Stat-6 signaling pathway and not Interleukin-1 mediates multi-walled carbon nanotube-induced lung fibrosis in mice: insights from an adverse outcome pathway framework

Abstract

BackgroundThe accumulation of MWCNTs in the lung environment leads to inflammation and the development of disease similar to pulmonary fibrosis in rodents. Adverse Outcome Pathways (AOPs) are a framework for defining and organizing the key events that comprise the biological changes leading to undesirable events. A putative AOP has been developed describing MWCNT-induced pulmonary fibrosis; inflammation and the subsequent healing response induced by inflammatory mechanisms have been implicated in disease progression.The objective of the present study was to address a key data gap in this AOP: empirical data supporting the essentiality of pulmonary inflammation as a key event prior to fibrosis. Specifically, Interleukin-1 Receptor1 (IL-1R1) and Signal Transducer and Activator of Transcription 6 (STAT6) knock-out (KO) mice were employed to target inflammation and the subsequent healing response using MWCNTs as a model pro-fibrotic stressor to determine whether this altered the development of fibrosis.ResultsWild type (WT) C57BL/6, IL-1R1 (KO) or STAT6 KO mice were exposed to a high dose of Mitsui-7 MWCNT by intratracheal administration. Inflammation was assessed 24 h and 28 days post MWCNT administration, and fibrotic lesion development was assessed 28 days post MWCNT administration. MWCNT-induced acute inflammation was suppressed in IL-1R1 KO mice at the 24 h time point relative to WT mice, but this suppression was not observed 28 days post exposure, and IL-1R1 KO did not alter fibrotic disease development. In contrast, STAT6 KO mice exhibited suppressed acute inflammation and attenuated fibrotic disease in response to MWCNT administration compared to STAT6 WT mice. Whole genome analysis of all post-exposure time points identified a subset of differentially expressed genes associated with fibrosis in both KO mice compared to WT mice.ConclusionThe findings support the essentiality of STAT6-mediated signaling in the development of MWCNT-induced fibrotic disease. The IL-1R1 KO results also highlight the nature of the inflammatory response associated with MWCNT exposure, and indicate a system with multiple redundancies. These data add to the evidence supporting an existing AOP, and will be useful in designing screening strategies that could be used by regulatory agencies to distinguish between MWCNTs of varying toxicity.Electronic supplementary materialThe online version of this article (10.1186/s12989-017-0218-0) contains supplementary material, which is available to authorized users.

21st Century Tools for Nanotoxicology: Transcriptomic Biomarker Panel and Precision‐Cut Lung Slice Organ Mimic System for the Assessment of Nanomaterial‐Induced Lung Fibrosis

“…Transcriptomics tools have been successfully used as an alternative approach to comprehensively investigate the toxicological response induced by various classes of ENMs and to identify the properties of ENMs that are responsible for eliciting adverse effects. [7][8][9][10][11][12][13][14] However, in order for toxicogenomics data to be effectively applied in safety assessment or predictive toxicology, it is essential to identify and discriminate genes that define tissue defense mechanisms from those potentially critical for the progression of toxic outcomes and diseases.…”

“…This AOP clearly identifies the key events in the progression of lung fibrosis (the detailed AOP can be found at https://aopwiki.org/aops/173). [9,18] In the present study, we developed a 17-gene pro-fibrotic biomarker panel (here after referred to as PFS17) that is specifically predictive of lung fibrosis in mice. Previously published, publicly available gene expression microarray data from mouse lungs exposed to a variety of stressors that are known to cause lung disease (i.e., lung inflammation, emphysema, chronic obstructive pulmonary disease [COPD], lung fibrosis, and lung cancer) were used.…”

“…Table S2, Supporting Information, lists all datasets included in the validation set and the class prediction results. [9,12,14,[33][34][35][36][37][38][39][40][41][42][43][44] Figures 3 and 4 show heatmaps of all non-nano and nano studies included in the validation dataset. The validation set consisted of three bleomycin studies with 12 individual experimental conditions reflecting different exposure concentrations and post-exposure time points.…”

Microenvironmental Alterations in Carbon Nanotube-Induced Lung Inflammation and Fibrosis

“…The hallmark of acute inflammation is the immediately and strikingly induced recruitment, infiltration, and accumulation of inflammatory cells Ma, 2016b, 2019b). Neutrophils and macrophages are markedly recruited and infiltrate into lung tissues, examined by cell counting of BAL samples and immunostaining of specific cell markers on lung tissues, which indicates their critical roles as the frontline responders in the onset of acute inflammation induced by CNTs (Shvedova et al, 2005;Porter et al, 2013;Rydman et al, 2015;Nikota et al, 2017;Dong and Ma, 2018a). At this stage, M1 (traditionally activated) macrophages are the dominant macrophage population, as determined by the cell surface markers CD86 and MHC II.…”

“…The detailed analysis of their individual functions is emerging at the current stage. For instance, IL-1 signaling activated by IL-1α or IL-1β has been shown to promote acute inflammation, but not chronic inflammation or fibrosis, in the lung exposed to MWNT-7 MWCNTs, examined by using IL-1R KO mice (Rydman et al, 2015;Nikota et al, 2017). Insightful studies in this direction would identify the critical mediators among these rapidly induced factors to serve as functional biomarkers and therapeutic targets for lung inflammation and fibrosis.…”