2015 Progress Report: Airway Epithelium Organotypic Culture as a Platform for AdverseOutcomesPathway Assessment of Engineered Nanomaterials

EPA 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
Current 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, 2014 through November 30,2015
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text |  Recipients Lists
Research Category: Safer Chemicals , Health , Human Health

Objective:

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 also can 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. 

Progress Summary:

Experiment 1: Our initial work has focused on the evaluating toxicity to engineered silver nanoparticles (average diameter of 20nm with citrate coating). We have evaluated the response of normally differentiated mouse tracheal epithelial cell culture (MTEC) to both acute (24 hour) and subacute (4 hours daily for 5 days) exposure. Additionally, we evaluated cells isolated from both C57BL/6 mice and from A/J mice after 24 hours to evaluate the potential impact of genotype on response. We also evaluated whether sub-acute exposure to AgNP altered subsequent cellular response to a common allergen, house dust mite extract.

We found that acute AgNP exposure resulted in no significant cellular toxicity, as assessed by LDH release, and no significant inflammatory response as assessed by neutrophil chemokine expression. Epithelial cells did increase expression of metallothionein 1 and 2 in response to AgNP but there was minimal change in heme oxygenase-1 expression. Subacute nanosilver exposure similarly did not result in significant cellular toxicity or increased neutrophil chemokine release. AgNP exposure did not potentiate HDM-induced epithelial cell activation.

Experiment 2: We have evaluated the effect of AgNP exposure on macrophages. Both resident and recruited macrophages are important components of host defense in response to lung infection or injury. Primary bone-marrow derived macrophages isolated from both C57BL/6 and A/J mice were differentiated in CSF-1-containing medium for 7 days, after which they were exposed to varying concentrations of AgNP for 24 hours followed by assessment of cytotoxicity and inflammatory activation.

We found a dose-dependent impact of AgNP on cell viability that was associated on caspase activation. There was a significant strain effect with A/J macrophages being more resistant to AgNP-induced cell death. AgNP exposure also resulted in expression of pro-inflammatory cytokines and upregulation of heme oxygenase-1, metallothionein-1 and metallothionein-2.

Experiment 3: Primary lung mesenchymal cells were isolated from C57BL/6 mice by magnetic bead immunodepletion of CD45+, CD31+, CD326+ cells followed by immunoselection of PDGFRb+ and PDGFRb- cells. Cultured cells were then exposed to varying AgNP concentrations. We found significant dose-dependent cytotoxicity after 24-hour exposure in both types of lung mesenchymal cells. 

Conclusions to date:

  • Primary mouse differentiated airway epithelial cells are highly resistant to AgNP-induced cytotoxicity or inflammatory activation. 
  • Sub-acute AgNP exposure does not modify airway epithelial cell response to the common allergen, house dust mite. 
  • Both macrophages and sub-epithelial mesenchymal cells are susceptible to AgNP-induced cytotoxicity. 

Future Activities:

Assess the impact of MTEC differentiation towards a diseased phenotype. Addition of IL-13 to the defined medium used for air-liquid interface differentiation results in a disease phenotype characterized by increased expression of mucus-secreting Goblet cells. We will use this model to determine whether AgNP has an impact on this model of diseased epithelium, focusing on cytotoxicity, inflammatory activation, and upregulation of mucus-associated genes, such as Muc5ac.

Evaluate culture models that combine MTEC cells with apical chamber macrophages and/or basal chamber mesenchymal cells. We hypothesize that AgNP activation or killing of susceptible cell types, including macrophages and mesenchymal cells, will result in release of mediators that activate epithelial cells, resulting in basolateral release of neutrophil chemokines. 

Journal Articles:

No journal articles submitted with this report: View all 11 publications for this subproject

Supplemental Keywords:

Airway epithelial cells, air-liquid interface, organotypic culture, engineered nanosilver material, chronic airways disease, allergic airways disease, macrophages, mesenchymal cells 

Relevant Websites:

The Predictive Toxicology Center (PTC) for Organotypic Cultures Exit

Progress and Final Reports:

Original Abstract
  • 2016 Progress Report
  • 2017 Progress Report
  • 2018

  • 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