IL-17A neutralization fails to attenuate airway remodeling and potentiates a proinflammatory lung microenvironment in diacetyl-exposed rats.

Bronchiolitis obliterans (BO) is a devastating lung disease that can develop following inhalation exposure to certain chemicals. Diacetyl (DA) is one chemical commonly associated with BO development when inhaled at occupational levels. Previous studies in rats have shown that repetitive DA vapor exposures increased lung CD4+CD25+ T cells and bronchoalveolar (BAL) interleukin-17A (IL-17A) concentrations concurrent with the development of airway remodeling. We hypothesized that IL-17A neutralization would attenuate the severity of airway remodeling after repetitive DA vapor exposures. Sprague-Dawley rats were exposed to 200 parts-per-million DA vapor or filtered air (RA) for 6 h/day × 5 days and monitored for 2 wk postexposure. Treatment with IL-17A neutralization (αIL-17A) or IgG (control) began immediately following exposures and continued twice weekly until study's end. Lungs were harvested for histology, flow cytometry, and BAL analyses. Survival, oxygen saturations, and percent weight change decreased significantly in DA-exposed versus RA-exposed rats, but did not differ significantly between DA + αIL-17A versus DA + IgG. Similarly, the number nor severity of airway lesions did not differ significantly between DA + αIL-17A versus DA + IgG rats despite the percentage of lung regulatory T cells increasing with decreased BAL IL-17A concentrations. Ashcroft scoring of the distal lung parenchyma suggested worse parenchymal remodeling in DA + αIL-17A versus DA + IgG rats with increased expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and nuclear factor-kappa B (NF-κB). Collectively, IL-17A neutralization in DA-exposed rats failed to attenuate airway remodeling with increased expression of pro-inflammatory cytokines TNF-α, IL-1ß, and NF-κB.NEW & NOTEWORTHY Interleukin-17A (IL-17A) neutralization has shown benefit previously in preclinical models of transplant-associated bronchiolitis obliterans (BO), yet it remains unknown whether IL-17A neutralization has similar benefit for other forms of BO. Here, IL-17A neutralization fails to prevent severe airway remodeling in rats exposed repetitively to the flavoring chemical diacetyl, and instead, promotes a proinflammatory microenvironment with increased expression of TNF-α, IL-1ß, and NF-κB within the lung.

Influence of the irradiated pulmonary microenvironment on macrophage and T cell dynamics.

BACKGROUND: The lung is sensitive to radiation, increasing normal tissue toxicity risks following radiation therapy. Adverse outcomes include pneumonitis and pulmonary fibrosis, which result from dysregulated intercellular communication within the pulmonary microenvironment. Although macrophages are implicated in these pathogenic outcomes, the impact of their microenvironment is not well understood. MATERIALS AND METHODS: C57BL/6J mice received 6Gyx5 irradiation to the right lung. Macrophage and T cell dynamics were investigated in ipsilateral right lungs, contralateral left lungs and non-irradiated control lungs 4-26wk post exposure. Lungs were evaluated by flow cytometry, histology and proteomics. RESULTS: Following uni-lung irradiation, focal regions of macrophage accumulation were noted in both lungs by 8wk, however by 26wk fibrotic lesions were observed only in ipsilateral lungs. Infiltrating and alveolar macrophages populations expanded in both lungs, however transitional CD11b + alveolar macrophages persisted only in ipsilateral lungs and expressed lower CD206. Concurrently, arginase-1 + macrophages accumulated in ipsilateral but not contralateral lungs at 8 and 26wk post exposure, while CD206 + macrophages were absent from these accumulations. While radiation expanded CD8 + T cells in both lungs, T regulatory cells only increased in ipsilateral lungs. Unbiased proteomics analysis of immune cells revealed a substantial number of differentially expressed proteins in ipsilateral lungs when compared to contralateral lungs and both differed from non-irradiated controls. CONCLUSIONS: Pulmonary macrophage and T cell dynamics are impacted by the microenvironmental conditions that develop following radiation exposure, both locally and systemically. While macrophages and T cells infiltrate and expand in both lungs, they diverge phenotypically depending on their environment.

Using Serum Cystatin C to Predict Acute Kidney Injury Following Infant Cardiac Surgery.

Acute kidney injury (AKI) following cardiopulmonary bypass (CPB) is associated with increased morbidity and mortality. Serum Cystatin C (CysC) is a novel biomarker synthesized by all nucleated cells that may act as an early indicator of AKI following infant CPB. Prospective observational study of infants (< 1 year) requiring CPB during cardiac surgery. CysC was measured at baseline and 12, 24, 48, and 72 h following CPB initiation. Each post-op percent difference in CysC (e.g. %CysC12h) from baseline was calculated. Clinical variables along with urine output (UOP) and serum creatinine (SCr) were followed. Subjects were divided into two groups: AKI and non-AKI based upon the Kidney Disease Improving Global Outcomes (KDIGO) classification. AKI occurred in 41.9% (18) of the 43 infants enrolled. Patient demographics and baseline CysC levels were similar between groups. CysC levels were 0.97 ± 0.28 mg/L over the study period, and directly correlated with SCr (R = 0.71, p < 0.0001). Although absolute CysC levels were not significant between groups, the %CysC12h was significantly greater in the AKI group (AKI: - 16% ± 22% vs. Non-AKI - 28% ± 9% mg/L; p = 0.003). However, multivariate analysis demonstrated that a lower UOP (Odds Ratio:0.298; 95% CI 0.073, 0.850; p = 0.02) but not %CysC12h was independently associated with AKI. Despite a significant difference in the %CysC12h, only UOP was independently associated with AKI. Larger studies of a more homogenous population are needed to understand these results and to explore the variability in this biomarker seen across institutions.

Prevention of Radiation-Induced Bladder Injury: A Murine Study Using Captopril.

PURPOSE: Pelvic radiation therapy (RT) can cause debilitating bladder toxicities but few clinical interventions exist to prevent injury or alleviate symptoms. From a large genome-wide association study in patients with prostate cancer it was previously reported that SNPs tagging AGT, part of the renin-angiotensin system (RAS), correlated with patient-reported late hematuria, identifying a potential targetable pathway to prevent RT-induced bladder injury. To investigate this association, we performed a preclinical study to determine whether RAS modulation protected the bladder against RT injury. METHODS AND MATERIALS: C57BL/6 male mice were treated with an oral angiotensin converting enzyme inhibitor (ACEi: 0.3g/L captopril) 5 days before focal bladder X-irradiation with either single dose (SD) 30 Gy or 3 fractions of 8 Gy (8 Gy × 3 in 5 days). RT was delivered using XStrahl SARRP Muriplan CT-image guidance with parallel-opposed lateral beams. ACEi was maintained for 20 weeks post RT. Bladder toxicity was assessed using assays to identify local injury that included urinalysis, functional micturition, bladder-released exosomes, and histopathology, as well as an assessment of systemic changes in inflammatory-mediated circulating immune cells. RESULTS: SD and fractionated RT increased urinary frequency and reduced the volume of individual voids at >14 weeks, but not at 4 weeks, compared with nonirradiated animals. Urothelial layer width was positively correlated with mean volume of individual voids (P = .0428) and negatively correlated with number of voids (P = .028), relating urothelial thinning to changes in RT-mediated bladder dysfunction. These chronic RT-induced changes in micturition patterns were prevented by captopril treatment. Focal bladder irradiation significantly increased the mean particle count of urine extracellular vesicles and the monocyte and neutrophil chemokines CCL2 and MIP-2, and the proportions of circulating inflammatory-mediated neutrophils and monocytes, which was also prevented by captopril. Exploratory transcriptomic analysis of bladder tissue implicated inflammatory and erythropoietic pathways. CONCLUSIONS: This study demonstrated that systemic modulation of the RAS protected against and alleviated RT-induced late bladder injury but larger confirmatory studies are needed.

Neonatal hypoxic ischemic encephalopathy increases acute kidney injury urinary biomarkers in a rat model.

Hypoxic ischemic encephalopathy (HIE) is associated with acute kidney injury (AKI) in neonates with birth asphyxia. This study aimed to utilize urinary biomarkers to characterize AKI in an established neonatal rat model of HIE. Day 7 Sprague-Dawley rat pups underwent HIE using the Rice-Vannucci model (unilateral carotid ligation followed by 120 mins of 8% oxygen). Controls included no surgery and sham surgery. Weights and urine for biomarkers (NGAL, osteopontin, KIM-1, albumin) were collected the day prior, daily for 3 days post-intervention, and at sacrifice day 14. Kidneys and brains were processed for histology. HIE pups displayed histological evidence of kidney injury including damage to the proximal tubules, consistent with resolving acute tubular necrosis, and had significantly elevated urinary levels of NGAL and albumin compared to sham or controls 1-day post-insult that elevated for 3 days. KIM-1 significantly increased for 2 days post-HIE. HIE did not significantly alter osteopontin levels. Seven days post-start of experiment, controls were 81.2% above starting weight compared to 52.1% in HIE pups. NGAL and albumin levels inversely correlated with body weight following HIE injury. The AKI produced by the Rice-Vannucci HIE model is detectable by urinary biomarkers, which can be used for future studies of treatments to reduce kidney injury.

Diacetyl Vapor Inhalation Induces Mixed, Granulocytic Lung Inflammation with Increased CD4+CD25+ T Cells in the Rat.

Diacetyl (DA) is a highly reactive alpha diketone associated with flavoring-related lung disease. In rodents, acute DA vapor exposure can initiate an airway-centric, inflammatory response. However, this immune response has yet to be fully characterized in the context of flavoring-related lung disease progression. The following studies were designed to characterize the different T cell populations within the lung following repetitive DA vapor exposures. Sprague-Dawley rats were exposed to 200 parts-per-million DA vapor for 5 consecutive days × 6 h/day. Lung tissue and bronchoalveolar lavage fluid (BALF) were analyzed for changes in histology by H&E and Trichrome stain, T cell markers by flow cytometry, total BALF cell counts and differentials, BALF IL17a and total protein immediately, 1 and 2 weeks post-exposure. Lung histology and BALF cell composition demonstrated mixed, granulocytic lung inflammation with bronchial lymphoid aggregates at all time points in DA-exposed lungs compared to air controls. While no significant change was seen in percent lung CD3+, CD4+, or CD8+ T cells, a significant increase in lung CD4+CD25+ T cells developed at 1 week that persisted at 2 weeks post-exposure. Further characterization of this CD4+CD25+ T cell population identified Foxp3+ T cells at 1 week that failed to persist at 2 weeks. Conversely, BALF IL-17a increased significantly at 2 weeks in DA-exposed rats compared to air controls. Lung CD4+CD25+ T cells and BALF IL17a correlated directly with BALF total protein and inversely with rat oxygen saturations. Repetitive DA vapor exposure at occupationally relevant concentrations induced mixed, granulocytic lung inflammation with increased CD4+CD25+ T cells in the rat lung.

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.

Saving normal tissues - a goal for the ages.

Almost since the earliest utilization of ionizing radiation, many within the radiation community have worked toward either preventing (i.e. protecting) normal tissues from unwanted radiation injury or rescuing them from the downstream consequences of exposure. However, despite over a century of such investigations, only incremental gains have been made toward this goal and, with certainty, no outright panacea having been found. In celebration of the 60th anniversary of the International Journal of Radiation Biology and to chronicle the efforts that have been made to date, we undertook a non-rigorous survey of the articles published by normal tissue researchers in this area, using those that have appeared in the aforementioned journal as a road map. Three 'snapshots' of publications on normal tissue countermeasures were taken: the earliest (1959-1963) and most recent (2013-2018) 5-year of issues, as well as a 5-year intermediate span (1987-1991). Limiting the survey solely to articles appearing within International Journal of Radiation Biology likely reduced the number of translational studies interrogated given the basic science tenor of this particular publication. In addition, by taking 'snapshots' rather than considering the entire breadth of the journal's history in this field, important papers that were published during the interim periods were omitted, for which we apologize. Nonetheless, since the journal's inception, we observed that, during the chosen periods, the majority of studies undertaken in the field of normal tissue countermeasures, whether investigating radiation protectants, mitigators or treatments, have focused on agents that interfere with the physical, chemical and/or biological effects known to occur during the acute period following whole body/high single dose exposures. This relatively narrow approach to the reduction of normal tissue effects, especially those that can take months, if not years, to develop, seems to contradict our growing understanding of the progressive complexities of the microenvironmental disruption that follows the initial radiation injury. Given the analytical tools now at our disposal and the enormous benefits that may be reaped in terms of improving patient outcomes, as well as the potential for offering countermeasures to those affected by accidental or mass casualty exposures, it appears time to broaden our approaches to developing normal tissue countermeasures. We have no doubt that the contributors and readership of the International Journal of Radiation Biology will continue to contribute to this effort for the foreseeable future.

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.

Histologic and biochemical alterations predict pulmonary mechanical dysfunction in aging mice with chronic lung inflammation.

Both aging and chronic inflammation produce complex structural and biochemical alterations to the lung known to impact work of breathing. Mice deficient in surfactant protein D (Sftpd) develop progressive age-related lung pathology characterized by tissue destruction/remodeling, accumulation of foamy macrophages and alteration in surfactant composition. This study proposes to relate changes in tissue structure seen in normal aging and in chronic inflammation to altered lung mechanics using a computational model. Alterations in lung function in aging and Sftpd -/- mice have been inferred from fitting simple mechanical models to respiratory impedance data (Zrs), however interpretation has been confounded by the simultaneous presence of multiple coexisting pathophysiologic processes. In contrast to the inverse modeling approach, this study uses simulation from experimental measurements to recapitulate how aging and inflammation alter Zrs. Histologic and mechanical measurements were made in C57BL6/J mice and congenic Sftpd-/- mice at 8, 27 and 80 weeks of age (n = 8/group). An anatomic computational model based on published airway morphometry was developed and Zrs was simulated between 0.5 and 20 Hz. End expiratory pressure dependent changes in airway caliber and recruitment were estimated from mechanical measurements. Tissue elements were simulated using the constant phase model of viscoelasticity. Baseline elastance distribution was estimated in 8-week-old wild type mice, and stochastically varied for each condition based on experimentally measured alteration in elastic fiber composition, alveolar geometry and surfactant composition. Weighing reduction in model error against increasing model complexity allowed for identification of essential features underlying mechanical pathology and their contribution to Zrs. Using a maximum likelihood approach, alteration in lung recruitment and diminished elastic fiber density were shown predictive of mechanical alteration at airway opening, to a greater extent than overt acinar wall destruction. Model-predicted deficits in PEEP-dependent lung recruitment correlate with altered lung lining fluid composition independent of age or genotype.

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.

Editor's Highlight: CCR2 Regulates Inflammatory Cell Accumulation in the Lung and Tissue Injury following Ozone Exposure.

Ozone-induced lung injury is associated with an accumulation of activated macrophages in the lung. Chemokine receptor CCR2 mediates the migration of inflammatory monocytes/macrophages to sites of tissue injury. It is also required for monocyte egress from the bone marrow. In the present studies, we analyzed the role of CCR2 in inflammatory cell trafficking to the lung in response to ozone. Treatment of mice with ozone (0.8 ppm, 3 h) resulted in increases in proinflammatory CCR2+ macrophages in the lung at 24 h, as well as proinflammatory CD11b + Ly6CHi and iNOS+ macrophages at 24 and 48 h. Mannose receptor+ anti-inflammatory macrophages were also observed in the lung 24 and 48 h post-ozone. Loss of CCR2 was associated with reduced numbers of proinflammatory macrophages in the lung and decreased expression of the proinflammatory cytokines, IL-1ß and TNFα. Decreases in anti-inflammatory CD11b + Ly6CLo macrophages were also observed in lungs of CCR2-/- mice treated with ozone, whereas mannose receptor+ macrophage accumulation was delayed; conversely, CX3CL1 and CX3CR1 were upregulated. Changes in lung macrophage subpopulations and inflammatory gene expression in CCR2-/- mice were correlated with reduced ozone toxicity and oxidative stress, as measured by decreases in bronchoalveolar lavage protein content and reduced lung expression of heme-oxygenase-1, 4-hydroxynonenal and cytochrome b5. These data demonstrate that CCR2 plays a role in both pro- and anti-inflammatory macrophage accumulation in the lung following ozone exposure. The fact that ozone-induced lung injury and oxidative stress are reduced in CCR2-/- mice suggests more prominent effects on proinflammatory macrophages.

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.

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.

Age-related increases in ozone-induced injury and altered pulmonary mechanics in mice with progressive lung inflammation.

In these studies we determined whether progressive pulmonary inflammation associated with aging in surfactant protein D (Sftpd)-/- mice leads to an exacerbated response to ozone. In Sftpd-/- mice, but not wild-type (WT) mice, age-related increases in numbers of enlarged vacuolated macrophages were observed in the lung, along with alveolar wall rupture, type 2 cell hyperplasia, and increased bronchoalveolar lavage protein and cell content. Numbers of heme oxygenase+ macrophages also increased with age in Sftpd-/- mice, together with classically (iNOS+) and alternatively (mannose receptor+, YM-1+, or galectin-3+) activated macrophages. In both WT and Sftpd-/- mice, increasing age from 8 to 27 wk was associated with reduced lung stiffness, as reflected by decreases in resistance and elastance spectra; however, this response was reversed in 80-wk-old Sftpd-/- mice. Ozone exposure (0.8 ppm, 3 h) caused increases in lung pathology, alveolar epithelial barrier dysfunction, and numbers of iNOS+ macrophages in 8- and 27-wk-old Sftpd-/-, but not WT mice at 72 h postexposure. Conversely, increases in alternatively activated macrophages were observed in 8-wk-old WT mice following ozone exposure, but not in Sftpd-/- mice. Ozone also caused alterations in both airway and tissue mechanics in Sftpd-/- mice at 8 and 27 wk, but not at 80 wk. These data demonstrate that mild to moderate pulmonary inflammation results in increased sensitivity to ozone; however, in senescent mice, these responses are overwhelmed by the larger effects of age-related increases in baseline inflammation and lung injury.

Prolonged injury and altered lung function after ozone inhalation in mice with chronic lung inflammation.

Surfactant protein-D (Sftpd) is a pulmonary collectin important in down-regulating macrophage inflammatory responses. In these experiments, we analyzed the effects of chronic macrophage inflammation attributable to loss of Sftpd on the persistence of ozone-induced injury, macrophage activation, and altered functioning in the lung. Wild-type (Sftpd(+/+)) and Sftpd(-/-) mice (aged 8 wk) were exposed to air or ozone (0.8 parts per million, 3 h). Bronchoalveolar lavage (BAL) fluid and tissue were collected 72 hours later. In Sftpd(-/-) mice, but not Sftpd(+/+) mice, increased BAL protein and nitrogen oxides were observed after ozone inhalation, indicating prolonged lung injury and oxidative stress. Increased numbers of macrophages were also present in BAL fluid and in histologic sections from Sftpd(-/-) mice. These cells were enlarged and foamy, suggesting that they were activated. This conclusion was supported by findings of increased BAL chemotactic activity, and increased expression of inducible nitric oxide synthase in lung macrophages. In both Sftpd(+/+) and Sftpd(-/-) mice, inhalation of ozone was associated with functional alterations in the lung. Although these alterations were limited to central airway mechanics in Sftpd(+/+) mice, both central airway and parenchymal mechanics were modified by ozone exposure in Sftpd(-/-) mice. The most notable changes were evident in resistance and elastance spectra and baseline lung function, and in lung responsiveness to changes in positive end-expiratory pressure. These data demonstrate that a loss of Sftpd is associated with prolonged lung injury, oxidative stress, and macrophage accumulation and activation in response to ozone, and with more extensive functional changes consistent with the loss of parenchymal integrity.