Abstract Background Tumour-associated macrophages (TAMs) play critical roles within the tumour microenvironment regulating immune evasion and therapeutic response. Previously, we have shown that the combination of Checkpoint kinase 1 inhibitor (CHK1i) with a subclinical dose of hydroxyurea (LDHU) reprograms the tumour immune microenvironment to a pro-inflammatory status. Methods We investigated a tumour-restricted Fcgr4 (Cd16.2) expressing macrophage population in multiple murine tumour models and the impact of CHK1i+LDHU on this population, using conventional and imaging flow cytometry as well as single-cell sequencing. Results Transcriptional profiling using CITE-seq and single-cell RNA sequencing reveals that Fcgr4⁺ TAMs closely resemble Fcgr4⁻ TAMs but display modest enrichment of interferon-associated and inflammatory gene programs, consistent with a functionally biased state rather than a distinct lineage. Importantly, we show that a highly tumour selective CHK1i+LDHU therapy shifts TAMs toward a more inflammatory phenotype while preserving dominant immunosuppressive features. Depletion of CSF1R⁺ macrophages enhanced CD8⁺ T cell activation without influencing tumour growth but significantly augmented therapeutic efficacy of CHK1i+LDHU. Conclusion Together, these findings define a novel TAM population and establish how targeted therapy reshapes, but does not fully overcome, TAM-mediated immune regulation.

The anabolic effect of Parathyroid Hormone (PTH 1-34) is a potent anti-osteoporosis treatment. In rat bone, the chemokine MCP1 (also known as CCL2) is induced about 250-fold occurring within 1 h of PTH injection and is the highest induced gene. In mice, we showed previously that global knockout (KO) of MCP1 blocked the anabolic effect of PTH. In the present work, we used cre-lox genetic models to generate cell lineage specific MCP1 KO in the osteoblast/osteocyte lineage by using the type 1 collagen promoter (Col1A1) to drive cre recombinase in mice carrying loxP sites flanking the MCP1 gene (MCP1f/f). In contrast to a 2.6 standard deviation (SD) increase in total bone in the proximal tibial metaphysis (p = 4 × 10-9) in control mice, there was no response to anabolic PTH in mice in which the MCP1 gene was deleted (MCP1f/f Col-cre+, p = 0.6). We then tested whether ovariectomy (OVX) induced bone loss could be suppressed by simultaneous anabolic PTH treatment (OVX-PTH) in such animals. In control animals, OVX resulted in a 2.0 SD decrease in bone density. OVX-PTH treated animals had bone density 1.6 SD higher than baseline untreated and 3.6 SD increased from the low OVX level. In contrast, the anabolic PTH effect was significantly blunted (p = 0.01) in OVX MCP1f/f Col-cre + animals with a 1.4 SD change. These data suggest a role for osteoblast/osteocyte expressed MCP1 in the anabolic effect of PTH.

Interleukin 34 (IL34) and colony-stimulating factor 1 (CSF1) signal through a shared receptor (CSF1R) to control macrophage survival, differentiation, and function. Here, we describe the impact of loss-of-function mutation in the rat Il34 gene. In contrast to IL34 mutant mice, macrophages within squamous epithelia (Langerhans cells) were not significantly depleted in Il34 -/- rats. In the brain, microglia and brain-associated macrophages were selectively depleted in grey matter. A gradient of microglial density in the Il34 -/- cortex suggests that CSF1 can diffuse outwards from the corpus callosum. Microglial loss was not associated with detectable neuropathology or altered gene expression in the cortex, hippocampus, and thalamus aside from selective loss of microglia-expressed transcripts. In the adenine diet model of renal interstitial fibrosis, both Il34 and Csf1 were induced. The absence of IL34 led to a significant reduction in macrophage recruitment compared with controls, but pathology was unaffected. We suggest that IL34 and CSF1 provide overlapping signals to sustain microglia and to direct macrophage recruitment and repair tissue injury in the periphery.

Hematological toxicity disrupts cancer treatment delivery and contributes to treatment-related mortality. Reducing hematopoietic injury and/or promoting bone marrow (BM) regeneration could improve treatment outcomes and survivorship. CD169+ BM-resident macrophages (Mφ), resilient to total body irradiation (TBI), promote reformation of niches that support hematopoietic stem cells (HSCs) and blood reconstitution. During BM recovery from TBI, the spleen supports extramedullary hematopoiesis, also associated with macrophage resilience and altered red pulp macrophage phenotype. We hypothesize that (1) BM-resident Mφ are resilient to a broad range of high-risk cancer therapies, and 2) their resilience and density are critical for re-establishing hematopoiesis, including preservation of the HSC pool. To test this, CD169-ZsGreen macrophage reporter mice were treated with a murine chronic lymphoid leukemia (CLL) immunotherapy regimen (anti-CD20 antibody + fludarabine + cyclophosphamide) known to cause severe neutropenia. Hematopoietic injury and recovery were assessed over 21 days. Flow cytometry of BM at day 2 post-treatment confirmed >90% reduction in mature hematopoietic cells and 83% reduction in HSCs. In contrast, F4/80+ BM-resident Mφ remained unaffected, whereas CD68 and MertK+ Mφ increased at day 2 and day 21. By day 14, BM HSCs increased to 35%, reflecting active blood cell repopulation. In the spleen, HSCs and progenitors increased by 9% and 38%, respectively, indicating extramedullary hematopoiesis. Notably, 28% of red pulp F4/80+ Mφ acquired CD169 expression by day 14, mimicking the phenotype shift seen after lethal TBI. This suggests that splenic macrophages may adopt BM-like roles to support extramedullary haematopoiesis. Overall, CLL-treatment resilience of Mφ in BM and spleen may play a key role in recovery from therapy-induced hematological toxicity.

Inflammatory bowel diseases (IBDs) are chronic inflammatory conditions of the gastrointestinal tract. Anemia is the most common and debilitating comorbidity in IBD and leads to chronic fatigue, poor quality of life and increased rates of hospitalization. We investigated the mechanisms of anemia and drugs targeting these mechanisms to reduce anemia associated with IBD. Our study discovered that both patients and mice with IBD and ulcerative colitis (induced by 3% dextran sodium sulfate [DSS] in drinking water) displayed endotoxemia. We also found a positive correlation between anemia and endotoxemia in patients with IBD. Mice with colitis were anemic with reduced quantities of red blood cells, hemoglobin and hematocrit. Medullary erythropoiesis was reduced and partially compensated by extramedullary erythropoiesis in the spleen in mice with colitis.We have previously shown that endotoxins inhibit medullary erythropoiesis indirectly via erythroblastic islands macrophages (EBI Mφ), which express CD169 antigen and endotoxin receptor TLR4. To investigate the role of EBI Mφ on colitis-induced anemia, we specifically deleted TLR4 gene in CD169+ Mφ. Cd169Cre:Tlr4fl/fl mice did not develop anemia and showed reduced colitis severity in response to DSS suggesting that endotoxemia may contribute to both anemia and gut inflammation in IBD. We then investigated the effect of inhibitors targeting endotoxin (colistin sulfate/polymyxin E) or its receptor TLR4 (C34), we found that colistin sulfate reduced the severity of colitis with reduced gut inflammation and reduced anemia. In conclusion, we show for the first time that endotoxins leaking from the inflamed gut may play a crucial role in IBD and associated anemia and that drugs targeting endotoxins protect against IBD-associated anemia.

Colony stimulating factor 1 receptor (CSF1R) is required for the differentiation and maintenance of many mononuclear phagocyte lineage cells and is a definitive marker of this lineage in humans and mice. CSF1R signaling is activated by two cognate ligands CSF1 and IL34. In mice, the expression of CSF1 and IL34 is spatially regulated in homeostasis and increased in inflammatory conditions. Whereas CSF1 expression is ubiquitous, IL34 expression is predominantly observed in the epidermis and central nervous system. Accordingly, IL34-deficient mice lack Langerhan's cells and grey matter microglia. CSF1-deficient mice lack many tissue macrophage populations, but microglia and Langerhan's cells are present. In other species, including humans and rats, IL34 is more broadly expressed, raising the possibility that IL34 plays distinct roles, or that there is more redundancy between CSF1R ligands, in species other than mice. We generated and characterised a novel IL34 knockout rat. IL34 KO rats are phenotypically indistinguishable from wildtype littermates from birth up to 2 years of age. Similar to mice, grey matter microglia are significantly reduced in IL34-deficient animals in a dose dependent manner (+/+ > +/- > -/-). The loss of grey matter microglia was not associated with any observable histological pathology or behavioural abnormalities. IL34 KO rats also have a reduction in the number of Langerhan's cells, but do not completely lack them, unlike IL34 KO mice. Blood and bone marrow monocytes and all other tissue macrophage populations examined were indistinguishable between IL34 KO rats and littermate controls. To investigate the role of IL34 in tissue injury we utilised an adenine diet-induced chronic kidney disease (CKD) model, since Il34 is relatively highly expressed in the rat kidney. Both Il34 and Csf1 were induced in chronically injured kidneys. IL34 KO rats had reduced macrophage numbers in injured kidneys, but the KO did not affect pathology at peak injury or recovery after cessation of the adenine diet. We conclude that IL34 is largely redundant for the maintenance of monocyte-macrophage lineage cells and normal physiological homeostasis at steady state in rats.

Macrophage and osteoclast proliferation, differentiation and survival are regulated by colony-stimulating factor 1 receptor (CSF1R) signaling. Osteopetrosis associated with Csf1 and Csf1r mutations has been attributed to the loss of osteoclasts and deficiency in bone resorption. Here, we demonstrate that homozygous Csf1r mutation in rat leads to delayed postnatal skeletal ossification associated with substantial loss of osteal macrophages in addition to osteoclasts. Osteosclerosis and site-specific skeletal abnormalities were reversed by intraperitoneal transfer of wild-type bone marrow cells (bone marrow cell transfer, BMT) at weaning. Following BMT, IBA1+ macrophages were detected before TRAP+ osteoclasts at sites of ossification restoration. These observations extend evidence that osteal macrophages independently contribute to bone anabolism and are required for normal postnatal bone growth and morphogenesis. Characterization of macrophage/osteoclast distribution in Csf1r knockouts plus recovery dynamics after normal bone marrow cell transfer reveals macrophage independent contributions to postnatal skeletal development.

Inflammatory bowel diseases (IBD), which include Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory conditions of the gastrointestinal tract and highly prevalent in Australia and USA. Anemia is debilitating and a common complication of IBD associated with chronic fatigue, poor quality of life and increased rate of hospitalization. However, current treatments for anemia in IBD are limited to iron supplementation which has limited efficacy. In this study, we investigated the cause of anemia in IBD patients and in a mouse colitis model. We have previously shown that endotoxins/lipopolysaccharides (LPS) inhibit bone marrow (BM) erythropoiesis in a TLR4- and MyD88-dependent manner in mice. Therefore, we first measured blood plasma concentrations of endotoxins in IBD patients and mouse model of acute colitis. Blood endotoxin concentrations were significantly increased relative to healthy controls in IBD patients (~4-fold in UC and ~2.2-fold in CD patients) and in mice with colitis induced by sodium dextran sulphate (DSS) (~4-fold). This is consistent with disruption of the intestinal barrier observed in IBD patients and DSS-treated mice allowing translocation of endotoxins produced by the gut bacterial flora into the circulation. A positive correlation between anemia and endotoxemia was found in IBD patients. To investigate the effect of gut inflammation on erythropoiesis, we measured the markers of erythropoiesis in mice with acute colitis induced by 3% DSS in drinking water. Mice with acute colitis had significant anemia with reduced number of red blood cells (RBCs; controls mean 9.0 x 1012 ± 0.7 /L vs DSS-treated 7.5 x 1012± 1.1 /L), hemoglobin (HgB; mean 140 ± 9.1 g/L vs 117 g/L ± 15 g/L) and hematocrit (HCT; mean 0.41 ± 0.02 vs 0.34 ± 0.05) compared to control mice. In line with this, we found that acute colitis in mice caused a marked whitening of the bone marrow (BM) with reduced number of erythrocytes (mean 4.3 x 106 ± 1.8 erythrocytes/femur vs 2.3 x 106 ± 1.4 erythrocytes/femur), accompanied by shortened RBC half-life (t1/2 = 19.7 ± 2.9 days in controls vs t1/2 = 12.8 ± 4.4 days in DSS treated mice) in the blood and increased blood concentration of interferon-γ (mean 4.7 pg/mL ± 0.8 vs 7.6 pg/mL ± 2.7). Interferon- γ is known to reduce RBC lifespan by increasing their clearance in the spleen. Acute colitis increased RBC size (~1.15-fold increase in red cell width and ~ 3.5-fold increase in blood reticulocytes) consistent with colitis induced anemia. This reduced medullary erythropoiesis was compensated in part by extramedullary erythropoiesis in the spleen. Patients with IBD had increased levels of hepcidin (~4-fold increase) and interleukin -6 (IL-6; ~1.3-fold increase) in their blood. However, mice with acute colitis only showed an increase in blood IL-6 concentration (~9-fold increase) but no change in hepcidin, suggesting that there is a combination of both iron-deficiency anemia and anemia of inflammation in these settings. We have previously shown that endotoxin inhibits erythropoiesis indirectly by deregulating erythroblastic islands macrophages (EBI Mφ) which express the CD169/Siglec1 antigen and endotoxin receptor TLR4. To investigate the role of EBI Mφ on colitis-induced anemia, we specifically deleted TLR-4 in CD169+ Mφ by crossing Tlr4flox/flox mice with Siglec1Cre mice (expressing Cre recombinase in CD169+ tissue Mφ). Mice lacking Tlr4 gene specifically in CD169+ Mφ did not develop anemia in response to DSS with normal blood RBC, HgB, HCT, reticulocytes numbers, BM erythrocyte numbers and no extramedullary erythropoiesis in the spleen compared to mice with Tlr4 gene in CD169+ macrophages. In addition to the protective effect of Tlr4 deletion in CD169+ Mφ on anemia, we found that these mice were also protected from colitis with improved body weight, colon length and histological scores suggesting that endotoxemia may contribute to both anemia and colitis in IBD. In conclusion, we show for the first time that inflammation via endotoxins may play a crucial role in anemia in IBD and there is a correlation between anemia and endotoxemia in IBD.

Apoptosis is crucial for tissue homeostasis and organ development. In bone, apoptosis is recognized to be a main fate of osteoblasts, yet the relevance of this process remains underexplored. Using our murine model with inducible Caspase 9, the enzyme that initiates intrinsic apoptosis, we triggered apoptosis in a proportion of mature osteocalcin (OCN+) osteoblasts and investigated the impact on postnatal bone development. Osteoblast apoptosis stimulated efferocytosis by osteal macrophages. A five-week stimulation of OCN+ osteoblast apoptosis in 3-week-old male and female mice significantly enhanced vertebral bone formation while increasing osteoblast precursors. A similar treatment regimen to stimulate osterix+ cell apoptosis had no impact on bone volume or density. The vertebral bone accrual following stimulation of OCN+ osteoblast apoptosis did not translate in improved mechanical strength due to disruption of the lacunocanalicular network. The observed bone phenotype was not influenced by changes in osteoclasts but was associated with stimulation of macrophage efferocytosis and vasculature formation. Phenotyping of efferocytic macrophages revealed a unique transcriptomic signature and expression of factors including VEGFA. To examine whether macrophages participated in the osteoblast precursor increase following osteoblast apoptosis, macrophage depletion models were employed. Depletion of macrophages via clodronate-liposomes and the CD169-diphtheria toxin receptor mouse model resulted in marked reduction in leptin receptor+ and osterix+ osteoblast precursors. Collectively, this work demonstrates the significance of osteoblast turnover via apoptosis and efferocytosis in postnatal bone formation. Importantly, it exposes the potential of targeting this mechanism to promote bone anabolism in the clinical setting.

In this spotlight, we review technical issues that compromise single-cell analysis of tissue macrophages, including limited and unrepresentative yields, fragmentation and generation of remnants, and activation during tissue disaggregation. These issues may lead to a misleading definition of subpopulations of macrophages and the expression of macrophage-specific transcripts by unrelated cells. Recognition of the technical limitations of single-cell approaches is required in order to map the full spectrum of tissue-resident macrophage heterogeneity and assess its biological significance.

As part of tissue homeostasis, endothelial progenitors (EPCs) residing within the endothelium contribute to vascular maintenance and regeneration through self-renewal and differentiation. In various stem cell niche, macrophages (MΦ) help modulate progenitor stemness and behavior and have been reported to regulate blood vessel development and remodeling. Recently, subsets of perivascular macrophage (PVMΦ) were identified in the blood vessel vicinity during tissue homeostasis and wound healing. Nevertheless, the physiological role of these PVMΦ on EPC function in homeostasis and wound re-vascularisation remains elusive. In this study, we investigated the interaction and regulatory function of PVMΦ on EPCs' stemness in the murine aorta and skin using an endothelial-specific lineage tracing model, Cdh5CreERT/Rosa-ZsGreen. Imaging flow cytometry analyses tracking remanent PVMΦ bodies on endothelial cells revealed that PVMΦ preferentially formed physical interaction with endothelial cells that displayed mesenchymal properties in the healthy aorta (18.81% ± 13.13%) and skin (10.02% ± 0.92%). In situ characterization showed that the F4/80+Lyve1+ PVMΦ were in the adventitial area of the aorta, and resided within the lower dermis of the dorsal skin. MΦ depletion using clodronate liposomes doubled aorta EPC proportion (p=0.0079) in vivo, where it displayed increased proliferative potential with reduced clonogenic capacity in vitro. Functionally, attenuating macrophage-derived Wnt activity using Wlsfl/flLysM-Cre mice elevated EPC cell number by two-fold on day 5 post cutaneous injury without affecting other endothelial subpopulations. Overall, our data indicate that PVMΦ constitute part of the vascular niche by regulating EPCs' quiescence and self-renewal, potentially via paracrine Wnt signaling.

Neurogenic heterotopic ossifications (NHOs) form in periarticular muscles after severe spinal cord (SCI) and traumatic brain injuries. The pathogenesis of NHO is poorly understood with no effective preventive treatment. The only curative treatment remains surgical resection of pathological NHOs. In a mouse model of SCI-induced NHO that involves a transection of the spinal cord combined with a muscle injury, a differential gene expression analysis revealed that genes involved in inflammation such as interleukin-1β (IL-1β) were overexpressed in muscles developing NHO. Using mice knocked-out for the gene encoding IL-1 receptor (IL1R1) and neutralizing antibodies for IL-1α and IL-1β, we show that IL-1 signaling contributes to NHO development after SCI in mice. Interestingly, other proteins involved in inflammation that were also overexpressed in muscles developing NHO, such as colony-stimulating factor-1, tumor necrosis factor, or C-C chemokine ligand-2, did not promote NHO development. Finally, using NHO biopsies from SCI and TBI patients, we show that IL-1β is expressed by CD68+ macrophages. IL-1α and IL-1β produced by activated human monocytes promote calcium mineralization and RUNX2 expression in fibro-adipogenic progenitors isolated from muscles surrounding NHOs. Altogether, these data suggest that interleukin-1 promotes NHO development in both humans and mice. © 2021 American Society for Bone and Mineral Research (ASBMR).

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.

The cells of origin of neurogenic heterotopic ossifications (NHOs), which develop frequently in the periarticular muscles following spinal cord injuries (SCIs) and traumatic brain injuries, remain unclear because skeletal muscle harbors two progenitor cell populations: satellite cells (SCs), which are myogenic, and fibroadipogenic progenitors (FAPs), which are mesenchymal. Lineage-tracing experiments using the Cre recombinase/LoxP system were performed in two mouse strains with the fluorescent protein ZsGreen specifically expressed in either SCs or FAPs in skeletal muscles under the control of the Pax7 or Prrx1 gene promoter, respectively. These experiments demonstrate that following muscle injury, SCI causes the upregulation of PDGFRα expression on FAPs but not SCs and the failure of SCs to regenerate myofibers in the injured muscle, with reduced apoptosis and continued proliferation of muscle resident FAPs enabling their osteogenic differentiation into NHOs. No cells expressing ZsGreen under the Prrx1 promoter were detected in the blood after injury, suggesting that the cells of origin of NHOs are locally derived from the injured muscle. We validated these findings using human NHO biopsies. PDGFRα+ mesenchymal cells isolated from the muscle surrounding NHO biopsies could develop ectopic human bones when transplanted into immunocompromised mice, whereas CD56+ myogenic cells had a much lower potential. Therefore, NHO is a pathology of the injured muscle in which SCI reprograms FAPs to undergo uncontrolled proliferation and differentiation into osteoblasts.

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.

Background 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. Methods 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. Results 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. Conclusion These results indicate that CSF1-Fc conditioning could represent a therapeutic strategy to overcome poor HSC mobilisation and subsequently improve HSC transplantation outcomes.

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.