Background: Graphene, a monolayer of carbon, is an engineered nanomaterial (ENM) with physical and chemical properties that may offer application advantages over other carbonaceous ENMs, such as carbon nanotubes (CNT). The goal of this study was to comparatively assess pulmonary and systemic toxicity of graphite nanoplates, a member of the graphene-based nanomaterial family, with respect to nanoplate size. Methods: Three sizes of graphite nanoplates [20 μm lateral (Gr20), 5 μm lateral (Gr5), and <2 μm lateral (Gr1)] ranging from 8-25 nm in thickness were characterized for difference in surface area, structure,, zeta potential, and agglomeration in dispersion medium, the vehicle for in vivo studies. Mice were exposed by pharyngeal aspiration to these 3 sizes of graphite nanoplates at doses of 4 or 40 μg/mouse, or to carbon black (CB) as a carbonaceous control material. At 4 h, 1 day, 7 days, 1 month, and 2 months post-exposure, bronchoalveolar lavage was performed to collect fluid and cells for analysis of lung injury and inflammation. Particle clearance, histopathology and gene expression in lung tissue were evaluated. In addition, protein levels and gene expression were measured in blood, heart, aorta and liver to assess systemic responses. Results: All Gr samples were found to be similarly composed of two graphite structures and agglomerated to varying degrees in DM in proportion to the lateral dimension. Surface area for Gr1 was approximately 7-fold greater than Gr5 and Gr20, but was less reactive reactive per m

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.

Introduction Stroke‐associated pneumonia (SAP) is a major cause of mortality in patients who have suffered from severe ischemic stroke. Although multifactorial in nature, stroke‐induced immunosuppression plays a key role in the development of SAP. Previous studies using a murine model of transient middle cerebral artery occlusion (tMCAO) have shown that focal ischemic stroke induction results in functional defects of lymphocytes in the spleen, thymus, and peripheral blood, leading to spontaneous bacterial infection in the lungs without inoculation. However, how ischemic stroke alters immune cell niche and the expression of cytokines and chemokines in the lungs has not been fully characterized. Methods Ischemic stroke was induced in mice by tMCAO. Immune cell profiles in the brain and the lungs at 24‐ and 72‐hour time points were compared by flow cytometric analysis. Cytokine and chemokine expression in the lungs were determined by multiplex bead arrays. Tissue damage and bacterial burden in the lungs following tMCAO were evaluated. Results Ischemic stroke increases the percentage of alveolar macrophages, neutrophils, and CD11b+ dendritic cells, but reduces the percentage of CD4+ T cells, CD8+ T cells, B cells, natural killer cells, and eosinophils in the lungs. The alteration of immune cell niche in the lungs coincides with a significant reduction in the levels of multiple chemokines in the lungs, including CCL3, CCL4, CCL5, CCL17, CCL20, CCL22, CXCL5, CXCL9, and CXCL10. Spontaneous bacterial infection and tissue damage following tMCAO, however, were not observed. Conclusion This is the first report to demonstrate a significant reduction of lymphocytes and multiple proinflammatory chemokines in the lungs following ischemic stroke in mice. These findings suggest that ischemic stroke directly impacts pulmonary immunity.

Signal tranducer and activator of transcription 5 (STAT5) plays a critical role in mediating cellular responses following cytokine stimulation. STAT proteins critically signal via the formation of dimers, but additionally, STAT tetramers serve key biological roles, and we previously reported their importance in T and natural killer (NK) cell biology. However, the role of STAT5 tetramerization in autoimmune-mediated neuroinflammation has not been investigated. Using the STAT5 tetramer-deficient Stat5a-Stat5b N-domain double knockin (DKI) mouse strain, we report here that STAT5 tetramers promote the pathogenesis of experimental autoimmune encephalomyelitis (EAE). The mild EAE phenotype observed in DKI mice correlates with the impaired extravasation of pathogenic T-helper 17 (Th17) cells and interactions between Th17 cells and monocyte-derived cells (MDCs) in the meninges. We further demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF)–mediated STAT5 tetramerization regulates the production of CCL17 by MDCs. Importantly, CCL17 can partially restore the pathogenicity of DKI Th17 cells, and this is dependent on the activity of the integrin VLA-4. Thus, our study reveals a GM-CSF-STAT5 tetramer-CCL17 pathway in MDCs that promotes autoimmune neuroinflammation.