The Transcriptional Repressor Gfi1 Plays a Critical Role in the Development of NKT1- and NKT2-Type iNKT Cells

<div><p>Gfi1 plays an important role in the development and maintenance of many hematopoietic linage cells. However, the impact of <i>Gfi1</i>-deficiency on the iNKT cell differentiation remains unclear. We herein demonstrate a critical role of Gfi1 in regulating the development of iNKT cell subsets. In the thymus of T cell-specific <i>Gfi1</i>-deficient mice, iNKT cells normally developed up to stage 2, while the number of stage 3 NK1.1^pos iNKT cells was significantly reduced. Furthermore, CD4^pos iNKT cells were selectively reduced in the peripheral organs of T cell-specific <i>Gfi1</i>-deficient mice. The α-GalCer-dependent production of IFN-γand Th2 cytokines, but not IL-17A, was severely reduced in T cell-specific <i>Gfi1</i>-deficient mice. In addition, a reduction of the α-GalCer-induced anti-tumor activity was observed in <i>Gfi1</i>-deficient mice. These findings demonstrate the important role of Gfi1 in regulating the development and function of NKT1- and NKT2-type iNKT cell subsets.</p></div

A Novel Small Compound SH-2251 Suppresses Th2 Cell-Dependent Airway Inflammation through Selective Modulation of Chromatin Status at the <i>Il5</i> Gene Locus

<div><p>IL-5 is a key cytokine that plays an important role in the development of pathological conditions in allergic inflammation. Identifying strategies to inhibit IL-5 production is important in order to establish new therapies for treating allergic inflammation. We found that SH-2251, a novel thioamide-related small compound, selectively inhibits the differentiation of IL-5-producing Th2 cells. SH-2251 inhibited the induction of active histone marks at the <i>Il5</i> gene locus during Th2 cell differentiation. The recruitment of RNA polymerase II, and following expression of the Th2 cell-specific intergenic transcripts around the <i>Il5</i> gene locus was also inhibited. Furthermore, Th2 cell-dependent airway inflammation in mice was suppressed by the oral administration of SH-2251. Gfi1, a transcriptional repressor, was identified as a downstream target molecule of SH-2251 using a DNA microarray analysis. The Gfi1 expression dramatically decreased in SH-2251-treated Th2 cells, and the SH-2251-mediated inhibition of IL-5-producing Th2 cell differentiation was restored by transduction of <i>Gfi1</i>. Therefore, our study unearthed SH-2251 as a novel therapeutic candidate for allergic inflammation that selectively inhibits active histone marks at the <i>Il5</i> gene locus.</p></div

Reduction of IFN-γand Th2 cytokine production in <i>Gfi1</i>-deficient iNKT cells.

<p>The results of the quantitative RT-PCR analysis of cytokine mRNA in iNKT cells from the spleen <b>(A)</b>, liver <b>(B)</b> and lung <b>(C)</b> of WT and <i>Gfi1</i>-deficient mice. The iNKT cells were purified by FACS sorting and stimulated with PMA plus ionomycin for 2h. The results are presented relative to the mRNA expression of <i>18s</i> ribosomal RNA with the standard deviation (n = 3). NS; not significant, *P<0.05, **P<0.01 (Student’s <i>t</i>-test). <b>(D)</b> The results of the intracellular staining of the indicated cytokines in iNKT cells of the indicated organs. iNKT cells were purified by FACS sorting, and stimulated with PMA plus ionomycin in the presence of monensin for 4 h. The mean fluorescence intensities of the indicated cytokine stainings are shown. <b>(E)</b> The results of the intracellular staining of IL-4/IFN-γin the iNKT cells of the spleen and liver. iNKT cells were purified by FACS sorting, and stimulated with PMA plus ionomycin in the presence of monensin for 4 h.</p

Gfi1 controls the transition from stage 2 to stage 3 during thymic iNKT cell development.

<p><b>(A)</b> The results of the quantitative RT-PCR analysis of the <i>Gfi1</i> mRNA expression in thymic iNKT cells. The developmental stage of iNKT cells was defined by the expression of CD24, CD44 and NK1.1. The results are presented relative to the mRNA expression of <i>18s</i> ribosomal RNA with the standard deviation (n = 3). <b>(B, C, D</b> and <b>E)</b> The results of the flow cytometric analyses of thymic iNKT cells in <i>Gfi1</i> KO mice. A typical CD3εand α-GalCer-loaded CD1d tetramers pattern is shown <b>(B)</b>. The typical pattern of CD24 and α-GalCer-loaded CD1d tetramers (left) and the absolute numbers with the standard deviation of stage 0 (CD24^pos) iNKT cells (right, n = 3) is indicated <b>(C)</b>. The CD1d-tetramer^pos thymocytes were enriched using a magnetic cell sorter and stained with anti-CD24 and anti-TCRVβ8 mAbs. The typical staining pattern of CD24 and α-GalCer-loaded CD1d tetramers (left), TCRVβ8 gated on CD24^high, α-GalCer-loaded CD1d tetramer^pos cells (middle), and the absolute numbers with the standard deviation of Vβ8^pos, CD24^high, and α-GalCer-loaded CD1d tetramer^pos iNKT cells (right, n = 3) are indicated <b>(D)</b>. The typical pattern of CD24 and NK1.1 gated on α-GalCer-loaded CD1d tetramer-positive iNKT cells (left) and the absolute numbers with the standard deviation of stage 1–2 (CD24^lowNK1.1^neg) iNKT cells and stage 3 (CD24^lowNK1.1^pos) iNKT cells (right, n = 3) is indicated <b>(E)</b>. <b>(F)</b> The typical pattern of CD44 and NK1.1 gated on α-GalCer-loaded CD1d tetramer-positive iNKT cells (left) and the absolute numbers with the standard deviation of stage 1 (CD44^lowNK1.1^neg), stage 2 (CD44^highNK1.1^neg) and stage 3 (CD44^highNK1.1^pos) iNKT cells (right, n = 3) is indicated. <b>(G)</b> The results of the quantitative RT-PCR analysis of the <i>Il17rb</i> mRNA expression in WT and <i>Gfi1</i>-deficient thymic iNKT cells. The results are presented relative to the mRNA expression of <i>18s</i> ribosomal RNA with the standard deviation (n = 3). <b>(H)</b> The absolute numbers of CD4^posCD8^pos, CD4^posCD8^neg, CD4^negCD8^pos and CD4^negCD8^neg iNKT cells in WT and <i>Gfi1</i> KO mice (n = 5 per group). NS; not significant, *P<0.05, **P<0.01 (Student’s <i>t</i>-test).</p

The expression profile of transcriptional regulators in <i>Gfi1</i>-deficient iNKT cells.

<p>The results of the quantitative RT-PCR analysis of transcriptional regulators in WT and <i>Gfi1</i>-deficient iNKT cells from the thymus <b>(A)</b>, spleen <b>(B)</b>, liver <b>(C)</b> and lung <b>(D)</b>. Each population was purified by FACS sorting. The results are presented relative to the mRNA expression of <i>18s</i> ribosomal RNA with the standard deviation (n = 3). <b>(E)</b> The results of the intracellular FACS analysis of Eomes, Gata3, Plzf, Rorγt and T-bet in iNKT cells from the indicated organs of WT and <i>Gfi1</i>-deficient mice. Three independent experiments were performed with similar results. NS; not significant, *P<0.05, **P<0.01 (Student’s <i>t</i>-test).</p

Decreased CD4^pos iNKT cell numbers in the peripheral organs of T cell-specific <i>Gfi1</i>-deficient mice.

<p><b>(A)</b> The results of the flow cytometric analyses of iNKT cells from the lung, liver and spleen of T cell-specific <i>Gfi1</i>-deficient (<i>Gfi1</i> KO) mice. <b>(B)</b> The absolute number of iNKT cells in the lung, liver and spleen of wild-type (WT) and <i>Gfi1</i> KO mice (n = 4 for each group). <b>(C)</b> The results of the flow cytometric analyses of the CD4 and CD8 expression on WT and Gfi1 KO iNKT cells in the lung, liver and spleen. <b>(D)</b> The absolute number with the standard deviation of CD4^pos (CD4 SP) and CD4^negCD8^neg (DN) iNKT cells in the lung, liver and spleen of WT and <i>Gfi1</i> KO mice (n = 5 for each group). NS; not significant, *P<0.05, **P<0.01 (Student’s <i>t</i>-test).</p

The induction of active histone marks at the <i>Il5</i> gene locus is inhibited by SH-2251.

<p><b>(A)</b>, Naïve CD4 T cells were cultured under Th2-conditions for five days in the presence of the indicated concentration of SH-2251, and a ChIP assay was performed with the indicated antibodies. The relative intensity (/Input) is shown with the standard deviation. <b>(B)</b>, The global patterns of histones H3K4me3 and H3K27ac at the Th2 cytokine gene loci were determined using ChIP-sequencing. <b>(C)</b>, The indicated histone modification status around the <i>Il5</i> gene locus in the SH-2251-treated Th2 cells was determined using a manual ChIP assay. The relative intensity (/Input) is shown with the standard deviation. <b>(D)</b>, Recruitment of RNA polymerase II around the <i>Il5</i> gene locus (upper panel) was determined using a manual ChIP assay. The relative intensity (/Input) is shown with the standard deviation. The transcripts around the <i>Il5</i> gene in the SH-2251-treated Th2 cells (lower panel) were determined using quantitative RT-PCR. The relative intensity (/<i>Hprt</i>) is shown with the standard deviation. Four independent experiments (A, C and D) were performed with similar results.</p

The development of iNKT1 (Plzf^lowT-bet^high), iNKT2 (Plzf^highT-bet^lowh) and iNKT17 (Rorγt^high) cells in <i>Gfi1</i>-deficient mice.

<p>iNKT cells were purified by FACS sorting and the intracellular staining of the indicated transcription factors was performed. The expression of Plzf and T-bet in the thymus <b>(A)</b> and the indicated peripheral organs <b>(C)</b>, and the patterns of Plzf and Rorγt in the thymus <b>(B)</b> and the indicated peripheral organs <b>(D)</b> are shown.</p

OVA-induced airway inflammation is attenuated by oral administration of SH-2251.

<p><b>(A)</b>, Decreased infiltration of eosinophils in the BAL fluid of asthmatic SH-2251-administered mice. The absolute numbers of eosinophils (Eos.), neutrophils (Neu.), lymphocytes (Lym.) and macrophages (Mac.) in the BAL fluid are shown with standard deviations (n = 5 per group). *<i>P</i><0.01 and **<i>P</i><0.001 by ANOVA and the Bonferroni-test. (B), Quantitative RT-PCR of <i>Il4</i>, <i>Il5</i> and <i>Il13</i> mRNA in the BAL fluid cells of vehicle and SH-2251-administered mice. (C), Quantitative RT-PCR of <i>Il4</i>, <i>Il5</i>, <i>Il13</i> and <i>Ifnγ</i> mRNA in the lung CD4 T cells of vehicle and SH-2251-administered mice. (n = 5 per group). (D), Cytokine production from lung CD4 T cells of vehicle and SH-2251-administered mice stimulated <i>in vitro</i>. The lung CD4 T cells were stimulated with immobilized anti-TCR-β mAb for 48 hours and the concentrations of cytokines in the culture supernatants were determined using ELISA. The lungs were fixed and stained with hematoxylin and eosin (E, left) or periodic acid-Schiff reagent (F). The scale bars represent 500 µm. The numbers of infiltrated leukocytes in the peribronchiolar regions are shown (mean cell numbers/mm²) (E, right). Three independent experiments were performed with similar results. Student's <i>t</i>-test was used for the statistical analyses. *<i>P</i><0.05 and **<i>P</i><0.01 (B, C, D and E)</p

The expression and functions of Gata3 are not impaired by treatment with SH-2251.

<p><b>(A)</b>, The mRNA expression of Gata3 in the SH-2251-trearted Th2 cells was determined using quantitative RT-PCR. The relative intensity (/<i>Hprt</i>) is shown with the standard deviation. <b>(B)</b>, The protein expression level of Gata3 was determined with immunoblotting. The nuclear (Gata3) and cytoplasmic (α-Tubulin) lysates with a three fold serial dilution were used. Three independent experiments (A and B) were performed with similar results. <b>(C)</b>, The global patterns of Gata3 binding at the Th2 cytokine gene loci (upper panel) and the <i>Il5</i> gene locus (lower panel) were determined using ChIP-sequencing with an anti-Gata3 pAb. The locations of PCR primer pairs (triangle) used in a manual ChIP assay are also listed. <b>(D)</b>, The binding of Gata3 around the <i>Il5</i> gene locus (left) panel and the V_A enhancer (V_A E) and intronic enhancer (IE) regions of the <i>Il4</i> gene locus (right panel) in the SH-2251-treated Th2 cells was determined using a manual ChIP assay. The relative intensity (/Input) is shown with the standard deviation. Three independent experiments were performed with similar results. <b>(E)</b>, The effects of SH-2251 on the Gata3-dependent transcriptional activation of the <i>Il5</i> promoter were determined using a Dual luciferase assay. The mean and standard deviation of the relative luciferase activity of three different experiments are shown. Stim: PMA (30 ng/ml)+dbcAMP (100 µM). Four independent experiments (A, B, D and E) were performed with similar results.</p