Christopher B. Massa

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.

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.

Pentoxifylline attenuates nitrogen mustard-induced acute lung injury, oxidative stress and inflammation

Nitrogen mustard (NM) is a toxic alkylating agent that causes damage to the respiratory tract. Evidence suggests that macrophages and inflammatory mediators including tumor necrosis factor (TNF)α contribute to pulmonary injury. Pentoxifylline is a TNFα inhibitor known to suppress inflammation. In these studies, we analyzed the ability of pentoxifylline to mitigate NM-induced lung injury and inflammation. Exposure of male Wistar rats (150-174 g; 8-10 weeks) to NM (0.125 mg/kg, i.t.) resulted in severe histopathological changes in the lung within 3d of exposure, along with increases in bronchoalveolar lavage (BAL) cell number and protein, indicating inflammation and alveolar-epithelial barrier dysfunction. This was associated with increases in oxidative stress proteins including lipocalin (Lcn)2 and heme oxygenase (HO)-1 in the lung, along with pro-inflammatory/cytotoxic (COX-2(+) and MMP-9(+)), and anti-inflammatory/wound repair (CD163+ and Gal-3(+)) macrophages. Treatment of rats with pentoxifylline (46.7 mg/kg, i.p.) daily for 3d beginning 15 min after NM significantly reduced NM-induced lung injury, inflammation, and oxidative stress, as measured histologically and by decreases in BAL cell and protein content, and levels of HO-1 and Lcn2. Macrophages expressing COX-2 and MMP-9 also decreased after pentoxifylline, while CD163+ and Gal-3(+) macrophages increased. This was correlated with persistent upregulation of markers of wound repair including pro-surfactant protein-C and proliferating nuclear cell antigen by Type II cells. NM-induced lung injury and inflammation were associated with alterations in the elastic properties of the lung, however these were largely unaltered by pentoxifylline. These data suggest that pentoxifylline may be useful in treating acute lung injury, inflammation and oxidative stress induced by vesicants.

Acute chlorine gas exposure produces transient inflammation and a progressive alteration in surfactant composition with accompanying mechanical dysfunction

Acute Cl2 exposure following industrial accidents or military/terrorist activity causes pulmonary injury and severe acute respiratory distress. Prior studies suggest that antioxidant depletion is important in producing dysfunction, however a pathophysiologic mechanism has not been elucidated. We propose that acute Cl2 inhalation leads to oxidative modification of lung lining fluid, producing surfactant inactivation, inflammation and mechanical respiratory dysfunction at the organ level. C57BL/6J mice underwent whole-body exposure to an effective 60ppm-hour Cl2 dose, and were euthanized 3, 24 and 48h later. Whereas pulmonary architecture and endothelial barrier function were preserved, transient neutrophilia, peaking at 24h, was noted. Increased expression of ARG1, CCL2, RETLNA, IL-1b, and PTGS2 genes was observed in bronchoalveolar lavage (BAL) cells with peak change in all genes at 24h. Cl2 exposure had no effect on NOS2 mRNA or iNOS protein expression, nor on BAL NO3(-) or NO2(-). Expression of the alternative macrophage activation markers, Relm-α and mannose receptor was increased in alveolar macrophages and pulmonary epithelium. Capillary surfactometry demonstrated impaired surfactant function, and altered BAL phospholipid and surfactant protein content following exposure. Organ level respiratory function was assessed by forced oscillation technique at 5 end expiratory pressures. Cl2 exposure had no significant effect on either airway or tissue resistance. Pulmonary elastance was elevated with time following exposure and demonstrated PEEP refractory derecruitment at 48h, despite waning inflammation. These data support a role for surfactant inactivation as a physiologic mechanism underlying respiratory dysfunction following Cl2 inhalation.

Low-dose AgNPs reduce lung mechanical function and innate immune defense in the absence of cellular toxicity

Abstract Multiple studies have examined the direct cellular toxicity of silver nanoparticles (AgNPs). However, the lung is a complex biological system with multiple cell types and a lipid-rich surface fluid; therefore, organ level responses may not depend on direct cellular toxicity. We hypothesized that interaction with the lung lining is a critical determinant of organ level responses. Here, we have examined the effects of low dose intratracheal instillation of AgNPs (0.05 μg/g body weight) 20 and 110 nm diameter in size, and functionalized with citrate or polyvinylpyrrolidone. Both size and functionalization were significant factors in particle aggregation and lipid interaction in vitro. One day post-intratracheal instillation lung function was assessed, and bronchoalveolar lavage (BAL) and lung tissue collected. There were no signs of overt inflammation. There was no change in surfactant protein-B content in the BAL but there was loss of surfactant protein-D with polyvinylpyrrolidone (PVP)-stabilized particles. Mechanical impedance data demonstrated a significant increase in pulmonary elastance as compared to control, greatest with 110 nm PVP-stabilized particles. Seven days post-instillation of PVP-stabilized particles increased BAL cell counts, and reduced lung function was observed. These changes resolved by 21 days. Hence, AgNP-mediated alterations in the lung lining and mechanical function resolve by 21 days. Larger particles and PVP stabilization produce the largest disruptions. These studies demonstrate that low dose AgNPs elicit deficits in both mechanical and innate immune defense function, suggesting that organ level toxicity should be considered.

The role of inducible nitric oxide synthase for interstitial remodeling of alveolar septa in surfactant protein D-deficient mice.

Surfactant protein D (SP-D) modulates the lungs immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd(-/-)) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd(-/-) mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd(-/-) mice. These changes were reduced in DiNOS, and compared with Sftpd(-/-) mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd(-/-). Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces.

Toxicodynamics of Rigid Polystyrene Microparticles on Pulmonary Gas Exchange in Mice: Implications for Microemboli-based Drug Delivery Systems

The toxicodynamic relationship between the number and size of pulmonary microemboli resulting from uniformly sized, rigid polystyrene microparticles (MPs) administered intravenously and their potential effects on pulmonary gas exchange were investigated. CD-1 male mice (6-8 weeks) were intravenously administered 10, 25 and 45 μm diameter MPs. Oxygen hemoglobin saturation in the blood (SpO(2)) was measured non-invasively using a pulse oximeter while varying inhaled oxygen concentration (F(I)O(2)). The resulting data were fit to a physiologically based non-linear mathematical model that estimates 2 parameters: ventilation-perfusion ratio (V(A)/Q) and shunt (percentage of deoxygenated blood returning to systemic circulation). The number of MPs administered prior to a statistically significant reduction in normalized V(A)/Q was dependent on particle size. MP doses that resulted in a significant reduction in normalized V(A)/Q one day post-treatment were 4000, 40,000 and 550,000 MPs/g for 45, 25 and 10 μm MPs, respectively. The model estimated V(A)/Q and shunt returned to baseline levels 7 days post-treatment. Measuring SpO(2) alone was not sufficient to observe changes in gas exchange; however, when combined with model-derived V(A)/Q and shunt early reversible toxicity from pulmonary microemboli was detected suggesting that the model and physical measurements are both required for assessing toxicity. Moreover, it appears that the MP load required to alter gas exchange in a mouse prior to lethality is significantly higher than the anticipated required MP dose for effective drug delivery. Overall, the current results indicate that the microemboli-based approach for targeted pulmonary drug delivery is potentially safe and should be further explored.

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.

World Trade Center (WTC) dust exposure in mice is associated with inflammation, oxidative stress and epigenetic changes in the lung

Exposure to World Trade Center (WTC) dust has been linked to respiratory disease in humans. In the present studies we developed a rodent model of WTC dust exposure to analyze lung oxidative stress and inflammation, with the goal of elucidating potential epigenetic mechanisms underlying these responses. Exposure of mice to WTC dust (20μg, i.t.) was associated with upregulation of heme oxygenase-1 and cyclooxygenase-2 within 3days, a response which persisted for at least 21days. Whereas matrix metalloproteinase was upregulated 7days post-WTC dust exposure, IL-6RA1 was increased at 21days; conversely, expression of mannose receptor, a scavenger receptor important in particle clearance, decreased. After WTC dust exposure, increases in methylation of histone H3 lysine K4 at 3days, lysine K27 at 7days and lysine K36, were observed in the lung, along with hypermethylation of Line-1 element at 21days. Alterations in pulmonary mechanics were also observed following WTC dust exposure. Thus, 3days post-exposure, lung resistance and tissue damping were decreased. In contrast at 21days, lung resistance, central airway resistance, tissue damping and tissue elastance were increased. These data demonstrate that WTC dust-induced inflammation and oxidative stress are associated with epigenetic modifications in the lung and altered pulmonary mechanics. These changes may contribute to the development of WTC dust pathologies.

Exposure to Silver Nanospheres Leads to Altered Respiratory Mechanics and Delayed Immune Response in an in Vivo Murine Model

Here we examine the organ level toxicology of both carbon black (CB) and silver nanoparticles (AgNP). We aim to determine metal-specific effects to respiratory function, inflammation and potential interactions with lung lining fluid (LLF). C57Bl6/J male mice were intratracheally instilled with saline (control), low (0.05 μg/g) or high (0.5 μg/g) doses of either AgNP or CB 15 nm nanospheres. Lung histology, cytology, surfactant composition and function, inflammatory gene expression, and pulmonary function were measured at 1, 3, and 7 days post-exposure. Acutely, high dose CB resulted in an inflammatory response, increased neutrophilia and cytokine production, without alteration in surfactant composition or respiratory mechanics. Low dose CB had no effect. Neither low nor high dose AgNPs resulted in an acute inflammatory response, but there was an increase in work of breathing. Three days post-exposure with CB, a persistent neutrophilia was noted. High dose AgNP resulted in an elevated number of macrophages and invasion of lymphocytes. Additionally, AgNP treated mice displayed increased expression of IL1B, IL6, CCL2, and IL10. However, there were no significant changes in respiratory mechanics. At day 7, inflammation had resolved in AgNP-treated mice, but tissue stiffness and resistance were significantly decreased, which was accompanied by an increase in surfactant protein D (SP-D) content. These data demonstrate that the presence of metal alters the response of the lung to nanoparticle exposure. AgNP-surfactant interactions may alter respiratory function and result in a delayed immune response, potentially due to modified airway epithelial cell function.