutrients are the body's primary defense against the oxidative stress of secondhand smoke (SHS). Micronutrient levels have been associated with lung function; decreased levels of vitamin C and ß-carotene have been associated with SHS exposure in children. We sought to determine the association between SHS exposure and micronutrient levels in children. METHODS: Data from the 2003-2004 National Health and Nutrition Examination Survey (NHANES) were analyzed. Serum cotinine levels were categorized into no (<0.015 ng/mL), moderate (0.015 to <2.0 ng/mL), and high (2.0-15.0 ng/mL) smoke exposure; t-tests determined associations between exposure and levels of micronutrients. Significant bivariate associations were tested further using linear regression. RESULTS: In all, 2,218 children, aged 6-18 years, were included (response rate of 82%); 17% had no, 76% moderate, and 7% high exposure. Children with no exposure had higher levels of vitamin A, C, and E, cis- and trans-ß-carotene, and folate, while levels of vitamins B(6), B(12), and D did not differ. In regression analysis, higher cotinine levels were negatively associated with levels of vitamin C (ß = -.03; p < .01), cis-ß-carotene (ß = -.04; p < .01), trans-ß-carotene (ß = -.7; p < .01), folate (ß = -.5; p < .001) and vitamin A (ß = -.6; p < .01). CONCLUSIONS: Children exposed to SHS have lower levels of antioxidants controlling for dietary and supplement intake. This antioxidant depletion may increase systemic inflammation and sensitivity to other oxidant stresses. Parents should be counseled on these specific risks from SHS exposure for their children, and the importance of smoking cessation and eliminating children's exposure to tobacco smoke.