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186 Assessment of Plaque Macrophage Phenotype in situ by Multicolour Fluorescence Microscopy
  1. Jessica Johnston,
  2. Adrienn Angyal,
  3. Mabruka Alfaidi,
  4. Sheila Francis,
  5. Endre Kiss-Toth
  1. University of Sheffield

Abstract

Background Macrophages are a heterogeneous and diverse population of cells with an important role in immunity and also in the development of diseases including atherosclerosis. Macrophages exhibit a spectrum of phenotypes, from pro-inflammatory or M1/ ‘classically activated’ to anti-inflammatory or M2/ ‘alternatively activated’ cells with wound healing properties. These cells are highly plastic and can switch from one phenotype to another depending on environmental cues. Despite considerable work aiming to characterise the polarisation of macrophages in disease, there have been few studies that have elucidated macrophage phenotype in situ and our knowledge of their complexity in vivo is only partial. Here we wished to establish a robust analysis pipeline to assess macrophage phenotype in aortic root plaques of high-fat diet fed mice. This approach will provide a unique insight into macrophage phenotype within the plaque and will advance our understanding of plaque development and phenotypic switching of macrophages in vivo.

Methods Using multicolour immunohistochemistry, formalin-fixed paraffin embedded (FFPE) aortic root sections from high-fat diet fed (ApoE–/– and LDLR–/–) mice were simultaneously stained for the pan macrophage marker Mac-3 (CD107b), iNOS (M1 marker) and Arginase I (M2 marker) in situ, respectively. Plaques from aortic roots were imaged using multi-colour fluorescence microscopy (Leica AF6000). The resulting images were analysed by Image J. Briefly, individual Mac-3 positive cells were selected as the region of interest (ROI) and corresponding iNOS and ArgI positive staining was evaluated. The analysis allowed consideration for the spectrum of M1/M2 associated marker expression and enabled characterisation of individual cells based on staining intensity. Using this approach it is possible to reveal and quantify complex populations of plaque macrophages in situ.

Results We have successfully optimised a simultaneous staining protocol for a pan macrophage marker and M1/M2 associated markers and quantified differential expression of these markers in individual macrophages. We have created a robust, semi-automated pipeline to analyse plaque macrophage phenotype andvalidated this approach by comparing macrophage phenotypes in LDLR-/- vs. ApoE-/- models of experimental atherosclerosis.

Conclusions/implications We envisage that our platform provides a novel tool to gain an in-depth understanding of macrophage phenotype in atherosclerosis and we will use this to elucidate the action of modulators of macrophage polarisation in vivo.

  • Macrophage
  • Polarisation
  • Immunohistochemistry

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