Role of the protease-activated receptor-2 (PAR2) in the exacerbation of house dust mite-induced murine allergic lung disease by multi-walled carbon nanotubes.

BACKGROUND: Pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) has been reported to exert strong pro-inflammatory and pro-fibrotic adjuvant effects in mouse models of allergic lung disease. However, the molecular mechanisms through which MWCNTs exacerbate allergen-induced lung disease remain to be elucidated. We hypothesized that protease-activated receptor 2 (PAR2), a G-protein coupled receptor previously implicated in the pathogenesis of various diseases including pulmonary fibrosis and asthma, may play an important role in the exacerbation of house dust mite (HDM) allergen-induced lung disease by MWCNTs. METHODS: Wildtype (WT) male C57BL6 mice and Par2 KO mice were exposed to vehicle, MWCNTs, HDM extract, or both via oropharyngeal aspiration 6 times over a period of 3 weeks and were sacrificed 3-days after the final exposure (day 22). Bronchoalveolar lavage fluid (BALF) was harvested to measure changes in inflammatory cells, total protein, and lactate dehydrogenase (LDH). Lung protein and RNA were assayed for pro-inflammatory or profibrotic mediators, and formalin-fixed lung sections were evaluated for histopathology. RESULTS: In both WT and Par2 KO mice, co-exposure to MWCNTs synergistically increased lung inflammation assessed by histopathology, and increased BALF cellularity, primarily eosinophils, as well as BALF total protein and LDH in the presence of relatively low doses of HDM extract that alone produced little, if any, lung inflammation. In addition, both WT and par2 KO mice displayed a similar increase in lung Cc1-11 mRNA, which encodes the eosinophil chemokine CCL-11, after co-exposure to MWCNTs and HDM extract. However, Par2 KO mice displayed significantly less airway fibrosis as determined by quantitative morphometry compared to WT mice after co-exposure to MWCNTs and HDM extract. Accordingly, at both protein and mRNA levels, the pro-fibrotic mediator arginase 1 (ARG-1), was downregulated in Par2 KO mice exposed to MWCNTs and HDM. In contrast, phosphorylation of the pro-inflammatory transcription factor NF-κB and the pro-inflammatory cytokine CXCL-1 was increased in Par2 KO mice exposed to MWCNTs and HDM. CONCLUSIONS: Our study indicates that PAR2 mediates airway fibrosis but not eosinophilic lung inflammation induced by co-exposure to MWCNTs and HDM allergens.

Pulmonary exposure of mice to ammonium perfluoro(2-methyl-3-oxahexanoate) (GenX) suppresses the innate immune response to carbon black nanoparticles and stimulates lung cell proliferation.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) have been associated with respiratory diseases in humans, yet the mechanisms through which PFAS cause susceptibility to inhaled agents is unknown. Herein, we investigated the effects of ammonium perfluoro(2-methyl-3-oxahexanoate) (GenX), an emerging PFAS, on the pulmonary immune response of mice to carbon black nanoparticles (CBNP). We hypothesized that pulmonary exposure to GenX would increase susceptibility to CBNP through suppression of innate immunity. METHODS: Male C57BL/6 mice were exposed to vehicle, 4 mg/kg CBNP, 10 mg/kg GenX, or CBNP and GenX by oropharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) was collected at 1 and 14 days postexposure for cytokines and total protein. Lung tissue was harvested for histopathology, immunohistochemistry (Ki67 and phosphorylated (p)-STAT3), western blotting (p-STAT3 and p-NF-κB), and qRT-PCR for cytokine mRNAs. RESULTS: CBNP increased CXCL-1 and neutrophils in BALF at both time points evaluated. However, GenX/CBNP co-exposure reduced CBNP-induced CXCL-1 and neutrophils in BALF. Moreover, CXCL-1, CXCL-2 and IL-1ß mRNAs were increased by CBNP in lung tissue but reduced by GenX. Western blotting showed that CBNP induced p-NF-κB in lung tissue, while the GenX/CBNP co-exposed group displayed decreased p-NF-κB. Furthermore, mice exposed to GenX or GenX/CBNP displayed increased numbers of BALF macrophages undergoing mitosis and increased Ki67 immunostaining. This was correlated with increased p-STAT3 by western blotting and immunohistochemistry in lung tissue from mice co-exposed to GenX/CBNP. CONCLUSIONS: Pulmonary exposure to GenX suppressed CBNP-induced innate immune response in the lungs of mice yet promoted the proliferation of macrophages and lung epithelial cells.

Femtosecond pulsed laser microscopy: a new tool to assess the in vitro delivered dose of carbon nanotubes in cell culture experiments.

BACKGROUND: In vitro models are widely used in nanotoxicology. In these assays, a careful documentation of the fraction of nanomaterials that reaches the cells, i.e. the in vitro delivered dose, is a critical element for the interpretation of the data. The in vitro delivered dose can be measured by quantifying the amount of material in contact with the cells, or can be estimated by applying particokinetic models. For carbon nanotubes (CNTs), the determination of the in vitro delivered dose is not evident because their quantification in biological matrices is difficult, and particokinetic models are not adapted to high aspect ratio materials. Here, we applied a rapid and direct approach, based on femtosecond pulsed laser microscopy (FPLM), to assess the in vitro delivered dose of multi-walled CNTs (MWCNTs). METHODS AND RESULTS: We incubated mouse lung fibroblasts (MLg) and differentiated human monocytic cells (THP-1) in 96-well plates for 24 h with a set of different MWCNTs. The cytotoxic response to the MWCNTs was evaluated using the WST-1 assay in both cell lines, and the pro-inflammatory response was determined by measuring the release of IL-1ß by THP-1 cells. Contrasting cell responses were observed across the MWCNTs. The sedimentation rate of the different MWCNTs was assessed by monitoring turbidity decay with time in cell culture medium. These turbidity measurements revealed some differences among the MWCNT samples which, however, did not parallel the contrasting cell responses. FPLM measurements in cell culture wells revealed that the in vitro delivered MWCNT dose did not parallel sedimentation data, and suggested that cultured cells contributed to set up the delivered dose. The FPLM data allowed, for each MWCNT sample, an adjustment of the measured cytotoxicity and IL-1ß responses to the delivered doses. This adjusted in vitro activity led to another toxicity ranking of the MWCNT samples as compared to the unadjusted activities. In macrophages, this adjusted ranking was consistent with existing knowledge on the impact of surface MWCNT functionalization on cytotoxicity, and might better reflect the intrinsic activity of the MWCNT samples. CONCLUSION: The present study further highlights the need to estimate the in vitro delivered dose in cell culture experiments with nanomaterials. The FPLM measurement of the in vitro delivered dose of MWCNTs can enrich experimental results, and may refine our understanding of their interactions with cells.

The pulmonary toxicity of carboxylated or aminated multi-walled carbon nanotubes in mice is determined by the prior purification method.

BACKGROUND: Inhalation of multi-walled carbon nanotubes (MWCNTs) poses a potential risk to human health. In order to safeguard workers and consumers, the toxic properties of MWCNTs need to be identified. Functionalization has been shown to either decrease or increase MWCNT-related pulmonary injury, depending on the type of modification. We, therefore, investigated both acute and chronic pulmonary toxicity of a library of MWCNTs derived from a common pristine parent compound (NC7000). METHODS: MWCNTs were thermally or chemically purified and subsequently surface functionalized by carboxylation or amination. To evaluate pulmonary toxicity, male C57BL6 mice were dosed via oropharyngeal aspiration with either 1.6 or 4 mg/kg of each MWCNT type. Mitsui-7 MWCNT was used as a positive control. Necropsy was performed at days 3 and 60 post-exposure to collect bronchoalveolar lavage fluid (BALF) and lungs. RESULTS: At day 3 all MWCNTs increased the number of neutrophils in BALF. Chemical purification had a greater effect on pro-inflammatory cytokines (IL-1ß, IL-6, CXCL1) in BALF, while thermal purification had a greater effect on pro-fibrotic cytokines (CCL2, OPN, TGF-ß1). At day 60, thermally purified, carboxylated MWCNTs had the strongest effect on lymphocyte numbers in BALF. Thermally purified MWCNTs caused the greatest increase in LDH and total protein in BALF. Furthermore, the thermally purified and carboxyl- or amine-functionalized MWCNTs caused the greatest number of granulomatous lesions in the lungs. The physicochemical characteristics mainly associated with increased toxicity of the thermally purified derivatives were decreased surface defects and decreased amorphous content as indicated by Raman spectroscopy. CONCLUSIONS: These data demonstrate that the purification method is an important determinant of lung toxicity induced by carboxyl- and amine-functionalized MWCNTs.

Susceptibility Factors in Chronic Lung Inflammatory Responses to Engineered Nanomaterials.

Engineered nanomaterials (ENMs) are products of the emerging nanotechnology industry and many different types of ENMs have been shown to cause chronic inflammation in the lungs of rodents after inhalation exposure, suggesting a risk to human health. Due to the increasing demand and use of ENMs in a variety of products, a careful evaluation of the risks to human health is urgently needed. An assessment of the immunotoxicity of ENMs should consider susceptibility factors including sex, pre-existing diseases, deficiency of specific genes encoding proteins involved in the innate or adaptive immune response, and co-exposures to other chemicals. This review will address evidence from experimental animal models that highlights some important issues of susceptibility to chronic lung inflammation and systemic immune dysfunction after pulmonary exposure to ENMs.

Inhalation exposure to multi-walled carbon nanotubes alters the pulmonary allergic response of mice to house dust mite allergen.

Background: Increasing evidence from rodent studies indicates that inhaled multi-walled carbon nanotubes (MWCNTs) have harmful effects on the lungs. In this study, we examined the effects of inhalation exposure to MWCNTs on allergen-induced airway inflammation and fibrosis. We hypothesized that inhalation pre-exposure to MWCNTs would render mice susceptible to developing allergic lung disease induced by house dust mite (HDM) allergen. Methods: Male B6C3F1/N mice were exposed by whole-body inhalation for 6 h a day, 5 d a week, for 30 d to air control or 0.06, 0.2, and 0.6 mg/m3 of MWCNTs. The exposure atmospheres were agglomerates (1.4-1.8 µm) composed of MWCNTs (average diameter 16 nm; average length 2.4 µm; 0.52% Ni). Mice then received 25 µg of HDM extract by intranasal instillation 6 times over 3 weeks. Necropsy was performed at 3 and 30 d after the final HDM dose to collect serum, bronchoalveolar lavage fluid (BALF), and lung tissue for histopathology. Results: MWCNT exposure at the highest dose inhibited HDM-induced serum IgE levels, IL-13 protein levels in BALF, and airway mucus production. However, perivascular and peribronchiolar inflammatory lesions were observed in the lungs of mice at 3 d with MWCNT and HDM, but not MWCNT or HDM alone. Moreover, combined HDM and MWCNT exposure increased airway fibrosis in the lungs of mice. Conclusions: Inhalation pre-exposure to MWCNTs inhibited HDM-induced TH2 immune responses, yet this combined exposure resulted in vascular inflammation and airway fibrosis, indicating that MWCNT pre-exposure alters the immune response to allergens.

STAT1-dependent and -independent pulmonary allergic and fibrogenic responses in mice after exposure to tangled versus rod-like multi-walled carbon nanotubes.

BACKGROUND: Pulmonary toxicity of multi-walled carbon nanotubes (MWCNTs) is influenced by physicochemical characteristics and genetic susceptibility. We hypothesized that contrasting rigidities of tangled (t) versus rod-like (r) MWCNTs would result in differing immunologic or fibrogenic responses in mice and that these responses would be exaggerated in transgenic mice lacking the signal transducer and activator of transcription-1 (STAT1), a susceptible mouse model of pulmonary fibrosis. METHODS: Male wild type (Stat1 +/+ ) and STAT1-deficient (Stat1 -/- ) mice were exposed to 4 mg/kg tMWCNTs, rMWCNTs, or vehicle alone via oropharyngeal aspiration and evaluated for inflammation at one and 21 days post-exposure via histopathology, differential cell counts, and cytokine levels in bronchoalveolar lavage fluid (BALF). Granuloma formation, mucous cell metaplasia, and airway fibrosis were evaluated by quantitative morphometry. Airway epithelial cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation. Cytokine protein levels in BALF and serum IgE levels were measured by ELISA. Lung protein Smad2/3 levels and activation were measured by Western blot. Lung mRNAs were measured by PCR. RESULTS: There was a 7-fold difference in rigidity between tMWCNTs and rMWCNTs as determined by static bending ratio. Both MWCNT types resulted in acute inflammation (neutrophils in BALF) after one-day post-exposure, yet only rMWCNTs resulted in chronic inflammation at 21 days as indicated by neutrophil influx and larger granulomas. Both MWCNTs induced BrdU uptake in airway epithelial cells, with the greatest proliferative response observed in rMWCNT-exposed mice after one-day. Only rMWCNTs induced mucous cell metaplasia, but this index was not different between genotypes. Stat1 -/- mice had higher levels of baseline serum IgE than Stat1 +/+ mice. Greater airway fibrosis was observed with rMWCNTs compared to tMWCNTs, and exaggerated airway fibrosis was seen in the Stat1 -/- mouse lungs with rMWCNTs but not tMWCNTs. Increased fibrosis correlated with elevated levels of TGF-ß1 protein levels in the BALF of Stat1 -/- mice exposed to rMWCNTs and increased lung Smad2/3 phosphorylation. CONCLUSIONS: Rigidity plays a key role in the toxicity of MWCNTs and results in increased inflammatory, immunologic, and fibrogenic effects in the lung. STAT1 is an important protective factor in the fibroproliferative response to rMWCNTs, regulating both induced TGF-ß1 production and Smad2/3 phosphorylation status. Therefore, both rigidity and genetic susceptibility should be major considerations for risk assessment of MWCNTs.

Atomic layer deposition coating of carbon nanotubes with zinc oxide causes acute phase immune responses in human monocytes in vitro and in mice after pulmonary exposure.

BACKGROUND: Atomic layer deposition (ALD) is a method for applying conformal nanoscale coatings on three-dimensional structures. We hypothesized that surface functionalization of multi-walled carbon nanotubes (MWCNTs) with polycrystalline ZnO by ALD would alter pro-inflammatory cytokine expression by human monocytes in vitro and modulate the lung and systemic immune response following oropharyngeal aspiration in mice. METHODS: Pristine (U-MWCNTs) were coated with alternating doses of diethyl zinc and water over increasing ALD cycles (10 to 100 ALD cycles) to yield conformal ZnO-coated MWCNTs (Z-MWCNTs). Human THP-1 monocytic cells were exposed to U-MWCNTs or Z-MWCNTs in vitro and cytokine mRNAs measured by Taqman real-time RT-PCR. Male C57BL6 mice were exposed to U- or Z-MWCNTs by oropharyngeal aspiration (OPA) and lung inflammation evaluated at one day post-exposure by histopathology, cytokine expression and differential counting of cells in bronchoalveolar lavage fluid (BALF) cells. Lung fibrosis was evaluated at 28 days. Cytokine mRNAs (IL-6, IL-1ß, CXCL10, TNF-α) in lung, heart, spleen, and liver were quantified at one and 28 days. DNA synthesis in lung tissue was measured by bromodeoxyuridine (BrdU) uptake. RESULTS: ALD resulted in a conformal coating of MWCNTs with ZnO that increased proportionally to the number of coating cycles. Z-MWCNTs released Zn(+2) ions in media and increased IL-6, IL-1ß, CXCL10, and TNF-α mRNAs in THP-1 cells in vitro. Mice exposed to Z-MWCNTs by OPA had exaggerated lung inflammation and a 3-fold increase in monocytes and neutrophils in BALF compared to U-MWCNTs. Z-MWCNTs, but not U-MWCNTs, induced IL-6 and CXCL10 mRNA and protein in the lungs of mice and increased IL-6 mRNA in heart and liver. U-MWCNTs but not Z-MWCNTs stimulated airway epithelial DNA synthesis in vivo. Lung fibrosis at 28 days was not significantly different between mice treated with U-MWCNT or Z-MWCNT. CONCLUSIONS: Pulmonary exposure to ZnO-coated MWCNTs produces a systemic acute phase response that involves the release of Zn(+2), lung epithelial growth arrest, and increased IL-6. ALD functionalization with ZnO generates MWCNTs that possess increased risk for human exposure.

Expert consensus on an in vitro approach to assess pulmonary fibrogenic potential of aerosolized nanomaterials.

The increasing use of multi-walled carbon nanotubes (MWCNTs) in consumer products and their potential to induce adverse lung effects following inhalation has lead to much interest in better understanding the hazard associated with these nanomaterials (NMs). While the current regulatory requirement for substances of concern, such as MWCNTs, in many jurisdictions is a 90-day rodent inhalation test, the monetary, ethical, and scientific concerns associated with this test led an international expert group to convene in Washington, DC, USA, to discuss alternative approaches to evaluate the inhalation toxicity of MWCNTs. Pulmonary fibrosis was identified as a key adverse outcome linked to MWCNT exposure, and recommendations were made on the design of an in vitro assay that is predictive of the fibrotic potential of MWCNTs. While fibrosis takes weeks or months to develop in vivo, an in vitro test system may more rapidly predict fibrogenic potential by monitoring pro-fibrotic mediators (e.g., cytokines and growth factors). Therefore, the workshop discussions focused on the necessary specifications related to the development and evaluation of such an in vitro system. Recommendations were made for designing a system using lung-relevant cells co-cultured at the air-liquid interface to assess the pro-fibrogenic potential of aerosolized MWCNTs, while considering human-relevant dosimetry and NM life cycle transformations. The workshop discussions provided the fundamental design components of an air-liquid interface in vitro test system that will be subsequently expanded to the development of an alternative testing strategy to predict pulmonary toxicity and to generate data that will enable effective risk assessment of NMs.

Role of signal transducer and activator of transcription 1 in murine allergen-induced airway remodeling and exacerbation by carbon nanotubes.

Asthma is characterized by a T helper type 2 phenotype and by chronic allergen-induced airway inflammation (AAI). Environmental exposure to air pollution ultrafine particles (i.e., nanoparticles) exacerbates AAI, and a concern is possible exacerbation posed by engineered nanoparticles generated by emerging nanotechnologies. Signal transducer and activator of transcription (STAT) 1 is a transcription factor that maintains T helper type 1 cell development. However, the role of STAT1 in regulating AAI or exacerbation by nanoparticles has not been explored. In this study, mice with whole-body knockout of the Stat1 gene (Stat1(-/-)) or wild-type (WT) mice were sensitized to ovalbumin (OVA) allergen and then exposed to multiwalled carbon nanotubes (MWCNTs) by oropharygneal aspiration. In Stat1(-/-) and WT mice, OVA increased eosinophils in bronchoalveolar lavage fluid, whereas MWCNTs increased neutrophils. Interestingly, OVA sensitization prevented MWCNT-induced neutrophilia and caused only eosinophilic inflammation. Stat1(-/-) mice displayed increased IL-13 in bronchoalveolar lavage fluid at 1 day compared with WT mice after treatment with OVA or OVA and MWCNTs. At 21 days, the lungs of OVA-sensitized Stat1(-/-) mice displayed increased eosinophilia, goblet cell hyperplasia, airway fibrosis, and subepithelial apoptosis. MWCNTs further increased OVA-induced goblet cell hyperplasia, airway fibrosis, and apoptosis in Stat1(-/-) mice at 21 days. These changes corresponded to increased levels of profibrogenic mediators (transforming growth factor-ß1, TNF-α, osteopontin) but decreased IL-10 in Stat1(-/-) mice. Finally, fibroblasts isolated from the lungs of Stat1(-/-) mice produced significantly more collagen mRNA and protein in response to transforming growth factor-ß1 compared with WT lung fibroblasts. Our results support a protective role for STAT1 in chronic AAI and exacerbation of remodeling caused by MWCNTs.

Genetic susceptibility to interstitial pulmonary fibrosis in mice induced by vanadium pentoxide (V2O5).

Interstitial lung diseases (ILDs) are characterized by injury, inflammation, and scarring of alveoli, leading to impaired function. The etiology of idiopathic forms of ILD is not understood, making them particularly difficult to study due to the lack of appropriate animal models. Consequently, few effective therapies have emerged. We developed an inbred mouse model of ILD using vanadium pentoxide (V2O5), the most common form of a transition metal found in cigarette smoke, fuel ash, mineral ores, and steel alloys. Pulmonary responses to V2O5, including dose-dependent increases in lung permeability, inflammation, collagen content, and dysfunction, were significantly greater in DBA/2J mice compared to C57BL/6J mice. Inflammatory and fibrotic responses persisted for 4 mo in DBA/2J mice, while limited responses in C57BL/6J mice resolved. We investigated the genetic basis for differential responses through genetic mapping of V2O5-induced lung collagen content in BXD recombinant inbred (RI) strains and identified significant linkage on chromosome 4 with candidate genes that associate with V2O5-induced collagen content across the RI strains. Results suggest that V2O5 may induce pulmonary fibrosis through mechanisms distinct from those in other models of pulmonary fibrosis. These findings should further advance our understanding of mechanisms involved in ILD and thereby aid in identification of new therapeutic targets.

Nickel nanoparticles cause exaggerated lung and airway remodeling in mice lacking the T-box transcription factor, TBX21 (T-bet).

BACKGROUND: Nickel nanoparticles (NiNPs) are increasingly used in a variety of industrial applications, including the manufacturing of multi-walled carbon nanotubes (MWCNTs). While occupational nickel exposure is a known cause of pulmonary alveolitis, fibrosis, and cancer, the health risks of NiNPs are not well understood, especially in susceptible individuals such as asthmatics. The T-box transcription factor Tbx21 (T-bet) maintains Th1 cell development and loss of T-bet is associated with a shift towards Th2 type allergic airway inflammation that characterizes asthma. The purpose of this study was to determine the role of T-bet in susceptibility to lung remodeling by NiNPs or MWCNTs. METHODS: Wild-type (WT) and T-bet-/- mice were exposed to NiNPs or MWCNTs (4 mg/kg) by oropharyngeal aspiration (OPA). Necropsy was performed at 1 and 21 days. Bronchoalveolar lavage fluid (BALF) was collected for differential counting of inflammatory cells and for measurement of cytokines by ELISA. The left lung was collected for histopathology. The right lung was analyzed for cytokine or mucin (MUC5AC and MUC5B) mRNAs. RESULTS: Morphometry of alcian-blue/periodic acid Schiff (AB/PAS)-stained lung tissue showed that NiNPs significantly increased mucous cell metaplasia in T-bet-/- mice at 21 days (p < 0.001) compared to WT mice, and increased MUC5AC and MUC5B mRNAs (p < 0.05). MWCNTs also increased mucous cell metaplasia in T-bet-/- mice, but to a lesser extent than NiNPs. Chronic alveolitis was also increased by NiNPs, but not MWCNTs, in T-bet-/- mice compared to WT mice at 21 days (P < 0.001). NiNPs also increased IL-13 and eosinophils (p < 0.001) in BALF from T-bet-/- mice after 1 day. Interestingly, the chemokine CCL2 in the BALF of T-bet-/- mice was increased at 1 and 21 days (p < 0.001 and p < 0.05, respectively) by NiNPs, and to a lesser extent by MWCNTs at 1 day. Treatment of T-bet-/- mice with a monoclonal anti-CCL2 antibody enhanced NiNP-induced mucous cell metaplasia and MUC5AC mRNA levels (p < 0.05), yet marginally reduced NiNP-induced alveolitis. CONCLUSION: These findings identify T-bet as a potentially important susceptibility factor for NiNP exposure and to a lesser extent for MWCNT exposure, and suggests that individuals with asthma are at greater risk.

Inflammasome activation in airway epithelial cells after multi-walled carbon nanotube exposure mediates a profibrotic response in lung fibroblasts.

BACKGROUND: In vivo studies have demonstrated the ability of multi-walled carbon nanotubes (MWCNT) to induce airway remodeling, a key feature of chronic respiratory diseases like asthma and chronic obstructive pulmonary disease. However, the mechanism leading to remodeling is poorly understood. Particularly, there is limited insight about the role of airway epithelial injury in these changes. OBJECTIVES: We investigated the mechanism of MWCNT-induced primary human bronchial epithelial (HBE) cell injury and its contribution in inducing a profibrotic response. METHODS: Primary HBE cells were exposed to thoroughly characterized MWCNTs (1.5-24 µg/mL equivalent to 0.37-6.0 µg/cm2) and MRC-5 human lung fibroblasts were exposed to 1:4 diluted conditioned medium from these cells. Flow cytometry, ELISA, immunostainings/immunoblots and PCR analyses were employed to study cellular mechanisms. RESULTS: MWCNT induced NLRP3 inflammasome dependent pyroptosis in HBE cells in a time- and dose-dependent manner. Cell death and cytokine production were significantly reduced by antioxidants, siRNA to NLRP3, a caspase-1 inhibitor (z-WEHD-FMK) or a cathepsin B inhibitor (CA-074Me). Conditioned medium from MWCNT-treated HBE cells induced significant increase in mRNA expression of pro-fibrotic markers (TIMP-1, Tenascin-C, Procollagen 1, and Osteopontin) in human lung fibroblasts, without a concomitant change in expression of TGF-beta. Induction of pro-fibrotic markers was significantly reduced when IL-1ß, IL-18 and IL-8 neutralizing antibodies were added to the conditioned medium or when conditioned medium from NLRP3 siRNA transfected HBE cells was used. CONCLUSIONS: Taken together these results demonstrate induction of a NLRP3 inflammasome dependent but TGF-beta independent pro-fibrotic response after MWCNT exposure.

House Dust Mite Extract Forms a Der p 2 Corona on Multi-Walled Carbon Nanotubes: Implications for Allergic Airway Disease.

Multi-walled carbons nanotubes (MWCNTs) are used in materials for the construction, automotive, and aerospace industries. Workers and consumers are exposed to these materials via inhalation. Existing recommended exposure limits are based on MWCNT exposures that do not take into account more realistic co-exposures. Our goal was to understand how a common allergen, house dust mites, interacts with pristine MWCNTs and lung fluid proteins. We used gel electrophoresis, western blotting, and proteomics to characterize the composition of the allergen corona formed from house dust mite extract on the surface of MWCNTs. We found that the corona is dominated by der p 2, a protein associated with human allergic responses to house dust mites. Der p 2 remains adsorbed on the surface of the MWCNTs following subsequent exposures to lung fluid proteins. The high concentration of der p 2, localized on surface of MWCNTs, has important implications for house dust mite-induced allergies and asthma. This research provides a detailed characterization of the complex house dust mite-lung fluid protein coronas for future cellular and in vivo studies. These studies will help to address the molecular and biochemical mechanisms underlying the exacerbation of allergic lung disease by nanomaterials.

Role of cyclooxygenase-2 in exacerbation of allergen-induced airway remodeling by multiwalled carbon nanotubes.

The emergence of nanotechnology has produced a multitude of engineered nanomaterials such as carbon nanotubes (CNTs), and concerns have been raised about their effects on human health, especially for susceptible populations such as individuals with asthma. Multiwalled CNTs (MWCNTs) have been shown to exacerbate ovalbumin (OVA)-induced airway remodeling in mice. Moreover, cyclooxygenase-2 (COX-2) has been described as a protective factor in asthma. We postulated that COX-2-deficient (COX-2(-/-)) mice would be susceptible to MWCNT-induced exacerbations of allergen-induced airway remodeling, including airway inflammation, fibrosis, and mucus-cell metaplasia (i.e., the formation of goblet cells). Wild-type (WT) or COX-2(-/-) mice were sensitized to OVA to induce allergic airway inflammation before a single dose of MWCNTs (4 mg/kg) delivered to the lungs by oropharyngeal aspiration. MWCNTs significantly increased OVA-induced lung inflammation and mucus-cell metaplasia in COX-2(-/-) mice compared with WT mice. However, airway fibrosis after exposure to allergen and MWCNTs was no different between WT and COX-2(-/-) mice. Concentrations of certain prostanoids (prostaglandin D2 and thromboxane B2) were enhanced by OVA or MWCNTs in COX-2(-/-) mice. No differences in COX-1 mRNA concentrations were evident between WT and COX-2(-/-) mice treated with OVA and MWCNTs. Interestingly, MWCNTs significantly enhanced allergen-induced cytokines involved in Th2 (IL-13 and IL-5), Th1 (CXCL10), and Th17 (IL-17A) inflammatory responses in COX-2(-/-) mice, but not in WT mice. We conclude that exacerbations of allergen-induced airway inflammation and mucus-cell metaplasia by MWCNTs are enhanced by deficiencies in COX-2, and are associated with the activation of a mixed Th1/Th2/Th17 immune response.

Nickel nanoparticles enhance platelet-derived growth factor-induced chemokine expression by mesothelial cells via prolonged mitogen-activated protein kinase activation.

Pleural diseases (fibrosis and mesothelioma) are a major concern for individuals exposed by inhalation to certain types of particles, metals, and fibers. Increasing attention has focused on the possibility that certain types of engineered nanoparticles (NPs), especially those containing nickel, might also pose a risk for pleural diseases. Platelet-derived growth factor (PDGF) is an important mediator of fibrosis and cancer that has been implicated in the pathogenesis of pleural diseases. In this study, we discovered that PDGF synergistically enhanced nickel NP (NiNP)-induced increases in mRNA and protein levels of the profibrogenic chemokine monocyte chemoattractant protein-1 (MCP-1 or CCL2), and the antifibrogenic IFN-inducible CXC chemokine (CXCL10) in normal rat pleural mesothelial 2 (NRM2) cells in vitro. Carbon black NPs (CBNPs), used as a negative control NP, did not cause a significant increase in CCL2 or CXCL10 in the absence or presence of PDGF. NiNPs prolonged PDGF-induced phosphorylation of the mitogen-activated protein kinase family termed extracellular signal-regulated kinases (ERK)-1 and -2 for up to 24 hours, and NiNPs also synergistically increased PDGF-induced hypoxia-inducible factor (HIF)-1α protein levels in NRM2 cells. Inhibition of ERK-1,2 phosphorylation with the mitogen-activated protein kinase kinase (MEK) inhibitor, PD98059, blocked the synergistic increase in CCL2, CXCL10, and HIF-1α levels induced by PDGF and NiNPs. Moreover, the antioxidant, N-acetyl-L-cysteine (NAC), significantly reduced HIF-1α, ERK-1,2 phosphorylation, and CCL2 protein levels that were synergistically increased by the combination of PDGF and NiNPs. These data indicate that NiNPs enhance the activity of PDGF in regulating chemokine production in NRM2 cells through a mechanism involving reactive oxygen species generation and prolonged activation of ERK-1,2.

Over-expression of human endosulfatase-1 exacerbates cadmium-induced injury to transformed human lung cells in vitro.

Environmental exposure to cadmium is known to cause damage to alveolar epithelial cells of the lung, impair their capacity to repair, and result in permanent structural alterations. Cell surface heparan sulfate proteoglycans (HSPGs) can modulate cell responses to injury through their interactions with soluble effector molecules. These interactions are often sulfate specific, and the removal of sulfate groups from HS side chains could be expected to influence cellular injury, such as that caused by exposure to cadmium. The goal of this study was to define the role 6-O-sulfate plays in cellular responses to cadmium exposure in two pulmonary epithelial cancer cell lines (H292 and A549) and in normal human primary alveolar type II (hAT2) cells. Sulfate levels were modified by transduced transient over-expression of 6-O-endosulfatase (HSulf-1), a membrane-bound enzyme which specifically removes 6-O-sulfate groups from HSPG side chains. Results showed that cadmium decreased cell viability and activated apoptosis pathways at low concentrations in hAT2 cells but not in the cancer cells. HSulf-1 over-expression, on the contrary, decreased cell viability and activated apoptosis pathways in H292 and A549 cells but not in hAT2 cells. When combined with cadmium, HSulf-1 over-expression further decreased cell viability and exacerbated the activation of apoptosis pathways in the transformed cells but did not add to the toxicity in hAT2 cells. The finding that HSulf-1 sensitizes these cancer cells and intensifies the injury induced by cadmium suggests that 6-O-sulfate groups on HSPGs may play important roles in protection against certain environmental toxicants, such as heavy metals.

Multi-walled carbon nanotubes induce COX-2 and iNOS expression via MAP kinase-dependent and -independent mechanisms in mouse RAW264.7 macrophages.

BACKGROUND: Carbon nanotubes (CNTs) are engineered graphene cylinders with numerous applications in engineering, electronics and medicine. However, CNTs cause inflammation and fibrosis in the rodent lung, suggesting a potential human health risk. We hypothesized that multi-walled CNTs (MWCNTs) induce two key inflammatory enzymes in macrophages, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), through activation of extracellular signal-regulated kinases (ERK1,2). METHODS: RAW264.7 macrophages were exposed to MWCNTs or carbon black nanoparticles (CBNPs) over a range of doses and time course. Uptake and subcellular localization of MWCNTs was visualized by transmission electron microscopy (TEM). Protein levels of COX-2, iNOS, and ERK1,2 (total ERK and phosphorylated ERK) were measured by Western blot analysis. Prostaglandin-E(2) (PGE(2)) and nitric oxide (NO) levels in cell supernatants were measured by ELISA and Greiss assay, respectively. RESULTS: MWCNTs, but not CBNPs, induced COX-2 and iNOS in a time- and dose-dependent manner. COX-2 and iNOS induction by MWCNTs correlated with increased PGE(2) and NO production, respectively. MWCNTs caused ERK1,2 activation and inhibition of ERK1,2 (U0126) blocked MWCNT induction of COX-2 and PGE2 production, but did not reduce the induction of iNOS. Inhibition of iNOS (L-NAME) did not affect ERK1,2 activation, nor did L-NAME significantly decrease COX-2 induction by MWCNT. Nickel nanoparticles (NiNPs), which are present in MWCNTs as a residual catalyst, also induced COX-2 via ERK-1,2. However, a comparison of COX-2 induction by MWCNTs containing 4.5 and 1.8% Ni did not show a significant difference in ability to induce COX-2, indicating that characteristics of MWCNTs in addition to Ni content contribute to COX-2 induction. CONCLUSION: This study identifies COX-2 and subsequent PGE(2) production, along with iNOS induction and NO production, as inflammatory mediators involved in the macrophage response to MWCNTs. Furthermore, our work demonstrates that COX-2 induction by MWCNTs in RAW264.7 macrophages is ERK1,2-dependent, while iNOS induction by MWCNTs is ERK1,2-independent. Our data also suggest contributory physicochemical factors other than residual Ni catalyst play a role in COX-2 induction to MWCNT.

Synergistic induction of IL-6 production in human bronchial epithelial cells in vitro by nickel nanoparticles and lipopolysaccharide is mediated by STAT3 and C/EBPß.

We previously reported that delivery of nickel nanoparticles (NiNPs) and bacterial lipopolysaccharide (LPS) into the lungs of mice synergistically increased IL-6 production and inflammation, and male mice were more susceptible than female mice. The primary goal of this study was to utilize an in vitro human lung epithelial cell model (BEAS-2B) to investigate the intracellular signaling mechanisms that mediate IL-6 production by LPS and NiNPs. We also investigated the effect of sex hormones on NiNP and LPS-induced IL-6 production in vitro. LPS and NiNPs synergistically induced IL-6 mRNA and protein in BEAS-2B cells. TPCA-1, a dual inhibitor of IKK-2 and STAT3, blocked the synergistic increase in IL-6 caused by LPS and NiNPs, abolished STAT3 activation, and reduced C/EBPß. Conversely, SC144, an inhibitor of the gp130 component of the IL-6 receptor, enhanced IL-6 production induced by LPS and NiNPs. Treatment of BEAS-2B cells with sex hormones (17ß-estradiol, progesterone, or testosterone) or the anti-oxidant NAC, had no effect on IL-6 induction by LPS and NiNPs. These data suggest that LPS and NiNPs induce IL-6 via STAT3 and C/EBPß in BEAS-2B cells. While BEAS-2B cells are a suitable model to study mechanisms of IL-6 production, they do not appear to be suitable for studying the effect of sex hormones.

Pulmonary endpoints (lung carcinomas and asbestosis) following inhalation exposure to asbestos.

Lung carcinomas and pulmonary fibrosis (asbestosis) occur in asbestos workers. Understanding the pathogenesis of these diseases is complicated because of potential confounding factors, such as smoking, which is not a risk factor in mesothelioma. The modes of action (MOA) of various types of asbestos in the development of lung cancers, asbestosis, and mesotheliomas appear to be different. Moreover, asbestos fibers may act differentially at various stages of these diseases, and have different potencies as compared to other naturally occurring and synthetic fibers. This literature review describes patterns of deposition and retention of various types of asbestos and other fibers after inhalation, methods of translocation within the lung, and dissolution of various fiber types in lung compartments and cells in vitro. Comprehensive dose-response studies at fiber concentrations inhaled by humans as well as bivariate size distributions (lengths and widths), types, and sources of fibers are rarely defined in published studies and are needed. Species-specific responses may occur. Mechanistic studies have some of these limitations, but have suggested that changes in gene expression (either fiber-catalyzed directly or by cell elaboration of oxidants), epigenetic changes, and receptor-mediated or other intracellular signaling cascades may play roles in various stages of the development of lung cancers or asbestosis.