Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.

Pro-inflammatory Aorta-Associated Macrophages Are Involved in Embryonic Development of Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) are generated from specialized endothelial cells of the embryonic aorta. Inflammatory factors are implicated in regulating mouse HSC development, but which cells in the aorta-gonad-mesonephros (AGM) microenvironment produce these factors is unknown. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation were involved in the AGM HSC-generative microenvironment. CyTOF analysis of CD45+ AGM cells revealed predominance of two hematopoietic cell types, mannose-receptor positive macrophages and mannose-receptor negative myeloid cells. We show here that macrophage appearance in the AGM was dependent on the chemokine receptor Cx3cr1. These macrophages expressed a pro-inflammatory signature, localized to the aorta, and dynamically interacted with nascent and emerging intra-aortic hematopoietic cells (IAHCs). Importantly, upon macrophage depletion, no adult-repopulating HSCs were detected, thus implicating a role for pro-inflammatory AGM-associated macrophages in regulating the development of HSCs.

Airway Epithelial Cell-Derived Colony Stimulating Factor-1 Promotes Allergen Sensitization.

Airway epithelial cells (AECs) secrete innate immune cytokines that regulate adaptive immune effector cells. In allergen-sensitized humans and mice, the airway and alveolar microenvironment is enriched with colony stimulating factor-1 (CSF1) in response to allergen exposure. In this study we found that AEC-derived CSF1 had a critical role in the production of allergen reactive-IgE production. Furthermore, spatiotemporally secreted CSF1 regulated the recruitment of alveolar dendritic cells (DCs) and enhanced the migration of conventional DC2s (cDC2s) to the draining lymph node in an interferon regulatory factor 4 (IRF4)-dependent manner. CSF1 selectively upregulated the expression of the chemokine receptor CCR7 on the CSF1R+ cDC2, but not the cDC1, population in response to allergen stimuli. Our data describe the functional specification of CSF1-dependent DC subsets that link the innate and adaptive immune responses in T helper 2 (Th2) cell-mediated allergic lung inflammation.

Tumor progression is independent of tumor-associated macrophages in cell lineage-based mouse models of glioblastoma.

Macrophage targeting therapies have had limited clinical success in glioblastoma (GBM). Further understanding the GBM immune microenvironment is critical for refining immunotherapeutic approaches. Here, we use genetically engineered mouse models and orthotopic transplantation-based GBM models with identical driver mutations and unique cells of origin to examine the role of tumor cell lineage in shaping the immune microenvironment and response to tumor-associated macrophage (TAM) depletion therapy. We show that oligodendrocyte progenitor cell lineage-associated GBMs (Type 2) recruit more immune infiltrates and specifically monocyte-derived macrophages than subventricular zone neural stem cell-associated GBMs (Type 1). We then devise a TAM depletion system that offers a uniquely robust and sustained TAM depletion. We find that extensive TAM depletion in these cell lineage-based GBM models affords no survival benefit. Despite the lack of survival benefit of TAM depletion, we show that Type 1 and Type 2 GBMs have unique molecular responses to TAM depletion. In sum, we demonstrate that GBM cell lineage influences TAM ontogeny and abundance and molecular response to TAM depletion.

Colony stimulating factor 1 receptor (Csf1r) expressing cell ablation in mafia (macrophage-specific Fas-induced apoptosis) mice alters monocyte landscape and atherosclerotic lesion characteristics.

Macrophage infiltration and accumulation in the atherosclerotic lesion are associated with plaque progression and instability. Depletion of macrophages from the lesion might provide valuable insights into plaque stabilization processes. Therefore, we assessed the effects of systemic and local macrophage depletion on atherogenesis. To deplete monocytes/macrophages we used atherosclerosis-susceptible Apoe- /- mice, bearing a MaFIA (macrophage-Fas-induced-apoptosis) suicide construct under control of the Csf1r (CD115) promotor, where selective apoptosis of Csf1r-expressing cells was induced in a controlled manner, by administration of a drug, AP20187. Systemic induction of apoptosis resulted in a decrease in lesion macrophages and smooth-muscle cells. Plaque size and necrotic core size remained unaffected. Two weeks after the systemic depletion of macrophages, we observed a replenishment of the myeloid compartment. Myelopoiesis was modulated resulting in an expansion of CSF1Rlo myeloid cells in the circulation and a shift from Ly6chi monocytes toward Ly6cint and Ly6clo populations in the spleen. Local apoptosis induction led to a decrease in plaque burden and macrophage content with marginal effects on the circulating myeloid cells. Local, but not systemic depletion of Csf1r+ myeloid cells resulted in decreased plaque burden. Systemic depletion led to CSF1Rlo-monocyte expansion in blood, possibly explaining the lack of effects on plaque development.

Csf1rb regulates definitive hematopoiesis in zebrafish.

In vertebrates, hematopoietic stem and progenitor cells (HSPCs) are capable of self-renewal and continuously replenishing all mature blood lineages throughout life. However, the molecular signaling regulating the maintenance and expansion of HSPCs remains incompletely understood. Colony-stimulating factor 1 receptor (CSF1R) is believed to be the primary regulator for the myeloid lineage but not HSPC development. Here, we show a surprising role of Csf1rb, a zebrafish homolog of mammalian CSF1R, in preserving the HSPC pool by maintaining the proliferation of HSPCs. Deficiency of csf1rb leads to a reduction in both HSPCs and their differentiated progenies, including myeloid, lymphoid and erythroid cells at early developmental stages. Likewise, the absence of csf1rb conferred similar defects upon HSPCs and leukocytes in adulthood. Furthermore, adult hematopoietic cells from csf1rb mutants failed to repopulate immunodeficient zebrafish. Interestingly, loss-of-function and gain-of-function assays suggested that the canonical ligands for Csf1r in zebrafish, including Csf1a, Csf1b and Il34, were unlikely to be ligands of Csf1rb. Thus, our data indicate a previously unappreciated role of Csf1r in maintaining HSPCs, independently of known ligands.

A kinase-dead Csf1r mutation associated with adult-onset leukoencephalopathy has a dominant inhibitory impact on CSF1R signalling.

Amino acid substitutions in the kinase domain of the human CSF1R gene are associated with autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). To model the human disease, we created a disease-associated mutation (pGlu631Lys; E631K) in the mouse Csf1r locus. Homozygous mutation (Csf1rE631K/E631K) phenocopied the Csf1r knockout, with prenatal mortality or severe postnatal growth retardation and hydrocephalus. Heterozygous mutation delayed the postnatal expansion of tissue macrophage populations in most organs. Bone marrow cells from Csf1rE631K/+mice were resistant to CSF1 stimulation in vitro, and Csf1rE631K/+ mice were unresponsive to administration of a CSF1-Fc fusion protein, which expanded tissue macrophage populations in controls. In the brain, microglial cell numbers and dendritic arborisation were reduced in Csf1rE631K/+ mice, as in patients with ALSP. The microglial phenotype is the opposite of microgliosis observed in Csf1r+/- mice. However, we found no evidence of brain pathology or impacts on motor function in aged Csf1rE631K/+ mice. We conclude that heterozygous disease-associated CSF1R mutations compromise CSF1R signalling. We speculate that leukoencephalopathy associated with dominant human CSF1R mutations requires an environmental trigger and/or epistatic interaction with common neurodegenerative disease-associated alleles.

CSF1R inhibition depletes brain macrophages and reduces brain virus burden in SIV-infected macaques.

Perivascular macrophages (PVMs) and, to a lesser degree, microglia are targets and reservoirs of HIV and simian immunodeficiency virus (SIV) in the brain. Previously, we demonstrated that colony-stimulating factor 1 receptor (CSF1R) in PVMs was upregulated and activated in chronically SIV-infected rhesus macaques with encephalitis, correlating with SIV infection of PVMs. Herein, we investigated the role of CSF1R in the brain during acute SIV infection using BLZ945, a brain-penetrant CSF1R kinase inhibitor. Apart from three uninfected historic controls, nine Indian rhesus macaques were infected acutely with SIVmac251 and divided into three groups (n = 3 each): an untreated control and two groups treated for 20-30 days with low- (10 mg/kg/day) or high- (30 mg/kg/day) dose BLZ945. With the high-dose BLZ945 treatment, there was a significant reduction in cells expressing CD163 and CD206 across all four brain areas examined, compared with the low-dose treatment and control groups. In 9 of 11 tested regions, tissue viral DNA (vDNA) loads were reduced by 95%-99% following at least one of the two doses, and even to undetectable levels in some instances. Decreased numbers of CD163+ and CD206+ cells correlated significantly with lower levels of vDNA in all four corresponding brain areas. In contrast, BLZ945 treatment did not significantly affect the number of microglia. Our results indicate that doses as low as 10 mg/kg/day of BLZ945 are sufficient to reduce the tissue vDNA loads in the brain with no apparent adverse effect. This study provides evidence that infected PVMs are highly sensitive to CSF1R inhibition, opening new possibilities to achieve viral clearance.

IL-34, the rationale for its expression in physiological and pathological conditions.

IL-34 is a cytokine that shares one of its receptors with CSF-1. It has long been thought that CSF-1 receptor (CSF-1R) receives signals only from CSF-1, but the identification of IL-34 reversed this stereotype. Regardless of low structural homology, IL-34 and CSF-1 emanate similar downstream signaling through binding to CSF-1R and provoke similar but different physiological events afterward. In addition to CSF-1R, protein-tyrosine phosphatase (PTP)-ζ and Syndecan-1 were also identified as IL-34 receptors and shown to be at play. Although IL-34 expression is limited to particular tissues in physiological conditions, previous studies have revealed that it is upregulated in several diseases. In cancer, IL-34 is produced by several types of tumor cells and contributes to therapy resistance and disease progression. A recent study has demonstrated that tumor cell-derived IL-34 abrogates immunotherapy efficacy through myeloid cell remodeling. On the other hand, IL-34 expression is downregulated in some brain and dermal disorders. Despite accumulating insights, our understanding of IL-34 may not be even close to its nature. This review aims to comprehensively describe the physiological and pathological roles of IL-34 based on its similarity and differences to CSF-1 and discuss the rationale for its disease-dependent expression pattern.

Clinical evaluation of colony-stimulating factor 1 receptor inhibitors.

Signaling through colony-stimulating factor 1 receptor (CSF1R) regulates the development, differentiation, and activation of mononuclear phagocytic cells. Inhibition of this pathway provides an opportunity for therapeutic intervention in diseases in which these cells play a pathogenic role, including cancers, inflammation, fibrosis, and others. Multiple monoclonal antibodies and small molecule inhibitors targeting CSF1R or its known ligands CSF1 and IL-34 have been clinically tested and are generally well tolerated with side effects associated with on-target macrophage inhibition or depletion. To date, clinical activity of CSF1R inhibitors has been primarily observed in diffuse-type tenosynovial giant cell tumors, a disease characterized by genetic alterations in CSF1 leading to dysregulated CSF1R signaling. Expanded development into novel indications such as chronic graft vs host disease may provide new opportunities to further explore areas where a role for CSF1R dependent monocytes and macrophages has been established. This review presents key findings from the clinical development of 12 CSF1/CSF1R targeted therapies as monotherapy or in combination with immune checkpoint inhibitors and chemotherapy.

Colony stimulating factors in the nervous system.

Although traditionally seen as regulators of hematopoiesis, colony-stimulating factors (CSFs) have emerged as important players in the nervous system, both in health and disease. This review summarizes the cellular sources, patterns of expression and physiological roles of the macrophage (CSF-1, IL-34), granulocyte-macrophage (GM-CSF) and granulocyte (G-CSF) colony stimulating factors within the nervous system, with a particular focus on their actions on microglia. CSF-1 and IL-34, via the CSF-1R, are required for the development, proliferation and maintenance of essentially all CNS microglia in a temporal and regional specific manner. In contrast, in steady state, GM-CSF and G-CSF are mainly involved in regulation of microglial function. The alterations in expression of these growth factors and their receptors, that have been reported in several neurological diseases, are described and the outcomes of their therapeutic targeting in mouse models and humans are discussed.

CSF-1 maintains pathogenic but not homeostatic myeloid cells in the central nervous system during autoimmune neuroinflammation.

The receptor for colony stimulating factor 1 (CSF-1R) is important for the survival and function of myeloid cells that mediate pathology during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). CSF-1 and IL-34, the ligands of CSF-1R, have similar bioactivities but distinct tissue and context-dependent expression patterns, suggesting that they have different roles. This could be the case in EAE, given that CSF-1 expression is up-regulated in the CNS, while IL-34 remains constitutively expressed. We found that targeting CSF-1 with neutralizing antibody halted ongoing EAE, with efficacy superior to CSF-1R inhibitor BLZ945, whereas IL-34 neutralization had no effect, suggesting that pathogenic myeloid cells were maintained by CSF-1. Both anti­CSF-1 and BLZ945 treatment greatly reduced the number of monocyte-derived cells and microglia in the CNS. However, anti­CSF-1 selectively depleted inflammatory microglia and monocytes in inflamed CNS areas, whereas BLZ945 depleted virtually all myeloid cells, including quiescent microglia, throughout the CNS. Anti­CSF-1 treatment reduced the size of demyelinated lesions and microglial activation in the gray matter. Lastly, we found that bone marrow­derived immune cells were the major mediators of CSF-1R­dependent pathology, while microglia played a lesser role. Our findings suggest that targeting CSF-1 could be effective in ameliorating MS pathology, while preserving the homeostatic functions of myeloid cells, thereby minimizing risks associated with ablation of CSF-1R­dependent cells.

Long-term reprogramming of primed microglia after moderate inhibition of CSF1R signaling.

In acute neuroinflammation, microglia activate transiently, and return to a resting state later on. However, they may retain immune memory of such event, namely priming. Primed microglia are more sensitive to new stimuli and develop exacerbated responses, representing a risk factor for neurological disorders with an inflammatory component. Strategies to control the hyperactivation of microglia are, hence, of great interest. The receptor for colony stimulating factor 1 (CSF1R), expressed in myeloid cells, is essential for microglia viability, so its blockade with specific inhibitors (e.g. PLX5622) results in significant depletion of microglial population. Interestingly, upon inhibitor withdrawal, new naïve microglia repopulate the brain. Depletion-repopulation has been proposed as a strategy to reprogram microglia. However, substantial elimination of microglia is inadvisable in human therapy. To overcome such drawback, we aimed to reprogram long-term primed microglia by CSF1R partial inhibition. Microglial priming was induced in mice by acute neuroinflammation, provoked by intracerebroventricular injection of neuraminidase. After 3-weeks recovery, low-dose PLX5622 treatment was administrated for 12 days, followed by a withdrawal period of 7 weeks. Twelve hours before euthanasia, mice received a peripheral lipopolysaccharide (LPS) immune challenge, and the subsequent microglial inflammatory response was evaluated. PLX5622 provoked a 40%-50% decrease in microglial population, but basal levels were restored 7 weeks later. In the brain regions studied, hippocampus and hypothalamus, LPS induced enhanced microgliosis and inflammatory activation in neuraminidase-injected mice, while PLX5622 treatment prevented these changes. Our results suggest that PLX5622 used at low doses reverts microglial priming and, remarkably, prevents broad microglial depletion.

Inhibiting CSF1R alleviates cerebrovascular white matter disease and cognitive impairment.

White matter abnormalities, related to poor cerebral perfusion, are a core feature of small vessel cerebrovascular disease, and critical determinants of vascular cognitive impairment and dementia. Despite this importance there is a lack of treatment options. Proliferation of microglia producing an expanded, reactive population and associated neuroinflammatory alterations have been implicated in the onset and progression of cerebrovascular white matter disease, in patients and in animal models, suggesting that targeting microglial proliferation may exert protection. Colony-stimulating factor-1 receptor (CSF1R) is a key regulator of microglial proliferation. We found that the expression of CSF1R/Csf1r and other markers indicative of increased microglial abundance are significantly elevated in damaged white matter in human cerebrovascular disease and in a clinically relevant mouse model of chronic cerebral hypoperfusion and vascular cognitive impairment. Using the mouse model, we investigated long-term pharmacological CSF1R inhibition, via GW2580, and demonstrated that the expansion of microglial numbers in chronic hypoperfused white matter is prevented. Transcriptomic analysis of hypoperfused white matter tissue showed enrichment of microglial and inflammatory gene sets, including phagocytic genes that were the predominant expression modules modified by CSF1R inhibition. Further, CSF1R inhibition attenuated hypoperfusion-induced white matter pathology and rescued spatial learning impairments and to a lesser extent cognitive flexibility. Overall, this work suggests that inhibition of CSF1R and microglial proliferation mediates protection against chronic cerebrovascular white matter pathology and cognitive deficits. Our study nominates CSF1R as a target for the treatment of vascular cognitive disorders with broader implications for treatment of other chronic white matter diseases.

Proinflammatory microglial activation impairs in vitro cortical tissue repair via zinc-dependent ADAM17 cleavage of the CSF-1 receptor.

Infection and subsequent inflammatory processes negatively impact prognosis in individuals with traumatic brain injury (TBI). Tissue repair following TBI is tightly regulated by microglia, promoting or, importantly, preventing astrocyte-mediated repair processes, depending on the activation state of the neuroimmune cells. This study investigated the poorly understood mechanism linking proinflammatory microglia activation and astrocyte-mediated tissue repair using an in vitro mechanical injury model in mixed cortical cultures of rat neurons and glia. We hypothesized that proinflammatory activation disrupts the microglial response to colony-stimulating factor 1 (CSF-1), which stimulates microglia migration and proliferation, both essential for astrocyte-mediated tissue repair. Following mechanical damage, cultures were treated with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) to induce a proinflammatory state. Immunocytochemical and biochemical analyses were used to evaluate glial repair. Proinflammatory activation dramatically impeded wound closure, reducing microglial levels via upregulation of the zinc-dependent disintegrin and metalloprotease 17 (ADAM17), leading to the cleavage of the CSF-1 receptor (CSF-1R). Indeed, pharmacological inhibition of ADAM17 effectively promoted wound closure during inflammation. Moreover, zinc chelation prevented ADAM17-mediated cleavage of CSF-1R and induced the release of trophic factors, dramatically improving tissue recovery. Our findings strongly identify ADAM17 as a primary regulator of CSF-1R-mediated signaling and establish a mechanism defining the association between pro-inflammatory microglial activation and tissue repair following injury.

Assessing Chitinases and Neurofilament Light Chain as Biomarkers for Adult-Onset Leukodystrophies.

Leukodystrophies represent a large and complex group of inherited disorders affecting the white matter of the central nervous system. Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a rare leukodystrophy which still needs the proper identification of diagnostic, prognostic, and monitoring biomarkers. The aim of this study was to determine the diagnostic and prognostic value of chitinases and neurofilament light chain as biomarkers for ALSP. A cross-sectional study was performed to analyze cerebrospinal fluid levels of chitinases (chitotriosidase and chitinase 3-like 2) and neurofilament light chain in five different groups: (i) normal health individuals; (ii) patients with definitive diagnosis of ALSP and genetic confirmation; (iii) asymptomatic patients with CSF1R variants; (iv) patients with other adult-onset leukodystrophies; and (v) patients with amyotrophic lateral sclerosis (external control group). Chitinase levels showed a statistical correlation with clinical assessment parameters in ALSP patients. Chitinase levels were also distinct between ALSP and the other leukodystrophies. Significant differences were noted in the levels of chitinases and neurofilament light chain comparing symptomatic (ALSP) and asymptomatic individuals with CSF1R variants. This study is the first to establish chitinases as a potential biomarker for ALSP and confirms neurofilament light chain as a good biomarker for primary microgliopathies.

Phagocytosis Checkpoints in Glioblastoma: CD47 and Beyond.

Glioblastoma multiforme (GBM) is one of the deadliest human cancers with very limited treatment options available. The malignant behavior of GBM is manifested in a tumor which is highly invasive, resistant to standard cytotoxic chemotherapy, and strongly immunosuppressive. Immune checkpoint inhibitors have recently been introduced in the clinic and have yielded promising results in certain cancers. GBM, however, is largely refractory to these treatments. The immune checkpoint CD47 has recently gained attention as a potential target for intervention as it conveys a "don't eat me" signal to tumor-associated macrophages (TAMs) via the inhibitory SIRP alpha protein. In preclinical models, the administration of anti-CD47 monoclonal antibodies has shown impressive results with GBM and other tumor models. Several well-characterized oncogenic pathways have recently been shown to regulate CD47 expression in GBM cells and glioma stem cells (GSCs) including Epidermal Growth Factor Receptor (EGFR) beta catenin. Other macrophage pathways involved in regulating phagocytosis including TREM2 and glycan binding proteins are discussed as well. Finally, chimeric antigen receptor macrophages (CAR-Ms) could be leveraged for greatly enhancing the phagocytosis of GBM and repolarization of the microenvironment in general. Here, we comprehensively review the mechanisms that regulate the macrophage phagocytosis of GBM cells.

Colony stimulating factor-1 receptor drives glomerular parietal epithelial cell activation in focal segmental glomerulosclerosis.

Parietal epithelial cells (PECs) are kidney progenitor cells with similarities to a bone marrow stem cell niche. In focal segmental glomerulosclerosis (FSGS) PECs become activated and contribute to extracellular matrix deposition. Colony stimulating factor-1 (CSF-1), a hematopoietic growth factor, acts via its specific receptor, CSF-1R, and has been implicated in several glomerular diseases, although its role on PEC activation is unknown. Here, we found that CSF-1R was upregulated in PECs and podocytes in biopsies from patients with FSGS. Through in vitro studies, PECs were found to constitutively express CSF-1R. Incubation with CSF-1 induced CSF-1R upregulation and significant transcriptional regulation of genes involved in pathways associated with PEC activation. Specifically, CSF-1/CSF-1R activated the ERK1/2 signaling pathway and upregulated CD44 in PECs, while both ERK and CSF-1R inhibitors reduced CD44 expression. Functional studies showed that CSF-1 induced PEC proliferation and migration, while reducing the differentiation of PECs into podocytes. These results were validated in the Adriamycin-induced FSGS experimental mouse model. Importantly, treatment with either the CSF-1R-specific inhibitor GW2580 or Ki20227 provided a robust therapeutic effect. Thus, we provide evidence of the role of the CSF-1/CSF-1R pathway in PEC activation in FSGS, paving the way for future clinical studies investigating the therapeutic effect of CSF-1R inhibitors on patients with FSGS.

Amino acids suppress macropinocytosis and promote release of CSF1 receptor in macrophages.

The internalization of solutes by macropinocytosis provides an essential route for nutrient uptake in many cells. Macrophages increase macropinocytosis in response to growth factors and other stimuli. To test the hypothesis that nutrient environments modulate solute uptake by macropinocytosis, this study analyzed the effects of extracellular amino acids on the accumulation of fluorescent fluid-phase probes in murine macrophages. Nine amino acids, added individually or together, were capable of suppressing macropinocytosis in murine bone marrow-derived macrophages stimulated with the growth factors colony stimulating factor 1 (CSF1) or interleukin 34, both ligands of the CSF1 receptor (CSF1R). The suppressive amino acids did not inhibit macropinocytosis in response to lipopolysaccharide, the chemokine CXCL12, or the tumor promoter phorbol myristate acetate. Suppressive amino acids promoted release of CSF1R from cells and resulted in the formation of smaller macropinosomes in response to CSF1. This suppression of growth factor-stimulated macropinocytosis indicates that different nutrient environments modulate CSF1R levels and bulk ingestion by macropinocytosis, with likely consequences for macrophage growth and function.

Intraperitoneal transfer of wild-type bone marrow repopulates tissue macrophages in the Csf1r knockout rat without contributing to monocytopoiesis.

Homozygous null mutation of the Csf1r gene (Csf1rko) in rats leads to the loss of most tissue macrophage populations and pleiotropic impacts on postnatal growth and organ maturation, leading to early mortality. The phenotype can be reversed by intraperitoneal transfer of WT BM cells (BMT) at weaning. Here, we used a Csf1r-mApple transgenic reporter to track the fate of donor-derived cells. Following BMT into Csf1rko recipients, mApple+ve cells restored IBA1+ tissue macrophage populations in every tissue. However, monocytes, neutrophils, and B cells in the BM, blood, and lymphoid tissues remained of recipient (mApple-ve ) origin. An mApple+ve cell population expanded in the peritoneal cavity and invaded locally in the mesentery, fat pads, omentum, and diaphragm. One week after BMT, distal organs contained foci of mApple+ve , IBA1-ve immature progenitors that appeared to proliferate, migrate, and differentiate locally. We conclude that rat BM contains progenitor cells that are able to restore, replace, and maintain all tissue macrophage populations in a Csf1rko rat directly without contributing to the BM progenitor or blood monocyte populations.