Seeing the whole macrophage: A nuclear reporter redefines tissue-resident macrophage quantification, isolation and profiling — ASN Events
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  2. Poster Session 2
  3. Seeing the whole macrophage: A nuclear reporter redefines tissue-resident macrophage quantification, isolation and profiling

Seeing the whole macrophage: A nuclear reporter redefines tissue-resident macrophage quantification, isolation and profiling (#231)

Jintao Guo 1 , Hollie J Vaughan 2 , Eva H Doyle 2 , Jiadong Mao 3 , Fang Ming Choo 1 , Simranpreet Kaur 1 , Alyssa H Cull 4 , Maria Kasherman 5 , Dylan Carter-Cusack 1 , Katharine M Irvine 1 , Kim M Summers 1 , David A Hume 1 , Kelli PA MacDonald 1 , Jarny Choi 6 , Susan M Millard 1 , Samanta A Mariani 2 , Allison R Pettit 1
  1. Mater Research Institute - The University of Queensland, Woolloongabba, QLD, Australia
  2. Centre for Inflammation Research, The University of Edinburgh, Edinburgh, Scotland, United Kingdom
  3. Melbourne Integrative Genomics, The University of Melbourne, Melbourne, Victoria, Australia
  4. University of York, York, England, United Kingdom
  5. Microscopy Core Facility, Translational Research Insitute, Brisbane, Queensland, Australia
  6. St Vincent's Institute of Medical Research and Faculty of Medicine, The University of Melbourne, Melbourne, Victoria, Australia

The molecular specialization underpinning tissue-resident macrophage (TRM) functional specialization in hematopoietic and skeletal tissues remains poorly defined. A major barrier is that conventional single-cell preparation methods fragment TRMs, resulting in poor recovery of intact cells and generation of protein- and RNA-containing macrophage remnants that adhere to non-macrophage cells. This confounds downstream single-cell analyses and can lead to substantial underappreciated errors in data interpretation. To overcome this challenge, we generated CD169-Cre × floxed nuclear GFP (nGFP) reporter mice and validated nGFP as a highly restricted, robust reporter for TRM (including osteoclasts), spanning embryonic development through ageing. This model enabled accurate ex vivo identification and in situ quantification of intact TRMs. Nuclear-based enumeration revealed that TRM abundance has been substantially overestimated by conventional approaches, while cell size and complexity have been underestimated, and permitted resolution that age-associated changes in TRM reflect increased size rather than previously assumed number. Optimised dissociation protocols combined with imaging-based sorting markedly improved recovery and diversity of bona fide nGFP⁺ TRMs from haematopoietic tissues. Additionally, nGFP profiling showed potential for accurate ex vivo identification of osteomorphs. Bulk RNA sequencing demonstrated that nGFP⁺ TRMs generated higher-fidelity transcriptional profiles than conventionally sorted populations, exposing contamination in public datasets and challenging the accuracy of current cell-identification and clustering approaches. Importantly, ubiquitous TRM functional programs overwhelmed specialization signatures, suggesting that current sensitivity of single-cell and spatial technologies will struggle to resolve TRM molecular specialization without intentional experimental design. These findings establish a best-in-class framework for accurate TRM quantification, isolation, and transcriptional profiling.