Journal: Immunity

Article Title: The Short Chain Fatty Acid Butyrate Imprints an Antimicrobial Program in Macrophages

doi: 10.1016/j.immuni.2018.12.018

Figure Lengend Snippet: Increase of Antimicrobial LC3-Associated Immune Defense in Butyrate Macrophages (A) Extracellular acidification rate (ECAR) measured at steady state in control and butyrate macrophages. Data represent the mean of nine biological replicates from three independent experiments. (B–D) Quantification of glycolysis (B), glycolytic capacity (C) and glycolytic reserve (D). (E) Heatmap of metabolites that were significantly higher or lower in control and butyrate macrophages as detected by mass-spectrometry (left panel). Results are from five healthy donors. Right panel: Fold change of all significantly higher or lower metabolites in control macrophages (black closed circles) and butyrate macrophages (red closed circles). (F) AMPK phosphorylation (Thr172) measured by ELISA (n = 4 individual donors). (G) Percentage of pS6 (left) and representative blot of phosphorylation and quantification (right) of the ribosomal protein S6 and β-actin in control and butyrate macrophages at steady state. (H) Gentamicin protection assay performed on control macrophages, butyrate macrophages, and butyrate macrophages treated for 2 h with the mTOR activator MHY1485 (20 mM). (I) Representative immunoblot of the expression of LC3-II, P62, and β-actin at steady state or after 2 h infection with Salmonella . (J and K) Protein quantification performed by ImageJ of LC3-II (I) and P62 (J) compared to β-actin. (L) Degradation of GFP- Salmonella and LC3 induction was assessed by confocal microscopy. Representative images and quantification of GFP fluorescence and LC3 accumulation as outlined in Figure S7 C. Data from six independent donors in two independent experiments. Scale bar 5 μm. Each dot represents one donor. Statistical significance was determined using Mann-Whitney U test ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Please also see and .

Article Snippet: Proteins were detected using primary antibodies against LC3B (D11, Cell signaling), β-actin (13E5, Cell signaling), phospho-S6 ribosomal protein (Ser235 and/or 236) (D57.2.2E, Cell signaling), S100A8 (EPR3554, Abcam), acetylated-H3 (ab47915, Abcam) and acetylated-H4 (EPR16606, Abcam), P62 lck (clone 3/P62, BD bioscience) and detected using HRP-conjugated secondary antibodies.

Techniques: Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Infection, Confocal Microscopy, Fluorescence, MANN-WHITNEY

Journal: Immunity

Article Title: The Short Chain Fatty Acid Butyrate Imprints an Antimicrobial Program in Macrophages

doi: 10.1016/j.immuni.2018.12.018

Figure Lengend Snippet: Induction of Antimicrobial Activity by Butyrate in Macrophages In Vivo (A) WT mice received sodium butyrate in drinking water (150 mM final concentration) or PBS control for 7 days. At day 7, colonic segments were digested, macrophages were isolated by flow cytometry sorting and a gentamicin protection assay was performed. Each dot represents macrophages pooled from ten mice. Four independent experiments are shown. (B) Mouse bone marrow progenitor cells from mice gavaged with sodium butyrate or with PBS were differentiated into macrophages in the presence of M-CSF or with M-CSF with butyrate as a positive control. A gentamycin assay was performed at day 7 of differentiation. (C and D) Mice received butyrate or PBS 5 days prior to oral infection with Salmonella typhymurium def aroA (1 × 10 9 bacteria/mouse). 2 days post-infection bacterial dissemination was assessed in MLN, spleen, liver (C), and caecum (D). Each dot represents a mouse. (E) Colitis score of control and butyrate-treated mice either uninfected or infected with Salmonella . (F) Representative H&E stained colon sections from control and butyrate-treated mice either uninfected or infected with Salmonella (original magnification 100x). (G and H) Mice were treated with 150 mM sodium butyrate or with PBS 3 days prior and every other day after oral infection with Citrobacter rodentium (1 × 10 9 bacteria/mouse). Mice were weighed daily. Lines represents mean of 3 mice (G). At day 7 post infection bacterial dissemination was assessed in the spleen and in the liver (H). Each dot represents a mouse. Statistical significance was determined using Mann-Whitney U test ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Please also see Figure S6 .

Article Snippet: Proteins were detected using primary antibodies against LC3B (D11, Cell signaling), β-actin (13E5, Cell signaling), phospho-S6 ribosomal protein (Ser235 and/or 236) (D57.2.2E, Cell signaling), S100A8 (EPR3554, Abcam), acetylated-H3 (ab47915, Abcam) and acetylated-H4 (EPR16606, Abcam), P62 lck (clone 3/P62, BD bioscience) and detected using HRP-conjugated secondary antibodies.

Techniques: Activity Assay, In Vivo, Concentration Assay, Isolation, Flow Cytometry, Positive Control, Infection, Staining, MANN-WHITNEY

Journal: Immunity

Article Title: The Short Chain Fatty Acid Butyrate Imprints an Antimicrobial Program in Macrophages

doi: 10.1016/j.immuni.2018.12.018

Figure Lengend Snippet:

Article Snippet: Proteins were detected using primary antibodies against LC3B (D11, Cell signaling), β-actin (13E5, Cell signaling), phospho-S6 ribosomal protein (Ser235 and/or 236) (D57.2.2E, Cell signaling), S100A8 (EPR3554, Abcam), acetylated-H3 (ab47915, Abcam) and acetylated-H4 (EPR16606, Abcam), P62 lck (clone 3/P62, BD bioscience) and detected using HRP-conjugated secondary antibodies.

Techniques: Enzyme-linked Immunosorbent Assay, Generated, Expressing

Journal: bioRxiv

Article Title: Cell Programmed Nutrient Partitioning in the Tumor Microenvironment

doi: 10.1101/2020.08.10.238428

Figure Lengend Snippet: a-b , Representative OCR ( a ) and ECAR ( b ) tracings of MC38 tumor cell fractions with indicated injections of oligomycin (O), FCCP (F), and rotenone and antimycin A (R/AA). c-d Basal mitochondrial OCR ( c ) and cellular ECAR ( d ) of MC38 tumor fractions (n=5 mice). e , Unsupervised cluster analysis of differentially expressed metabolic mRNA transcripts of whole tumor, CD45-cancer cell, CD45+ CD11b+ F4/80hi TAM, CD45+ CD11b+ F4/80lo myeloid cell, CD45+ CD3+ CD8a+ T cell, and CD45+ CD3+ CD8a-(CD4+) T cell flow-sorted MC38 tumor populations (n=2 mice). f-g , Glucose transporter ( f ) and hexokinase ( g ) mRNA transcript levels of indicated MC38 tumor populations (n=2 mice). Dotted line approximates limit of detection. hi , Representative flow cytometry plots ( h ) and quantification ( i ) of pS6 levels in indicated MC38 tumor and spleen populations. j-k , Representative flow cytometry histograms ( j ) and quantification ( k ) of pS6 levels in cancer cells (CD45-CA9+), myeloid cells (CD45+ CD11b+ CD14+), T cells (CD45+ CD3+), and other immune cells (CD45+ CD3-CD14-) from patient ccRCC tumor and PBMC (n=4 patients). Each data point represents a biological replicate and error bars are SEM. a-d and f-g are representative of at least two independent experiments. P values were calculated using Welch’s 2-tailed t-test for (c-d) and Brown-Forsythe ANOVA test for (h, j). * p <0.05, ** p <0.01, *** p <0.001. ECAR: extracellular acidification rate; FCCP: Trifluoromethoxy carbonylcyanide phenylhydrazone; FMO: fluorescence minus one; MFI: median fluorescence intensity; OCR: oxygen consumption rate; PBMC: peripheral blood mononuclear cells; pS6: phosphorylated ribosomal protein S6 (Ser235/236).

Article Snippet: The anti-mouse and cross-reactive antibodies used were: CD45 BV510 (40-F11, Biolegend 103138), B220 e450 (RA3-6B2, ThermoFisher 48-0452-82), CD11b e450 (M1/70, ThermoFisher 48-0112-82), CD11b FITC (M1/70, Biolegend 101206), CD8a AF488 (53-6.7, Biolegend 100723), Ly6C FITC (HK1.4, Biolegend 128006), CD11c PE (N418, BioLegend 117308), FOXP3 PE (FJK-16s, ThermoFisher 12-5773-82), pS6 Ser235/236 PE (D57.2.2E, Cell Signaling 5316S), CD4 PerCP-Cy5.5 (RM4-5, BioLegend 100540), Ly6G PerCP-Cy5.5 (1A8, BioLegend 127616), F4/80 PE-Cy7 (BM8, BioLegend 123114), NKp46 PE-Cy7 (29A1.4, BioLegend 137618), CD3 PE-Cy7 (17A2, BioLegend 100220), CD3 FITC (17A2, BioLegend 100204), CD3 APC (17A2, BioLegend 100236), CD206 APC (C068C2, BioLegend 141708), GLUT1 AF647 (EPR3915, Abcam ab195020), EPCAM PE (G8.8, BioLegend 118206), Thy1.1 PerCP-Cy5.5 (HIS51, ThermoFisher 45-090082), CD45 PE (30-F11, ThermoFisher 12-0451-83), Ly6C BV570 (HK1.4, BioLegend 128030), CD68 BV605 (FA-11, BioLegend 137021).

Techniques: Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Cell Programmed Nutrient Partitioning in the Tumor Microenvironment

doi: 10.1101/2020.08.10.238428

Figure Lengend Snippet: a , Sorting gates of MC38 tumor cells used for mRNA transcript analyses. b , Expression of selected cell identity markers in flow sorted MC38 tumor cell population. Dotted line approximates limit of detection. c-d , GLUT1 levels determined by flow cytometry in MC38 ( c ) and CT26 ( d ) tumor populations. e, Quantification of pS6 levels determined by flow cytometry in indicated CT26 tumor and spleen populations. Each data point represents a biological replicate and error bars are SEM. c-e are representative of independent experiments performed at least twice. P values were calculated using the Brown-Forsythe ANOVA test. *** p <0.001.

Article Snippet: The anti-mouse and cross-reactive antibodies used were: CD45 BV510 (40-F11, Biolegend 103138), B220 e450 (RA3-6B2, ThermoFisher 48-0452-82), CD11b e450 (M1/70, ThermoFisher 48-0112-82), CD11b FITC (M1/70, Biolegend 101206), CD8a AF488 (53-6.7, Biolegend 100723), Ly6C FITC (HK1.4, Biolegend 128006), CD11c PE (N418, BioLegend 117308), FOXP3 PE (FJK-16s, ThermoFisher 12-5773-82), pS6 Ser235/236 PE (D57.2.2E, Cell Signaling 5316S), CD4 PerCP-Cy5.5 (RM4-5, BioLegend 100540), Ly6G PerCP-Cy5.5 (1A8, BioLegend 127616), F4/80 PE-Cy7 (BM8, BioLegend 123114), NKp46 PE-Cy7 (29A1.4, BioLegend 137618), CD3 PE-Cy7 (17A2, BioLegend 100220), CD3 FITC (17A2, BioLegend 100204), CD3 APC (17A2, BioLegend 100236), CD206 APC (C068C2, BioLegend 141708), GLUT1 AF647 (EPR3915, Abcam ab195020), EPCAM PE (G8.8, BioLegend 118206), Thy1.1 PerCP-Cy5.5 (HIS51, ThermoFisher 45-090082), CD45 PE (30-F11, ThermoFisher 12-0451-83), Ly6C BV570 (HK1.4, BioLegend 128030), CD68 BV605 (FA-11, BioLegend 137021).

Techniques: Expressing, Flow Cytometry