Fig 1: K. pneumoniae SUMOylation decrease in macrophages is mediated by TLR4-dependent type I interferon and let-7 miRNAs. (A) Immunoblot analysis of SUMO1 and tubulin levels in lysates of immortalized bone marrow-derived macrophages (iBMDMs) derived from C57BL/6 mice (WT) or Tlr4 knockout mice (tlr4−/−) infected with Kp52145 for the indicated time points. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (LI-COR) and normalized to α-tubulin. The graph represents fold change compared to wild-type (WT) iBMDM noninfected control cells. **, P ≤ 0.01; *, P ≤ 0.05 by one-way ANOVA with Bonferroni’s multiple-comparison test. (B) Immunoblot analysis of SUMO1 and tubulin levels in lysates of iBMDM cells derived from wild-type (WT) mice or MyD88 knockout mice (myd88−/−) infected with Kp52145 for the indicated time points. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (LI-COR) and normalized to α-tubulin. The graph represents fold change compared to wild-type (WT) iBMDM noninfected control cells. ***, P ≤ 0.001 by one-way ANOVA with Bonferroni’s multiple-comparison test. (C) Immunoblot analysis of SUMO1 and tubulin levels in lysates of iBMDM cells derived from WT mice or TRAM/TRIF double knockout mice (tram/trif−/−) infected with Kp52145 for the indicated time points. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (LI-COR) and normalized to α-tubulin. The graph represents fold change compared to wild-type (WT) iBMDM noninfected control cells. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05 by one-way ANOVA with Bonferroni’s multiple-comparison test. (D) Type I IFN levels determined in the supernatants of iBMDM wild-type (WT) or tlr4−/− cells left untreated (n.i.) or infected for 3 h with Kp52145. The reporter cell line B16-Blue IFN-α/β was used for the quantification of levels of SEAP produced upon stimulation of the supernatants with the detection medium QUANTI-Blue and presented as IFN-α/β U ml−1. IFN-β mRNA levels were assessed by qPCR and are presented as fold change against untreated wild-type (WT) cell levels. Values are presented as the means ± SDs from three independent experiments measured in duplicates. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; n.s., not significant by one-way ANOVA with Tukey’s multiple-comparison test. (E) Immunoblot analysis of SUMO1 and tubulin levels in lysates of BMDM cells derived from wild-type (WT) or IFNAR1 knockout mice (ifnar1−/−) infected with Kp52145 for the indicated time points or left untreated (n.i.). SUMO1 smears were quantified from three independent experiments using Image Studio Lite (LI-COR) and normalized to α-tubulin. The graph represents fold change compared to wild-type (WT) BMDM noninfected control cells. ***, P ≤ 0.001; **, P ≤ 0.01; * ≤ 0.05 by one-way ANOVA with Bonferroni’s multiple-comparison test. (F) ifnb1, ifit1, and isg15 mRNA levels, assessed by qPCR, in MH-S cells left untreated (n.i.) or infected for 3 h with the K. pneumoniae strains indicated. Values are presented as the means ± SDs from three independent experiments measured in duplicates. ***, P ≤ 0.001 by one-way ANOVA with Tukey’s multiple-comparison test. (G) let-7 mRNA levels, assessed by qPCR, in BMDM cells derived from wild-type (WT) or ifnar1−/− mice infected with Kp52145 for 1 h or left untreated (n.i.). Values are presented as the means ± SDs from three independent experiments measured in duplicates. ***, P ≤ 0.001 versus WT n.i. determined using two way-ANOVA with Holm-Sidak’s multiple-comparison test. (H) Immunoblot analysis of SUMO1 and tubulin levels in lysates of let-7 miRNA antagomir-transfected MH-S cells infected with Kp52145 for 1 h or left untreated (n.i.). Negative, Caenorhabditis elegans control sequence with minimal sequence identity in mouse cells. SUMO1 smears were quantified from three independent experiments using Image Studio Lite (LI-COR) and normalized to α-tubulin. The graph represents fold change compared to negative-transfected noninfected control cells. ***, P ≤ 0.001 by one-way ANOVA with Bonferroni’s multiple-comparison test. In panels A, B, C, E, and H, data are representative of at least three independent experiments.
Fig 2: MEF2A controls inflammatory gene expression in macrophages(A) Heatmap showing the behavior of LPS-inducible genes (n = 312; STAR Methods) in WT or Mef2a-/- macrophages. The defined set of MEF2A-dependent genes (n = 94; STAR Methods) is highlighted in red. Colors represent row-normalized percentages of gene expression. Selected gene names are shown on the right, and color legends and clone IDs are shown at the bottom. Data are from three biological replicates. Pearson correlation > 0.93 for all replicates.(B) IFN-ß release by WT or Mef2a-/- iMacs stimulated with LPS. Genotypes and IDs of the individual clones are shown. The bar plot represents mean ± SD. Data are from three biological replicates. ****p < 0.0001 (two-way ANOVA).(C) Gene set enrichment analysis (GSEA) of IFN-a-induced genes (gene set) in ranked gene lists obtained by comparing LPS-stimulated Mef2a-/- versus WT iMacs. Normalized enrichment score (NES) and p value are shown.(D) qRT-PCR analysis of Ifnb1 and Relb expression in BMDMs upon CRISPR-Cas9 targeting of the indicated putative Ifnb1 enhancers, named according to their distance to the transcription start site (TSS). Dot plots represent mean ± SD. Data are from three biological replicates. Numbers indicate p values for the corresponding comparisons (two-way ANOVA).(E) qRT-PCR analysis of Ifnb1 and Relb expression by iMac clones upon CRISPR-Cas9-mediated editing of the MEF2A binding site within the +7.25-kb Ifnb1 enhancer. Dot plots represent mean ± SD. Data are from three biological replicates. Numbers indicate p values for the corresponding comparisons (two-way ANOVA).(F) Luciferase reporter activity of the +7.25-kb Ifnb1 enhancer upon mutagenesis of the MEF2A TFBs in BMDMs treated with LPS or LPS+PGE2. a.u., arbitrary unit. The bar plot represents mean ± SD. Data are from three biological replicates. Numbers indicate p values for the corresponding comparisons (two-way ANOVA).(G) GSEA of PGE2-sensitive or -resistant genes (gene sets) in ranked gene lists obtained comparing LPS-stimulated Mef2a-/- versus WT iMacs. NES and p value are shown for each plot.(H) Boxplot showing mean expression values of PGE2-sensitive (MEF2A-dependent or not MEF2A-dependent STAR Methods) or resistant genes in WT or MEF2A-deficient iMacs under the indicated conditions. Data are from three biological replicates. Pearson correlation > 0.93 for all replicates. Numbers indicate p values for the corresponding comparisons (Mann-Whitney U test).(I) Line plot showing expression of PGE2-sensitive or -resistant genes as percent ratio of unstimulated MEF2A-deficient versus WT iMacs. Lines represent individual MEF2A-deficient clones.See also Figure S6.
Fig 3: MEF2A controls PGE2-sensitive inflammatory gene enhancers(A) Motif enrichment analysis showing top-ranking motifs identified within pre-existing or de novo OCRs at PGE2-sensitive or resistant enhancers. Putative cognate TF families and associated p values and q values are shown.(B) ChIP-seq signal intensities for the indicated TFs at pre-existing or de novo OCRs within PGE2-sensitive or -resistant enhancers under the indicated conditions. Numbers denote p values (Mann-Whitney U test) for the indicated comparisons.(C) Heatmap showing the intensity of ChIP-seq signals for the indicated TFs under the indicated conditions at pre-existing or de novo OCRs within PGE2-sensitive or -resistant enhancers. Signal intensities are represented over a 2-kb genomic region spanning the ATAC-seq peak summit. Legends are shown at the bottom.(D) IGV snapshot showing read coverage of the indicated datasets at the Ifnb1 genomic locus in WT (black) or Mef2a-/- (orange) iMacs under the indicated experimental conditions.(E) Motif enrichment analysis showing top-ranking motifs identified within basal or LPS-inducible MEF2A-dependent enhancers. Putative cognate TF families and associated p values and q values are shown.(F) Boxplot showing mean intensity values within PGE2-sensitive (blue) or resistant (gray) enhancers in the indicated datasets, obtained in WT or MEF2A-deficient iMacs for the indicated conditions. Data are from two biological replicates. Pearson correlation > 0.82 for all replicates. Numbers denote p values (Mann-Whitney U test) for the indicated comparisons.(G) H3K27Ac ChIP-seq signal intensities within MEF2A-dependent (orange) and -independent (black) enhancers. Data are from two biological replicates. Pearson correlation > 0.94 for all replicates. Numbers denote p values (Mann-Whitney U test) for the indicated comparisons.See also Figure S5.
Fig 4: STING degradation and termination of signalling is dependent on ubiquitination APrimary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 4.5 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown are representative of three independent experiments.BPrimary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 25 μg/ml DMXAA for 1, 2 or 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown are representative of three independent experiments.CPrimary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated or treated with 10 μg/ml 2′3′‐cGAM(PS)2 for 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown are representative of three independent experiments.DPrimary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated (UT) or treated with 25 μg/ml DMXAA for 1, 2 or 4 h. Cells were fixed and stained for p‐STING (white) and the nucleus (DAPI; blue), before Z stack images were acquired on the LSM980 confocal microscope. Images are displayed as a maximum intensity projection (MIP) of Z stack images. Scale bar = 5 μm. Data shown are representative of three independent experiments.E, FThe size (i.e. mean radius) (E) and mean intensity (F) of individual p‐STING punctate regions was quantified using CellProfiler. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using two‐way ANOVA using Bonferroni's multiple comparisons test, where *P < 0.05, ****P < 0.0001. A representative experiment used for quantification is shown in (D).G–IPrimary BMDMs were treated with 2 μM TAK243 or DMSO vehicle control for 30 min before being either left untreated (UT) or treated with 25 μg/ml DMXAA or 10 μg/ml 2′3′‐cGAM(PS)2 for 4 h. Cells were lysed for RNA purification and the expression of Ifnb1 (G), Isg15 (H) and Il6 (I) was analysed by qPCR. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using two‐way ANOVA using Bonferroni's multiple comparisons test, where *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.J Sting −/− iBMDMs expressing either WT hSTING or hSTING‐N154S were left untreated or treated with 1 μg/ml doxycycline (Dox) for 2 h. Cells were then washed and left to recover for a further 8 h in the absence or presence of 2 μM TAK243. Cells were lysed for immunoblot with indicated antibodies. Data shown are representative of three independent experiments. Source data are available online for this figure.
Fig 5: MEF2A controls IFN I induction by nucleic acids or pathogens and is targeted by PGE2 partly via ERK5(A) qRT-PCR analysis of Ifnb1, Cxcl10, or Irf1 in WT or Mef2a-/- iMacs stimulated for 4 h with poly(I:C) (red, top) or DMXAA (blue, bottom). Genotypes and IDs of the individual clones are shown. Bar plots represent mean ± SD. Data are from three biological replicates. ****p < 0.0001 (two-way ANOVA).(B) IFN-ß release by WT or Mef2a-/- iMacs stimulated with poly(I:C) (red, top) or DMXAA (blue, bottom). Genotypes and IDs of the individual clones are shown. Bar plots represent mean ± SD. Data are from three biological replicates. ****p < 0.0001 (two-way ANOVA).(C) Experimental layout of in vitro infection of iMacs with VSV, BCG, or M.tb.(D) IFN-ß release by WT (black) or Mef2a-/- (green) iMacs infected with VSV, BCG, or M.tb. MOI values are reported for each plot. Dot plots represent mean ± SD. Data are from two or three biological replicates. ****p < 0.0001, **p < 0.005 (two-way ANOVA test).(E–G) qRT-PCR analysis of Ifnb1, Cxcl10, or Il12b in WT (black) or Mef2a-/- (green) iMacs infected with VSV (E), BCG (F), or M.tb (G). MOI values are reported for each plot. Dot plots represent mean ± SD. Data are from two or three biological replicates. ****p < 0.0001, ***p < 0.001 (two-way ANOVA).(H and I) Western blot analyses of HDAC5 and loading control (VCL) in BMDMs stimulated with PGE2 for the indicated times (H) or in BMDMs upon CRISPR-Cas9-mediated targeting of Hdac5 (I).(J) qRT-PCR analysis of PGE2-sensitive and -resistant genes in WT (black) or HDAC5-deficient (orange) BMDMs stimulated as indicated. Dot plots represent mean ± SD. Data from three biological replicates. ***p < 0.001, **p < 0.01, *p < 0.05 (unpaired t test).(K) Western blot analyses for phosphorylated ERK5 (Thr218/Tyr220) as well as ERK5 and VCL as loading controls in BMDMs stimulated as indicated.(L) qRT-PCR analysis of PGE2-sensitive and resistant genes in WT (black) or Mapk7-/- (orange) iMacs clones stimulated as indicated. Genotypes and IDs of the individual clones are shown. Dot plots represent mean ± SD. Data are from three biological replicates. ****p < 0.0001, ***p < 0.001 (unpaired t test).(M) Western blot analyses of ERK5 and VCL in WT and Mapk7-/- iMacs clones. Genotypes and IDs of the individual clones are shown.(N) IFN-ß release by WT (black) and Mapk7-/- (orange) iMacs stimulated as indicated. Dot plot represents mean ± SD. Data are from three biological replicates. ****p < 0.0001 (two-way ANOVA).See also Figure S7.
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