Journal: Cell death and differentiation

Article Title: Acetylation licenses Th1 cell polarization to constrain Listeria monocytogenes infection.

doi: 10.1038/s41418-022-01017-9

Figure Lengend Snippet: Fig. 1 Epigenetic compound library screening identifies HDAC6 as an inhibitor of Th1 cell differentiation. A Relative expression of Ifng in CD4+ T cells on day 3 of induction into Th1 cell subsets treated with epigenetic compounds (black dots). The top eight compounds that induced expression of Ifng by more than sixteen-fold over that induced by vehicle (mock) treatment are listed on the right. Blue dots, compounds that preferentially target HDAC. B qPCR analysis of the mRNA level of Ifng in CD4+ T cells on day 3 of induction into Th1 cell subsets in the absence or presence of HDACs inhibitors. TSA, trichostatin. Representative fluorescence-activated cell sorting (FACS) plots (C) and population frequencies (D) of Th1 cells polarized in the presence or absence of the indicated inhibitors are shown. E Listeria CFUs per spleen (left) and liver (right) of WT mice infected with 1 ×104 L. monocytogenes i.v. and treated with vehicle saline (mock), TSA (1 mg/kg), Nex A (3 mg/kg) or Ricolinostat (3 mg/kg) accordingly i.p. Mice were sacrificed for the following analysis on day 5 post infection. Colored dots represent individual mice (n = 6). Bars represent the mean log CFU/organ. F Survival rate of mice from Fig. 1E (n = 7). G, H Representative FACS plots (G) and cell number (H) of CD4+ IFNγ+ T cells in the spleen and liver of mice from Fig. 1E are shown. The data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001, n.s means not significant (Student’s t test).

Article Snippet: Naïve CD4+ T cells were isolated by magnetic sorting with a Miltenyi Biotec CD4+ CD62L+ T Cell Isolation kit, according to the manufacturer’s directions (Miltenyi Biotec, 130-106-643).

Techniques: Drug discovery, Cell Differentiation, Expressing, FACS, Infection, Saline

Journal: Cell death and differentiation

Article Title: Acetylation licenses Th1 cell polarization to constrain Listeria monocytogenes infection.

doi: 10.1038/s41418-022-01017-9

Figure Lengend Snippet: Fig. 2 Hdac6-deficient mice are resistant to Listeria infection. Representative FACS plots (A) and population frequencies (B) of Th1 cells polarized from wild-type (WT) or Hdac6–/– naïve CD4+ T cells are shown. C Listeria CFUs per spleen (left) and liver (right) of WT and Hdac6−/−

Article Snippet: Naïve CD4+ T cells were isolated by magnetic sorting with a Miltenyi Biotec CD4+ CD62L+ T Cell Isolation kit, according to the manufacturer’s directions (Miltenyi Biotec, 130-106-643).

Techniques: Infection

Journal: Cell death and differentiation

Article Title: Acetylation licenses Th1 cell polarization to constrain Listeria monocytogenes infection.

doi: 10.1038/s41418-022-01017-9

Figure Lengend Snippet: Fig. 3 HDAC6 and CBP reversibly modify the acetylation of STAT4. Coimmunoprecipitation (CoIP) and immunoblot analysis of HEK293T cells (A) transfected with the indicated constructs or primary CD4+ T cells (B). Immunoblot analysis of IL-12-treated CD4+ T cells (C) or polarized CD4+ T cells (D) after immunoprecipitation with an anti-STAT4 antibody. E qPCR analysis of the mRNA level of Ifng in CD4+ T cells on day 3 of induction into Th1 cell subsets in the absence or presence of HATs inhibitors. F Representative FACS plots of Th1 cells polarized in the presence or absence of HATs inhibitors are shown. G Immunoblot analysis of HEK293T cells transfected with the indicated constructs followed by immunoprecipitation with an anti-myc antibody. H CoIP and immunoblot analysis of primary CD4+ T cells treated with or without IL-12. I Representative FACS plots of Th1 cells polarized from CBP-knockdown and control naïve CD4+ T cells are shown. J Immunoblot analysis of STAT4 acetylation in HEK293T cells transfected with the indicated constructs. K Immunoblot analysis of STAT4 acetylation incubated in an in vitro system. The data are representative of three independent experiments with similar results or shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student’s t test).

Article Snippet: Naïve CD4+ T cells were isolated by magnetic sorting with a Miltenyi Biotec CD4+ CD62L+ T Cell Isolation kit, according to the manufacturer’s directions (Miltenyi Biotec, 130-106-643).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Knockdown, Control, Incubation, In Vitro

Journal: Cell death and differentiation

Article Title: Acetylation licenses Th1 cell polarization to constrain Listeria monocytogenes infection.

doi: 10.1038/s41418-022-01017-9

Figure Lengend Snippet: Fig. 4 Acetylation of STAT4-K667 is required for Th1 cell differentiation. A The IRF-luciferase reporter activity assay was performed in HEK293T cells transfected with empty vector (EV), WT or mutant STAT4 constructs. B Mass spectrometry analysis of purified STAT4 protein revealed the acetylation of STAT4 at two lysine residues. C Sequence alignment showing that the acetylated lysine residue 667 of STAT4 is conserved in both humans and mice species, as well as in the STAT protein family. D Immunoblot analysis of STAT4-K667 acetylation in HEK293T cells transfected with the indicated constructs. E Immunoblot analysis of polarized CD4+ T cells after immunoprecipitation with an anti-STAT4 antibody. F Immunoblot analysis of STAT4-K667 acetylation in CD4+ T cells pretreated with PBS or SGC-CBP30 (1 μM) for 12 h followed by IL-12 treatment or not. G Immunoblot analysis of STAT4-K667 acetylation in CD4+ T cells primed into Th1 cells in the absence or presence of the indicated inhibitors. H Mass spectrometry analysis of acetylated STAT4-K667 peptide which were incubated with HDAC6 or not under in vitro system. I, J Representative FACS plots (I) and population frequencies (J) of Th1 cells polarized from naïve Stat4−/−CD4+

Article Snippet: Naïve CD4+ T cells were isolated by magnetic sorting with a Miltenyi Biotec CD4+ CD62L+ T Cell Isolation kit, according to the manufacturer’s directions (Miltenyi Biotec, 130-106-643).

Techniques: Cell Differentiation, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Construct, Mass Spectrometry, Sequencing, Residue, Western Blot, Immunoprecipitation, Incubation, In Vitro

Journal: Cell death and differentiation

Article Title: Acetylation licenses Th1 cell polarization to constrain Listeria monocytogenes infection.

doi: 10.1038/s41418-022-01017-9

Figure Lengend Snippet: Fig. 5 Acetylation of STAT4-K667 is required for phosphorylation of Y693 and STAT4 nuclear translocation. A Immunoblot analysis of STAT4-Y693 phosphorylation levels in A1.1 cells stably transfected with STAT4-constructs treated with or without IL-12. B, C Immunoblot analysis of STAT4-Y693 phosphorylation levels in HEK 293 T cells transfected with the indicated constructs. Immunoblot analysis of STAT4-Y693 phosphorylation levels in control or CBP-knockdown (D) or HDAC6-knockdown (E) CD4+ T cells treated with IL-12 or not. F Left, immunoblot analysis of STAT4 acetylation and phosphorylation levels in WT or HDAC6-deficient CD4+ T cells treated with or without IL-12. Right, quantification of the relative modified STAT4 level from three independent experiments. G Mean flour intensity of phosphorylated STAT4-Y693 in WT and Hdac6−/−naïve CD4+ T cells on day 4 of induction into Th1 cell subsets. H Mean flour intensity of acetylated STAT4-K667 in CD4+

Article Snippet: Naïve CD4+ T cells were isolated by magnetic sorting with a Miltenyi Biotec CD4+ CD62L+ T Cell Isolation kit, according to the manufacturer’s directions (Miltenyi Biotec, 130-106-643).

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Stable Transfection, Transfection, Construct, Control, Knockdown

Journal: Cell death and differentiation

Article Title: Acetylation licenses Th1 cell polarization to constrain Listeria monocytogenes infection.

doi: 10.1038/s41418-022-01017-9

Figure Lengend Snippet: Fig. 6 Stat4K667R mice display impaired Th1 cell differentiation and are more susceptible to Listeria infection. Representative FACS plots (A) and population frequencies (B) of Th1 cells polarized from WT, Stat4K667R or Stat4–/– naïve CD4+ T cells are shown. C Top 100 differentially expressed genes from RNA-seq among WT, KO (Stat4–/–) and KR (Stat4K667R) Th1 cells. D Volcano plot of significantly altered genes between WT and KR Th1 cells. E Hierarchical clustering of Pearson correlations of DESeq2 normalized counts for the RNA-seq libraries among WT, KO and KR Th1 cells. Colors represent the Pearson correlation coefficient. F GSEA plots of KR Th1 cells show downregulation of Th1 cell differentiation. G GO analysis of WT and KR Th1 cells for the top differentially regulated gene sets. H Listeria CFUs per spleen (left) and liver (right) of WT, Stat4−/−and Stat4K667R mice infected with 1 ×104 L. monocytogenes (n = 6). Representative FACS plots (I) and cell number (J) of CD4+ IFNγ+ T cells in the spleen and liver of mice from Fig. 6H are shown. The data are representative of three independent experiments with similar results or shown as the mean ± SD of three independent experiments. ***p < 0.001 (Student’s t test).

Article Snippet: Naïve CD4+ T cells were isolated by magnetic sorting with a Miltenyi Biotec CD4+ CD62L+ T Cell Isolation kit, according to the manufacturer’s directions (Miltenyi Biotec, 130-106-643).

Techniques: Cell Differentiation, Infection, RNA Sequencing

Journal: Cell death and differentiation

Article Title: Acetylation licenses Th1 cell polarization to constrain Listeria monocytogenes infection.

doi: 10.1038/s41418-022-01017-9

Figure Lengend Snippet: Fig. 7 HDAC6 inhibitors reduce Listeria infection in a STAT4-K667 dependent manner. Representative FACS plots (A) and population frequencies (B) of Th1 cells polarized from WT or Stat4K667R naïve CD4+ T cells are shown. WT or Stat4K667R naïve CD4+ T cells were cultured in the absence or presence of HDACs inhibitors. C qPCR analysis of the mRNA level of Ifng in the CD4+ T cells from Fig. 7A. D Listeria CFUs per spleen (left) and liver (right) of WT, Stat4−/−and Stat4K667R mice infected with L. monocytogenes i.v. and treated with vehicle saline, TSA, Nex A or Ricolinostat accordingly i.p. (n = 6). E Schematic diagram of Th1 polarization induced by STAT4-K667 acetylation during Listeria infection. The data are representative of three independent experiments with similar results or shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student’s t test).

Article Snippet: Naïve CD4+ T cells were isolated by magnetic sorting with a Miltenyi Biotec CD4+ CD62L+ T Cell Isolation kit, according to the manufacturer’s directions (Miltenyi Biotec, 130-106-643).

Techniques: Infection, Cell Culture, Saline