Developing Alternative Models to Evaluate the Impact of Nanomaterials on Neutrophils during the Stimulation and Resolution of Inflammation
Lead Research Organisation:
Heriot-Watt University
Department Name: Sch of Energy, Geosci, Infrast & Society
Abstract
Activation of the immune system (inflammation) is essential to protect the body from harm caused by infection (e.g. bacteria), chemicals or pollution. Immune cells (white blood cells) are responsible for recognising these harmful stimuli and if possible, destroying them. Different types of immune cells vary with regards to their appearance, what stimuli they recognise, and how quickly they respond (hours vs days). Neutrophils (a type of immune cell) are part of the first line of defence; they travel in blood and patrol organs. Following recognition of a harmful stimulus neutrophils accumulate in the affected organ (e.g. lungs, liver) causing inflammation. This inflammation can last for a few hours or persist for weeks to years. In order to stop an inflammatory response neutrophils die. Failure of neutrophil responses to resolve (recover) can cause illness, as during inflammation neutrophils release molecules which destroy harmful stimuli but unfortunately may also damage the body. Toxicology studies investigate if substances cause harmful effects in humans, including inflammation.
Nanomaterials (NMs) are substances that are very small (<100 nanometers), which for comparison, are hundreds of times smaller than the width of human hair. NMs can be made from many different elements and can vary in terms of size, shape and other characteristics so that there are tens of thousands of different NMs that can be produced. The small size of NMs introduces new properties to materials e.g. gold NMs are red/purple in colour. These new properties allow used in diverse products (e.g. clothing, medicines, cosmetics) leading to exposure of humans to NMs. As NMs are a new type of substance it is important to assess their safety. There is evidence that some NMs can cause inflammation which may lead to adverse health effects.
Current regulations (laws) require laboratory experiments to use animals (rodents) to assess neutrophil accumulation in organs over time as an indicator of how toxic NMs are. Many studies therefore focus on neutrophil accumulation as a marker of toxicity. More studies are needed to assess whether inflammation resolves. If inflammation resolves then NMs can be regarded as being of low toxicity. As NM use increases there is more demand for rodent studies to assess their potential toxicity. However, rodent studies are expensive, time consuming and there are ethical concerns about the use of rodents for scientific research and regulatory testing. The availability of good alternatives to rodent models for NM toxicity testing will reduce or replace rodent use. In vitro experiments study the response of neutrophils in the laboratory, without requiring rodents. Cells can be isolated from human blood (termed primary cells). Primary cells have a short lifetime and require humans to provide blood samples. Neutrophil cell lines, which live longer, and can be purchased, however they may not respond in the same way as primary cells. In vitro experiments can be criticised as they only look at the response of one cell type. For the immune system to function properly communication between different cell types in the body is required. Zebrafish (ZF) allow inflammatory responses to be evaluated in an intact non-rodent and so can be employed to reduce rodent use.
Our project will identify approaches to investigate the ability of NMs to stimulate neutrophil responses in the laboratory and will identify if cells and ZF are good alternatives to rodents. We will compare our results to findings previously obtained from rodent studies to identify whether cells and ZF provide a good prediction of the rodent response. If they behave similarly then the scientific community may use these alternatives to decrease the numbers of rodents used to assess NM safety. Importantly, these alternatives are not restricted to testing NM safety and could be used to test the toxicity of a wider range of pollutants, chemicals (e.g. drugs) and microbes.
Nanomaterials (NMs) are substances that are very small (<100 nanometers), which for comparison, are hundreds of times smaller than the width of human hair. NMs can be made from many different elements and can vary in terms of size, shape and other characteristics so that there are tens of thousands of different NMs that can be produced. The small size of NMs introduces new properties to materials e.g. gold NMs are red/purple in colour. These new properties allow used in diverse products (e.g. clothing, medicines, cosmetics) leading to exposure of humans to NMs. As NMs are a new type of substance it is important to assess their safety. There is evidence that some NMs can cause inflammation which may lead to adverse health effects.
Current regulations (laws) require laboratory experiments to use animals (rodents) to assess neutrophil accumulation in organs over time as an indicator of how toxic NMs are. Many studies therefore focus on neutrophil accumulation as a marker of toxicity. More studies are needed to assess whether inflammation resolves. If inflammation resolves then NMs can be regarded as being of low toxicity. As NM use increases there is more demand for rodent studies to assess their potential toxicity. However, rodent studies are expensive, time consuming and there are ethical concerns about the use of rodents for scientific research and regulatory testing. The availability of good alternatives to rodent models for NM toxicity testing will reduce or replace rodent use. In vitro experiments study the response of neutrophils in the laboratory, without requiring rodents. Cells can be isolated from human blood (termed primary cells). Primary cells have a short lifetime and require humans to provide blood samples. Neutrophil cell lines, which live longer, and can be purchased, however they may not respond in the same way as primary cells. In vitro experiments can be criticised as they only look at the response of one cell type. For the immune system to function properly communication between different cell types in the body is required. Zebrafish (ZF) allow inflammatory responses to be evaluated in an intact non-rodent and so can be employed to reduce rodent use.
Our project will identify approaches to investigate the ability of NMs to stimulate neutrophil responses in the laboratory and will identify if cells and ZF are good alternatives to rodents. We will compare our results to findings previously obtained from rodent studies to identify whether cells and ZF provide a good prediction of the rodent response. If they behave similarly then the scientific community may use these alternatives to decrease the numbers of rodents used to assess NM safety. Importantly, these alternatives are not restricted to testing NM safety and could be used to test the toxicity of a wider range of pollutants, chemicals (e.g. drugs) and microbes.
Technical Summary
We will develop methods using existing in vitro neutrophil and transgenic zebrafish (ZF) embryo models to assess the initiation, progression and resolution of inflammation. The alternative models will be applied to test the hazard potential of nanomaterials (NMs) of varied physicochemical properties, focusing on NMs for which in vivo rodent pulmonary data already exists in order to assess the relevance and limitations of the alternative models.
HL60 neutrophil-like cell line and primary neutrophils (isolated from human blood) will be exposed to NMs (copper oxide, carbon nanotubes, silver, zinc oxide, organic pigments) of varied physicochemical properties (e.g. composition, size and shape). NM cytotoxicity will be ranked and sub-lethal concentrations identified to investigate the mechanism underlying NM mediated neutrophil activation by NMs including; intracellular signaling via calcium and ROS, oxidative stress, cytokine production, respiratory burst and phagocytic function, as well as cell death including netosis and apoptosis. Research using ZF embryos will be conducted in UK Home Office approved facilities using non-protected early life history stages (age<120h post-fertilisation). Neutrophil responses to NMs will be visualised and quantified over time in transgenic ZF embryos (with fluorescent neutrophils) following micoinjection, at a range of doses. The mechanism underlying neutrophil activation and resolution in ZF embryos will be investigated via assessment of cytokine (e.g. CXCL8) and antioxidant expression, ROS production, calmodulin expression and neutrophil apoptosis. A range of transgenic ZF embryos are available to further probe the involvement of specific cellular and molecular events in the activation and resolution of NM inflammatory responses (e.g. CXCL8 knockout). Data obtained will be compared to existing in vivo data using in vitro-in vivo extrapolation (IVIVE), to explore the relevance of the alternative models.
HL60 neutrophil-like cell line and primary neutrophils (isolated from human blood) will be exposed to NMs (copper oxide, carbon nanotubes, silver, zinc oxide, organic pigments) of varied physicochemical properties (e.g. composition, size and shape). NM cytotoxicity will be ranked and sub-lethal concentrations identified to investigate the mechanism underlying NM mediated neutrophil activation by NMs including; intracellular signaling via calcium and ROS, oxidative stress, cytokine production, respiratory burst and phagocytic function, as well as cell death including netosis and apoptosis. Research using ZF embryos will be conducted in UK Home Office approved facilities using non-protected early life history stages (age<120h post-fertilisation). Neutrophil responses to NMs will be visualised and quantified over time in transgenic ZF embryos (with fluorescent neutrophils) following micoinjection, at a range of doses. The mechanism underlying neutrophil activation and resolution in ZF embryos will be investigated via assessment of cytokine (e.g. CXCL8) and antioxidant expression, ROS production, calmodulin expression and neutrophil apoptosis. A range of transgenic ZF embryos are available to further probe the involvement of specific cellular and molecular events in the activation and resolution of NM inflammatory responses (e.g. CXCL8 knockout). Data obtained will be compared to existing in vivo data using in vitro-in vivo extrapolation (IVIVE), to explore the relevance of the alternative models.
Planned Impact
We are committed to ensuring that our research has a 3Rs legacy and that the alternative models developed during the project are widely adopted by end users, internationally. We propose that cell (in vitro) and transgenic zebrafish (ZF) embryo models can be used as alternatives to rodent testing when investigating the involvement of the activation and resolution of neutrophil driven inflammatory responses in the toxicity, efficacy or pathogenicity of diverse substances (e.g. pollutants, particles, chemicals, pharmaceuticals, pathogens (e.g. bacteria)).
In the proposed research the alternative models will be applied to nanomaterials (NMs). The relatively large number of NMs on the market and under development means that an increasing emphasis will be placed on the use of reliable, predictive alternative models when assessing their safety to improve implementation of the 3Rs. Currently, assessment of NM hazard often relies on in vivo (rodent) testing. However whilst the majority of in vivo rodent studies use neutrophil accumulation as an indicator of NM hazard, few studies use alternative models to test the neutrophil response to NMs or assess the mode of action of NM toxicity to this key cell type. Existing studies have assessed NM inflammogenicity following exposure via several routes (e.g. inhalation, intratracheal instillation, injection, ingestion), and use an average of 68 animals/study (PubMed Search, March 2016). Despite it representing a significant gap in knowledge in nanotoxicology very few studies have assessed NM hazard following inhalation, the preferred testing system of regulators.
We propose that a reduction in animal use (by at least 20%) could be realised immediately if the alternative models used in this study were taken up by our own laboratories and the wider, international nanotoxicology community. This would require that a tiered testing strategy was adopted (in accordance with REACH guidance) whereby NM hazard testing would commence via testing of inflammogenicity using neutrophils in vitro, and then ZF embryo models before progressing to in vivo rodent studies. If alternative models identify NMs as being highly toxic these NMs may not proceed to in vivo testing to improve NM prioritisation. As a wider range of doses, time points and NMs can be tested using alternative models their use could facilitate the design of more focused in vivo (rodent) studies to reduce animal use via a reduced requirement for dose ranging studies, and a reduction in doses, time points and NMs tested in each in vivo study. We will perform an in vivo-in vitro extrapolation (IVIVE) to better understand the relevance and limitations of the cell and ZF embryo models, to increase end user confidence. Letters of support from the nanotoxicology community demonstrate the usefulness of the models and the willingness of end users to adopt the models. Widespread adoption of the models to other research disciplines could lead to further reductions or replacements in animal testing to those described.
Cell culture facilities are widely available, but a barrier to the widespread use of ZF embryos may be access to ZF. However, in the UK alone there are ZF facilities in at least 9 locations. The transgenic ZF embryos used in the study are already available but have not been previously applied to assess NM inflammogenicity. Similarly, whilst in vitro models are routinely used in nanotoxicology studies, there are a lack of studies which have assessed the response of neutrophils to NMs in vitro.
End users include academia, regulators, policy makers, funders, NGOs, industry and the general public. The support of national and international regulators and policy makers concerned with assessing and managing chemicals will encourage widespread use and validation of the models. A number of dissemination strategies will communicate findings to stakeholders and encourage acceptance and adoption of the models.
In the proposed research the alternative models will be applied to nanomaterials (NMs). The relatively large number of NMs on the market and under development means that an increasing emphasis will be placed on the use of reliable, predictive alternative models when assessing their safety to improve implementation of the 3Rs. Currently, assessment of NM hazard often relies on in vivo (rodent) testing. However whilst the majority of in vivo rodent studies use neutrophil accumulation as an indicator of NM hazard, few studies use alternative models to test the neutrophil response to NMs or assess the mode of action of NM toxicity to this key cell type. Existing studies have assessed NM inflammogenicity following exposure via several routes (e.g. inhalation, intratracheal instillation, injection, ingestion), and use an average of 68 animals/study (PubMed Search, March 2016). Despite it representing a significant gap in knowledge in nanotoxicology very few studies have assessed NM hazard following inhalation, the preferred testing system of regulators.
We propose that a reduction in animal use (by at least 20%) could be realised immediately if the alternative models used in this study were taken up by our own laboratories and the wider, international nanotoxicology community. This would require that a tiered testing strategy was adopted (in accordance with REACH guidance) whereby NM hazard testing would commence via testing of inflammogenicity using neutrophils in vitro, and then ZF embryo models before progressing to in vivo rodent studies. If alternative models identify NMs as being highly toxic these NMs may not proceed to in vivo testing to improve NM prioritisation. As a wider range of doses, time points and NMs can be tested using alternative models their use could facilitate the design of more focused in vivo (rodent) studies to reduce animal use via a reduced requirement for dose ranging studies, and a reduction in doses, time points and NMs tested in each in vivo study. We will perform an in vivo-in vitro extrapolation (IVIVE) to better understand the relevance and limitations of the cell and ZF embryo models, to increase end user confidence. Letters of support from the nanotoxicology community demonstrate the usefulness of the models and the willingness of end users to adopt the models. Widespread adoption of the models to other research disciplines could lead to further reductions or replacements in animal testing to those described.
Cell culture facilities are widely available, but a barrier to the widespread use of ZF embryos may be access to ZF. However, in the UK alone there are ZF facilities in at least 9 locations. The transgenic ZF embryos used in the study are already available but have not been previously applied to assess NM inflammogenicity. Similarly, whilst in vitro models are routinely used in nanotoxicology studies, there are a lack of studies which have assessed the response of neutrophils to NMs in vitro.
End users include academia, regulators, policy makers, funders, NGOs, industry and the general public. The support of national and international regulators and policy makers concerned with assessing and managing chemicals will encourage widespread use and validation of the models. A number of dissemination strategies will communicate findings to stakeholders and encourage acceptance and adoption of the models.