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

Katherine S. Duke, Alexia J. Taylor-Just, Mark D. Ihrie, Kelly A. Shipkowski, Elizabeth A. Thompson, Erinn C. Dandley, Gregory N. Parsons, James C. Bonner

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. 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. 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-β1 protein levels in the BALF of Stat1 (-/-) mice exposed to rMWCNTs and increased lung Smad2/3 phosphorylation. 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-β1 production and Smad2/3 phosphorylation status. Therefore, both rigidity and genetic susceptibility should be major considerations for risk assessment of MWCNTs.