Abstract
Macrophages are a heterogeneous cell population playing a pivotal role in tissue homeostasis and inflammation, and their phenotype strongly depends on the micromilieu. Despite its increasing importance as a translational animal model for human diseases, there is a considerable gap of knowledge with respect to macrophage polarization in dogs. The present study comprehensively investigated the morphologic, phenotypic, and transcriptomic characteristics of unstimulated (M0), M1- (GM-CSF, LPS, IFNγ-stimulated) and M2- (M-CSF, IL-4-stimulated)-polarized canine blood-derived macrophages in vitro. Scanning electron microscopy revealed distinct morphologies of polarized macrophages with formation of multinucleated cells in M2-macrophages, while immunofluorescence employing literature-based prototype-antibodies against CD16, CD32, iNOS, MHC class II (M1-markers), CD163, CD206, and arginase-1 (M2-markers) demonstrated that only CD206 was able to discriminate M2-macrophages from both other phenotypes, highlighting this molecule as a promising marker for canine M2-macrophages. Global microarray analysis revealed profound changes in the transcriptome of polarized canine macrophages. Functional analysis pointed out that M1-polarization was associated with biological processes such as “respiratory burst”, whereas M2-polarization was associated with processes such as “mitosis”. Literature-based marker gene selection revealed only minor overlaps in the gene sets of the dog compared to prototype markers of murine and human macrophages. Biomarker selection using supervised clustering suggested latexin (LXN) and membrane-spanning 4-domains, subfamily A, member 2 (MS4A2) to be the most powerful predicting biomarkers for canine M1- and M2-macrophages, respectively. Immunofluorescence for both markers demonstrated expression of both proteins by macrophages in vitro but failed to reveal differences between canine M1 and M2-macrophages. The present study provides a solid basis for future studies upon the role of macrophage polarization in spontaneous diseases of the dog, a species that has emerging importance for translational research.
Highlights
Circulating peripheral blood mononuclear cells (PBMCs) play an important role during both the steady state and inflammation
T tests revealed that the number of M2-macrophages was significantly higher than M0-macrophages (p = 0.02), while comparisons between M0and M1-macrophages and M1-and M2-macrophages failed to reach the level of significance (p = 0.30 and p = 0.06, respectively)
Even though in vitro data on macrophage polarization doubtlessly cannot be extrapolated to in vivo situations and the paradigm of M1-/M2-polarization represents a simplified approach, which depicts only two extremes of macrophage heterogeneity [8, 48,49,50], this initial reductionistic in vitro approach will provide a basis for future investigations on the role of macrophage polarization in both healthy and diseased dogs
Summary
Circulating peripheral blood mononuclear cells (PBMCs) play an important role during both the steady state and inflammation. Functional diversity and plasticity are hallmarks of macrophages [2, 3] Together they represent a heterogeneous cell population of the mononuclear phagocyte system playing a pivotal role in tissue homeostasis, inflammation, host defense, and tissue repair [4, 5]. Activated M1-macrophages develop after exposure to proinflammatory stimuli such as interferon γ (IFNγ), lipopolysaccharide (LPS), or tumor necrosis factor (TNF). Subsequent to such stimulation, M1-macrophages release pro-inflammatory cytokines, reactive oxygen species (ROS), and nitric oxide (NO) [8]. Gene expression profiling has been applied as a sophisticated technique to detect the underlying molecular mechanisms following macrophage activation in murine and human cells [11,12,13,14]. Current comparative studies strongly support the observation of marked interspecies differences and variability between mice and humans indicating that about 50% of polarization specific markers are selectively expressed in only one of both species [13, 14]
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