Journal: Redox Biology

Article Title: Itaconate promotes a wound resolving phenotype in pro-inflammatory macrophages

doi: 10.1016/j.redox.2022.102591

Figure Lengend Snippet: 4-OI upregulates pro-resolution macrophage markers and reduces collagen uptake by human blood monocyte derived macrophages (hMDM). A-C) qPCR of cyclin dependent kinase inhibitor 1A ( CDKN1A ) (A) , transforming growth factor beta-1 ( TGFB1 ) (B) and MMP8 (C) mRNA levels after 24 h of LPS stimulation with and without 4-OI, normalised to an untreated control (dashed line) ( n = 5 , paired t -test). D) Collagen uptake by flow cytometry of macrophages that were incubated for 24 h in FITC-collagen-coated wells with 4-OI, LPS or both ( n = 3 donors, one-way ANOVA with a Dunnette's multiple comparison test). E) FITC-labelled zymosan uptake by macrophages after 24 h of incubation with LPS, 4-OI or both ( n = 6, one-way ANOVA with a Dunnett's test for multiple comparison). F) Alexa Fluor 647-labelled ovalbumin (OVA-647) uptake by macrophages after 24 h of incubation with LPS, 4-OI or both ( n = 6, one-way ANOVA with a Dunnett's test for multiple comparison). G-H) Representative flow cytometry histograms and quantifications of surface levels of collagen receptors and M2-macrophage markers CD11c ( G ), CD206 ( H ), and CD36 ( I ) in hMDMs treated with 4-OI for 24 h ( n = 6 donors, paired t-tests). Data points show individual donors.

Article Snippet: Next CD11c-APC (BD, 559877), HLA-DR-APC (BD, 559866), CD80-APC (BioLegend, 305220), or CD206-APC (BioLegend, 321110) antibodies were added (0.5 μl/10 5 cells) for 30 min in 2% human serum in PBS.

Techniques: Derivative Assay, Flow Cytometry, Incubation, Comparison

Journal: EMBO Reports

Article Title: Multinucleation resets human macrophages for specialized functions at the expense of their identity

doi: 10.15252/embr.202256310

Figure Lengend Snippet: Venn diagram showing the trancriptomic comparison between genes upregulated in mononucleated IFN‐γ vs. IL‐4 stimulated cells (designated as UP [IFN‐γ mono vs. IL‐4 mono ]) and in multinucleated LGCs vs. FBGCs (designated as UP [IFN‐γ multi vs. IL‐4 multi ]). The most significant group‐specific pathways (BioPlanet 2019) are shown with arrows. The right panel shows the 5 most significant pathways for commonly upregulated 1,286 transcripts. Relevant pathways to IFN‐ γ are shown in red. Venn diagram showing the trancriptomic comparison between genes upregulated in mononucleated IL‐4 vs. IFN‐γ stimulated cells (designated as UP [IL‐4 mono vs. IFN‐γ mono ]) and in multinucleated FBGCs vs. LGCs (designated as UP [IL‐4 multi vs. IFN‐γ multi ]). The most significant group‐specific pathways are shown with arrows whereas. The right panel shows the 5 most significant pathways for commonly upregulated 1,027 transcripts. Relevant pathways to IL‐4 are shown in red. Data information: n = 6 donors (LGCs), n = 7 donors (FBGCs). MRC1 , CSF1R , TLR2 , STAB1 and SUCNR1 relative expression measured by qRT–PCR in LGCs (upper), FBGCs (middle) and osteoclasts (lower), in sorted mononuclear and multinucleated cells; at least n = 6 donors. Error bars are mean ± SD; significance tested by paired t‐test; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Cells were then briefly centrifuged, re‐suspended in ice‐cold sorting buffer, incubated with FcR block (Miltenyi Biotec, Cat # 130059901) and stained with human anti‐MRC1 or anti‐B7‐H3 (APC, Biolegend, MRC1, Cat # 321110 and B7‐H3, Cat # 351006).

Techniques: Comparison, Expressing, Quantitative RT-PCR

Journal: EMBO Reports

Article Title: Multinucleation resets human macrophages for specialized functions at the expense of their identity

doi: 10.15252/embr.202256310

Figure Lengend Snippet: Venn diagram showing the commonly downregulated transcripts ( n = 191) as a result of multinucleation in LGCs, FBGCs and osteoclasts (in gray). KEGG and BioPlanet 2019 pathway analyses on the 191 commonly downregulated genes. Pathways in red are shared between KEGG and BioPlanet. MRC1 surface marker expression (red) acquired by ImageStream in mononuclear (mono) and multinucleated (multi) LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) represent normalized MRC1 mean fluorescence intensity (MFI), measured by ImageStream; n = 4 donors. Representative MRC1 (left panel) and CSF1R (right panel) immunofluorescence in mononuclear and multinucleated osteoclasts stained for DAPI (blue). Note the dim MRC1 and CSF1R in multinucleated osteoclasts (dashed lines) compared to surrounding mononucleated ones. CSF1R , MRC1 , TLR2 and FOS relative expression measured by qRT–PCR for LGCs (upper), FBGCs (middle) and osteoclasts (lower), following the cell membrane fusion regulator DC‐STAMP knockdown. si‐Ctrl, scrambled siRNA; si‐DC‐STAMP, DC‐STAMP siRNA; at least n = 5 donors. Data information: Error bars are mean ± SD; significance tested by paired t ‐test; * P < 0.05; ** P < 0.01, *** P < 0.001. Scale bars, 20 μm (C) and 100 μm (D). Source data are available online for this figure.

Article Snippet: Cells were then briefly centrifuged, re‐suspended in ice‐cold sorting buffer, incubated with FcR block (Miltenyi Biotec, Cat # 130059901) and stained with human anti‐MRC1 or anti‐B7‐H3 (APC, Biolegend, MRC1, Cat # 321110 and B7‐H3, Cat # 351006).

Techniques: Marker, Expressing, Staining, Fluorescence, Immunofluorescence, Quantitative RT-PCR, Membrane

Journal: EMBO Reports

Article Title: Multinucleation resets human macrophages for specialized functions at the expense of their identity

doi: 10.15252/embr.202256310

Figure Lengend Snippet: Protein–protein interaction (PPI) network of 191 commonly downregulated genes in LGCs, FBGCs and osteoclasts illustrated by STRING (high confidence score = 0.9, only connected nodes are shown). ImageStream gating for mononuclear and multinucleated cells shown in LGCs. The gating strategy for FBGCs and osteoclasts is similar. ImageStream showing bright field, nuclei staining (Hoechst), and MRC1 (red) staining in mononuclear and multinucleated LGCs, FBGCs and osteoclasts. MRC1 and CSF1R immunofluorescence quantification in mononuclear and multinucleated LGCs (upper), FBGCs (middle) and osteoclasts (lower); n = 2 donors (biological replicates), n > 48 technical replicates per cell type, condition (mono or multi) and surface marker (MRC1 or CSF1R). DC‐STAMP expression following its knockdown in LGCs (left) and FBGCs (right). si‐Ctrl, scrambled siRNA; si‐DC‐STAMP, DC‐STAMP siRNA; n = 7 donors. Fusion index following DC‐STAMP knockdown in human LGCs (upper panel) and FBGCs (lower panel); n = 4 donors. Fusion was measured in cells stained with Giemsa in both conditions (right panel). Data information: Error bars are mean ± SD; significance tested by unpaired (D) and paired (E, F) t ‐test; ** P < 0.01; **** P < 0.0001; scale bar, 20 μm (C), 100 μm (F).

Article Snippet: Cells were then briefly centrifuged, re‐suspended in ice‐cold sorting buffer, incubated with FcR block (Miltenyi Biotec, Cat # 130059901) and stained with human anti‐MRC1 or anti‐B7‐H3 (APC, Biolegend, MRC1, Cat # 321110 and B7‐H3, Cat # 351006).

Techniques: Staining, Immunofluorescence, Marker, Expressing

Journal: EMBO Reports

Article Title: Multinucleation resets human macrophages for specialized functions at the expense of their identity

doi: 10.15252/embr.202256310

Figure Lengend Snippet: Venn diagram showing the uniquely differentially expressed transcripts in LGCs (in gray). KEGG and BioPlanet 2019 pathway analyses on the differentially expressed transcripts in LGCs only. Pathways in red are in common in KEGG and BioPlanet or relevant for LGC function. Giemsa stained LGCs (left), FBGCs (middle) and osteoclasts (right) differentiated from PBMCs. Note the cell cluster formation in LGCs only. CD3 immunofluorescence (green) in PBMC‐derived LGCs. Hoechst (gray) and phalloidin (red) staining show the nuclei and cytoskeleton, respectively. Merged (CD3, Hoechst, phalloidin) image shows CD3 + T cells in the granuloma‐like clusters. B7‐H3 surface marker expression (red) acquired by ImageStream in mononuclear and multinucleated LGCs, FBGCs and osteoclasts stained for Hoechst. Bar graphs (lower panel) show normalized B7‐H3 mean fluorescence intensity (MFI), measured by ImageStream; n = 4 donors. Error bars are mean ± SD; significance tested by paired t‐test; ** P < 0.01; ns, non‐significant. Data information: Scale bars, 100 μm (C and D) and 20 μm (E). Source data are available online for this figure.

Article Snippet: Cells were then briefly centrifuged, re‐suspended in ice‐cold sorting buffer, incubated with FcR block (Miltenyi Biotec, Cat # 130059901) and stained with human anti‐MRC1 or anti‐B7‐H3 (APC, Biolegend, MRC1, Cat # 321110 and B7‐H3, Cat # 351006).

Techniques: Staining, Immunofluorescence, Derivative Assay, Marker, Expressing, Fluorescence

Journal: EMBO Reports

Article Title: Multinucleation resets human macrophages for specialized functions at the expense of their identity

doi: 10.15252/embr.202256310

Figure Lengend Snippet: CD3 immunofluorescence in PBMC‐derived FBGCs (upper panel) and osteoclasts (lower panel). Hoechst (gray) and phalloidin (red) staining show the nuclei and cytoskeleton, respectively. LGCs show increased membrane expression of B7‐H3. ImageStream showing bright field, nuclei staining (Hoechst), and B7‐H3 (red) staining in mononuclear and multinucleated LGCs, FBGCs and osteoclasts. Data information: Scale bar, 20 μm.

Article Snippet: Cells were then briefly centrifuged, re‐suspended in ice‐cold sorting buffer, incubated with FcR block (Miltenyi Biotec, Cat # 130059901) and stained with human anti‐MRC1 or anti‐B7‐H3 (APC, Biolegend, MRC1, Cat # 321110 and B7‐H3, Cat # 351006).

Techniques: Immunofluorescence, Derivative Assay, Staining, Membrane, Expressing

Journal: EMBO Reports

Article Title: Multinucleation resets human macrophages for specialized functions at the expense of their identity

doi: 10.15252/embr.202256310

Figure Lengend Snippet: Primers used in qRT–PCR experiments.

Article Snippet: Cells were then briefly centrifuged, re‐suspended in ice‐cold sorting buffer, incubated with FcR block (Miltenyi Biotec, Cat # 130059901) and stained with human anti‐MRC1 or anti‐B7‐H3 (APC, Biolegend, MRC1, Cat # 321110 and B7‐H3, Cat # 351006).

Techniques:

Journal: Frontiers in Immunology

Article Title: YTHDF1 Negatively Regulates Treponema pallidum -Induced Inflammation in THP-1 Macrophages by Promoting SOCS3 Translation in an m6A-Dependent Manner

doi: 10.3389/fimmu.2022.857727

Figure Lengend Snippet: YTHDF1 regulates M2 macrophage polarization and suppresses the secretion of inflammatory cytokines in THP-1 cells infected with TP. (A, B) Flow cytometry profile of the expression of M1 and M2 markers (CD86 and CD206) in THP-1 cells infected with TP. (C-D) The concentrations of inflammatory cytokines (TNF-α and IL-1β) in the supernatants of THP-1 cells treated with TP. Data are shown as the mean ± S.D. **P < 0.01, ***P < 0.001,****P < 0.0001.

Article Snippet: Single-cell suspensions were prepared from cultured THP-1 cells and incubated with anti-CD206 (374208) and anti-CD86 (321110) antibodies (both from Abcam) for 30 min at 4°C for cell-surface staining.

Techniques: Infection, Flow Cytometry, Expressing