Journal: bioRxiv

Article Title: Modulation of macrophage inflammatory function through selective inhibition of the epigenetic reader protein SP140

doi: 10.1101/2020.08.10.239475

Figure Lengend Snippet: 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 1h 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 , c) FACS analysis was performed in IFN-γ(M1) polarized macrophages to assess: (b) the frequency of CD64 + macrophages (left) and CD64 protein expression intensity (right) and (c) the frequency of CD206 + macrophages (left) and CD206 protein expression intensity, n=3. For FACS data, the count was normalized to mode. (d) M1 polarized macrophages were pretreated for 1h with 1% DMSO or with 0.04 µM GSK761. The cells were then stimulated for 4h with 100 ng/mL LPS and Customized RT Profiler PCR Arrays was performed, the scatter plot illustrates the differentially expressed genes (2-fold change), n=4 to 5. (e) M1 polarized macrophages were pretreated for 1h with 1% DMSO or with an increasing concentration of GSK761 (0.01, 0.04, 0.12, 0.37. 1.11 µM). The cells were then stimulated for 24h 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 using ProInflammatory 7-Plex MSD kit, n=4 to 7. The Y axis indicates the fold change in cytokine protein expression relative to DMSO control. (f) CD14 + cells were isolated from inflamed CD mucosa. The cells were then incubated ex vivo for 4h with either 0.1% DMSO or 0.04 µM GSK761. Relative gene expression of TNF, IL6, IL10 and CD64 were measured using qPCR, n=2. 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.

Article Snippet: Cytokine expression in the supernatant of DMSO- or GSK761-treated M1 macrophages (LPS-stimulated or unstimulated) were measured using electro-chemiluminescence assays (Meso Scale Discovery [MSD])-Human ProInflammatory 7-Plex Tissue Culture Kit (IFN-γ, IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF) according to manufacturer’s protocols and analyzed on an MSD 1250 Sector Imager 2400 (Mesoscale).

Techniques: Expressing, Marker, Concentration Assay, Isolation, Incubation, Ex Vivo