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. 2010 Sep 15;185(6):3489-97.
doi: 10.4049/jimmunol.0903610. Epub 2010 Aug 11.

V体育官网 - Conditional deletion of histone deacetylase 1 in T cells leads to enhanced airway inflammation and increased Th2 cytokine production

Reinhard Grausenburger  1 , Ivan Bilic, Nicole Boucheron, Gordin Zupkovitz, Lamia El-Housseiny, Roland Tschismarov, Yu Zhang, Martina Rembold, Martin Gaisberger, Arnulf Hartl, Michelle M Epstein, Patrick Matthias, Christian Seiser, Wilfried Ellmeier
Affiliations

Conditional deletion of histone deacetylase 1 in T cells leads to enhanced airway inflammation and increased Th2 cytokine production

Reinhard Grausenburger et al. J Immunol. .

Abstract

Chromatin modifications, such as reversible histone acetylation, play a key role in the regulation of T cell development and function. However, the role of individual histone deacetylases (HDACs) in T cells is less well understood. In this article, we show by conditional gene targeting that T cell-specific loss of HDAC1 led to an increased inflammatory response in an in vivo allergic airway inflammation model. Mice with HDAC1-deficient T cells displayed an increase in all critical parameters in this Th2-type asthma model, such as eosinophil recruitment into the lung, mucus hypersecretion, parenchymal lung inflammation, and enhanced airway resistance VSports手机版. This correlated with enhanced Th2 cytokine production in HDAC1-deficient T cells isolated from diseased mice. In vitro-polarized HDAC1-deficient Th2 cells showed a similar enhancement of IL-4 expression, which was evident already at day 3 of Th2 differentiation cultures and restricted to T cell subsets that underwent several rounds of cell divisions. HDAC1 was recruited to the Il4 gene locus in ex vivo isolated nonstimulated CD4(+) T cells, indicating a direct control of the Il4 gene locus. Our data provide genetic evidence that HDAC1 is an essential HDAC that controls the magnitude of an inflammatory response by modulating cytokine expression in effector T cells. .

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Figures

FIGURE 1
FIGURE 1
Targeting strategy for the generation of a conditional Hdac1 allele. A, Schematic map of the endogenous Hdac1 locus (upper part, Hdac1+), the targeted Hdac1 locus after homologous recombination (upper middle part), the targeted Hdac1 locus after Cre recombinase-mediated deletion of the neomycin cassette (lower middle part; “floxed” allele, Hdac1F), and the Hdac1 locus after Cre-mediated deletion of exon 6 (lower part, Hdac1D), as previously shown (17). The filled arrowheads indicate loxP sites. B, PCR analysis of DNA isolated from Hdac1+/F (wt), Hdac1F (F), or Hdac1Δ (D) CD4+ T cells. The location of the genotyping primers A, B, and C is shown at the left. All three primers were used in one PCR reaction to detect the various alleles. wt, 487 bp; F, 567 bp; Δ, 535 bp. C, PCR analysis of DNA isolated from Hdac1F (F) and Hdac1Δ (Δ) double-negative, DP, and CD4 SP thymocytes.
FIGURE 2
FIGURE 2
Conditional deletion of HDAC1 using transgenic Cd4-Cre does not affect thymocyte development or the generation of peripheral T cells. A, CD4/CD8 expression on thymocytes from Hdac1F and Hdac1Δ mice. B, Total thymocytes and thymocyte subset cell numbers in Hdac1F (n = 4) and Hdac1Δ (n = 7) mice. Data are mean ± SD. C, Immunoblot analysis showing the expression of HDAC1, HDAC2, HDAC3, HDAC6, HDAC7, and HDAC9 in sorted DP and CD4SP thymocytes, in splenic CD4+ T cells, CD8+ T cells, and B220+ B cells from Hdac1F and Hdac1Δ mice. Whole-cell lysates from 3 × 106 cells were used for each population. β-actin levels were used as loading control. Data are representative of two independent experiments. D, CD3/B220 expression profiles of Hdac1F and Hdac1Δ splenocytes. E, CD4/CD8 expression profiles of Hdac1F and Hdac1Δ splenocytes. F, Cell numbers of total splenocytes and of CD3+, B220+, CD3+CD4+, and CD3+CD8+ subsets in Hdac1F (n = 4) and Hdac1Δ (n = 5) mice. Data are mean ± SD. G, CD44/CD62L expression pattern on peripheral splenic CD4+ T cells from Hdac1F and Hdac1Δ mice. H, CD25/Foxp3 expression pattern on peripheral splenic CD4+ T cells from Hdac1F and Hdac1Δ mice. A, D and E, Data are representative of at least four Hdac1F and 7 Hdac1Δ mice. The numbers indicate the percentage of cells in the quadrant. G and H, Data are representative of three mice analyzed. The numbers indicate the percentage of cells in the indicated regions.
FIGURE 3
FIGURE 3
Enhanced allergic airway disease in HDAC1-deficient mice. Hdac1F and Hdac1Δ mice were immunized i.p. twice with OVA, followed by OVA aerosol challenges (OVA; n = 5). Untreated Hdac1F and Hdac1Δ mice were used as control (con; n = 3). A, BAL differential cell count. Mean ± SEM are shown. B, Eosinophil numbers at specified areas of lung parenchyma. Luna-stained lung tissue sections were counted with a 100× oil-immersion objective. C, Representative photomicrographs of lung sections. Arrows indicate fuchsia-stained mucus in epithelial goblet cells. Scale bar, 50 μm; original magnification ×400. D, Grading of mucus production. The distribution of goblet cells in the central and peripheral airways was scored on periodic-acid Schiff reagent-stained sections. Grade 0, no goblet cells; grade 1, >20%; grade 2, 20–40%; grade 3, 41–60%; grade 4, 61–80%; and grade 5; >80% goblet cells. E, Representative photomicrographs of H&E-stained lung tissue sections. Areas of highest-density inflammation are shown. Arrows indicate infiltrates. Scale bar, 200 μm; original magnification ×100. F, OVA-specific IgG1 titers of sera harvested 2 d after the last OVA-aerosol challenge. Mean ± SEM is shown. Data in A–F are representative of three independent experiments. Airway resistance (G) and dynamic compliance (H) in response to MCh. The bsl value was set as 1, and values relative to baseline are shown. Data show summary of eight independent mice per genotype. *p < 0.05; **p < 0.01, unpaired Student t test. AW, airways; bsl, baseline; BV, blood vessel; CA, central airways; CL, central lung; con, control; E, eosinophil; L, lymphocyte; M, macrophage; MCh, metacholine; MU, mucus; N, neutrophil; PA, peripheral airways; PL, peripheral lung.
FIGURE 4
FIGURE 4
Enhanced Th2 cytokine production in diseased Hdac1Δ mice. A, Lung cells from imm or nonimmunized con Hdac1F and Hdac1Δ mice (CD45.2+) were isolated and stimulated for 8 h with gamma-irradiated CD45.1+ OVA-loaded splenocytes, as described in Materials and Methods. Dot plots show representative CD44 versus intracellular IL-5 (left panel) or IL-13 (right panel) on gated CD4+CD45.2+ cells. Numbers in the quadrants indicate the percentage of cells in the respective quadrant. B, Summary of all individual CD4+CD45.2+ lung cells analyzed as described in A. Experiments were performed in two independent batches with at least three mice per experiment. Each dot represents a mouse. The p values were calculated using an unpaired Student t test. C, Lung cells from imm or nonimmunized Hdac1F and Hdac1Δ mice were isolated and stimulated for 5 h with PMA/ionomycin as described in Materials and Methods. Dot plots show CD44 versus intracellular IL-4 (upper panel), IL-5 (middle panel), or IL-13 (lower panel) expression on CD4+ CD45.2+ cells. Each dot plot shows the pool of lung cells isolated from three mice (lung cells from mice were pooled prior to activation and staining). One representative experiment of two is shown. D, IL-4, IL-5, and IL-13 cytokine production of OVA-restimulated CD8+ T cell-depleted splenocytes from imm or nonimmunized con Hdac1F and Hdac1Δ mice. Cytokine levels in the supernatant were determined 72 h after restimulation. Mean ± SEM are shown. Data are the summary of three (for IL-4 and Il-5) or two (for IL-13) independent experiments with at least four mice per experiment for diseased mice (n = 13 for IL-4 and IL-5; n = 9 for IL-13) and at least two mice per experiment for control mice (n = 7 for IL-4 and IL-5; n = 5 for IL-13). *p < 0.05, unpaired Student t test. Δ, Hdac1Δ; con, control; F, Hdac1F ; imm, immunized and diseased; n.s., not significant.
FIGURE 5
FIGURE 5
Th1 and Th2 cells show augmented cytokine production in the absence of HDAC1. Relative cytokine levels in the supernatant of polarized Th2 (A) and Th1 (B) cells. In each experiment, Hdac1F cytokine levels were set as 1 (= 100%), and the relative level of Hdac1Δ Th2 or Th1 T cells was calculated. Each dot represents one independent experiment. Cytokine levels were determined with ELISA. The p values were calculated using a Wilcoxon signed-rank test. The cytokine levels in individual batches of Hdac1F Th1 cultures were in the range of 9.9–1420 ng/ml for IFN-γ and 1.5–2.7 ng/ml for IL-13. For Hdac1F Th2 cultures, the cytokines levels were in the range of 5.1–21.9 ng/ml for IL-4, 0.6–2.3 ng/ml for IL-5, 0.7–17 ng/ml for IL-10, and 4.5–10.9 ng/ml for IL-13. n.d., not detectable.
FIGURE 6
FIGURE 6
Elevated cytokine expression after 3 d of Th2-polarizing conditions. A, CFSE-labeled Hdac1F and Hdac1Δ CD4+ T cells were cultured under Th2-polarizing conditions for 3 d and restimulated with PMA/ionomycin for 4 h. Intracellular IL-4 (upper panels) and IL-2 (middle panels) cytokine expression was determined. The rectangle outlined in bold in the dot plots indicates gating regions to determine the percentage (the numbers next to the rectangle) of cytokine-positive CD4+ T cells, whereas the vertical lines in the rectangle separate the populations that underwent zero, one, two, three, four, and five rounds of cell division. The numbers above the CFSE graph (lower panels) indicate the percentage of cells that underwent zero, one, two, three, four, and five cell divisions. B, Summary of all experiments (n = 6) performed as described in A. The percentage of IL-4– and IL-2–expressing CD4+ T cells after zero, one, two, three, four, and five cell divisions are shown in the upper and middle panel, respectively, whereas the percentage of CD4+ T cells that underwent zero, one, two, three, four, and five cell divisions is shown in the lower panel. The p values were calculated using the Student t test (paired, two-tailed). Δ, Hdac1Δ CD4+ T cells; F, Hdac1F CD4+ T cells.
FIGURE 7
FIGURE 7
HDAC1 binds to several cis-regulatory sites at the Th2 gene complex in nonstimulated CD4+ T cells. A, Upper panel, Map of the Il4 and Il13 gene loci indicating the location of exons, the identified DNase I HSs, and the neighboring Kif3a and Rad50 genes (45). Horizontal arrows above the genes indicate the transcriptional orientation. Lower panel, Semi-quantitative PCR analysis of chromatin immunopre-cipitated with an HDAC1 Ab from sorted primary F and Δ CD4+ T cells. The location of the 11 amplicons within known HSs spanning the Il4 and Il13 genes are indicated by the lines. The precipitation is compared with the 1:80 dilution of input DNA. One representative PCR amplification (from experiment 1) of three independent ChIP assays is shown, and the summary for all experiments is shown below. Boxed amplicons indicate similar results observed in all three ChIP experiments. B, Quantitative real-time PCR analysis of chromatin immunoprecipitated with an HDAC1 Ab from sorted primary Hdac1F (F) and Hdac1Δ (Δ) CD4+ T cells. The location of the 11 amplicons is shown in A. Data represent the summary of two independent experiments. +, binding; −, no binding; Δ, Hdac1Δ; F, Hdac1F; nd, not done.
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