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Estrogen receptor α contributes to T cell–mediated autoimmune inflammation by promoting T cell activation and proliferation

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Science Signaling  17 Apr 2018:
Vol. 11, Issue 526, eaap9415
DOI: 10.1126/scisignal.aap9415
  • Fig. 1 ERα deletion within T cells limits their pathogenic potential in a murine colitis model.

    (A to D) Rag1−/− mice received 400,000 sorted naïve CD4+CD45RBhiCD25 T cells isolated from ERαfl/fl (filled squares) or CD4-creERαfl/fl (open circles) mice. (A) Mice were weighed weekly to monitor the onset of colitis. Data are pooled from 22 ERαfl/fl mice and 23 CD4-creERαfl/fl mice from five independent experiments. (B) Serum tumor necrosis factor (TNF), interleukin-6 (IL-6), interferon-γ (IFN-γ), IL-12p70, monocyte chemoattractant protein-1 (MCP-1), and IL-10 were measured using a murine inflammation cytokine bead array at the time of sacrifice. Data are pooled from 15 ERαfl/fl mice and 18 CD4-creERαfl/fl mice from four independent experiments. (C) Colon sections from mice that received the indicated T cells were subjected to hematoxylin and eosin staining and clinical scoring. Data are histological images representative of 15 ERαfl/fl mice (filled squares) and 18 CD4-creERαfl/fl mice (open circles) from four independent experiments. In mice receiving ERαfl/fl T cells, there is diffuse inflammation in the mucosa, and the inflammation extends multifocally to the submucosa (arrows). The lamina propria (bar) contains colonic glands, and within each gland, there are numerous goblet cells (arrows). Original magnification, ×40; scale bar, 200 μm. (D) Development of colitis was assessed by monitoring the distribution of inflammation, degree of inflammation, and extent of erosion/ulceration. Cumulative histopathologic scores are also shown. *P < 0.05 and **P < 0.01 by two-tailed Student’s t test (A and B) or two-tailed Mann-Whitney test (C).

  • Fig. 2 ERα deletion within T cells limits their accumulation and inflammatory cytokine production in a T cell transfer model of colitis.

    (A to F) Rag1−/− mice received 400,000 sorted naïve CD4+CD45RBhiCD25 T cells isolated from ERαfl/fl mice or CD4-creERαfl/fl mice. (A) Spleens were harvested at about 9 weeks after T cell transfer and weighed. Data are means ± SEM from 11 ERαfl/fl mice and 13 CD4-creERαfl/fl mice from three independent experiments. Percentage (B) and total cell numbers (C) of spleen and MLN CD4 T cells (TCRβ+CD4+) were quantitated. Data are means ± SEM from 19 ERαfl/fl mice and 23 CD4-creERαfl/fl mice from five independent experiments. (D to F) Spleens and MLNs were harvested, and the proportions of IFN-γ+ and IL-17A+ effectors within the gated TCRβ+CD4+ population were analyzed. Representative flow cytometry plots (D) gated CD4+TCRβ+ T cells from the spleens of colitic mice from five independent experiments. Quantified data (E and F) are means ± SEM of 19 ERαfl/fl mice and 23 CD4-creERαfl/fl mice. *P < 0.05 and **P < 0.01 by two-tailed Student’s t test. SSC, side scatter.

  • Fig. 3 Transcriptomics analysis reveals reduced expression of genes involved in T cell activation in CD4-creERαfl/fl T cells.

    (A to C) RNA sequencing (RNA-seq) analysis of transcripts detected in ERαfl/fl and CD4-creERαfl/fl CD4+CD3+ T cells isolated from the spleens of colitic Rag1−/− recipient mice. Data are representative of two biological replicates. (A) Scatter plot of differentially expressed genes where orange triangles represent the genes that were increased in expression and blue diamonds represent genes that were decreased in expression in CD4+ T cells from mice receiving CD4-creERαfl/fl T cells, as compared to ERαfl/fl T cells. (B) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway gene enrichment of differentially expressed genes from ERαfl/fl and CD4-creERαfl/fl cells that were isolated from spleens of Rag1−/− recipient mice. Rich factor is measured as the ratio of number of differentially expressed genes annotated in this pathway term to number of all genes annotated in this pathway term. (C) Heatmap displaying the log fold change of differentially expressed genes from CD4-creERαfl/fl versus ERαfl/fl CD4+ T cells.

  • Fig. 4 ERα regulates T cell activation.

    (A) CD69 expression was assessed by flow cytometry on naïve T cells activated for 1 day with the indicated concentrations of anti-CD3 alone (left), anti-CD3, and the indicated concentrations of anti-CD28 (middle and right). Data are means ± SEM of three biological replicates. (B) Flow cytometry plots of CD69 and CD25 expression on ERαfl/fl and CD4-creERαfl/fl T cells after 1 day of culture with the indicated concentrations of anti-CD3 and anti-CD28. Data are representative of three independent experiments. (C) Western blot analysis of the indicated proteins in ERαfl/fl and CD4-creERαfl/fl T cells that were activated for 3 days. Left: Blots are representative of three experiments. Right: Normalized band intensity data are means ± SEM. (D) Western blot analysis of the indicated proteins in CD4-creERαfl/fl T cells transduced with retrovirus-expressing control vector (pRV) or ERα-expressing plasmid (pRV-Esr1) that were activated for 3 days. Left: Blots are representative of three independent experiments. Right: Normalized band intensity data are means ± SEM. *P < 0.1 and **P < 0.05 by paired Student’s t test.

  • Fig. 5 ERα influences T cell survival.

    (A) RNA-seq of Pdcd4 transcript in ERαfl/fl and CD4-creERαfl/fl splenic CD4+ T cells from colitic Rag1−/− recipient mice. Data are means ± SEM of fragments per kilobase of transcript per million mapped reads (FPKM) values from two biological replicates. (B and C) ERαfl/fl and CD4-creERαfl/fl CD4 T cells were activated for 3 days. (B) Cellular viability was assessed by annexin V and propidium iodide (PI) staining. Top: Flow cytometry plots are representative of three independent experiments. Bottom: Quantified data are means ± SEM. (C) Bcl2 expression was assessed by intracellular staining. Left: Flow cytometry plots are representative of three independent experiments. Right: Quantified data are means ± SEM. (D) Mitochondrial membrane potential was determined by staining ERαfl/fl and CD4-creERαfl/fl CD4 T cells that were activated for 40 min or 1 day with TMRE. Top: Flow cytometry plots are representative of three independent experiments. Bottom: Quantified data are means ± SEM. **P < 0.05 by paired Student’s t test.

  • Fig. 6 ERα contributes to T cell proliferation and is required in lymphopenia-induced expansion of CD4+ T Cells.

    (A) In vitro proliferation of CellTrace Violet–labeled ERαfl/fl and CD4-creERαfl/fl T cells stimulated with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 4 and 5 days. Right: Representative flow cytometry plots from three independent experiments. Left: Quantified data are means ± SEM. (B) Naïve CD4+ T cells were isolated from CD4-creERαfl/fl and littermate control mice. Cells were labeled with CellTrace Violet or CellTrace Yellow dye, mixed at a 1:1 ratio, and cultured together under iTreg conditions. Dye dilution was detected on day 4. Data are representative flow cytometry plots of one mouse per group from three independent experiments. (C) In vivo proliferation of carboxyfluorescein succinimidyl ester (CFSE)–labeled wild-type (WT) CD45.1+ mice and CD45.2+ CD4-creERαfl/fl CD4+ T cells transferred into Rag1−/− recipient mice. An aliquot of input cells was retained to confirm the starting ratio (input). Spleens and MLNs of recipients were harvested for analysis on day 10 or 14 after injection. Right: Flow cytometry plots of CFSE dilution for WT (CD45.1+) and CD4-creERαfl/fl (CD45.2+) T cells within the same host on day 10 are representative of three independent experiments. Left: Ratio of WT and CD4-creERαfl/fl CD4+ T cells was quantified by staining for CD45.1 and CD45.2. Representative data from one experiment are means ± SEM of three mice per group on day 10 and five mice per group on day 14. *P < 0.1 by paired Student’s t test.

  • Fig. 7 ERα modulates iTreg cell differentiation and function.

    (A) FoxP3 expression was assessed by flow cytometry of CD4-creERαfl/fl and control ERαfl/fl naïve CD4+ T cells from female mice that were activated with anti-CD3 and anti-CD28 alone (TH0) or under iTreg-inducing conditions for 3 days with or without exogenous 17β-estradiol (E2; 10 nM). Left: Flow cytometry plots are representative of three independent experiments. Right: Quantified data are means ± SEM. (B) Western blot analysis of the indicated proteins in ERαfl/fl and CD4-creERαfl/fl T cells activated for 3 days. Left: Blots are representative of three experiments. Right: Normalized band intensity data are means ± SEM. (C) Western blot analysis of CD4-creERαfl/fl CD4 T cells transduced with either control vector (pRV) or ERα-expressing vector (pRV-Esr1) and cultured under iTreg-polarizing conditions. Left: Blots are representative of three experiments. Right: Normalized band intensity data are means ± SEM. (D) RNA-seq analysis of the indicated transcripts in ERαfl/fl and CD4-creERαfl/fl splenic CD4+ T cells from colitic Rag1−/− recipient mice. FPKM data are means ± SEM from two biological replicates. (E) Intracellular Foxp3 staining of ERαfl/fl and CD4-creERαfl/fl CD4+ T cells that were harvested from the lamina propria (LP) of colitic Rag1−/− recipient mice. Right: Flow cytometry plots are representative of two independent experiments. Left: Quantified data are means ± SEM of one mouse per group. *P < 0.1 and **P < 0.05 by paired Student’s t test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/526/eaap9415/DC1

    Fig. S1. Detection of Esr1 deletion or overexpression.

    Fig. S2. ERα is not essential for T cell development.

    Fig. S3. ERα deletion within T cells limits colonic inflammation in a murine colitis model.

    Fig. S4. ERα deletion within T cells limits skin inflammation in a murine T cell transfer model.

    Fig. S5. Activation of CD4-creERαfl/fl CD4+ T cells and control T cells with a high concentration of anti-CD3 and varying doses of anti-CD28.

    Fig. S6. Analysis of selected mRNAs in splenocytes.

    Fig. S7. Oxygen consumption by ERαfl/fl and CD4-creERαfl/fl CD4+ T cells.

    Table S1. Transcriptomics analysis of gene expression changes in CD4-creERαfl/fl and control CD4+ T cells.

  • Supplementary Materials for:

    Estrogen receptor α contributes to T cell–mediated autoimmune inflammation by promoting T cell activation and proliferation

    Imran Mohammad, Inna Starskaia, Tamas Nagy, Jitao Guo, Emrah Yatkin, Kalervo Väänänen, Wendy T. Watford,* Zhi Chen*

    *Corresponding author. Email: watfordw{at}uga.edu (W.T.W.); zchen{at}utu.fi (Z.C.)

    This PDF file includes:

    • Fig. S1. Detection of Esr1 deletion or overexpression.
    • Fig. S2. ERα is not essential for T cell development.
    • Fig. S3. ERα deletion within T cells limits colonic inflammation in a murine colitis model.
    • Fig. S4. ERα deletion within T cells limits skin inflammation in a murine T cell transfer model.
    • Fig. S5. Activation of CD4-creERαfl/fl CD4+ T cells and control T cells with a high concentration of anti-CD3 and varying doses of anti-CD28.
    • Fig. S6. Analysis of selected mRNAs in splenocytes.
    • Fig. S7. Oxygen consumption by ERαfl/fl and CD4-creERαfl/fl CD4+ T cells.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Transcriptomics analysis of gene expression changes in CD4-creERαfl/fl and control CD4+ T cells.

    © 2018 American Association for the Advancement of Science

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