Pulmonary exposure to multiwalled carbon nanotubes (MWCNTs) causes indirect systemic inflammation through unknown pathways. MWCNTs translocate only minimally from the lungs into the systemic circulation, suggesting that extrapulmonary toxicity may be caused indirectly by lung-derived factors entering the circulation. To assess a role for MWCNT-induced circulating factors in driving neuroinflammatory outcomes, mice were acutely exposed to MWCNTs (10 or 40 μg/mouse) via oropharyngeal aspiration. At 4 h after MWCNT exposure, broad disruption of the blood-brain barrier (BBB) was observed across the capillary bed with the small molecule fluorescein, concomitant with reactive astrocytosis. However, pronounced BBB permeation was noted, with frank albumin leakage around larger vessels (>10 μm), overlain by a dose-dependent astroglial scar-like formation and recruitment of phagocytic microglia. As affirmed by elevated inflammatory marker transcription, MWCNT-induced BBB disruption and neuroinflammation were abrogated by pretreatment with the rho kinase inhibitor fasudil. Serum from MWCNT-exposed mice induced expression of adhesion molecules in primary murine cerebrovascular endothelial cells and, in a wound-healing in vitro assay, impaired cell motility and cytokinesis. Serum thrombospondin-1 level was significantly increased after MWCNT exposure, and mice lacking the endogenous receptor CD36 were protected from the neuroinflammatory and BBB permeability effects of MWCNTs. In conclusion, acute pulmonary exposure to MWCNTs causes neuroinflammatory responses that are dependent on the disruption of BBB integrity.nanoparticle | blood-brain barrier | microglia | thrombospondin-1 | multiwalled carbon nanotube

Inhalation of multi walled carbon nanotubes (MWCNT) causes systemic effects including vascular inflammation, endothelial dysfunction, and acute phase protein expression. MWCNTs translocate only minimally beyond the lungs, thus cardiovascular effects thereof may be caused by generation of secondary biomolecular factors from MWCNT-pulmonary interactions that spill over into the systemic circulation. Therefore, we hypothesized that induced matrix metalloproteinase-9 (MMP-9) is a generator of factors that, in turn, drive vascular effects through ligand-receptor interactions with the multiligand pattern recognition receptor, CD36. To test this, wildtype (WT; C57BL/6) and MMP-9−/− mice were exposed to varying doses (10 or 40 μg) of MWCNTs via oropharyngeal aspiration and serum was collected at 4 and 24 h postexposure. Endothelial cells treated with serum from MWCNT-exposed WT mice exhibited significantly reduced nitric oxide (NO) generation, as measured by electron paramagnetic resonance, an effect that was independent of NO scavenging. Serum from MWCNT-exposed WT mice inhibited acetylcholine (ACh)-mediated relaxation of aortic rings at both time points. Absence of CD36 on the aortic rings (obtained from CD36-deficient mice) abolished the serum-induced impairment of vasorelaxation. MWCNT exposure induced MMP-9 protein levels in both bronchoalveolar lavage and whole lung lysates. Serum from MMP-9−/− mice exposed to MWCNT did not diminish the magnitude of vasorelaxation in naïve WT aortic rings, although a modest right shift of the ACh dose-response curve was observed in both MWCNT dose groups relative to controls. In conclusion, pulmonary exposure to MWCNT leads to elevated MMP-9 levels and MMP-9-dependent generation of circulating bioactive factors that promote endothelial dysfunction and decreased NO bioavailability via interaction with vascular CD36.

Pulmonary toxicity studies on carbon nanotubes focus primarily on as-produced materials and rarely are guided by a life cycle perspective or integration with exposure assessment. Understanding toxicity beyond the as-produced, or pure native material, is critical, due to modifications needed to overcome barriers to commercialization of applications. In the first series of studies, the toxicity of as-produced carbon nanotubes and their polymer-coated counterparts was evaluated in reference to exposure assessment, material characterization, and stability of the polymer coating in biological fluids. The second series of studies examined the toxicity of aerosols generated from sanding polymer-coated carbon-nanotube-embedded or neat composites. Postproduction modification by polymer coating did not enhance pulmonary injury, inflammation, and pathology or in vitro genotoxicity of as-produced carbon nanotubes, and for a particular coating, toxicity was significantly attenuated. The aerosols generated from sanding composites embedded with polymer-coated carbon nanotubes contained no evidence of free nanotubes. The percent weight incorporation of polymer-coated carbon nanotubes, 0.15% or 3% by mass, and composite matrix utilized altered the particle size distribution and, in certain circumstances, influenced acute in vivo toxicity. Our study provides perspective that, while the number of workers and consumers increases along the life cycle, toxicity and/or potential for exposure to the as-produced material may greatly diminish.

Background The mechanisms driving systemic effects consequent pulmonary nanoparticle exposure remain unclear. Recent work has established the existence of an indirect process by which factors released from the lung into the circulation promote systemic inflammation and cellular dysfunction, particularly on the vasculature. However, the composition of circulating contributing factors and how they are produced remains unknown. Evidence suggests matrix protease involvement; thus, here we used a well-characterized multi-walled carbon nanotube (MWCNT) oropharyngeal aspiration model with known vascular effects to assess the distinct contribution of nanoparticle-induced peptide fragments in driving systemic pathobiology. Results Data-independent mass spectrometry enabled the unbiased quantitative characterization of 841 significant MWCNT-responses within an enriched peptide fraction, with 567 of these factors demonstrating significant correlation across animal-paired bronchoalveolar lavage and serum biofluids. A database search curated for known matrix protease substrates and predicted signaling motifs enabled identification of 73 MWCNT-responsive peptides, which were significantly associated with an abnormal cardiovascular phenotype, extracellular matrix organization, immune-inflammatory processes, cell receptor signaling, and a MWCNT-altered serum exosome population. Production of a diverse peptidomic response was supported by a wide number of upregulated matrix and lysosomal proteases in the lung after MWCNT exposure. The peptide fraction was then found bioactive, producing endothelial cell inflammation and vascular dysfunction ex vivo akin to that induced with whole serum. Results implicate receptor ligand functionality in driving systemic effects, exemplified by an identified 59-mer thrombospondin fragment, replete with CD36 modulatory motifs, that when synthesized produced an anti-angiogenic response in vitro matching that of the peptide fraction. Other identified peptides point to integrin ligand functionality and more broadly to a diversity of receptor-mediated bioactivity induced by the peptidomic response to nanoparticle exposure. Conclusion The present study demonstrates that pulmonary-sequestered nanoparticles, such as multi-walled carbon nanotubes, acutely upregulate a diverse profile of matrix proteases, and induce a complex peptidomic response across lung and blood compartments. The serum peptide fraction, having cell-surface receptor ligand properties, conveys peripheral bioactivity in promoting endothelial cell inflammation, vasodilatory dysfunction and inhibiting angiogenesis. Results here establish peptide fragments as indirect, non-cytokine mediators and putative biomarkers of systemic health outcomes from nanoparticle exposure.

The strain 68-1 rhesus cytomegalovirus (RhCMV)–based vaccine for simian immunodeficiency virus (SIV) can stringently protect rhesus macaques (RMs) from SIV challenge by arresting viral replication early in primary infection. This vaccine elicits unconventional SIV-specific CD8 + T cells that recognize epitopes presented by major histocompatibility complex (MHC)–II and MHC-E instead of MHC-Ia. Although RhCMV/SIV vaccines based on strains that only elicit MHC-II– and/or MHC-Ia–restricted CD8 + T cells do not protect against SIV, it remains unclear whether MHC-E–restricted T cells are directly responsible for protection and whether these responses can be separated from the MHC-II–restricted component. Using host microRNA (miR)–mediated vector tropism restriction, we show that the priming of MHC-II and MHC-E epitope–targeted responses depended on vector infection of different nonoverlapping cell types in RMs. Selective inhibition of RhCMV infection in myeloid cells with miR-142–mediated tropism restriction eliminated MHC-E epitope–targeted CD8 + T cell priming, yielding an exclusively MHC-II epitope–targeted response. Inhibition with the endothelial cell–selective miR-126 eliminated MHC-II epitope–targeted CD8 + T cell priming, yielding an exclusively MHC-E epitope–targeted response. Dual miR-142 + miR-126–mediated tropism restriction reverted CD8 + T cell responses back to conventional MHC-Ia epitope targeting. Although the magnitude and differentiation state of these CD8 + T cell responses were generally similar, only the vectors programmed to elicit MHC-E–restricted CD8 + T cell responses provided protection against SIV challenge, directly demonstrating the essential role of these responses in RhCMV/SIV vaccine efficacy.

Boron nitride nanotubes (BNNTs) are an emerging engineered nanomaterial attracting significant attention due to superior electrical, chemical and thermal properties. Currently, the toxicity profile of this material is largely unknown. Commercial grade BNNTs are composed of a mixture (BNNT-M) of ∼50-60% BNNTs, and ∼40-50% impurities of boron and hexagonal boron nitride. We performed acute in vitro and in vivo studies with commercial grade BNNT-M, dispersed by sonication in vehicle, in comparison to the extensively studied multiwalled carbon nanotube-7 (MWCNT-7). THP-1 wild-type and NLRP3-deficient human monocytic cells were exposed to 0-100 µg/ml and C57BL/6 J male mice were treated with 40 µg of BNNT-M for in vitro and in vivo studies, respectively. In vitro, BNNT-M induced a dose-dependent increase in cytotoxicity and oxidative stress. This was confirmed in vivo following acute exposure increase in bronchoalveolar lavage levels of lactate dehydrogenase, pulmonary polymorphonuclear cell influx, loss in mitochondrial membrane potential and augmented levels of 4-hydroxynonenal. Uptake of this material caused lysosomal destabilization, pyroptosis and inflammasome activation, corroborated by an increase in cathepsin B, caspase 1, increased protein levels of IL-1β and IL-18 both in vitro and in vivo. Attenuation of these effects in NLRP3-deficient THP-1 cells confirmed NLRP3-dependent inflammasome activation by BNNT-M. BNNT-M induced a similar profile of inflammatory pulmonary protein production when compared to MWCNT-7. Functionally, pretreatment with BNNT-M caused suppression in bacterial uptake by THP-1 cells, an effect that was mirrored in challenged alveolar macrophages collected from exposed mice and attenuated with NLRP3 deficiency. Analysis of cytokines secreted by LPS-challenged alveolar macrophages collected after in vivo exposure to dispersions of BNNT-M showed a differential macrophage response. The observed results demonstrated acute inflammation and toxicity in vitro and in vivo following exposure to sonicated BNNT-M was in part due to NLRP3 inflammasome activation.

Welding fume is a complex mixture of different potentially cytotoxic and genotoxic metals, such as chromium (Cr), manganese (Mn), nickel (Ni), and iron (Fe). Documented health effects have been observed in workers exposed to welding fume. The objective of the study was to use an animal model to identify potential biomarkers of epigenetic changes (e.g., changes in telomere length, DNA methylation) in isolated peripheral blood mononuclear cells (PBMCs) after exposure to different welding fumes. Male Sprague-Dawley rats were exposed by intratracheal instillation (ITI) of 2.0 mg/rat of gas metal arc-mild steel (GMA-MS) or manual metal arc-stainless steel (MMA-SS) welding fume. Vehicle controls received sterile saline by ITI. At 4 h, 14 h, 1 d, 3 d, 10 d, and 30 d, bronchoalveolar lavage (BAL) was performed to assess lung inflammation. Whole blood was collected, and PBMCs were isolated. Dihydroethidium (DHE) fluorescence and 4-hydroxylnonenal protein adduct (P-HNE) formation were measured in PBMCs to assess reactive oxygen species (ROS) production. DNA alterations in PBMCs were determined by evaluating changes in DNA methylation and telomere length. Metal composition of the two fumes was different: MMA-SS (41 % Fe, 29 % Cr, 17 % Mn, 3 % Ni) versus GMA-MS (85 % Fe, 14 % Mn). The more soluble and chemically complex MMA-SS sample induced a more persistent and greater inflammatory response compared to the other groups. Also, oxidative stress markers increased at 24 h in the PBMCs recovered from the MMA-SS group compared to other group. No significant differences were observed when comparing DNA methylation between the welding fume and control groups at any of the time points, whereas the MMA-SS sample significantly increased telomere length at 1 and 30 d after a single exposure compared to the other groups. These findings suggest that genotoxic metals in MMA-SS fume (e.g., Cr and Ni), that are absent in the GMA-MS fume, may enhance lung toxicity, as well as induce markers of oxidative stress and increase telomere length in PBMCs. Importantly, the measurement of telomere length in cells isolated from peripheral blood may serve as a potential biomarker of response in the assessment of toxicity associated with welding fumes.

Welding generates a complex aerosol of incidental nanoparticles and cytotoxic metals, such as chromium (Cr), manganese (Mn), nickel (Ni), and iron (Fe). The goal was to use both in vivo and in vitro methodologies to determine the mechanisms by which different welding fumes may damage the lungs. Sprague-Dawley rats were treated by intratracheal instillation (ITI) with 2.0 mg of gas metal arc-mild steel (GMA-MS) or manual metal arc-stainless steel (MMA-SS) fumes or saline (vehicle control). At 1, 3, and 10 days, bronchoalveolar lavage (BAL) was performed to measure lung toxicity. To assess molecular mechanisms of cytotoxicity, RAW264.7 cells were exposed to both welding fumes for 24 h (0-100 μg/ml). Fume composition was different: MMA-SS (41% Fe, 29% Cr, 17% Mn, 3% Ni) versus GMA-MS (85% Fe, 14% Mn). BAL indicators of lung injury and inflammation were increased by MMA-SS at all time points and by GMA-MS at 3 and 10 days after exposure. RAW264.7 cells exposed to MMA-SS had elevated generation of reactive oxygen species (ROS), protein-HNE (P-HNE) adduct formation, activation of ERK1/2, and expression of cyclooxygenase-2 (COX-2) compared to GMA-MS and control. Increased generation of ROS due to MMA-SS exposure was confirmed by increased expression of Nrf2 and heme oxygenase-1 (HO-1). Results of in vitro studies provide evidence that stainless steel welding fume mediate inflammatory responses via activation of ROS/P-HNE/ERK1/2/Nrf2 signaling pathways. These findings were corroborated by elevated expression of COX-2, Nrf2, and HO-1 in homogenized lung tissue collected 1 day after in vivo exposure.