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

Abstract

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.

Mechanisms of carbon nanotube‐induced pulmonary fibrosis: a physicochemical characteristic perspective

“…Cell membrane disruption also causes leakage of cellular constituents (e.g., reactive oxygen species, enzymes, cytokines) that can cause injury to surrounding cells and tissues. Rod-like MWCNTs also result in mucous cell metaplasia alone while tangled MWCNTs do not 31–34 . While CNT length is an important determinant of clearance rate from the lungs, length alone does not necessarily determine CNT persistence 25,35 .…”

“…M2 macrophages also produce more TGF-β1 in response to stimuli. Interestingly, a rod-like MWCNT treatment via oropharyngeal aspiration results in a more Th2 response as indicated via mucous cell metaplasia and/or eosinophilia 31,34 . M2 macrophages produce Th2 cytokines, like IL-4, IL-5, and IL-13 which drive the adaptive immune response and are upregulated in bleomycin-induced fibrosis 105 .…”

“…STAT1 knockout mice exhibit an increased susceptibility to bleomycin induced fibrosis 112 . Furthermore, these STAT1 knockout mice experience MWCNT-induced exacerbation of fibrosis and asthma demonstrating STAT1 as protective against fibrosis 33,34 . Specifically, STAT1 was found to negatively regulate TGF-β1 and its downstream signaling molecule Smad2/3 phosphorylation and activation 34 .…”

The Toxicology of Engineered Nanomaterials in Asthma

“…Rod-like MWCNTs, which are more rigid, are known to be more toxic than other more flexible MWCNTs, and are able to cause AHR, mucus secretion, eosinophilia and T H 2 cytokine release (IL-4, IL-13) [72]. These finding are supported by another study, which compared rod-like and the more flexible tangled MWCNTs, finding that rod-like but not tangled MWCNTs induced IL-4, airway mucus production, and elevated serum IgE levels [73]. In vitro , MWCNTs are able to activate the NLRP3 inflammasome in human bronchial epithelial cells, indicating a potential to cause airway remodeling, a key feature of asthma [74].…”

“…Additionally, IL-1, IL-6, IL-12 and especially IL-10 are significantly increased in the BALF of MWCNT-exposed mice [71]. The physicochemical properties of different types of CNTs have varying effects on the type of immune response they elicit, as examined by two studies [72, 73]. Rod-like MWCNTs, which are more rigid, are known to be more toxic than other more flexible MWCNTs, and are able to cause AHR, mucus secretion, eosinophilia and T H 2 cytokine release (IL-4, IL-13) [72].…”