An efficient method to isolate Kupffer cells eliminating endothelial cell contamination and selective bias.

Multicolor flow cytometry and cell sorting are powerful immunologic tools for the study of hepatic mϕ, yet there is no consensus on the optimal method to prepare liver homogenates for these analyses. Using a combination of mϕ and endothelial cell reporter mice, flow cytometry, and confocal imaging, we have shown that conventional flow-cytometric strategies for identification of Kupffer cells (KCs) leads to inclusion of a significant proportion of CD31hi endothelial cells. These cells were present regardless of the method used to prepare cells for flow cytometry and represented endothelium tightly adhered to remnants of KC membrane. Antibodies to endothelial markers, such as CD31, were vital for their exclusion. This result brings into focus recently published microarray datasets that identify high expression of endothelial cell-associated genes by KCs compared with other tissue-resident mϕ. Our studies also revealed significant and specific loss of KCs among leukocytes with commonly used isolation methods that led to enrichment of proliferating and monocyte-derived mϕ. Hence, we present an optimal method to generate high yields of liver myeloid cells without bias for cell type or contamination with endothelial cells.

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.

Long-lived self-renewing bone marrow-derived macrophages displace embryo-derived cells to inhabit adult serous cavities.

Peritoneal macrophages are one of the most studied macrophage populations in the body, yet the composition, developmental origin and mechanisms governing the maintenance of this compartment are controversial. Here we show resident F4/80(hi)GATA6(+) macrophages are long-lived, undergo non-stochastic self-renewal and retain cells of embryonic origin for at least 4 months in mice. However, Ly6C(+) monocytes constitutively enter the peritoneal cavity in a CCR2-dependent manner, where they mature into short-lived F4/80(lo)MHCII(+) cells that act, in part, as precursors of F4/80(hi)GATA6(+) macrophages. Notably, monocyte-derived F4/80(hi) macrophages eventually displace the embryonic population with age in a process that is highly gender dependent and not due to proliferative exhaustion of the incumbent embryonic population, despite the greater proliferative activity of newly recruited cells. Furthermore, although monocyte-derived cells acquire key characteristics of the embryonic population, expression of Tim4 was impaired, leading to cumulative changes in the population with age.

CSF1 Restores Innate Immunity After Liver Injury in Mice and Serum Levels Indicate Outcomes of Patients With Acute Liver Failure.

BACKGROUND & AIMS: Liver regeneration requires functional liver macrophages, which provide an immune barrier that is compromised after liver injury. The numbers of liver macrophages are controlled by macrophage colony-stimulating factor (CSF1). We examined the prognostic significance of the serum level of CSF1 in patients with acute liver injury and studied its effects in mice. METHODS: We measured levels of CSF1 in serum samples collected from 55 patients who underwent partial hepatectomy at the Royal Infirmary Edinburgh between December 2012 and October 2013, as well as from 78 patients with acetaminophen-induced acute liver failure admitted to the Royal Infirmary Edinburgh or the University of Kansas Medical Centre. We studied the effects of increased levels of CSF1 in uninjured mice that express wild-type CSF1 receptor or a constitutive or inducible CSF1-receptor reporter, as well as in chemokine receptor 2 (Ccr2)-/- mice; we performed fate-tracing experiments using bone marrow chimeras. We administered CSF1-Fc (fragment, crystallizable) to mice after partial hepatectomy and acetaminophen intoxication, and measured regenerative parameters and innate immunity by clearance of fluorescent microbeads and bacterial particles. RESULTS: Serum levels of CSF1 increased in patients undergoing liver surgery in proportion to the extent of liver resected. In patients with acetaminophen-induced acute liver failure, a low serum level of CSF1 was associated with increased mortality. In mice, administration of CSF1-Fc promoted hepatic macrophage accumulation via proliferation of resident macrophages and recruitment of monocytes. CSF1-Fc also promoted transdifferentiation of infiltrating monocytes into cells with a hepatic macrophage phenotype. CSF1-Fc increased innate immunity in mice after partial hepatectomy or acetaminophen-induced injury, with resident hepatic macrophage as the main effector cells. CONCLUSIONS: Serum CSF1 appears to be a prognostic marker for patients with acute liver injury. CSF1 might be developed as a therapeutic agent to restore innate immune function after liver injury.