Analysis of the impact of CSF-1 administration in adult rats using a novel Csf1r-mApple reporter gene.

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.

Irvine KM ，Caruso M ，Cestari MF ，Davis GM ，Keshvari S ，Sehgal A ，Pridans C ，Hume DA ... -  《-》

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 ΔCsf1-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 ΔCsf1r-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.

Hawley CA ，Rojo R ，Raper A ，Sauter KA ，Lisowski ZM ，Grabert K ，Bain CC ，Davis GM ，Louwe PA ，Ostrowski MC ，Hume DA ，Pridans C ，Jenkins SJ ... -  《-》

A Transgenic Line That Reports CSF1R Protein Expression Provides a Definitive Marker for the Mouse Mononuclear Phagocyte System.

The proliferation, differentiation, and survival of cells of the mononuclear phagocyte system (MPS; progenitors, monocytes, macrophages, and classical dendritic cells) are controlled by signals from the M-CSF receptor (CSF1R). Cells of the MPS lineage have been identified using numerous surface markers and transgenic reporters, but none is both universal and lineage restricted. In this article, we report the development and characterization of a CSF1R reporter mouse. A FusionRed (FRed) cassette was inserted in-frame with the C terminus of CSF1R, separated by a T2A-cleavable linker. The insertion had no effect of CSF1R expression or function. CSF1R-FRed was expressed in monocytes and macrophages and absent from granulocytes and lymphocytes. In bone marrow, CSF1R-FRed was absent in lineage-negative hematopoietic stem cells, arguing against a direct role for CSF1R in myeloid lineage commitment. It was highly expressed in marrow monocytes and common myeloid progenitors but significantly lower in granulocyte-macrophage progenitors. In sections of bone marrow, CSF1R-FRed was also detected in osteoclasts, CD169+ resident macrophages, and, consistent with previous mRNA analysis, in megakaryocytes. In lymphoid tissues, CSF1R-FRed highlighted diverse MPS populations, including classical dendritic cells. Whole mount imaging of nonlymphoid tissues in mice with combined CSF1R-FRed/Csf1r-EGFP confirmed the restriction of CSF1R expression to MPS cells. The two markers highlight the remarkable abundance and regular distribution of tissue MPS cells, including novel macrophage populations within tendon and skeletal muscle and underlying the mesothelial/serosal/capsular surfaces of every major organ. The CSF1R-FRed mouse provides a novel reporter with exquisite specificity for cells of the MPS.

Grabert K ，Sehgal A ，Irvine KM ，Wollscheid-Lengeling E ，Ozdemir DD ，Stables J ，Luke GA ，Ryan MD ，Adamson A ，Humphreys NE ，Sandrock CJ ，Rojo R ，Verkasalo VA ，Mueller W ，Hohenstein P ，Pettit AR ，Pridans C ，Hume DA ... -  《-》

The MacBlue binary transgene (csf1r-gal4VP16/UAS-ECFP) provides a novel marker for visualisation of subsets of monocytes, macrophages and dendritic cells and responsiveness to CSF1 administration.

Sauter KA ，Pridans C ，Sehgal A ，Bain CC ，Scott C ，Moffat L ，Rojo R ，Stutchfield BM ，Davies CL ，Donaldson DS ，Renault K ，McColl BW ，Mowat AM ，Serrels A ，Frame MC ，Mabbott NA ，Hume DA ... -  《PLoS One》

Protein kinase A inhibition of macrophage maturation is accompanied by an increase in DNA methylation of the colony-stimulating factor 1 receptor gene.

Macrophage colony-stimulating factor 1 (CSF-1) plays a critical role in the differentiation of mononuclear phagocytes from bone marrow precursors, and maturing monocytes and macrophages exhibit increased expression of the CSF-1 receptor, CSF1R. The expression of CSF1R is tightly regulated by transcription factors and epigenetic mechanisms. We previously showed that prostaglandin E2 and subsequent activation of protein kinase A (PKA) inhibited CSF1R expression and macrophage maturation. Here, we examine the DNA methylation changes that occur at the Csf1r locus during macrophage maturation in the presence or absence of activated PKA. Murine bone marrow cells were matured to macrophages by incubating cells with CSF-1-containing conditioned medium for up to 6 days in the presence or absence of the PKA agonist 6-bnz-cAMP. DNA methylation of Csf1r promoter and enhancer regions was assayed by bisulphite pyrosequencing. DNA methylation of Csf1r decreased during normal macrophage maturation in concert with an increase in Csf1r mRNA expression. Treatment with the PKA agonist inhibited Csf1r mRNA and protein expression, and increased DNA methylation at the Csf1r promoter. This was associated with decreased binding of the transcription factor PU.1 to the Csf1r promoter. Treatment with the PKA agonist inhibited the responsiveness of macrophages to CSF-1. Levels of endogenous PKA activity decreased during normal macrophage maturation, suggesting that attenuation of this signalling pathway contributes to the increase in CSF1R expression during macrophage maturation. Together, these results demonstrate that macrophage maturation is accompanied by Csf1r hypomethylation, and illustrates for the first time the ability of PKA to increase Csf1r DNA methylation.

Zasłona Z ，Scruggs AM ，Peters-Golden M ，Huang SK ... -  《-》