Figure 1.
Phenotype of M-CSF and GM-CSF MΦ.
(A) Cell culture images and cytospin slides of M-CSF and GM-CSF MΦ on day+7 of culture before polarization. Images of cell cultures and cytospins were taken at a 20X magnification with an EVOS microscope (AMG) and at a 40X magnification using a Olympus BX43 microscope and CellSens 1.7 Software (Olympus), respectively. Adobe Photoshop CS5.1 (Adobe Systems) was used to adjust brightness and contrast. (B) Flow cytometric measurement of surface marker expression depicted as mean fluorescence intensity (MFI) relative to the isotype control in box plots for M-CSF and GM-CSF MΦ (4-5 donors). White bars indicate resting MΦ, grey bars indicate IFN-γ stimulated M1 MΦ, and black bars indicate IL-4+IL13 stimulated M2 MΦ. Box plots represent the median and interquartile range, whiskers indicate the minimum and maximum, respectively. * p < 0.05.
Figure 2.
Expression of M1 and M2 marker genes.
In all these figures resting MΦ expression levels are indicated by the dotted line. (A) M-CSF (white box plots) and GM-CSF MΦ (grey box plots) were analyzed for mRNA expression of M1 genes. Data are expressed as fold differences of polarized MΦ relative to resting MΦ (6-8 donors). (B) Fold expression of M1 genes in GM-CSF MΦ relative to M-CSF MΦ (4 donors). White bars indicate resting MΦ, grey bars indicate IFN-γ stimulated M1 MΦ, and black bars indicate IL-4+IL13 stimulated M2 MΦ. (C) M2 gene expression in polarized MΦ relative to resting MΦ (7-8 donors). (D) Fold expression of M2 genes in GM-CSF MΦ relative M-CSF MΦ (4 donors). Bars are as described above in (B). Box plots represent the median and interquartile range; whiskers indicate the minimum and maximum, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3.
Expression and functional analysis of chromatin modifying enzymes (CME).
In Figures A-C resting MΦ expression levels are indicated by the dotted line. (A) M-CSF (white box plots) and GM-CSF MΦ (grey box plots) were analyzed for mRNA expression of CME. Data are expressed as fold differences of polarized MΦ relative to resting MΦ (6-8 donors). Box plots represent the median and interquartile range, whiskers indicate the minimum and maximum, respectively. (B) Nuclear extracts of M-CSF (white bars) and GM-CSF MΦ (grey bars) were analyzed for H3K4-methyltransferase activity 24h after polarization (3 donors each). Data are expressed as relative absorbance of polarized MΦ relative to resting MΦ. Bars represent the median with range. (C) Genomic DNA of M-CSF (5 donors) and GM-CSF MΦ (4 donors) was analyzed for total DNA methylation 24h after polarization. Total DNA methylation is depicted as absorbance of polarized MΦ as relative to resting MΦ. Bars represent the median with range. (D) M-CSF MΦ and GM-CSF MΦ were polarized for 72h and stained for intracellular flow cytometry. MFI of H3K4me3 relative to the secondary antibody stain is shown for M-CSF and GM-CSF MΦ (5 donors). White bars indicate resting MΦ, grey bars indicate IFN-γ stimulated M1 MΦ, and black bars indicate IL-4+IL13 stimulated M2 MΦ. (E) MFI of H3K27me3 relative to the secondary antibody stain (4-5 donors). Bar codes are as described in (D). (F) Cytospin slides of M-CSF MΦ, polarized for 24h, were stained with anti-MLLc (upper panel) and anti-H3K4me3 (lower panel). Slides were analyzed using an Olympus BX43 microscope (Olympus) with a X-Cite®120Q excitation light source (Lumen Dynamics) and CellSens1.7 Software (Olympus). Adobe Photoshop CS5.1 (Adobe Systems) was used to adjust brightness and contrast. * p < 0.05, ** p < 0.01.
Figure 4.
ChIP analysis for gene promoter-specific H3K4 and H3K27 trimethylation.
For ChIP analysis M-CSF MΦ were polarized for 48h and the promoter regions of CXCL10, ALOX15 and MRC1 were analyzed for enrichment of H3K4me3 and H3K27me3. Rabbit polyclonal IgG was used as negative control. Data are expressed as percent of input DNA. Data from 3 independent donors for control IgG (white bars), H3K4me3 (grey bars) and H3K27me3 (black bars) are shown.
Figure 5.
M-CSF MΦ were polarized for 24h and analyzed for SMYD3 mRNA expression of M1. Data is expressed as fold differences of polarized MΦ relative to resting MΦ. Resting MΦ expression levels are indicated by the dotted line. (5 donors). Box plots represent the median and interquartile range; whiskers indicate the minimum and maximum, respectively. * p < 0.05.
Figure 6.
Effect of targeting MLL-Menin interaction on CXCL10 protein expression.
Flow cytometric analysis of CXCL10 expression in M1 MΦ, pre-treated for 24h with 40µM of the MLL-Menin inhibitor MI-2-2 or DMSO 1:500. Results are shown as % of CXCL10 positive cells (left panel) and as MFI relative to the isotype control (right panel). Box plots represent the median and interquartile range, whiskers indicate the minimum and maximum, respectively.
Figure 7.
Phenotype of polarized M-CSF and GM-CSF MΦ.
MΦ that were differentiated in M-CSF (A) and GM-CSF (B) were further polarized using IFN-γ or IL-4+IL-13. Characteristic features of M-CSF vs. GM-CSF MΦ are depicted in the upper part. Additional changes found in this study, induced by the polarization process are shown in the lower section with characteristic genes, surface markers and epigenetic changes for M-CSF and GM-CSF M1and M2 MΦ.