1015 – APPEARANCES CAN BE DECEIVING: MACROPHAGE FRAGMENTATION CONFOUNDS EX VIVO HAEMATOPOIETIC TISSUE ANALYSIS

Elucidation of the molecular signatures that define hematopoietic tissue resident macrophage specialisation has been challenging. There are no validated markers that differentiate the specialised macrophage subsets in bone marrow (BM) that support erythropoiesis, bone homeostasis and hematopoietic stem cell (HSC) niches. We took an unbiased ex vivo approach to characterise macrophage subsets in mouse BM, spleen and lymph node using a flow cytometry marker panel which allowed analysis of all mature leucocytes, red blood cells and hematopoietic stem and progenitor cells (HSPC) in combination with in situ verified macrophage markers. Despite readily detectable F4/80 staining we were unable to identify any population in hematopoietic tissues that definitively represented intact macrophages. Imaging flow cytometry and confocal microscopy showed macrophage marker staining was derived from membrane-bound subcellular remnants associated with unrelated cell types. Remnant-restricted macrophage membrane markers, cytoplasmic reporters and mRNA were detected in non-macrophage cell populations including HSPC. Of note, HSC-associated detection of a Csf1r-reporter as well as anti-F4/80 and VCAM-1 staining were entirely attributable to membrane-bound subcellular remnants. Distinct marker expression on macrophage subsets within spleen verified that the profile of remnant binding reflected in vivo cell-cell interactions. Macrophage remnant attachment was reduced in Siglec1 deficient mice with frequency of F4/80+ BM events reduced by over 50% in HSPC and neutrophils yet unchanged in lymphocytes. Analysis of published RNA-seq data confirmed that macrophage fragmentation is a general phenomenon in disaggregated hematopoietic tissues. Overall, we have shown that abundant tissue macrophages are absent/under-represented in hematopoietic tissue cell suspensions. Detection of macrophage remnant-restricted cytoplasmic and membrane contents on other cells has confounded interpretation of ex vivo analyses and results in misattribution of macrophage-expressed genes to non-macrophage cells.