Fig. 5
GSK761 affects macrophage polarization and cytokine production in peripheral blood and CD mucosal macrophages. Human primary CD14+ monocytes were differentiated with 20 ng/mL M-CSF for 3 days. The cells where then washed with PBS and treated with 0.1% DMSO or 0.04 μM GSK761 for 1 h prior to 3 days polarization to “M1” and “M2” macrophages phenotypes with 100 ng/mL INF-γ or 40 ng/mL IL-4, respectively (GSK761 and DMSO were not washed and kept in the culture during the 3 days of polarization). a Gene expression of the inflammatory marker CD64 (“M1” marker) and anti-inflammatory marker CD206 (“M2” marker) was measured by qPCR in IFN-γ (“M1”) and IL-4 (“M2”) polarized macrophages and b–e FACS analysis was performed in IFN-γ (“M1”) polarized macrophages. b Scatter plots illustrating the frequency of CD64+ cells. c A graph bar summarizing the frequency of CD64+ macrophages in 3 donors (left) and CD64 protein expression intensity (right). d Scatter plots illustrating the frequency of CD206+ cells. e A graph bar summarizing the frequency of CD206+ macrophages in 3 donors (left) and CD206 protein expression intensity, n=3. f “M1” polarized macrophages were pretreated for 1 h with 1% DMSO (n=4) or with 0.04 μM GSK761 (n=5). The cells were then stimulated for 4 h with 100 ng/mL LPS and Customized RT2 Profiler PCR Arrays was performed, the scatter plot illustrates the differentially expressed genes (2-fold change). g “M1” polarized macrophages were pretreated for 1 h with 1% DMSO or with an increasing concentration of GSK761 (0.01, 0.04, 0.12, 0.37, and 1.11 μM). The cells were then stimulated for 24 h with 100 ng/mL LPS. Protein levels of TNF, IL-6, IL-1β, IL-10, IL-8, and IL-12p70 were measured in the supernatant, n=4–7. The Y axis indicates the fold change in cytokine protein expression relative to DMSO control. h CD14+ cells were isolated from inflamed CD mucosa. The cells were then incubated ex vivo for 4 h with either 0.1% DMSO or 0.04 μM GSK761. Relative gene expression of TNF, IL6, IL10, and CD64 were measured using qPCR, n=4. The Y axis indicates the fold change in mRNA level relative to DMSO control. Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001