Table 1.
List of antibodies used for immunofluorescence.
Fig 1.
Polarization-dependent morphological differences in canine M0-, M1-, and M2-macrophage cultures.
A) In scanning electron microscopy, unstimulated macrophages (M0; day 7) obtain a small and roundish morphology, lacking cytoplasmic extensions. B) M1-treated macrophages (day 7) are characterized by an enlarged amoeboid cell shape with roundish cell bodies and numerous delicate cytoplasmic extensions on the cellular surface. C; D) M2-treated macrophage cultures (day 7) demonstrate a marked heterogeneity with two dominating cell types. Large “spindeloid” macrophages with an elongated cell body and cytoplasmic extensions on the apical ends of the cell bodies (C). Second, in M2-cultures, numerous multinucleated giant cells (MNGs) with abundant cytoplasmic projections on the cellular surface are present (D). E-H) Dot plot diagrams depicting the morphological changes of macrophage cultures (n = 3) following stimulation (M0, M1, M2) over the time course as calculated with a mixed ANOVA with post-hoc alpha adjustment (Tukey-Kramer) and significance level at p≤0.05 (asterisks). E) Small/roundish macrophages dominate in untreated M0 cell cultures and their relative percentage is significantly higher in M0 when compared to M2 at days 5 and 7. F) Amoeboid macrophages display a statistical trend of predominance in M1 compared to M0 at day 5 and 7. G) The relative percentage of spindeloid macrophages is significantly increased in M2 at day 5 compared to M1. H) MNGs are almost exclusively observed in M2-macrophages at the end of the culturing period and their percentage is significantly higher at day 7 in M2 compared to both M0 and M1.
Fig 2.
Immunofluorescence staining of in vitro cultured canine M0-, M1-, and M2-macrophages labeled with prototypic literature-based antibodies for the M1- (CD16, CD32, MHC class II, and iNOS) and M2-phenotype (CD163, CD206, and arginase 1), respectively.
A-C) Low to moderate membranous staining of M0-, M1-, and M2-macrophages for CD16. D-F) Likewise, CD32 shows a low to moderate staining in all three treatment conditions. G-I) M0-, M1-, and M2-macrophages demonstrate a moderate to high membranous staining with CD163. J-L) Intense membranous staining of M0-, M1-, and M2-macrophages with an anti-MHC class II antibody. M-O) Strong intracytoplasmic labeling of macrophages in all treatments (polarity 0, 1, 2) for inducible nitric oxide synthase (iNOS). P-R) High intracytoplasmic expression of arginase 1 in small/roundish, amoeboid, and spindeloid macrophages as well as in multinucleated giant cells (MNGs). a-f) Statistical evaluation of the mean expression percentages of prototypic M1-/M2-markers evaluated in 5 dogs and related to the polarization state of the macrophages (polarity 0, 1, 2). Note that, except for MHC class II, none of the remaining tested antigens (CD16, CD32, iNOS, CD163, and arginase-1) were differently expressed between canine M0-, M1-, and M2-macrophages (* = p≤0.05; Kruskal-Wallis-Test with pairwise Mann-Whitney-U-Tests). Scale bars = 100 μm. Nuclear counterstaining with bisbenzimide.
Fig 3.
Phenotypical characterization of canine M0-, M1-, and M2-macrophages.
A) Low membranous expression of CD206 antigen by small/roundish M0-macrophages. B) Moderate membranous staining of amoeboid M1-macrophages for CD206. C) Intense membranous expression of CD206 antigen by M2-macrophages. Scale bars = 100 μm. Nuclear counterstaining with bisbenzimide. D) The mean percentage of CD206-immunopositive cells is significantly higher in M2-macrophages as compared to both M1- and M0-macrophages (* = p≤0.05; Kruskal-Wallis-Test with pair-wise Mann-Whitney-U-Tests). E) The log2-transformed expression values of the probe set encoding for the gene CD206 is similarly significantly higher in M2-macrophages compared to both M1- and M0-macrophages (* = p≤0.05; Kruskal-Wallis-Test with pair-wise Mann-Whitney-U-Tests), thus confirming the results of the immunofluorescence investigation.
Table 2.
Summarized results of the functional annotation of the pairwise comparisons of differentially expressed probe sets (DEPs) in canine M0-, M1-, and M2-macrophages.
Table 3.
List and subgrouping of the top hits of highly differentially expressed genes (fold change ≥ 50 or ≤ -50) in canine M1- vs. M0-macrophages.
Table 4.
List and subgrouping of the top hits of highly differentially expressed genes (fold change ≥ 50 or ≤ -50) in canine M2- vs. M0-macrophages.
Table 5.
List and subgrouping of the top hits of highly differentially expressed genes (fold change ≥ 50 or ≤ -50) in M2- vs. M1-macrophages.
Fig 4.
Hierarchical clustering analysis.
Unsupervised hierarchical clustering analysis of the median centered, log2-transformed expression values of 6358 differentially expressed probe sets in canine polarized macrophages as obtained by TM4 MultiExperimentViewer with default settings (Euclidean distance; complete linkage), depicted on a color scale from red (relatively high expression) to green (relatively low expression). A) The analysis identified 9 distinct clusters (I-IX) based on similarities as well as differences in the expression intensity of canine polarized macrophages. Two out of these 9 clusters (VIII and VI) visually displayed an expression profile that was clearly associated with either the M1- or M2-phenotype. B) Functional annotation of the M1-cluster (magnified from A) using Web-based Gene Set Analysis Toolkit (WebGestalt) identified the enriched biological process category “respiratory burst” (adjusted p-value≤0.05). C) The M2-cluster (magnified from A) is associated with enriched biological GO terms such as “M phase of mitotic cell cycle” and “mitotic spindle organization” (adjusted p-value≤0.05).
Fig 5.
Comparative evaluation of canine M1- and M2-associated differentially expressed genes (M1 = 404; M2 = 700) with established literature-based human and murine orthologous genes (A) and detection of polarization-specific biomarkers employing supervised clustering with a K-nearest-neighbors algorithm and correlation-based feature selection using Prophet (B, C). A) The Venn diagram depicts the numbers and intersections of differential and common canine M1- and M2-genes with literature-based human and murine genes. The majority of literature-based M1- (65 genes = 57+8) and M2-markers (58 genes = 44+11+3) did not comply with the present microarray data upon canine macrophages. However, overlapping expression of 8 genes for the M1-phenotype and 11 genes for the M2-phenotype was identified. The genes of the intersections, not specifically mentioned in the figure, are listed in S4 Table B) Biomarker selection detected 4 highly specific probe sets for annotated by the mammalian carboxypeptidase inhibitor latexin (LXN) to be highly correlated to the M1-phenotype. The boxplots depict the significantly enhanced log2-transformed expression values for LXN in M1-macrophages as compared to both M0- and M2-macrophages (p = 0.002), as well as between M0- and M2-macrophages (p = 0.002). C) For the M2-phenotype, the most significant predicted biomarker was high affinity receptor for IgE, i.e. membrane-spanning 4-domains, subfamily A, member 2 (MS4A2). The expression data of M2-macrophages show significantly higher log2-transformed expression values as compared to both M0- and M1-macrophages (p = 0.002) as well as between M0- and M1-macrophages (p = 0.002). Asterisks indicate significance (Kruskal-Wallis-Test with subsequent pairwise Mann-Whitney-U-Tests).
Table 6.
Top 50 candidate M1-macrophage associated probesets, which were upregulated in M1- vs. M0-macrophages and simultaneously downregulated in M2- vs. M1-macrophages.
Table 7.
Top 50 candidate M2-macrophage associated probesets, which were upregulated in M2- vs. M0-macrophages and simultaneously upregulated in M2- vs. M1-macrophages.
Fig 6.
Protein expression of the predicted biomarkers latexin and MS4A2 in canine M0-, M1-, and M2-macrophages.
A) Expression of latexin in non-stimulated canine M0-macrophages. B) Expression of latexin in canine M1-macrophages. C) Expression of latexin in canine M2-macrophages. D) Expression of MS4A2 in canine M0-macrophages. E) Expression of MS4A2 in canine M1-macrophages. F) Expression of MS4A2 in canine M2-macrophages. A-F) Scale bars = 100 μm. Nuclear counterstaining with bisbenzimide. G) Dot plots illustrating a significantly higher percentage of immunopositive cells in canine M1- and M2-macrophages as compared to M0-macrophages (One-factorial ANOVA with group-wise t-tests, astrerisk = p<0.05). H) Dot plot showing lack of statistical differences in the percentage of immunopositive cells for MS4A2 in canine M0-, M1-, and M2-macrophages.