CSF1R-dependent macrophages control postnatal somatic growth and organ maturation.

Homozygous mutation of the Csf1r locus (Csf1rko) in mice, rats and humans leads to multiple postnatal developmental abnormalities. To enable analysis of the mechanisms underlying the phenotypic impacts of Csf1r mutation, we bred a rat Csf1rko allele to the inbred dark agouti (DA) genetic background and to a Csf1r-mApple reporter transgene. The Csf1rko led to almost complete loss of embryonic macrophages and ablation of most adult tissue macrophage populations. We extended previous analysis of the Csf1rko phenotype to early postnatal development to reveal impacts on musculoskeletal development and proliferation and morphogenesis in multiple organs. Expression profiling of 3-week old wild-type (WT) and Csf1rko livers identified 2760 differentially expressed genes associated with the loss of macrophages, severe hypoplasia, delayed hepatocyte maturation, disrupted lipid metabolism and the IGF1/IGF binding protein system. Older Csf1rko rats developed severe hepatic steatosis. Consistent with the developmental delay in the liver Csf1rko rats had greatly-reduced circulating IGF1. Transfer of WT bone marrow (BM) cells at weaning without conditioning repopulated resident macrophages in all organs, including microglia in the brain, and reversed the mutant phenotypes enabling long term survival and fertility. WT BM transfer restored osteoclasts, eliminated osteopetrosis, restored bone marrow cellularity and architecture and reversed granulocytosis and B cell deficiency. Csf1rko rats had an elevated circulating CSF1 concentration which was rapidly reduced to WT levels following BM transfer. However, CD43hi non-classical monocytes, absent in the Csf1rko, were not rescued and bone marrow progenitors remained unresponsive to CSF1. The results demonstrate that the Csf1rko phenotype is autonomous to BM-derived cells and indicate that BM contains a progenitor of tissue macrophages distinct from hematopoietic stem cells. The model provides a unique system in which to define the pathways of development of resident tissue macrophages and their local and systemic roles in growth and organ maturation.

Regulation of gene expression in the bovine blastocyst by colony-stimulating factor 2 is disrupted by CRISPR/Cas9-mediated deletion of CSF2RA.

Colony-stimulating factor 2 (CSF2) functions in the reproductive tract to modulate the function of the preimplantation embryo. The ß subunit of the CSF2 receptor (CSF2RB) is not expressed in the embryo, and signal transduction is therefore different than for myeloid cells where the receptor is composed of α (CSF2RA) and ß subunits. Here, we produced embryos in which exons 5 and 6 of CSF2RA were disrupted using the CRISPR/Cas 9 system to test whether CSF2RA signaling was essential for actions of CSF2 in the bovine embryo. Wild-type and CSF2RA knockout embryos were treated with 10 ng/mL CSF2 or vehicle at day 5 of development. Blastocysts were harvested at day 8 to determine transcript abundance of 90 genes by real-time polymerase chain reaction (PCR). Responses in female blastocysts were examined separately from male blastocysts because actions of CSF2 are sex-dependent. For wild-type embryos, CSF2 altered expression of 10 genes in females and 20 in males. Only three genes were affected by CSF2 in a similar manner for both sexes. Disruption of CSF2RA prevented the effect of CSF2 on expression for 9 of 10 CSF2-regulated genes in females and 19 of 20 genes in males. The results confirm the importance of CSF2RA for regulation of gene expression by CSF2 in the blastocyst.

GM-CSF intrinsically controls eosinophil accumulation in the setting of allergic airway inflammation.

BACKGROUND: Eosinophils are a therapeutic target in asthmatic patients, and GM-CSF has been suggested to control various aspects of eosinophil biology, including development, function, and survival. However, to date, the role of GM-CSF signaling in eosinophils in vivo is largely unclear. OBJECTIVE: We sought to elucidate the role of GM-CSF signaling in asthmatic inflammation. METHODS: Wild-type and GM-CSF receptor α (Csf2ra)-deficient mice reconstituted with Csf2ra-proficient alveolar macrophages were subjected to different models of airway inflammation to evaluate the effect of GM-CSF signaling deficiency on asthmatic inflammation in general and on eosinophils in particular. RESULTS: We demonstrate that GM-CSF signaling, although being largely dispensable for eosinophil development at steady state, intrinsically promotes accumulation of eosinophils in the lung during allergic airway inflammation. In contrast, chitin-induced eosinophil accumulation in the peritoneal cavity occurs independent of GM-CSF, indicating organ specificity. We show that GM-CSF induces chemokinesis and promotes eosinophil survival in vitro, which likely contribute to eosinophil accumulation in the airways in vivo. CONCLUSION: GM-CSF intrinsically promotes eosinophil accumulation in the setting of pulmonary allergic inflammation.

Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus.

We have produced Csf1r-deficient rats by homologous recombination in embryonic stem cells. Consistent with the role of Csf1r in macrophage differentiation, there was a loss of peripheral blood monocytes, microglia in the brain, epidermal Langerhans cells, splenic marginal zone macrophages, bone-associated macrophages and osteoclasts, and peritoneal macrophages. Macrophages of splenic red pulp, liver, lung, and gut were less affected. The pleiotropic impacts of the loss of macrophages on development of multiple organ systems in rats were distinct from those reported in mice. Csf1r-/- rats survived well into adulthood with postnatal growth retardation, distinct skeletal and bone marrow abnormalities, infertility, and loss of visceral adipose tissue. Gene expression analysis in spleen revealed selective loss of transcripts associated with the marginal zone and, in brain regions, the loss of known and candidate novel microglia-associated transcripts. Despite the complete absence of microglia, there was little overt phenotype in brain, aside from reduced myelination and increased expression of dopamine receptor-associated transcripts in striatum. The results highlight the redundant and nonredundant functions of CSF1R signaling and of macrophages in development, organogenesis, and homeostasis.

Genetic Models of Macrophage Depletion.

Macrophages are a heterogeneous population of innate immune cells and are distributed in most adult tissues. Certain tissue-resident macrophages with a prenatal origin, together with postnatal monocyte-derived macrophages, serve as the host scavenger system to eliminate invading pathogens, malignant cells, senescent cells, dead cells, cellular debris, and other foreign substances. As a key member of the mononuclear phagocyte system, macrophages play essential roles in regulation of prenatal development, tissue homeostasis, and disease progression. Over the past two decades, considerable efforts have been made to generate genetic models of macrophage ablation in mice. These models support investigations of the precise functions of tissue-specific macrophages under physiological and pathological conditions. Herein, we overview the currently available mouse strains for in vivo genetic ablation of macrophages and discuss their respective advantages and limitations.

Regulation of lymphangiogenesis in the diaphragm by macrophages and VEGFR-3 signaling.

Lymphatic vessels play important roles in fluid drainage and in immune responses, as well as in pathological processes including cancer progression and inflammation. While the molecular regulation of the earliest lymphatic vessel differentiation and development has been investigated in much detail, less is known about the control and timing of lymphatic vessel maturation in different organs, which often occurs postnatally. We investigated the time course of lymphatic vessel development on the pleural side of the diaphragmatic muscle in mice, the so-called submesothelial initial diaphragmatic lymphatic plexus. We found that this lymphatic network develops largely after birth and that it can serve as a reliable and easily quantifiable model to study physiological lymphangiogenesis in vivo. Lymphangiogenic growth in this tissue was highly dependent on vascular endothelial growth factor receptor (VEGFR)-3 signaling, whereas VEGFR-1 and -2 signaling was dispensable. During diaphragm development, macrophages appeared first in a linearly arranged pattern, followed by ingrowth of lymphatic vessels along these patterned lines. Surprisingly, ablation of macrophages in colony-stimulating factor-1 receptor (Csf1r)-deficient mice and by treatment with a CSF-1R-blocking antibody did not inhibit the general lymphatic vessel development in the diaphragm but specifically promoted branch formation of lymphatic sprouts. In agreement with these findings, incubation of cultured lymphatic endothelial cells with conditioned medium from P7 diaphragmatic macrophages significantly reduced LEC sprouting. These results indicate that the postnatal diaphragm provides a suitable model for studies of physiological lymphangiogenic growth and maturation, and for the identification of modulators of lymphatic vessel growth.

Treatment of Adult Primary Alveolar Proteinosis.

Pulmonary alveolar proteinosis (PAP) is a rare disease characterized by the accumulation of surfactant-like lipoproteinaceous material in the distal air spaces and terminal bronchi, which may lead to impaired gas exchange. This accumulation of surfactant is due to decreased clearance by the alveolar macrophages. Its primary, most common form, is currently considered an autoimmune disease. Better knowledge of the causes of PAP have led to the emergence of alternatives to whole lung lavage, although this is still considered the treatment of choice. Most studies are case series, often with limited patient numbers, so the level of evidence is low. Since the severity of presentation and clinical course are variable, not all patients will require treatment. Due to the low level of evidence, some objective criteria based on expert opinion have been arbitrarily proposed in an attempt to define in which patients it is best to initiate treatment.

Gene correction of human induced pluripotent stem cells repairs the cellular phenotype in pulmonary alveolar proteinosis.

RATIONALE: Hereditary pulmonary alveolar proteinosis (hPAP) caused by granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor α-chain (CSF2RA) deficiency is a rare, life-threatening lung disease characterized by accumulation of proteins and phospholipids in the alveolar spaces. The disease is caused by a functional insufficiency of alveolar macrophages, which require GM-CSF signaling for terminal differentiation and effective degradation of alveolar proteins and phospholipids. Therapeutic options are extremely limited, and the pathophysiology underlying the defective protein degradation in hPAP alveolar macrophages remains poorly understood. OBJECTIVES: To further elucidate the cellular mechanisms underlying hPAP and evaluate novel therapeutic strategies, we here investigated the potential of hPAP patient-derived induced pluripotent stem cell (PAP-iPSCs) derived monocytes and macrophages. METHODS: Patient-specific PAP-iPSCs were generated from CD34(+) bone marrow cells of a CSF2RA-deficient patient with PAP. We assessed pluripotency, chromosomal integrity, and genetic correction of established iPSC lines. On hematopoietic differentiation, genetically corrected or noncorrected monocytes and macrophages were investigated in GM-CSF-dependent assays. MEASUREMENTS AND MAIN RESULTS: Although monocytes and macrophages differentiated from noncorrected PAP-iPSCs exhibited distinct defects in GM-CSF-dependent functions, such as perturbed CD11b activation, phagocytic activity, and STAT5 phosphorylation after GM-CSF exposure and lack of GM-CSF uptake, these defects were fully repaired on lentiviral gene transfer of a codon-optimized CSF2RA-cDNA. CONCLUSIONS: These data establish PAP-iPSC-derived monocytes and macrophages as a valid in vitro disease model of CSF2RA-deficient PAP, and introduce gene-corrected iPSC-derived monocytes and macrophages as a potential autologous cell source for innovative therapeutic strategies. Transplantation of such cells to patients with hPAP could serve as a paradigmatic proof for the potential of iPSC-derived cells in clinical gene therapy.

Use of induced pluripotent stem cells to recapitulate pulmonary alveolar proteinosis pathogenesis.

RATIONALE: In patients with pulmonary alveolar proteinosis (PAP) syndrome, disruption of granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling is associated with pathogenic surfactant accumulation from impaired clearance in alveolar macrophages. OBJECTIVES: The aim of this study was to overcome these barriers by using monocyte-derived induced pluripotent stem (iPS) cells to recapitulate disease-specific and normal macrophages. METHODS: We created iPS cells from two children with hereditary PAP (hPAP) caused by recessive CSF2RA(R217X) mutations and three normal people, differentiated them into macrophages (hPAP-iPS-Mφs and NL-iPS-Mφs, respectively), and evaluated macrophage functions with and without gene-correction to restore GM-CSF signaling in hPAP-iPS-Mφs. MEASUREMENTS AND MAIN RESULTS: Both hPAP and normal iPS cells had human embryonic stem cell-like morphology, expressed pluripotency markers, formed teratomas in vivo, had a normal karyotype, retained and expressed mutant or normal CSF2RA genes, respectively, and could be differentiated into macrophages with the typical morphology and phenotypic markers. Compared with normal, hPAP-iPS-Mφs had impaired GM-CSF receptor signaling and reduced GM-CSF-dependent gene expression, GM-CSF- but not M-CSF-dependent cell proliferation, surfactant clearance, and proinflammatory cytokine secretion. Restoration of GM-CSF receptor signaling corrected the surfactant clearance abnormality in hPAP-iPS-Mφs. CONCLUSIONS: We used patient-specific iPS cells to accurately reproduce the molecular and cellular defects of alveolar macrophages that drive the pathogenesis of PAP in more than 90% of patients. These results demonstrate the critical role of GM-CSF signaling in surfactant homeostasis and PAP pathogenesis in humans and have therapeutic implications for hPAP.

Cutting edge: granulocyte-macrophage colony-stimulating factor is the major CD8+ T cell-derived licensing factor for dendritic cell activation.

During priming, CD8(+) T lymphocytes can induce robust maturation of dendritic cells (DCs) in a CD40-independent manner by secreting licensing factor(s). In this study, we isolate this so-far elusive licensing factor and identify it, surprisingly, as GM-CSF. This provides a new face for an old factor with a well-known supporting role in DC development and recruitment. Signaling through the GM-CSFR in ex vivo-purified DCs upregulated the expression of costimulatory molecules more efficiently than did any tested TLR agonist and provided a positive feedback loop in the stimulation of CD8(+) T cell proliferation. Combined with a variety of microbial stimuli, GM-CSF supports the formation of potent "effector" DCs capable of secreting a variety of proinflammatory cytokines that guide the differentiation of T cells during the immune response.

Rapid cell senescence and apoptosis in lymphocytes and granulocytes and absence of GM-CSF receptor in congenital dysgranulopoietic neutropenia.

A girl with congenital dysgranulopoietic neutropenia (CDN) and her non-neutropenic mother with aphthae (A) were investigated. Apoptosis in lymphocytes and granulocytes of both patients (mother A+) were documented by high annexin and electron microscopic morphology. CD11b/CD18 of the daughter's granulocytes ranged between low to normal while that of the mother changed between very low to high levels through A(-) to A(+) periods. In both patients, CD11b/CD18 on lymphocytes were high; GM-CSF receptor was negative; CD4-/CD8- lymphocytes were high and the leukocytes which showed abnormal cell cycle were stained by senescence associated beta-galactosidase. We think that increased apoptosis and rapid cell senescence of leukocytes underlies the pathophysiology of CDN.

Granulocyte macrophage colony stimulating factor (GM-CSF) activity is regulated by a GM-CSF binding molecule in Wallerian degeneration following injury to peripheral nerve axons.

The hematopoietic factor and inflammatory cytokine GM-CSF is involved in PNS and CNS injury and disease, and in macrophage and microglia function regulation. We presently document that injury to PNS axons induces in vivo production of GM-CSF-inhibitor and GM-CSF-augmenter activities. GM-CSF-inhibitor activity was detected in extract and conditioned medium (CM) of injured PNS but not in extract of intact PNS, and was removed from CM by GM-CSF affinity chromatography, suggesting it is carried by a secreted GM-CSF binding molecule. CM further displayed GM-CSF-augmenter activity along with GM-CSF-inhibitor activity but at contrasting concentrations; augmentation at lowest and inhibition at highest. GM-CSF activity is thus regulated during Wallerian degeneration (WD); augmenter activity characterizes the onset and inhibitor activity the later stages of WD.

IL-12 alone and in synergy with IL-18 inhibits osteoclast formation in vitro.

IL-12, like IL-18, was shown to potently inhibit osteoclast formation in cultures of cocultures of murine osteoblast and spleen cells, as well as in adult spleen cells treated with M-CSF and receptor activator of NF-kappaB ligand (RANKL). Neither IL-12 nor IL-18 was able to inhibit RANKL-induced osteoclast formation in cultured RAW264.7 cells, demonstrating that IL-12, like IL-18, was unable to act directly on osteoclastic precursors. IL-12, like IL-18, was found to act by T cells, since depletion of T cells from the adult spleen cell cultures ablated the inhibitory action of IL-12 and addition of either CD4 or CD8 T cells from C57BL/6 mice to RANKL-stimulated RAW264.7 cultures permitted IL-12 or IL-18 to be inhibitory. Additionally, IL-12 was still able to inhibit osteoclast formation in cocultures with osteoblasts and spleen cells from either GM-CSF R(-/-) mice or IFN-gamma R(-/-) mice, indicating that neither GM-CSF nor IFN-gamma was mediating osteoclast inhibition in these cultures. Combined, IL-18 and IL-12 synergistically inhibited osteoclast formation at concentrations 20- to 1000-fold less, respectively, than when added individually. A candidate inhibitor could not be demonstrated using neutralizing Abs to IL-4, IL-10, or IL-13 or from mRNA expression profiles among known cytokine inhibitors of osteoclastogenesis in response to IL-12 and IL-18 treatment, although the unknown inhibitory molecule was determined to be secreted from T cells.

Polyethylene glycol-modified GM-CSF expands CD11b(high)CD11c(high) but notCD11b(low)CD11c(high) murine dendritic cells in vivo: a comparative analysis with Flt3 ligand.

Dendritic cells (DC) are potent APCs that can be characterized in the murine spleen as CD11b(high)CD11c(high) or CD11b(low)CD11c(high). Daily injection of mice of Flt3 ligand (FL) into mice transiently expands both subsets of DC in vivo, but the effect of administration of GM-CSF on the expansion of DC in vivo is not well defined. To gain further insight into the role of GM-CSF in DC development and function in vivo, we treated mice with polyethylene glycol-modified GM-CSF (pGM-CSF) which has an increased half-life in vivo. Administration of pGM-CSF to mice for 5 days led to a 5- to 10-fold expansion of CD11b(high)CD11c(high) but not CD11b(low)CD11c(high) DC. DC from pGM-CSF-treated mice captured and processed Ag more efficiently than DC from FL-treated mice. Although both FL- and pGM-CSF-generated CD11b(high)CD11c(high) DC were CD8alpha-, a greater proportion of these DC from pGM-CSF-treated mice were 33D1+ than from FL-treated mice. CD11b(low)CD11c(high) DC from FL-treated mice expressed high levels of intracellular MHC class II. DC from both pGM-CSF- and FL-treated mice expressed high levels of surface class II, low levels of the costimulatory molecules CD40, CD80, and CD86 and were equally efficient at stimulating allogeneic and Ag-specific T cell proliferation in vitro. The data demonstrate that treatment with pGM-CSF in vivo preferentially expands CD11b(high)CD11c(high) DC that share phenotypic and functional characteristics with FL-generated CD11b(high)CD11c(high) DC but can be distinguished from FL-generated DC on the basis of Ag capture and surface expression of 33D1.

Defective expression of granulocyte-macrophage colony-stimulating factor/interleukin-3/interleukin-5 receptor common beta chain in children with acute myeloid leukemia associated with respiratory failure.

Deficiency of the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin-3 (IL-3)/IL-5 receptors common beta chain (betac) is a cause of fatal respiratory failure. betac deficiency manifests as pulmonary alveolar proteinosis (PAP). PAP has heterogenous etiologies that may be genetic or aquired. Some cases of PAP have been reported to be associated with hematologic malignancies such as acute myeloid leukemia (AML). In mice, the PAP phenotype was generated by targeted deletion of the gene for betac and can be treated by transplantation of wild-type bone marrow into betac -/- mice. Thus, our findings in betac -/- mice provide evidence for a causal relationship between the lung disease and the hematopoietic system. We describe here expression defects of betac or betac plus GM-CSF receptor alpha chain (GM-CSFR alpha) in 3 pediatric patients with AML and PAP symptoms. All of the patients' leukemic cells failed to express normal levels of betac. The leukemic cells of patients no. 2 and 3 additionally lacked the expression of GM-CSFR alpha, as shown by flow cytometry. Strikingly reduced or absent function of betac was demonstrated in clonogenic progenitor assays with absent colony-forming unit (CFU) growth after GM-CSF or IL-3 stimulation. The response to growth factors acting via a growth factor receptor distinct from the GM-CSF/IL-3/IL-5 system (recombinant human granulocyte colony-stimulating factor [rhG-CSF]) was normal. After antileukemic treatment, the pulmonary symptoms resolved and betac or betac plus GM-CSFR alpha expression was normal. Our findings provide evidence that a defect in the expression of betac or betac plus GM-CSFR alpha on AML blasts can be associated with respiratory failure in patients with AML.

Myeloid development is selectively disrupted in PU.1 null mice.

The ets family transcription factor PU.1 is expressed in monocytes/macrophages, neutrophils, mast cells, B cells, and early erythroblasts, but not in T cells. We have recently shown that PU.1 gene disruption results in mice with no detectable monocytes/macrophages and B cells but T-cell development is retained. Although neutrophil development occurred in these mice, it was delayed and markedly reduced. We now proceed to demonstrate that PU. 1 null hematopoietic cells fail to proliferate or form colonies in response to macrophage colony-stimulating factor (M-CSF), granulocyte CSF (G-CSF), and granulocyte/macrophage CSF (GM-CSF). In contrast, PU.1 null cells did proliferate and form colonies in response to interleukin-3 (IL-3), although the response was reduced as compared with control littermates. Compared with control cells, PU.1 null cells had minimal expression of G- and GM-CSF receptors and no detectable M-CSF receptors. The size of individual myeloid colonies produced from PU.1 null primitive and committed myeloid progenitors in the presence of IL-3, IL-6, and stem cell factor (SCF) were reduced compared with controls. Under these conditions, PU.1 null progenitors produced neutrophils but not monocytes/macrophages. These observations suggest that PU.1 gene disruption induces additional cell-autonomous effects that are independent of the alterations in myeloid growth factor receptor expression. Our results demonstrate that PU.1 gene disruption affects a number of developmentally regulated hematopoietic processes that can, at least in part, explain the changes in myeloid development and reduction in myeloid and neutrophil expansion observed in PU.1 null mice.

The biological consequences of excess GM-CSF levels in transgenic mice also lacking high-affinity receptors for GM-CSF.

GM-CSF transgenic mice were crossed with mice with homozygous inactivation of the gene encoding the common beta chain (beta c) of the GM-CSF receptor to produce mice with constitutively elevated GM-CSF levels but no high-affinity GM-CSF receptors. GM-CSF transgenic beta c -/- mice had exceptionally elevated serum GM-CSF levels but failed to develop the abnormal peritoneal cell population, eye destruction or tissue lesions characteristic of GM-CSF transgenic beta c +/+ mice. The alveolar proteinosis of beta c -/- mice was not altered in GM-CSF transgenic beta c -/- mice. Levels of GM-CSF mRNA in transgenic GM-CSF beta c -/- were elevated but lower than in transgenic beta +/+ mice and the higher serum GM-CSF levels were traced in part to the longer serum half-life of GM-CSF in beta c -/- than in beta c +/+ mice although urinary loss of GM-CSF was higher in beta c -/- than in +/+ mice. The data indicate that the transgenic phenotype was due to stimulation by GM-CSF and not an insertional effect, that low-affinity receptors are not capable of initiating tissue pathology even in the presence of excess GM-CSF levels and that autocrine production of GM-CSF by GM-CSF-responsive cells also fails to induce changes in these cells. The results support current dogma that the action of polypeptide regulators is mediated exclusively by activation of high-affinity membrane receptors.

Human pulmonary alveolar proteinosis associated with a defect in GM-CSF/IL-3/IL-5 receptor common beta chain expression.

Pulmonary alveolar proteinosis (PAP) is a heterogeneous disorder of genetic or acquired etiologies. In some cases congenital PAP is associated with hereditary surfactant protein (SP)-B deficiency. To date, the molecular defect in the majority of patients with PAP has not been identified. In mice, PAP has been generated by targeted deletion of the genes for either the GM-CSF/IL-3/IL-5 receptor common beta chain (beta c) or GM-CSF. Here, we describe an expression defect of beta c in three of seven pediatric patients with PAP and in one patient with severe lung disease suspected to be PAP. The patients failed to express normal levels of beta c as shown by flow cytometry. Strikingly reduced or absent function of beta c was demonstrated by ligand binding studies and progenitor clonogenic assays. Analysis of beta c DNA revealed a point mutation from proline to threonine at codon 602 in one patient. Our findings provide evidence that a defect in the expression of a hematopoietic cytokine receptor is associated with human PAP.