BackgroundPulmonary 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.MethodsMale 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.ResultsThere 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-β1 protein levels in the BALF of Stat1 −/− mice exposed to rMWCNTs and increased lung Smad2/3 phosphorylation.ConclusionsRigidity 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-β1 production and Smad2/3 phosphorylation status. Therefore, both rigidity and genetic susceptibility should be major considerations for risk assessment of MWCNTs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-017-0207-3) contains supplementary material, which is available to authorized users.

Nickel nanoparticles (NiNPs) are increasingly used in nanotechnology applications, yet information on sex differences in NiNP-induced lung disease is lacking. The goal of this study was to explore mechanisms of susceptibility between male and female mice after acute or subchronic pulmonary exposure to NiNPs. For acute exposure, male and female mice received a single dose of NiNPs with or without LPS by oropharyngeal aspiration and necropsied 24 hours later. For subchronic exposure, mice received NiNPs with or without LPS six times over 3 weeks prior to necropsy. After acute exposure to NiNPs and LPS, male mice had elevated cytokines (CXCL1 and IL-6) and more neutrophils in bronchoalveolar lavage fluid (BALF), along with greater STAT3 phosphorylation in lung tissue. After subchronic exposure to NiNPs and LPS, male mice exhibited increased monocytes in BALF. Moreover, subchronic exposure of male mice to NiNP only induced higher CXCL1 and CCL2 in BALF along with increased alveolar infiltrates and CCL2 in lung tissue. STAT1 in lung tissue was induced by subchronic exposure to NiNPs in females but not males. Males had a greater induction of IL-6 mRNA in liver after acute exposure to NiNPs and LPS, and greater CCL2 mRNA in liver after subchronic NiNP exposure. These data indicate that susceptibility of males to acute lung inflammation involves enhanced neutrophilia with increased CXCL1 and IL-6/STAT3 signaling, whereas susceptibility to subchronic lung inflammation involves enhanced monocytic infiltration with increased CXCL1 and CCL2. STAT transcription *

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-1β, IL-6, CXCL1) in BALF, while thermal purification had a greater effect on pro-fibrotic cytokines (CCL2, OPN, TGF-β1). 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.

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 hours a day, 5 days a week, for 30 days to air control or 0.06, 0.2 and 0.6 mg/m 3 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 days 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 days 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 T H 2 immune responses, yet this combined exposure resulted in vascular inflammation and airway fibrosis, indicating that MWCNT pre-exposure alters the immune response to allergens.

The fiber-like shape of multi-walled carbon nanotubes (MWCNTs) is reminiscent of asbestos, suggesting they pose similar health hazards when inhaled, including pulmonary fibrosis and mesothelioma. Mice deficient in the tumor suppressor p53 are susceptible to carcinogenesis. However, the chronic pathological effect of MWCNTs delivered to the lungs of p53 heterozygous (p53+/−) mice has not been investigated. We hypothesized that p53+/− mice would be susceptible to lung tumor development after exposure to either tangled (t-) or rod-like (r-) MWCNTs. Wild type (p53+/+) or p53+/− mice were exposed to MWCNTs (1 mg/kg) via oropharyngeal aspiration weekly over 4 consecutive weeks and evaluated for cellular and pathologic outcomes 11-months post initial exposure. No lung or pleural tumors were observed in p53+/+ or p53+/− mice exposed to either t- or rMWCNTs. In comparison to tMWCNTs, the rMWCNTs induced the formation of larger granulomas, a greater number of lymphoid aggregates and greater epithelial cell hyperplasia in terminal bronchioles in both p53+/− and p53+/+ mice. A constitutively larger area of CD45R+/CD3+ lymphoid tissue was observed in p53+/− mice compared to p53+/+ mice. Importantly, p53+/− mice had larger granulomas induced by rMWCNTs as compared to p53+/+ mice. These findings indicate that a combination of p53 deficiency and physicochemical characteristics including nanotube geometry are factors in susceptibility to MWCNT-induced lymphoid infiltration and granuloma formation.

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