Pettit, A. R. | bioRxiv Science

bioRxiv2021

Fragmentation of macrophages during isolation confounds analysis of single cell preparations from mouse hematopoietic tissues

Mouse hematopoietic tissues contain abundant and heterogeneous populations of tissue-resident macrophages attributed trophic functions in control of immunity, hematopoiesis and bone homeostasis. A systematic strategy to characterise macrophage subsets in mouse bone marrow (BM), spleen and lymph node, unexpectedly revealed macrophage surface marker staining typically emanated from membrane-bound subcellular remnants associated with unrelated cell types. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters and mRNA were all detected in non-macrophage cell populations including isolated stem and progenitor cells. The profile of macrophage remnant association reflects adhesive interactions between macrophages and other cell types in vivo. Applying this knowledge, reduced macrophage remnant attachment to BM granulocytes in Siglec1 deficient mice was associated with compromised emergency granulocytosis, revealing a function for Siglec1-dependent granulocyte-macrophage interactions. Analysis of published RNA-seq data for purified macrophage and non-macrophage populations indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single cell analysis of disaggregated tissues.

immunology↗

bioRxiv2020

Stable colony stimulating factor 1 fusion protein treatment increases HSC pool and enhances their mobilisation in mice.

Prior chemotherapy and/or underlying morbidity commonly leads to poor mobilisation of hematopoietic stem cells (HSC) for transplantation in cancer patients. Increasing the number of available HSC prior to mobilisation is a potential strategy to overcome this deficiency. Resident bone marrow (BM) macrophages are essential for maintenance of niches that support HSC and enable engraftment in transplant recipients. Here we examined potential of donor treatment with colony stimulating factor-1 (CSF1) to modify the BM niche and expand the potential HSC pool for autologous transplantation. We administrated CSF1 Fc fusion protein (CSF1-Fc) to naive C57Bl/6 mice and assessed the impacts on HSC number and function and overall haematopoiesis. Outcomes were assessed by in situ imaging and ex vivo flow cytometry with functional validation by colony formation and competitive transplantation assay. CSF1-Fc treatment caused a transient expansion of monocyte-macrophage cells within BM and spleen at the expense of BM B lymphopoiesis and hematopoietic stem and progenitor cell (HSPC) homeostasis. During the recovery phase after cessation of CSF1-Fc treatment, normalisation of haematopoiesis was accompanied by an increase in the total available HSPC pool. In the spleen, increased HSC was associated with expression of the BM niche marker CD169 in red pulp macrophages. Pre-treatment with CSF1-Fc increased the number and reconstitution potential of HSPC in blood following a HSC mobilising regimen of granulocyte colony stimulating factor (G-CSF) treatment. These results indicate that CSF1-Fc conditioning could represent a therapeutic strategy to overcome poor HSC mobilisation and subsequently improve autologous or heterologous HSC transplantation outcomes. Key points1) Recovery from Fc-modified colony stimulating factor-1 (CSF1-Fc) treatment was accompanied by an increase in total haematopoietic stem cells. 2) Pre-conditioning with CSF1-Fc increased the reconstitution potential of blood after haematopoietic stem cell mobilisation.

cell biology↗