Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a. Heatmap of IRF family members expression in NK cells throughout the course of MCMV infection represented as z-score of log2 normalized counts based on RNA-seq. b. Representative histogram of IRF4 expression in WT and Irf4−/− NK cells on day 2 PI (n = 4 biological replicates). c. Tracks (top) show chromatin accessibility dynamics of the Irf4 locus in Ly49H+ NK cells at days 0, 2, 4, and 7 PI as assessed by ATAC-seq. Graphs (bottom) show normalized counts for each peak indicated. d. Tables of enriched known motifs of highly accessible regions (log2FC > 1 & padj < 0.05) on day 4 PI versus day 2 PI from ATAC-seq data of Ly49H+ NK cells after MCMV infection. e. Heatmap of motif score from de novo motif analysis on highly accessible regions (log2FC > 1 & padj < 0.05) during day 0, 2, 4, and 7 transition based on ATAC-seq data of Ly49H+ NK cells after MCMV infection. f. IRF4 expression of sorted naïve splenic NK cells after an overnight stimulation with the indicated stimuli. IRF4 induction is displayed as fold change of IRF4 MFI over unstimulated condition (n = 6 biological replicates per condition). Two-way ANOVA test adjusted for multiple comparisons was used for statistical analysis. g. Representative histogram of IRF4 expression upon indicated stimulation gated on human CD56bright NK cells. Data are presented as paired fold change of IRF4 MFI compared to unstimulated condition (n = 8 donors per condition). Two-way ANOVA test adjusted for multiple comparisons was used for statistical analysis. h. UMAP embedding of scATAC-seq data from in vitro stimulated human NK cells (sorted on CD3−CD14−CD19−CD7+NKG2C+) from HCMV− donors. i. Coverage plot of the IRF4 locus from in vitro stimulated human NK cells as in (H).

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Control, Expressing, Infection, RNA Sequencing, In Vitro

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, Scatter-plot comparing the expression of IRF family members that are differentially expressed on days 2 and 4 PI versus day 0 in Ly49H+ NK cells by RNA-seq analysis. Points highlighted in red and blue indicate genes that are upregulated and downregulated, respectively, on day 2 versus day 0 and day 4 versus day 0 (Padj < 0.05). b, Kinetics of RNA-seq normalized reads for Irf4 transcripts in Ly49H+ NK cells throughout the course of MCMV infection (n = 2 biological replicates per time point). c, Representative flow cytometric histogram of IRF4 expression in splenic Ly49H+ NK cells in MCMV-infected mice (left) and fold change of IRF4 mean fluorescent intensity (MFI) compared to uninfected mice (right) (n = 3 biological replicates for day 0, 2 and 3 and n = 2 biological replicates for day 4). d, UMAP embedding of scATAC-seq data from human NK cells of HCMV+ or HCMV− healthy donors. e, Coverage plot of the IRF4 locus from defined human NK cell subsets as in d. f, RNA-seq reads for IRF4 expression from human NK cell subsets represented as fragments per kilobase of transcript per million mapped reads (FPKM) (n = 5). CD56bright (CD56brightCD16dim), early CD56dim (CD56dimNKG2A+KIR−CD57−), CD56dim (CD56dimNKG2A−self-KIR+CD57+), adaptive NKG2C+ (CD56dimCD16+NKG2C+CD7lowNKp30lowCD57+). g, Representative histogram of IRF4 expression of sorted naive splenic NK cells after an overnight stimulation with the indicated stimuli. IRF4 induction is displayed as FC of IRF4 MFI over unstimulated condition (n = 3 per group). h, Volcano plot of RNA-seq data from human NK cells stimulated with HCMV UL-40 peptide + IL-12/18 versus control peptide. Red and blue points represent DEGs (Padj < 0.05 and |log2FC| > 0.5) in stimulated and control conditions, respectively. Data are represented as mean ± s.e.m. and are representative of, or pooled from, at least two independent experiments. A two-tailed unpaired t-test was used for g. Statistical analysis was not derived from groups with n < 3.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Expressing, RNA Sequencing, Infection, Control, Two Tailed Test, Derivative Assay

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, GSEA pathway analysis of DEGs between WT and Irf4−/−Ly49H+ cells on day 7 PI, from scRNA-seq experiment in Fig. 4. b, Representative histograms of CD98 expression between naive splenic WT and Irf4−/−Ly49H+ NK cells (dashed lines) or day 5 PI (solid lines). Data are represented as FC gMFI from WT cells taken from day 5 MCMV-infected mice (n = 8 biological replicates). c, Representative histograms of CD71 expression between naive splenic WT and Irf4−/−Ly49H+ NK cells (dashed lines) or day 5 PI (solid lines). Data are represented as FC gMFI from WT cells taken from day 5 MCMV-infected mice (n = 8 biological replicates). d, Representative histogram and gMFI of kynurenine uptake by splenic WT or Irf4−/−Ly49H+ NK cells taken from day 5 PI. Kyn + lysine (Lys) was added as a non-competitive control and Kyn + leucine (Leu) acted as a competitive control (n = 4 biological replicates in each condition). e, Representative histogram and gMFI of transferrin uptake assay from naive or day 5 PI splenic WT or Irf4−/−Ly49H+ NK cells (n = 11 biological replicates). Data are represented as mean ± s.e.m. and are representative of or pooled from at least two independent experiments. A two-tailed paired t-test was performed for b–e and adjusted for multiple comparisons for d.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Expressing, Infection, Control, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, b Representative histograms and quantification of IFN-γ production (A) and LAMP-1 (also known as CD107a) (B) from either naive WT (gray) or Irf4−/− (orange) NK cells upon in vitro stimulation with nothing, IL-12 + IL-18, PMA + Ionomycin, or PMA + Ionomycin + IL-12 for 3 hours. c–f. Representative histograms (upper panels) and quantifications (bottom panels) of CD25, Gzmb, CD69, and IFN-γ expression gated on Ly49H+ NK cells from MCMV-infected WT:Irf4−/− mBMC on day 2 PI. Data are represented as mean ± SEM and are representative of or pooled from at least two independent experiments. n = 3 biological replicates per group. Paired two-tailed t-test was performed unless stated otherwise.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: In Vitro, Expressing, Infection, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a–c. Number of NK cells in blood (A), liver (B), and spleen (C) of WT (n = 8), Irf4+/− (n = 4), or Irf4−/− (n = 6) mice at 8 weeks old. d–f. Maturation status based on the expression of CD27 and CD11b of WT (n = 8), Irf4+/− (n = 4), or Irf4−/− (n = 5 for blood, and n = 6 for liver and spleen) mice in the blood (D), liver (E), and spleen (F) at 8 weeks old. g. Percentage of Ly49H+ NK cells within NK cells (Lineage-NK1.1+ CD49b+) of WT (n = 8), Irf4+/− (n = 4), or Irf4−/− (n = 6) mice at 8 weeks old. h. Experimental schematic of WT:Irf4−/− mixed bone-marrow chimera (mBMC) generation. i–k. Percentage of NK cells of WT or Irf4−/− mice from WT:Irf4−/− mBMC in the blood (n = 60 biological replicates) (I) and spleen (n = 21 biological replicates) ( J) after 8 weeks (I-J), 16 weeks (n = 3 biological replicates) or 6 months (n = 5 biological replicates) post-transplant (K). l. Representative of flow plots of CD62L versus CD27 of either WT or Irf4−/− NK cells from WT:Irf4−/− mBMC in the blood and spleen 8 weeks post-transplant. Data are represented as percentage of each subset within total NK cells of each genotype (n = 22 biological replicates in blood, and n = 13 biological replicates in spleen). m. Representative of flow plots of CD11b versus CD27 of either WT or Irf4−/− NK cells from WT:Irf4−/− mBMC in the blood and spleen 8 weeks post-transplant. Data are represented as percentage of each subset within total NK cells of each genotype (n = 22 biological replicates in blood, and n = 16 biological replicates in spleen). n. Histogram and percentage quantification of Ly49H+ NK cells within splenic NK cells of either WT or Irf4−/− NK cells from WT:Irf4−/− mBMC (n = 24 biological replicates). Data are represented as mean ± SEM and are representative of or pooled from at least two independent experiments. Unpaired (A-G) and paired (I-N) two-tailed t-tests were performed.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Expressing, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a. Experimental schematic of adoptive transfer of WT and Irf4−/− NK cells into Rag2−/− Il2rg−/− mice. b. Fold change chimerism between WT and Irf4−/− NK cells on day 7 over day 0 (pre-transfer) in blood, liver, and spleen (n = 3 biological replicates in blood, and n = 6 biological replicates in liver and spleen). c. Representative histogram of IRF4 expression between transferred WT and Irf4−/− NK cells on day 7 post-transfer into Rag2−/− Il2rg−/−. d. Representative flow plots of CD27 and CD11b expression between WT and Irf4−/− NK cells on day 7 post-transfer. Data are represented as percentage of each subset within WT or Irf4−/− NK cells in indicated tissues (n = 3 biological replicates). e. Histogram of CD122 and quantification of CD122 MFI between WT and Irf4−/− NK cells on day 7 post-transfer in the spleen (n = 9 biological replicates). f. Histogram of CD132 and quantification of CD132 MFI between WT and Irf4−/− NK cells on day 7 post-transfer in the spleen (n = 9 biological replicates). g. Histogram of pSTAT5 and quantification of pSTAT5 MFI between WT and Irf4−/− NK cells on day 7 post-transfer in the spleen (n = 9 biological replicates). H-J. h–j. Quantification of Ki-67 staining in the blood and spleen between WT and Irf4−/− NK cells (H, n = 5 biological replicates per group), BIM and BCL2 MFI from splenic NK cells (I-J, n = 9 biological replicates). Data are represented as mean ± SEM and are representative of or pooled from at least two independent experiments. Paired two-tailed t-test was performed unless stated otherwise.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Adoptive Transfer Assay, Expressing, Staining, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, Kaplan–Meier survival curves from Rag2−/− Il2rg−/− mice transferred with WT Ly49H+ NK, Irf4−/−Ly49H+ NK or no NK cells (n as indicated). b, Percentage of Ly49H+ NK cells from adoptive co-transfer of WT (CD45.1) and Irf4−/− (CD45.2) NK cells into Ly49H-deficient mice from the blood throughout the course of MCMV infection (left) and percent chimerism of WT versus Irf4−/− transferred Ly49H+ NK cells (right) (n = 4 biological replicates). c, Representative flow plots gated on transferred WT (CD45.1) and Irf4−/− (CD45.2) Ly49H+ NK cells on day 7 in different tissues (left) and the percent chimerism of WT and Irf4−/− NK cells within transferred Ly49H+ NK cells between different tissues (n = 4 biological replicates per group). d, Representative histogram of CTV dilution on day 0 and day 4 PI gated on transferred Ly49H+ NK cells (left). Dashed gray and orange lines represent WT and Irf4−/− NK cells on day 0, respectively; solid gray and orange lines represent WT and Irf4−/− NK cells on day 4 PI, respectively. Percentage of transferred NK cells that have divided at least once (n = 5 biological replicates per group) (right). e, Representative histogram of BIM expression on day 5 PI from splenic WT or Irf4−/−Ly49H+ NK cells (left) and BIM gMFI (right) (n = 4 biological replicates). gMFI, geometric mean fluorescence intensity. f, Percentage of FLICA+ (marking activated caspase) WT or Irf4−/−Ly49H+ NK cells taken from day 5 PI WT:Irf4−/− mBMC splenocytes upon ex vivo treatment with ABT199 (BCL2 inhibitor) (n = 3 biological replicates in each condition). Data are represented as mean ± s.e.m. and are representative of, or pooled from, at least two independent experiments. For statistical testing, a log-rank test was used for a and a two-tailed paired t-test was used for b–f and adjusted for multiple comparisons for b and c.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Infection, Expressing, Fluorescence, Ex Vivo, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, Representative flow plots showing CD62L and CD27 expression of transferred WT or Irf4−/−Ly49H+ NK cells on day 7 PI (left). Percentage of NK cell subset based on CD62L and CD27 expression of transferred cells on day 7 PI in the blood, liver and spleen (right) (n = 5 biological replicates in blood and spleen and n = 4 biological replicates in liver). b, Representative histogram and percentages of Ly6C, CX3CR1 and KLRG1 of transferred WT or Irf4−/− Ly49H+ NK cells on day 7 PI from spleens of MCMV-infected mice (n = 5 per group). c, Fold change of chimerism between splenic WT and Irf4−/−Ly49H+ NK cells upon infection of WT:Irf4−/− mBMC with MCMV from day 0, 2, 4, 5 and 7 PI (n = 6 biological replicates for day 0 and 7, n = 5 biological replicates for day 2, n = 7 and 8 biological replicates for day 4 and 5, respectively). d, Kinetics of different subsets of splenic WT and Irf4−/−Ly49H+ NK cells from WT:Irf4−/− mBMC during MCMV infection setting based on CD62L and CD27 expression from day 0 to 7 PI (n = 6 biological replicates for day 0 and 7, n = 7 biological replicates for day 2 and n = 8 biological replicates for day 4 and 5, respectively). Data are represented as mean ± s.e.m. and are representative of or pooled from at least two independent experiments. A two-tailed paired t-test was performed for b–d and adjusted for multiple comparisons for c and d.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Expressing, Infection, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, WT and Irf4−/− Ly49H+ NK cells were sorted on day 0, 2, 4 and 7 following MCMV infection and single-cell RNA-sequencing performed on multiplexed samples. UMAP embedding of scRNA-seq colored by Louvain clusters (kn = 30). b, Top 50 DEGs between WT and Irf4−/−Ly49H+ NK cells during MCMV shown as z score of normalized reads. c, PAGA network analysis based on scRNA-seq data from WT versus Irf4−/−Ly49H+ NK cells scRNA-seq. d, UMAP embedding of WT and Irf4−/−Ly49H+ NK cells on day 7 from clusters 1 and 3 in a. e, Volcano plot of scRNA-seq as determined by MAST between WT and Irf4−/−Ly49H+ NK cells on day 7 PI. Blue and yellow points represent significant genes (FDR < 0.05) that are upregulated in WT or Irf4−/−Ly49H+ NK cells, respectively. Highlighted genes are effector genes and/or factors known to be important for NK cell differentiation/maturation. f, Dot plot of scRNA-seq for maturation markers between WT and Irf4−/−Ly49H+ NK cells on day 7 PI shown as mean normalized expression and percent of cell fraction within the group. g, UMAP plot from d as MPEC or SLEC gene signature module scores.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Infection, RNA Sequencing, Cell Differentiation, Expressing

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a. UMAP embedding of WT (left) or Irf4−/− (right) of Ly49H+ NK cells colored by genotypes and days post-infection. b. Proportion of each genotype from each time points per Louvain clusters. c. Volcano plot of scRNA-seq as determined by MAST between WT and Irf4−/− Ly49H+ NK cells on day 0, 2, 4 and 7 PI. Blue and yellow points represent significant genes (FDR < 0.05) that are upregulated in WT or Irf4−/− Ly49H+ NK cells, respectively. d. Number of differentially expressed genes between WT or Irf4−/− Ly49H+ NK cells from each time point as identified by MAST. e. Violin plot quantification of normalized reads for MYC transcript by scRNA-seq between WT and Irf4−/− Ly49H+ NK cells. f. Representative histogram of Myc protein by flow cytometry between WT (gray) and Irf4−/− (orange) Ly49H+ NK cells on day 5 PI. g. Violin plot quantification of MAGIC-imputed reads for CD71 (Tfrc), IRP1 (Aco1), and IRP2 (Ireb2) on day 7 PI between WT (blue) and Irf4−/− (yellow) Ly49H+ NK cells by scRNA-seq.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Infection, Flow Cytometry

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, Normalized count heat map of IRF4 binding upon anti-NK1.1 + cytokines (IFN-α + IL-12/18 + IL-2/15) stimulation or IL-15 alone at +0.5 kb from the peak summit (n = 12,344 peaks). b, IGV tracks showing IRF4-binding signal between unstimulated (IL-15 alone, gray) or stimulated (anti-NK1.1 + cytokines, orange) conditions in Bcl2l11, Sell, Tfrc and Ireb2 loci. Regions highlighted in dashed boxes represent called IRF4 peaks by MACS2 in stimulated condition. c, Top: Venn diagram comparing IRF4-bound genes in stimulated condition and DEGs between WT versus Irf4−/−Ly49H+ NK cells on day 7 PI from scRNA-seq. Bottom: distribution of IRF4-bound DEGs between WT versus Irf4−/−Ly49H+ NK shown as fold change as analyzed by MAST. d, Proportion of IRF4 genome-wide occupancy within the exon, intergenic, intronic and promoter regions. e, Enriched de novo motifs found by HOMER on IRF4-bound regions upon stimulation. P values were calculated by HOMER (Methods). f, Metacoverage of IRF4-bound accessible regions that contain the indicated motifs in Ly49H+ NK cells upon MCMV infection as assessed by ATAC-seq. Data are shown as median of normalized counts. g, Top: the z score of DEGs that contain AICE motifs in Ly49H+ NK cells during MCMV infection. Bottom: average of scaled log-normalized counts of AICE-motif-containing genes enriched in either WT or Irf4−/−Ly49H+ NK cells (Methods) throughout the course of MCMV. Adjusted P values (Padj) were calculated by ‘geseca’ function of the fgsea package (Methods).

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Binding Assay, Genome Wide, Infection

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a. Gating strategy for sorting WT or Irf4−/− Ly49H+ NK cells. b. Gating strategy for phenotypic analysis of Ly49H+ NK cells after lymphocytes, singlets and live cells cleanup (as depicted in Extended Data Fig. 7a). c. Gating strategy for human NK cells.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques:

Journal: Advanced Science

Article Title: Mechanisms of Baicalin Alleviates Intestinal Inflammation: Role of M1 Macrophage Polarization and Lactobacillus amylovorus

doi: 10.1002/advs.202415948

Figure Lengend Snippet: Baicalin inhibits LPS‐induced M1 macrophage polarization in vitro. A) Macrophages in CON, LPS, and BL + LPS group. B) mRNA expression of STAT1 , STAT2 , STAT3 , and TLR2 in CON, LPS, and BL + LPS group ( n = 4). C) mRNA expression of TLR4 , IRF4 , IRF7 , IRF8 , IRF9 , CD86 , IL‐1β , NLRP3 , IL‐12 , and MMP9 in CON, LPS, and BL + LPS group ( n = 4). D) Immunofluorescence analyses of CD86, IL‐1β, NLRP3, and TLR4 protein expression in RAW264.7 cells. Nuclei are counterstained with DAPI (blue). (E) Mean gray value of CD86, IL‐1β, NLRP3, and TLR4 in immunofluorescence analyses ( n = 3).

Article Snippet: The primary antibodies used were obtained from the following suppliers: anti‐ACTB (Sangon Biotech, #D110001), NLRP3 (Beyotime, #AF2155), IL‐1β (Bioss, #bs‐25615R), Occludin (Sangon Biotech, #D110001), CD163 (Sangon Biotech, #D260965), STAT2 (Proteintech, #16674‐1‐AP), TLR4 (Beyotime, #AF8187), Foxp3 (Beyotime, #AF6927), RORγt (Bioss, #bs‐10647R), IL‐17F (Bioss, #bs‐7333R), CTLA4 (ABclonal, #A13966), IRF4 (Proteintech, #66451‐1‐Ig), IRF7 (Proteintech, #22392‐1‐AP), and TLR9 (Beyotime, #AF8193).

Techniques: In Vitro, Expressing, Immunofluorescence

Journal: Advanced Science

Article Title: Mechanisms of Baicalin Alleviates Intestinal Inflammation: Role of M1 Macrophage Polarization and Lactobacillus amylovorus

doi: 10.1002/advs.202415948

Figure Lengend Snippet: Lactobacillus amylovorus SKLAN202301ZF regulates macrophage polarization and T cell differentiation. A) mRNA expression of STAT1 , STAT2 , STAT3 , TLR2 , TLR3 , TLR4 , IRF7 , and IRF8 ( n = 12). B) mRNA expression of IRF9 , PIAS1 , PIAS3 , CD226 , Unc93b1 , RORγt , CD86 , and CD163 ( n = 12). C) mRNA expression of NLRP3 , IL‐1β , IL‐17A , IL‐17F , IL‐18 , CCL17 , MMP9 ( n = 12). D) Western blotting was conducted to measure the expression of CTLA4, IRF4, IRF7, TLR9, FOXP3, RORγt, IL‐1β, NLRP3, and ACTB in the colon, E) and the ratio of cleaved to full‐length forms for these proteins was calculated ( n = 4). ACTB was used as a loading control.

Article Snippet: The primary antibodies used were obtained from the following suppliers: anti‐ACTB (Sangon Biotech, #D110001), NLRP3 (Beyotime, #AF2155), IL‐1β (Bioss, #bs‐25615R), Occludin (Sangon Biotech, #D110001), CD163 (Sangon Biotech, #D260965), STAT2 (Proteintech, #16674‐1‐AP), TLR4 (Beyotime, #AF8187), Foxp3 (Beyotime, #AF6927), RORγt (Bioss, #bs‐10647R), IL‐17F (Bioss, #bs‐7333R), CTLA4 (ABclonal, #A13966), IRF4 (Proteintech, #66451‐1‐Ig), IRF7 (Proteintech, #22392‐1‐AP), and TLR9 (Beyotime, #AF8193).

Techniques: Cell Differentiation, Expressing, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Immunomodulatory effect of NEDD8-activating enzyme inhibition in Multiple Myeloma: upregulation of NKG2D ligands and sensitization to Natural Killer cell recognition

doi: 10.1038/s41419-021-04104-w

Figure Lengend Snippet: A SKO-007(J3) cells were cotransfected with 5 µg of the indicated luciferase reporter vector and 1 µg of pRL-TK. After electroporation cells were treated with MLN4924 (200 nM) for 48 h, harvested and protein extracts were prepared for the Luciferase and Renilla assays. Results are expressed as Relative Luciferase activity normalized to protein concentration as well as to Renilla activity produced off the internal control plasmid and represent the mean value (±SEM) of four independent experiments (* P < 0.05). A schematic representation of the progressive MICA promoter deletions used in these experiments is shown. B Real-time PCR analysis of total mRNA obtained from SKO-007(J3) cells, untreated or treated with the indicated concentrations of MLN4924 for 48 h. Data, expressed as fold change units, were normalized to GAPDH, and were referred to the untreated sample considered as calibrator, represent the mean (±SEM) of four experiments (* P < 0.05). C Western-blot analysis of IRF4 in SKO-007(J3) cells untreated or treated with MLN4924 for 48 h. β-Actin was used as protein-loading control. One representative western-blot out of three independent experiments is shown. Densitometric analysis of the reported Western blot is shown. D Real-time PCR analysis of total mRNA obtained from purified CD138 + cells untreated or treated with MLN4924 (400 nM) as described above for 48 h in complete medium supplemented with 20 ng/ml IL-3 and 2 ng/ml IL-6. Data expressed as fold change units, were normalized to GAPDH, and referred to the untreated sample considered as calibrator. E , F Lysates of MM cells purified from two patients (CD138 + cells) untreated or treated with MLN4924 (200 and 400 nM) for 48 h in complete medium supplemented with 20 ng/ml IL-3 and 2 ng/ml IL-6, were subjected to Western Blotting using anti-IRF4 and anti-βActin antibodies. Densitometric analysis of normalized IRF4/Actin is shown.

Article Snippet: Reverse transcription was carried out in a 25 μl reaction volume with 2 μg of total RNA according to the manufacturer’s protocol for M-MLV reverse transcriptase (Promega, Madison, Wisconsin, USA). cDNAs were amplified (TaqMan assays) in triplicate with primers for MICA (Hs00792195_m1), MICB (Hs00792952_m1), IRF4 (Hs01056533_m1), IKZF1 (Hs00958474_m1), IKZF3 (Hs00232635_m1), MEIS2 (Hs00542638_m1), CRBN (Hs00372271_m1), GAPDH (Hs02758991_g1) conjugated to the fluorochrome FAM (Applied Biosystems, Foster City, California, USA).

Techniques: Luciferase, Plasmid Preparation, Electroporation, Activity Assay, Protein Concentration, Produced, Control, Real-time Polymerase Chain Reaction, Western Blot, Purification

Journal: Cell Death & Disease

Article Title: Immunomodulatory effect of NEDD8-activating enzyme inhibition in Multiple Myeloma: upregulation of NKG2D ligands and sensitization to Natural Killer cell recognition

doi: 10.1038/s41419-021-04104-w

Figure Lengend Snippet: A MLN4924 (200 nM) stabilized the expression of pIkBα and B ) repressed nuclear translocation of p65/RelA in SKO-007(J3) cells treated for 48 h. Data are representative of one out of two independent experiments. Densitometric analyses of normalized pIkBα or p65/Actin are shown. C IRF4 mRNA levels were quantified by Real-time PCR in SKO-007(J3) cells overexpressing a dominant-negative form of IkBα (IkBα-DN) or the empty control vector pMSCV-Neo. Data, expressed as fold change units, were normalized to GAPDH, and referred to the pMSCV-Neo infected cells considered as calibrator and represent the mean (± SEM) of 3 experiments (* P < 0.05). D SKO-007(J3) cells were transiently cotransfected with the indicated MICA promoter deletion and an expression vector for IkBα-DN (S32A/S36A) or the empty control vector (pRc-CMV). Results are expressed as Relative Luciferase activity normalized to protein concentration as well as to Renilla activity produced off the internal control plasmid and represent the mean value (±SEM) of three independent experiments (* P < 0.05). E – F MICA mRNA and cell surface expression were analyzed in SKO-007(J3) cells overexpressing a dominant-negative form of IkBα (IkBα-DN) as described above. Data shown in ( E ), expressed as fold change units, were normalized to GAPDH and referred to the pMSCV-Neo infected cells considered as calibrator, represent the mean (±SEM) of 3 experiments (* P < 0.05). A representative FACS analysis of MICA expression in infected cells is shown in ( F ).

Article Snippet: Reverse transcription was carried out in a 25 μl reaction volume with 2 μg of total RNA according to the manufacturer’s protocol for M-MLV reverse transcriptase (Promega, Madison, Wisconsin, USA). cDNAs were amplified (TaqMan assays) in triplicate with primers for MICA (Hs00792195_m1), MICB (Hs00792952_m1), IRF4 (Hs01056533_m1), IKZF1 (Hs00958474_m1), IKZF3 (Hs00232635_m1), MEIS2 (Hs00542638_m1), CRBN (Hs00372271_m1), GAPDH (Hs02758991_g1) conjugated to the fluorochrome FAM (Applied Biosystems, Foster City, California, USA).

Techniques: Expressing, Translocation Assay, Real-time Polymerase Chain Reaction, Dominant Negative Mutation, Control, Plasmid Preparation, Infection, Luciferase, Activity Assay, Protein Concentration, Produced

Journal: Cell Death & Disease

Article Title: Immunomodulatory effect of NEDD8-activating enzyme inhibition in Multiple Myeloma: upregulation of NKG2D ligands and sensitization to Natural Killer cell recognition

doi: 10.1038/s41419-021-04104-w

Figure Lengend Snippet: A , C Real-time PCR analysis of total mRNA obtained from SKO-007(J3) cells, untreated or treated with the indicated concentrations of MLN4924 for 48 h. Data, expressed as fold change units, were normalized to GAPDH, and referred to the untreated cells considered as calibrator, represent the mean ± SEM of 3 independent experiments (* P < 0.05). B , D Western blot analysis of CRBN ( B ) and MEIS2 ( D ) in SKO-007(J3) cells untreated or treated with MLN4924 for 48/72 h. β-Actin was used as protein-loading control. Data are representative of one out of two independent experiments. Densitometric analyses of the reported Western blots are shown. E MICA cell surface expression was analyzed by flow cytometry on SKO-007(J3) cells treated with MLN4924 (200 nM) and/or Lenalidomide (5 µM) following the indicated scheme. (–) and (—) indicate respectively the treatment with vehicle for 24 h and 48 h. After the first 24 h of MLN4924 or vehicle treatment, cells were washed twice in complete medium, reseeded, and stimulated with Lenalidomide or vehicle for additional 48 h. Representative overlays are shown. F Histograms represent the average of the mean fluorescence intensity (MFI) values of the indicated ligand (with treatment-specific isotype control MFI subtracted out). The MFI of MICA is representative of at least four independent experiments as shown in ( E ) and statistical significance was evaluated by paired Student t test (* P < 0.05). G Real-time PCR analysis of total mRNA obtained from SKO-007(J3) cells, untreated or treated as described above with MLN4924 (200 nM) and/or Lenalidomide (5 µM). Data, expressed as fold change units, were normalized to GAPDH, and referred to the untreated sample considered as calibrator, represent the mean (±SEM) of 3 experiments (* P < 0.05). H Western blot analysis of IRF4 in SKO-007(J3) cells untreated or treated with MLN4924 (200 nM) for 24 h and/or Lenalidomide (5 µM) for 48 h. β-Actin was used as protein-loading control. Data are representative of one out of two independent experiments. Densitometric analysis of the reported Western blot is shown. I Model: inhibition of neddylation and regulation of MICA/B expression on MM cells. The present study provides evidence that inhibition of neddylation increases NKG2D ligand MICA and MICB expression in MM via transcriptional and post-translational mechanisms respectively and suggests possible cooperation with IMiDs in this pathway.

Article Snippet: Reverse transcription was carried out in a 25 μl reaction volume with 2 μg of total RNA according to the manufacturer’s protocol for M-MLV reverse transcriptase (Promega, Madison, Wisconsin, USA). cDNAs were amplified (TaqMan assays) in triplicate with primers for MICA (Hs00792195_m1), MICB (Hs00792952_m1), IRF4 (Hs01056533_m1), IKZF1 (Hs00958474_m1), IKZF3 (Hs00232635_m1), MEIS2 (Hs00542638_m1), CRBN (Hs00372271_m1), GAPDH (Hs02758991_g1) conjugated to the fluorochrome FAM (Applied Biosystems, Foster City, California, USA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Control, Expressing, Flow Cytometry, Fluorescence, Inhibition

Journal: Advanced Science

Article Title: Targeting Immunoproteasome in Polarized Macrophages Ameliorates Experimental Emphysema Via Activating NRF1/2‐P62 Axis and Suppressing IRF4 Transcription

doi: 10.1002/advs.202405318

Figure Lengend Snippet: ONX‐0914 Suppresses M1 Polarization via NRF1 and NRF2‐P62 Signaling Pathway. A) Differential binding of transcription factors between M1+ONX‐0914 and M1 predicted by TOBIAS. This plot indicates a significant increase in NRF1 (MAFG::Nfe2l1) and NRF2 (MAF::Nfe2l2) binding score following ONX‐0914 treatment. B) Mouse genome NRF1 and NRF2 motifs obtained from JASPAR 2022 database. C) Gene Ontology enrichment analysis of genes upregulated by ONX‐0914 treatment in M1 phenotype of PMs. The y‐axis represents the GO terms, and the x‐axis indicates the gene count. D) Heatmaps displaying proteasome‐related genes, the NRF1 downstream regulated genes, in M0, M0+ONX‐0914, M1, and M1+ONX‐0914 group (n = 4). E) Volcano plot showing DEGs between ONX‐0914‐treated and untreated M1 AMs. Genes meeting dual thresholds of FDR <0.05 and log 2 (fold change) >1 are highlighted in red, indicating significant upregulation or downregulation after ONX‐0914 treatment. F) Protein expression level of NRF1 analyzed by Western blotting in ONX‐0914‐treated M1 AMs. Data was obtained from 3 independent samples in each group. G) Heatmaps displaying anti‐oxidative stress‐related genes, the NRF2 downstream regulated genes, in M0, M0+ONX‐0914, M1, and M1+ONX‐0914 group of PMs (n = 4). H) Representative IF images of NRF2 after applying 0.2 µM ONX‐0914 to AMs for 24 h. I) After AMs were pretreated with 0.2 µM ONX‐0914 for 6 h, and then stimulated with 250 µg mL −1 dosage of CSE for 24 h, the gene Nfe2l2 was examined by RT‐qPCR (n = 3). J and K) Transcriptional changes in genes associated with NRF1‐increased binding sites (J) and NRF2‐increased binding sites (K) after ONX‐0914 treatment in M1 of AMs. The plot demonstrates that the regulatory activity of upregulated genes significantly surpasses that of downregulated genes (n = 3). L) After silencing the expression of NRF1 with siRNA and treating M1‐polarized AMs with 0.2 µM ONX‐0914, the expression of Nfe2l1, Sqstm1 , and M1 marker genes ( Nos2, Il12b ) were detected by RT‐qPCR (n = 3). M) After silencing the expression of NRF2 with siRNA and treating M1‐polarized AMs with 0.2 µM ONX‐0914, the expression of Nfe2l2, Sqstm1 , and M1 marker genes ( Nos2, Il12b, Il1b, Tnf ) were detected by RT‐qPCR (n = 3). N) Integrative Genomics Viewer (IGV) plot illustrates the binding landscape of the NRF2 transcription factor to Sqstm1 in M0, M0+ONX‐0914, M1 and M1+ONX‐0914. The upper 4 tracks display the signal intensity of NRF2 binding to Sqstm1 , and the lower 4 tracks display the specific locations where NRF2 binding peaks were identified, which suggest the potential regulatory roles in Sqstm1 gene expression after ONX‐0914 treatment in M1 macrophages. O) CUT&Tag‐qPCR shows that the binding affinity of NRF2 to Sqstm1 enhancer and promoter is significantly increased under ONX‐0914 treatment in M1 macrophages. P) RT‐qPCR analysis detected Nfe2l2 , Sqstm1 and M1 marker genes Nos2 , Il12b , Il1b , and Il6 in each group (n = 3). After silencing the expression of NRF2 with siRNA and overexpression of P62 with p3xFLAG‐P62 var2 plasmid, 0.2 µM ONX‐0914 was used to treat M1‐polarized AMs, and the expression of Nfe2l2 , Sqstm1 , and M1 marker genes ( Nos2 , Il12b , Il1b and Il6 ) were detected by RT‐qPCR (n = 3). Q) After silencing the expression of NRF2 with siRNA and using 0.2 µM ONX‐0914 pretreated CSE (250 µg mL −1 ) stimulation of AMs, Sqstm1 and M1 marker genes ( Nos2, Il12b, Il1b, Tnf ) were detected by RT‐qPCR (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The plasmids used in this study included p3xFLAG‐P62 var2 (#204576, Addgene) and pMIG‐IRF4 (#58987, Addgene).

Techniques: Binding Assay, Expressing, Western Blot, Quantitative RT-PCR, Activity Assay, Marker, Gene Expression, Over Expression, Plasmid Preparation

Journal: Cancer Immunology Research

Article Title: The IKZF1–IRF4/IRF5 Axis Controls Polarization of Myeloma-Associated Macrophages

doi: 10.1158/2326-6066.cir-20-0555

Figure Lengend Snippet: Figure 4. Lenalidomide changes the polarization state by interfering with the IRF4/IRF5 homeostasis. A, ChIP sequencing (ChIP-seq) analysis demonstrates the IKZF1 binding profile at the IRF4, IRF5, and IRF3 promoters. ChIP-seq data were from the ENCODE project (GM12878). Protein levels of IKZF1, IRF4, IRF5, IRF3, and b-actin were determined by Western blotting in M2-like macrophages after treatment with increasing concentrations of lenalidomide (as indicated; B) and after different incubation periods with 1 mmol/L lenalidomide (C). D, Protein levels of IKZF1, IRF4, IRF5, IRF3, and b-actin were determined in M2-like macrophages after treatment with either lenalidomide (1 mmol/L) or specific siRNA. Lane M shows the protein marker. E, M2-like macrophages were treated for 72 hours with lenalidomide (blue, 1 mmol/L) or untreated (orange). ChIP using antibodies against tri-methyl-histone H3 (K4), tri-methyl-histone H3 (K27), or normal IgG as control was performed. Enrichment in the proximal region of the IRF4 or IRF5 promoter was quantified by qPCR (n ¼ 3). TSS, transcription start site. F, Macrophages (defined as CD163þCD15) from patients with multiple myeloma before (orange, n ¼ 12) and during IMiD therapy (blue, n ¼ 10) were analyzed by flow cytometry for expression of IRF4 or IRF5. MFI, median fluorescence intensity. Error bars show SEM (, P < 0.05; , P < 0.01).

Article Snippet: Western blotting and chromatin immunoprecipitation Antibodies specific for IKZF1 (14859S, Cell Signaling Technology), IRF3 (11904S, Cell Signaling Technology), IRF4 (4299S, Cell Signaling Technology), IRF5 (13496S, Cell Signaling Technology), c-Jun (60A8, Cell Signaling Technology), NF-kB p65 (D14E12, Cell Signaling Technology), Tri-Methyl-Histone H3 (K4) (9751P, Cell Signaling Technology), Tri-Methyl-Histone H3 (K27) (9733S, Cell Signaling Technology), IgG (2729P, Cell Signaling Technology), Tubulin (Abcam, ab7291), b-actin (AC-15, Abcam), goat anti-mouse IgGHRP (Dako, P0447), and goat anti-rabbit IgG-HRP (Cell Signaling Technology) were used for Western blotting and chromatin immunoprecipitation (ChIP).

Techniques: ChIP-sequencing, Binding Assay, Western Blot, Incubation, Marker, Control, Cytometry, Expressing

Journal: Cancer Immunology Research

Article Title: The IKZF1–IRF4/IRF5 Axis Controls Polarization of Myeloma-Associated Macrophages

doi: 10.1158/2326-6066.cir-20-0555

Figure Lengend Snippet: Figure 5. Lenalidomide treatment of CrbnI391V mice promotes the tumoricidal activity of macrophages. A, B6.CrbnI391V mice received daily intraperitoneal (i.p.) injections with either DMSO or lenalidomide (len; 10 mg/kg body weight, in PBS containing 10% DMSO) for 7 days. Subsequently, mice were sacrificed, and BM was extracted from femur, tibia, and hip bone and analyzed in vitro. B, BM macrophages (Ly6G, CD115þ, and F4/80þ) from solvent-treated mice (n ¼ 5, orange) or lenalidomide-treated mice (n ¼ 7, blue) were analyzed by flow cytometry for the expression of IKZF1, IRF4, and IRF5. C, Expression of effector molecules by BM macrophages (solvent treated: orange, n ¼ 5 and lenalidomide treated: blue, n ¼ 7) was measured by qPCR [iNOS and arginase (Arg1); arbitrary units (A.U.)] or by flow cytometry (PD-L1). Horizontal lines show mean values. Isolated BM macrophages from solvent-treated mice or lenalidomide-treated mice were coincubated with labeled myeloma cells (5TGM1, red; D) or with labeled lymphoma cells (291PC, red; E) in an E:T of 1:1 in the presence or absence of a CD38- or CD20-specific antibody, respectively. After 3 hours, ADCP was analyzed by confocal microscope. Macrophages were stained with CD11b (green). Magnifications (630) present representative areas from one of five independent experiments performed with different mice. Scale bar, 20 mm. Error bars show SEM (, P < 0.05; , P < 0.01).

Article Snippet: Western blotting and chromatin immunoprecipitation Antibodies specific for IKZF1 (14859S, Cell Signaling Technology), IRF3 (11904S, Cell Signaling Technology), IRF4 (4299S, Cell Signaling Technology), IRF5 (13496S, Cell Signaling Technology), c-Jun (60A8, Cell Signaling Technology), NF-kB p65 (D14E12, Cell Signaling Technology), Tri-Methyl-Histone H3 (K4) (9751P, Cell Signaling Technology), Tri-Methyl-Histone H3 (K27) (9733S, Cell Signaling Technology), IgG (2729P, Cell Signaling Technology), Tubulin (Abcam, ab7291), b-actin (AC-15, Abcam), goat anti-mouse IgGHRP (Dako, P0447), and goat anti-rabbit IgG-HRP (Cell Signaling Technology) were used for Western blotting and chromatin immunoprecipitation (ChIP).

Techniques: Activity Assay, In Vitro, Solvent, Cytometry, Expressing, Isolation, Labeling, Microscopy, Staining