Nicole Boucheron

Conditional Deletion of Histone Deacetylase 1 in T Cells Leads to Enhanced Airway Inflammation and Increased Th2 Cytokine Production

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. 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.

The Role of Tec Family Kinases in Myeloid Cells

Members of the Tec kinase family (Bmx, Btk, Itk, Rlk and Tec) are primarily expressed in the hematopoietic system and form, after the Src kinase family, the second largest class of non-receptor protein tyrosine kinases. During lymphocyte development and activation Tec kinases have important functions in signaling pathways downstream of the antigen receptors. Tec family kinases are also expressed in cells of the myeloid lineage. However, with the exception of mast cells and platelets, their biological role in the myeloid system is only poorly understood. This review summarizes the current knowledge about the function of Tec family kinases in hematopoietic cells of the myeloid lineage.

The transcriptional regulator PLZF induces the development of CD44 high memory phenotype T cells

Transcriptional pathways controlling the development of CD44hi memory phenotype (MP) T cells with “innate-like” functions are not well understood. Here we show that the BTB (bric-a-brac, tramtrack, broad complex) domain-containing protein promyelocytic leukemia zinc finger (PLZF) is expressed in CD44hi, but not in CD44lo, CD4+ T cells. Transgenic expression of PLZF during T cell development and in CD4+ and CD8+ T cells induced a T cell intrinsic program leading to an increase in peripheral CD44hi MP CD4+ and CD8+ T cells and a corresponding decrease of naïve CD44lo T cells. The MP CD4+ and CD8+ T cells produced IFNγ upon PMA/ionomycin stimulation, thus showing innate-like function. Changes in the naïve versus memory-like subset distribution were already evident in single-positive thymocytes, indicating PLZF-induced T cell developmental alterations. In addition, CD1d-restricted natural killer T cells in PLZF transgenic mice showed impaired development and were severely reduced in the periphery. Finally, after anti-CD3/CD28 stimulation, CD4+ transgenic T cells showed reduced IL-2 and IFNγ production but increased IL-4 secretion as a result of enhanced IL-4 production of the CD44hiCD62L+ subset. Our data indicate that PLZF is a novel regulator of the development of CD44hi MP T cells with a characteristic partial innate-like phenotype.

Cd8 enhancer E8I and Runx factors regulate CD8α expression in activated CD8+ T cells

Cd8a and Cd8b1 coreceptor gene (Cd8) expression is tightly controlled during T-cell development by the activity of five Cd8 enhancers (E8I–E8V). Here we demonstrate a unique transcriptional program regulating CD8 expression during CD8+ effector T-cell differentiation. The Cd8 enhancer E8I and Runx/core-binding factor-β (CBFβ) complexes were required for the establishment of this regulatory circuit, because E8I-, Runx3-, or CBFβ-deficient CD8+ T cells down-regulated CD8α expression during activation. This finding correlated with enhanced repressive histone marks at the Cd8a promoter in the absence of E8I, and the down-regulation of CD8α expression could be blocked by treating E8I-, Runx3-, or CBFβ-deficient CD8+ T cells with the histone deacetylase inhibitor trichostatin A. Moreover, Runx/CBFβ complexes bound the Cd8ab gene cluster in activated CD8+ T cells, suggesting direct control of the Cd8a locus. However, CD8+ effector T cells maintained high levels of CD8α when CBFβ was conditionally deleted after activation. Thus, our data suggest an E8I- and Runx3/CBFβ-dependent epigenetic programming of the Cd8a locus during T-cell activation, leading to Runx/CBFβ complex-independent maintenance of CD8α expression in effector T cells.

Cross-Talk Between Interferon-γ and Hedgehog Signaling Regulates Adipogenesis

OBJECTIVE T cells and level of the cytokine interferon-γ (IFN-γ) are increased in adipose tissue in obesity. Hedgehog (Hh) signaling has been shown to potently inhibit white adipocyte differentiation. In light of recent findings in neurons that IFN-γ and Hh signaling cross-talk, we examined their potential interaction in the context of adipogenesis. RESEARCH DESIGN AND METHODS We used Hh reporter cells, cell lines, and primary adipocyte differentiation models to explore costimulation of IFN-γ and Hh signaling. Genetic dissection using Ifngr1−/− and Stat1−/− mouse embryonic fibroblasts, and ultimately, anti–IFN-γ neutralization and expression profiling in obese mice and humans, respectively, were used to place the findings into the in vivo context. RESULTS T-cell supernatants directly inhibited hedgehog signaling in reporter and 3T3-L1 cells. Intriguingly, using blocking antibodies, Ifngr1−/− and Stat1−/− cells, and simultaneous activation of Hh and IFN-γ signaling, we showed that IFN-γ directly suppresses Hh stimulation, thus rescuing adipogenesis. We confirmed our findings using primary mouse and primary human (pre)adipocytes. Importantly, robust opposing signals for Hh and T-cell pathways in obese human adipose expression profiles and IFN-γ depletion in mice identify the system as intact in adipose tissue in vivo. CONCLUSIONS These results identify a novel antagonistic cross-talk between IFN-γ and Hh signaling in white adipose tissue and demonstrate IFN-γ as a potent inhibitor of Hh signaling.

CD4 + T cell lineage integrity is controlled by the histone deacetylases HDAC1 and HDAC2

Molecular mechanisms that maintain lineage integrity of helper T cells are largely unknown. Here we show histone deacetylases 1 and 2 (HDAC1 and HDAC2) as crucial regulators of this process. Loss of HDAC1 and HDAC2 during late T cell development led to the appearance of major histocompatibility complex (MHC) class II–selected CD4+ helper T cells that expressed CD8-lineage genes such as Cd8a and Cd8b1. HDAC1 and HDAC2–deficient T helper type 0 (TH0) and TH1 cells further upregulated CD8-lineage genes and acquired a CD8+ effector T cell program in a manner dependent on Runx-CBFβ complexes, whereas TH2 cells repressed features of the CD8+ lineage independently of HDAC1 and HDAC2. These results demonstrate that HDAC1 and HDAC2 maintain integrity of the CD4 lineage by repressing Runx-CBFβ complexes that otherwise induce a CD8+ effector T cell–like program in CD4+ T cells.

Transcriptional signatures of Itk-deficient CD3+, CD4+ and CD8+ T-cells

BackgroundThe Tec-family kinase Itk plays an important role during T-cell activation and function, and controls also conventional versus innate-like T-cell development. We have characterized the transcriptome of Itk-deficient CD3+ T-cells, including CD4+ and CD8+ subsets, using Affymetrix microarrays.ResultsThe largest difference between Itk-/- and Wt CD3+ T-cells was found in unstimulated cells, e.g. for killer cell lectin-like receptors. Compared to anti-CD3-stimulation, anti-CD3/CD28 significantly decreased the number of transcripts suggesting that the CD28 co-stimulatory pathway is mainly independent of Itk. The signatures of CD4+ and CD8+ T-cell subsets identified a greater differential expression than in total CD3+ cells. Cyclosporin A (CsA)-treatment had a stronger effect on transcriptional regulation than Itk-deficiency, suggesting that only a fraction of TCR-mediated calcineurin/NFAT-activation is dependent on Itk. Bioinformatic analysis of NFAT-sites of the group of transcripts similarly regulated by Itk-deficiency and CsA-treatment, followed by chromatin-immunoprecipitation, revealed NFATc1-binding to the Bub1, IL7R, Ctla2a, Ctla2b, and Schlafen1 genes. Finally, to identify transcripts that are regulated by Tec-family kinases in general, we compared the expression profile of Itk-deficient T-cells with that of Btk-deficient B-cells and a common set of transcripts was found.ConclusionTaken together, our study provides a general overview about the global transcriptional changes in the absence of Itk.

The AMP analog AICAR modulates the Treg/Th17 axis through enhancement of fatty acid oxidation

T cells must tightly regulate their metabolic processes to cope with varying bioenergetic demands depending on their state of differentiation. The metabolic sensor AMPK is activated in states of low energy supply andmodulates cellular metabolism toward a catabolic state. Although this enzyme isknown tobeparticularly active in regulatory T (Treg) cells, its impact on T helper (Th)‐cell differentiation is poorly understood. We investigated the impact of several AMPK activators on Treg‐cell differentiation and found that the direct activator AICAR (5‐aminoimidazole‐4‐carboxamide ribonucleotide), but not the indirect activators metformin and 2‐deoxyglucose, strongly enhanced Treg‐cell induction by specifically enhancing Treg‐cell expansion. Conversely, Th17 generation was impaired by the agent. Further investigation of the metabolic background of our observations revealed that AICAR enhanced both cellular mitochondrogenesis and fatty acid uptake. Consistently, increased Treg induction was entirely reversible on inhibition of fatty acid oxidation, thus confirming the dependence of AICARs effects on metabolic pathways alterations. Translating our findings to an in vivo model, we found that the substance enhanced Treg cell generation on IL‐2 complex–induced immune stimulation. We provide a previously unrecognized insight into the delicate interplay between immune cell function and metabolism and delineate a potential novel strategy for metabolism‐targeting immunotherapy.—Gualdoni, G. A., Mayer, K. A., Göschl, L., Boucheron, N., Ellmeier, W., Zlabinger, G. J. The AMP analog AICAR modulates the Treg/Th17 axis through enhancement of fatty acid oxidation. FASEB J. 30, 3800–3809 (2016) www.fasebj.org

The protein tyrosine kinase Tec regulates mast cell function.

Mast cells play crucial roles in a variety of normal and pathophysiological processes and their activation has to be tightly controlled. Here, we demonstrate that the protein tyrosine kinase Tec is a crucial regulator of murine mast cell function. Tec was activated upon FcεRI stimulation of BM‐derived mast cells (BMMC). The release of histamine in the absence of Tec was normal in vitro and in vivo; however, leukotriene C4 levels were reduced in Tec−/− BMMC. Furthermore, the production of IL‐4 was severely impaired, and GM‐CSF, TNF‐α and IL‐13 levels were also diminished. Finally, a comparison of WT, Tec−/−, Btk−/− and Tec−/−Btk−/− BMMC revealed a negative role for Btk in the regulation of IL‐4 production, while for the efficient production of TNF‐α, IL‐13 and GM‐CSF, both Tec and Btk were required. Our results demonstrate a crucial role for Tec in mast cells, which is partially different to the function of the well‐characterized family member Btk.

The Protein Tyrosine Kinase Tec Regulates a CD44highCD62L− Th17 Subset

The generation of Th17 cells has to be tightly controlled during an immune response. In this study, we report an increase in a CD44highCD62L− Th17 subset in mice deficient for the protein tyrosine kinase Tec. CD44highCD62L− Tec−/− CD4+ T cells produced enhanced IL-17 upon activation, showed increased expression levels of IL-23R and RORγt, and IL-23–mediated expansion of Tec−/− CD4+ T cells led to an increased production of IL-17. Tec−/− mice immunized with heat-killed Streptococcus pneumoniae displayed increased IL-17 expression levels in the lung postinfection with S. pneumoniae, and this correlated with enhanced pneumococcal clearance and reduced lung inflammation compared with Tec+/+ mice. Moreover, naive Tec−/− OT-II CD4+ T cells produced higher levels of IL-17 when cultured with OVA peptide-loaded bone marrow-derived dendritic cells that have been previously activated with heat-killed S. pneumoniae. Taken together, our data indicated a critical role for Tec in T cell-intrinsic signaling pathways that regulate the in vivo generation of CD44highCD62L− effector/memory Th17 populations.