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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., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology.)

The differentiation of resident intestinal macrophages from blood monocytes depends upon signals from the macrophage colony-stimulating factor receptor (CSF1R). Analysis of genome-wide association studies (GWAS) indicates that dysregulation of macrophage differentiation and response to microorganisms contributes to susceptibility to chronic inflammatory bowel disease (IBD). Here, we analyzed transcriptomic variation in monocyte-derived macrophages (MDM) from affected and unaffected sib pairs/trios from 22 IBD families and 6 healthy controls. Transcriptional network analysis of the data revealed no overall or inter-sib distinction between affected and unaffected individuals in basal gene expression or the temporal response to lipopolysaccharide (LPS). However, the basal or LPS-inducible expression of individual genes varied independently by as much as 100-fold between subjects. Extreme independent variation in the expression of pairs of HLA-associated transcripts (HLA-B/C, HLA-A/F and HLA-DRB1/DRB5) in macrophages was associated with HLA genotype. Correlation analysis indicated the downstream impacts of variation in the immediate early response to LPS. For example, variation in early expression of IL1B was significantly associated with local SNV genotype and with subsequent peak expression of target genes including IL23A, CXCL1, CXCL3, CXCL8 and NLRP3. Similarly, variation in early IFNB1 expression was correlated with subsequent expression of IFN target genes. Our results support the view that gene-specific dysregulation in macrophage adaptation to the intestinal milieu is associated with genetic susceptibility to IBD., (© 2024. The Author(s).)

Macrophages regulate metabolic homeostasis in health and disease. Macrophage colony-stimulating factor (CSF1)-dependent macrophages contribute to homeostatic control of the size of the liver. This study aimed to determine the systemic metabolic consequences of elevating circulating CSF1. Acute administration of a CSF1-Fc fusion protein to mice led to monocytosis, increased resident tissue macrophages in the liver and all major organs, and liver growth. These effects were associated with increased hepatic glucose uptake and extensive mobilization of body fat. The impacts of CSF1 on macrophage abundance, liver size, and body composition were rapidly reversed to restore homeostasis. The effects of CSF1 on metabolism were independent of several known endocrine regulators and did not impact the physiological fasting response. Analysis using implantable telemetry in metabolic cages revealed progressively reduced body temperature and physical activity with no change in diurnal food intake. These results demonstrate the existence of a dynamic equilibrium between CSF1, the mononuclear phagocyte system, and control of liver-to-body weight ratio, which in turn controls systemic metabolic homeostasis. This novel macrophage regulatory axis has the potential to promote fat mobilization, without changes in appetence, which may have novel implications for managing metabolic syndrome. NEW & NOTEWORTHY CSF1 administration expands tissue macrophages, which transforms systemic metabolism. CSF1 drives fat mobilization and glucose uptake to support liver growth. The effects of CSF1 are independent of normal hormonal metabolic regulation. The effects of CSF1 are rapidly reversible, restoring homeostatic body composition. CSF1-dependent macrophages and liver size are coupled in a dynamic equilibrium.

Macrophages contribute to many aspects of development and homeostasis, innate and acquired immunity, immunopathology, and tissue repair. Every tissue contains an abundant resident macrophage population. Inflammatory stimuli promote the recruitment of monocytes from the blood and their adaptation promotes the removal of the stimulus and subsequent restoration of normal tissue architecture. Dysregulation of this response leads to chronic inflammation and tissue injury. In many tissues, their differentiation and survival are dependent on the colony stimulating factor 1 receptor (CSF1R) signalling axis, which is highly conserved across all vertebrates. Complete loss of either CSF1R or its cognate ligands, colony stimulating factor 1 (CSF1), and interleukin 34 (IL-34), results in the loss of many tissue-resident macrophage populations. This provides a useful paradigm to study macrophages.There are many tools used to visualize tissue-resident macrophages and their precursors, monocytes, in mice and humans. Particularly in mice there are genetic tools available to delete, enhance and manipulate monocytes and macrophages and their gene products to gain insight into phenotype and function. The laboratory rat has many advantages as an experimental model for the understanding of human disease, but the analytical resources are currently more limited than in mice. Here, we describe available genetic models, antibodies, and immunohistochemistry (IHC) methods that may be used to visualize tissue-resident macrophages in rats., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023 by The American Society of Hematology.)

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., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

The mononuclear phagocyte system (MPS) was defined in the early 1970s as a family of cells including progenitors, monocytes in the circulation, and resident tissue macrophages. They arise during development in three waves, in the yolk sac, fetal liver, and bone marrow. Fate-mapping studies using conditional reporter genes and regulated expression of cre recombinase have led to the view that most resident tissue macrophage populations are established during embryonic development and maintained in the adult by self-renewal with minimal input from bone marrow progenitors or blood monocytes. The interpretation of fate-mapping studies depends upon multiple assumptions: (i) that expression of cre recombinase has no effect on monocyte-macrophage homeostasis, (ii) that tamoxifen is a neutral agonist, (iii) that life in an SPF animal facility reflects the normal life course of a mouse, and (iv) that the C57Bl/6J inbred mouse is a generalizable model and the biology of the MPS is unaffected by mouse genetic background or species. This review summarizes evidence that questions each of these assumptions and concludes that fate-mapping studies may over-estimate the longevity and relative contribution of fetal-derived cells to resident tissue macrophage populations. In the opinion of the author, the original concept of the MPS does not require revision., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Germinal centers (GCs) that form within lymphoid follicles during antibody responses are sites of massive cell death. Tingible body macrophages (TBMs) are tasked with apoptotic cell clearance to prevent secondary necrosis and autoimmune activation by intracellular self antigens. We show by multiple redundant and complementary methods that TBMs derive from a lymph node-resident, CD169-lineage, CSF1R-blockade-resistant precursor that is prepositioned in the follicle. Non-migratory TBMs use cytoplasmic processes to chase and capture migrating dead cell fragments using a "lazy" search strategy. Follicular macrophages activated by the presence of nearby apoptotic cells can mature into TBMs in the absence of GCs. Single-cell transcriptomics identified a TBM cell cluster in immunized lymph nodes which upregulated genes involved in apoptotic cell clearance. Thus, apoptotic B cells in early GCs trigger activation and maturation of follicular macrophages into classical TBMs to clear apoptotic debris and prevent antibody-mediated autoimmune diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

Macrophage colony-stimulating factor (CSF1) is the primary growth factor required for the control of monocyte and macrophage differentiation, survival, proliferation and renewal. Although the cDNAs encoding multiple isoforms of human CSF1 were cloned in the 1980s, and recombinant proteins were available for testing in humans, CSF1 has not yet found substantial clinical application. Here we present an overview of CSF1 biology, including evolution, regulation and functions of cell surface and secreted isoforms. CSF1 is widely-expressed, primarily by cells of mesenchymal lineages, in all mouse tissues. Cell-specific deletion of a floxed Csf1 allele in mice indicates that local CSF1 production contributes to the maintenance of tissue-specific macrophage populations but is not saturating. CSF1 in the circulation is controlled primarily by receptor-mediated clearance by macrophages in liver and spleen. Administration of recombinant CSF1 to humans or animals leads to monocytosis and expansion of tissue macrophage populations and growth of the liver and spleen. In a wide variety of tissue injury models, CSF1 administration promotes monocyte infiltration, clearance of damaged cells and repair. We suggest that CSF1 has therapeutic potential in regenerative medicine., (Copyright © 2021. Published by Elsevier Ltd.)

The central nervous system hosts parenchymal macrophages, known as microglia, and non-parenchymal macrophages, collectively termed border-associated macrophages (BAMs). Microglia, but not BAMs, were reported to be absent in mice lacking a conserved Csf1r enhancer: the fms -intronic regulatory element (FIRE). However, it is unknown whether FIRE deficiency also impacts BAM arrival and/or maintenance . Here, we show that macrophages in the ventricular system of the brain, including Kolmer's epiplexus macrophages, are absent in Csf1r mice. Stromal choroid plexus BAMs are also considerably reduced. During normal development, we demonstrate that intracerebroventricular macrophages arrive from embryonic day 10.5, and can traverse ventricular walls in embryonic slice cultures. In ΔFIRE/ΔFIRE embryos, the arrival of both primitive microglia and intracerebroventricular macrophages was eliminated, whereas the arrival of cephalic mesenchyme and stromal choroid plexus BAMs was only partially restricted. Our results provide new insights into the development and regulation of different CNS macrophage populations.Csf1r ΔFIRE/ΔFIRE embryos, the arrival of both primitive microglia and intracerebroventricular macrophages was eliminated, whereas the arrival of cephalic mesenchyme and stromal choroid plexus BAMs was only partially restricted. Our results provide new insights into the development and regulation of different CNS macrophage populations., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The mononuclear phagocyte system (MPS) is a family of cells including progenitors, circulating blood monocytes, resident tissue macrophages, and dendritic cells (DCs) present in every tissue in the body. To test the relationships between markers and transcriptomic diversity in the MPS, we collected from National Center for Biotechnology Information Gene Expression Omnibus (NCBI-GEO) a total of 466 quality RNA sequencing (RNA-seq) data sets generated from mouse MPS cells isolated from bone marrow, blood, and multiple tissues. The primary data were randomly downsized to a depth of 10 million reads and requantified. The resulting data set was clustered using the network analysis tool BioLayout. A sample-to-sample matrix revealed that MPS populations could be separated based upon tissue of origin. Cells identified as classical DC subsets, cDC1s and cDC2s, and lacking Fcgr1 (encoding the protein CD64) were contained within the MPS cluster, no more distinct than other MPS cells. A gene-to-gene correlation matrix identified large generic coexpression clusters associated with MPS maturation and innate immune function. Smaller coexpression gene clusters, including the transcription factors that drive them, showed higher expression within defined isolated cells, including monocytes, macrophages, and DCs isolated from specific tissues. They include a cluster containing Lyve1 that implies a function in endothelial cell (EC) homeostasis, a cluster of transcripts enriched in intestinal macrophages, and a generic lymphoid tissue cDC cluster associated with Ccr7. However, transcripts encoding Adgre1, Itgax, Itgam, Clec9a, Cd163, Mertk, Mrc1, Retnla, and H2-a/e (encoding class II major histocompatibility complex [MHC] proteins) and many other proposed macrophage subset and DC lineage markers each had idiosyncratic expression profiles. Coexpression of immediate early genes (for example, Egr1, Fos, Dusp1) and inflammatory cytokines and chemokines (tumour necrosis factor [Tnf], Il1b, Ccl3/4) indicated that all tissue disaggregation and separation protocols activate MPS cells. Tissue-specific expression clusters indicated that all cell isolation procedures also co-purify other unrelated cell types that may interact with MPS cells in vivo. Comparative analysis of RNA-seq and single-cell RNA-seq (scRNA-seq) data from the same lung cell populations indicated that MPS heterogeneity implied by global cluster analysis may be even greater at a single-cell level. This analysis highlights the power of large data sets to identify the diversity of MPS cellular phenotypes and the limited predictive value of surface markers to define lineages, functions, or subpopulations., Competing Interests: The authors have declared that no competing interests exist.

Intestinal macrophages are the largest group of mononuclear phagocytes in the body and play a role in intestinal innate immunity, neuroimmune interactions and maintaining intestinal homeostasis. Conversely, they also are implicated in numerous pathologies of the gastrointestinal tract, such as postoperative ileus and inflammatory bowel disease. As a result, macrophages could be potential therapeutic targets. To date, there are limited studies on the morphology and distribution of macrophages in the equine gastrointestinal tract (GIT). The aim of this study was to identify the location and abundance of resident macrophages in the equine GIT using CD163 as an immunohistochemical marker. Tissue samples were obtained post-mortem from 14 sites along the gastrointestinal tracts of 10 horses free from gastrointestinal disease; sample sites extended from the stomach to the small colon. CD163 +ve cells were present in all regions of the equine GIT from stomach to small colon. CD163 +ve cells were also identified in all tissue layers of the intestinal wall, namely, mucosa, submucosa, muscularis externa (ME), myenteric plexus and serosa. Consistent with a proposed function in regulation of intestinal motility, CD163 +ve cells were regularly distributed within the ME, with accumulations closely associated with the myenteric plexus and effector cells such as neurons and the interstitial cells of Cajal (ICC)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Macrophage colony-stimulating factor (CSF1) is an essential growth factor to control the proliferation, differentiation and survival of cells of the macrophage lineage in vertebrates. We have previously produced a recombinant chicken CSF1-Fc fusion protein and administrated it to birds which produced a substantial expansion of tissue macrophage populations. To further study the biology of CSF1 in the chicken, here we generated anti-chicken CSF1 antibodies (ROS-AV181 and 183) using CSF1-Fc as an immunogen. The specific binding of each monoclonal antibody was confirmed by ELISA, Western blotting and immunohistochemistry on tissue sections. Using the anti-CSF1 antibodies, we show that chicken bone marrow derived macrophages (BMDM) express CSF1 on their surface, and that the level appears to be regulated further by exogenous CSF1. By capture ELISA circulating CSF1 levels increased transiently in both layer and broiler embryos around the day of hatch. The levels of CSF1 in broilers was higher than in layers during the first week after hatch. Antibody ROS-AV183 was able to block CSF1 biological activity in vitro and treatment of hatchlings using this neutralising antibody in vivo impacted on some tissue macrophage populations, but not blood monocytes. After anti-CSF1 treatment, CSF1R-transgene reporter expressing cells were reduced in the bursa of Fabricius and cecal tonsil and TIM4 + Kupffer cells in the liver were almost completely ablated. Anti-CSF1 treatment also produced a reduction in overall bone density, trabecular volume and TRAP + osteoclasts. Our novel neutralising antibody provides a new tool to study the roles of CSF1 in birds., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Macrophages are present in large numbers in every tissue in the body where they play critical roles in development and homeostasis. They exhibit remarkable phenotypic and functional diversity, underpinning their adaptation to specialized roles in each tissue niche. CSF1, signaling through the CSF1 receptor, which is restricted to monocyte-macrophage lineage cells in adults, is a critical growth factor controlling macrophage proliferation, differentiation, and many aspects of mature macrophage function. We have generated a macrophage reporter rat, utilizing a construct containing elements of the mouse Csf1r promoter and the highly conserved Fms intronic regulatory element to drive mApple fluorescent protein expression. Csf1r-mApple was robustly expressed in monocyte-macrophage lineage cells in rat bone marrow (BM), peripheral blood, and tissues, with detectable expression in granulocytes and B cells and no evidence of expression in hematopoietic precursors or non-hematopoietic cells. Here, we use the Csf1r-mApple transgene to highlight and dissect the abundance and heterogeneity of rat tissue macrophage populations, and to demonstrate parallel increases in blood monocytes and multiple tissue macrophage populations, including BM, liver, spleen, and lung, in response to CSF1 treatment in vivo. The Csf1r-mApple rat is a novel tool enabling analysis of rat macrophages in situ by direct imaging and providing an additional phenotypic marker to facilitate exploration of rat tissue macrophage phenotypic and functional heterogeneity., (©2019 Society for Leukocyte Biology.)

Mϕ proliferation, differentiation, and survival are controlled by signals from the Mϕ CSF receptor (CSF1R). Mono-allelic gain-of-function mutations in CSF1R in humans are associated with an autosomal-dominant leukodystrophy and bi-allelic loss-of-function mutations with recessive skeletal dysplasia, brain disorders, and developmental anomalies. Most of the phenotypes observed in these human disease states are also observed in mice and rats with loss-of-function mutations in Csf1r or in Csf1 encoding one of its two ligands. Studies in rodent models also highlight the importance of genetic background and likely epistatic interactions between Csf1r and other loci. The impacts of Csf1r mutations on the brain are usually attributed solely to direct impacts on microglial number and function. However, analysis of hypomorphic Csf1r mutants in mice and several other lines of evidence suggest that primary hydrocephalus and loss of the physiological functions of Mϕs in the periphery contribute to the development of brain pathology. In this review, we outline the evidence that CSF1R is expressed exclusively in mononuclear phagocytes and explore the mechanisms linking CSF1R mutations to pleiotropic impacts on postnatal growth and development., (© 2019 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.)

The proliferation, differentiation and survival of mononuclear phagocytes depend on signals from the receptor for macrophage colony-stimulating factor, CSF1R. The mammalian Csf1r locus contains a highly conserved super-enhancer, the fms-intronic regulatory element (FIRE). Here we show that genomic deletion of FIRE in mice selectively impacts CSF1R expression and tissue macrophage development in specific tissues. Deletion of FIRE ablates macrophage development from murine embryonic stem cells. Csf1r ΔFIRE/ΔFIRE mice lack macrophages in the embryo, brain microglia and resident macrophages in the skin, kidney, heart and peritoneum. The homeostasis of other macrophage populations and monocytes is unaffected, but monocytes and their progenitors in bone marrow lack surface CSF1R. Finally, Csf1r ΔFIRE/ΔFIRE mice are healthy and fertile without the growth, neurological or developmental abnormalities reported in Csf1r -/- rodents. Csf1r ΔFIRE/ΔFIRE mice thus provide a model to explore the homeostatic, physiological and immunological functions of tissue-specific macrophage populations in adult animals.

The mononuclear phagocyte system (MPS) is defined as a cell lineage in which committed marrow progenitors give rise to blood monocytes and tissue macrophages. Here, we discuss the concept of self-proscribed macrophage territories and homeostatic regulation of tissue macrophage abundance through growth factor availability. Recent studies have questioned the validity of the MPS model and argued that tissue-resident macrophages are a separate lineage seeded during development and maintained by self-renewal. We address this issue; discuss the limitations of inbred mouse models of monocyte-macrophage homeostasis; and summarize the evidence suggesting that during postnatal life, monocytes can replace resident macrophages in all major organs and adopt their tissue-specific gene expression. We conclude that the MPS remains a valid and accurate framework for understanding macrophage development and homeostasis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 The Authors.)

The F4/80 antigen, encoded by the Adgre1 locus, has been widely-used as a monocyte-macrophage marker in mice, but its value as a macrophage marker in other species is unclear, and has even been questioned. ADGRE1 is a seven transmembrane G protein-coupled receptor with an extracellular domain containing repeated Epidermal Growth Factor (EGF)-like calcium binding domains. Using a new monoclonal antibody, we demonstrated that ADGRE1 is a myeloid differentiation marker in pigs, absent from progenitors in bone marrow, highly-expressed in mature granulocytes, monocytes, and tissue macrophages and induced by macrophage colony-stimulating factor (CSF1) treatment in vivo . Based upon these observations, we utilized RNA-Seq to assess the expression of ADGRE1 mRNA in bone marrow or monocyte-derived macrophages (MDM) and alveolar macrophages from 8 mammalian species including pig, human, rat, sheep, goat, cow, water buffalo, and horse. ADGRE1 mRNA was expressed by macrophages in each species, with inter-species variation both in expression level and response to lipopolysaccharide (LPS) stimulation. Analysis of the RNA-Seq data also revealed additional exons in several species compared to current Ensembl annotations. The ruminant species and horses appear to encode a complete duplication of the 7 EGF-like domains. In every species, Sashimi plots revealed evidence of exon skipping of the EGF-like domains, which are highly-variable between species and polymorphic in humans. Consistent with these expression patterns, key elements of the promoter and a putative enhancer are also conserved across all species. The rapid evolution of this molecule and related ADGRE family members suggests immune selection and a role in pathogen recognition.

Distinct subsets of resident tissue macrophages are important in hematopoietic stem cell niche homeostasis and erythropoiesis. We used a myeloid reporter gene ( Csf1r -eGFP) to dissect the persistence of bone marrow and splenic macrophage subsets following lethal irradiation and autologous hematopoietic stem cell transplantation in a mouse model. Multiple recipient bone marrow and splenic macrophage subsets survived after autologous hematopoietic stem cell transplantation with organ-specific persistence kinetics. Short-term persistence (5 weeks) of recipient resident macrophages in spleen paralleled the duration of extramedullary hematopoiesis. In bone marrow, radiation-resistant recipient CD169 + resident macrophages and erythroid-island macrophages self-repopulated long-term after transplantation via autonomous cell division. Posttransplant peak expansion of recipient CD169 + resident macrophage number in bone marrow aligned with the persistent engraftment of phenotypic long-term reconstituting hematopoietic stem cells within bone marrow. Selective depletion of recipient CD169 + macrophages significantly compromised the engraftment of phenotypic long-term reconstituting hematopoietic stem cells and consequently impaired hematopoietic reconstitution. Recipient bone marrow resident macrophages are essential for optimal hematopoietic stem cell transplantation outcomes and could be an important consideration in the development of pretransplant conditioning therapies and/or chemoresistance approaches., (© 2018 by The American Society of Hematology.)

Several lines of evidence link macrophage activation and inflammation with (monoaminergic) nervous systems in the etiology of depression. IFN treatment is associated with depressive symptoms, whereas anti-TNFα therapies elicit positive mood. This study describes the actions of 2 monoaminergic antidepressants (escitalopram, nortriptyline) and 3 anti-inflammatory drugs (indomethacin, prednisolone, and anti-TNFα antibody) on the response of human monocyte-derived macrophages (MDMs) from 6 individuals to LPS or IFN-α. Expression profiling revealed robust changes in the MDM transcriptome (3294 genes at P < 0.001) following LPS challenge, whereas a more limited subset of genes (499) responded to IFNα. Contrary to published reports, administered at nontoxic doses, neither monoaminergic antidepressant significantly modulated the transcriptional response to either inflammatory challenge. Each anti-inflammatory drug had a distinct impact on the expression of inflammatory cytokines and on the profile of inducible gene expression-notably on the regulation of enzymes involved in metabolism of tryptophan. Inter alia, the effect of anti-TNFα antibody confirmed a predicted autocrine stimulatory loop in human macrophages. The transcriptional changes were predictive of tryptophan availability and kynurenine synthesis, as analyzed by targeted metabolomic studies on cellular supernatants. We suggest that inflammatory processes in the brain or periphery could impact on depression by altering the availability of tryptophan for serotonin synthesis and/or by increasing production of neurotoxic kynurenine., (©2018 Society for Leukocyte Biology.)

Colony-stimulating factor 1 (CSF1) controls the growth and differentiation of macrophages.CSF1R signaling has been implicated in the maintenance of the intestinal stem cell niche and differentiation of Paneth cells, but evidence of expression of CSF1R within the crypt is equivocal. Here we show that CSF1R-dependent macrophages influence intestinal epithelial differentiation and homeostasis. In the intestinal lamina propria CSF1R mRNA expression is restricted to macrophages which are intimately associated with the crypt epithelium, and is undetectable in Paneth cells. Macrophage ablation following CSF1R blockade affects Paneth cell differentiation and leads to a reduction of Lgr5 + intestinal stem cells. The disturbances to the crypt caused by macrophage depletion adversely affect the subsequent differentiation of intestinal epithelial cell lineages. Goblet cell density is enhanced, whereas the development of M cells in Peyer's patches is impeded. We suggest that modification of the phenotype or abundance of macrophages in the gut wall alters the development of the intestinal epithelium and the ability to sample gut antigens.

Glucocorticoids act by binding to the glucocorticoid receptor (GR), which binds to specific motifs within enhancers of target genes to activate transcription. Previous studies have suggested that GRs can promote interactions between gene promoters and distal elements within target loci. In contrast, we demonstrate here that glucocorticoid addition to mouse bone-marrow-derived macrophages produces very rapid chromatin unfolding detectable by fluorescence in situ hybridization (FISH) at loci associated with GR binding. Rapid chromatin decompaction was generally not dependent on transcription at those loci that are known to be inducible in both mouse and human macrophages and was sustained for up to 5 days following ligand removal. Chromatin decompaction was not dependent upon persistent GR binding, which decayed fully after 24 hr. We suggest that sustained large-scale chromatin reorganization forms an important part of the response to glucocorticoid and might contribute to glucocorticoid sensitivity and resistance., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: Postoperative ileus (POI) is assumed to result from myeloid cells infiltrating the intestinal muscularis externa (ME) in patients undergoing abdominal surgery. In the current study, we investigated the role of infiltrating monocytes in a murine model of intestinal manipulation (IM)-induced POI in order to clarify whether monocytes mediate tissue damage and intestinal dysfunction or they are rather involved in the recovery of gastrointestinal (GI) motility., Design: IM was performed in mice with defective monocyte migration to tissues (C-C motif chemokine receptor 2, Ccr2 -/ - mice) and wild-type (WT) mice to study the role of monocytes and monocyte-derived macrophages (MΦs) during onset and resolution of ME inflammation., Results: At early time points, IM-induced GI transit delay and inflammation were equal in WT and Ccr2 - / - mice. However, GI transit recovery after IM was significantly delayed in Ccr2 - / - mice compared with WT mice, associated with increased neutrophil-mediated immunopathology and persistent impaired neuromuscular function. During recovery, monocyte-derived MΦs acquire pro-resolving features that aided in the resolution of inflammation. In line, bone marrow reconstitution and treatment with MΦ colony-stimulating factor 1 enhanced monocyte recruitment and MΦ differentiation and ameliorated GI transit in Ccr2 - / - mice., Conclusion: Our study reveals a critical role for monocyte-derived MΦs in restoring intestinal homeostasis after surgical trauma. From a therapeutic point of view, our data indicate that inappropriate targeting of monocytes may increase neutrophil-mediated immunopathology and prolong the clinical outcome of POI, while future therapies should be aimed at enhancing MΦ physiological repair functions., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

The FANTOM5 consortium used cap analysis of gene expression (CAGE) to analyze the time course of gene expression over development from 11 days postcoitum (dpc) to adult in 16 developing organs and the whole body of the mouse. Every tissue in the body contains a large number of resident macrophages that initially infiltrate the embryo from the yolk sac. These cells contribute to organogenesis, and their functions diversify during development as they acquire tissue-specific adaptations. In each of the FANTOM5 time courses, the expression of known macrophage-specific genes, including CSF1 receptor ( Csf1r ), epidermal growth factor-like module-containing mucin-like hormone receptor-like 1 ( Emr1 ), and mer receptor tyrosine kinase ( Mertk ), was readily detectable and increased with time. We reasoned that genes expressed by macrophages would be strongly correlated in their expression with these known markers and might vary between tissues. We used the network analysis tool, Miru, to extract the sets of coexpressed genes from the time course and identified a core set of coexpressed genes attributable to embryonic macrophages, including some, such as dehydrogenase/reductase 3 ( Dhrs3 ), that may have unique functions in development. The FANTOM5 data also detected the appearance of tissue-specific macrophage-expressed genes, such as T cell Ig and mucin domain-containing 4 ( Timd4 ) and V-set and Ig domain-containing 4 ( Vsig4 ) in liver and sialic acid-binding Ig-like lectin 5 ( Siglec5 ) in lung, and confirmed that macrophage content increases with time in each organ as the proliferative phases end, and tissue-specific gene-expression increases. The FANTOM5 data are available on a comprehensive browser (http://fantom.gsc.riken.jp/zenbu/), which provides a resource for the study of macrophage transcriptional regulation and roles in mouse development., (© Society for Leukocyte Biology.)

The FANTOM5 consortium utilised cap analysis of gene expression (CAGE) to provide an unprecedented insight into transcriptional regulation in human cells and tissues. In the current study, we have used CAGE-based transcriptional profiling on an extended dense time course of the response of human monocyte-derived macrophages grown in macrophage colony-stimulating factor (CSF1) to bacterial lipopolysaccharide (LPS). We propose that this system provides a model for the differentiation and adaptation of monocytes entering the intestinal lamina propria. The response to LPS is shown to be a cascade of successive waves of transient gene expression extending over at least 48 hours, with hundreds of positive and negative regulatory loops. Promoter analysis using motif activity response analysis (MARA) identified some of the transcription factors likely to be responsible for the temporal profile of transcriptional activation. Each LPS-inducible locus was associated with multiple inducible enhancers, and in each case, transient eRNA transcription at multiple sites detected by CAGE preceded the appearance of promoter-associated transcripts. LPS-inducible long non-coding RNAs were commonly associated with clusters of inducible enhancers. We used these data to re-examine the hundreds of loci associated with susceptibility to inflammatory bowel disease (IBD) in genome-wide association studies. Loci associated with IBD were strongly and specifically (relative to rheumatoid arthritis and unrelated traits) enriched for promoters that were regulated in monocyte differentiation or activation. Amongst previously-identified IBD susceptibility loci, the vast majority contained at least one promoter that was regulated in CSF1-dependent monocyte-macrophage transitions and/or in response to LPS. On this basis, we concluded that IBD loci are strongly-enriched for monocyte-specific genes, and identified at least 134 additional candidate genes associated with IBD susceptibility from reanalysis of published GWA studies. We propose that dysregulation of monocyte adaptation to the environment of the gastrointestinal mucosa is the key process leading to inflammatory bowel disease.

In mammals, the inducible cytokine interleukin 10 is a feedback negative regulator of inflammation. To determine the extent to which this function is conserved in birds, recombinant chicken IL-10 was expressed as a secreted human Ig Fc fusion protein (chIL-10-Fc) and used to immunise mice. Five monoclonal antibodies (mAb) which specifically recognise chicken IL-10 were generated and characterised. Two capture ELISA assays were developed which detected native chIL-10 secreted from chicken bone marrow-derived macrophages (chBMMs) stimulated with lipopolysaccharide (LPS). Three of the mAbs detected intracellular IL-10. This was detected in only a subset of the same LPS-stimulated chBMMs. The ELISA assay also detected massive increases in circulating IL-10 in chickens challenged with the coccidial parasite, Eimeria tenella. The same mAbs neutralised the bioactivity of recombinant chIL-10. The role of IL-10 in feedback control was tested in vitro. The neutralising antibodies prevented IL-10-induced inhibition of IFN-γ synthesis by mitogen-activated lymphocytes and increased nitric oxide production in LPS-stimulated chBMMs. The results confirm that IL-10 is an inducible feedback regulator of immune response in chickens, and could be the target for improved vaccine efficacy or breeding strategies., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Expression of Csf1r in adults is restricted to cells of the macrophage lineage. Transgenic reporters based upon the Csf1r locus require inclusion of the highly conserved Fms-intronic regulatory element for expression. We have created Csf1r-EGFP transgenic sheep via lentiviral transgenesis of a construct containing elements of the mouse Fms-intronic regulatory element and Csf1r promoter. Committed bone marrow macrophage precursors and blood monocytes express EGFP in these animals. Sheep monocytes were divided into three populations, similar to classical, intermediate, and nonclassical monocytes in humans, based upon CD14 and CD16 expression. All expressed EGFP, with increased levels in the nonclassical subset. Because Csf1r expression coincides with the earliest commitment to the macrophage lineage, Csf1r-EGFP bone marrow provides a tool for studying the earliest events in myelopoiesis using the sheep as a model., (Copyright © 2016 The Authors.)

Monocytes and macrophages are professional phagocytes that occupy specific niches in every tissue of the body. Their survival, proliferation, and differentiation are controlled by signals from the macrophage colony-stimulating factor receptor (CSF-1R) and its two ligands, CSF-1 and interleukin-34. In this review, we address the developmental and transcriptional relationships between hematopoietic progenitor cells, blood monocytes, and tissue macrophages as well as the distinctions from dendritic cells. A huge repertoire of receptors allows monocytes, tissue-resident macrophages, or pathology-associated macrophages to adapt to specific microenvironments. These processes create a broad spectrum of macrophages with different functions and individual effector capacities. The production of large transcriptomic data sets in mouse, human, and other species provides new insights into the mechanisms that underlie macrophage functional plasticity.

Phenotypic differences between individuals and species are controlled in part through differences in expression of a relatively conserved set of genes. Genes expressed in the immune system are subject to especially powerful selection. We have investigated the evolution of both gene expression and candidate enhancers in human and mouse macrophages exposed to glucocorticoid (GC), a regulator of innate immunity and an important therapeutic agent. Our analyses revealed a very limited overlap in the repertoire of genes responsive to GC in human and mouse macrophages. Peaks of inducible binding of the GC receptor (GR) detected by chromatin immunoprecipitation-Seq correlated with induction, but not repression, of target genes in both species, occurred at distal regulatory sites not promoters, and were strongly enriched for the consensus GR-binding motif. Turnover of GR binding between mice and humans was associated with gain and loss of the motif. There was no detectable signal of positive selection at species-specific GR binding sites, but clear evidence of purifying selection at the small number of conserved sites. We conclude that enhancer divergence underlies the difference in transcriptional activation after GC treatment between mouse and human macrophages. Only the shared inducible loci show evidence of selection, and therefore these loci may be important for the subset of responses to GC that is shared between species., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Background: The FANTOM5 consortium used Cap Analysis of Gene Expression (CAGE) tag sequencing to produce a comprehensive atlas of promoters and enhancers within the human and mouse genomes. We reasoned that the mapping of these regulatory elements to the pig genome could provide useful annotation and evidence to support assignment of orthology., Results: For human transcription start sites (TSS) associated with annotated human-mouse orthologs, 17% mapped to the pig genome but not to the mouse, 10% mapped only to the mouse, and 55% mapped to both pig and mouse. Around 17% did not map to either species. The mapping percentages were lower where there was not clear orthology relationship, but in every case, mapping to pig was greater than to mouse, and the degree of homology was also greater. Combined mapping of mouse and human CAGE-defined promoters identified at least one putative conserved TSS for >16,000 protein-coding genes. About 54% of the predicted locations of regulatory elements in the pig genome were supported by CAGE and/or RNA-Seq analysis from pig macrophages., Conclusions: Comparative mapping of promoters and enhancers from humans and mice can provide useful preliminary annotation of other animal genomes. The data also confirm extensive gain and loss of regulatory elements between species, and the likelihood that pigs provide a better model than mice for human gene regulation and function.

We have identified differences in gene expression in macrophages grown from the bone marrow of male and female chickens in recombinant chicken M-CSF (CSF1). Cells were profiled with or without treatment with bacterial LPS for 24 h. Approximately 600 transcripts were induced by prolonged LPS stimulation to an equal extent in the male and female macrophages. Many transcripts encoded on the Z chromosome were expressed ∼1.6-fold higher in males, reflecting a lack of dosage compensation in the homogametic sex. A smaller set of W chromosome-specific genes was expressed only in females. LPS signaling in mammals is associated with induction of type 1 IFN-responsive genes. Unexpectedly, because IFNs are encoded on the Z chromosome of chickens, unstimulated macrophages from the female birds expressed a set of known IFN-inducible genes at much higher levels than male cells under the same conditions. To confirm that these differences were not the consequence of the actions of gonadal hormones, we induced gonadal sex reversal to alter the hormonal environment of the developing chick and analyzed macrophages cultured from male, female, and female sex-reversed embryos. Gonadal sex reversal did not alter the sexually dimorphic expression of either sex-linked or IFN-responsive genes. We suggest that female birds compensate for the reduced dose of inducible IFN with a higher basal set point of IFN-responsive genes., (Copyright © 2015 The Authors.)

Macrophages are a major cell type in tissue homeostasis and contribute to both pathology and resolution in all acute and chronic inflammatory diseases ranging from infections, cancer, obesity, atherosclerosis, autoimmune disorders to neurodegenerative diseases such as Alzheimer's disease. The cellular and functional diversity of macrophages depends upon tightly regulated transcription. The innate immune system is under profound evolutionary selection. There is increasing recognition that human macrophage biology differs very significantly from that of commonly studied animal models, which therefore can have a limited predictive value. Here we report on the newest findings on transcriptional control of macrophage activation, and how we envision integrating studies on transcriptional and epigenetic regulation, and more classical approaches in murine models. Moreover, we provide new insights into how we can learn about transcriptional regulation in the human system from larger efforts such as the FANTOM (Functional Annotation of the Mammalian Genome) consortium., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We have generated the first transgenic chickens in which reporter genes are expressed in a specific immune cell lineage, based upon control elements of the colony stimulating factor 1 receptor (CSF1R) locus. The Fms intronic regulatory element (FIRE) within CSF1R is shown to be highly conserved in amniotes and absolutely required for myeloid-restricted expression of fluorescent reporter genes. As in mammals, CSF1R-reporter genes were specifically expressed at high levels in cells of the macrophage lineage and at a much lower level in granulocytes. The cell lineage specificity of reporter gene expression was confirmed by demonstration of coincident expression with the endogenous CSF1R protein. In transgenic birds, expression of the reporter gene provided a defined marker for macrophage-lineage cells, identifying the earliest stages in the yolk sac, throughout embryonic development and in all adult tissues. The reporter genes permit detailed and dynamic visualisation of embryonic chicken macrophages. Chicken embryonic macrophages are not recruited to incisional wounds, but are able to recognise and phagocytose microbial antigens., (© 2014. Published by The Company of Biologists Ltd.)

The mononuclear phagocyte system (MPS) is a family of functionally related cells including bone marrow precursors, blood monocytes, and tissue macrophages. We review the evidence that macrophages and dendritic cells (DCs) are separate lineages and functional entities, and examine whether the traditional view that monocytes are the immediate precursors of tissue macrophages needs to be refined based upon evidence that macrophages can extensively self-renew and can be seeded from yolk sac/foetal liver progenitors with little input from monocytes thereafter. We review the role of the growth factor colony-stimulating factor (CSF)1, and present a model consistent with the concept of the MPS in which local proliferation and monocyte recruitment are connected to ensure macrophages occupy their well-defined niche in most tissues., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Macrophages respond to the TLR4 agonist LPS with a sequential transcriptional cascade controlled by a complex regulatory network of signaling pathways and transcription factors. At least two distinct pathways are currently known to be engaged by TLR4 and are distinguished by their dependence on the adaptor molecule MyD88. We have used gene expression microarrays to define the effects of each of three variables--LPS dose, LPS versus IFN-β and -γ, and genetic background--on the transcriptional response of mouse BMDMs. Analysis of correlation networks generated from the data has identified subnetworks or modules within the macrophage transcriptional network that are activated selectively by these variables. We have identified mouse strain-specific signatures, including a module enriched for SLE susceptibility candidates. In the modules of genes unique to different treatments, we found a module of genes induced by type-I IFN but not by LPS treatment, suggesting another layer of complexity in the LPS-TLR4 signaling feedback control. We also observe that the activation of the complement system, in common with the known activation of MHC class 2 genes, is reliant on IFN-γ signaling. Taken together, these data further highlight the exquisite nature of the regulatory systems that control macrophage activation, their likely relevance to disease resistance/susceptibility, and the appropriate response of these cells to proinflammatory stimuli., (© 2014 Society for Leukocyte Biology.)

Macrophages contribute to innate and acquired immunity as well as many aspects of homeostasis and development. Studies of macrophage biology and function in birds have been hampered by a lack of definitive cell surface markers. As in mammals, avian macrophages proliferate and differentiate in response to CSF1 and IL34, acting through the shared receptor, CSF1R. CSF1R mRNA expression in the chicken is restricted to macrophages and their progenitors. To expedite studies of avian macrophage biology, we produced an avian CSF1R-Fc chimeric protein and generated a monoclonal antibody (designated ROS-AV170) against the chicken CSF1R using the chimeric protein as immunogen. Specific binding of ROS-AV170 to CSF1R was confirmed by FACS, ELISA and immunohistochemistry on tissue sections. CSF1 down-regulated cell surface expression of the CSF1R detected with ROS-AV170, but the antibody did not block CSF1 signalling. Expression of CSF1R was detected on the surface of bone marrow progenitors only after culture in the absence of CSF1, and was induced during macrophage differentiation. Constitutive surface expression of CSF1R distinguished monocytes from other myeloid cells, including heterophils and thrombocytes. This antibody will therefore be of considerable utility for the study of chicken macrophage biology., (Copyright © 2013. Published by Elsevier Ltd.)

Macrophages (MΦs) colonize tissues during inflammation in two distinct ways: recruitment of monocyte precursors and proliferation of resident cells. We recently revealed a major role for IL-4 in the proliferative expansion of resident MΦs during a Th2-biased tissue nematode infection. We now show that proliferation of MΦs during intestinal as well as tissue nematode infection is restricted to sites of IL-4 production and requires MΦ-intrinsic IL-4R signaling. However, both IL-4Rα-dependent and -independent mechanisms contributed to MΦ proliferation during nematode infections. IL-4R-independent proliferation was controlled by a rise in local CSF-1 levels, but IL-4Rα expression conferred a competitive advantage with higher and more sustained proliferation and increased accumulation of IL-4Rα(+) compared with IL-4Rα(-) cells. Mechanistically, this occurred by conversion of IL-4Rα(+) MΦs from a CSF-1-dependent to -independent program of proliferation. Thus, IL-4 increases the relative density of tissue MΦs by overcoming the constraints mediated by the availability of CSF-1. Finally, although both elevated CSF1R and IL-4Rα signaling triggered proliferation above homeostatic levels, only CSF-1 led to the recruitment of monocytes and neutrophils. Thus, the IL-4 pathway of proliferation may have developed as an alternative to CSF-1 to increase resident MΦ numbers without coincident monocyte recruitment.

Broad-spectrum inhibitors of histone deacetylases (HDACs) constrain Toll-like receptor (TLR)-inducible production of key proinflammatory mediators. Here we investigated HDAC-dependent inflammatory responses in mouse macrophages. Of the classical Hdacs, Hdac7 was expressed at elevated levels in inflammatory macrophages (thioglycollate-elicited peritoneal macrophages) as compared with bone marrow-derived macrophages and the RAW264 cell line. Overexpression of a specific, alternatively spliced isoform of Hdac7 lacking the N-terminal 22 amino acids (Hdac7-u), but not the Refseq Hdac7 (Hdac7-s), promoted LPS-inducible expression of Hdac-dependent genes (Edn1, Il-12p40, and Il-6) in RAW264 cells. A novel class IIa-selective HDAC inhibitor reduced recombinant human HDAC7 enzyme activity as well as TLR-induced production of inflammatory mediators in thioglycollate-elicited peritoneal macrophages. Both LPS and Hdac7-u up-regulated the activity of the Edn1 promoter in an HDAC-dependent fashion in RAW264 cells. A hypoxia-inducible factor (HIF) 1 binding site in this promoter was required for HDAC-dependent TLR-inducible promoter activity and for Hdac7- and HIF-1α-mediated trans-activation. Coimmunoprecipitation assays showed that both Hdac7-u and Hdac7-s interacted with HIF-1α, whereas only Hdac7-s interacted with the transcriptional repressor CtBP1. Thus, Hdac7-u positively regulates HIF-1α-dependent TLR signaling in macrophages, whereas an interaction with CtBP1 likely prevents Hdac7-s from exerting this effect. Hdac7 may represent a potential inflammatory disease target.

PTPRJ/CD148 is a tyrosine phosphatase that has tumour suppressor-like activity. Quantitative PCR of various cells and tissues revealed that it is preferentially expressed in macrophage-enriched tissues. Within lymphoid tissues immunohistochemistry revealed that PTPRJ/CD148 co-localised with F4/80, indicating that macrophages most strongly express the protein. Macrophages express the highest basal level of ptprj, and this is elevated further by treatment with LPS and other Toll-like receptor ligands. In contrast, CSF-1 treatment reduced basal and stimulated Ptprj expression in human and mouse cells, and interferon also repressed Ptprj expression. We identified a 1006 nucleotide long noncoding RNA species, Ptprj-as1 that is transcribed antisense to Ptprj. Ptprj-as1 was highly expressed in macrophage-enriched tissue and was transiently induced by Toll-like receptor ligands with a similar time course to Ptprj. Finally, putative transcription factor binding sites in the promoter region of Ptprj were identified.

Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, contribute to tissue integrity as well as to innate and adaptive immune defense. Emerging evidence for labor division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organization of this cellular network are not well defined. Here we report a fate-mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX(3)CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue-resident macrophage populations, including liver Kupffer cells and lung alveolar, splenic, and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that short-lived Ly6C(+) monocytes constitute obligatory steady-state precursors of blood-resident Ly6C(-) cells and that the abundance of Ly6C(+) blood monocytes dynamically controls the circulation lifespan of their progeny., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Monocytes and macrophages differentiate from progenitor cells under the influence of colony-stimulating factors. Genome-scale data have enabled the identification of the set of genes that distinguishes macrophages from other cell types and the ways in which thousands of genes are regulated in response to pathogen challenge. Although there has been a focus on a small subset of lineage-enriched transcription factors, such as PU.1, more than one-half of the transcription factors in the genome can be expressed in macrophage lineage cells under some state of activation, and they interact in a complex network. The network architecture is conserved across species, but many of the target genes evolve rapidly and differ between mouse and human. The data and publication deluge related to macrophage biology require the development of new analytical tools and ways of presenting information in an accessible form.

Evolutionary change in gene expression is generally considered to be a major driver of phenotypic differences between species. We investigated innate immune diversification by analyzing interspecies differences in the transcriptional responses of primary human and mouse macrophages to the Toll-like receptor (TLR)-4 agonist lipopolysaccharide (LPS). By using a custom platform permitting cross-species interrogation coupled with deep sequencing of mRNA 5' ends, we identified extensive divergence in LPS-regulated orthologous gene expression between humans and mice (24% of orthologues were identified as "divergently regulated"). We further demonstrate concordant regulation of human-specific LPS target genes in primary pig macrophages. Divergently regulated orthologues were enriched for genes encoding cellular "inputs" such as cell surface receptors (e.g., TLR6, IL-7Rα) and functional "outputs" such as inflammatory cytokines/chemokines (e.g., CCL20, CXCL13). Conversely, intracellular signaling components linking inputs to outputs were typically concordantly regulated. Functional consequences of divergent gene regulation were confirmed by showing LPS pretreatment boosts subsequent TLR6 responses in mouse but not human macrophages, in keeping with mouse-specific TLR6 induction. Divergently regulated genes were associated with a large dynamic range of gene expression, and specific promoter architectural features (TATA box enrichment, CpG island depletion). Surprisingly, regulatory divergence was also associated with enhanced interspecies promoter conservation. Thus, the genes controlled by complex, highly conserved promoters that facilitate dynamic regulation are also the most susceptible to evolutionary change.

Mouse bone marrow-derived macrophages (BMDM) grown in M-CSF (CSF-1) have been used widely in studies of macrophage biology and the response to TLR agonists. We investigated whether similar cells could be derived from the domestic pig using human rCSF-1 and whether porcine macrophages might represent a better model of human macrophage biology. Cultivation of pig bone marrow cells for 5-7 d in presence of human rCSF-1 generated a pure population of BMDM that expressed the usual macrophage markers (CD14, CD16, and CD172a), were potent phagocytic cells, and produced TNF in response to LPS. Pig BMDM could be generated from bone marrow cells that had been stored frozen and thawed so that multiple experiments can be performed on samples from a single animal. Gene expression in pig BMDM from outbred animals responding to LPS was profiled using Affymetrix microarrays. The temporal cascade of inducible and repressible genes more closely resembled the known responses of human than mouse macrophages, sharing with humans the regulation of genes involved in tryptophan metabolism (IDO, KYN), lymphoattractant chemokines (CCL20, CXCL9, CXCL11, CXCL13), and the vitamin D3-converting enzyme, Cyp27B1. Conversely, in common with published studies of human macrophages, pig BMDM did not strongly induce genes involved in arginine metabolism, nor did they produce NO. These results establish pig BMDM as an alternative tractable model for the study of macrophage transcriptional control.

Macrophages play a major role in tissue remodelling during development, wound healing and tissue homeostasis, and are central to innate immunity and to the pathology of tissue injury and inflammation. Given this fundamental role in many aspects of biological function, an enormous wealth of information has accumulated on these fascinating cells in the literature and other public repositories. With the escalation of genome-scale data derived from macrophages and related haematopoietic cell types, there is a growing need for an integrated resource that seeks to compile, organise and analyse our collective knowledge of macrophage biology. Here we describe a community-driven web-based resource, macrophages.com that aims to provide a portal onto various types of Omics data to facilitate comparative genomic studies, promoter and transcriptional network analyses, models of macrophage pathways together with other information on these cells. To this end, the website combines public and in-house analyses of expression data with pre-analysed views of co-expressed genes as supported by the network analysis tool BioLayout Express(3D), as well as providing access to maps of pathways active in macrophages. Macrophages.com also provides access to an extensive image library of macrophages in adult/embryonic tissue sections prepared from normal and transgenic mice. In addition, the site links to the Human Protein Atlas database so as to provide direct access to protein expression patterns in human macrophages. Finally, an integrated gene-centric portal provides the tools for rapid promoter analysis studies based on a comprehensive set of CAGE-derived transcription start site (TSS) sequences in human and mouse genomes as generated by the Functional Annotation of Mammalian genomes (FANTOM) projects initiated by the RIKEN Omics Science Center. Our aim is to continue to grow the macrophages.com resource using publicly available data, as well as in-house generated knowledge. In so doing we aim to provide a user-friendly community website and a community portal for access to comprehensive sets of macrophage-related data., (Crown Copyright © 2011. Published by Elsevier GmbH. All rights reserved.)

The murine mononuclear phagocyte (MNP) system comprises a diverse population of cells, including monocytes, dendritic cells (DC) and macrophages. Derived from the myeloid haematopoietic lineage, this group of cells express a variety of well characterized surface markers. Expression of the integrin alpha X (Itgax, CD11c) is commonly used to identify classical DC, and similarly expression of colony stimulating factor 1 receptor (Csf1r, CD115) to identify macrophages. We have characterized the expression of these markers using a variety of transgenic mouse models. We confirmed previous observations of Itgax expression in anatomically defined subsets of MNPs in secondary lymphoid organs, including all MNPs identified within the germinal centres. The majority of MNPs in the intestinal lamina propria and lung express Itgax. All mucosal Itgax expressing cells also express Csf1r suggesting Csf1-dependent haematopoietic derivation. This double-positive population included germinal centre MNPs. These data reveal that Itgax expression alone does not specifically define classical DC. These results suggest more cautious interpretation of Itgax-dependent experimentation and direct equation with uniquely DC-mediated activities, particularly in the functioning of non-lymphoid MNPs within the intestinal lamina propria., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Colony-stimulating factor (CSF)-1 controls the survival, proliferation, and differentiation of macrophages, which are recognized as scavengers and agents of the innate and the acquired immune systems. Because of their plasticity, macrophages are endowed with many other essential roles during development and tissue homeostasis. We present evidence that CSF-1 plays an important trophic role in postnatal organ growth and kidney repair. Notably, the injection of CSF-1 postnatally enhanced kidney weight and volume and was associated with increased numbers of tissue macrophages. Moreover, CSF-1 promotes postnatal renal repair in mice after ischemia-reperfusion injury by recruiting and influencing macrophages toward a reparative state. CSF-1 treatment rapidly accelerated renal repair with tubular epithelial cell replacement, attenuation of interstitial fibrosis, and functional recovery. Analysis of macrophages from CSF-1-treated kidneys showed increased expression of insulin-like growth factor-1 and anti-inflammatory genes that are known CSF-1 targets. Taken together, these data suggest that CSF-1 is important in kidney growth and the promotion of endogenous repair and resolution of inflammatory injury., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)