Csf1r-mApple transgene expression and ligand binding in vivo reveal dynamics of CSF1R expression within the mononuclear phagocyte system

CSF1 is the primary growth factor controlling macrophage numbers, but whether expression of the CSF1 receptor differs between discrete populations of mononuclear phagocytes remains unclear. We have generated a Csf1r-mApple transgenic fluorescent reporter mouse that, in combination with lineage tracing, Alexa Fluor 647-labeled CSF1-Fc and CSF1, and a modified Delta Csf1-enhanced cyan fluorescent protein (ECFP) transgene that lacks a 150 bp segment of the distal promoter, we have used to dissect the differentiation and CSF1 responsiveness of mononuclear phagocyte populations in situ. Consistent with previous Csf1r-driven reporter lines, Csf1r-mApple was expressed in blood monocytes and at higher levels in tissue macrophages, and was readily detectable in whole mounts or with multiphoton microscopy. In the liver and peritoneal cavity, uptake of labeled CSF1 largely reflected transgene expression, with greater receptor activity in mature macrophages than monocytes and tissue-specific expression in conventional dendritic cells. However, CSF1 uptake also differed between subsets of monocytes and discrete populations of tissue macrophages, which in macrophages correlated with their level of dependence on CSF1 receptor signaling for survival rather than degree of transgene expression. A double Delta Csf1r-ECFP-Csf1r-mApple transgenic mouse distinguished subpopulations of microglia in the brain, and permitted imaging of interstitial macrophages distinct from alveolar macrophages, and pulmonary monocytes and conventional dendritic cells. The Csf1r-mApple mice and fluorescently labeled CSF1 will be valuable resources for the study of macrophage and CSF1 biology, which are compatible with existing EGFP-based reporter lines

Species-Specific Transcriptional Regulation of Genes Involved in Nitric Oxide Production and Arginine Metabolism in Macrophages

Activated mouse macrophages metabolize arginine via NO synthase (NOS2) to produce NO as an antimicrobial effector. Published gene expression datasets provide little support for the activation of this pathway in human macrophages. Generation of NO requires the coordinated regulation of multiple genes. We have generated RNA-sequencing data from bone marrow–derived macrophages from representative rodent (rat), monogastric (pig and horse), and ruminant (sheep, goat, cattle, and water buffalo) species, and analyzed the expression of genes involved in arginine metabolism in response to stimulation with LPS. In rats, as in mice, LPS strongly induced Nos2, the arginine transporter Slc7a2, arginase 1 (Arg1), GTP cyclohydrolase (Gch1), and argininosuccinate synthase (Ass1). None of these responses was conserved across species. Only cattle and water buffalo showed substantial NOS2 induction. The species studied also differed in expression and regulation of arginase (ARG2, rather than ARG1), and amino acid transporters. Variation between species was associated with rapid promoter evolution. Differential induction of NOS2 and ARG2 between the ruminant species was associated with insertions of the Bov-A2 retrotransposon in the promoter region. Bov-A2 was shown to possess LPS-inducible enhancer activity in transfected RAW264.7 macrophages. Consistent with a function in innate immunity, NO production and arginine metabolism vary greatly between species and differences may contribute to pathogen host restriction

Macrophage colony-stimulating factor (CSF1) controls monocyte production and maturation and the steady-state size of the liver in pigs

Macrophage colony-stimulating factor (CSF1) is an essential growth and differentiation factor for cells of the macrophage lineage. To explore the role of CSF1 in steady-state control of monocyte production and differentiation and tissue repair, we previously developed a bioactive protein with a longer half-life in circulation by fusing pig CSF1 with the Fc region of pig IgG1a. CSF1-Fc administration to pigs expanded progenitor pools in the marrow and selectively increased monocyte numbers and their expression of the maturation marker CD163. There was a rapid increase in the size of the liver, and extensive proliferation of hepatocytes associated with increased macrophage infiltration. Despite the large influx of macrophages, there was no evidence of liver injury and no increase in circulating liver enzymes. Microarray expression profiling of livers identified increased expression of macrophage markers, i.e., cytokines such as TNF, IL1, and IL6 known to influence hepatocyte proliferation, alongside cell cycle genes. The analysis also revealed selective enrichment of genes associated with portal, as opposed to centrilobular regions, as seen in hepatic regeneration. Combined with earlier data from the mouse, this study supports the existence of a CSF1-dependent feedback loop, linking macrophages of the liver with bone marrow and blood monocytes, to mediate homeostatic control of the size of the liver. The results also provide evidence of safety and efficacy for possible clinical applications of CSF1-Fc

A Csf1r-EGFP transgene provides a novel marker for monocyte subsets in sheep

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

Targeting the macrophage in equine post-operative ileus

Post-operative ileus (POI) is the functional inhibition of propulsive intestinal motility which is a frequent occurrence following abdominal surgery in the horse and in humans. Rodent and human-derived data have shown that manipulation-induced activation of the resident muscularis externa (ME) macrophages in the intestine contributes to the pathophysiology of the disease. Most studies of the disease, specifically in the horse, have focussed on identification of risk factors, descriptive studies of the disease or the assessment of the efficacy of various therapeutic and prophylactic interventions. As a result, the proposed pathogenesis of equine POI is largely reliant on the translation of data from rodent models. The aims of this thesis were to identify macrophage populations in the normal equine gastrointestinal tract (GIT) and to study equine macrophage activation by stimulating equine bone marrow-derived macrophages (eqBMDMs) with lipopolysaccharide (LPS) as a model for intestinal macrophage activation. Firstly, the normal population of macrophages in the equine GIT was determined. Using CD163 as an immunohistochemical marker for macrophages. CD163+ve cells were present in all tissue layers of the equine intestine: mucosa, submucosa, ME and serosa. CD163+ve cells were regularly distributed within the ME, with accumulations adjacent to the myenteric plexus, and therefore to intestinal motility effector cells such as neurons and the Interstitial Cells of Cajal. The differentiation and survival of intestinal macrophages depends upon signals from the macrophage colony-stimulating factor (CSF-1) receptor. LPS translocation from the gut lumen is thought to be a key activator of ME macrophages. To provide a model for gut macrophages, a protocol was optimised to produce pure populations of equine bone marrow-derived macrophages (eqBMDMs) by cultivation of equine bone marrow in CSF-1. Macrophage functionality was assessed using microscopy, flow cytometry and phagocytosis assays. EqBMDMs responded to LPS stimulation with increases in expression of positive control genes, tumour necrosis factor alpha (TNF-α) and Indoleamine 2,3-dioxygenase (IDO1). The same mRNA was subjected to transcriptomic (RNA-Seq) analysis. Differential gene expression and network cluster analysis demonstrated an inflammatory response characterised by the production of pro-inflammatory cytokines such as interleukin 1 beta (IL-1β) and interleukin 6 (IL-6). However, in contrast to rodent macrophages, eqBMDMs failed to produce nitric oxide in response to LPS, showing species-specific variation in innate immune biology. Using these data, we compared gene expression in normal equine intestine and in intestine from horses undergoing abdominal surgery for colic (abdominal pain). Horses undergoing abdominal surgery showed evidence of increased expression of IL-1β, IL-6 and TNF-α in the mucosa and ME when compared to control tissue. Horses with post-operative reflux (POR), a clinical sign of POI, had increased gene expression of IL-1β, IL-6 and TNF-α compared to horses that did not develop POR following abdominal surgery. These preliminary data suggest that there is macrophage activation within the ME of the intestine during abdominal surgery in the horse, and that a greater activation state is present in horses that subsequently develop POR. The final part of this study was to investigate the effect of a long-acting form of CSF- 1, an Fc fusion protein (CSF1-Fc), as a potential treatment for POI using a mouse model. This work, performed in collaboration with another research group, found that mice lacking the C-C chemokine receptor type 2 (CCR2) gene, which is required for monocyte recruitment into tissues, had a longer recovery period following intestinal manipulation (IM) than wild type (WT) mice. With the administration of CSF1-Fc, infiltration of neutrophils to the ME was reduced and the number of macrophages in the ME was increased in both WT and CCR2-/- mice following IM. Administration of CSF1-Fc in CCR2-/- mice improved recovery of gastrointestinal transit three days following IM, to the same extent as WT mice. Network cluster analysis and RT-qPCR of the ME revealed clusters of genes induced and downregulated by CSF1-Fc, with increased expression of anti-inflammatory and pro-resolving genes after IM in WT and CCR2-/- mice following treatment with CSF1-Fc

Erratum: Pleiotropic impacts of macrophage ands microglial deficiency on development in rats with targeted mutation of the Csf1r locus (Journal of Immunology (2019) 201 (2683-2699) DOI: 10.4049/jimmunol.1701783)

In the analysis of gene expression in the brain, the sample labels for the pituitary gland and olfactory bulb were reversed. As a consequence, the clusters in Fig. 10B were mislabeled. A corrected version of Fig. 10 is shown below, along with the corrected figure legend. These corrections have been made to the online version of the article, which now differs from the print version as originally published. A corrected version of Supplemental Table I has also been published online. The current online supplemental material therefore differs from what was originally published online. The relevant text in the Results section under the heading “Network analysis of gene expression in the brain of Csf1r-deficient rats” now reads as follows: Fig. 10B shows the network graph for the combined analysis of the four brain regions; gene lists are provided in Supplemental Table I. The largest cluster was cluster 1 (4283 genes), containing pituitary gland-associated genes. Other region-specific clusters were cluster 3 (olfactory bulb; 878 genes), cluster 5 (hippocampus; 513 genes), and cluster 7 (striatum; 384 genes). The second largest cluster (cluster 2; 1253 genes) contained genes that were more highly expressed in both hippocampus and striatum. Two other main clusters also shared highly expressed genes between tissues: cluster 4 (olfactory bulb, striatum, and hippocampus; 757 genes) and cluster 6 (pituitary gland and olfactory bulb; 471 genes). None of these clusters showed any evidence of genotype association. (Figure Presented)

Deletion of a Csf1r enhancer selectively impacts CSF1R expression and development of tissue macrophage populations.

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. Csf1rΔFIRE/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, Csf1rΔFIRE/ΔFIRE mice are healthy and fertile without the growth, neurological or developmental abnormalities reported in Csf1r-/- rodents. Csf1rΔFIRE/ΔFIRE mice thus provide a model to explore the homeostatic, physiological and immunological functions of tissue-specific macrophage populations in adult animals