Immune cell effects of environmental chemicals after in vitro exposure: a high-dimentional single-cell mass cytometry approach
The immune system plays a role in many large public health diseases, like allergy, asthma, autoimmunity, cancer and others. Environmental chemicals are known to affect the immune system, and there is an urgent need within toxicology to identify chemicals with immune-modulating properties. The Adverse Outcome Pathway (AOP) framework is the state-of the-art tool  to apply mechanistic understanding of toxicity pathways in regulatory decisions. The vast knowledge gaps in AOPs, and the lack of in vitro screening tests, of adverse immune effects demonstrate the urgent need for mechanistic studies. Novel avenues are needed to identify predictive biomarkers for immunotoxic effects and to develop new assays for toxicity screening.
Recent advances in single cell proteomics by CyTOF (Cytometry by Time Of Flight mass spectrometry, i.e. mass cytometry) permits the simultaneous detection of 40-50 metal isotope-tagged antibody specificities (i.e. trillions of marker combinations) in a single tube. Thus, classification of cell subtypes and their activation status, and simultaneous detection of functional markers like intracellular cytokines, signalling pathways and proliferation is possible. This mass cytometry approach is particularly powerful in identifying new combinations of characteristics that are easily overlooked in traditional (supervised) analyses. Combined with unsupervised mathematical algorithms on a systems level, mass cytometry data is therefore a powerful tool to explore and investigate the breadth and depth of the cell system with single cell resolution. The Norwegian Institute of Public Health have recently acquired a CyTOF instrument.
We hypothesize that mass cytometry will allow for detection of direct immunotoxicity and identification of specific mechanisms and cell signatures, since it enable simultaneous profiling of cell phenotypes, signaling pathways and functional events in high numbers of single cells. This knowledge will allow for future development of more focused test systems, enabling high-throughput tests for immunotoxicity. Identification of biomarkers of effect (cell populations or functional signatures associated with exposure) will also allow use in human cohort studies, providing a mechanistic/causal link in studies reporting associations between exposure and health outcomes.
We will perform mass cytometric detection of cell phenotypes and functionality after in vitro exposure of blood cells to a selection of known and suspected immunotoxic chemicals. In addition, we will add immune-stimulating agents that either induce cell signaling broadly (PMA/ionomycin) or more specifically (such as LPS, ConA, Poly(I:C), TLR ligands ). Assessing the effects of chemicals in the presence of immune stimulants will reveal chemical interactions with the functionality or “fitness” of the cells. Unsupervised analyses of the high-dimensional data will allow for identification of the most frequently affected and the most susceptible subpopulations and functional parameters/signatures impacted by the chemicals. For each chemical, the data set will provide a mechanistic blueprint or signature of functional events. This information will be important for future development of robust immunotxicity tests and identification of biomarkers.
Folkehelseinstituttet, Oslo (Unni C Nygaard, Hege Hjertholm, Berit Granum, Hubert Dirven, Anette K Bølling).
In collaboration with Stanford University and Karolinska Institutet for the newest biostatistical analyses.
Students are invited to apply for a MSc project to work on this topic. Minimum period is 12 months. Experience with lab work and experience or interest in biostatistics is needed.
1. Villeneuve, D.L., et al., Adverse Outcome Pathway (AOP) Development I: Strategies and Principles. Toxicol Sci, 2014. 142(2): p. 312-20.
2. Bendall, S.C., et al., Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science, 2011. 332(6030): p. 687-96.