Multiple sclerosis (MS) is an autoimmune disease involving demyelination and axonal damage in the central nervous system (CNS). In this study, we investigated pathological changes in the lumbar ...spinal cord of C57BL/6 mice induced with progressive experimental autoimmune encephalomyelitis (EAE) disease using 9.4-T magnetic resonance imaging (MRI). Multiparametric MRI measurements including MR spectroscopy, diffusion tensor imaging (DTI) and volumetric analyses were applied to detect metabolic changes in the CNS of EAE mice. Compared with healthy mice, EAE mice showed a significant reduction in N-acetyl aspartate and increases in choline, glycine, taurine and lactate. DTI revealed a significant reduction in fractional anisotropy and axial diffusivity and an increase in radial diffusivity in the lumbar spinal cord white matter (WM), while in the grey matter (GM), fractional anisotropy increased. High-resolution structural imaging also revealed lumbar spinal cord WM hypertrophy and GM atrophy. Importantly, these MRI changes were strongly correlated with EAE disease scoring and pathological changes in the lumbar (L2-L6), particularly WM demyelination lesions and aggregation of immune cells (microglia/macrophages and astrocytes) in this region. This study identified changes in MRI biomarker signatures that can be useful for evaluating the efficacy of novel drugs using EAE models in vivo.
The proliferation, differentiation, and survival of cells of the mononuclear phagocyte system (MPS; progenitors, monocytes, macrophages, and classical dendritic cells) are controlled by signals from ...the M-CSF receptor (CSF1R). Cells of the MPS lineage have been identified using numerous surface markers and transgenic reporters, but none is both universal and lineage restricted. In this article, we report the development and characterization of a CSF1R reporter mouse. A FusionRed (FRed) cassette was inserted in-frame with the C terminus of CSF1R, separated by a T2A-cleavable linker. The insertion had no effect of CSF1R expression or function. CSF1R-FRed was expressed in monocytes and macrophages and absent from granulocytes and lymphocytes. In bone marrow, CSF1R-FRed was absent in lineage-negative hematopoietic stem cells, arguing against a direct role for CSF1R in myeloid lineage commitment. It was highly expressed in marrow monocytes and common myeloid progenitors but significantly lower in granulocyte-macrophage progenitors. In sections of bone marrow, CSF1R-FRed was also detected in osteoclasts, CD169
resident macrophages, and, consistent with previous mRNA analysis, in megakaryocytes. In lymphoid tissues, CSF1R-FRed highlighted diverse MPS populations, including classical dendritic cells. Whole mount imaging of nonlymphoid tissues in mice with combined CSF1R-FRed/
-EGFP confirmed the restriction of CSF1R expression to MPS cells. The two markers highlight the remarkable abundance and regular distribution of tissue MPS cells, including novel macrophage populations within tendon and skeletal muscle and underlying the mesothelial/serosal/capsular surfaces of every major organ. The CSF1R-FRed mouse provides a novel reporter with exquisite specificity for cells of the MPS.
Mouse hematopoietic tissues contain abundant tissue-resident macrophages that support immunity, hematopoiesis, and bone homeostasis. A systematic strategy to characterize macrophage subsets in mouse ...bone marrow (BM), spleen, and lymph node unexpectedly reveals that macrophage surface marker staining emanates from membrane-bound subcellular remnants associated with unrelated cells. Intact macrophages are not present within these cell preparations. The macrophage remnant binding profile reflects interactions between macrophages and other cell types in vivo. Depletion of CD169+ macrophages in vivo eliminates F4/80+ remnant attachment. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters, and mRNA are all detected in non-macrophage cells including isolated stem and progenitor cells. Analysis of RNA sequencing (RNA-seq) data, including publicly available datasets, indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single-cell analysis of disaggregated hematopoietic tissues. Hematopoietic tissue macrophage fragmentation undermines the accuracy of macrophage ex vivo molecular profiling and creates opportunity for misattribution of macrophage-expressed genes to non-macrophage cells.
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•Genuine macrophages are absent from hematopoietic tissue single-cell preparations•Fragmented remnants of macrophages containing cytoplasm adhere to other cells•Macrophage remnants convolute hematopoietic tissue flow cytometry analysis•Remnant-derived macrophage-RNA is misattributed to other cells in RNA-seq data
Millard et al. demonstrate intact macrophages are absent in hematopoietic tissue single-cell suspensions as they are disrupted during preparation. Macrophage remnants containing membrane and intracellular contents remain attached to other cells within the suspensions. This results in misassignment of macrophage identity and misattribution of macrophage-expressed genes.
Vincristine is an important component of many regimens used for pediatric and adult malignancies, but it causes a dose-limiting sensorimotor neuropathy for which there is no effective treatment. This ...study aimed to delineate the neuro-inflammatory mechanisms contributing to the development of mechanical allodynia and gait disturbances in a murine model of vincristine-induced neuropathy, as well as to identify novel treatment approaches. Here, we show that vincristine-induced peripheral neuropathy is driven by activation of the NLRP3 inflammasome and subsequent release of interleukin-1β from macrophages, with mechanical allodynia and gait disturbances significantly reduced in knockout mice lacking NLRP3 signaling pathway components, or after treatment with the NLRP3 inhibitor MCC950. Moreover, treatment with the IL-1 receptor antagonist anakinra prevented the development of vincristine-induced neuropathy without adversely affecting chemotherapy efficacy or tumor progression in patient-derived medulloblastoma xenograph models. These results detail the neuro-inflammatory mechanisms leading to vincristine-induced peripheral neuropathy and suggest that repurposing anakinra may be an effective co-treatment strategy to prevent vincristine-induced peripheral neuropathy.
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 modified recombinant colony-stimulating factor 1 (CSF1) to influence the HSC niche and expand the HSC pool for autologous transplantation.
We administered an acute treatment regimen of CSF1 Fc fusion protein (CSF1-Fc, daily injection for 4 consecutive days) to naive C57Bl/6 mice. Treatment impacts on macrophage and HSC number, HSC function and overall hematopoiesis were assessed at both the predicted peak drug action and during post-treatment recovery. A serial treatment strategy using CSF1-Fc followed by granulocyte colony-stimulating factor (G-CSF) was used to interrogate HSC mobilisation impacts. Outcomes were assessed by in situ imaging and ex vivo standard and imaging 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 hematopoiesis was accompanied by an increase in the total available HSPC pool. Multiple approaches confirmed that CD48
CD150
HSC do not express the CSF1 receptor, ruling out direct action of CSF1-Fc on these cells. In the spleen, increased HSC was associated with expression of the BM HSC niche macrophage marker CD169 in red pulp macrophages, suggesting elevated spleen engraftment with CD48
CD150
HSC was secondary to CSF1-Fc macrophage impacts. Competitive transplant assays demonstrated that pre-treatment of donors with CSF1-Fc increased the number and reconstitution potential of HSPC in blood following a HSC mobilising regimen of G-CSF treatment.
These results indicate that CSF1-Fc conditioning could represent a therapeutic strategy to overcome poor HSC mobilisation and subsequently improve HSC transplantation outcomes.
Macrophage-targeted therapies, including macrophage colony-stimulating factor 1 (CSF1), have been shown to have pro-repair impacts post-fracture. Preclinical/clinical applications of CSF1 have been ...expedited by development of chimeric CSF1-Fc which has extended circulating half-life. Here, we used mouse models to investigate the bone regenerative potential of CSF1-Fc in healthy and osteoporotic fracture. We also explored whether combination of CSF1-Fc with interleukin (IL)-4 provided additional fracture healing benefit in osteopenic bone. Micro-computed tomography, in situ histomorphometry, and bone mechanical parameters were used to assess systemic impacts of CSF1-Fc therapy in naive mice (male and female young, adult and geriatric). An intermittent CSF1-Fc regimen was optimized to mitigate undesirable impacts on bone resorption and hepatosplenomegaly, irrespective of age or gender. The intermittent CSF1-Fc regimen was tested in a mid-diaphyseal femoral fracture model in healthy bones with treatment initiated 1-day post-fracture. Weekly CSF1-Fc did not impact osteoclasts but increased osteal macrophages and improved fracture strength. Importantly, this treatment regimen also improved fracture union and strength in an ovariectomy-model of delayed fracture repair. Combining CSF1-Fc with IL-4 initiated 1-week post-fracture reduced the efficacy of CSF1-Fc. This study describes a novel strategy to specifically achieve bone regenerative actions of CSF1-Fc that has the potential to alleviate fragility fracture morbidity and mortality.
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.
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