Lori A. Bramble

The adjuvant effect of ambient particulate matter is closely reflected by the particulate oxidant potential

Background It has been demonstrated that ambient particulate matter (PM) can act as an adjuvant for allergic sensitization. Redox-active organic chemicals on the particle surface play an important role in PM adverse health effects and may determine the adjuvant effect of different particle types according to their potential to perturb redox equilibrium in the immune system. Objectives We determined whether the adjuvant effect of ambient fine particles versus ultrafine particles (UFPs) is correlated to their prooxidant potential. Methods We have established an intranasal sensitization model that uses ambient PM as a potential adjuvant for sensitization to ovalbumin (OVA), which enhances the capacity for secondary OVA challenge to induce allergic airway inflammation. Results UFPs with a greater polycyclic aromatic hydrocarbon (PAH) content and higher oxidant potential enhanced OVA sensitization more readily than did fine particles. This manifests as enhanced allergic inflammation upon secondary OVA challenge, leading to eosinophilic inflammation and mucoid hyperplasia starting at the nasal turbinates all the way down to the small pulmonary airways. The thiol antioxidant N-acetyl cysteine was able to suppress some of these sensitization events. Conclusions The adjuvant effects of ambient UFP is determined by their oxidant potential, which likely plays a role in changing the redox equilibrium in the mucosal immune system.

Interlaboratory Evaluation of Rodent Pulmonary Responses to Engineered Nanomaterials: The NIEHS Nano GO Consortium

Background: Engineered nanomaterials (ENMs) have potential benefits, but they also present safety concerns for human health. Interlaboratory studies in rodents using standardized protocols are needed to assess ENM toxicity. Methods: Four laboratories evaluated lung responses in C57BL/6 mice to ENMs delivered by oropharyngeal aspiration (OPA), and three labs evaluated Sprague-Dawley (SD) or Fisher 344 (F344) rats following intratracheal instillation (IT). ENMs tested included three forms of titanium dioxide (TiO2) [anatase/rutile spheres (TiO2-P25), anatase spheres (TiO2-A), and anatase nanobelts (TiO2-NBs)] and three forms of multiwalled carbon nanotubes (MWCNTs) [original (O), purified (P), and carboxylic acid “functionalized” (F)]. One day after treatment, bronchoalveolar lavage fluid was collected to determine differential cell counts, lactate dehydrogenase (LDH), and protein. Lungs were fixed for histopathology. Responses were also examined at 7 days (TiO2 forms) and 21 days (MWCNTs) after treatment. Results: TiO2-A, TiO2-P25, and TiO2-NB caused significant neutrophilia in mice at 1 day in three of four labs. TiO2-NB caused neutrophilia in rats at 1 day in two of three labs, and TiO2-P25 and TiO2-A had no significant effect in any of the labs. Inflammation induced by TiO2 in mice and rats resolved by day 7. All MWCNT types caused neutrophilia at 1 day in three of four mouse labs and in all rat labs. Three of four labs observed similar histopathology to O-MWCNTs and TiO2-NBs in mice. Conclusions: ENMs produced similar patterns of neutrophilia and pathology in rats and mice. Although interlaboratory variability was found in the degree of neutrophilia caused by the three types of TiO2 nanoparticles, similar findings of relative potency for the three types of MWCNTs were found across all laboratories, thus providing greater confidence in these interlaboratory comparisons.

Ambient particulate air pollution induces oxidative stress and alterations of mitochondria and gene expression in brown and white adipose tissues

BackgroundPrior studies have demonstrated a link between air pollution and metabolic diseases such as type II diabetes. Changes in adipose tissue and its mitochondrial content/function are closely associated with the development of insulin resistance and attendant metabolic complications. We investigated changes in adipose tissue structure and function in brown and white adipose depots in response to chronic ambient air pollutant exposure in a rodent model.MethodsMale ApoE knockout (ApoE-/-) mice inhaled concentrated fine ambient PM (PM < 2.5 μm in aerodynamic diameter; PM2.5) or filtered air (FA) for 6 hours/day, 5 days/week, for 2 months. We examined superoxide production by dihydroethidium staining; inflammatory responses by immunohistochemistry; and changes in white and brown adipocyte-specific gene profiles by real-time PCR and mitochondria by transmission electron microscopy in response to PM2.5 exposure in different adipose depots of ApoE-/- mice to understand responses to chronic inhalational stimuli.ResultsExposure to PM2.5 induced an increase in the production of reactive oxygen species (ROS) in brown adipose depots. Additionally, exposure to PM2.5 decreased expression of uncoupling protein 1 in brown adipose tissue as measured by immunohistochemistry and Western blot. Mitochondrial number was significantly reduced in white (WAT) and brown adipose tissues (BAT), while mitochondrial size was also reduced in BAT. In BAT, PM2.5 exposure down-regulated brown adipocyte-specific genes, while white adipocyte-specific genes were differentially up-regulated.ConclusionsPM2.5 exposure triggers oxidative stress in BAT, and results in key alterations in mitochondrial gene expression and mitochondrial alterations that are pronounced in BAT. We postulate that exposure to PM2.5 may induce imbalance between white and brown adipose tissue functionality and thereby predispose to metabolic dysfunction.

Ambient ultrafine particles provide a strong adjuvant effect in the secondary immune response: implication for traffic-related asthma flares.

We have previously demonstrated that intranasal administration of ambient ultrafine particles (UFP) acts as an adjuvant for primary allergic sensitization to ovalbumin (OVA) in Balb/c mice. It is important to find out whether inhaled UFP exert the same effect on the secondary immune response as a way of explaining asthma flares in already-sensitized individuals due to traffic exposure near a freeway. The objective of this study is to determine whether inhalation exposure to ambient UFP near an urban freeway could enhance the secondary immune response to OVA in already-sensitized mice. Prior OVA-sensitized animals were exposed to concentrated ambient UFP at the time of secondary OVA challenge in our mobile animal laboratory in Los Angeles. OVA-specific antibody production, airway morphometry, allergic airway inflammation, cytokine gene expression, and oxidative stress marker were assessed. As few as five ambient UFP exposures were sufficient to promote the OVA recall immune response, including generating allergic airway inflammation in smaller and more distal airways compared with the adjuvant effect of intranasally instilled UFP on the primary immune response. The secondary immune response was characterized by the T helper 2 and IL-17 cytokine gene expression in the lung. In summary, our results demonstrated that inhalation of prooxidative ambient UFP could effectively boost the secondary immune response to an experimental allergen, indicating that vehicular traffic exposure could exacerbate allergic inflammation in already-sensitized subjects.

DNA-PKcs mutations in dogs and horses: allele frequency and association with neoplasia.

Previously, spontaneous genetic immunodeficiencies in mice, Arabian foals, and recently in Jack Russell terriers have been ascribed to defects in DNA-PKcs (catalytic subunit of the DNA dependent protein kinase) expression. In severe combined immunodeficiency (SCID) foals, a 5 bp deletion at codon 9480 results in a frameshift and a 967 amino acid deletion from the C terminus (including the entire PI3 kinase domain) and an unstable mutant protein. In SCID mice, a single base pair mutation results in a premature stop codon and deletion of 83 amino acids; as in SCID foals, the mutant protein is unstable. Here, we define the mutation within the canine DNA-PKcs gene that results in SCID. In this case, a point mutation results in a stop codon at nucleotide 10,828 and premature termination at a position 517 amino acids before the normal C terminus resulting in a functionally null allele. Thus, this is the third documentation of a spontaneous germline mutation in the C terminus of DNA-PKcs. Emerging data implicate DNA repair factors as potential tumor suppressors. Here, we have ascertained the carrier frequency of the defective DNA-PKcs genes in Arabian horses and in Jack Russell terriers. Our data indicate (in good agreement with a previous report) that the carrier frequency of the equine SCID allele is approximately 8%; in contrast, the carrier frequency of the canine SCID allele is less than 1.1%. We also assessed the frequency of the equine SCID allele in a series of 295 tumors from Arabian horses. We find a statistically significant correlation between the development of a virally induced tumor (sarcoid) and heterozygosity for the equine SCID allele. These data provide further support for an emerging consensus: that DNA-PK may normally act as a tumor suppressor through its caretaker role in maintaining chromosomal stability.

Adjuvant effects of ambient particulate matter monitored by proteomics of bronchoalveolar lavage fluid

Ambient particulate matter (PM) from air pollution is associated with exacerbation of asthma. The immunological basis for the adjuvant effects of PM is still not well understood. The generation of ROS and the resulting oxidative stress has been identified as one of the major mechanisms. Using a new intranasal sensitization model in which ambient PM is used as an adjuvant to enhance allergic inflammation (Li et al., Environ. Health Perspect. 2009, 117, 1116–1123), a proteomics approach was applied to study the adjuvant effects of ambient PM. The enhanced in vivo adjuvant effect of ultrafine particles correlates with a higher in vitro oxidant potential and a higher content of redox‐cycling organic chemicals. Bronchoalveolar lavage fluid proteins from normal and sensitized mice were resolved by 2‐DE, and identified by MS. Polymeric immunoglobulin receptor, complement C3, neutrophil gelatinase‐associated lipocalin, chitinase 3‐like protein 3, chitinase 3‐like protein 4, and acidic mammalian chitinase demonstrated significantly enhanced up‐regulation by UFP with a polycyclic aromatic hydrocarbon content and a higher oxidant potential. These proteins may be the important specific elements targeted by PM in air pollution through the ability to generate ROS in the immune system, and may be involved in allergen sensitization and asthma pathogenesis.

Role of hypoxia-inducible factor 1α in modulating cobalt-induced lung inflammation

Hypoxia plays an important role in development, cellular homeostasis, and pathological conditions, such as cancer and stroke. There is also growing evidence that hypoxia is an important modulator of the inflammatory process. Hypoxia-inducible factors (HIFs) are a family of proteins that regulate the cellular response to oxygen deficit, and loss of HIFs impairs inflammatory cell function. There is little known, however, about the role of epithelial-derived HIF signaling in modulating inflammation. Cobalt is capable of eliciting an allergic response and promoting HIF signaling. To characterize the inflammatory function of epithelial-derived HIF in response to inhaled cobalt, a conditional lung-specific HIF1alpha, the most ubiquitously expressed HIF, deletion mouse, was created. Control mice showed classic signs of metal-induced injury following cobalt exposure, including fibrosis and neutrophil infiltration. In contrast, HIF1alpha-deficient mice displayed a Th2 response that resembled asthma, including increased eosinophilic infiltration, mucus cell metaplasia, and chitinase-like protein expression. The results suggest that epithelial-derived HIF signaling has a critical role in establishing a tissues inflammatory response, and compromised HIF1alpha signaling biases the tissue towards a Th2-mediated reaction.

COMPARATIVE MICROARRAY ANALYSIS AND PULMONARY CHANGES IN BROWN NORWAY RATS EXPOSED TO OVALBUMIN AND CONCENTRATED AIR PARTICULATES

The interaction between air particulates and genetic susceptibility has been implicated in the pathogenesis of asthma. The overall objective of this study was to determine the effects of inhalation exposure to environmentally relevant concentrated air particulates (CAPs) on the lungs of ovalbumin (ova) sensitized and challenged Brown Norway rats. Changes in gene expression were compared with lung tissue histopathology, morphometry, and biochemical and cellular parameters in bronchoalveolar lavage fluid (BALF). Ova challenge was responsible for the preponderance of gene expression changes, related largely to inflammation. CAPs exposure alone resulted in no significant gene expression changes, but CAPs and ova-exposed rodents exhibited an enhanced effect relative to ova alone with differentially expressed genes primarily related to inflammation and airway remodeling. Gene expression data was consistent with the biochemical and cellular analyses of the BALF, the pulmonary pathology, and morphometric changes when comparing the CAPs-ova group to the air-saline or CAPs-saline group. However, the gene expression data were more sensitive than the BALF cell type and number for assessing the effects of CAPs and ova versus the ova challenge alone. In addition, the gene expression results provided some additional insight into the TGF-beta-mediated molecular processes underlying these changes. The broad-based histopathology and functional genomic analyses demonstrate that exposure to CAPs exacerbates rodents with allergic inflammation induced by an allergen and suggests that asthmatics may be at increased risk for air pollution effects.

Neonatal epithelial hypoxia inducible factor-1α expression regulates the response of the lung to experimental asthma

Allergic airway disease is characterized by a T helper type 2 cell-mediated airway inflammation and airway hyperresponsiveness. Little is known about the role of hypoxia-mediated signaling in the progression of the disease. To address this knowledge gap, a mouse model was created in which doxycycline exposure induces the functional deletion of hypoxia inducible factor-1α from alveolar type II and Clara cells of the lung. When hypoxia inducible factor-1α deletion was induced during the early postnatal development period of the lung, the mice displayed an enhanced response to the ovalbumin model of allergic airway disease. These hypoxia inducible factor-1α-deficient mice exhibit increased cellular infiltrates, eosinophilia in the lavage fluid and parenchyma, and T helper type 2 cytokines, as compared with ovalbumin-treated control mice. Moreover, these hypoxia inducible factor-1α-deficient mice display increased airway resistance when compared with their control counterparts. Interestingly, if the loss of hypoxia inducible factor-1α was induced in early adulthood, the exacerbated phenotype was not observed. Taken together, these results suggest that epithelial hypoxia inducible factor-1α plays an important role in establishing the innate immunity of the lung and epithelial-specific deficiency in the transcription factor, during early postnatal development, increases the severity of inflammation and functional airway resistance, following ovalbumin challenge. Finally, these results might explain some of the chronic respiratory pathology observed in premature infants, especially those that receive supplemental oxygen. This early hyperoxic exposure, from normal ambient and supplemental oxygen, would presumably inhibit normal hypoxia inducible factor-1α signaling, mimicking the functional deletion described.

Repeated ozone exposure exacerbates insulin resistance and activates innate immune response in genetically susceptible mice

Abstract Background: Inhaled ozone (O3) has been demonstrated as a harmful pollutant and associated with chronic inflammatory diseases such as diabetes and vascular disorders. However, the underlying mechanisms by which O3 mediates harmful effects are poorly understood. Objectives: To investigate the effect of O3 exposure on glucose intolerance, immune activation and underlying mechanisms in a genetically susceptible mouse model. Methods: Diabetes-prone KK mice were exposed to filtered air (FA), or O3 (0.5 ppm) for 13 consecutive weekdays (4 h/day). Insulin tolerance test (ITT) was performed following the last exposure. Plasma insulin, adiponectin, and leptin were measured by ELISA. Pathologic changes were examined by H&E and Oil-Red-O staining. Inflammatory responses were detected using flow cytometry and real-time PCR. Results: KK mice exposed to O3 displayed an impaired insulin response. Plasma insulin and leptin levels were reduced in O3-exposed mice. Three-week exposure to O3 induced lung inflammation and increased monocytes/macrophages in both blood and visceral adipose tissue. Inflammatory monocytes/macrophages increased both systemically and locally. CD4 + T cell activation was also enhanced by the exposure of O3 although the relative percentage of CD4 + T cell decreased in blood and adipose tissue. Multiple inflammatory genes including CXCL-11, IFN-γ, TNFα, IL-12, and iNOS were up-regulated in visceral adipose tissue. Furthermore, the expression of oxidative stress-related genes such as Cox4, Cox5a, Scd1, Nrf1, and Nrf2, increased in visceral adipose tissue of O3-exposed mice. Conclusions: Repeated O3 inhalation induces oxidative stress, adipose inflammation and insulin resistance.