Imaging flow cytometry reveals that granulocyte colony-stimulating factor treatment causes loss of erythroblastic islands in the mouse bone marrow

Tay, J. S. H.; Bisht, Kavita; McGirr, Crystal; Millard, Susan; Pettit, Allison R.; Winkler, Ingrid G.; Levesque, Jean‐Pierre

doi:10.1016/j.exphem.2020.02.003

Cited by 26 publications

(34 citation statements)

References 22 publications

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“…As pro-erythroblast and erythroblasts mature in EBIs containing a central Mφ, we next evaluated the effect of LPS on EBIs in the BM by in-flight imaging flow cytometry to directly visualize and quantify EBIs and their central Mφ ( 38 ). In saline-treated mouse BM, we could detect typical EBIs with at least 5 Ter119 + CD71 + erythroblasts and reticulocytes rosetted around a central F4/80 + VCAM-1 + CD169 + EBI Mφ ( Figures 5B–D ) as previously reported ( 28 , 37 , 38 ). Remarkably, EBIs were disrupted in the BM 48 h after two successive daily LPS injections as suggested by the very low frequency of cell aggregates containing Ter119 + CD71 + erythroblasts rosetted around a central Mφ ( Figures 5B–D ).…”

Section: Resultsmentioning

confidence: 99%

“…We have previously reported that administration of G-CSF, a widely used cytokine to mobilize HSCs in the clinic ( 63 ), causes similar suppression of medullary erythropoiesis ( 20 ) and reduces EBI frequency in the BM ( 38 ). It has also been shown that LPS increases splenic hematopoiesis in G-CSF-dependent manner however, whether increased splenic hematopoiesis was mediated through HSC mobilization into the blood or extramedullary hematopoiesis was not investigated ( 49 ).…”

Section: Resultsmentioning

confidence: 99%

“…Extraction and staining of BM aggregates was performed as described previously ( 37 , 38 ). In brief, harvested femurs were gently flushed multiple times with a 25G needle and 1 mL syringe containing ice cold IMDM supplemented with 20% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L -glutamine (all from Thermo Fisher Scientific, Waltham, MA) to dislodge most of the BM cells.…”

Section: Methodsmentioning

confidence: 99%

“…Cell suspensions were washed twice in PBS containing 2% newborn calf serum (NCS) and stained with Hoechst33342 (Hoe), CD169-FITC, CD71-PE, CD11b-PECF594, anti-Ter119-PercPCy5.5, anti-VCAM-1-PECy7, anti-F4/80-APC and anti-LY6G-APCCy7 antibodies ( Supplementary Table 1 ). Samples were acquired within 6 h of preparation, using INSPIRE software on an Amnis ImageStream X Mk II equipped with 405, 488, and 642 nm lasers (Luminex, Austin, TX) and detected and quantified using IDEAS 6.2 software (Luminex) as previously described ( 38 ). The number of EBIs per 10 million BM cells ( N 10 mil ) was calculated by the following formula:

, where N raw = number of EBIs counted on IDEAS software, C femur = number of BM cells per femur, P femur = proportion of femur (by volume) stained with antibodies, and P analyzed = volumetric proportion of BM sample run.…”

Section: Methodsmentioning

confidence: 99%

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Bacterial Lipopolysaccharides Suppress Erythroblastic Islands and Erythropoiesis in the Bone Marrow in an Extrinsic and G- CSF-, IL-1-, and TNF-Independent Manner

et al. 2020

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Anemia of inflammation (AI) is the second most prevalent anemia after iron deficiency anemia and results in persistent low blood erythrocytes and hemoglobin, fatigue, weakness, and early death. Anemia of inflammation is common in people with chronic inflammation, chronic infections, or sepsis. Although several studies have reported the effect of inflammation on stress erythropoiesis and iron homeostasis, the mechanisms by which inflammation suppresses erythropoiesis in the bone marrow (BM), where differentiation and maturation of erythroid cells from hematopoietic stem cells (HSCs) occurs, have not been extensively studied. Here we show that in a mouse model of acute sepsis, bacterial lipopolysaccharides (LPS) suppress medullary erythroblastic islands (EBIs) and erythropoiesis in a TLR-4- and MyD88-dependent manner with concomitant mobilization of HSCs. LPS suppressive effect on erythropoiesis is indirect as erythroid progenitors and erythroblasts do not express TLR-4 whereas EBI macrophages do. Using cytokine receptor gene knock-out mice LPS-induced mobilization of HSCs is G-CSF-dependent whereas LPS-induced suppression of medullary erythropoiesis does not require G- CSF-, IL- 1-, or TNF-mediated signaling. Therefore suppression of medullary erythropoiesis and mobilization of HSCs in response to LPS are mechanistically distinct. Our findings also suggest that EBI macrophages in the BM may sense innate immune stimuli in response to acute inflammation or infections to rapidly convert to a pro-inflammatory function at the expense of their erythropoietic function.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Bacterial Lipopolysaccharides Suppress Erythroblastic Islands and Erythropoiesis in the Bone Marrow in an Extrinsic and G- CSF-, IL-1-, and TNF-Independent Manner

et al. 2020

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“…CD169 is expressed by HSC 5,7 and erythroid 39,40 niche macrophages in the BM. Given both the sustained increase in spleen size and shift in splenic red pulp macrophage phenotype to express CD169 5,7 , we assessed whether CSF1-Fc treatment induced extramedullary haematopoiesis and/or impacted HSPC populations in BM, spleen and liver at 7 and 14 days post treatment.…”

Section: Csf1-fc Treatment Transiently Increased Hspc Populations In mentioning

confidence: 99%

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

Kaur

Sehgal

et al. 2020

Preprint

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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.

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Imaging flow cytometry data analysis using convolutional neural network for quantitative investigation of phagocytosis

Mochalova

Kotov

Lifanov

et al. 2021

Biotech & Bioengineering

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Macrophages play an important role in the adaptive immune system. Their ability to neutralize cellular targets through Fc receptor-mediated phagocytosis has relied upon immunotherapy that has become of particular interest for the treatment of cancer and autoimmune diseases. A detailed investigation of phagocytosis is the key to the improvement of the therapeutic efficiency of existing medications and the creation of new ones. A promising method for studying the process is imaging flow cytometry (IFC) that acquires thousands of cell images per second in up to 12 optical channels and allows multiparametric fluorescent and morphological analysis of samples in the flow. However, conventional IFC data analysis approaches are based on a highly subjective manual choice of masks and other processing parameters that can lead to the loss of valuable information embedded in the original image. Here, we show the application of a Faster region-based convolutional neural network (CNN) for accurate quantitative analysis of phagocytosis using imaging flow cytometry data. Phagocytosis of erythrocytes by peritoneal macrophages was chosen as a model system. CNN performed automatic high-throughput processing of datasets and demonstrated impressive results in the identification and classification of macrophages and erythrocytes, despite the variety of shapes, sizes, intensities, and textures of cells in images. The developed procedure allows determining the number of phagocytosed cells, disregarding cases with a low probability of correct classification. We believe that CNN-based approaches will enable powerful in-depth investigation of a wide range of biological processes and will reveal the intricate nature of heterogeneous objects in images, leading to completely new capabilities in diagnostics and therapy.

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Imaging flow cytometry reveals that granulocyte colony-stimulating factor treatment causes loss of erythroblastic islands in the mouse bone marrow

Cited by 26 publications

References 22 publications

Bacterial Lipopolysaccharides Suppress Erythroblastic Islands and Erythropoiesis in the Bone Marrow in an Extrinsic and G- CSF-, IL-1-, and TNF-Independent Manner

Bacterial Lipopolysaccharides Suppress Erythroblastic Islands and Erythropoiesis in the Bone Marrow in an Extrinsic and G- CSF-, IL-1-, and TNF-Independent Manner

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

Imaging flow cytometry data analysis using convolutional neural network for quantitative investigation of phagocytosis

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