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Substrate Stiffness-Dependent Carbon Nanotube-Induced Lung Fibrogenesis.

ABSTRACT: Most living tissues exhibit the specific stiffness, which has been known to have profound influence on cell behaviors, yet how the stiffness affects cellular responses to engineered nanomaterials has not been elucidated. Particularly, discrepancies exist between in vitro and in vivo nanotoxicological studies. Here, we investigated the effects of substrate stiffness on the fibrogenic responses of normal human lung fibroblasts (NHLFs) to multiwalled carbon nanotubes (MWCNTs). NHLFs were grown on polyacrylamide (PAAm) hydrogels with the stiffness comparable to that of human normal and fibrotic lung tissues, and treated with MWCNTs for various time. The fibrogenic responses, including cell proliferation, reactive oxygen species production, and collagen I expression, of NHLFs to MWCNTs were observed to be regulated by substrate stiffness in a time-dependent manner. NHLFs generally were rounded on soft hydrogels and required a long treatment time to exhibit fibrogenic responses, while on stiff hydrogels the cells were well-spread with defined stress fibers and short-time MWCNTs treatment sufficiently induced the fibrogenic responses. Mechanistic studies showed that MWCNTs induced fibrogenesis of NHLFs through promoting expression and phosphorylation of focal adhesion kinase (FAK), while attenuating intracellular tension in the cells on stiff gels could increase MWCNTs uptake and thus elevate the induced fibrogenic responses. Moreover, we proposed a time-stiffness superposition principle to describe the equivalent effects of treatment time and substrate stiffness on nanomaterials-induced fibrogenesis, which suggested that increasing substrate stiffness expedited fibrogenesis and shed light on the rational design of in vitro models for nanotoxicological study.

SUBMITTER: Wang K

PROVIDER: S-EPMC6724206 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Substrate Stiffness-Dependent Carbon Nanotube-Induced Lung Fibrogenesis.

Wang Kai K Shi Lin L Linthicum Will W Man Kun K He Xiaoqing X Wen Qi Q Rojanasakul Liying Wang LW Rojanasakul Yon Y Yang Yong Y

Nano letters 20190805 8

Most living tissues exhibit the specific stiffness, which has been known to have profound influence on cell behaviors, yet how the stiffness affects cellular responses to engineered nanomaterials has not been elucidated. Particularly, discrepancies exist between <i>in vitro</i> and <i>in vivo</i> nanotoxicological studies. Here, we investigated the effects of substrate stiffness on the fibrogenic responses of normal human lung fibroblasts (NHLFs) to multiwalled carbon nanotubes (MWCNTs). NHLFs w ...[more]

PMID: 31369708

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Project description:BackgroundMulti-walled carbon nanotubes (MWCNTs) represent a human health risk as mice exposed by inhalation display pulmonary fibrosis. Production of IL-1? via inflammasome activation is a mechanism of MWCNT-induced acute inflammation and has been implicated in chronic fibrogenesis. Mice sensitized to allergens have elevated T-helper 2 (Th2) cytokines, IL-4 and IL-13, and are susceptible to MWCNT-induced airway fibrosis. We postulated that Th2 cytokines would modulate MWCNT-induced inflammasome activation and IL-1? release in vitro and in vivo during allergic inflammation.MethodsTHP-1 macrophages were primed with LPS, exposed to MWCNTs and/or IL-4 or IL-13 for 24 hours, and analyzed for indicators of inflammasome activation. C57BL6 mice were sensitized to house dust mite (HDM) allergen and MWCNTs were delivered to the lungs by oropharyngeal aspiration. Mice were euthanized 1 or 21 days post-MWCNT exposure and evaluated for lung inflammasome components and allergic inflammatory responses.ResultsPriming of THP-1 macrophages with LPS increased pro-IL-1? and subsequent exposure to MWCNTs induced IL-1? secretion. IL-4 or IL-13 decreased MWCNT-induced IL-1? secretion by THP-1 cells and reduced pro-caspase-1 but not pro-IL-1?. Treatment of THP-1 cells with STAT6 inhibitors, either Leflunomide or JAK I inhibitor, blocked suppression of caspase activity by IL-4 and IL-13. In vivo, MWCNTs alone caused neutrophilic infiltration into the lungs of mice 1 day post-exposure and increased IL-1? in bronchoalveolar lavage fluid (BALF) and pro-caspase-1 immuno-staining in macrophages and airway epithelium. HDM sensitization alone caused eosinophilic inflammation with increased IL-13. MWCNT exposure after HDM sensitization increased total cell numbers in BALF, but decreased numbers of neutrophils and IL-1? in BALF as well as reduced pro-caspase-1 in lung tissue. Despite reduced IL-1? mice exposed to MWCNTs after HDM developed more severe airway fibrosis by 21 days and had increased pro-fibrogenic cytokine mRNAs.ConclusionsThese data indicate that Th2 cytokines suppress MWCNT-induced inflammasome activation via STAT6-dependent down-regulation of pro-caspase-1 and suggest that suppression of inflammasome activation and IL-1? by an allergic lung microenvironment is a mechanism through which MWCNTs exacerbate allergen-induced airway fibrosis.

Project description:Carbon nanotubes were among the earliest products of nanotechnology and have many potential applications in medicine, electronics, and manufacturing. The low density, small size, and biological persistence of carbon nanotubes create challenges for exposure control and monitoring and make respiratory exposures to workers likely. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to 24, 48 and 96 ?g/cm(2) single-walled carbon nanotubes (SWCNT). To investigate mitotic spindle aberrations at concentrations anticipated in exposed workers, primary and immortalized human airway epithelial cells were exposed to SWCNT for 24-72 h at doses equivalent to 20 weeks of exposure at the Permissible Exposure Limit for particulates not otherwise regulated. We have now demonstrated fragmented centrosomes, disrupted mitotic spindles and aneuploid chromosome number at those doses. The data further demonstrated multipolar mitotic spindles comprised 95% of the disrupted mitoses. The increased multipolar mitotic spindles were associated with an increased number of cells in the G2 phase of mitosis, indicating a mitotic checkpoint response. Nanotubes were observed in association with mitotic spindle microtubules, the centrosomes and condensed chromatin in cells exposed to 0.024, 0.24, 2.4 and 24 ?g/cm(2) SWCNT. Three-dimensional reconstructions showed carbon nanotubes within the centrosome structure. The lower doses did not cause cytotoxicity or reduction in colony formation after 24h; however, after three days, significant cytotoxicity was observed in the SWCNT-exposed cells. Colony formation assays showed an increased proliferation seven days after exposure. Our results show significant disruption of the mitotic spindle by SWCNT at occupationally relevant doses. The increased proliferation that was observed in carbon nanotube-exposed cells indicates a greater potential to pass the genetic damage to daughter cells. Disruption of the centrosome is common in many solid tumors including lung cancer. The resulting aneuploidy is an early event in the progression of many cancers, suggesting that it may play a role in both tumorigenesis and tumor progression. These results suggest caution should be used in the handling and processing of carbon nanotubes.

Dataset Information

Substrate Stiffness-Dependent Carbon Nanotube-Induced Lung Fibrogenesis.

Publications

Substrate Stiffness-Dependent Carbon Nanotube-Induced Lung Fibrogenesis.

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