2017 Progress Report: Airway Epithelium Organotypic Culture as a Platform for AdverseOutcomesPathway Assessment of Engineered NanomaterialsEPA Grant Number: R835738C001
Subproject: this is subproject number 001 , established and managed by the Center Director under grant R835738
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Predictive Toxicology Center for Organotypic Cultures and Assessment of AOPs for Engineered Nanomaterials
Center Director: Faustman, Elaine
Title: Airway Epithelium Organotypic Culture as a Platform for AdverseOutcomesPathway Assessment of Engineered Nanomaterials
Investigators: Altemeier, William
Institution: University of Washington
EPA Project Officer: Klieforth, Barbara I
Project Period: December 1, 2014 through November 30, 2018 (Extended to November 30, 2019)
Project Period Covered by this Report: December 1, 2016 through November 30,2017
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text | Recipients Lists
Research Category: Safer Chemicals , Health , Human Health
The overall goal of this project is to utilize mouse lung organotypic culture systems to better evaluate for cellular and organ toxicity to relevant engineered nanoparticles. The lungs are a major route of exposure to environmental and occupational compounds, and the airway epithelium is the primary surface for initial contact and management of inhaled exogenous materials. This project therefore focuses on using primary epithelial cells differentiated at an air-liquid system as the basis for modeling. This cell system represents an organotypic model system consisting of a combination of ciliated epithelium and club (Clara) secretory cells. Furthermore, altering the defined culture medium can skew the cell phenotype towards a mucus secretory cell type (aka goblet cells) to model chronic airway diseases. The culture system can also be combined with stromal cells in the basal chamber and/or macrophages in the apical chamber to further extend the relevance of the model system.
Specific Aim 1: Determine proliferation response of undifferentiated mTEC cells (basal epithelial cells) in response to the proposed engineered nanomaterials. Determine whether nanomaterials alter the relative proportions of ciliated epithelial cells, Club cells, and mucus secreting or goblet cells during differentiation at ALI in the presence or absence of IL-13, which skews differentiation towards a “disease phenotype” characterized by increase in mucus producing cells.
Specific Aim 2: Evaluate undifferentiated mTEC cultures, and mTEC cultures differentiated at ALI with or without IL-13 for evidence of oxidative stress and ER stress following acute and chronic engineered nanomaterial exposure. Assess cellular uptake of nanomaterials and cellular toxicity.
Specific Aim 3: Determine the temporal pattern of inflammatory gene expression in undifferentiated, normally differentiated, and IL-13 skewed differentiated mTEC culture following nanomaterial exposure. Determine the role of oxidative stress by examining cellular response in the presence of exogenous antioxidants. Determine the role of Toll-like receptor (TLR) activation by contrasting cellular responses of mTEC culture isolated from normal mice and mice lacking the essential TLR adapter protein, MyD88.
Revised goal – Confirm translational relevance of select genes, using primary human airway epithelial cell culture, differentiated with and without IL13. Confirming genes in human cells confirms the relevance of selected genes in screening applications, using mouse cells.
Specific Aim 4: Determine whether mTEC culture isolated from two different laboratory mouse strains, A/J and C57BL/6 have varying responses to nanomaterial exposure.
Experiment 1: We have evaluated response to AgNP in cultures, using cells isolated from both A/J and C57BL/6 mice, which were differentiated in the presence of IL13. We compared results of 24-hour acute exposure versus sub-acute 4 hours/day for 1 week. There were not significant differences from exposure duration in terms of cytotoxicity and barrier integrity. Differences were observed, dependent on dose of AgNP and strain from which cells were isolated.
Experiment 2: Samples were collected from 24-hour exposure to AgNP in A/J and C57BL/6 mice differentiated with and without IL13 for dosimetry analysis by ICP-MS. These results will be used to identify benchmark dose and will be included in AOP analysis.
Experiment 3: RNA was isolated and quality checked from IL13-diferentiated airway epithelial cells exposed to AgNP. Cells were from both A/J and C57BL6 mice. RNA has been submitted for BioSpyder transcriptional analysis through the PTC core. Results will be incorporated into AOP development.
Experiment 4: A protocol has been implemented for isolation and cryopreservation of human bronchial epithelial cells. Briefly, bronchial rings are collected from deceased donor airways prior to transplantation. Cells are isolated, expanded and cryopreserved. Basic epidemiologic data (age, biological sex, smoking history) are collected. Cryopreserved cells recover and can be differentiated at air-liquid interface to form organotypic airway culture. 20 samples have been processed. These will be used for human confirmation of select genes identified by BioSpyder analysis.
Conclusions to date:
IL13 differentiated cells (modeling chronic airway disease) are more susceptible to AgNP-induced cytotoxicity and epithelial barrier dysfunction as compared with normally differentiated cells. This suggests that individuals with chronic airways disease (e.g., asthma, COPD) may be at heightened risk to occupational or environmental exposure to engineered silver nanoparticles.
Cells from A/J mice are more susceptible to AgNP-induced cytotoxicity as compared with cells from C57BL6 mice. These data suggest that genetic background modulates host response to engineered silver nanoparticles. Identification of specific genetic loci, which confer risk, would provide mechanisms for assessing occupational risk of specific individuals. The next step in this experiment would be to repeat exposure studies in cells isolated from a mouse diversity panel to allow identification of specific QTLs and candidate genes within the QTLs.
Human airway epithelial cells can be isolated from discarded surgical material, cryopreserved, and recovered for preparation of airway organotypic culture systems. This will allow confirmation of select differentially regulated genes from mouse studies that are used in AOP generation.
Complete dosimetry studies and BioSpyder analysis of AgNP-treated organotypic culture systems.
Develop AOP in response to data from #1.
Confirm key findings in human airway organotypic culture.
Complete assessment of cadmium exposure to organotypic culture systems differentiated with IL13 and perform BioSpyder and dosimetry studies for AOP planning.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
|Other subproject views:||All 11 publications||3 publications in selected types||All 3 journal articles|
|Other center views:||All 134 publications||39 publications in selected types||All 38 journal articles|
||Nolin JD, Lai Y, Ogden HL, Manicone AM, Murphy RC, An D, Frevert CW, Ghomashchi F, Naika GS, Gelb MH, Gauvreau GM, Piliponsky AM, Altemeier WA, Hallstrand TS. Secreted PLA2 group X orchestrates innate and adaptive immune responses to inhaled allergen. JCI Insight 2017;2(21):94929 (18 pp.).||
||Scoville DK, Botta D, Galdanes K, Schmuck SC, White CC, Stapleton PL, Bammler TK, MacDonald JW, Altemeier WA, Hernandez M, Kleeberger SR, Chen LC, Gordon T, Kavanagh TJ. Genetic determinants of susceptibility to silver nanoparticle-induced acute lung inflammation in mice. FASEB Journal 2017;31(10):4600-4611.||
Supplemental Keywords:Airway, lung, engineered nanomaterials, asthma, chronic obstructive lung disease, COPD
Progress and Final Reports:Original Abstract
Main Center Abstract and Reports:R835738 Predictive Toxicology Center for Organotypic Cultures and Assessment of AOPs for Engineered Nanomaterials
Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R835738C001 Airway Epithelium Organotypic Culture as a Platform for AdverseOutcomesPathway Assessment of Engineered Nanomaterials
R835738C002 Organotypic Model of Human Kidney as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
R835738C003 Organotypic Models of Mammalian Liver as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
R835738C004 Organotypic Model of Testis as a Platform for Adverse Outcomes Pathway Assessment of Engineered Nanomaterials
R835738C005 Integrating Liver, Kidney and Testis Nanomaterial Toxicity using the Adverse Outcome Pathway Approach