Oral Presentation Australasian Cytometry Society 41st Annual Conference

Using suspension and imaging mass cytometry to track changes to haematopoiesis and cellular infiltration in neuropathology (24250)

Thomas M Ashhurst 1 , Darren A Cox 2 , Paula Niewold 2 , Zheng L Ling 2 , Nicholas J. C. King 1 , Adrian L Smith 3
  1. Sydney Cytometry Facility, Discipline of Pathology, and Ramaciotti Facility for Human Systems Biology, The University of Sydney and Centenary Institute, Sydney, NSW, Australia
  2. Pathology, The University of Sydney, Sydney, NSW, Australia
  3. Sydney Cytometry Facility and Ramaciotti Facility for Human Systems Biology, The University of Sydney and Centenary Institute, Sydney, NSW, Australia

Viral infection of the central nervous system (CNS) with West Nile virus (WNV) or Zika virus (ZIKV) stimulates a rapid influx of bone marrow (BM)-derived monocytes/macrophages into the parenchyma, that ultimately result in fatal pathology in the mouse. We have previously shown that in various inflammatory scenarios, a systemic and disease-driven re-organisation of haematopoietic output is responsible for the mobilisation of these pathogenic monocytes from the BM. However, whilst these cells are derived from the BM, little is known about the signalling events that result in the mobilisation of these cells in response to CNS infection. As such, we performed antibody blocking and genetic knock out studies to assess the impact of these interventions on inflammatory haematopoiesis. To do this we utilised high-dimensional flow and mass cytometry to investigate the haematopoietic compartments in the CNS and BM. In order to analyse the resultant datasets, we developed an analysis pipeline for clustering (using FlowSOM) and dimensionality reduction (using tSNE) to manage very large datasets. Although our suspension cytometry approaches enable comprehensive analysis of the immune system in a variety of disease situations, the dissociation of tissues to generate a single cell suspension eliminates all meaningful microenvironment and architectural data from samples. Because cellular behaviour is influence by the surrounding microenvironment, and not by phenotype alone, a consideration of cellular neighbourhoods is an important factor in understanding immunity. As such, we incorporated an Imaging Mass Cytometer (IMC) into our workflow to examine sections of CNS or BM, and examined the data using image analysis tools (such as HistoCAT). This approach allowed us to perform high-dimensional profiling whilst maintaining microenvironment data. The results of our study reveal key factors involved in the mobilisation of the immune in response to inflammation.